U.S. patent application number 17/543245 was filed with the patent office on 2022-05-26 for compositions and methods for cancer therapy.
This patent application is currently assigned to UNIVERSITY OF IOWA RESEARCH FOUNDATION. The applicant listed for this patent is UNIVERSITY OF IOWA RESEARCH FOUNDATION. Invention is credited to Ivana Frech, Guido Tricot, Fenghuang Zhan.
Application Number | 20220160679 17/543245 |
Document ID | / |
Family ID | 1000006137627 |
Filed Date | 2022-05-26 |
United States Patent
Application |
20220160679 |
Kind Code |
A1 |
Zhan; Fenghuang ; et
al. |
May 26, 2022 |
COMPOSITIONS AND METHODS FOR CANCER THERAPY
Abstract
The invention provides compositions and methods to treat
relapsed multiple myeloma with pharmacological ascorbic acid or a
pharmaceutically acceptable salt thereof, and one or more
anti-cancer therapies.
Inventors: |
Zhan; Fenghuang; (Iowa City,
IA) ; Frech; Ivana; (Iowa City, IA) ; Tricot;
Guido; (Iowa City, IA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
UNIVERSITY OF IOWA RESEARCH FOUNDATION |
Iowa City |
IA |
US |
|
|
Assignee: |
UNIVERSITY OF IOWA RESEARCH
FOUNDATION
Iowa City
IA
|
Family ID: |
1000006137627 |
Appl. No.: |
17/543245 |
Filed: |
December 6, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16307240 |
Dec 5, 2018 |
11298338 |
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PCT/US2017/036146 |
Jun 6, 2017 |
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17543245 |
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62346271 |
Jun 6, 2016 |
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62447293 |
Jan 17, 2017 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/496 20130101;
A61K 45/06 20130101; A61P 35/00 20180101; A61K 31/381 20130101;
A61K 31/4184 20130101; A61K 31/5377 20130101; A61K 9/0019 20130101;
A61K 31/198 20130101; A61K 31/375 20130101 |
International
Class: |
A61K 31/375 20060101
A61K031/375; A61K 31/198 20060101 A61K031/198; A61P 35/00 20060101
A61P035/00; A61K 45/06 20060101 A61K045/06; A61K 31/381 20060101
A61K031/381; A61K 31/4184 20060101 A61K031/4184; A61K 31/496
20060101 A61K031/496; A61K 31/5377 20060101 A61K031/5377; A61K 9/00
20060101 A61K009/00 |
Claims
1. A method of treating a hyperproliferative disorder associated
with high intracellular iron comprising administering
pharmacological ascorbic acid or a pharmaceutically acceptable salt
thereof and melphalan, wherein the pharmacological ascorbic acid is
administered at a dose of about 15 g-100 g, wherein the
hyperproliferative disorder is relapsed multiple myeloma.
2. The method of claim 1, wherein the melphalan is administered at
a dosage of about 2 mg/m.sup.2 to 200 mg/m.sup.2.
3. The method of claim 1, wherein the melphalan is administered at
a dosage of about 50 mg/m.sup.2 and 100 mg/m.sup.2.
4. The method of claim 1, wherein the pharmacological ascorbic acid
and the melphalan are administered simultaneously or
sequentially.
5. The method of claim 1, further comprising administering a
proteasome inhibitor.
6. The method of claim 5, wherein the pharmacological ascorbic
acid, the melphalan and the proteasome inhibitor are administered
simultaneously or sequentially in any order.
7. The method of claim 1, further comprising administering an
anti-cancer therapy.
8. The method of claim 7, wherein the anti-cancer therapy is
immunotherapy or biologic therapy.
Description
RELATED APPLICATION
[0001] This application is a continuation of U.S. patent
application Ser. No. 16/307,240, filed Dec. 5, 2018, which is a 35
U.S.C. .sctn. 371 application of International Application Serial
No. PCT/US2017/036146 that was filed on Jun. 6, 2017, and claims
priority to U.S. Provisional Application No. 62/346,271 that was
filed on Jun. 6, 2016, and U.S. Provisional Application No.
62/447,293 that was filed on Jan. 17, 2017. The entire content of
the applications referenced above are hereby incorporated by
reference.
BACKGROUND
[0002] Most treatment plans for patients with cancer include
surgery, radiation therapy, and/or chemotherapy. Early clinical
trials were performed for the use of vitamin C (ascorbic acid) to
treat cancer. But epidemiological studies evaluating the
association between the intake of vitamin C and cancer risk
produced inconsistent results. (Luo, et al., Association between
vitamin C intake and lung cancer: a dose-response meta-analysis,
Sci Rep. 2014 Aug. 22; 4:6161). Other studies determined that no
significant differences were noted between the ascorbate-treated
and placebo-treated groups for symptoms, performance status, or
survival (Moertel C G, Fleming T R, Creagan E T, Rubin J, O'Connell
M J, Ames M M. High-dose vitamin C versus placebo in the treatment
of patients with advanced cancer who have had no prior
chemotherapy. A randomized double-blind comparison. N Engl J Med.
1985; 312(3):137-41; Creagan E T, Moertel C G, O'Fallon J R, Schutt
A J, O'Connell M J, Rubin J, Frytak S. Failure of high-dose vitamin
C (ascorbic acid) therapy to benefit patients with advanced cancer.
A controlled trial. N Engl J Med. 1979; 301(13):687-90). There is a
need for more efficacious cancer treatments with minimal side
effects.
SUMMARY
[0003] The present invention provides in certain embodiments a
method of treating a hyperproliferative disorder associated with
high intracellular iron comprising administering pharmacological
ascorbic acid (PAA) or a pharmaceutically acceptable salt thereof.
As used herein the term "high iron" means that the intracellular
free iron concentration is greater than the in a corresponding
non-tumor cell.
[0004] The present invention provides in certain embodiments a
method of reducing toxic effects of melphalan in a patient in need
thereof comprising administering pharmacological ascorbic acid
(PAA) or a pharmaceutically acceptable salt thereof.
[0005] The present invention provides in certain embodiments a
method of treating multiple myeloma, including smoldering multiple
myeloma, comprising administering pharmacological ascorbic acid
(PAA) or a pharmaceutically acceptable salt thereof.
[0006] The present invention provides in certain embodiments a use
of the combination of pharmacological ascorbic acid (PAA) or a
pharmaceutically acceptable salt thereof and melphalan in the
preparation of a medicament for the treatment of a
hyperproliferative disorder in a mammal.
[0007] The present invention provides in certain embodiments a kit
comprising pharmacological ascorbic acid (PAA) or a
pharmaceutically acceptable salt thereof and melphalan, a
container, and a package insert or label indicating the
administration of the PAA and with melphalan for treating a
hyperproliferative disorder.
[0008] The present invention provides in certain embodiments a
product comprising pharmacological ascorbic acid (PAA) and
melphalan as a combined preparation for separate, simultaneous or
sequential use in the treatment of a hyperproliferative
disorder.
[0009] The present invention provides in certain embodiments a
therapeutic composition comprising a combination of (a)
pharmacological ascorbic acid (PAA) or a pharmaceutically
acceptable salt thereof and (b) an alkylating agent. In certain
embodiments, the therapeutic composition lacks a chelator, such as
ethylene diamine tetraacetic acid (EDTA).
[0010] The present invention provides in certain embodiments, a
method of administering to a mammalian cell having downregulated
expression of Ferroportin 1 (Fpn1) as compared with its normal
counterpart cell an expression-modulating agent, comprising
contacting the mammalian cell with pharmacological ascorbic acid
(PAA) or a pharmaceutically acceptable salt thereof.
[0011] The present invention provides in certain embodiments, a
method of administering to a mammalian cell having upregulated
expression of enhancer of zeste 2 (EZH2) as compared with its
normal counterpart cell an expression-modulating agent, comprising
contacting the mammalian cell with an inhibitor of EZH2.
[0012] The present invention provides in certain embodiments, a
method of administering to a mammalian cell having upregulated
expression of Thyroid Hormone Receptor Interactor Protein 13
(TRIP13) as compared with its normal counterpart cell an
expression-modulating agent, comprising contacting the mammalian
cell with pharmacological ascorbic acid (PAA) or a pharmaceutically
acceptable salt thereof and/or with an inhibitor of TRIP13.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIGS. 1A-1G. Pharmacologic ascorbic acid selectively kills
tumor cells in MM and synergistically acts with melphalan in vivo.
(FIG. 1A) CD138+ tumor cells and CD138- non-tumor cells were
treated with either PAA (1, 2, 4, 8, 20 mM) or PBS (control) from
MM patients and (FIG. 1B) and (FIG. 1C) respectively from SMM and
MGUS patients. (FIG. 1D) Xenografted NOD.C.gamma.-Rag1 mice were
treated with PAA and in combination or not with melphalan,
carfizomib and bortezomib. After one-week injection of ARP1 cells,
mice were treated with either PAA (4 mg/kg) injected
intraperitoneal once a day, 5 days every week for 3 weeks.
Melphalan (3 mg/kg) was injected intraperitoneal once a day, 2 days
a week for 3 weeks. Carfizomib (3 mg/kg) was injected by in vein
once a day, 2 days every week for 3 weeks. Total flux indicates
quantification of luciferase intensity (tumor burden) of mice pre-
and post-PAA treatment at different time points. (FIG. 1E &
FIG. 1F) Tumor burden was analyzed in ARP1 NOD.C.gamma.-Rag1 mice
treated with PAA and with or without different doses of melphalan
(1, 3, 5 mg/kg). (FIG. 1G) Treatment-related survival curve of
mice. The log-rank test was performed and indicated that mouse
survivals among these groups are significantly different
(p<0.001) and PAA when combined with low dose of melphalan
extends MM mouse survival.
[0014] FIGS. 2A-2D. Pharmacologic ascorbic acid targets reactive
oxidative species and labile iron pool. (FIG. 2A) OCI-MY5 WT cells
were incubated with or without catalase (100 U/ml), NAC (15 mM) or
DFO (200 .mu.M) for 3 hrs following treatment with PAA. PAA was
washed away after 1 hr treatment and cell viability was determined
24 hrs later. (FIG. 2B) OCI-MY5 EV and OCI-MY5 OE-Fpn1 were treated
with or without PAA (0-20 mM). PAA was washed away after 1 hr and
cell viability was measured 24 hrs later. OCI-MY5 EV (FIG. 2C) and
OCI-MY5 OE-Fpn1 (FIG. 2D) were incubated with or without iron
(Fe-NTA (FE), 100 .mu.M). After 18 hrs cells were treated with or
without DFO (200 .mu.M) for 3 hrs followed by PAA treatment for 1
hr and cell viability was measured as described in FIG. 2A.
[0015] FIGS. 3A-3B. Pharmacologic ascorbic acid induces
mitochondria-mediated apoptosis in MM cells. (FIG. 3A) Transmission
electron microscopy of OCI-MY5 WT cells treated with or without PAA
(4 mM). After 1 hr incubation, PAA was washed away and cells were
fixed for TEM after 60 min and 120 min. Red boxes represent zooming
image of mitochondria in OCI-MY5 WT cells (left) and OCI-MY5 WT
cells treated with PA (right). (FIG. 3B) OCI-MY5 WT cells were
treated with or without PAA. After 1 hr, PAA was washed away and
cells were lysed at the specified times and RIP1, RIP3, Caspase 3,
Caspase 8, Caspase 9, and .beta.-actin levels were examined by
western blots.
[0016] FIGS. 4A-4E. Pharmacologic ascorbic acid induces AIF1
release from mitochondria. (FIG. 4A) Top bar graph represents
OCI-MY5 shRNA-Scramble and shRNA-AIF1 cells incubated with
doxycycline to knockdown AIF1 for 2 days. Bottom bar graph
represents OCI-MY5 EV and OCI-MY5 OE-AIF1 cells. All cells were
treated without or with PAA at the specified concentrations. After
1 hr treatment, PAA was washed away and cells viability was
measured after 24 hrs. Knockdown and overexpression of AIF1 was
confirmed by western blots. (FIG. 4B) Schematic representation of
PAA inducing AIF1 cleavage, release and nuclear translocation in MM
tumor cells. (FIG. 4C) OCI-MY5 WT cells with or without PAA. After
1 hr PAA was washed away and cells were incubated with melphalan
(Mel, 0-80 .mu.M) for 4 hrs then lysed. AIF1, .beta.-actin and
.gamma.-H2AX levels were analyzed by western blots. (FIG. 4D)
OCI-MY5 WT cells were incubated with or without DFO (200 .mu.M) for
3 hrs followed PAA (2 mM) treatment. After 1 hr PAA was washed away
and cells were lysed. AIF1 and .beta.-actin levels were analyzed by
western blots. (FIG. 4E) Electron microscope shows AIF1
immunolabeling staining of OCI-MY5 WT cells treated without (left)
or with (right) PAA (2 mM). N, M, C respectively represent nucleus,
mitochondria and cytoplasm. Blue arrows indicate the nuclear
membrane and red arrowheads indicate AIF1 gold beads in cytoplasm
or mitochondria. Black arrowheads indicate AIF1 gold beads in
nuclei.
[0017] FIG. 5. Pie chart of patients' diagnosis.
[0018] FIGS. 6A-6B. Box charts of iron transporter expression
profiles show dysregulation between normal plasma cells and MM
cells. The 22 normal plasma cell (NPC), 44 MGUS, and 351 newly
diagnosed MM samples are distributed along the x-axis and the log
2-transformed Affymetrix Signal is plotted on the y-axis. The top,
bottom, and middle lines of each box correspond to the 75th
percentile (top quartile), 25th percentile (bottom quartile), and
50th percentile (median) of the log 2-transformed Affymetrix Signal
for each gene, respectively. The whiskers extend from the 10th
percentile (bottom decile) and top 90th percentile (top decile).
The One-Way ANOVA tests for differences in expression of each gene
across the groups are: TfR1, p<0.001; FPN1, p<0.001.
[0019] FIG. 7. Combination of PAA with melphalan does not change
body weight. Six groups of ARP1 xenografted NOD.C.gamma.-Rag1 mice
were treated with PAA and with or without melphalan (1, 3, and 5
mg/kg) and body weight was determined at the specified time.
[0020] FIG. 8. Combination of PAA with melphalan increases MM mouse
survival. An IVIS shows ARP1 cell growth in xenografted
NOD.C.gamma.-Rag1 mice with or without PAA treatment (4 g/kg, i.p.
once a day, 5 days a week for 3 weeks). Total flux after PAA
treatment was normalized with pre-PAA treatment and indicates
quantification of luciferase intensity of mice post-PAA treatment.
ARP1 xenografted NOD.C.gamma.-Rag1 mice were treated with PAA or
melphalan alone or in combination (1, 3, and 5 mg/kg). Total flux
indicates quantification of luciferase intensity of mice pre- and
post-PAA treatment at different time points.
[0021] FIGS. 9A-9B. BCS does not block PAA anti-cancer activity.
(FIG. 9A) OCI-MY5 EV and OE cells were incubated with or without
BCS (10 .mu.M) for 3 hrs following PAA treatment (4 mM). PAA was
washed away after 1 hr and cell viability was determined 24 hrs
later. (FIG. 9B) Total RNA was extracted from OCI-MY5 EV and OE
cells and Fpn1 mRNA was analyzed by Real time RT-PCR.
[0022] FIG. 10. Iron Homeostasis is altered in Myeloma Patients.
Supervised cluster analysis of iron signature genes in normal
plasma (Normal) and Multiple myeloma cells. Arrow indicates
SLC40A1, the Fpn1 gene.
[0023] FIG. 11: Ferroportin 1 is Downregulated in MM Tumor Cells.
Scatter plots depict the Affymetrix signal of Fpn1 in normal plasma
cells (NPCs), MGUS, newly diagnosed multiple myeloma (MM; TT2
cohort), and multiple myeloma cell lines (MMCL). One-way ANOVA was
performed and identified the p<0.0001 among these four groups.
The p value presented in the figure was obtained by comparison
between NPC and indicated group, respectively.
[0024] FIGS. 12A and 12B. Low Expression of Ferroportin 1 is Linked
to Poor Patients Outcome in MM. (FIGS. 12A-B) Survival analysis
were performed based on Fpn1 expression in different cohorts. The
EFS (FIG. 12A) and OS (FIG. 12B) were performed in the TT2
cohort.
[0025] FIG. 13. Ferroportin 1 Regulates MM Intracellular Iron.
Cells overexpressing Fpn1 (FPN1) have lower intracellular LIP than
wild type cells (EV).
[0026] FIGS. 14A and 14B. Iron Retention Promotes Tumor Development
and Progression. 5TGM1-Fpn1 KaLwRij mice were administrated with or
without doxycycline and dextran-iron as indicated 1 week after cell
injection. (FIG. 14A) Kaplan-Meier showed the survival curves, and
pvalue was analyzed by the logrank test. (FIG. 14B) tumor burden
was measured by the ELISA assay, and the significance (p<0.0001)
was determined by one-way ANOVA.
[0027] FIG. 15. Iron Uptake and Efflux in non-Tumor and Multiple
Myeloma Tumor Cell. Left: Non-tumor cells show low levels of
transferrin receptor (iron uptake) and high levels of ferroportin
(iron efflux) to maintain low levels of cytosolic iron (ferritin,
iron storage). Right: Multiple Myeloma cells show higher level of
transferrin receptor and lower level of Ferroportin 1 leading to
higher cytosolic iron (ferritin) and free iron.
[0028] FIG. 16. Transferrin Receptor is Upregulated in MM Tumor
Cells. Affymetrix signal of TFRC in normal plasma cells (NPC), MGUS
and multiple myeloma (MM).
[0029] FIG. 17. Repression of Transferrin Receptor Leads to
SLC22A17 Upregulation in MM. Affymetrix signal of SLC22A17,
lipocalin-2 receptor, in MM patients' tumor cells either with high
or low TFRC expression.
[0030] FIG. 18. Schematic Representation of Co-Culture Between
Macrophages and MM Tumor Cell.
[0031] FIGS. 19A and 19B. Iron Retention Promotes Tumor Development
and Progression. FIG. 19A. Affymetrix signal of Fpn1 and EZH2 in
normal plasma cells (NPCs), newly diagnosed multiple myeloma (MM;
TT2 cohort) and low and high risk. p<0.0001 among these four
groups. FIG. 19B. Putative binding site of EZH2 on Fpn1 promoter.
Promoter was analyzed using whole genome association study
(GWAS).
[0032] FIG. 20. Schematic flow for the identification of candidates
Fpn1 repressor in MM.
[0033] FIG. 21. Model for Hepcidin-Mediated Ferroportin 1
Internalization, Degradation and Transcriptional Repression.
[0034] FIG. 22. Induction of Ferroptosis in MM Cells by Erastin.
Erastin blocks, via mitochondria, the cell's antioxidative defenses
and ultimately leads to an irondependent, oxidative cell death.
[0035] FIG. 23. Erastin Inhibits MM Cells Growth. KMS11, ARK and
ARP1 cells were treated with 10 .mu.M Erastin with or without
ferrostatin (Fer-1) at 1 .mu.M for 48 hours. Cell proliferation was
measured by PrestoBlue assay and normalized to control cells to
calculated growth inhibition.
[0036] FIG. 24: Pharmacological Ascorbic Acid Selectively Kills MM
Tumor Cells. CD138+ tumor cells and CD138- non-tumor cells from MM
patients were treated with either PAA (8, 20 mM) or PBS (control)
and cell viability was analyzed after 24 hours.
[0037] FIG. 25. Pharmacological Ascorbic Acid Anti-Cancer Activity
is Iron-Dependent. Xenografted NOD.C.gamma.-Rag1 mice were injected
with ARP1 cells. After one-week, mice were treated with either PAA
(4 mg/kg) injected intraperitoneal once a day, PAA and in
combination or not with DFO (100 mg/Kg, twice a week,
intraperitoneal) and DFO alone. Total flux indicates quantification
of luciferase intensity (tumor burden) of mice before (top panel)
and after treatment (bottom panel).
[0038] FIGS. 26A-26C. TRIP13 expression is increased in a subset of
newly diagnosed MM samples which link to a poor prognosis by GEP.
(A) The heatmap presents the expression of TRIP13 and other 9 CIN
genes related to MM drug resistance in 22 healthy subjects (NPC),
44 subjects with MGUS, 351 patients with newly diagnosed MM and 9
human MM cells lines (MMCL). Note: blue and pink (red) colors
represent lower or higher median expression across all samples
respectively. (B & C) High TRIP13 expression is linked to a
poor prognosis in myeloma. Kaplan-Meier analyses of event-free
survival (B) and overall survival (C) revealed inferior outcomes
from 351 cases in the TT2 trial.
[0039] FIGS. 27A-27F. Increased TRIP13 induces MM cell
proliferation and drug resistance. (A) The expression of TRIP13
mRNA and proteins is increased in MM cell lines ARP1, H929 and
OCI-MY5 with TRIP13 overexpression (OE) compared to the control
cells (EV). (B) Cell proliferation of ARP1, OCI-MY5 and H929 with
TRIP13-OE as well as their counterparts transfected with empty
vectors (EV) were counted for 3 consecutive days (p<0.05). (C
& D) Knockdown of TRIP13 (shRNA) inhibits MM cell growth
compared to the control (Scramble) in a xenograft mouse model using
ARP1 MM cells (C) and quantified (D). (E & F) Cell viabilities
of ARP1 cells with TRIP13-OE or EV were counted with indicated
concentrations of Bortezomib (E) or Etoposide (F) after 24 h.
[0040] FIGS. 28A-28C. TRIP13 is an oncogene. (A) NIH3T3 cells
transfected with empty vector (EV) or mouse TRIP13 (mTRIP13) were
assessed by anchorage-independent colony formations in soft agar.
(B) Images of NIH3T3 cells transfected with EV or mTRIP13 were
shown in soft agar under microscope with bright field and green
fluorescence (.times.4). (C) NIH3T3 cells transfected with EV or
mTRIP13 were subcutaneously injected into NOD Rag1.sup.null mice
and assessed for tumor formation at day 15.
[0041] FIGS. 29A-29F. Overview of the principal experimental model
system used for the studies in Aim 1A. (A) Schematic illustration
of adoptive B cell transfer from young, tumor-free CD45.2.sup.+
C.IL6iMyc mice. B cells are isolated (left), and genetically
modified in vitro (center), and transferred to sub-lethally
irradiated (4Gy) C.CD45.1.sup.+ congenic mice. (B) Flow cytometry
contour plots 138 days post B-cell transfer, indicating the
presence of CD45.2.sup.+CD138.sup.+ plasma cell tumors (PCT) in the
bone marrow of a CD45.1.sup.+ Balb/c (designated `C`) mouse. (C)
Histopathology of a representative CD45.2.sup.+ tumor (from a lymph
node in this case) from a CD45.1.sup.+ C mouse (H&E;
63.times.). (D) Tumor propagation in vivo. Shown is a serum
electropherogram containing the M-spike of a mouse harboring a
primary (G0) CD45.2.sup.+ PCT (lane 2) and the same M-spike (red
box) from a `C` mouse 5 weeks after transfer of one million tumor
cells (lane 3). A serum sample from a normal `C` mouse was included
as control (lane 1). (E) .mu.CT analysis of the femur of a
tumor-bearing mouse. Many osteolytic lesions generating a
moth-eaten pattern are striking. (F) Kaplan-Meier curve indicating
the survival advantage (p<0.001, log-rank test) of IL-6 knockout
mice (IL-6.sup.-/-; 210 days median tumor onset) compared to normal
`C` mice (122 days), both reconstituted with Myc-transgenic
CD45.2.sup.+ B cells.
[0042] FIGS. 30A-30E. TRIP13 enhances tumor development. (A) The
construct of p1026.times. vector including a LCK promoter and E.mu.
enhancer (red * are stop codons of TRIP13 and human growth hormone
gene (HGx). (B) The double transgenic TRIP13/E.mu.-Myc mice show a
short survival compared to E.mu.-Myc mice. (C) An example of
enlarged spleen (yellow arrow) and lymph nodes (red arrows) from a
representative Tg TRIP13/E.mu.-Myc mouse. (D & E)
Histopathology of a representative tumor (from a lymph node in this
case) from a Tg TRIP13/E.mu.-Myc mouse (H&E).
[0043] FIGS. 31A-31C. Preliminary analysis of the TRIP13 network in
pre-malignant B cells from Tg TRIP13/E.mu.-Myc mice and E.mu.-Myc
mice. (A) Volcano plot indicating the magnitude (abscissa) and
statistical significance (ordinate) of the expression changes seen
in 1,900 genes from RNA-seq of B cells from two types of transgenic
mice (p<0.001). (B) GSEA of B cells using RNA-seq that
distinguishes Tg TRIP13/E.mu.t-Myc mice from Tg E.mu.-Myc mice as
input. The 10 most significant pathways are presented rank ordered
in accordance with the corresponding pathway scores. The inhibitors
or activators listed in the right side are corresponding to the
pathways with the references from #1.about.#10. * means the
inhibitor is used in clinical trials. #9 (p53) and #10 (PTEN)
pathways are negatively correlated with TRIP13 expression (see FIG.
32). (C) Strategy for identifying TRIP13 oncogenic signaling
pathways.
[0044] FIGS. 32A-32B. Multiple pathways are enriched in transgenic
TRIP13 pre-malignant B cells. (A) Bar views show TRIP13 expression
in B cells collected at 6 weeks Tg TRIP13/E.mu.-Myc mice and
E.mu.-Myc mice. (B) GSEAs show the c-Myc, EZH2, p53 and PTEN
pathways are dysregulated by TRIP13.
[0045] FIGS. 33A-33E. TRIP13 binds and interacts with AIF1. (A)
HEK293 cells is used to construct with stable expression of TRIP13
tagged with HA and 3.times.FLAG; TRIP13 binding proteins are pulled
down by HA antibodies and then by FLAG antibodies for mass
spectrometry analysis. (B) Co-immunoprecipitation using HA
antibodies to pull-down TRIP13 binding proteins is performed, and
western blots show the binding of AIF1 and TRIP13 proteins in 293T
and MM cell line ARP1. (C) Fractionation and western blots show
TRIP13 expresses in both cytoplasm and mitochondria; AIF1 expresses
in mitochondrial. (D) Western blots show expression of AIF1 in
TRIP13-OE ARP1 cells. (E) Quantification of AIF1 protein expression
from (D) in cytoplasm and nucleus of TRIP13-OE ARP1 cells.
[0046] FIG. 34. TRIP13 contains conserved AAA.sup.+ sequence
motifs. Schematic representation of ATPase motifs in TRIP13 and
their mutants. ATPase mutants will be generated by single amino
acid change in Walker A (G184A) and B (E253Q). Nucleotide binding
(R385A) and catalytic (W221A) mutants will be generated by single
amino acid substitution in the Pore Loop and Sensor 2 motifs. A
deletion lacking of the ATPase domain including Sensor 1 motif will
be generated.
[0047] FIG. 35. MM treatment schema at the U of Iowa. D-PACE:
Dexamethasone with infusion of cisplatin, doxorubicin,
cyclophosphamide, and etoposide. Arrows indicate time points for
laboratory investigations. Tx: transplantation.
[0048] FIGS. 36A-36C. Increased TRIP13 links to drug resistance in
primary MM samples. (A) TRIP13 expression is upregulated in MM
cells derived at diagnosis and relapse compared to normal plasma
cells (NPC). GEP was performed in plasma cells from 22 normal
donors, 351 newly diagnosed MMs and 90 relapsed MMs. (B) TRIP13
increases in sequential primary MM samples from 9 MM patients (36
samples). Red color for a gene indicates expression above the
median and blue color indicates expression below the median. (C)
Top 100 genes highly correlated with TRIP13 expression in newly
diagnosed MM samples. The Heatmap shows 50-positive and 50-negative
genes between TRIP13-high (n=88) and TRIP-13-low (n=88) MM
samples.
[0049] FIGS. 37A-37G. PAA overcomes TRIP13-induced drug resistance
in MM cells. (A) TRIP13-OE ARP1 cells are resistant to bortezomib.
Cell viability showed that ARP1 MM cells with or without TRIP13-OE
were treated with different doses of botezomib in ARP1-OE and the
control cells ARP1-EV. (B) TRIP13-OE ARP1 cells are sensitive to
pharmacological ascorbic acid (PAA). Cell viability showed ARP1-OE
and ARP1-EV cells treated with different doses of PAA. (C) PAA
selectively kills primary MM cells. Bar-view presents cell
viability between CD138.sup.+ tumor cells and CD138.sup.- non-tumor
cells treated with either PAA (8, 20 mM) or PBS (control) from 9 MM
patients (p<0.01). (D) PAA targets reactive oxidative species
and labile iron pool: OCI-MY5 WT cells were incubated with or
without catalase (100 U/mL), N-acetyl cysteine (NAC, 15 mM) or
deferoxamine (DFO, 200 .mu.M) for 3 h following treatment with PAA.
PAA was washed away after 1 h treatment and cell viability was
determined 24 h later. (E) PAA induces AIF1 cleavage: OCI-MY5 WT
cells were incubated with or without PAA. After 1 h PAA was washed
away and cells were incubated with melphalan (Mel, 0-80 .mu.M) for
4 h. AIF1, .beta.-actin and .gamma.-H2AX levels were analyzed by
western blots. (F) PAA acts synergistically with melphalan in vivo.
Xenografted ARP1 MM cells injected in NOD.C.gamma.-Rag1 mice were
treated with PAA and melphalan alone or in combination.
Kaplan-Meier curves show that mouse survivals among these groups
are significantly different (p<0.001) and that PAA, when
combined with low dose of melphalan, extends MM mouse survival. (G)
Electron microscopy shows AIF1 immunolabeling stain of OCI-MY5 WT
cells treated without (up) or with (bottom) PAA (2 mM). N, M, C
respectively represent nucleus, mitochondria and cytoplasm. Blue
arrows indicate the nuclear membrane and red arrowheads indicate
AIF1 gold beads in cytoplasm or mitochondria. Black arrowheads
indicate AIF1 gold beads in nuclei.
[0050] FIGS. 38A-38D. TRIP13 regulates iron genes' expression and
increases cellular iron. (A) Bar-views show the expression of
TRIP13, Tfrc, and Fpn1 in pre-malignant B cells derived from Tg
TRIP13/E.mu.t-Myc and Tg E.mu.-Myc mice. (B) Dot-plots show the
expression of TFRC and FPN1 between primary MM samples with
low-TRIP13 expression (n=50) and High-TRIP13 expression (n=50). (C)
Western blots show increased Ferritin in TRIP13-OE ARP1 MM cells.
(D) Western blots show nuclear AIF1 expression with or without PAA
or Bortezomib (Bor) treatment in ARP1 MM cell line.
[0051] FIG. 39. Schematic representation of PAA action in TRIP13
cells. In brief, TRIP13 cells show increased levels of redox-active
iron (1.) due to increased ferritin level. Once cells are treated
with PAA, it reacts with Fe.sup.2+ and by its oxidation will
generated --OH (2.). PAA-mediated cellular oxidative damage leads
to AIF1 cleavage (3.) from mitochondria. AIF1 cleavage form gets
released in the cytoplasm (4.) and subsequently (5.) translocate to
the nucleus inducing apoptosis (6.) and cell death.
DETAILED DESCRIPTION
[0052] The present invention provides in certain embodiments a
therapeutic composition comprising a combination of (a)
pharmacological ascorbic acid (PAA) or a pharmaceutically
acceptable salt thereof; and (b) an alkylating agent.
[0053] In certain embodiments, the alkylating agent is melphalan or
bendamustine.
[0054] In certain embodiments, the alkylating agent is
melphalan.
[0055] The present invention provides in certain embodiments a
method of treating a hyperproliferative disorder associated with
high intracellular iron comprising administering pharmacological
ascorbic acid (PAA) or a pharmaceutically acceptable salt
thereof.
[0056] In certain embodiments, the PAA is administered at a dosage
of about 15 g-100 g. In certain embodiments, the PAA is
administered at a dosage of about 45 g-90 g. In certain
embodiments, the PAA is administered at a dosage of about 45, 46,
47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,
64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,
81, 82, 83, 84, 85, 86, 87, 88, 89, or 90 grams.
[0057] In certain embodiments, the PAA is administered by infusion
two times per week.
[0058] In certain embodiments, the method further comprises
administering an alkylating agent.
[0059] In certain embodiments, the alkylating agent is melphalan or
bendamustine.
[0060] In certain embodiments, the alkylating agent is
melphalan.
[0061] In certain embodiments, the melphalan is administered at a
dosage of about 2 mg/m.sup.2 and 200 mg/m.sup.2.
[0062] In certain embodiments, the melphalan is administered at a
dosage of about 50 mg/m.sup.2 and 100 mg/m.sup.2.
[0063] In certain embodiments the melphalan is administered at a
dosage of about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,
63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,
80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,
97, 98, 99, 100 mg/m.sup.2.
[0064] In certain embodiments, the PAA and the alkylating agent are
administered simultaneously.
[0065] In certain embodiments, the PAA and the alkylating agent are
administered sequentially.
[0066] In certain embodiments, the administration of the PAA begins
about 1 to about 10 days before administration of the alkylating
agent.
[0067] In certain embodiments, the administration of the alkylating
agent begins about 1 to about 10 days before administration of the
PAA.
[0068] In certain embodiments, the administration of the PAA and
alkylating agent begin on the same day.
[0069] In certain embodiments, the PAA is administered about less
than four hours prior to the administration of the alkylating
agent.
[0070] The present invention provides in certain embodiments a
method of treating a hyperproliferative disorder associated with
high intracellular iron comprising administering pharmacological
ascorbic acid (PAA) or a pharmaceutically acceptable salt thereof
and a proteasome inhibitor.
[0071] In certain embodiments, the proteasome inhibitor is
carfizomib.
[0072] In certain embodiments, the carfizomib is administered at a
dosage of about 2 mg/m.sup.2 to 200 mg/m.sup.2.
[0073] In certain embodiments, the carfizomib is administered at a
dosage of about 50 mg/m.sup.2 to 100 mg/m.sup.2.
[0074] In certain embodiments, the proteasome inhibitor (e.g.,
carfizomib) is administered at a dose of 56 mg/m.sup.2 on days 1,
8, 15 and 22 of each 4-week cycle.
[0075] In certain embodiments, the PAA and the proteasome inhibitor
are administered simultaneously.
[0076] In certain embodiments, the PAA and the proteasome inhibitor
are administered sequentially.
[0077] In certain embodiments, the administration of the PAA begins
about 1 to about 10 days before administration of the proteasome
inhibitor.
[0078] In certain embodiments, the administration of the proteasome
inhibitor begins about 1 to about 10 days before administration of
the PAA.
[0079] In certain embodiments, the administration of the PAA and
proteasome inhibitor begin on the same day.
[0080] In certain embodiments, the PAA is administered about less
than four hours prior to the administration of the proteasome
inhibitor.
[0081] In certain embodiments, the method further comprises
administering an anti-cancer therapy.
[0082] In certain embodiments, the anti-cancer therapy is
immunotherapy or biologic therapy.
[0083] In certain embodiments, the hyperproliferative disorder
associated with high iron is multiple myeloma, smoldering multiple
myeloma, ovarian cancer, pancreatic cancer, neuroblastoma,
rhabdomyosarcoma, or breast cancer.
[0084] In certain embodiments, the hyperproliferative disorder
associated with high iron is multiple myeloma, including smoldering
multiple myeloma.
[0085] The present invention provides in certain embodiments a
method of reducing toxic effects of melphalan in a patient in need
thereof comprising administering pharmacological ascorbic acid
(PAA) or a pharmaceutically acceptable salt thereof.
[0086] The present invention provides in certain embodiments a
method of treating multiple myeloma, including smoldering multiple
myeloma, comprising administering pharmacological ascorbic acid
(PAA) or a pharmaceutically acceptable salt thereof.
[0087] In certain embodiments, the PAA is administered at a dosage
of about 15-100 g.
[0088] In certain embodiments, the PAA is administered at a dosage
of about 45 g-90 g infusion.
[0089] In certain embodiment's, the PAA is administered by infusion
two times per week.
[0090] The present invention provides in certain embodiments a use
of the combination of pharmacological ascorbic acid (PAA) or a
pharmaceutically acceptable salt thereof and melphalan in the
preparation of a medicament for the treatment of a
hyperproliferative disorder in a mammal.
[0091] In certain embodiments, the present invention provides the
use of the combination of pharmacological ascorbic acid (PAA) or a
pharmaceutically acceptable salt thereof and carfizomib in the
preparation of a medicament for the treatment of a
hyperproliferative disorder in a mammal.
[0092] The present invention provides in certain embodiments a kit
comprising pharmacological ascorbic acid (PAA) or a
pharmaceutically acceptable salt thereof and melphalan, a
container, and a package insert or label indicating the
administration of the PAA and with melphalan for treating a
hyperproliferative disorder.
[0093] In certain embodiments, the present invention provides a kit
comprising pharmacological ascorbic acid (PAA) or a
pharmaceutically acceptable salt thereof and carfizomib, a
container, and a package insert or label indicating the
administration of the PAA and with carfizomib for treating a
hyperproliferative disorder.
[0094] The present invention provides in certain embodiments a
product comprising pharmacological ascorbic acid (PAA) and
melphalan as a combined preparation for separate, simultaneous or
sequential use in the treatment of a hyperproliferative
disorder.
[0095] In certain embodiments, the PAA is administered for more
than a month. In certain embodiments, the PAA is administered for
more than a year.
[0096] In certain embodiments, the PAA is administered at a dosage
of at least 75 g/day and the alkylating agent is administered at a
dosage of at least 35 mg/day.
[0097] In certain embodiments, the PAA is administered
intravenously.
[0098] In certain embodiments, the PAA is administered at a dosage
of at least 50 g/infusion.
[0099] The present invention provides in certain embodiments a
therapeutic composition comprising a combination of (a)
pharmacological ascorbic acid (PAA) or a pharmaceutically
acceptable salt thereof; and (b) an alkylating agent.
[0100] In certain embodiments, the alkylating agent is melphalan or
bendamustine.
[0101] In certain embodiments, the alkylating agent is
melphalan.
[0102] The present invention provides in certain embodiments a
therapeutic composition comprising a combination of (a)
pharmacological ascorbic acid (PAA) or a pharmaceutically
acceptable salt thereof; and (b) a proteasome inhibitor.
[0103] In certain embodiments, the proteasome inhibitor is
carfizomib.
[0104] The present invention provides in certain embodiments, a
method of administering to a mammalian cell having downregulated
expression of Ferroportin 1 (Fpn1) as compared with its normal
counterpart cell an expression-modulating agent, comprising
contacting the mammalian cell with pharmacological ascorbic acid
(PAA) or a pharmaceutically acceptable salt thereof.
[0105] The present invention provides in certain embodiments, a
method of administering to a mammalian cell having upregulated
expression of enhancer of zeste 2 (EZH2) as compared with its
normal counterpart cell an expression-modulating agent, comprising
contacting the mammalian cell with an inhibitor of EZH2.
[0106] In certain embodiments, the inhibitor of EZH2 is DZNep or
GSK343.
[0107] In certain embodiments, the method further comprises
contacting the mammalian cell pharmacological ascorbic acid (PAA)
or a pharmaceutically acceptable salt thereof.
[0108] The present invention provides in certain embodiments, a
method of administering to a mammalian cell having upregulated
expression of Thyroid Hormone Receptor Interactor Protein 13
(TRIP13) as compared with its normal counterpart cell an
expression-modulating agent, comprising contacting the mammalian
cell with pharmacological ascorbic acid (PAA) or a pharmaceutically
acceptable salt thereof and/or with an inhibitor of TRIP13.
[0109] Pharmaceutical Ascorbic Acid (PAA)
[0110] Vitamin C is a highly effective and non-toxic anti-oxidant
that can be used to protect the body against oxidative stress
including free radicals. As used herein, a reference to ascorbic
acid includes the anionic component, ascorbate whether as an acid
or one of the pharmaceutically acceptable salts thereof, such as
sodium ascorbate and calcium ascorbate, all of which are included
in a reference to CGMP "ascorbic acid" or "ascorbate."
[0111] Injectable pharmacological ascorbic acid (PAA), or vitamin
C, has recently re-emerged as a promising anti-cancer therapy.
Studies in a variety of cancer cell types, both in cell culture and
animal models, have demonstrated selective (relative to normal
cells) cancer cell killing as well as selective sensitization of
cancer cells to standard of care therapies when combined with
injectable pharmacological ascorbate. PAA's selective toxicity to
cancer cells appears to be dependent upon the presence of redox
active metal ions (such as iron), which are capable of receiving
and donating electrons during the oxidation of ascorbate to form
hydrogen peroxide.
[0112] Patients with a variety of cancer types are currently
receiving intravenous pharmacological ascorbate in combination with
standard cancer therapies in clinical trials to determine
pharmacological ascorbate's clinical safety and efficacy.
Pharmacological ascorbate has recently been shown in tissue culture
models and animal modes to increase the sensitivity of tumor cells
to chemotherapy and radiation therapy. In addition, phase I
clinical trials assessing the tolerability of pharmacological
ascorbate in a variety of cancer types have been well
tolerated.
[0113] Pharmacological doses of ascorbate (resulting in plasma
concentrations>10 mM) can be achieved by intravenous (IV)
administration and have been shown to be safe and well tolerated in
both animals and humans. (Welsh et al., Pharmacological ascorbate
with gemcitabine for the control of metastatic and node-positive
pancreatic cancer (PACMAN): results from a phase I clinical trial.
Cancer Chemother Pharmacol. 2013 March; 71(3):765-775; Ma et al.,
High-Dose Parenteral Ascorbate Enhanced Chemosensitivity of Ovarian
Cancer and Reduced Toxicity of Chemotherapy. Sci Transl Med. 2014
Feb. 5; 6(222):222ra18-222ra18). Recent in vitro experiments
demonstrate that pharmacological ascorbate is selectively toxic to
cancer cells, whereas normal cells are unaffected (preliminary
results). (Du et al., Mechanisms of ascorbate-induced cytotoxicity
in pancreatic cancer Clin Cancer Res. 2010 Jan. 15; 16(2):509-20
PMID: 20068072). High ascorbate concentrations in cancer cells
appear to selectively induce the formation of H.sub.2O.sub.2 via
the catalytic oxidation of ascorbate in the presence of redox
active metals such as iron (Fe). (Chen et al., Pharmacologic
ascorbic acid concentrations selectively kill cancer cells: action
as a pro-drug to deliver hydrogen peroxide to tissues. Proc Natl
Acad Sci USA. 2005 Sep. 20; 1 02(38):13604-13609.) Because cancer
cells are believed to have higher concentrations of labile redox
active metal ions due to increased steady-state levels of
superoxide, pharmacological ascorbate will selectively increase
H.sub.2O.sub.2 in lung cancer cells, relative to normal lung cells,
thereby increasing the sensitivity of NSCLC to chemo-radiation
therapy by increasing oxidative stress (preliminary results).
[0114] The method of the present invention comprises the treatment
of cancer by administering sufficient amounts of ascorbic acid to
raise the concentration of ascorbic acid in the patient's plasma
above a level that is cytotoxic to the cancer tumor cells. In
certain embodiments, ascorbate is administered so as to reach a
blood level of at least about 20 mM. Doses of 75 g/infusion or
greater are typically able to achieve this concentration.
[0115] Inhibitors of EZH2
[0116] In certain embodiments, the inhibitor of EZH2 is DZNep or
GSK343.
[0117] Inhibitors of TRIP13
[0118] In certain embodiments, the inhibitor of TRIP13 is
P5091.
[0119] Anti-Cancer Therapy
[0120] As used herein, the term "anti-cancer therapy" includes
therapeutic agents that kill cancer cells; slow tumor growth and
cancer cell proliferation; and ameliorate or prevent one or more of
the symptoms of cancer. For example, the term "anti-cancer therapy"
includes an anti-cancer therapy that enhances DNA damage in cancer
cells. In certain embodiments, the anti-cancer therapy is standard
immunotherapy or biologic therapy.
[0121] Alkylating Agents. Alkylating agents are a class of
chemotherapy drugs that bind to DNA and prevent proper DNA
replication. They have chemical groups that can form permanent
covalent bonds with nucleophilic substances in the DNA. In certain
embodiments, the alkylating agent is melphalan or bendamustine.
[0122] Additive Agents
[0123] In certain embodiments, the combination further comprises an
inhibition agent that inhibits glucose and/or hydroperoxide
metabolism. In certain embodiments, the inhibition agent is
Buthionine sulfoximine, Auranofin, 2-deoxyglucose, other inhibitors
of glutathione and/or thioredoxin metabolism, inhibitors of
catalase, sulfasalazine, other inhibitors of cysteine transport,
inhibitors of glucose transport, diets that limit glucose and other
simple sugars such as ketogenic diets.
[0124] Hyperproliferative Diseases
[0125] In certain embodiments of the methods described above, the
cancer is breast cancer, prostate cancer, lung cancer, pancreas
cancer, head and neck cancer, ovarian cancer, brain cancer, colon
cancer, hepatic cancer, skin cancer, leukemia, melanoma,
endometrial cancer, neuroendocrine tumors, carcinoids,
neuroblastoma, glioma, tumors arising from the neural crest,
lymphoma, myeloma, or other malignancies characterized by aberrant
mitochondrial hydroperoxide metabolism. In certain embodiments, the
cancer is the above cancers that are not curable or not responsive
to other therapies. In certain embodiments, the cancer is multiple
myeloma, smoldering multiple myeloma, ovarian cancer, pancreatic
cancer, neuroblastoma, rhabdomyosarcoma, or breast cancer.
[0126] Compositions and Methods of Administration
[0127] The method of the present invention comprises the treatment
of cancer by administering sufficient amounts of ascorbic acid to
raise the concentration of ascorbic acid in the patient's plasma
above a level that is cytotoxic to the cancer tumor cells, in
combination with an alkylating agent (such as melphalan), and
optionally with an additional anti-cancer therapy.
[0128] The present invention provides a method for increasing the
anticancer effects of an alkylating agent (such as melphalan),
optionally in conjunction with conventional cancer therapy (i.e.,
radio- and/or chemo-therapy) on cancerous cells in a mammal. In
certain embodiments, the method comprises contacting the cancerous
cell with an effective amount of pharmaceutical ascorbic acid (PAA)
or a pharmaceutically acceptable salt thereof and an alkylating
agent (such as melphalan), and optionally administering an
additional conventional cancer therapy modality. In certain
embodiments, the additional cancer therapy is chemotherapy. In
certain embodiments, the PAA and alkylating agent are administered
sequentially to a mammal rather than in a single composition. In
certain embodiments, the mammal is a human.
[0129] In certain embodiments of the methods described above, the
composition does not significantly inhibit viability of comparable
non-cancerous cells.
[0130] In certain embodiments of the methods described above, the
tumor is reduced in volume by at least 10%. In certain embodiments,
the tumor is reduced by any amount between 1-100%. In certain
embodiments, the tumor uptake of molecular imaging agents, such as
fluorine-18 deoxyglucose, fluorine-18 thymidine or other suitable
molecular imaging agent, is reduced by any amount between 1-100%.
In certain embodiments the imaging agent is fluorine-18
deoxyglucose, fluorine-18 thymidine or other suitable molecular
imaging agent. In certain embodiments, the mammal's symptoms (such
as flushing, nausea, fever, or other maladies associated with
cancerous disease) are alleviated.
[0131] Administration of a compound as a pharmaceutically
acceptable acid or base salt may be appropriate. Examples of
pharmaceutically acceptable salts are organic acid addition salts
formed with acids which form a physiological acceptable anion, for
example, tosylate, methanesulfonate, acetate, citrate, malonate,
tartrate, succinate, benzoate, ascorbate, .alpha.-ketoglutarate,
and .alpha.-glycerophosphate. Suitable inorganic salts may also be
formed, including hydrochloride, sulfate, nitrate, bicarbonate, and
carbonate salts.
[0132] Pharmaceutically acceptable salts may be obtained using
standard procedures well known in the art, for example by reacting
a sufficiently basic compound such as an amine with a suitable acid
affording a physiologically acceptable anion. Alkali metal (for
example, sodium, potassium or lithium) or alkaline earth metal (for
example calcium) salts of carboxylic acids can also be made.
[0133] Ascorbate, alkylating agents and anti-cancer agents can be
formulated as pharmaceutical compositions and administered to a
mammalian host, such as a human patient in a variety of forms
adapted to the chosen route of administration, i.e., orally or
parenterally, by intravenous, intramuscular, topical or
subcutaneous routes.
[0134] Thus, the present compounds may be systemically
administered, e.g., intravenously, in combination with a
pharmaceutically acceptable vehicle such as an inert diluent or an
assimilable edible carrier. They may be enclosed in hard or soft
shell gelatin capsules, may be compressed into tablets, or may be
incorporated directly with the food of the patient's diet. The
amount of active compound in such therapeutically useful
compositions is such that an effective dosage level will be
obtained.
[0135] Of course, any material used in preparing any unit dosage
form should be pharmaceutically acceptable and substantially
non-toxic in the amounts employed. In addition, the active compound
may be incorporated into sustained-release preparations and
devices.
[0136] The active compound may also be administered intravenously
or intraperitoneally by infusion or injection. Solutions of the
active compound or its salts can be prepared in water, optionally
mixed with a nontoxic surfactant. Dispersions can also be prepared
in glycerol, liquid polyethylene glycols, triacetin, and mixtures
thereof and in oils. Under ordinary conditions of storage and use,
these preparations contain a preservative to prevent the growth of
microorganisms.
[0137] The pharmaceutical dosage forms suitable for injection or
infusion can include sterile aqueous solutions or dispersions or
sterile powders comprising the active ingredient which are adapted
for the extemporaneous preparation of sterile injectable or
infusible solutions or dispersions, optionally encapsulated in
liposomes. In all cases, the ultimate dosage form should be
sterile, fluid and stable under the conditions of manufacture and
storage. The liquid carrier or vehicle can be a solvent or liquid
dispersion medium comprising, for example, water, ethanol, a polyol
(for example, glycerol, propylene glycol, liquid polyethylene
glycols, and the like), vegetable oils, nontoxic glyceryl esters,
and suitable mixtures thereof. The proper fluidity can be
maintained, for example, by the formation of liposomes, by the
maintenance of the required particle size in the case of
dispersions or by the use of surfactants. The prevention of the
action of microorganisms can be brought about by various
antibacterial and antifungal agents, for example, parabens,
chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In
many cases, it will be preferable to include isotonic agents, for
example, sugars, buffers or sodium chloride. Prolonged absorption
of the injectable compositions can be brought about by the use in
the compositions of agents delaying absorption, for example,
aluminum monostearate and gelatin.
[0138] Sterile injectable solutions are prepared by incorporating
the active compound in the required amount in the appropriate
solvent with various other ingredients enumerated above, as
required, followed by filter sterilization. In the case of sterile
powders for the preparation of sterile injectable solutions, the
preferred methods of preparation are vacuum drying and the freeze
drying techniques, which yield a powder of the active ingredient
plus any additional desired ingredient present in the previously
sterile-filtered solutions.
[0139] For topical administration, the present compounds may be
applied in pure form, i.e., when they are liquids. However, it may
be desirable to administer them to the skin as compositions or
formulations, in combination with a dermatologically acceptable
carrier, which may be a solid or a liquid.
[0140] Useful solid carriers include finely divided solids such as
talc, clay, microcrystalline cellulose, silica, alumina and the
like. Useful liquid carriers include water, alcohols or glycols or
water-alcohol/glycol blends, in which the present compounds can be
dissolved or dispersed at effective levels, optionally with the aid
of non-toxic surfactants. Adjuvants such as fragrances and
additional antimicrobial agents can be added to optimize the
properties for a given use. The resultant liquid compositions can
be applied from absorbent pads, used to impregnate bandages and
other dressings, or sprayed onto the affected area using pump-type
or aerosol sprayers.
[0141] Thickeners such as synthetic polymers, fatty acids, fatty
acid salts and esters, fatty alcohols, modified celluloses or
modified mineral materials can also be employed with liquid
carriers to form spreadable pastes, gels, ointments, soaps, and the
like, for application directly to the skin of the user.
[0142] The dosage of the ascorbate, alkylating agent(s) and the
anti-cancer agent will vary depending on age, weight, and condition
of the subject. Treatment may be initiated with small dosages
containing less than optimal doses, and increased until a desired,
or even an optimal effect under the circumstances, is reached. In
general, the dosage is about 75-100 g per infusion Higher or lower
doses, however, are also contemplated and are, therefore, within
the confines of this invention. A medical practitioner may
prescribe a small dose and observe the effect on the subject's
symptoms. Thereafter, he/she may increase the dose if suitable. In
general, the ascorbate, alkylating agent(s) and the anti-cancer
agent are administered at a concentration that will afford
effective results without causing any unduly harmful or deleterious
side effects, and may be administered either as a single unit dose,
or if desired in convenient subunits administered at suitable
times.
[0143] A pharmaceutical composition of the invention is formulated
to be compatible with its intended route of administration. For
example, the therapeutic agent may be introduced directly into the
cancer of interest via direct injection. Additionally, examples of
routes of administration include parenteral, e.g., intravenous,
slow infusion, intradermal, subcutaneous, oral (e.g., ingestion or
inhalation), transdermal (topical), transmucosal, and rectal
administration depending on the location of the tumor. Such
compositions typically comprise the PBA or pharmaceutically
acceptable salt thereof and the anti-cancer agent and a
pharmaceutically acceptable carrier. As used herein,
"pharmaceutically acceptable carrier" is intended to include any
and all solvents, dispersion media, coatings, antibacterial and
anti-fungal agents, isotonic and absorption delaying agents, and
the like, compatible with pharmaceutical administration, and a
dietary food-based form. The use of such media and agents for
pharmaceutically active substances is well known in the art and
food as a vehicle for administration is well known in the art.
[0144] Solutions or suspensions can include the following
components: a sterile diluent such as water for injection, saline
solution (e.g., phosphate buffered saline (PBS)), fixed oils, a
polyol (for example, glycerol, propylene glycol, and liquid
polyetheylene glycol, and the like), glycerine, or other synthetic
solvents; antibacterial and antifungal agents such as parabens,
chlorobutanol, phenol, ascorbic acid, thimerosal, and the like;
antioxidants such as ascorbic acid or sodium bisulfite; alkylating
agents such as melphalan; buffers such as acetates, citrates or
phosphates and agents for the adjustment of tonicity such as sodium
chloride or dextrose. The proper fluidity can be maintained, for
example, by the use of a coating such as lecithin, by the
maintenance of the required particle size in the case of dispersion
and by the use of surfactants. In many cases, it will be preferable
to include isotonic agents, for example, sugars, polyalcohols such
as mannitol or sorbitol, and sodium chloride in the composition.
Prolonged administration of the injectable compositions can be
brought about by including an agent that delays absorption. Such
agents include, for example, aluminum monostearate and gelatin. The
parenteral preparation can be enclosed in ampules, disposable
syringes, or multiple dose vials made of glass or plastic.
[0145] It may be advantageous to formulate compositions in dosage
unit form for ease of administration and uniformity of dosage.
Dosage unit form as used herein refers to physically discrete units
suited as unitary dosages for an individual to be treated; each
unit containing a predetermined quantity of active compound
calculated to produce the desired therapeutic effect in association
with the required pharmaceutical carrier. The dosage unit forms of
the invention are dependent upon the amount of a compound necessary
to produce the desired effect(s). The amount of a compound
necessary can be formulated in a single dose, or can be formulated
in multiple dosage units. Treatment may require a one-time dose, or
may require repeated doses.
[0146] "Systemic delivery," as used herein, refers to delivery of
an agent or composition that leads to a broad biodistribution of an
active agent within an organism. Some techniques of administration
can lead to the systemic delivery of certain agents, but not
others. Systemic delivery means that a useful, preferably
therapeutic, amount of an agent is exposed to most parts of the
body. To obtain broad biodistribution generally requires a blood
lifetime such that the agent is not rapidly degraded or cleared
(such as by first pass organs (liver, lung, etc.) or by rapid,
nonspecific cell binding) before reaching a disease site distal to
the site of administration. Systemic delivery of lipid particles
can be by any means known in the art including, for example,
intravenous, subcutaneous, and intraperitoneal. In a preferred
embodiment, systemic delivery of lipid particles is by intravenous
delivery.
[0147] "Local delivery," as used herein, refers to delivery of an
active agent directly to a target site within an organism. For
example, an agent can be locally delivered by direct injection into
a disease site, other target site, or a target organ such as the
liver, heart, pancreas, kidney, and the like.
[0148] The term "mammal" refers to any mammalian species such as a
human, mouse, rat, dog, cat, hamster, guinea pig, rabbit,
livestock, and the like.
[0149] The terms "treat" and "treatment" refer to both therapeutic
treatment and prophylactic or preventative measures, wherein the
object is to prevent or decrease an undesired physiological change
or disorder, such as the development or spread of cancer. For
purposes of this invention, beneficial or desired clinical results
include, but are not limited to, alleviation of symptoms,
diminishment of extent of disease, stabilized (i.e., not worsening)
state of disease, delay or slowing of disease progression,
amelioration or palliation of the disease state, and remission
(whether partial or total), whether detectable or undetectable.
"Treatment" can also mean prolonging survival as compared to
expected survival if not receiving treatment. Those in need of
treatment include those already with the condition or disorder as
well as those prone to have the condition or disorder or those in
which the condition or disorder is to be prevented.
[0150] The invention will now be illustrated by the following
non-limiting Examples.
Example 1
Efficacy of Lower-Dose of Melphalan Plus Pharmacological Ascorbic
Acid as New Therapy for Multiple Myeloma
[0151] High-dose chemotherapies to treat multiple myeloma (MM) can
be life-threatening due to toxicities to normal cells and there is
a need to target only tumor cells and/or lower standard drug dosage
without losing efficacy. We show that pharmacologically-dosed
ascorbic acid (PAA) in the presence of iron leads to the formation
of highly reactive oxygen species (ROS) resulting in cell death.
PAA selectively killed CD138.sup.+ MM tumor cells derived from MM
and smoldering MM (SMM) but not from undetermined significane
(MGUS) MGUS patients. PAA alone or combination with carfizomib or
melphalan inhibits tumor formation in MM xenograft mice. This is
first report on PAA efficacy on primary cancer cells in vitro and
in vivo.
[0152] Multiple myeloma (MM) is a plasma cell neoplasm. Four active
classes of drugs including glucocorticoids, DNA alkylators
(melphalan), proteasome inhibitors (bortezomib and carfizomib) and
immunomodulatory agents (thalidomide, lenalidomide, and
pomalidomide), combined with or without Autologous Stem Cell
Transplantation (ASCT) have led to complete remissions (CRs) in the
large majority of newly diagnosed patients with MM (Alexanian, R.,
et al. Value of novel agents and intensive therapy for patients
with multiple myeloma. Bone marrow transplantation 49, 422-425
(2014); Fu, C., et al. Therapeutic effects of autologous
hematopoietic stem cell transplantation in multiple myeloma
patients. Exp Ther Med 6, 977-982 (2013); Terpos, E., et al. VTD
consolidation, without bisphosphonates, reduces bone resorption and
is associated with a very low incidence of skeletal-related events
in myeloma patients post ASCT. Leukemia 28, 928-934 (2014); Wang,
L., Xu, Y. L. & Zhang, X. Q. Bortezomib in combination with
thalidomide or lenalidomide or doxorubicin regimens for the
treatment of multiple myeloma: a metaanalysis of 14 randomized
controlled trials. Leukemia & lymphoma 55, 1479-1488 (2014);
Sonneveld, P., et al. Bortezomib-based versus nonbortezomib-based
induction treatment before autologous stem-cell transplantation in
patients with previously untreated multiple myeloma: a
meta-analysis of phase III randomized, controlled trials. Journal
of clinical oncology: official journal of the American Society of
Clinical Oncology 31, 3279-3287 (2013); Gay, F., et al. Bortezomib
induction, reduced-intensity transplantation, and lenalidomide
consolidation-maintenance for myeloma: updated results. Blood 122,
1376-1383 (2013); Liu, J., et al. Determining the optimal time for
bortezomib-based induction chemotherapy followed by autologous
hematopoietic stem cell transplant in the treatment of multiple
myeloma. Chin J Cancer Res 25, 166-174 (2013); Bergsagel, P. L.
Where we were, where we are, where we are going: progress in
multiple myeloma. American Society of Clinical Oncology educational
book/ASCO. American Society of Clinical Oncology. Meeting, 199-203
(2014).). These treatments have greatly improved patient
progression-free and overall survival. However, there are at least
three major problems limiting the administration of these agents:
1. All these drugs target both tumor and non-tumor cells; 2.
Increased hematologic toxicity has been identified by combining
alkylators with either IMIDs; and 3. High doses of the DNA
alkalating agent, such as melphalan, have strong cytotoxicity on
gut epithelial cells and hematopoietic stem cells. One way to deal
with non-selective toxicity of high dose melphalan is to combine it
with another agent which very specifically targets tumor cells and
therefore allows a decrease in melphalan dose without loss of
efficacy.
[0153] In the 1970s, Cameron and Pauling reported that high doses
of vitamin C increased survival of patients with cancer (Cameron,
E. & Pauling, L. Supplemental ascorbate in the supportive
treatment of cancer: Prolongation of survival times in terminal
human cancer. Proceedings of the National Academy of Sciences of
the United States of America 73, 3685-3689 (1976); Cameron, E.
& Pauling, L. Supplemental ascorbate in the supportive
treatment of cancer: reevaluation of prolongation of survival times
in terminal human cancer. Proceedings of the National Academy of
Sciences of the United States of America 75, 4538-4542 (1978)).
Recently, reports have shown that pharmacologically dosed ascorbic
acid (PAA) 20.about.80 folds higher than physiologically dosed
ascorbate, administered intravenously, has potent anti-cancer
activity and its role as a novel anti-cancer therapy is being
studied at the University of Iowa and in other centers. In the
presence of catalytic metal ions like iron, PAA administered
intravenously exerts pro-oxidant effects leading to the formation
of highly reactive oxygen species (ROS), resulting in cell death
(Du, J., Cullen, J. J. & Buettner, G. R. Ascorbic acid:
chemistry, biology and the treatment of cancer. Biochimica et
biophysica acta 1826, 443-457 (2012); Ma, Y., et al. High-dose
parenteral ascorbate enhanced chemosensitivity of ovarian cancer
and reduced toxicity of chemotherapy. Science translational
medicine 6, 222ra218 (2014); Yun, J., et al. Vitamin C selectively
kills KRAS and BRAF mutant colorectal cancer cells by targeting
GAPDH. Science 350, 1391-1396 (2015); Chen, Q., et al. Ascorbate in
pharmacologic concentrations selectively generates ascorbate
radical and hydrogen peroxide in extracellular fluid in vivo.
Proceedings of the National Academy of Sciences of the United
States of America 104, 8749-8754 (2007); Chen, Q., et al.
Pharmacologic ascorbic acid concentrations selectively kill cancer
cells: action as a pro-drug to deliver hydrogen peroxide to
tissues. Proceedings of the National Academy of Sciences of the
United States of America 102, 13604-13609 (2005)). In a previous
study, it was reported that the labile iron pool (LIP) is
significantly elevated in MM cells, suggesting that PAA treatment
should target MM cells quite selectively (Gu, Z., et al. Decreased
ferroportin promotes myeloma cell growth and osteoclast
differentiation. Cancer research 75, 2211-2221 (2015)). The higher
LIP is the direct result of the low expression of the only known
mammalian cellular iron exporter, Ferroportin 1 (Fpn1), in MM as
demonstrated. These findings led to the current hypothesis that PAA
might specifically target MM cells with high iron content and may
also act synergistically in combination with commonly used MM
therapies.
[0154] Methods
[0155] Patients and Mice
[0156] Peripheral-blood samples or bone marrow aspirates were
obtained from patients with monoclonal gammopathy of undetermined
significance (MGUS), smoldering multiple myeloma (SMM), and
multiple myeloma (MM). Written informed consent was obtained from
all the participants. The study was approved by the institutional
review board at the University of Iowa. NOD.C.gamma.-Rag1 mice
(Jackson laboratory, Bar Harbor, Me.) were bred and maintained in
compliance with the guidelines of the institutional animal care at
the University of Iowa.
[0157] Gene Expression
[0158] Gene expression profiling (GEF) has been described
previously (Zhan et al., The molecular classification of multiple
myeloma. Blood 108, 2020 (Sep. 15, 2006); Shaughnessy, Jr. et al.,
A validated gene expression model of high-risk multiple myeloma is
defined by deregulated expression of genes mapping to chromosome 1.
Blood 109, 2276 (Mar. 15, 2007)). GEP access number of normal
plasma cell (NPC), MGUS, and primary myeloma samples is
GSE2658.
[0159] Pharmacological Ascorbic Acid Viability Assay
[0160] Pharmacological Ascorbic Acid (PAA) was kindly provided by
Dr. Garry R. Buettner (University of Iowa). CD138.sup.+ MM cells
and CD138.sup.- non-MM cells were isolated from MGUS, SMM, and MM
patient samples using anti-CD138 immunomagnetic beads (Miltenyl
Biotec, Auburn, Calif.). Cells were cultured with or without PAA at
the described concentration for 1 hr. After incubation, the cells
were washed and cultured up to 24 h. Cell counts and viable cell
number were determined using Trypan Blue staining.
[0161] Human Myeloma Xenografts Mice
[0162] NOD.C.gamma.-Rag1 mice 6-8 weeks old (Jackson laboratory,
Bar Harbor, Me.) were injected intravenously with ARP1 MM cells
(1.times.10.sup.6) expressing luciferase. After one-week injection
of ARP1 cells, mice were treated with either PAA (4 mg/kg) injected
intraperitoneal once a day, 5 days every week for 3 weeks.
Melphalan (3 mg/kg) was injected intraperitoneal once a day, 2 days
a week for 3 weeks (Sanchez, E., et al. Serum B-cell maturation
antigen is elevated in multiple myeloma and correlates with disease
status and survival. British journal of haematology 158, 727-738
(2012).) Carfizomin (3 mg/kg) was injected by in vein once a day, 2
days every week for 3 weeks (Eda, H., et al. A novel Bruton's
tyrosine kinase inhibitor CC-292 in combination with the proteasome
inhibitor carfilzomib impacts the bone microenvironment in a
multiple myeloma model with resultant antimyeloma activity.
Leukemia 28, 1892-1901 (2014)). Bortezomib (3 mg/kg) was injected
intraperitoneal once a day, 2 days a week for 3 weeks. The mice
were euthanized when humane endpoint was reached.
[0163] In Vivo Imaging System (IVIS)
[0164] Xenogen IVIS-200 an in vivo imaging system (IVIS) was used
to analyze tumor burden and was indicated by quantification of
luciferase intensity of mice pre- and post-treatments.
[0165] Cell Culture
[0166] Human myeloma cell lines (ARP1, OCI-MY5 and their derivative
cell lines) were cultured in RPMI 1640 (Invitrogen, Carlsbad,
Calif.), supplemented with 10% heat-inactivated FBS (Invitrogen),
penicillin (100 IU/mL), and streptomycin (100 .mu.g/mL) in a
humidified incubator at 37.degree. C. and 5% CO.sub.2/95% air. To
increase cellular iron concentration, ferric nitrilotriacetate
(Fe-NTA) was used.
[0167] Western Blotting
[0168] Cells were harvested and lysed with lysis buffer: 150 mM
NaCl, 10 mM EDTA, 10 mM Tris, pH7.4, 1% X-100 Triton. Cell lysates
were subjected to SDS-PAGE, transferred onto a pure nitrocellulose
membrane (BioRad), and blocked with 5% fat-free milk. Primary
antibodies for immunoblotting included: anti-AIF1 (1:1000, Cell
Signaling), anti-RIP (1:1000, Santa Cruz Biotechnology), anti-RIP3
(1:1000, Cell Signaling), anti-Caspase3 (1:1000, Cell Signaling),
anti-Caspase 8 (1:1000, Cell Signaling), anti-Caspase 9 (1:1000,
Cell Signaling) Phosphorylated .gamma.H2AX (1:1000, Enzo Life
Sciences), and .beta.-actin (1:1000, Cell Signaling) as loading
control. Membranes were incubated with horseradish peroxidase
(HRP)-conjugated anti-mouse secondary antibody (1:10,000, Santa
Cruz Biotechnology, cat #: sc-2005) or anti-rabbit secondary
antibody (1:10,000, AnaSpec Inc., cat #: AS-28177) for 1 h and
chemi-luminescence signals were detected by HRP substrate (EMD
Millipore).
[0169] Statistical Analyses
[0170] GEP data were analyzed by One-Way Anova test using log 2
transformed Affymetrix Signals and presented by boxplot. The
comparisons of tumor burden were analyzed either by student t-test
(2 groups) or by One-Way Anova test (>2 groups). Kaplan-Meier
method was performed for survival with the use of SPSS 16.0
software (SPSS, Chicago, Ill.). Two-tailed p value at an alpha
level of 0.05 was considered to indicate statistical significance.
Graphs were generated using Prism 6 software.
[0171] Electron Microscopy
[0172] Electron microscopy was performed by the Central Microscopy
Research Facility personnel at the University of Iowa. Images were
captured on JEOL JEM 1230.
[0173] Results
[0174] Pharmacological Ascorbic Acid (PAA) Selectively Kills
Myeloma Tumor Cells
[0175] The response to PAA of both CD138.sup.+ primary MM cells
(high cytosolic iron) and CD138.sup.- non-myeloma bone marrow (BM)
cells obtained from 13 patients was analyzed. The 13 patients
included 2 monoclonal gammopathy of undetermined significance
(MGUS), 2 smoldering MM (SMM) and 9 MM patients. Patient
demographic, disease characteristics and therapy are listed in
Table 1 and FIG. 5.
TABLE-US-00001 TABLE 1 M-com- Plasma ponent Stage Cells Subject
Disease Age Sex type (ISS) (%) Cytogenetics Last treatment 1 MGUS
58 F IgA nd 5.0 Hyperdiploid NT Kappa karyotype p53 amplification 3
MM 65 M IgG I 20 Hyperdiploid D-PACE Kappa karyotype 4 MM 38 M IgG
II 2.0 Hyperdiploid Carfilzomib Kappa karyotype Dexamethasone
Lenalidomide 5 MM 62 F IgG I 4.0 1q amplification Melphalan Lambda
t (14;16) (q32;q23) VTD 6 MM 62 M IgG III 80 Hypodiploid VDT Lambda
karyotype 1q amplification 7 MM 79 F IgG III 10 1q amplification
Dexamethasone Kappa p53 amplification Lenalidomide t (4;14)
(p16;q32) 8 MM 59 M IgG II 5.0 1q amplification Bortezomib Lambda t
(4;14) (p16;q32) Lenalidomide 9 MM 56 F Lambda II <1 Hypodiploid
RVD Light karyotype Chain ONLY 11 SMM 48 M IgA nd 6.0 Hypodiploid
NT Kappa karyotype 1q amplification t (4;14) (p16;q32) 12 SMM 60 M
IgG nd 15 Hypodiploid NT Lambda karyotype 1q amplification 13 MM 49
F IgA II 60 Hyperdiploid Bortezomib Lambda karyotype Dexamethasone
14 MGUS 65 F IgG nd 5 Normal FISH NT Lambda 15 MM 61 F IgG I 17 13q
deletion Bortezomib Kappa 1q amplification Dexamethasone t
(11;14)(q13;q32)
The survival of CD138.sup.+ cells in vitro was significantly
decreased following PAA treatment in all 9 MM (FIG. 1A, grey bars).
In contrast, no significant change of cell viability was observed
in CD138.sup.- BM cells from the same patients (FIG. 1A, black
bars). The same effect of PAA was also observed in the SMM patients
(FIG. 1B). However, almost no response to PAA was detected in
CD138.sup.+ cells from the 2 MGUS patients (FIG. 1C). It was
predicted that this would be the case because MGUS patients have
much lower cytosolic iron compared to MM patients (FIGS. 2A-2B) as
the consequence of lower expression of transferrin receptor 1, the
cellular iron receptor-mediated importer (FIG. 6A), and higher
expression of Ferroportin 1 (Fpn1), the iron exporter (FIG.
2B).
[0176] Pharmacological Ascorbic Acid Decreases Melphalan Doses in
Myeloma Treatment
[0177] To confirm the capacity of PAA to induce MM cell death in
vivo, ARP1 MM cells expressing luciferase were injected
intravenously into NOD.C.gamma.-Rag1 (n=6) mice. Three days later,
half of the injected mice were treated for 15 days with PAA (4
mg/kg, once per day, IP) and the other half with saline as
controls. An in vivo imaging system (IVIS) showed that tumor
progression was significantly delayed in mice treated with PAA
(data not shown). These data support the concept that PAA also
targets MM cells effectively in vivo. To investigate whether PAA
may be effective in killing MM cells when combined with currently
used MM therapies, we treated mice with melphalan or carfilzomib or
bortezomib. Seven combinations (control, PAA, melphalan,
carfilzomib, melphalan+PAA, carfilzomib+PAA and bortezomib) were
tested in vivo (Sanchez, E., et al. Serum B-cell maturation antigen
is elevated in multiple myeloma and correlates with disease status
and survival. British journal of haematology 158, 727-738 (2012);
Eda, H., et al. A novel Bruton's tyrosine kinase inhibitor CC-292
in combination with the proteasome inhibitor carfilzomib impacts
the bone microenvironment in a multiple myeloma model with
resultant antimyeloma activity. Leukemia 28, 1892-1901 (2014)).
Compared to the control group, all treatments inhibited MM cell
growth significantly (p<0.05) (FIG. 1D). Within the single agent
treatments, melphalan only showed a higher decrease in tumor burden
when compared to PAA treatment and other single agents tested.
However, the combination of melphalan plus PAA showed greater tumor
burden reduction than either drug alone, suggesting a synergistic
activity between the two drugs. Bortezomib was not given in
combination with PAA because ascorbic acid directly inactivates
bortezomib by forming a tight and reversible complex through its
vicinal diol group (Perrone, G., et al. Ascorbic acid inhibits
antitumor activity of bortezomib in vivo. Leukemia 23, 1679-1686
(2009); Harvey, R. D., Nettles, J., Wang, B., Sun, S. Y. &
Lonial, S. Commentary on Perrone et al.: "Vitamin C: not for
breakfast anymore . . . if you have myeloma". Leukemia 23,
1939-1940 (2009)).
[0178] The clinical objective of this study was to determine if PAA
addition would allow a decrease in melphalan dose without losing
its efficacy. Therefore, mice were treated with 3 different doses
of melphalan (1, 3, and 5 mg/kg) plus PAA. Tumor burden at three
weeks of treatment showed that single agent melphalan also at the
lowest dose was able to inhibit tumor growth better than PAA alone
(FIGS. 1E & F). Further, the presence of tumor at the highest
dose of melphalan was detected only after four weeks confirming
that the high dose had greater antitumor effect. In contrast, no
difference in outcome was observed when melphalan was combined with
PAA even at the lowest dose. Reduction of mouse weight was not
observed suggesting lack of acute inflammation (FIG. 7). Tumor
burden was almost undetectable in mice treated with any of the
three combinational therapies (FIG. 1F). Survival curves confirmed
that high doses of single agent melphalan (3 and 5 mg/kg) extended
MM mouse survival (FIG. 1G) better than PAA alone. However, the
combination of PAA with lowdose melphalan (1 mg/kg) extended MM
mouse survival significantly compared with low-dose melphalan alone
(FIG. 1G; p<0.05). Importantly, no survival differences were
observed between low and high doses of melphalan when given in
combination with PAA (FIG. 1G and FIG. 8).
[0179] The Therapeutic Effect of Pharmacological Ascorbic Acid
Depends on Cellular Iron and Reactive Oxygen Species
[0180] We subsequently asked whether PAA was selectively killing MM
tumor cells by generating ROS, we treated OCI-MY5 MM wild-type (WT)
cells with N-acetyl cystein (NAC) or catalase. Both catalase and
NAC are commonly used anti-oxidant agents. OCI-MY5 cells pretreated
with NAC and catalase became resistant to PAA even at high doses
(FIG. 2A). Importantly, adding deferoxamine (DFO), an iron
chelator, to OCI-MY5 cells before PAA treatment was also sufficient
to prevent PAA-induced cellular death (FIG. 2A) but
bathocuproinedisulfonic acid disodium salt (BCS), a selective
copper chelator, was not able to block cellular death (FIG. 9A)
suggesting that iron is essential for PAA to achieve its
anti-cancer activity. We reasoned that high cytosolic iron would
catalyze PAA auto-oxidation leading to cell death. Because MM tumor
cells have a higher labile iron pool (LIP) than nontumor cells, we
hypothesized that PAA anti-cancer effect is dependent on LIP. We
have previously showed that Fpn1, the only known mammalian iron
exporter, is down-regulated in MM cells at the expression levels
leading to higher LIP. We next sought to determine if higher Fpn1
levels in MM tumor cells could also block cell death mediated by
PAA. We overexpressed and confirmed Fpn1 expression by qRT-PCR in
OCI-MY5 cells (FIG. 9B). We noticed that 4 mM PAA was able to kill
OCI-MY5 EV cells but not to OE-Fpn1 cells (FIG. 2B). Five-fold
greater concentration of PAA (20 mM) was required to successfully
kill OE-Fpn1 cells.
[0181] Since the overexpression of Fpn1 in OCI-MY5 cells inhibits
PAA anti-cancer activity, we next explored whether iron
supplementation was able to restore sensitivity to PAA. Iron
pre-treatment caused a rapid decrease in cells viability of OCI-MY5
EV cells (FIG. 2C) and the same effect was obtained in OCI-MY5
OE-Fpn1 cells (FIG. 2D). Consistent with our hypothesis, DFO, an
iron chelator, (FIGS. 2C & D) abolished the ability of PAA to
reduce cells viability in both EV and OE-Fpn1 OCI-MY5 cells
pre-treated with iron.
[0182] Pharmacological Ascorbic Acid Induces Both Necrosis and
Apoptosis in Myeloma Cells
[0183] To determine the type of cell death induced by PAA, we
performed transmission electron microscopy (TEM) experiments. FIG.
3A showed that in OCI-MY5 WT cells incubated with 4 mM PAA for one
hour and then left for another two hours, PAA induced early
necrosis (FIG. 3A, 60 mins) followed by late apoptosis (FIG. 3A,
120 mins). OCI-MY5 WT cells untreated appeared healthy and the
mitochondria had visible cristae. However, once cells were treated
with PAA, mitochondria started to swell and the cristae
disappeared, but no remarkable chromatin condensation was
identified (FIG. 3A, 60 mins). In a later stage, chromatin
condensation was seen in almost all cells, while mitochondrial
membranes disappeared and most of cellular organelles were degraded
(FIG. 3A, 120 mins), consistent with apoptosis. Apoptosis can be
induced by extrinsic stimuli through membrane death receptors or by
intrinsic stimuli through mitochondrial signaling pathways
(Hengartner, M. O. The biochemistry of apoptosis. Nature 407,
770-776 (2000); Kurokawa, M. & Kornbluth, S. Caspases and
kinases in a death grip. Cell 138, 838-854 (2009)). Our results
further indicated that PAA induced mitochondria-mediated apoptosis
with marked increase in caspases 3, 8, and 9 activity (FIG. 3B).
All three caspases were cleaved after 60 mins post-PAA treatment.
However, because necrosis was seen at earlier time points after PAA
treatment we also hypothesized that extrinsic stimuli might be
involved in PAA-mediated cell death and we tested the activation of
receptor interacting protein kinase 1 and 3 (RIP1 and RIP3) 24.
FIG. 3B indicated that RIP1 and RIP3 were cleaved.
[0184] Apoptosis-inducing Factor 1 Plays a Critical Role in
Pharmacological Ascorbic Acid-Induced Myeloma Cell Death
[0185] We subsequently tried to determine the molecular pathway by
which PAA induced mitochondria-mediated apoptosis. Our hypothesis
was that increased mitochondrial permeabilization was the trigger
for the death signal transduction machinery. We focused our
attention on apoptosis-inducing factor 1 (AIF1), because AIF1
induces cell death in a caspase-dependent and caspase-independent
manners (Nikoletopoulou, V., Markaki, M., Palikaras, K. &
Tavernarakis, N. Crosstalk between apoptosis, necrosis and
autophagy. Biochimica et biophysica acta 1833, 3448-3459 (2013)).
We firstly evaluate if PAA induced MM cell death depends on AIF1 at
least partially. We generated OCI-MY5 cells with AIF1 knockdown
(shRNA-AIF1) or overexpression (OE-AIF1). The viability of OCI-MY5
AIF1-shRNA cells (FIG. 4A, top bar graph) was significantly higher
than those cells expressing scrambled sequence after PAA treatment
(FIG. 4A, top bar graph), while OCI-MY5 OE-AIF1 showed
significantly less viability (FIG. 4A, bottom bar graph) than cells
transfected with empty vector (EV) when treated with PAA (FIG. 4A,
bottom bar graph). It is widely accepted that AIF1 must be cleaved
and released from the mitochondria to the cytoplasm and then
translocate to the nucleus to induce chromatolysis leading to cell
death (FIG. 4B) (Sevrioukova, I. F. Apoptosis-inducing factor:
structure, function, and redox regulation. Antioxidants & redox
signaling 14, 2545-2579 (2011)). We thus examined if PAA induced
AIF1 cleavage. OCI-MY5 cells treated with PAA showed an increase in
the AIF1 cleaved form by western blotting (FIG. 4C). Interestingly,
melphalan was not able to induce AIF1 cleavage in OCI-MY5 cells
(FIG. 4C). We hypothesized that the AIF1 cleavage was mediated by
PAA reacting with LIP to form ROS. Thus, we incubated OCI-MY5 cells
with or without DFO followed by PAA treatment. AIF1 was not cleaved
after PAA incubation in OCI-MY5 cells pretreated with DFO
confirming the crucial role of LIP in this process (FIG. 4D, white
arrow). We also tested the level of phosphorylated .gamma.-H2AX, a
biomarker for DNA double-stand breaks, after PAA and melphalan
treatment, and determined that PAA and high dose of melphalan
induced .gamma.-H2AX. However, a lower dose of melphalan with PAA
was also able to induce .gamma.-H2AX (FIG. 4C). These data support
our earlier in vivo data (FIG. 1F) that combination of PAA and
melphalan at lower dose inhibits tumor formation as the same level
or greater than melphalan alone. Cellular localization of AIF1 was
examined by immunolabeling electron microscope with and without PAA
treatment in OCI-MY5 cells. This staining revealed that AIF1
localizes not only in the mitochondria, as seen in untreated cells
(FIG. 4E, left panel), but also in cytoplasm and nuclei in
PAA-treated OCI-MY5 cells (FIG. 4E, right panel). These results
indicate that PAA by reacting with LIP and generating ROS induces
mitochondria-mediated apoptosis in which AIF1 cleavage is important
for cell death.
[0186] Discussion
[0187] High-dose vitamin C has been studies in multiple cancers and
has shown controversial clinical effects (Cameron, E. &
Pauling, L. Supplemental ascorbate in the supportive treatment of
cancer: Prolongation of survival times in terminal human cancer.
Proceedings of the National Academy of Sciences of the United
States of America 73, 3685-3689 (1976); Cameron, E. & Pauling,
L. Supplemental ascorbate in the supportive treatment of cancer:
reevaluation of prolongation of survival times in terminal human
cancer. Proceedings of the National Academy of Sciences of the
United States of America 75, 4538-4542 (1978); Creagan, E. T., et
al. Failure of high-dose vitamin C (ascorbic acid) therapy to
benefit patients with advanced cancer. A controlled trial. The New
England journal of medicine 301, 687-690 (1979); Moertel, C. G., et
al. High-dose vitamin C versus placebo in the treatment of patients
with advanced cancer who have had no prior chemotherapy. A
randomized double-blind comparison. The New England journal of
medicine 312, 137-141 (1985)). The contradictory clinical results
can be at least partially explained by different routes of vitamin
C administration applied, i.e., either orally or intravenously.
Recent reports indicate that a certain ROS concentration is
required for high-dose vitamin C to induce cytotoxicity in cancer
cells. The generation of ascorbyl- and H.sub.2O.sub.2 radicals by
PAA increases ROS stress in cancer cells. These studies including
preclinical and clinical were performed in solid tumors, such as
glioblastoma, pancreatic cancer, ovarian cancer, prostate cancer,
hepatoma, colon cancer, mesothelioma, breast cancer, bladder
cancer, and neuroblastoma. Reports are lacking to show that PAA can
be used as a pro-oxidant drug in the treatment of "liquid" tumors,
where tumor cells are surrounded by blood. This environmental
difference between solid tumor and blood cancer has the potential
to influence the PAA efficacy on cancer cell death even when given
at high doses, because ascorbic acid generated ROS are much easier
permeabilized in liquid tumor than in solid tumor. In this study,
we now report for the first time that PAA is very efficacious in
killing MM cells in vitro and in vivo models, which generated
levels of 20-40 mM ascorbate and 500 nM ascorbyl radicals after
intraperitoneal administration of 4 g ascorbate per kilogram of
body weight 38, in xenograft MM mice. These data suggest that PAA
may be a better therapeutic applied to blood cancers than solid
tumors because of the communication advantage between tumor cells
and blood plasma.
[0188] We have shown that FPN1 regulates iron export in MM cells
and LIP in vitro and in vivo. In addition, ferritin also regulates
LIP by sequestering free iron in an oxidized form to prevent
formation of free radicals. Our preliminary data show that
overexpression of FPN1 in MM cell line OCI-MY5 results in increased
viability compared to wild type cells after PAA treatment. We
hypothesize that Fpn1 expressing MM cells are less sensitive to PAA
because their cytosolic iron content is reduced by Fpn1. To test if
resistance to PAA is indeed due to low cytosolic iron content, we
depleted cytosolic iron by pre-incubating cells with an iron
chelator, deferoxamine (DFO). ARP1 MM cells pre-treated with DFO
(200 .mu.M, 3 hrs) followed by PAA treatment showed a higher
viability than cells not pre-treated with DFO. These results
strongly suggest that the mechanism of PAA killing of MM cells is
indeed iron-dependent. In addition, Fpn1 is significantly
down-regulated in CD138.sup.+ primary MM cells, while the iron
importer, transferrin receptor 1, is significantly upregulated in
CD138.sup.+ MM cells compared to normal plasma cells, further
supporting that MM cells have higher iron content than non-tumor
cells. PAA showed increased killing of MM cells derived from almost
all primary MM patients and smoldering MM, but not from MGUS
patients. These results suggest that PAA administration in SMM may
be able to prevent progression to symptomatic MM.
[0189] Though ROS and H.sub.2O.sub.2 are well known factors
mediating PAA-induced cancer cell death, a single molecular
mechanism cannot explain these observations, because multiple
pathways are involved in the downstream effects of ROS and
H.sub.2O.sub.2. Necrosis, casepase-dependent and
caspase-independent apoptosis, and autophagy were reported in
ascorbate induced cell death in different types of cancer. A recent
study by Yun and colleagues demonstrated that vitamin C selectively
kills KRAS and BRAF mutant colorectal cancer cells by targeting
GAPDH, but spares normal cells (Yun, J., et al. Vitamin C
selectively kills KRAS and BRAF mutant colorectal cancer cells by
targeting GAPDH. Science 350, 1391-1396 (2015)). Other molecular
mechanisms including ATP depletion and ATM-AMPK signaling have been
reported to explain PAA-induced cell death. In this study, TEM data
indicate that mitochondrial morphology and structure are
significantly altered after PAA treatment. Furthermore, AIF1 was
originally discovered as an intermembrane space (EMS) component of
mitochondria and characterized as a proapoptotic gene. Therefore,
we focused on AIF1 to explain PAA-induced MM cell death. The
proapoptotic AIF1 or truncated AIF1 (tAIF) is cleaved from the
full-length AIF1 by calpains and/or cathepsins after a
caspase-independent cell death insult. tAIF moves from the
mitochondria to the cytosol and nucleus, where it initiates
chromatolysis and caspase-dependent and caspase-independent cell
death. Our data show that PAA increases AIF1 cleavage and
translocation from mitochondria to cytoplasm and nucleus.
Overexpression of AIF1 in MM cells increases while knock-down of
AIF1 prevents PAA-induced MM cell death, indicating that AIF1 plays
a critical role in mediating PAA-induced MM cell death. Because the
mitochondrial apoptogenic factors such as cytochrome c and Bcl-2
family proteins are also important for the activation of caspases,
future work will have to determine if AIF1 is the major pathway
related to PAA activity in cancer cells as well as the exact
relationship with other mitochondrial apotogenetic factors. In
addition, the necrosis and apoptosis markers, such as RIP1/3 and
caspases 3/8/9, are cleaved after PAA administration. It is
therefore possible that PAA activates caspase 8 resulting in RIP1
cleavage and necrosis evidenced by strong caspase 8 cleavage after
a short-term treatment with PAA.
[0190] High oxidative stress and DNA damage activity are increased,
while the anti-oxidant enzyme levels are decreased in MM patients.
Several free radical drugs, such as As2O3 and ascorbic acid, have
been used to treat MM, in which As2O3 generates ROS while ascorbic
acid serves as an anti-oxidant agent. In MM preclinical and
clinical studies, ascorbate was used as an adjunct drug and showed
controversial results (Perrone, G., et al. Ascorbic acid inhibits
antitumor activity of bortezomib in vivo. Leukemia 23, 1679-1686
(2009); Harvey, R. D., Nettles, J., Wang, B., Sun, S. Y. &
Lonial, S. Commentary on Perrone et al.: "Vitamin C: not for
breakfast anymore . . . if you have myeloma". Leukemia 23,
1939-1940 (2009); Held, L. A., et al. A Phase I study of arsenic
trioxide (Trisenox), ascorbic acid, and bortezomib (Velcade)
combination therapy in patients with relapsed/refractory multiple
myeloma. Cancer investigation 31, 172-176 (2013); Sharma, M., et
al. A randomized phase 2 trial of a preparative regimen of
bortezomib, high-dose melphalan, arsenic trioxide, and ascorbic
acid. Cancer 118, 2507-2515 (2012); Nakano, A., et al. Delayed
treatment with vitamin C and N-acetyl-L-cysteine protects Schwann
cells without compromising the anti-myeloma activity of bortezomib.
International journal of hematology 93, 727-735 (2011); Takahashi,
S. Combination therapy with arsenic trioxide for hematological
malignancies. Anti-cancer agents in medicinal chemistry 10, 504-510
(2010); Sharma, A., Tripathi, M., Satyam, A. & Kumar, L. Study
of antioxidant levels in patients with multiple myeloma. Leukemia
& lymphoma 50, 809-815 (2009); Qazilbash, M. H., et al. Arsenic
trioxide with ascorbic acid and high-dose melphalan: results of a
phase II randomized trial. Biology of blood and marrow
transplantation. Journal of the American Society for Blood and
Marrow Transplantation 14, 1401-1407 (2008)). However, none of
these tests used pharmacological doses of ascorbate and intravenous
administration. It has been reported that ascorbate directly
inactivates proteasome inhibitor by forming a tight but reversible
complex through its vicinal diol group (Perrone, G., et al.
Ascorbic acid inhibits antitumor activity of bortezomib in vivo.
Leukemia 23, 1679-1686 (2009); Harvey, R. D., Nettles, J., Wang,
B., Sun, S. Y. & Lonial, S. Commentary on Perrone et al.:
"Vitamin C: not for breakfast anymore . . . if you have myeloma".
Leukemia 23, 1939-1940 (2009)). This dose of ascorbate in the
combination with bortezomib is at a physiological level which has
anti-oxidant effect. It will be interesting to test if high dose
ascorbate, which functions as a pro-oxidant agent, can increase
bortezomib efficacy in MM treatment.
[0191] Our findings complement reported studies and further address
the mechanism of action using clinical samples in which we observed
that PAA only kill tumor cells with high iron content, suggesting
that iron is the initiator of PAA cytotoxicity. In addition,
combination of PAA with standard therapeutic drugs, such as
melphalan, may significantly reduce the dose of melphalan needed,
because high dose melphalan is very toxic not only to tumor cells
but also to normal tissues, such as hematopoietic stem cell and
epithelial cells in the gut. The efficacy of high dose melphalan by
itself is clearly dose-dependent. Combined treatment of reduced
dose melphalan with PAA achieved a significantly longer
progression-free survival than the same dose of melphalan alone.
These data also suggest that the bone marrow suppression induced by
high-dose melphalan can be ameliorated by the combination of PAA
with lower dose of melphalan because of the lack of toxicity of PAA
on normal cells with low iron content.
Example 2
Implication of Iron in Multiple Myeloma Tumor Biology and
Progression
[0192] Multiple myeloma (MM) is a plasma cell neoplasm. Novel
drugs, such as proteasome inhibitors and immunomodulatory agents,
combined with Autologous Stem Cell Transplantation have led to
complete remissions in a majority of newly diagnosed patients with
MM. These treatments are not aimed at specific molecular targets
and often result in increased toxicity and decreased therapeutic
efficacy, therefore, development of novel target therapies is
urgent. Recent reports have shown that iron induces cancer
development and is associated with progression and poor prognosis
in several malignancies. It has recently been discovered that iron
plays an important role in MM tumor development and progression. In
particular, it was observed that alterations of iron homeostasis
are key metabolic changes in MM patients. Ferroportin 1 (Fpn1)
expression, the only known iron efflux pump in mammalian cells, is
significantly downregulated in MM cells compared with their normal
counterparts. In normal cells, Fpn1 is mainly regulated
post-translationally by hepcidin resulting in its degradation. Low
expression of Fpn1 results in an increased labile iron pool in
tumor cells. Importantly, low expression of Fpn1 has been linked to
poor prognosis in primary MM samples using gene expression
profiles. Similar outcomes have been reported in breast cancer
studies.
[0193] The present Example characterizes iron homeostasis in MM
cells and its role in tumor cell development and progression. Five
novel discoveries have laid the groundwork for these studies: (1)
Multiple signature genes related to iron homeostasis are
dysregulated in MM. (2) The expression of Fpn1 is downregulated in
MM cells and its downregulation is negatively correlated with
patient outcome. (3) Fpn1 regulates intracellular iron in MM cells
using in vitro and in vivo models. (4) Restoring expression of Fpn1
suppresses MM cell growth both in vitro and in vivo. And (5)
Pharmacological modulation of MM cellular iron prevents tumor
progression in vivo. The results suggest that iron is not only a
hallmark for disease progression but also could serve as a target
for therapy in MM.
[0194] Introduction
[0195] Multiple Myeloma (MM) is a plasma cell tumor and the second
most common blood-derived malignancy in the US. Clinical outcomes
of patients with MM are extremely heterogeneous, with survival
ranging from only several months to more than 15 years. In addition
to genetic heterogeneity, increasing evidence suggests that iron
metabolism in cancer cells accounts for the divergent clinical
outcomes. The expression of proteins involved in maintaining
cellular iron balance was analyzed and it was discovered that iron
homeostatic mechanisms are altered in MM. Particularly, in
different MM stages, Fpn1 is less expressed leading to high
intracellular labile iron pool (LIP). Fpn1 is the receptor for the
hormone hepcidin (Hamp). Increased hepcidin expression induces
impaired iron utilization and results in normochromic/normocytic
anemia in many diseases, including MM. Fpn1 expression also is
negatively correlated with patient outcomes. Fpn1 encodes a
multiple transmembrane domain protein that transfers cellular iron
to the plasma, which regulates the exit of iron from cell. It has
been reported that Fpn1 is downregulated in breast cancer cells
when compared to their normal counterparts (Pinnix Z K, Miller L D,
Wang W, D'Agostino R, Jr., Kute T, Willingham M C, et al.
Ferroportin and iron regulation in breast cancer progression and
prognosis. Science translational medicine 2010 Aug. 4; 2(43):
43ra56). Consistent with the low levels of Fpn1 expression, the
breast cancer cells showed a markedly higher LIP than the
non-malignant breast epithelial cells. Iron metabolism is emerging
as a key metabolic hallmark of cancer. In normal cells, Fpn1 is
mainly regulated post-translationally by hepcidin resulting in its
degradation. Studies suggest that iron dysregulated is not only a
biomarker for prognosis but also could serve as a target for
treatment in MM. Cancer cells tend to enhance cellular iron
availability, resulting in increased cellular proliferation. MM
cells exhibit different iron needs when compared to normal
differentiated plasma cells. The studies described in this
application focus on how iron distribution is regulated and how its
changes affect MM tumor cells biology. Further, because of these
metabolic alterations, targeting the specific iron needs of MM
cells can be of therapeutic value.
[0196] The studies in this Example investigate the molecular basis
of iron regulation in MM cells and its therapeutic implication. The
importance of iron in MM cells is critical because subtle changes
in iron balance influence tumor development, progression and
treatment in multiple ways. Finally, information gained from this
study is relevant to dysregulated iron metabolism in other forms of
cancer.
[0197] Specific treatment for the dysregulated iron metabolism in
cancer cells is lacking, because the critical regulation mechanisms
of iron homeostasis remain largely unknown.
[0198] Iron Homeostasis is Altered in Multiple Myeloma Cells.
[0199] Gene expression analysis of iron-regulatory genes in the MM
malignant cells from 351 newly diagnosed patients (Total Therapy 2,
TT2) shows a deregulation in cellular iron homeostasis signaling
when compared to 22 normal plasma cells. Of the 61 signature genes
related to iron metabolism (131 probe sets), 29 genes were
significantly deregulated by comparison of normal plasma cells to
MM samples (FIG. 10). The expression of these 29 genes was then
correlated with patient outcome in the TT2 cohort and Fpn1 was
found to be the gene mostly associated with an inferior outcome in
MM. Fpn1 expression was significantly lower in plasma cells derived
from MM patients compared to those derived from patients with
monoclonal gammopathy of undetermined significance (MGUS, benign
monoclonal gammmopathy) and healthy donors (p<0.0001, FIG.
11).
[0200] Low-Expression of Fpn1 is Linked to Poor Outcome in MM.
[0201] Survival analysis was performed using Kaplan-Meier test in
three different data sets. Consistent with the low Fpn1 expression
in the aggressive MM subgroups, decreased Fpn1 in the 351 TT2
cohort showed that about 60% of such cases showed a short
event-free survival (EFS) (FIG. 12A, p<0.001) and also an
inferior overall survival (OS) (p<0.001, FIG. 12B). Similar
results were observed from HOVON65 49 and APEX50 cohorts including
288 newly diagnosed MM samples and 264 relapsed MM samples
respectively (Gu Z, Wang H, Xia J, Yang Y, Jin Z, Xu H, et al.
Decreased ferroportin promotes myeloma cell growth and osteoclast
differentiation. Cancer research 2015 Jun. 1; 75(11):
2211-2221).
[0202] Fpn1 Regulates Intracellular Iron In Vitro and In Vivo in MM
Cells.
[0203] To test whether Fpn1 regulates iron efflux in MM cells, the
labile iron pool (LIP) was measured with fluorescent metallosensor
calcein. ARP1 and OCI-MY5 cells overexpressing Fpn1 had
significantly lower LIP compared to their EV counterparts (FIG.
13).
[0204] Iron retention promotes tumor development and progression in
vivo. The 5TGM1-KaLwRij model was further analyzed to test the role
of Fpn1-inducing iron retention on MM progression in vivo.
Real-time PCR confirmed that 5TGM1 MM cells had much lower
expression of Fpn1 than normal bone marrow plasma cells in KaLwRij
mice. The coding region of Fpn1 cDNA in a doxycycline inducible
lentiviral construct was stably transduced with lentivirus into the
5TGM1 cells, in which the expression of Fpn1 was conditionally
induced upon addition of doxycycline. One week after transduced
5TGM1 cell injection, mice were administrated doxycycline and
dextran-iron to increase systemic iron content in the mouse body.
Overexpression of Fpn1 (induced by administration of doxycycline,
Dox) significantly extended mouse survival (FIG. 14A, +DOX open
square) compared to non-induced (-Dox triangle) group. Tumor burden
in this group was also reduced, assessed by mouse serum IgG2b level
(FIG. 14B, compare open circle with triangle). To confirm whether
inhibition of tumor formation by Fpn1 is because it enhances iron
efflux, iron was given in the drinking water to the above mice to
reverse the effect. 5TGM1 mice that received iron accelerated tumor
progression resulting in a shorter survival and higher tumor burden
than those without iron in their drinking water (FIG. 14A, compare
closed circle with triangle). Importantly, the effect of iron
administration on MM progression could be blocked if Fpn1 is
overexpressed in MM cells (FIG. 14A, compare square with open
circle). All findings, to date, show that MM cells exhibit
different iron needs when compared to normal differentiated plasma
cells, as schematized in FIG. 15.
[0205] Determination of how Myeloma Tumor Cells Uptake Iron from
the Bone Marrow Microenvironment.
[0206] Previous studies conducted in MM and in different blood and
solid tumors show that cancer cells differ from their non-malignant
counterparts in the levels and activity of multiple proteins
involved in iron homeostasis. These changes result in increased
intracellular iron levels facilitating to tumor proliferation.
Despite the mechanisms that tumor cells retain intracellular iron,
particularly in MM, remain unclear, the possible changes in iron
uptake may allow MM cells to accumulate iron from the
microenvironment. To depict these crucial changes in iron uptake
the following three possibilities are investigated: (1) if the
transferrin pathway is critical to increase intracellular iron in
MM cells; (2) if a transferrin-independent iron transport
mechanism, such as lipocalin-2, is involved in iron accumulation in
MM cells; and (3) if macrophages are the predominant iron
reservoir.
[0207] Determine if Transferrin Pathway is Critical for Iron Uptake
in Multiple Myeloma Tumor Cells.
[0208] The transferrin pathway plays a critical role for iron
acquisition by most cells. In the body, iron circulates bound to
transferrin (TF) which binds two atoms of ferric iron. Once the
TF-iron complex is formed, it binds to the transferrin receptor 1
(TFRC) present at the plasma membrane in many cells, then the new
complex TF-iron-TFRC is internalized by endocytosis. After iron is
released in the cytoplasm, the TF-TFRC complex recycles back to the
plasma membrane. The levels of TF and TFRC in normal cells are
relatively low to maintain a small pool of labile iron, however
some findings have reported that tumor cells have increased
expression of TFRC and this increase could be associated with
patients' poor prognosis. It was recently reported that
upregulation of TFRC may not only enhance the iron uptake but also
promote cell survival by activating other cellular signaling
pathways in breast cancer. Gene expression profiles show that MM
cells have higher expression of TFRC (FIG. 16) when compared with
normal plasma cells (NPC). TFRC also appears unregulated in MGUS
samples, a pre-MM disease, when compared to normal plasma cells.
Thus, it is hypothesized that TFRC is upregulated in MM tumor cells
to maintain higher pool of labile iron for MM development and
progression.
[0209] To ascertain the role of TFRC in iron uptake by MM tumor
cells, a lentiviral vector expressing TFRC shRNA is used to
knockdown TFRC expression. Two shRNA targeting different regions of
the TFRC transcript are designed and one scramble shRNA is used as
a control. RT-PCR and western blotting confirms the shRNA-mediated
efficiency suppression of TFRC. Using real-time PCR and western
blotting, any changes in the expression of components related to
iron metabolism such as iron storage factor ferritin and Fpn1 after
TFRC knockdown are detected. These changes are examined in the
presence or absence of an external iron source such as diferric
transferrin and/or ferric ammonium sulfate. Diferric transferrin
and ferric ammonium sulfate are commercially available and widely
used. Labile iron pool are measured using fluorescent metallosensor
calcein. It is anticipated that labile iron pool is constant or
decreased in the knockdown cells for TFRC if TFRC is a crucial for
iron uptake; if TFRC is not the only protein responsible for iron
uptake, an LIP increase in the TFRC shRNA cells when iron is added
in the cell culture media is also expected. If this is the case,
other(s) protein(s) could be responsible for the increase of
cellular iron pool. The cell survival effect of TFRC suppression by
colony formation assay is also analyzed. ARP1 and OCI-MY5
transduced with scramble or TFRC shRNA lentiviruses are mixed with
RPMI1640 media containing 10% FBS and 0.33% agar and layered on the
top of the base layer of 0.5% agar in each well of 6-well plate.
Half wells are treated with diferric transferrin and ferric
ammonium sulfate. Colony numbers are counted after approximately
2-3 weeks. All plates are pictured under a microscope and overall
numbers of colonies counted and quantified by Image J software.
[0210] Determine if a Transferrin-Independent Mechanism is Involved
in Iron Uptake in Multiple Myeloma Cells.
[0211] Recent studies pointed to a role of lipocalin-2 in
facilitating tumorigenesis in various solid cancers and trafficking
iron into cells in a transferrin receptor-independent manner. To
properly traffic iron, lipocalin-2 forms a complex with
iron-enriched mammalian siderophores (holo-lipocalin-2) and binds
to its cell surface receptor, SLC22A17. Once internalized
lipocalin-2 releases iron leading to a higher labile iron pool. It
is important to point out that SLC22A17 also binds apo-lipocalin-2
(a form not bound to siderophore) and in this case lipocalin-2 in
the cytoplasm acts as an iron chelator by transferring
intracellular iron to the extracellular compartment with consequent
reduction of labile iron pool in the cytoplasm. Interestingly, the
gene expression profile data from primary MM samples showed that
TFRC expression was not upregulated in all MM samples as might be
expected to maintain higher cytosolic iron (FIG. 10). This
observation suggests that MM tumor cells are also able to uptake
iron in a transferrin-independent manner. To test if this is true,
the SLC22A17 expression level in the two populations respectively
with high and low TFRC was analyzed. The results indicated that
SLC22A17 was upregulated in the MM tumor cells with low TFRC, while
SLC22A17 was downregulated in those highly expressing TFRC (FIG.
17).
[0212] To determine the role of SLC22A17 and lipocalin-2 in MM iron
uptake, the following experiment is performed. First, the
expression of SLC22A17 is measured in ARP1 and OCI-MY5 cells with
or without knockdown of TFRC by RT-PCR and western blotting. It is
expected that MM cells silenced TFRC will upregulate SLC22A17
expression if lipocalin-2 is involved in iron uptake. It is then
determined if MM cells silenced TFRC are able to increase their
cellular iron concentration after incubation with
lipocalin-2-iron-siderophore complex. Recombinant mouse lipocalin-2
is synthetized as a glutathione S-transferase fusion protein in the
BL21 strain of Escherichia coli (Stratagene, La Jolla, Calif.).
Briefly, ferric sulfate is added to the culture medium at 50 .mu.M.
The protein is isolated using glutathione-Sepharose 4B beads
(Amersham Biosciences), eluted with thrombin (Sigma-Aldrich), and
purified with gel filtration (Superdex 75; Amersham Biosciences).
Recombinant protein is mixed with iron-loaded and iron-unloaded
forms of a bacterial siderophore enterochelin (EMC
Microcollections, Tubingen, Germany) in phosphate-buffered saline
at room temperature for 60 min. Unbound siderophore is removed with
Microcon YM-10 (Millipore). The recombinant protein is added to the
culture media of MM cells silenced TFRC. Cellular iron
concentration will be measured by fluorescent metallosensor
calcein. It is expected that MM cells with low expression of TFRC
increases their iron content when incubated with the recombinant
protein and conclude that TFRC is not the only responsible protein
for iron uptake. An important control for these cells is the
incubation with transferrin-iron because under these conditions
only the control MM cells transduced with scramble lentiviruses are
able to increase their labile iron pool but not the TFRC silenced
MM cells.
[0213] Determine if Bone Marrow Macrophages are the Iron Reservoir
for Multiple Myeloma Cells.
[0214] MM cells are always in need of an iron reservoir in order to
fulfill their higher metabolic demand and support their growth and
progression. Under normal conditions, macrophages are considered
the "specialized iron cells" because they are able to acquire,
recycle, process, store and transport iron. Further, macrophages,
including those in a malignant setting, exhibit a remarkable
heterogeneity and functional plasticity by assuming an M1, iron
sequestration and tumor repression, or M2, iron release and tumor
promotion, phenotype. It is hypothesized that macrophages within MM
bone marrow microenvironment are the strong candidate as an iron
source for MM tumor cells. Interestingly, to support our
hypothesis, several studies have reported that M2 macrophages are
increased in MM patients. The following studies investigate whether
macrophages can be co-cultured with the mouse cell line 5TGM1 and
the iron trafficking under these conditions analyzed. For these
experiments, bone marrow macrophages are isolated from
C57BL/Kalwrij mice, which spontaneously develop myeloma in aging.
These bone marrow macrophages, isolated from mouse femurs, are
grown in RPMI 1640 media supplemented with 20% equine serum for 6
days and adherent cells are further cultured in RPMI 1640 with 20%
fetal bovine serum and 30% L-cell conditioned medium. L-cell
conditioned medium is used as source of colony stimulating factor
required for macrophage differentiation. Later, macrophages are
iron loaded with ferric ammonium citrate (FAC, 10 .mu.M iron) for
18 hours and after that iron is washed away for 18 hours to allow
them to export the iron via Fpn1. It is known that during iron
loading in macrophages Fpn1 is synthetized and goes to the cell
surface, and once iron is washed away from the medium Fpn1 exports
iron out from cells. In the co-culture experiments, this phenomenon
is taken advantage of to determine if the iron exported by Fpn1
from macrophages is taken by MM tumor cells. As a control that
macrophages uptake and later release iron, ferritin levels, the
cytosolic iron storage, are analyzed by western blotting and also
intracellular iron pool will be measured by fluorescent calcein as
described above. The anticipated result is that ferritin/iron pool
is higher when macrophages are incubated with iron but rapidly
decrease once iron is removed and Fpn1 starts exporting
intracellular iron into the extracellular compartment.
[0215] Once the experiments are completed that show that
macrophages are able to increase and release their cytosolic iron;
co-culture experiments with MM cells are performed. Briefly, three
conditions are evaluated: (1) macrophages without iron; (2)
macrophages incubated with iron; and (3) macrophages incubated with
iron for 18 hours and later iron washed away for another 18 hours.
This condition is run in duplicate with or without co-culture with
5TGM1 MM cells (FIG. 18). Cells are lysed and total RNA and
proteins are extracted from each condition. It is expected that
5TGM1 cells uptake iron from macrophages, cytosolic ferritin and
labile iron pool decreases in macrophages in which iron is washed
away and co-cultured with 5TGM1 cells.
[0216] Determine the Mechanisms that Lead to Transcriptional
Repression of Fpn1 in Multiple Myeloma Cells.
[0217] Data described herein show that Fpn1 expression in MM cells
is sharply downregulated and cytosolic iron is high. Regulation of
Fpn1 at the translational and posttranslational level is well
described but little is known about transcriptional regulation.
Through a systemic analysis of microarray data, it was identified
that the epigenetic modulator histone methyltransferase enhancer of
zeste 2 (EZH2) was negatively correlated with the Fpn1 expression
between normal with malignant plasma cells and low-risk and
high-risk MM samples (FIG. 19A). Since Fpn1 functions are tightly
related to iron and oxidative reaction, it also is hypothesized
that cellular iron and oxidants might regulate Fpn1 transcription.
To identify how Fpn1 is downregulated in MM cells, the following
three possibilities are investigated: (1) if low Fpn1 is a
consequence of epigenetic modification; (2) if high intracellular
iron content is involved in regulation of Fpn1 expression; and (3)
if high oxidants suppress Fpn1 transcription. These experiments
determine how transcription of Fpn1 is regulated in MM cells and to
offer insights for a potential clinical utility.
[0218] Determine if the Histone Methyltransferase Enhancer of Zeste
2 Suppresses Fpn1 Transcription.
[0219] Several studies have shown that epigenetic modifications
affecting specific pathways are important in the development and
treatment of MM. In MM, some of the epigenetic effects result in
repression of gene expression such as EZH2. EZH2 is a component of
the Polycomb Repressive Complex 2 (PRC2) which includes EZH2,
Suz12, and EED. It was found that EZH2 is dramatically upregulated
and shows an inverse correlation with Fpn1 expression using gene
expression profiles in primary MM samples (FIG. 19A). A putative
EZH2 binding site at the Fpn1 promoter was further discovered,
which overlaps with the mark of transcription start sites of active
genes (H3K4m3) and the mark of transcriptional silencer H3K27me3,
but not with the mark of transcriptional activator H3K27ac from the
UCSC-ENCODE (FIG. 19B). This suggested that EZH2 may repress Fpn1
expression. To prove the involvement of EZH2 in Fpn1 regulation, MM
cell lines ARP1 and OCI-MY5 are treated with commercially available
EZH2 inhibitors and Fpn1 levels are analyzed by Real Time-PCR
(RT-PCR).
[0220] The EZH2 inhibitors are an emerging class of therapeutics
with anticancer properties and several studies show that they can
decrease EZH2 protein levels. For these studies, the efficacy of
DZNep and GSK343 are tested. DZNep has been shown to inhibit EZH2
protein expression and subsequently reduce the trimethylation of
H3K27me3. GSK343 is a potent inhibitor of the histone H3K27
resulting in inhibition of EZH2 enzymatic activity. The above MM
samples are treated with EZH2 inhibitors at two doses 5 and 10
.mu.M for 24, 48 and 72 hours. For each dose and time of
incubation, total RNA is isolated and Fpn1 is analyzed by RT-PCR. A
critical control for this experiment is to monitor apoptosis
because it has been shown that EZH2 inhibitors induce cell death
via apoptosis. To eliminate the off-target issue, shRNA or
CRISPR-Cas9 is used to silence EZH2 in MM cells and the expression
of Fpn1 is analyzed by RT-PCR. It is expected that if EZH2 is
involved in regulation of Fpn1 transcription, Fpn1 mRNA should be
higher in cells treated with the inhibitors or knockdown of EZH2
when compared to the control cells. If this is the case, it is
examined if EZH2 directly binds to the promoter region of Fpn1 by
chromatin immunoprecipitation-qPCR (ChIP-qPCR) analysis. Cell
extracts from the above described cells are crosslinked and
sonicated to obtain DNA fragments with an average size of 0.3-0.5
kb. Protein-DNA complexes are immunoprecipitated using EZH2
antibody or IgG as a control, followed by the addition of Dynabeads
protein. The relative amount of Fpn1 promoter fragments containing
the EZH2 element is measured by real-time PCR with appropriate
primers for human Fpn1. These data may provide a strong evidence
that EZH2 is an epigenetic repressor of mFpn1 in MM cells.
[0221] Determine if Iron Mediates Fpn1 mRNA Decrease in MM
Cells.
[0222] Iron impacts the expression profile in all eukaryotic cells.
These effects can occur at the transcriptional and
post-transcriptional levels. Iron-mediated transcriptional
regulation has been less studied. These experiments focus on
determining if iron is involved in the downregulation of Fpn1 in MM
cells. Fpn mRNA contains a 5' iron-response element (5'IRE)
suggesting the Fpn1 is regulated post-transcriptionally. Others
have shown that Fpn1 is transcriptionally upregulated in wild type
macrophages when treated with iron. The gene expression profile is
initially analyzed by microarray of iron-fed wild type ARP1 MM
cells compared to untreated cells.
[0223] The following experimental procedures also are schematized
in FIG. 20. First, those genes are examine that show evidence of 3'
iron-responsive element (IRE) and an informatic approach is used to
determine if sites are available on Fpn1 promoter. A few candidates
are selected, these sites are mutated in a Fpn1 luciferase promoter
construct. The iron-sensitive elements within the promoter are
identified by making specific deletion(s). Second, it is explored
as to which sites affect Fpn1 transcription. It is determined if,
in an iron-dependent or iron-independent manner, the candidate
repressor sites on a Fpn1 promoter by ChIP-qPCR analysis. Third, it
is determined if knockdown of these possible repressors leads to
increased expression of Fpn1.
[0224] Determine if Fpn1 Expression in MM Cells is Suppressed by
Oxidants.
[0225] It was determined that cytosolic iron in MM cells is higher
(see FIG. 13). It is hypothesized that iron affects Fpn1
transcription through its effect on oxidation. An increase in
oxidants can trigger alterations in transcription through a number
of distinct mechanisms. Among the different possibilities,
oxidation and reduction regulate transcription factors. First, it
is determine if Fpn1 transcription in MM cells is altered by
anti-oxidants. MM cell lines ARP1 and OCI-MY5 are incubated with
iron and N-acetyl cysteine (NAC) or ascorbate and transcription of
target genes assayed by RT-PCR or by reporter constructs.
[0226] Levels of O.sub.2..sup.- and H.sub.2O.sub.2 are measured
using SOD-inhibitable and catalase-inhibitable dihydroethidium
(DHE) and 2',7'-dichlorodihydrofluorescein diacetate (H2DCF-DA)
oxidation. Mitochondrial localization of O.sub.2..sup.- signals are
determined using MitoSOX Red oxidation and MitoTracker Green
staining followed by confocal microscopy. Further confirmation that
the dye oxidation is mediated by mitochondrial reactive oxygen
species (ROS) utilizes adenoviruses overexpressing the
mitochondrial form of manganese superoxide dismutase (Ad-MnSOD) or
catalase (Ad-MitoCAT); these recombinant adenoviruses are available
through the Vector Core, University of Iowa. Adenovirus-mediated
increases in enzymatic activity of SOD/catalase are assayed
(Radiation and Free Radical Research Core, RFRRC, University of
Iowa). If the anti-oxidants increase Fpn1, then it can be concluded
that iron is acting through modification of redox status.
[0227] Determine if Hepcidin is Responsible for Fpn1
Transcriptional Repression.
[0228] It is known that hepcidin binds to Fpn1 and induces its
internalization and degradation and it has also been reported that
serologic hepcidin levels are higher in MM patients than healthy
controls. It is important to take into account that gene expression
profile showed that hepcidin expression (HAMP, see FIG. 10) is
decreased; however, this result does not preclude that hepcidin
does not regulate Fpn1 but only suggests that hepcidin seen in
patients' sera does not come from tumor cells. Therefore, it is
hypothesized that hepcidin by inducing Fpn1 internalization lead
also to transcription repression. The present studies determine if
decreased transcription of Fpn1 results from the loss of cell
surface Fpn1 by hepcidin. Previously it has been shown that cells
treated with dynasore, an inhibitor of dynamin-mediated
internalization or expression of a dominant negative K44A dynamin
mutant lacking of the GTPase activity prevents Fpn1 internalization
after hepcidin binding.
[0229] The first set of experiments are done to verify that in ARP1
and OCI-MY5 MM cell lines expressing Fpn1-GFP (GFP is integrated in
the Fpn gene) and dynamin mutant K44A, Fpn1 is not internalized
after incubation of hepcidin by immunofluorescence and western
blotting. It is predicted that the results will confirm that
dynamin is necessary for hepcidin-mediated Fpn1 internalization in
MM cells. Hepcidin is add to MM cells expressing Fpn1-GFP and
dynamin K44A or treated with dynasore, and it is determined if
endogenous Fpn1 transcription increases at different time courses
(FIG. 21). If Fpn1 levels do not change, it can be concluded that
hepcidin does not control Fpn1 transcription in MM cells. If Fpn1
levels decrease further, it can be concluded that hepcidin is
involved in the transcriptional repression of Fpn1 in MM cells. An
important control is required to prove that hepcidin is involved in
Fpn1 downregulation. Fpn1 is measured in cells treated with
dynasore or expressing dynamin K44A in the absence of hepcidin to
rule out blocking cellular dynamin-mediated internalization does
not interfere with Fpn1 transcription.
[0230] Determine if Pharmacological Cellular Iron Modulations Serve
as New Therapeutic Approaches in Multiple Myeloma.
[0231] Data show that Fpn1 overexpression inhibits tumor growth in
a xenografted MM mouse model. These results suggest that modulating
intracellular iron may be used as a therapeutic approach for MM. In
the present experiments, both MM cell lines and primary MM samples
are used to develop novel treatment strategies by pharmacological
regulating iron homeostasis or "utilizing" high cytosolic iron
content.
[0232] Determine if Direct Iron Chelation Inhibits Tumor Growth in
a Xenografted MM Mouse Model.
[0233] One way to regulate cytosolic iron in MM cells is the direct
chelation of iron. Previous studies have shown that desferrioxamine
(DFO) has anti-cancer activity. However, these studies suggest that
the utility of DFO is limited due to its poor cell membrane
permeability and short half-life. Based on this information, it is
proposed to use two relatively new iron chelators for our
experiments, deferiprone (Ferriprox; ApoPharma, Toronto, Canada)
and deferasirox (Exjade; Novartis, Basel, Switzerland). These iron
chelators show more permeability and longer half-life when compared
to DFO. Recently deferasirox was reported to inhibit the growth of
myeloid leukemia cells in vitro and in vivo. It has also been
determined that deferasirox inhibits the growth of human lung
carcinoma xenographed mice.
[0234] The following experiments investigate the in vivo
anti-cancer activity of deferasirox and deferiprone in xenografted
MM mice. Human myeloma cell lines (ARP1 and OCI-MY5) with
luciferase expression are injected subcutaneously into each flank
of NOD-Rag/null gamma mice, tumor burdens will be monitored by
bioluminescence assay and tumor volumes as described previously.
Also, mice receive a single intraperitoneal injection with
dextran-iron (250 .mu.g per gram of body weight) to increase
systemic iron content in the mouse body. Iron accumulation is
monitored in these mice by measuring transferrin saturation using a
commercially available kit. Increased transferrin saturation
demonstrates that mice are absorbing iron. Subsequently, a group of
mice will be treated with an iron chelator (40 mg/kg by oral gavage
for 3 weeks). In this study, each group (control, iron, chelator 1,
chelator 1+iron, chelator 2, chelator 2+iron) include 3 mice with 6
tumors, thus a total of 36 mice are required (6 groups.times.3
mice/group.times.2 cells lines). It is expected that direct iron
chelation therapy delays tumor progression significantly in mice
and that longer MM mouse survival occurs when compared to the group
that was not treated with the chelators.
[0235] Determine if Induction of Ferroptosis Inhibits Tumor Growth
in a Xenografted MM Mouse Model.
[0236] Ferroptosis is a non-apoptotic form of cell death resulting
from an iron-dependent accumulation of lipid ROS and it has been
shown that ferroptosis facilitates the selective elimination of
some tumor cells. It has been discovered that erastin, a cell
permeable piperazinyl-quinazolinone compound, can induce
ferroptosis by binding the mitochondrial voltage-dependent anion
channels and altering its gating. Others have shown that
ferroptosis can be inhibited by iron chelation. The following
experiment investigate the anti-cancer activity of ferroptosis in a
xenograft MM mouse model. It has been shown that MM cells have high
cytosolic iron. Thus, it is hypothesized that injection of erastin
induces MM cells ferroptosis with consequent delay in tumor
progression and longer survival of MM mice. This proposed mechanism
is summarized in FIG. 22. Data support this hypothesis: erastin
treatment in MM cells (KMS11, ARK and ARP1) inhibits cellular
growth and this effect can be reversed when cells are treated with
a ferroptosis inhibitor ferrostatin (FIG. 23). Human MM cell lines
with low mFpn1 (ARP1 and OCI-MY5) are injected subcutaneously into
each flank of NOD-Rag/null gamma mice and tumor burden is monitored
by bioluminescence assay and tumor volumes as described previously.
Subsequently, the piperazine erastin (PE) water-soluble analog as
previously described is used for in vivo injections 72. The PE is
administrated subcutaneously at 60 mg/kg mouse weight twice per
week for 2 weeks according to published studies and 12 mice (2
groups.times.3 mice/group.times.2 MM cell lines) are required for
this study. The ferroptosis activity is monitored in mice by
analyzing expression of ferropotosis marker PTGS2 by RT-PCR and the
up- or downstream regulators, such as GPX4, p21, and p53
activation.
[0237] Determine if High Cytosolic Iron in MM Patients is
Targetable by Pharmacological Ascorbic Acid.
[0238] Recent studies have shown that pharmacological ascorbic acid
(PAA) selectively kills cancer cells while sparing the
non-malignant cells (FIG. 24) in primary tumor samples. Further, it
has been observed that PAA anticancer activity is iron-dependent.
In fact, PAA was not able to decrease tumor burden in mice
receiving injection at the same time with the iron chelator DFO
(FIG. 25). Therefore, it is hypothesized that high iron in MM
patients' tumor cells can be targetable by PAA anti-cancer
activity. The present studies assess the efficacy of PAA in
treating human primary MM cells collected at diagnosis and at
relapse using the NOD-Rag1.sup.null-hu mouse model. The
NOD-Rag1.sup.null-hu mouse model uniquely enables the study of
human primary MM cells in a human bone marrow microenvironment.
Briefly, human fetal long bones (tibias and femurs) from 18- to
21-week gestational fetuses are cut into two 10-mm pieces and
implanted subcutaneously, on either the left or right side of the
dorsum of NOD-Rag1.sup.null mice (one bone/mouse). Eight to 10
weeks after implantation of the bones, 1.5.times.10.sup.6
CD138.sup.+ MM cells sorted from newly diagnosed and relapsed
patients (9 for each set) are injected directly into the human
fetal bone. Each sample of myeloma cells are transferred to 3
NOD-Rag1.sup.null-hu mice. Before injection of MM cells, qRT-PCR is
performed to quantify the expression of Fpn1 in sorted CD138.sup.+
MM cells and CD138.sup.- cells (nonmalignant group). The efficacy
of PAA alone and in combination with 2 common drugs currently used
for MM treatment are studied: melphalan (Mel) and carfilzomib
(Cfz). Six combinations for each clinical sample (untreated, PAA,
Mel, Cfz, PAA+Mel and PAA+Cfz) are used. It is possible to purify
10.times.10.sup.6 MM cells from a newly diagnosed MM sample or from
a relapsed MM patient, respectively. Therefore, one sample is
sufficient to cover the six combinations outlined above. Drug
concentrations are PAA (4 mg/kg, twice a week, intraperitoneal, for
4 weeks), Mel (3 mg/kg, twice a week, intraperitoneal, on the same
days as PAA administration for 4 weeks) and Cfz (3 mg/kg, twice a
week, intraperitoneal, on the same days as PAA administration for 4
weeks). This study utilizes nine paired MM samples obtained at
diagnosis and in relapse. A total of 108 mice with equal
representation of mouse gender within each treatment group at each
time point are required. Tumor growth is monitored by measuring
human serum free light chains and M protein. Mice survival and time
to tumor recurrence time are compared among the above outlined
groups. Experiments are terminated when drug-treated mice reach
complete remission for three months or when control mice become
sick due to high tumor burden. The implanted femoral bone is be
processed for histology and histomorphometry.
[0239] Statistical Analysis:
[0240] Statistical analysis is performed to compare treatment
groups within each experiment with respect to the proportion of
mice. Power is estimated based on pairwise treatment group
comparisons performed with a simpler one-sided Fisher's exact test
at a single time point. Without treatment, the rate of tumor
development or relapse is conservatively estimated to be 95%.
Accordingly, the use of nine mice per group achieves 80% power to
detect a difference of at least 60% (95% vs 35%) between the
untreated and an active treatment group at the 5% significance
level. In addition, time to relapse or time to B lymphoma is
explored in a full analysis comparing treatment groups. Survival
curves are constructed using the Kaplan-Meier method and compared
between treatment groups using the log-rank test.
Example 3
TRIP13: A Novel Gene in Multiple Myeloma Tumorigenesis and
Progression
[0241] Multiple myeloma (MM) is a prototypical clonal B-cell
malignancy with a terminally differentiated plasma-cell (PC)
phenotype. Both genetics and exposure to carcinogens have been
considered etiologic in MM. The monoclonal gammopathy of
undetermined significance (MGUS) is a pre-MM disease and 1% of
patients with MGUS progresses to MM annually. Smoldering multiple
myeloma (SMM) is another asymptomatic plasma cell disorder that
carries a higher risk of progression to MM compared to MGUS. MM is
a difficult-to treat malignancy. High-dose chemotherapy, including
tandem autotransplants, in recently diagnosed MM patients has led
to complete remissions (CRs) in the large majority of newly
diagnosed patients with MM. However, many patients achieving CR
subsequently relapse, indicating that clinically significant
minimal residual disease (MRD) persists in CR. Elucidating the
mechanisms governing relapse is critical. Since little is known
about these molecular mechanism, further research to identify the
underlying driver genes is justified with the aim to develop novel
targeted therapies. Thyroid Hormone Receptor Interactor Protein 13
(TRIP13), one of the CIN genes, has been implicated in oncogenic
functions and drug resistance. TRIP13 is an AAA.sup.+-ATPase that
alters the conformation of client macromolecules and affects
cellular signaling.
[0242] Five novel discoveries have laid the groundwork for the
following studies. (1) TRIP13 transforms NIH3T3 fibroblasts to
tumor cells and enhances tumor progression in transgenic mice. (2)
Compared to normal and MGUS plasma cells, TRIP13 is highly
expressed in MM cells, surviving in complete remission, and is also
significantly increased in patients relapsing early after
transplantation. (3) High TRIP13 expression in MM samples at
diagnosis is associated with a poor prognosis in MM. (4) TRIP13
interacts with the apoptosis-inducing factor 1 (AIF1), which is
related to cell apoptosis and forms a promising pharmacological
tool 24. And (5) Treatment with pharmacological ascorbic acid (PAA)
overcomes TRIP13-induced MM cell drug resistance and selectively
kills MM cells in vitro and in vivo.
[0243] Introduction
[0244] Multiple myeloma (MM), originating from its precursors MGUS
and SMM, is the second most common hematological malignancy in the
United States. MM accounts for 10% of all hematological malignancy
and causes over 12,000 deaths in the United States annually. MM is
a cancer of plasma cells in the bone marrow associated with an
overproduction in most cases of a complete or partial monoclonal
(M)-protein. Monoclonal gammopathy of undetermined significance
(MGUS), a MM precursor, is an asymptomatic plasma cell dyscrasia
that is present in more than 3% of the general population older
than age. Smoldering multiple myeloma (SMM) is another asymptomatic
plasma cell disorder that carries a higher risk of progression to
MM compared to MGU. The MM literature supports a role for both
genetic and environmental factors in the progression of MM from its
precursor states, which are present in virtually all MM patients.
However, little is known about the mechanisms governing the
transition of MGUS/SMM to symptomatic MM.
[0245] Dysregulation of chromosomal stability genes causes drug
resistance and myeloma relapse. Drug resistance is a universal
problem with current MM therapies. Although the large majority of
MM patients achieve a complete remission, many patients suffer a
relapse die of their disease. Drug-resistance can be categorized as
de novo resistance and acquired resistance. De novo resistance is
likely genetic in nature while acquired resistance likely results
from a combination of cumulative mutations as a result of
inadequate treatment of a genetically unstable clone, and
cross-talk between MM cells and the bone marrow environment,
resulting in survival and proliferation. Previous work revealed
that high expression of chromosomal instability (CIN) genes (AURKA,
KIF4A, CEP55, RRM2, CCNB1, CDCl20, TRIP13, TOP2A, PBK and NEK2)
increases cell survival and drug resistance with consequent poor
outcome in MM and other cancers.
[0246] TRIP13 acts as an oncogene and is linked to sensitivity to
chemotherapy and disease relapse in myeloma. TRIP13 is an
AAA.sup.+-ATPase protein and is upregulated in multiple types of
human cancers. This enzyme contains a specific N-terminal domain
(NTD) responsible for localization and substrate recognition, and
one or two AAA.sup.+-ATPase modules that typically assemble into a
hexametric ring. It was found that TRIP13 transforms NIH3T3
fibroblasts to tumor cells and enhances tumor progression in
transgenic mice. High levels of TRIP13 activates the non-homologous
end joining (NHEJ) signaling pathway to repair doublestrand breaks
(DSBs), thereby leading to chromosomal instability (CIN), cancer
cell survival, metastasis, and enhanced drug resistance. Data
indicate that compared to MGUS and SMM plasma cells TRIP13 is
significantly increased in MM cells, during CR and in MM samples at
relapse early after treatment. Therefore, therapeutic targeting of
the TRIP13 pathway in patients with MM is very likely to be
effective in preventing progression from MGUS/SMM to MM and
relapse.
[0247] The experiments below were developed to determine novel
therapies to sensitize high-TRIP13 myeloma cells. First, a genetic
mouse model is used to further understand the role of TRIP13 and
its signaling pathways in MM disease development and progression,
and determine if TRIP13 is critical for tumorigenesis. Using a
systematic TAP-MS analysis, it was identified that TRIP13 binds to
AIF1. This interaction may explain why high TRIP13 increases cell
survival and drug resistance in MM. Second, it is investigated
whether TRIP13 sequesters AIF1 in mitochondria and/or cytosol and
prevents cell apoptosis induced by AIF1 nuclear translocation.
Third, the hypothesis is tested that modulation of reactive oxygen
species (ROS) by utilizing PAA eliminates MM tumor cells with high
levels of TRIP13. Previous work has shown that PAA has potent
clinical anti-cancer pro-oxidant activity. In vitro and in vivo
models have been developed to elucidate the role of TRIP13 in tumor
development and progression useful for the development of a novel
therapy approach directed at eradicating drug-resistant MM cells.
It is very likely that our findings will not be unique to MM, but
will also apply to other hematologic malignancies and solid
tumors.
[0248] The candidate gene TRIP13, which is increased in MM cells
and has been linked to drug resistance and poor prognosis, was
discovered by comprehensive analyses of the MM genome from 1,500
clinical samples by the inventors. Further, its oncogenic function
was determined by the transformation of normal fibroblasts into
tumor cells. Tissue-specific TRIP13 transgenic mice have been
generated that show enhanced B cell lymphoma progression (FIGS.
31A-B). To the best of our knowledge, this is the first report that
TRIP13 localizes in both mitochondria and cytosol and binds to
AIF1. A new genetically engineered MM mouse model, designated
C.IL6/iMyc is used in the studies described herein. The model
recapitulates key features of the human disease (e.g., serum
para-protein, osteolytic lesions, kidney disease) and lends itself
nicely to adoptive transfer of B cells. Treatment with high-dosed
ascorbic acid produces oxidative stress, which breaks the
interaction of TRIP13 with AIF1. This results in killing of MM
cells. New technologies, such as tandem affinity purification
followed by mass spectrometry (TAP-MS), RNA sequencing, chromatin
immunoprecipitation (ChIP)-sequencing, advanced biochemical assays,
adoptive B cell transplantation, and the FDG-PET-CT for mouse
imaging, are employed in the experiments described herein.
[0249] TRIP13, a CIN Gene, is Linked to a Poor Survival in MM.
[0250] Using sequential analyses of gene expression profiling (GEP)
in the same patient, 56 genes were identified, the expression of
which was significantly up-regulated compared to those at baseline
after intensive chemotherapy and at relapse, early after
transplantation. The major functional group including 10 genes with
a significant negative impact on survival (Hazard ratio
[HR]>=2), belongs to the well-established chromosomal
instability (CIN) signature (Zhou W, Yang Y, Xia J, Wang H, Salama
M E, Xiong W, et al. NEK2 induces drug resistance mainly through
activation of efflux drug pumps and is associated with poor
prognosis in myeloma and other cancers. Cancer Cell 2013 Jan. 14;
23(1): 48-62). Supervised clustering using the 10 CIN gene model,
was applied to plasma cells from 22 healthy donors (NPC), 44
patients with MGUS, 351 patients with newly diagnosed MM, and 9
human myeloma cell lines (MMCL) (FIG. 26A). The correlation between
gene expression and survival was determined by the p value and HR
at the best expression signal cut-off. TRIP13 was one of the most
significant genes associated with an inferior survival in
unadjusted log rank tests. As shown in FIGS. 26A-C, the top
quartile (25%) of MM patients with the highest TRIP13 expression
had a significantly inferior event free survival and overall
survival (FIGS. 26B & 26C, p<0.001) in Total Therapy 2 (TT2)
cohort.
[0251] Increased TRIP13 Expression Promotes Myeloma Cell Growth and
Drug Resistance.
[0252] To test the role of TRIP13 on MM cell growth, TRIP13 was
overexpressed by lentivirus-mediated TRIP13-cDNA transfection in
the MM cell lines ARP1, OCI-MY5, and H929 with low baseline
expression of TRIP13. The expression level of TRIP13 was verified
by RT-PCR and western blot (FIG. 27A). TRIP13 overexpression
significantly increased MM cell proliferation of ARP1, OCI-MY5, and
H929 MM cell lines (FIG. 27B). The effects of TRIP13-knockdown on
MM cell growth in vivo was next determined. ARP1 MM cells
transduced with TRIP13-shRNA or scrambled vectors were injected
subcutaneously into the abdomen of NOD-Rag1null mice. It was
observed that tumor size was significantly smaller in the
TRIP13-shRNA mice compared to those controls (FIGS. 27C & 27D).
To determine whether high expression of TRIP13 increases drug
resistance in MM cells, ARP1 MM cells were incubated with
bortezomib and etoposide, which are widely used in MM treatment. As
shown in FIG. 27E and FIG. 27F, treatment with bortezomib or
etoposide induced significantly less growth inhibition in TRIP13-OE
MM cells compared with EV controls (p<0.05).
[0253] TRIP13 is an Oncogene that Transforms Normal Fibroblasts to
Tumor Cells.
[0254] To determine whether TRIP13 functions as an oncogene,
malignant cellular transformation in NIH3T3 fibroblasts was
performed. NIH3T3 cells were transfected with mouse TRIP13
(mTRIP13) and empty vector (EV) and compared the formation of
anchorage-independent colonies in soft agar. After 2-week culture,
>20 colonies were observed in each well of the 6-well plates
with mTRIP13 overexpression, while virtually no colonies were
observed in wells with control cells (EV) (FIGS. 28A & 28B).
Next, 2.5.times.105 NIH3T3 cells with mTRIP13 overexpression or
empty vector were injected subcutaneously into each flank of
NOD-Rag1null mice (n=5 and repeat n=3) and evaluated for tumor
growth respectively. Tumor mass was palpable on Day15.about.19 for
mTRIP13 overexpressing cells, but no tumors were found after
injection of control cells with empty vector after 26 days. Of the
mice injected with mTRIP13 overexpressing cells, 6 of 8 (75%)
developed tumors (FIG. 28C). These results implicate that mTRIP13
has oncogenic capabilities.
[0255] Determination of the Role of TRIP13 in Myeloma
Pathogenesis.
[0256] Characterization of the Role of TRIP13 in Myeloma-Like Tumor
Development and Progression.
[0257] Recent work revealed that high expression of CIN genes,
including TRIP13, induces MM cell proliferation and drug
resistance. Data demonstrate that TRIP13 has an oncogenic function,
such that overexpression of TRIP13 in NIH3T3 cells induces tumor
transformation (FIG. 28A-C). Given that plasma cells in MM
originate from terminally differentiated B cells, a
lymphocyte-specific TRIP13-transgenic C57/BL6 mouse was generated
in which TRIP13 expression is controlled by the LCK promoter.
Although tumor formation was not observed in Tg TRIP13 mice, TRIP13
significantly promoted B cell tumor development by crossing with
E.mu.-Myc mice (FIGS. 30A-E), further suggesting that TRIP13 plays
an oncogenic role. Therefore, it is hypothesized that high TRIP13
enhances MM development and progression.
[0258] To test this hypothesis, two approaches are used that rely
on engineered over- or under-expression of RIP13 in a non-germline
mouse tumor model. First, a relatively inexpensive mouse model of
MM has been developed that enables rapid in vivo validation of
candidate MM genes (Tompkins V S, Rosean T R, Holman C J, DeHoedt
C, Olivier A K, Duncan K M, et al. Adoptive B-cell transfer mouse
model of human myeloma. Leukemia 2016 April; 30(4): 962-966). The
cornerstone of the method is adoptive B-cell transfer (FIG.
29A-29D). Briefly, Balb/c IL6/iMyc-double transgenic (TG) mice,
which develop spontaneous plasma cell tumors (PCTs) with 100%
penetrance are used as the source of mature CD45.2.sup.+
B-lymphocytes that are genetically "hard wired" to undergo
malignant transformation when transferred to CD45.1.sup.+ hosts,
where the CD45.2.sup.+ cells complete neoplastic transformation and
form PCTs. The donor B cells can be genetically modified in vitro
by retro- or lentiviral gene transduction. The new method affords
numerous scientific and practical advantages including: the use of
hosts genetically deficient in key factors of the MM
micro-environment (see FIG. 29F for an example); the generation of
"waves" of genetically tagged (CD45.2.sup.+) PCTs in CD45.1.sup.+
hosts in a predictable, timely, economic fashion (note that one
donor mouse suffices to reconstitute up to 30 hosts); and
combination of adoptive cell transfer with integrated micro-CT
imaging for studies of MM bone disease (FIG. 29E). Here, the newly
developed technology is employed to evaluate the role of TRIP13 in
PCT development and progression.
[0259] Generation of TRIP13-Silenced and TRIP13-Overexpressing
C.IL6/iMyc Mice.
[0260] The experimental model system depicted in FIG. 29A-29F is
used to evaluate the biological significance of TRIP13 in PCT
development and progression at sites of myeloma-like tumors in
mice. C.IL6/iMyc-TG CD45.2.sup.+ B220.sup.+ B cells are transduce
at age 6 weeks (.about.30 days earlier for detection of tumor) with
a lentiviral vector that co-expresses mouse TRIP13 and luciferase
(Luc): TRIP13.sup.OE cells. These B cells are also transduced with
a lentiviral vector that co-expresses scrambled control shRNA
(scrCON) or two different mouse TRIP13-targeted shRNAs (designated
TRIP13.sup.KD) and Luc. 45 CD45.1.sup.+ mice are reconstituted with
B cells in which TRIP13 expression is overexpressed (n=15,
TRIP13.sup.OE) or undetectable (n=15 for each shRNA to TRIP13), and
15 CD45.1.sup.+ mice with B cells infected with scrCON virus
(designated TRIP13.sup.WT because cells express mouse TRIP13 at
wild type [WT] levels). The TRIP13.sup.KD mice are given
doxycycline in their chow immediately after adoptive transfer to
achieve early downregulation of TRIP13 in the CD45.2.sup.+ B
cells.
[0261] Characterize Cancer Cells and MM Progression:
[0262] It has been shown that increased TRIP13 accelerates tumor
development and progression in the TRIP13/E.mu.-Myc mice (FIGS.
30A-E). The major goal of this experiment is to assess whether
TRIP13 is critical for MM development and progression. Therefore,
we will determine whether knockdown (KD) of TRIP13 in pre-malignant
B cells prevents PCT formation and progression. The TRIP13.sup.OE
and scrCON mice will serve as positive and negative controls
respectively. These mice will be observed for a period of 20
months. The growth of tumors in these mice will be monitored weekly
based on physical examination including body weight and health
status parameters and measurement of tumor burden by detection of
any serum paraprotein (M-spike) through serum protein
electrophoresis combined with the F18-PET-Scan. Time to tumor onset
will be recorded. A comprehensive, systematic approach to analysis
of the transgene in the mice is planned. This includes a complete
necropsy with particular emphasis on lymph nodes, spleen, and bone
marrow. Representative tissue samples from lymph nodes, spleen and
bone marrow are placed in a fixative, such as Bouin's or 10%
neutral buffered formalin. Cut sections of tissues, placed in
mounting medium and snap-frozen, are used for immunohistochemistry
testing. A large sample of whole blood (.about.1 ml) is collected
by heart puncture and used to measure serum protein (cytokines,
chemokines) levels and to isotype paraproteins (by ELISA).
[0263] Clonal Cytogenetics Karyotyping and Spectral Karyotyping
(SKY) Analysis.
[0264] The tumor cells from Tg mice (TRIP13.sup.KD, TRIP13.sup.OE,
and scrCON C.IL6/iMyc mice) Re-collected and grown in culture
medium RPMI1640 with 20% FBS. Cell growth Re arrested by colcemid
(4 .mu.l/ml). Metaphases from the first-passage tumor cells are
examined by "chromosome painting" with the use of commercially
available SKY probes for mouse (Vysis Inc). This technique serves
as a screen for chromosome number (gains or losses), inversion and
translocations.
[0265] Identify Genomic Changes Between TRIP13.sup.KD with
TRIP13.sup.OE and scrCON C.IL6/iMyc Mice.
The Illumina next generation whole genomic sequencing is used to
detect genomic instability, such as mutational and copy number
changes at the DNA level, induced by TRIP13 overexpression.
CD138.sup.+ MM cells from 10-15 tumors are collected from these
mice. Deeper whole-genome sequencing of tumor cells will be
performed. Gene mutations, chromosome amplifications, deletions,
and translocations are characterized by mapping on the mouse genome
browser (UCSC genomic browser GRCm38/mm10). Specific mutation
patterns, such as G=>A or C=>T, and C=>T 60, 61 that are
commonly observed in human MM are examined. Further, these findings
are compared with the mutation pattern and chromosome changes in
human MM patient samples. This may determine TRIP13 functions in
chromosomal instability (CIN). RNA-sequencing is also performed on
these mouse tissues.
[0266] Identify the Mechanisms by which TRIP13 Accelerates Tumor
Development and Progression.
[0267] TRIP13 accelerates tumor development and shortens mouse
survival in double Tg TRIP13/E.mu.-Myc mice compared to the control
E.mu.-Myc mice alone (FIGS. 30A-E). However, the mechanisms by
which TRIP13 exerts its oncogenic function are unknown.
Pre-malignant B cells (B220.sup.+) have been collected from both Tg
TRIP13/E.mu.-Myc and Tg E.mu.-Myc mice at age of 6 weeks, and
RNA-seq has been performed on these two groups of B cells. As shown
in FIG. 31A, more than 1,900 genes are differentially expressed
between these B cells (p<0.001). The TRIP13 signaling pathways
were analyzed using Gene Set Enrichment Analysis (GSEA). FIG. 31B
lists the 10 most significant pathways, which distinguished B cells
of the TRIP13/E.mu.-Myc mice from the E.mu.-Myc mice and can be
targeted by commercially available inhibitors. To further define
how TRIP13 functions as an oncogene, it is determined if inhibition
of these pathways delays or prevents TRIP13-induced tumor
development using in vitro, in vivo and primary MM samples.
[0268] TRIP13 Modifies the Transcriptional Profiles of E.mu.-Myc
Mice.
[0269] As shown in FIG. 30B and FIGS. 32A-B, c-Myc, PRC2-EZH2, p53
and PTEN signaling pathways are significantly activated or
inhibited by TRIP13. More than hundreds to thousands of genes have
been identified as potential binding targets of the transcription
factors (c-Myc, p53, and PTEN) or the epigenetic regulator (EZH2).
It is hypothesized that TRIP13 regulates the c-Myc, EZH2, p53 and
PTEN activity, which accelerates tumor onset and progression.
Therefore, ChIP-sequencing is performed in pre-malignant B cells
derived from Tg TRIP13/E.mu.-Myc mice and from the control
E.mu.-Myc mice at age 6 weeks (the same for RNA-seq in the FIGS.
31A-B). Briefly, DNA fragments with an average size of 0.3-0.5 kb
after crosslinking and sonication are immunoprecipitated using
anti-c-Myc, -EZH2, -p53 or -PTEN antibodies or IgG as a control.
The DNA fragments binding to these antibodies are identified by
sequencing on an Illumina HiSeq 2500 sequencer. Combined with
RNA-seq described above, the above proteins--targeted genes
regulated by TRIP13 in B cell lymphoma-genesis--are
identifiable.
[0270] Determination of Pathways for Tumorigenesis.
[0271] FIG. 31B shows the 10 most significant signaling pathways by
comparing pre-malignant B cells from Tg TRIP13/E.mu.-Myc versus
E.mu.-Myc mice. To determine which pathways activated by TRIP13
play a critical role in tumorigenesis, this function is
investigated from the approaches depicted in FIG. 31C.
[0272] 1) Soft agar assay for colony formation in NIH3T3 cells:
1.times.10.sup.4 NIH3T3 cells transduced with control vector or
murine TRIP13 (mTRIP13) and N-Ras (as the positive control) are
mixed with RPMI1640 media containing 10% FBS and 0.33% agar and
layered on top of the base layer of 0.5% agar in each well of
6-well plates. Half of the wells are treated with the 10 drugs
listed in FIG. 31B respectively. Colony numbers are counted after
approximately 2.about.3 weeks. All plates are imaged under
microscope and overall numbers of colonies in the pictures are
counted by the Image J software.
[0273] 2) NIH3T3 tumor transformation in vivo: For tumorigenesis
assay, the most five effective drugs related to TRIP13 signaling
pathways identified by the above-soft agar assays are tested.
2.5.times.10.sup.5 NIH/3T3 cells that co-express mTRIP13 and
luciferase (Luc): TRIP13.sup.OE cells or the control cells with Luc
will be injected subcutaneously into each side of the
NOD-Rag1.sup.null mice dorsa. Each group consists of 3 mice (total
mice n=30) including 6 tumors. Tumor incidence and the number of
tumor nodules from each group are counted and compared to each
other. Tumor burden is measured by Bioluminescence Assay. Tumor
length and width will also be gauged, and tumor volume will be
calculated as (length.times.width).times.0.5. For each time point,
results will be presented as the mean tumor volume.+-.SD for the
indicated mice.
[0274] 3) Tg TRIP13/E.mu.-Myc and E.mu.-Myc mice: Because this is a
faithful genetic model for TRIP13 signaling, three drugs identified
above from NIH3T3 tumor transformation in vivo are tested in this
model. Both Tg TRIP13/E.mu.t-Myc or E.mu.-Myc mice at age 50 days
are used for this study. 24 Tg TRIP13/E.mu.-Myc mice and 24
E.mu.-Myc mice are randomly assigned to one of four treatment
groups (three drugs and one control) with equal representation of
mouse gender. Blocking the pathways by the inhibitors should delay
the tumor formation in the Tg TRIP13/E.mu.-Myc mice and show less
impact in the E.mu.-Myc mice.
[0275] 4) Plasma cell tumor (PCT) in C.IL6/iMyc mice: Because this
is a genetic MM mouse model, the two most effective drugs are
tested as defined above. Similar to the description described above
in FIGS. 29A-F, CD45.2.sup.+ B220.sup.+ B cells from Tg C.IL6/iMyc
at age 6 weeks are reconstituted in CD45.1.sup.+ mice. Six mice are
required for each group and, a total of 18 mice are used for the
two drugs and a control.
[0276] 5) Expression and activity in MGUS, SMM MM at diagnosis, and
relapsed NM Finally, the most two most important pathways defined
above are evaluated in different stages of primary plasma cell
tumor samples. Ten samples of each stage of MGUS, SMM, newly
diagnosed MM, and relapsed MM are included in this study. The
targeted gene or these two signaling pathways targeted are
evaluated by qRT-PCR, western blotting, ELISA, and the molecular
assays to measure mRNA and protein levels, protein modification,
cellular localization, Cdk activity, kinase activity, and
ubiquitination activity, etc.
[0277] Preliminary data showed that increased TRIP13 enhances B
lymphomagenesis resulting in a shorter survival in Tg
TRIP13/E.mu.-Myc mice. Importantly, past experience evaluating the
collaboration of other genes (e.g., Bcl2 and IL-6) with c-Myc in
mouse B-cell and PCT development suggests that enforced expression
of mouse TRIP13 accelerates C.IL6/iMyc-dependent tumors. It is
predicted that compared to TRIP13 normal B cells, TRIP13OE B cells
undergo malignant transformation more rapidly and give rise to more
aggressive disease.
[0278] Characterize Molecular Mechanisms of TRIP13-Mediated Myeloma
Chemoresistance.
[0279] To define the molecular mechanism by which TRIP13 promotes
drug resistance and cell survival, the TAP-MS analysis was
performed to identify the interacting partners of TRIP13.
Interestingly, it was found and confirmed that TRIP13 binds to the
apoptosis-inducing factor 1(AIF1). Although AIF1 was considered to
mainly localize in mitochondria, it was further discovered that
TRIP13 localizes in both cytoplasm (main) and mitochondria, and
high TRIP13 decreases nuclear AIF1 protein (FIGS. 33A-E). AIF1 is a
mitochondrial FAD-dependent oxidoreductase that plays a vital role
in oxidative phosphorylation (OXPHOS) and redox metabolism in
normal and cancer cells. AIF1 was originally discovered as an
intermembrane space (IMS) component of mitochondria and
characterized as a pro-apoptotic gene. The pro-apoptotic AIF1 or
truncated AIF1 (tAIF) is cleaved from the full-length AIF1 by
calpains and/or cathepsins after a caspase independent cell death
insult. tAIF moves from the mitochondria to the cytoplasm and then
to the nucleus, where it initiates caspase-independent cell
apoptosis. Therefore, it is hypothesized that the interaction of
TRIP13 with AIF1 prevents AIF1 nuclear translocation resulting in
decreased myeloma cell apoptosis. TRIP13 shares a frequently
observed AAA.sup.+ ATPase architecture (FIG. 34).
[0280] Evaluate the Role of Interaction Between TRIP13 and AIF1 in
MM Cell Drug Resistance.
[0281] Structural Domains of TRIP13 for Interacting with AIF1.
[0282] TRIP13 contains a common AAA+ ATPase domain at the 3' and
conserved Walker A & B motifs. The ATPase domain is required
for diverse activities of AAAATPase proteins and the Walker A &
B motifs are required for ATP-binding activity. Using site-directed
mutagenesis, the following TRIP13 mutants are generated (FIG. 34):
G184A (TRIP13.sup.G184A), mutation localized in the Walker A motif;
W221A (TRIP13.sup.W221A), a mutant that has been previously
described showing higher Kcat than WT TRIP13; E253Q
(TRIP13.sup.E253Q), a mutant that will not be able to hydrolase
ATP; R385A (TRIP13.sup.R385A), a mutant unable to bind nucleotide.
Also, a D293-312 deletion mutant is generated in which the
conserved ATPase domain (TRIP13.sup..DELTA.293-312) has been
deleted. All the mutants are tagged with HA. HEK293T and MM cell
lines ARP1 and OCI-MY5 are transduced with different constructs
expressing with the HA-TRIP13.sup.WT or HA-TRIP13.sup.G184A,
HA-TRIP13.sup.W221A, or HA-TRIP13.sup.E253Q, HA-TRIP13.sup.R385A,
and HA-TRIP13.sup..DELTA.293-312. HA-Tag antibody is used to pull
down TRIP13 and its binding proteins. Western blot is used to
identify which TRIP13 domain binds to AIF1. The localization of
each mutant is determined by immunofluorescence using HA and
MytoTracker for mitochondrial localization.
[0283] Does TRIP13 Bind Directly to AIF1 and Affect Sensitivity to
Chemotherapy?
[0284] It has been shown that AIF1 binds to TRIP13 protein and
TRIP13 localizes in both mitochondria and cytoplasm of MM cells
(FIGS. 33A-33C). To determine whether the interaction between AIF1
and TRIP13 is direct, in vitro GST pull down assays are performed.
The GST-tagged TRIP13 is purified from bacteria using glutathione
beads. The purified GSTTRIP13 is incubated with recombinant AIF1
protein. The glutathione beads are washed and western blotting
analysis using the AIF1 antibody to detect whether AIF1 binds to
TRIP13 protein directly.
[0285] To investigate which domain of TRIP13 is required to
interact with AIF1 using GST-pull down assay, different GST-tagged
mutants of TRIP13 defined above are purified from bacteria and
incubated with full length recombinant AIF1 in vitro. The positive
interacting domain once identified are deleted from the full length
TRIP13 to generate a dominant-negative mutant .DELTA.TRIP13 that
should no longer be capable to interact with AIF1. WT-TRIP13 or
.DELTA.TRIP13 is then introduced to MM cell lines ARP1, H929 and
OCI-MY5 with inducible shRNA against 3'-UTR of endogenous TRIP13.
The endogenous TRIP13 is depleted by doxycycline administration.
Because it is expected that TRIP13 promotes cancer cell survival
and drug resistance through binding with AIF1, cell survival and
drug resistance induced by .DELTA.TRIP13 relative to the WT-TRIP13
is compared. (1) To assay the changes in G1-S progression, cells
are synchronized in M phase by nocodazole or in G0 by serum
starvation, released into cycle by drug removal, re-plated into
media with serum, and assayed at 2 hr intervals for rates of S
phase entry (via flow analysis of DNA content and BrdU positivity).
(2) DNA repair is assayed by treatment of cells with a pulse of
bleomycin to cause double stranded DNA breaks. Measurement of these
breaks by an alkaline "comet" assay, in which single cells are
subjected to electrophoresis and unrepaired DNA breaks, are
visualized as a "tail". Cell survival after DNA damage is
determined by a colony assay. (3) Notably, the IC50 for each drug
including bortezomib, melphalan, lenalidomide and dexamethasone is
determined in order to test if sensitivity is altered by changes in
TRIP13 and AIF1 status. Drug resistance is also evaluated by soft
agar clonogenic assays described in the FIGS. 28A-C. (4) Cell
viability is assayed using Resazurin (Life Technologies),
proliferation using colorimetric BrdU detection (Roche) and growth
in soft agar. The dependence of AIF1 in TRIP13-induced DNA repair,
cell growth and drug resistance is thereby determined in MM
cells.
[0286] Does TRIP13 Neutralize AIF1 in Myeloma Cells?
[0287] AIF1-mediated caspase-independent cell apoptosis depends on
the mitochondrial.fwdarw.cytosol.fwdarw.nuclear translocation. Data
in FIGS. 33D and 33E show that overexpression of TRIP13 decreases
nuclear AIF1 protein. To further determine if high TRIP13 inhibits
AIF1-mediated apoptosis, the subcellular localization of AIF1 after
an apoptotic insult is examined. The above ARP1 and OCI-MY5
transfected with WT-TRIP13 or .DELTA.TRIP13 (no binding domain with
AIF1) are transduced with AIF1-GFP. These MM cells are treated for
90 min with N-methyl-N-nitroso-N'-nitroguanidine (MNNG) 500 mM 92,
which is a carcinogen and mutagen and can trigger AIF1 to be
released from mitochondria and move to the nucleus. The relative
mitochondria/cytoplasm/nucleus distribution of AIF1 is evaluated by
both immunofluorescence confocal microscopy, cellular fractionation
assays, and transmission electron microscopy (TEM) as previously
described (FIGS. 36A-C) (Xia J, Xu H, Zhang X, Allamargot C,
Coleman K L, Nessler R, et al. Multiple Myeloma Tumor Cells are
Selectively Killed by Pharmacologically-dosed Ascorbic Acid.
EBioMedicine 2017 Feb. 16.).
[0288] Define TRIP13 Signaling Pathways Using Clinical Samples and
Genetic Mouse Models.
[0289] Biological samples. CD138+ MM cells from patient samples are
isolated using human anti-CD138+ antibody (FIG. 35).
[0290] Clinical Relevance of TRIP13 with AIF1 in Serial MM Samples
at Diagnosis, Remission and Relapse.
[0291] To determine the relevance of the interaction between TRIP13
with AIF1 in human MM disease, their expression and localization in
human primary sequential MM samples at the protein level is
evaluated. As we show in FIGS. 36A-C, MM cells collected in
remission (FIG. 36B) and relapse (FIG. 36A) show higher expression
of TRIP13 than those at diagnosis by GEP. In this study, about 30
serial MM biopsies collected at diagnosis, in remission (only
samples can be isolated enough MM cells) and relapse (HawkIRB
protocol 201302833; arrows in FIG. 35) are used.
Immunohistochemistry on bone marrow biopsies is performed using
anti-TRIP13, anti-AIF1, and anti-CD138 Abs in these serial MM
samples. It has been shown that AIF1 is increased in the cytosol
and decreased in the nucleus of TRIP13-OE MM cells (FIGS. 33D &
33E), suggesting a mechanism that TRIP13 sequesters AIF1 in the
mitochondria or cytosol to block its apoptosis function. Protein
levels and subcellular localization of TRIP13 and AIF1 are also
analyzed by cellular fractionation and TEM on CD138.sup.+ primary
MM cells sorted by flow cytometry. Western blots and/or TEM are
performed on a smaller number of selected tumors: (i) that
sufficiently represent each patient group studied, and (ii) for
which there is an adequate amount of isolated protein available.
The correlations between TRIP13 and AIF1 expression and
localization as well as with clinical stages and outcome are
analyzed.
[0292] Dissect the molecular regulation networks of TRIP13 using
patient samples and genetic mouse models. Two approaches are used:
1) Microarray data analysis of clinical samples: As we showed in
FIGS. 26 and 36, GEPs have been generated from 22 normal plasma
cells, 44 MGUSs, 550 newly diagnosed MMs, 59 from different
treatment stages including partial and complete remission, and 90
relapsed MM samples. The expression correlation of TRIP13 and its
signaling pathway-related transcriptome is analyzed using the
transcriptome data; Correlation and clustering methods are applied
to identify TRIP13 targets and regulatory networks. 2) RNA
sequencing on genetic mouse tissues: RNA-sequencing has been
performed in pre-malignant B cells between Tg TRIP13/E.mu.t-Myc and
E.mu.-Myc mice (FIGS. 32A-B). To further identify and confirm
upstream regulators or downstream effectors of TRIP13 in MM, deep
RNA-sequencing is also performed to detect differentially expressed
genes between tumors derived from TRIP13.sup.KD, TRIP13.sup.OE, and
scrCON C.IL6/iMyc mice (see discussion above). About 10 samples
from each group are collected. Briefly, CD138.sup.+ MM cells are
sorted out and 10,000.about.30,000 sorted cells will be used to
extract total RNA followed by cDNA synthesis/amplification using
the Clontech SMARTer kit for RNA-Seq experiment. Deep sequencing
will be performed using an Illumina HiSeq 2500 sequencer. Each
sample is sequenced to a depth of 100 million read pairs to ensure
sufficient depth for accurate detection of alternative transcripts.
Briefly, four analysis programs are used: (i) reads that pass
quality control are mapped to the genome by STAR; (ii)
featureCounts are used to estimate transcript expression level; and
(iii) the Deseq2 is used to determine differential expression; and
(iv) enriched pathways are analyzed by GSEA and Enrichr.
Integrative data analyses are performed on these both microarray
data and the deep-sequencing dataset.
[0293] Structure function studies are critical to identifying the
TRIP13 intermolecular interactions important for MM disease
biology. It is anticipated that specific residues or domains of
TRIP13 will be identified that bind to AIF1 and mediate
chemotherapy resistance. It is anticipated that wild-type TRIP13
will confer resistance to bortezomib, melphalan, lenalidomide and
dexamethasone treatment but that the mutant lacking binding to AIF1
will not. In human primary MM samples, it is predicted that TRIP13
will increase in remission and relapsed MM samples at the protein
level, but will negatively correlate with nuclear AIF1 expression
and patient outcome. Integrative analyses of RNA-sequencing data
from TRIP13KD, TRIP13OE, and scrCON C.IL6/iMyc mice and microarray
data from more than 1500 patient samples with clinical information,
should identify novel downstream signaling pathways and networks
that are associated with TRIP13-induced drug resistance in MM.
[0294] Develop Novel Therapies to Target High-TRIP13 Myeloma
Cells.
[0295] TRIP13 encodes an AAA.sup.+-ATPase enzyme but has received
little attention in cancer including MM. Studies have shown that
TRIP13 localizes in both mitochondria and cytosol and interacts
with AIF1 directly (FIGS. 33A-E). It was recently reported that
pharmacologically-dosed ascorbic acid (PAA), in the presence of
iron, leads to the formation of highly reactive oxygen species
(ROS) resulting in AIF1 cleavage and translocation from the
mitochondria to the nucleus, causing cell death (FIGS. 37A-G).
TRIP13 upregulates the iron importer: Transferrin Receptor (TFRC)
and downregulates the iron exporter: ferroportin (FPN1) resulting
in increased ferritin (a known marker of cytosolic iron) in MM
cells overexpressing TRIP13 (FIG. 38A-38C). Importantly, PAA
induces AIF1 nuclear translocation not only in TRIP13.sup.N MM
cells but also in TRIP13.sup.OE MM cells, whereas Bortezomib
treatment does not increase AIF1 nuclear localization (FIG. 38D).
Therefore, it is hypothesized that TRIP13.sup.high cells are
sensitive to PAA treatment by disrupting its interaction with AIF1
leading to increased apoptosis and are able to overcome
TRIP13-induced drug resistance. As described in FIG. 39, TRIP13
cells have increased cytosolic ferritin leading to high levels of
redox-active iron. In TRIP13-OE cells, PAA oxidizes by reacting
with iron. PAA autoxidation generates cellular oxidative damage
leading to AIF1 cleavage in the mitochondria with subsequently
translocation to the nucleus. AIF1 nuclear translocation induces
apoptosis and cell death. Based on this model, it is hypothesized
that PAA treatment is a valuable therapeutic approach to overcome
TRIP13-mediated drug-resistance in vivo.
[0296] Does Pharmacological Ascorbic Acid (PAA) Disrupt the
TRIP13-AIF1 Association and Lead to Nuclear Accumulation of
AIF1?
[0297] AIF1-mediated caspase-independent cell apoptosis is the
consequence of AIF1 translocation from the mitochondria to the
nucleus. Preliminary data show that PAA induces MM cell necrosis
and apoptosis and is partially dependent on AIF1 cleavage and
nuclear translocation (FIGS. 37E & 37G). To further determine
how PAA overcomes TRIP13-induced drug resistance in MM cells, the
MM cell lines ARP1, H929 and OCI-MY5 transfected with WT-TRIP13 or
.DELTA.TRIP13 (lacking binding domain with AIF1) are treated with
PAA at 1, 2, 4, and 8 mM for 60 min and cultured for another 16 h.
The relative mitochondria/cytoplasm/nuclei distribution of AIF1 is
evaluated by immunofluorescence confocal microscopy, cellular
fractionation assays, and TEM. The AIF1 cleavage is detected by
western blot. Chromatolysis is also evaluated in PAA treated MM
cells as described above. FIG. 39 summarizes the PAA action in
killing MM cells with high TRIP13 expression. Based on this data,
it is tested if PAA by targeting TRIP13/AIF1 interaction overcomes
TRIP13-induced drug resistance. Bortezomib is used as a negative
control in these experiments.
[0298] Investigate Therapeutic Effects of PAA in Doubly Tg
TRIP13/Ep-Myc Mice, which have Increased TRIP13 Expression and
Normal Immune System.
[0299] Double-transgenic TRIP13/E.mu.-Myc mice have recently been
generated that develop B cell lymphoma in the presence of a normal
immune system (FIGS. 30A-E). To investigate the effects of PAA on
established TRIP13/E.mu.-Myc lymphoma, paired
TRIP13.sup.OE/TRIP13.sup.N B lymphoma autografts, derived from two
genetic transgenic mouse models are used: TRIP13/E.mu.-Myc and
E.mu.-Myc. The mTORC1 inhibitor Everolimus (RAD001) is used as a
control, which showed a good efficacy in inhibiting tumor
development in the E.mu.-Myc mice. Each group will include nine
mice with equal representation of mouse gender. For lymphoma
transplantation from either Tg TRIP13/E.mu.-Myc or E.mu.-Myc mice,
a total of 2.5.times.10.sup.5 cryopreserved cells are thawed and
resuspended in sterile PBS before being introduced into syngeneic
recipient mice by tail vein injection for each mouse. 36 mice are
treated with PAA (4 mg/kg, i.p., twice a week for 4 weeks) or
Everolimus (5 mg/kg, oral gavage, once/week for 4 weeks) or
combination of PAA with Everolimus or no treatment. In addition to
FDG-PET scanning, mice are closely monitored for signs of tumor
development. This entails weekly determination of body weight,
health status parameters, and lymphadenopathy by palpation.
Peripheral blood lymphocytosis is monitored by serial blood tests
weekly. At necropsy, a representative set of tissues are harvested
for histopathology, immunological, molecular genetic and genomic
analyses. The TRIP13 signaling pathways are also evaluated by
qRT-PCR, western blot and the molecular assays described above to
assess mRNA and protein levels in tumor cells with or without PAA
treatment.
[0300] Determination of the Therapeutic Efficacy of PAA by
Analyzing Primary MM Cells at Diagnosis and in Relapse in the
NOD-Rag1null-Hu Mouse Model.
[0301] The efficacy of PAA in treating human primary MM cells
collected at diagnosis and at relapse using the
NOD-Rag1.sup.null-hu mouse model is assessed.
[0302] Human fetal bones are obtained from Advanced Bioscience
Resources. Briefly, human fetal long bones (tibias and femurs) from
18- to 21-week gestational fetuses are cut into two 10-mm pieces,
and implanted subcutaneously, on either left or right side of the
dorsum of NOD-Rag1null mice (one bone/mouse). Primary MM cells are
isolated from MM patients at diagnosis (low TRIP13) and in relapse
(high TRIP13) using CD138.sup.+ magnetic beads or flow cytometry.
The level of TRIP13 is assessed in each of these samples as
outlined above. At 6 to 8 weeks after implantation of bone, about
1.5.about.2.times.10.sup.6MM cells (CD138.sup.+) are injected
directly into the marrow cavity of each bone implanted into the
NOD-Rag1.sup.null-hu host. PAA is combined with melphalan in this
study, because the preliminary data in a MM cell line and other
murine models showed clearly that a synergistic effect when PAA is
combined with melphalan at a lower dose (FIGS. 37F & 37G). Four
treatment combinations for each sample are the following:
untreated, PAA, melphalan, and PAA+ melphalan. It is possible to
purify 10.times.10.sup.6 MM cells from a newly diagnosed MM sample
or from a relapsed MM patient, respectively. Therefore, one sample
is sufficient to cover the four combinations outlined above. Drug
concentration of the PAA is described above, and melphalan dosing
is 3 mg/kg (twice a week, i.p., on the same days as PAA
administration for 4 weeks). This study utilizes nine paired MM
samples obtained at diagnosis and in relapse. A total of 72 mice
with equal representation of mouse gender within each treatment
group at each time point are required. Tumor growth is monitored by
measuring human serum free light chains and M protein. Mice
survival and time to tumor recurrence time are compared among the
above outlined groups. M cell apoptosis is evaluated by double
staining with a CD138.sup.+ antibody and the deoxyuridine
triphosphate nick-end labeling (TUNEL) assay in the fixed fetal
bone sections. The number and size of bone lesions are determined
by X-ray and micro-CT. TRIP13 expression and activity are analyzed
as described above.
[0303] Statistical Analyses:
[0304] Statistical analysis is performed to compare treatment
groups within each experiment with respect to the proportion of
mice that develop B cell lymphomas, MM or relapse by the end of the
study. Power is estimated based on pairwise treatment group
comparisons performed with a simpler one-sided Fisher's exact test
at a single time point. Without treatment, the rate of tumor
development or relapse is conservatively estimated to be 95%.
Accordingly, the use of nine mice per group achieves 80% power to
detect a difference of at least 60% (95% vs 35%) between the
untreated and an active treatment group at the 5% significance
level. In addition, time to relapse or time to B lymphoma is
explored in a full analysis comparing treatment groups. Survival
curves are constructed using the Kaplan-Meier method and compared
between treatment groups using the log-rank test. It is anticipated
that PAA should break the interaction of TRIP13 with AIF1 and
induce AIF1 nuclear translocation in TRIP13-OE MM cells as depicted
in FIG. 39. It is predicted that it is possible to define the best
way to prevent TRIP13-mediated B cell lymphoma development and/or
MM disease progression. Considering that transgenic
TRIP13/E.mu.-Myc mice provide one of the most faithful experimental
model systems of TRIP13 signaling in mammalian cells currently
available, it is expected to gain insight into TRIP13-dependent
tumor inhibition. It is also anticipated that PAA delays or
prevents development of B cell lymphoma in Tg TRIP13/E.mu.-Myc
mice. It is predicted that the PAA kills primary MM cells
especially when using relapsed MM cells, which contain high
cellular iron. The combination with PAA should result in lower
dosage of commonly used drugs, such as melphalan, without losing
efficacy in the NOD-Rag1.sup.null-hu mice model when compared to
high-dose melphalan by itself.
[0305] All publications, patents and patent applications cited
herein are incorporated herein by reference. While in the foregoing
specification this invention has been described in relation to
certain embodiments thereof, and many details have been set forth
for purposes of illustration, it will be apparent to those skilled
in the art that the invention is susceptible to additional
embodiments and that certain of the details described herein may be
varied considerably without departing from the basic principles of
the invention.
[0306] The use of the terms "a" and "an" and "the" and similar
referents in the context of describing the invention are to be
construed to cover both the singular and the plural, unless
otherwise indicated herein or clearly contradicted by context. The
terms "comprising," "having," "including," and "containing" are to
be construed as open-ended terms (i.e., meaning "including, but not
limited to") unless otherwise noted. Recitation of ranges of values
herein are merely intended to serve as a shorthand method of
referring individually to each separate value falling within the
range, unless otherwise indicated herein, and each separate value
is incorporated into the specification as if it were individually
recited herein. All methods described herein can be performed in
any suitable order unless otherwise indicated herein or otherwise
clearly contradicted by context. The use of any and all examples,
or exemplary language (e.g., "such as") provided herein, is
intended merely to better illuminate the invention and does not
pose a limitation on the scope of the invention unless otherwise
claimed. No language in the specification should be construed as
indicating any non-claimed element as essential to the practice of
the invention.
[0307] Embodiments of this invention are described herein.
Variations of those embodiments may become apparent to those of
ordinary skill in the art upon reading the foregoing description.
The inventors expect skilled artisans to employ such variations as
appropriate, and the inventors intend for the invention to be
practiced otherwise than as specifically described herein.
Accordingly, this invention includes all modifications and
equivalents of the subject matter recited in the claims appended
hereto as permitted by applicable law. Moreover, any combination of
the above-described elements in all possible variations thereof is
encompassed by the invention unless otherwise indicated herein or
otherwise clearly contradicted by context.
* * * * *