nihms754039.pdf

A DNA-binding Molecule Targeting the Adaptive Hypoxic

Response in Multiple Myeloma has Potent Anti-tumor Activity

Veena S. Mysore

1,2

Jerzy Szablowski

Peter B. Dervan

, and

Patrick J. Frost

1,2,*

Greater Los Angeles Veteran Administration Healthcare System, Los Angeles, CA 90073

University of California, Los Angeles, Los Angeles, CA 90024

Division of Chemistry & Chemical Engineering, California Institute of Technology, Pasadena, CA

91125

Abstract

Multiple myeloma (MM) is incurable and invariably becomes resistant to chemotherapy. Although

the mechanisms remain unclear, hypoxic conditions in the bone marrow have been implicated in

contributing to MM progression, angiogenesis, and resistance to chemotherapy. These effects

occur via adaptive cellular responses mediated by hypoxia-inducible transcription factors (HIFs),

and targeting HIFs can have anti-cancer effects in both solid and hematological malignancies.

Here, it was found that in most myeloma cell lines tested, HIF1

, but not HIF2

expression was

oxygen dependent and this could be explained by the differential expression of the regulatory

prolyl-hydroxylase isoforms. The anti-MM effects of a sequence-specific DNA-binding pyrrole-

imidazole polyamide (HIF-PA), that disrupts the HIF heterodimer from binding to its cognate

DNA sequences, were also investigated. HIF-PA is cell permeable, localizes to the nuclei, and

binds specific regions of DNA with an affinity comparable to that of HIF transcription factors.

Most of the MM cells were resistant to hypoxia-mediated apoptosis, and HIF-PA treatment could

overcome this resistance

in vitro

. Using xenograft models, it was determined that HIF-PA

significantly decreased tumor volume and increased hypoxic and apoptotic regions within solid

tumor nodules and the growth of myeloma cells engrafted in the bone marrow. This provides a

rationale for targeting the adaptive cellular hypoxic response of the O

-dependent activation of

HIF

using polyamides.

Keywords

mTOR; multiple myeloma; hypoxia

Corresponding author: Patrick Frost, Ph.D, Department of Hematology-Oncology, Greater Los Angeles VA Healthcare System,

11301 Wilshire Blvd, Los Angeles, CA, 90073., Phone=310-478-3711 ext 40410, Fax=310-268-3190, Patrick.Frost@va.gov.

CONFLICT OF INTEREST

: The authors have no conflict of interest to report.

DISCLAIMER:

These contents do not necessarily represent the views of the US Department of Veterans Affairs or the US

Government

HHS Public Access

Author manuscript

Mol Cancer Res

. Author manuscript; available in PMC 2017 March 01.

Published in final edited form as:

Mol Cancer Res

. 2016 March ; 14(3): 253–266. doi:10.1158/1541-7786.MCR-15-0361.

Author Manuscript

INTRODUCTION

Multiple myeloma (MM) is an incurable disease of malignant plasma cells characterized by

high rates of relapse and resistance to drug therapies (

–

). The reasons why this disease is

so difficult to cure is unclear, but the bone marrow (BM) microenvironment is known to

confer critical growth and survival advantages that protect tumor cells from apoptosis-

inducing stressors (

). The BM is hypoxic (p0

~10–30 mmHg) when compared to most

other tissues (pO

~85–150 mmHg) (

) and paradoxically, while oxygen stress can kill

tumor cells (

), low-oxygen conditions also promote tumor progression (

), angiogenesis

(

), and resistance to chemotherapy (

). These pro-survival responses to low oxygen

tension are regulated by adaptive cellular responses mediated by several oxygen-sensitive

transcription factors; the most important of these being the hypoxia-inducible factors (HIFs)

(for review see (

)). HIFs are composed of a constitutively expressed

-subunit (HIF1

ARNT) and inducible

-subunits (HIF1

, 2

, and 3

) that are responsive to oxygen levels

and are regulated via proteosome-mediated degradation. Briefly, under “normoxic”

conditions, the HIF

subunits are hydroxylated by a number of closely related prolyl-

hydroxylase domain proteins (PHD1-3) that results in recognition of the

-subunit by the

von Hippel-Lindau tumor suppressor (VHL), and its subsequent ubiquination and rapid

degradation by the proteosome. This normally maintains the HIF

-subunits at very low

levels in the cell. Under hypoxic conditions (pO

<50 mmHg), the proline-hydroxylase

activity is inhibited and HIF

degradation is not initiated (

). This allows dimerization of

the

and

-subunits and the translocation and binding of HIF to the hypoxic response

elements (HRE), thereby inducing gene transcription of hypoxia related survival and

angiogenic factors.

It has been shown that heightened expression of pro-angiogenic factors (such as VEGF) and

increased microvessel density within myeloma tumors is strongly correlated with disease

development and progression, as well as being predictive of poor patient prognosis (

–

This provided a rationale for using VEGF-targeting drugs, such as bevacizumab (Avastin),

to attempt to inhibit angiogenesis and increase hypoxic stress in MM tumors, although only

modest anti-tumor effects were observed (

). These results call into question the overall

effectiveness of targeting angiogenesis as a mono-therapeutic strategy for treating MM in

the clinic. Noting that the increase of hypoxia within the tumor bed following inhibition of

VEGF results in the subsequent activation of the adaptive hypoxic response and induction of

survival factors may provide an explanation for these underwhelming effects. Under this

hypothesis, we would argue that these resultant hypoxic conditions are actually supportive

of MM progression (because low O

represents a natural component of the bone marrow

niche in which myeloma engraft) by maintaining hyperactive HIF activity. Thus, rather than

killing the tumor cells, inhibition of angiogenesis may actually facilitate a pro-survival

adaptive hypoxic response through HIF activation. If true, then targeting the specific HIF-

mediated response to hypoxia may be a more effective anti-myeloma therapy than targeting

VEGF or angiogenesis alone. In fact, there are a number of different hypoxia and HIF

targeting strategies currently being tested in MM with varying levels of success (

In this study, we tested the effects of inhibiting the hypoxic adaptive response using a

synthetically derived, sequence-specific DNA-binding Py-Im polyamide (PA) composed of

Mysore et al.

Page 2

Mol Cancer Res

. Author manuscript; available in PMC 2017 March 01.

Author Manuscript

the aromatic rings of N-methylpyrrole and N-methylimidazole amino acids designed to

recognize and interfere with HIF binding to HRE sequences within the minor grove of the

DNA helix (

). We found that under low oxygen culturing conditions, most myeloma cell

lines were relatively resistant to hypoxia-mediated apoptosis and that HIF1

, but not HIF2

was upregulated in an oxygen-dependent manner. Treatment of MM cells with our HIF-

polyamide (HIF-PA) overcame resistance to hypoxia-mediated apoptosis

in vitro

as well as

inhibited the transcription of multiple hypoxia-induced genes. We also found that

combination treatment with HIF-PA polyamides (to inhibit gene transcription) and the

mTOR inhibitor rapamycin (to inhibit gene translation) was markedly more effective at

overcoming resistance to hypoxia-mediated apoptosis in MM cells. In additional

experiments, we used xenograft models to study the anti-MM effects of Py-Im polyamide

treatment on MM tumors

in vivo

and found that Py-Im polyamides were well tolerated by

the mice and had a marked anti-tumor effect characterized by a significant increase in

hypoxia as well as concomitant increases in apoptotic and necrotic regions within solid

tumor nodules as well as inhibition of myeloma growth in tumors engrafted in the BM.

Altogether, these data suggest that sensitivity of myeloma to polyamide therapy may be

related to the inhibition of gene expression induced by the oxygen-dependent activation of

HIF1

(but not necessarily HIF2

) and provides a rationale for targeting the adaptive

hypoxic responses in MM using these compounds.

MATERIALS AND METHODS

Cell lines and reagents

All cell lines were purchased from ATCC and maintained at 37°C and 5% CO

(“normoxic”

condition) unless noted. The cell lines were validated using the Johns Hopkins Genetic Core

Research Facility (Baltimore, MD) and stock aliquots were stored under liquid nitrogen.

Testing for mycoplasma was performed using a mycoplasma PCR detection kit (Sigma-

Aldrich, St Louis, MO). Py-Im polyamides were synthesized by solid-phase methods on

Kaiser oxime resin (Nova Biochem, Billerica, MA) (

). The tested polyamide, HIF-PA,

targets the sequence 5

′

-WTWCGW-3

′

(W=A or T) and modulates a subset of hypoxia-

induced genes, whilst the control polyamide (CO-PA) recognizes the non-HRE sequence 5

′

WGGWCW-3

′

. Enzyme-linked immunosorbent assay (ELISA) kits specific for human

VEGF was purchased from R&D Systems (Minneapolis, MN). The Hypoxyprobe-1 kit was

purchased from HPI Inc (Burlington, MA). Cellular apoptosis was measured by flow

cytometry using a cleaved caspase-3 kit (BD Biosciences, San Jose, CA). Small inhibitory

RNA (siRNA) for HIF1

(Silencer Select siRNA ID# s6539, gene ID# 3091) and scrambled

control RNA (Silencer Select negative control #1 siRNA) were purchased from Ambion

(Grand Island, NY). Cells were transfected with siRNA using Lipofectamine-2000 (Life

Technologies, Grand Island, NY).

Immunoblots

Protein was isolated and western blot analysis was performed as described previously (

Nuclear and cytoplasm fractions were isolated using the Thermo Scientific NE-PER

™

Nuclear and Cytoplasmic Extraction Kit (Rockford, IL) following the manufacturers

instruction. HIF1

antibody (clone 54/HIF1) was purchased from BD Biosciences.

-tubulin

Mysore et al.

Page 3

Mol Cancer Res

. Author manuscript; available in PMC 2017 March 01.

Author Manuscript

(clone H-235), LaminA/C (clone 14/LaminAC), BCL-XL (clone H-5), Survivan (clone

D-8), BNIP3 (clone ANa40), BCL-2 (clone C-2), goat anti-mouse and goat anti-rabbit IgG

horseradish peroxidase-conjugated antibodies were purchased from Santa Cruz

Biotechnology (Dallas, TX). BID (rabbit polyclonal), BAX (rabbit polyclonal), AKT-total

(clone C67E7), AKT-S473 (clone D9E) and AKT-T308 (clone C31E5E) and the P70-total,

P70-T421/S424, and P70-T389 antibody kits were purchased from Cell Signaling

Technology (Danvers, MA). MCL-1 (clone 542808) was purchased from R&D systems.

REDD1 (clone 1G11) was purchased from Bethyl Laboratories (Montgomery, TX). The

EGLN1/PHD2 (rabbit polyclonal), EGLN3/PHD3 (mouse polyclonal), EGLN3/PHD3

positive control (an EGLN3/PHD3 over expressing lysate from HEK293T cells), HIF2

(rabbit polyclonal), and Factor inhibiting HIF1 (FIH) antibodies (clone 162C) were

purchased from NOVUS Biologicals (Littleton, CO).

Hypoxia-treatments

For induction of hypoxia, MM cells were cultured in a humidified Hypoxygen hypoxia

chamber (Grandpair, Heidelberg, Germany). Variable pO

levels were established in the

hypoxia chamber from 2-0.1% O

, and 5% CO

at 37 °C. Oxygen levels were regularly

tested and calibrated using the manufacturer’s protocol.

Generation of hypoxia response element luciferase-expressing (HRE-LUC) cell lines

HRE-LUC reporter MM cell lines (8226, U266, OPM-2) were generated by stably

transducing cells using the Cignal lentiviral kit (Qiagen, Valencia, CA) followed by

selection with hygromycin (350 mg/ml). For the orthotropic xenograft studies, other

luciferase-expressing 8226 cells (8226-LUC) were stably transfected with the pGL4.5

luciferase reporter vector (Promega, San Louis Obispo, CA) using an AMAXA Nucleofector

Kit (Lonza, Koln Germany) followed by selection with hygromycin (350 mg/ml). The

vitro

luciferase activity was confirmed and measured using the dual-luciferase reporter assay

kit (Promega, San Louis Obispo) in a luminometer. The

in vivo

luciferase activity was

measured using the VivoGlo luciferine substrate (Promega).

Real time PCR

Quantitative expression of VEGF was carried out by quantitative real time PCR. RNA was

isolated using Triazol (Life Technologies, Carlesbad, CA) and cDNA was synthesized using

cDNA synthesis kit (Life Technologies). VEGF mRNA was amplified using VEGF primers

(Life Technologies) in an ABI 7300 real time PCR machine (Life Technologies). The

Applied Biosystems® TaqMan® Array Human Hypoxia 96-well Plate was used to test for

changes in hypoxia signaling associated genes.

Animals

Male NOD/SCID or NOG mice (4–6 weeks old) were obtained from Jackson Laboratories

(Bar Harbor, ME, USA). All animal studies were conducted in accordance with protocols

approved by the Institutional Animal Care and Use Committed (IACUC) of the Greater Los

Angeles Veterans Administration Healthcare System (GLA-VAHS).

Mysore et al.

Page 4

Mol Cancer Res

. Author manuscript; available in PMC 2017 March 01.

Author Manuscript

SQ Xenograft Model

We used the murine myeloma xenograft model of Leblanc et al (

) with minor

modifications (

). The 8226 cells (10 × 10

cells/mouse) were mixed with matrigel (BD

biosciences) then injected subcutaneously into the right flank (200 μl/mouse) of the mice.

The mice were monitored and randomized into drug treated or control groups (10 mice/

group) when the tumor volume reached approximately 300–500 mm

. The Py-Im polyamide

solution was prepared as previously described (

). Mice were given HIF-PA injections IP

every other day for a total of 5 injections. The tumor volume was measured using calipers

(width and length) using the formula W

× (L/2) (

) every other day during the course

of the experiment.

Orthotopic Xenograft Model

8226-LUC cells were injected (10 × 10

cells/mouse) through the tail vein (200 μl/injection).

In our initial experiments, the localization of human 8226 myeloma cell lines in the bone

marrow was determined by two methods: (1) flow cytometry staining of bone marrow

aspirates from the femurs of mice using FITC labeled anti-human CD45 antibodies (BD

Biosciences) and (2) immunohistochemistry of femurs using anti-human CD45 antibodies.

Real time 8226-LUC engraftment in the bone marrow was measured in anesthetized mice

given an IP injection of VivoGlo luciferin substrate (100 mg/kg mouse) and then monitored

for luciferase activity in the skeleton using a Perkin-Elmer IVIS XRMS small animal

imaging system. Luciferase activity in the skeleton of mice challenged with 8226-LUC was

typically observed between day +15 and day +20 post challenge. Once a positive

bioluminescent signal was observed, the animals were randomized into groups (6–8 animals/

group) and were treated with HIF-PA or vehicle control as described above, except a total of

6 injections were given, rather than 5. The luciferase activity was measured twice/week as

described above and the change in average radiance (photons/sec/cm

/steradian) was

measured and analyzed using the LivingImage version 4.4 imaging software.

Immunohistochemistry

For studies in our SQ model, 24 hours after the last injections, the mice were euthanized and

the tumor mass was excised. The tumor was bisected using a razor blade: one half of the

tumor was immediately placed in 10% buffered formaldehyde overnight, and the other half

was flash frozen for protein extraction. Formaldehyde fixed tumors were embedded in

paraffin and cut into 5 μm-thick serial sections using standard histological procedures.

Immunohistochemical staining with cleaved caspase 3, and Hypoxyprobe (pimonidazole)

(HPI Inc) was conducted using standardized automated methods.

Morphometric Analysis

IHC analysis was performed on tissue sections with a Nikon Microphot-SA microscope

(Melville, NY, USA) equipped with plan-apochromat lenses (20X and 40X). A Diagnostic

Technologies digital camera, model SPOT-RT was used to capture images with a resolution

of 1520 × 1080 pixels. Fields were selected by reviewing the slides at low power by a

researcher (VM) blinded to the treatments. Multiple non-overlapping fields were identified

for analysis of regions of hypoxia/necrosis and apoptotic index at higher powers within these

Mysore et al.

Page 5

Mol Cancer Res

. Author manuscript; available in PMC 2017 March 01.

Author Manuscript

areas of interest. ImageJ software (NIH, USA) was used to measure percent of hypoxia

stained tumor sections. Final images for publication were prepared using Adobe Photoshop

software.

Oncomine Analysis

The expression level of

PHD2/EGLN1

and

PHD3/EGLN3

genes in normal, MGUS and MM

tumors were analyzed using Oncomine, a cancer microarray database and web-based data-

mining platform (

). In order to reduce our false discovery rate, we selected p<0.0001 as a

threshold. We analyzed the results for p-values and fold change of our genes of interest.

Statistics

Data was screened for consistency and quality by both graphical (histograms, scatter plots)

and analytical methods (descriptive statistics). Variables were analyzed using generalized

linear models (GLM) such as ANOVA and

-tests. The effect of combining HIF-PA with

rapamycin on induction of apoptosis was assessed by the median effect method using

Calcusyn Software Version 1.1 (Biosoft, Cambridge, United Kingdom). Combination

indices (CI) values were calculated using the most conservative assumption of mutually

nonexclusive drug interactions. CI values were calculated from median results of apoptosis

assays.

RESULTS

Myeloma cell lines are resistant to hypoxia-induced apoptosis

in vitro

Because patient MM tumor cells specifically engraft within the hypoxic BM

microenvironment, we anticipated that patient-derived MM cell lines would also exhibit

resistance to hypoxia-mediated apoptosis

in vitro

. This was confirmed by analyzing the

sensitivity of a panel of MM cell lines cultured under various pO

concentrations in

increments from 2% down to 0.1% O

and for various time points up to 72 hours. We chose

these experimental ranges because these values fell within the actual pO

ranges reported by

Spencer et al (

) from

in situ

measurement in mouse bone marrow. In their study, O

levels

in the marrow were found to be <32 mm Hg, but in some bone marrow niches, it could be as

low as 9.9 mm Hg (or about 1% O

with a range of 2 - 0.6% measured in the extra-vascular

spaces). We found that at O

levels greater than ~1%, only modest cytotoxicity was

observed, even when cells were cultured out to 72 hours. Only at very low oxygen

conditions (i.e. 0.5 – 0.1% O

), did we see significant levels of hypoxia-mediated apoptosis

of the MM cells tested when compared to the “normoxia-cultured” controls. As shown in

Fig 1A, 8226 and U266 cell lines were the most resistant to low pO

(~15–20% apoptosis),

whilst H929 and MM1.S were intermediately sensitive (~25–35% apoptosis) to hypoxia-

mediated apoptosis (all measured at 72 hours). In contrast, OPM-2 cells were the most

sensitive (>50% apoptosis) to low O

, and this effect occurred significantly earlier, by 48

hours.

Culturing cells under low pO

had different effects on the HIF

-subunits expression in the

nuclear and cytoplasmic fractions of the hypoxia-resistant (8226) and hypoxia-sensitive

(OPM-2) cell lines cultured under standard normoxic (~22% O

) or hypoxic (0.1% O

)

Mysore et al.

Page 6

Mol Cancer Res

. Author manuscript; available in PMC 2017 March 01.

Author Manuscript

conditions. As shown in Fig 1B (left panel), HIF1

was constitutively expressed in the

nuclear fraction of 8226 cells as well as significantly upregulated under hypoxic conditions

(measured at 24 hours). In contrast to 8226, baseline HIF1

was absent in OPM-2 cells, but

was strongly induced by low O

(Fig 1B, right panel). On the other hand, HIF2

was

constitutively expressed in both 8226 and OPM-2 cells and this was independent of O

levels (Fig 1B). The rapid upregulation of HIF1

in OPM-2 cells was confirmed using the

hypoxia mimic, CoCl

, which induced HIF1

expression by 1 hour and reached a maximum

by 18 hours (Fig 1C). A strong O

-dependent induction of HIF1

was also noted in MM1S,

H929, and U266 cell lines (Fig 1D), while HIF2

expression was independent of O

levels

in H929 and U266 cells, but not in MM1S cells. These findings are generally similar to

other reports describing HIF expression in MM cells (

). Low pO

(0.1% 24hrs) did not

affect the expression of the pro-survival factor Bcl-2, but did inhibit Bcl-xl and MCL-1 in

OPM-2 and 8226, whilst survivin was only downregulated in OPM-2 cells (Fig 1E).

Survivin has been reported to play a role in HIF-regulated survival of MM and may be an

important target for future studies (

). Low pO

also upregulated the pro-apoptotic factors,

BNIP3 (a known HIF target) and BID in both lines tested, but BAX was only upregulated in

OPM-2.

Since we observed differential oxygen-dependent expression patterns for the HIF

-subunits,

we next asked if expression in the PHD isoforms responsible for regulating the

-subunits

could explain these results. As seen in Fig 2A, expression of PHD2/EGNL1 was absent in

8226, but strongly expressed in OPM-2 cells. It has been reported that PHD2/EGLN1

preferentially hydroxylates HIF1

(

), and its absence would explain why 8226 cells

express constitutive HIF1

under normoxic conditions. Along similar lines, we also found

that PHD3/EGLN3, which preferentially hydroxylates HIF2

(

), was absent in both 8226

and OPM-2 cells, and that also explains the oxygen-independent expression of HIF2

both these cell lines. To ensure that our antibodies could identify PHD/EGLN3 antigens, we

included a positive internal EGLN3 control (EGLN3 overexpressing lysate from HEK293T

cells purchased from NOVUS) in our immunoblots. We also found that low pO

(0.1%)

significantly upregulated PHD2/EGLN1 protein expression, and probably this likely acts as

a negative feedback mechanism to return HIF back to basal levels when O

levels return

back to normal (

). Furthermore, hypoxia-mediated upregulation of PHD2/EGLN1 was

unaffected by treatment with HIF-PA. We also examined the expression of another O

dependent regulator of HIF1

, the factor inhibiting HIF-1 protein (FIH), and found that

there was no difference in expression under either normoxic or hypoxic conditions in the

cell lines we tested.

To further characterize the expression patterns of

PHD2/EGLN1

, we used the Oncomine

tool, a publically available cancer microarray database to query if there were differences in

gene expression in clinical specimens of MM or monoclonal gammopathy of undetermined

significance (MGUS) versus normal controls within two data sets; Zhan et al (N=78

samples) (

) and Agnelli et al (N=158) (

). As shown in Figure 2B,

PHD2/EGLN1

(top

panel) was significantly overexpressed in MM specimens versus normal specimens

(p<0.05), and in MGUS versus normal specimens in the Zhan study (p<0.05) but not in the

Agnelli study (p>0.05). On the other hand,

PHD3/EGLN3

(bottom panel) was not

Mysore et al.

Page 7

Mol Cancer Res

. Author manuscript; available in PMC 2017 March 01.

Author Manuscript

significantly different in MM and MGUS versus normal specimens (p>0.05) in either data

set, although there was a general downward trend of expression of this gene. Next, we used

the TaqMan® Array Human Hypoxia 96-well assay system to analyze for changes in a

subset of hypoxia-related genes in 8226 cells. As shown in Fig 2C, hypoxia increased the

relative expression of

HIF1

by 6 fold, but had only minor effects on

HIF2

expression,

mirroring the results shown in Fig 1B. Other genes associated with the HIF regulatory

pathway, such as

PHD2/EGLN1

cullen-2

and

EP300

, were induced, whilst

PHD3/EGLN3

and

E3 ubiquitin

were downregulated and which mirrored the results from Fig 2A and 2B

above. Not unexpectedly,

VEGF

and

angiopoieitin-4

gene expression, genes that are well

known to be sensitive to hypoxia, were upregulated by hypoxia.

HIF-targeting polyamide (HIF-PA) inhibits the adaptive hypoxic response in MM cells

We next tested the ability of HIF-PA to inhibit the cellular response to hypoxia using 8226

cells that had been stably transduced with a hypoxia response element (HRE)-luciferase

reporter construct (8226-HRE-LUC). As shown in Fig 3A, culturing 8226-HRE-LUC cells

under low pO

(0.1% O

, 24hr) resulted in a ~2 fold induction of luciferase activity over the

baseline “normoxic” controls. This hypoxia-mediated LUC induction was significantly

inhibited (p<0.05) by treatment with HIF-PA but not by a control polyamide (CO-PA) that

recognized a non-HRE sequence. To provide further support for our model, we knocked

down HIF1

expression in 8226-LUC cells using HIF1

siRNA (Fig 3B top panel). As we

expected, and as shown in Fig 3B (bottom panel), the hypoxia-mediated increase of LUC

activity in 8226-LUC reporter cells transfected with HIF1

siRNA also demonstrated a

significant inhibition of the hypoxia-induced LUC activity. These data support our

hypothesis that HIF-PA can specifically inhibit the HIF-mediated cellular response to

hypoxia in 8226 cells. Next we showed that HIF-PA could inhibit the expression of HIF-

mediated gene expression using real-time PCR to assay for the effect of HIF-PA treatment

on the expression VEGF RNA. It is well known that hypoxia upregulates VEGF

transcription in myeloma cells, and as expected, culturing 8226 cells under hypoxic

conditions (0.1% O

, 24 hrs) induced VEGF mRNA by ~3–4 fold (Fig 3C). Treatment with

HIF-PA significantly (p<0.05) inhibited this hypoxia-mediated upregulation of VEGM

mRNA in a dose dependent manner. This effect on mRNA was also mirrored by HIF-PA

mediated inhibition of VEGF protein (measured by ELISA) in the supernatant of these cells

(Fig 3D). Altogether, these data demonstrate that HIF-PA specifically and effectively

inhibits the adaptive hypoxic response in MM cells.

HIF-PA treatment overcomes MM cell resistance to hypoxia

We next asked if inhibiting the hypoxic response with HIF-PA could sensitize a panel of

MM cells to hypoxia-mediated killing. We assayed this using hypoxia resistant 8226 and

U266 cells and hypoxia sensitive MM1.S and OPM-2 cells that were cultured under

normoxic or hypoxic conditions (0.1% O

) in the presence of HIF-PA or a control

polyamide (CO-PA). HIF-PA had little effect on 8226 cells (Fig 4A) and U266 (Fig 4B)

cultured under standard conditions (white bars), but the treatment of 8226 and U266 cells

cultured under hypoxic conditions resulted in significant and dose-dependent hypoxia-

mediated killing (an increase from ~20% to ~60%) (black bars) (ANOVA *p<0.05). The

hypoxia “sensitive” MM1.S and OPM-2 cells (Fig 4C and D) were even more responsive to

Mysore et al.

Page 8

Mol Cancer Res

. Author manuscript; available in PMC 2017 March 01.

Author Manuscript

HIF-PA, (ANOVA *p<0.05) with a similar increase in apoptosis observed by only 24 hrs

when cultured under low O

conditions. The CO-PA compound had little, if any, effect on

the hypoxia-mediated apoptosis in any of the cell lines examined. These data support our

hypothesis that inhibiting the adaptive hypoxic response with HIF-PA overcomes MM

resistance to hypoxia-mediated apoptosis

in vitro

As an

in vivo

correlate of the above

in vitro

data, we tested HIF-PA activity using a

xenograft SQ MM tumor model in NOD/SCID mice (

). The mice were challenged

on the flank with 10 X10

8226 cells admixed with matrigel. Once a palpable tumor

developed (~15 days later), the mice were randomized into groups (N=10 mice/group) to be

given 5 IP injections of either HIF-PA (100 nmol/injection) or vehicle control every other

day (Fig 5A, arrows on X-axis indicate days of treatment). The HIF-PA treatments were

well tolerated, with only a small transient decrease in weight being observed, and induced a

rapid and significant inhibition of tumor growth in the HIF-PA-treated mice compared to

control mice (p<0.05). In order to confirm uptake of HIF-PA, we treated an additional group

of mice (N=2 mice/group) with a FITC-conjugated HIF-PA and measured drug uptake by

real-time fluorescent imaging. We observed some auto-fluorescence signal in the bladder

and gut (Supplemental Figure 1A, mice #1 and #2). However, in FITC-HIF-PA treated mice,

positive signals were also observed in the tumor nodules (Supplemental Figure 1A, note

arrow indicating tumor nodule in mouse #3 and #4). Labeled HIF-PA localization in the

nuclei was confirmed by fluorescent confocal microscopy of excised tumor sections,

costained with DAPI (Supplemental Figure 1B). This is consistent with the recent studies on

the uptake C

labeled Py-Im polyamides into tumors (

)

In order to characterize the in vivo effect of HIF-PA, we excised the tumor nodules 24 hrs

after the last injection and performed immunohistochemistry on serial sections stained either

for hypoxia (pimonidazole staining, left panels) or apoptosis (cleaved caspase 3, right

panels) (Fig 5B). Both vehicle control (top left panel) and HIF-PA (bottom left panel)

treated tumors had regions of hypoxia (brown stained areas), but the extent of hypoxia was

significantly greater in the HIF-PA treated tumors than in the control tumors (quantified in

Fig 5C). We also noted there were significantly greater areas of necrosis within the tumor

bed in HIF-PA treated tumors compared to control tumors as well as a strong physical

correlation between areas of hypoxia and presence of apoptosis in the HIF-PA-treated

tumors (see Fig 5B, bottom right panel). In contrast, apoptotic cells were evenly distributed

throughout the tumor bed in the control tumors (Fig 5B, top right panel) and were not

localized to a specific geographic region in the tumor. As shown in Fig 5C, we found that

the area of positive hypoxia staining in tumor sections (10 tumors/group, 10 fields/tumor)

was ~35% in nodules harvested from the HIF-PA treated mice, compared to about 18% in

the tumors harvested from mice treated with vehicle control (p<0.05). The apoptotic index

(AI) in both the normoxic or hypoxic regions of the tumor was determined by counting

number of apoptotic cells in these regions using corresponding serial sections (10 tumors/

group, 10 fields/region) stained with pimonidazole to identify the specific geographical

regions of interest. As shown in Fig 5D, there was an approximate 3–4 fold increase in

apoptotic cells located within the hypoxic regions of tumors from the HIF-PA treated mice

compared to hypoxic regions of the control tumors (P<0.05), whilst there was no difference

Mysore et al.

Page 9

Mol Cancer Res

. Author manuscript; available in PMC 2017 March 01.

Author Manuscript

in apoptotic index within “normoxic” regions of the tumor bed. Finally, we examined the

expression of VEGF in tumor lysates by ELISA (Fig 5E). As with our

in vitro

data (see Fig

3D), HIF-PA significantly inhibited VEGF expression by approximately 50% when

compared to control tumors. Altogether, this data supports our hypothesis that HIF-PA can

overcome resistance to hypoxia-mediated apoptosis

in vivo

by inhibiting the adaptive

hypoxic response.

As a more physiologically relevant model, we developed an orthotopic, “disseminated” bone

marrow-engrafted xenograft model based on the model developed by Miyakawa (

) using

LUC2-transfected 8226 (these cells use a different p4.5 LUC2 plasmid to drive luciferase

expression and thus are different than the 8226-HRE-LUC cells described above) allowing

us to perform real-time longitudinal studies on myeloma tumors engrafted in the BM. As

shown in Supplemental Fig 1A, NOG mice challenged with 8226LUC cells developed bone

marrow engrafted MM tumors that could be observed by bioluminescence (top panel) and

X-ray (bottom panel) analysis. Approximately 20–50% of the bone marrow cells from

inoculated mice were positive for human CD45 confirmed by flow cytometry using FITC-

conjugated anti-huCD45 antibody (Supplemental Fig 2B) and by IHC of

in situ

huCD45+

8226 cells in the mouse femurs (Supplemental Fig 2C). Gross histological analysis of the

mice didn’t show tumor formation in other tissues (i.e. liver, lung, spleen, or kidney).

We then asked if treatment with HIF-PA had an anti-MM effect on tumor cells in the bone

marrow. As shown in Fig 6A, approximately +20 days post challenge with 8226-LUC cell,

engraftment of LUC2+ cells in the skeleton was confirmed and the mice were then

randomized into treatment groups (6–8 mice/group). The mice were then give IP injections

of 100 nmol/injection of HIF-PA every other day (arrows indicate days of injection on day

+27, +29 +31, +34, +36 and +38) or vehicle control. The average radiance

(photons/sec/cm

/steradian) was measured at various time points using a Perkin Elmer

Lumina XRMS small animal imager (out to day +40). The treatment with HIF-PA

significantly inhibited MM tumor growth in the marrow (data is presented as average

radiance ± 95% confidence intervals) starting after the third injection and reaching statistical

significance by the last HIF-PA injection on day +38). It should be noted that this particular

HIF-PA treatment regimen was not able to delay the development of hind limb paralysis (the

main end point criteria) when compared to control animals, with ~50% of the animals

reaching their end point on day +59 (control mice) and day +62 (HIF-PA treated mice).

Representative images of some of the mice are shown for day +22, +35 and +40. We noted a

decrease in the luciferase activity as well as a general shrinkage of individual tumor foci in

the mice. Specifically, in control mice, tumor foci tended to grow and merge over time,

while in HIF-PA treated mice, the foci remain relatively small and isolated.

Effect of targeting the mTOR pathway on regulation of HIF-PA sensitivity

Since we have previously demonstrated that mammalian target of rapamycin (mTOR)

inhibitors kill MM cells

in vivo

, and this anti-MM effects is mediated, at least in part, by

inhibiting VEGF translation and

de novo

angiogenesis (

), we asked if there

would be a synergistic effect if we combined HIF-PA-mediated inhibition of hypoxia-

inducible gene transcription in combination with rapamycin-mediated inhibition of mTOR-

Mysore et al.

Page 10

Mol Cancer Res

. Author manuscript; available in PMC 2017 March 01.

Author Manuscript

mediated regulation of protein translation. As shown in Fig 7A, treatment of 8226 cells with

the mTOR inhibitor rapamycin had a modest effect on apoptosis in MM cells cultured under

normoxic or hypoxic conditions (0.1% O

, 72 hours). However, under low pO

conditions,

using median effect combination indices (CI) analysis for apoptosis induction, we found that

the combination treatment with rapamycin and HIF-PA resulted in synergistic drug

interactions with CI values <1 across several concentrations tested. Altogether, this suggests

that targeting both transcription and translation of hypoxia-induced genes may be an

effective anti-MM strategy (Fig 7A see grey and black bars and bracketed area). We also

asked if REDD1, a hypoxia-sensitive negative regulator of mTOR, was affected by low pO

in both 8226 and OPM-2 cells. As shown in Fig 7B, REDD1 was strongly induced in MM

cells. Furthermore, the functional effects of hypoxia on mTOR signaling demonstrated a

marked inhibition of phosphorylation of p70S6 kinase but only modest effects on AKT

phosphorylation. The effects of hypoxia-mediated inhibition on p70 was transient and

reversible in OPM-2 cells, with p70 (389) phosphorylation levels rapidly returning to normal

within 2 hrs following reoxygenation of the cells (Fig 7C).

Discussion

There is increasing evidence that low oxygen conditions are supportive of MM growth,

progression and the development of resistance to chemotherapy, and that this occurs via a

cellular adaptive hypoxic response mediated, at least in part, by the action of HIF-

transcriptional factors. Because of this and the development of more resistant tumor

phenotypes associated with hypoxia, there has been increasing interest in targeting HIF-

mediated gene transcription to overcome these effects (

). Hypoxia induces the expression

of about ~100–200 genes mostly related to metabolism, angiogenesis, and apoptosis (

), and in this study, we found that a sequence-specific DNA-binding oligomer (HIF-PA)

that is capable of binding to the HRE and inhibiting HIF-mediated gene transcription (

) can overcome MM resistance to hypoxia-mediated apoptosis

in vitro

and

in vivo

. We

also found that treating MM cells with a combination of HIF-PA, to inhibit gene

transcription, and rapamycin, to inhibit gene translation, had a strong synergistic cytotoxic

effect against MM tumor cells cultured under hypoxic conditions. Our results provide a

strong pre-clinical rationale for using polyamides to target the adaptive hypoxic response in

MM and may prove to be efficacious in treating myeloma tumors engrafted in the hypoxic

bone marrow microenvironment. We also note that the ability of HIF-PA to recognize and

bind to HRE sequences enables them to block both the HIF1

/HIF1

and HIF2

/HIF1

dimers.

We surveyed a panel of MM cell lines and found that the expression of the HIF1

-subunit

was generally absent under normal culturing conditions but was rapidly increased by low

levels. The exception to this was 8226 cells, which constitutively expressed HIF1

under normoxic conditions, although it was further upregulated by hypoxia. In contrast to

the HIF1

-subunit, HIF2

was constitutively expressed in all the cell lines studied (except

MM1S), and the expression levels were independent of O

levels. Of note, we did not

observe a correlation between the expression of the HIF1

-subunit and sensitivity to

hypoxia-mediated cytotoxicity in the cells we examined. These results are interesting

because they suggest that the HIF

-subunits are differentially regulated by low pO

levels in

Mysore et al.

Page 11

Mol Cancer Res

. Author manuscript; available in PMC 2017 March 01.

Author Manuscript

MM cells and that mechanisms other than just the expression of HIF1

regulates sensitivity

to hypoxia. In fact, despite their similarities, it is known that the

-subunits can mediate

different responses to hypoxia and tumorogenesis: HIF1

acts as a tumor suppressor gene

and inhibits tumor growth, whilst HIF2

acts as an oncogene and promotes tumor growth

(

). It has also been demonstrated that the different

-subunits have varying affinities for

hydroxylation by the different PHD isoforms, with PHD2/EGLN1 showing the highest

affinity for HIF1

(

) and PHD3/EGLN3 showing the highest affinity for HIF-2

(

). In

our study, we found that the expression patterns of PHD/EGLN isoforms could explain

HIF

-subunit expression in both 8226 and OPM-2 cells. Specifically, PHD2/EGLN1 was

expressed at very low levels in 8226, but not in OPM-2, which can explain why HIF1

was

constitutively expressed in the former but not the later. It is also likely that the absence of

PHD3/EGLN3 in MM cells results in the inability to hydroxylate HIF2

, thereby rendering

its expression independent of O

levels. Thus, it is likely that the expression and ability of

PHD isoforms to regulate the expression of the different

-subunits may have important

ramifications for disease progression and pathology in MM, such as what is observed in

patients with VHL-cancer syndrome (

). In support of this,

PHD3/EGLN3

, but not

PHD2/

EGLN1

silencing has been reported at a relatively high frequency for patients with MM,

Waldenström’s macroglobulinaemia and monoclonal gammopathy of undetermined

significance and that these patients have a poorer prognosis (

) suggesting that PHD/EGLN

activity is as important as HIF activity in regulating the adaptive hypoxic responses in MM,

and is something that we will pursue in future experiments.

There is a dynamic physiological process that exists between the metabolic needs of tumor

cell growth and the sufficiency of the vascular networks that are required to support these

needs. On one hand, the development of oxygen stress within the tumor inhibits cell division

and growth and induces cell death, but on the other hand, hypoxia activates the HIF-

mediated cellular responses that provide protective growth and survival advantages and that

can foster the development of tumor resistance to radiation and chemotherapy (

). In

this study, we found that HIF-PA was well tolerated by mice, localized to the nuclei of the

tumor xenografts, and could inhibit tumor growth. Histological analysis of HIF-PA treated

SQ xenografts indicated that tumor cytotoxicity was remarkably co-localized with regions of

ischemic stress in the tumor nodules and that VEGF expression was significantly inhibited.

While VEGF is clearly a factor in the induction of the HIF-PA-mediated killing of MM

tumor cells, we believe that VEGF is probably only one of many genes affected by targeting

the adaptive hypoxic response using HIF-PA (

). While our SQ model demonstrated some

efficacy of HIF-PA in reducing solid MM xenograft tumors, we recognize that this

particular model may not be physiologically relevant to clinical myeloma, which engrafts in

the bone marrow. Therefore, we also present data using an orthotopic MM xenograft model.

In this model, 8226 tumor cells localized to the mouse BM demonstrate a similar anti-tumor

response to HIF-PA treatment that mirrored the effects seen in our SQ model. Specifically,

we noted a decrease in luciferase activity measured by the change in average radiance, as

well as a general decrease in the size of the MM tumor foci engrafted in the bone marrow.

While HIF-PA had a statistically significant effect on orthotopic MM tumors, this did not

translate to a longer survival in the mice. We believe that this may be because our initial

HIF-PA treatment strategy was not the most effective dosing regimen for establishing and

Mysore et al.

Page 12

Mol Cancer Res

. Author manuscript; available in PMC 2017 March 01.

Author Manuscript

maintaining long term anti-myeloma responses in our orthotopic model. For example, HIF-

PA availability and clearance in the bone marrow still needs to be established. This, in fact,

has important ramifications for addressing the known limitations of these anti-angiogenesis

therapies (

), because HIF-PA directly targets the hypoxic response at the level of gene

transcription, rather than targeting a single downstream effector of hypoxia, such as VEGF.

Because of our previous

in vivo

studies (

) that demonstrated a correlation

between the anti-angiogenic effects of the mTOR inhibitor, temsirolimus, we initially

hypothesized that hypoxic stress alone would be sufficient to kill MM cells. However, we

found that MM cell lines are generally resistant to low O

, suggesting that hypoxia-induced

physiological stress alone cannot fully explain our results. In fact, myeloma cells that are the

most resistant to mTOR inhibition (e.g. 8226 and U266), are also the most the resistant to

hypoxia, whilst cells that are more sensitive to rapalogs, such as OPM-2 are the most

sensitive hypoxia-mediated killing (

). It is also known that hypoxia regulates the activity

of mTOR via the induction of REDD1 (

) although the relationship between hypoxia,

mTOR activation, and HIF expression/activation remain complex and is not fully

understood (

). This is further confounded by the presence of cap-independent, IRES-

mediated translational pathways (

) that may allow tumor cells to escape mTOR-

targeting therapies. Interestingly, we find that the combination of polyamides and rapamycin

can effectively synergize with each other to overcome resistance to hypoxia-mediated killing

of 8226 cells

in vitro

. Along another line of reasoning, we noted that in a recent study by

Maiso et al (

), hypoxic conditions conferred a striking resistance to bortezomib-mediated

apoptosis in MM cell lines and, critically, inhibiting HIF1

expression could restore

sensitivity. If true, then we hypothesized that HIF-PA would likely have a similar effect in

bortezomib-treated cells by inhibiting the adaptive hypoxic response and overcoming any

hypoxia-mediated resistance to this drug. However, despite our best efforts, we were unable

to replicate this phenomenon, and instead found that our results were more similar to those

reported by Hu et al (

) which found instead that bortezomib killed MM cell lines cultured

under hypoxic conditions. It is unclear why our experiments differed from those of Maiso et

al, but could reflect variations in how hypoxic conditions were established. Therefore, at

least in our hands, it remains to be shown if hypoxia confers resistance to chemotherapeutic

drugs and if HIF-PA can overcome and sensitize MM cells by inhibiting HIF-activity.

In summary, HIF and related hypoxic response factors are frequently upregulated in MM

tumors, and has been implicated in contributing to the development and prognosis of MM

(

). The induction of pro-angiogenic, proliferative, metastatic, and glycolytic genes by

HIF1 may also be involved in the development of chemotherapy resistant phenotypes (

). In this sense, HIF1, and in particular the expression of the different

-subunits, may

play dual roles in the survival and progression of MM, through the differential

-subunit

expression patterns and the genes that they activate. Thus, we argue that understanding the

role and mechanisms HIF1-mediated adaptive hypoxic response at the level of HIF1/DNA

binding could be clinically relevant for developing novel therapies against patient MM

engrafted in the hypoxic bone marrow environment.

Mysore et al.

Page 13

Mol Cancer Res

. Author manuscript; available in PMC 2017 March 01.

Author Manuscript

Supplementary Material

Refer to Web version on PubMed Central for supplementary material.

Acknowledgments

GRANT SUPPORT

This work was supported by a MERIT grant 1I01BX001532 to PJF, from the United States Department of Veterans

Affairs Biomedical Laboratory Research and Development Service and a NIH GM051747 grant to PBD

Literature Cited

1. Kumar SK, Rajkumar SV, Dispenzieri A, Lacy MQ, Hayman SR, Buadi FK, Zeldenrust SR, Dingli

D, et al. Improved survival in multiple myeloma and the impact of novel therapies. Blood. 2008;

111(5):2516–20. [PubMed: 17975015]

2. Lonial S, Mitsiades CS, Richardson PG. Treatment options for relapsed and refractory multiple

myeloma. Clin Cancer Res. 2011; 17(6):1264–77. [PubMed: 21411442]

3. Martin SK, Diamond P, Gronthos S, Peet DJ, Zannettino AC. The emerging role of hypoxia, HIF-1

and HIF-2 in multiple myeloma. Leukemia. 2011; 25(10):1533–42. [PubMed: 21637285]

4. Hideshima T, Chauhan D, Podar K, Schlossman RL, Richardson P, Anderson KC. Novel therapies

targeting the myeloma cell and its bone marrow microenvironment. Semin Oncol. 2001; 28(6):607–

12. [PubMed: 11740818]

5. Podar K, Chauhan D, Anderson KC. Bone marrow microenvironment and the identification of new

targets for myeloma therapy. Leukemia. 2009; 23(1):10–24. [PubMed: 18843284]

6. Spencer JA, Ferraro F, Roussakis E, Klein A, Wu J, Runnels JM, Zaher W, Mortensen LJ, et al.

Direct measurement of local oxygen concentration in the bone marrow of live animals. Nature.

2014; 508(7495):269–73. [PubMed: 24590072]

7. Hockel M, Vaupel P. Biological consequences of tumor hypoxia. Semin Oncol. 2001; 28(2 Suppl

8):36–41. [PubMed: 11395851]

8. Asosingh K, De Raeve H, de Ridder M, Storme GA, Willems A, Van Riet I, Van Camp B,

Vanderkerken K. Role of the hypoxic bone marrow microenvironment in 5T2MM murine myeloma

tumor progression. Haematologica. 2005; 90(6):810–7. [PubMed: 15951294]

9. Giatromanolaki A, Bai M, Margaritis D, Bourantas KL, Koukourakis MI, Sivridis E, Gatter KC.

Hypoxia and activated VEGF/receptor pathway in multiple myeloma. Anticancer Res. 2010; 30(7):

2831–6. [PubMed: 20683019]

10. Maiso P, Huynh D, Moschetta M, Sacco A, Aljawai Y, Mishima Y, Asara JM, Roccaro AM, et al.

Metabolic signature identifies novel targets for drug resistance in multiple myeloma. Cancer Res.

2015; 75(10):2071–82. [PubMed: 25769724]

11. Greer SN, Metcalf JL, Wang Y, Ohh M. The updated biology of hypoxia-inducible factor. Embo J.

2012; 31(11):2448–60. [PubMed: 22562152]

12. Fandrey J, Gorr TA, Gassmann M. Regulating cellular oxygen sensing by hydroxylation.

Cardiovascular research. 2006; 71(4):642–51. [PubMed: 16780822]

13. Vacca A, Ribatti D, Roncali L, Ranieri G, Serio G, Silvestris F, Dammacco F. Bone marrow

angiogenesis and progression in multiple myeloma. Br J Haematol. 1994; 87(3):503–8. [PubMed:

7527645]

14. Vacca A, Di Loreto M, Ribatti D, Di Stefano R, Gadaleta-Caldarola G, Iodice G, Caloro D,

Dammacco F. Bone marrow of patients with active multiple myeloma: angiogenesis and plasma

cell adhesion molecules LFA-1, VLA-4, LAM-1, and CD44. Am J Hematol. 1995; 50(1):9–14.

[PubMed: 7545353]

15. Rajkumar SV, Leong T, Roche PC, Fonseca R, Dispenzieri A, Lacy MQ, Lust JA, Witzig TE, et al.

Prognostic value of bone marrow angiogenesis in multiple myeloma. Clin Cancer Res. 2000; 6(8):

3111–6. [PubMed: 10955791]

Mysore et al.

Page 14

Mol Cancer Res

. Author manuscript; available in PMC 2017 March 01.

Author Manuscript

16. Kumar S, Witzig TE, Dispenzieri A, Lacy MQ, Wellik LE, Fonseca R, Lust JA, Gertz MA, et al.

Effect of thalidomide therapy on bone marrow angiogenesis in multiple myeloma. Leukemia.

2004; 18(3):624–7. [PubMed: 14749707]

17. White D, Kassim A, Bhaskar B, Yi J, Wamstad K, Paton VE. Results from AMBER, a randomized

phase 2 study of bevacizumab and bortezomib versus bortezomib in relapsed or refractory multiple

myeloma. Cancer. 2013; 119(2):339–47. [PubMed: 22811009]

18. Podar K, Anderson KC. A therapeutic role for targeting c-Myc/Hif-1-dependent signaling

pathways. Cell Cycle. 2010; 9(9):1722–8. [PubMed: 20404562]

19. Olenyuk BZ, Zhang GJ, Klco JM, Nickols NG, Kaelin WG Jr, Dervan PB. Inhibition of vascular

endothelial growth factor with a sequence-specific hypoxia response element antagonist. Proc Natl

Acad Sci U S A. 2004; 101(48):16768–73. [PubMed: 15556999]

20. Belitsky JM, Nguyen DH, Wurtz NR, Dervan PB. Solid-phase synthesis of DNA binding

polyamides on oxime resin. Bioorg Med Chem. 2002; 10(8):2767–74. [PubMed: 12057666]

21. Frost P, Moatamed F, Hoang B, Shi Y, Gera J, Yan H, Gibbons J, Lichtenstein A. In vivo

antitumor effects of the mTOR inhibitor CCI-779 against human multiple myeloma cells in a

xenograft model. Blood. 2004; 104(13):4181–7. [PubMed: 15304393]

22. LeBlanc R, Catley LP, Hideshima T, Lentzsch S, Mitsiades CS, Mitsiades N, Neuberg D,

Goloubeva O, et al. Proteasome inhibitor PS-341 inhibits human myeloma cell growth in vivo and

prolongs survival in a murine model. Cancer Res. 2002; 62(17):4996–5000. [PubMed: 12208752]

23. Raskatov JA, Nickols NG, Hargrove AE, Marinov GK, Wold B, Dervan PB. Gene expression

changes in a tumor xenograft by a pyrrole-imidazole polyamide. Proc Natl Acad Sci U S A. 2012;

109(40):16041–5. [PubMed: 22988074]

24. Frost P, Berlanger E, Mysore V, Hoang B, Shi Y, Gera J, Lichtenstein A. Mammalian target of

rapamycin inhibitors induce tumor cell apoptosis in vivo primarily by inhibiting VEGF expression

and angiogenesis. J Oncol. 2013; 2013:897025. [PubMed: 23533410]

25. Rhodes DR, Yu J, Shanker K, Deshpande N, Varambally R, Ghosh D, Barrette T, Pandey A, et al.

ONCOMINE: a cancer microarray database and integrated data-mining platform. Neoplasia. 2004;

6(1):1–6. [PubMed: 15068665]

26. Martin SK, Diamond P, Williams SA, To LB, Peet DJ, Fujii N, Gronthos S, Harris AL, et al.

Hypoxia-inducible factor-2 is a novel regulator of aberrant CXCL12 expression in multiple

myeloma plasma cells. Haematologica. 2010; 95(5):776–84. [PubMed: 20015878]

27. Hu Y, Kirito K, Yoshida K, Mitsumori T, Nakajima K, Nozaki Y, Hamanaka S, Nagashima T, et

al. Inhibition of hypoxia-inducible factor-1 function enhances the sensitivity of multiple myeloma

cells to melphalan. Mol Cancer Ther. 2009; 8(8):2329–38. [PubMed: 19671732]

28. Berra E, Benizri E, Ginouves A, Volmat V, Roux D, Pouyssegur J. HIF prolyl-hydroxylase 2 is the

key oxygen sensor setting low steady-state levels of HIF-1alpha in normoxia. EMBO J. 2003;

22(16):4082–90. [PubMed: 12912907]

29. Bishop T, Gallagher D, Pascual A, Lygate CA, de Bono JP, Nicholls LG, Ortega-Saenz P, Oster H,

et al. Abnormal sympathoadrenal development and systemic hypotension in PHD3−/− mice. Mol

Cell Biol. 2008; 28(10):3386–400. [PubMed: 18332118]

30. Fong GH, Takeda K. Role and regulation of prolyl hydroxylase domain proteins. Cell Death

Differ. 2008; 15(4):635–41. [PubMed: 18259202]

31. Zhan F, Barlogie B, Arzoumanian V, Huang Y, Williams DR, Hollmig K, Pineda-Roman M,

Tricot G, et al. Gene-expression signature of benign monoclonal gammopathy evident in multiple

myeloma is linked to good prognosis. Blood. 2007; 109(4):1692–700. [PubMed: 17023574]

32. Agnelli L, Mosca L, Fabris S, Lionetti M, Andronache A, Kwee I, Todoerti K, Verdelli D, et al. A

SNP microarray and FISH-based procedure to detect allelic imbalances in multiple myeloma: an

integrated genomics approach reveals a wide gene dosage effect. Genes Chromosomes Cancer.

2009; 48(7):603–14. [PubMed: 19396863]

33. Frost P, Shi Y, Hoang B, Lichtenstein A. AKT activity regulates the ability of mTOR inhibitors to

prevent angiogenesis and VEGF expression in multiple myeloma cells. Oncogene. 2007; 26:2255–

62. [PubMed: 17016437]

Mysore et al.

Page 15

Mol Cancer Res

. Author manuscript; available in PMC 2017 March 01.

Author Manuscript