Geoffrey Grandjean , Petrus De Jong , Brian James...

Targeting a glycolysis HIF-1 feed-forward mechanism

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Definition of a novel feed-forward mechanism for glycolysis-HIF1α signaling in hypoxic tumors

highlights adolase A as a therapeutic target

Geoffrey Grandjean 1, 2, 4, Petrus De Jong2,4, Brian James2, Mei Yee Koh2, Robert Lemos2, John

Kingston1, Alexander Aleshin2, Laurie A. Bankston2, Claudia P. Miller2, Eun Jeong Cho3,

Ramakrishna Edupuganti3, Ashwini Devkota3, Gabriel Stancu3, Robert C. Liddington2, Kevin

Dalby3 and Garth Powis2

1 Department of Experimental Therapeutics, University of Texas MD Anderson Cancer Center.

Houston, TX.

2 Cancer Center, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA

3 Department of Medicinal Chemistry, College of Pharmacy, University of Texas at Austin,

Austin, TX.

4 G Grandjean and P De Jong contributed equally to this article.

Type of manuscript: Research Article

Running title: Targeting a glycolysis HIF-1 feed-forward mechanism

Keywords: glycolysis, HIF-1, feed-forward, ALDOA, structure.

Communicating Author:

Garth Powis, D. Phil. Director, NCI-Designated Cancer Center Sanford Burnham Prebys Medical Discovery Institute 10901 North Torrey Pines Road La Jolla, CA 92037 Email: [email protected] Tel: 858.795.5195 Fax: 858.795.5490

Research. on February 1, 2019. © 2016 American Association for Cancercancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 3, 2016; DOI: 10.1158/0008-5472.CAN-16-0401

http://cancerres.aacrjournals.org/


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Word counts:

Abstract: 164

Text: (including figure legends, excluding references) 5,532

Figures: 7

Tables: 0

References: 30

Supplemental information file: 3 methods, 5 tables, 10 figures,





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Abstract:

The hypoxia-inducible transcription factor HIF1α drives expression of many glycolytic enzymes.

Here we show that hypoxic glycolysis, in turn, increases HIF1α transcriptional activity and

stimulates tumor growth, revealing a novel feed-forward mechanism of glycolysis-HIF1α

signaling. Negative regulation of HIF1α by AMPK1 is bypassed in hypoxic cells, due to ATP

elevation by increased glycolysis, thereby preventing phosphorylation and inactivation of the

HIF1α transcriptional co-activator p300. Notably, of the HIF1α activated glycolytic enzymes we

evaluated by gene silencing, aldolase A (ALDOA) blockade produced the most robust decrease

in glycolysis, HIF-1 activity and cancer cell proliferation. Furthermore, either RNAi-mediated

silencing of ALDOA or systemic treatment with a specific small molecule inhibitor of aldolase A

was sufficient to increase overall survival in a xenograft model of metastatic breast cancer. In

establishing a novel glycolysis-HIF-1α feed-forward mechanism in hypoxic tumor cell, our

results also provide a preclinical rationale to develop aldolase A inhibitors as a generalized

strategy to treat intractable hypoxic cancer cells found widely in most solid tumors.





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Introduction

Glycolysis is defined as the sequence of 10 enzymatic reactions converting glucose to

pyruvate, which is accompanied by release of energy in the form of ATP. In normal cells,

pyruvate then enters the mitochondrial carboxylic acid cycle in the presence of oxygen, or is

converted to lactic acid in its absence. Glycolysis is critical for providing rapidly-dividing normal

and cancer cells with energy and metabolic intermediates to synthesize cellular biomass (1,2).

Malignant transformation greatly increases aerobic glycolysis (the Warburg effect) (3), which

favors production of additional ATP and metabolites for biomass synthesis, and enables

uncontrolled proliferation. Solid tumors also have an abnormal vasculature that leads to poor

blood perfusion and hypoxia. A cancer cell’s response to hypoxia is mediated by the hypoxia-

inducible transcription factors HIF-1 and 2 (4), which increase the expression of numerous

survival factors, including genes that encode vascular endothelial growth factor (VEGF) (5,6).

HIF-1 also upregulates the transcription of several glycolytic enzymes (7). Both glycolysis and

HIF activity are critical for cancer cell survival, and have been proposed as therapeutic targets

for agents that inhibit tumor growth (8-11).

While HIF-1 is critical for tumor growth, in part by inducing glycolytic enzymes, our findings

suggest that glycolysis is necessary for maintaining HIF-1 activity. This constitutes a feed-

forward loop that promotes increased HIF-1 activity and glycolysis, which we show is mediated

by inhibition of the AMPK1/EA1 binding protein p300 pathway. HIF-1 is often studied in terms of

its protein levels, but we show here that HIF-1 may not be functionally active if glycolysis is

limiting. Inhibiting the glycolysis–HIF-1 feed-forward loop therefore offers a novel target for

blocking tumor energy and biomass production and the HIF-1 survival response. Although HIF-1

and glycolysis have previously been proposed as targets for cancer treatment, efforts to develop

inhibitors have been unsuccessful. Here, we used an unbiased approach to identify the





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glycolytic enzyme, fructose-biphosphatase aldolase A (ALDOA) as a key target for inhibiting

both glycolysis and HIF1 activity. By using an inhibitor that targets ALDOA, we found that

inhibition of ALDOA does indeed break the feed-forward loop, blocking both glycolysis and HIF-

1 activity in cells, with the prospect of inhibiting tumor growth in vivo.

Materials and Methods:

Creation of Stable and Inducible Cell Lines

MIA PaCa-2 and PANC-1 pancreatic, MDA-MB-231 metastatic breast, HT-29 colon and 786-

O renal cell carcinoma cancer cell lines were obtained in 2012 from ATCC (Manassas, VA). The

identity of each line was authenticated on arrival, for each frozen stock and at two month

intervals during culture by the Molecular Cytogenetics Facility, University of Texas MD

Anderson Cancer Center. Each cell line was stably transfected a pGL3 plasmid (Promega)

containing a 5x repeat of the hypoxia transcriptional response element (HRE) flanking a

luciferase reporter and a G418 selection marker (HRE-luc). The reporter plasmid was a gift of

Dr. R. Gillies (Moffitt Cancer Center, Tampa, FL). Following selection, pools of stably

transfected cells were generated and stored frozen for later use.

For conditional Aldolase A (ALDOA) knockdown in an in vivo murine model, four sequences

predicted to target ALDOA gene expression were selected from the Thermo Scientific

Dharmacon shRNA library and each was inserted in a TRIPZ lentiviral vector (Open

Biosystems, Huntsville, Alabama). The HRE luciferase MDA-MB-231 line described above was

transduced with shALDOA-expressing lentivirus, and stable lines were selected in puromycin in

96-well plates with one cell per well to generate clonal populations. Sequence identification for

use in both in vitro and in vivo experiments was determined by relative ALDOA by Western blot.

After puromycin- and G418-resistant clones were selected, shALDOA expression in cells was





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induced using 400 ng/ml doxycycline in both normoxia and hypoxia (1% O2) and for in vivo

tumors by feeding mice chow containing 625 mg/kg doxycycline (Harlan Laboratories,

Indianapolis, IN) to achieve ALDOA knockdown.

Cell Transfection

Transient siRNA reverse transfections were carried out for global siRNA screening using

XTremeGene (Roche, Basel, Switzerland) according to the manufacturer’s instructions with the

genome-wide SmartPool siRNA library from Dharmacon using the MIA PaCa-2 HRE luciferase

line. After identifying initial glycolysis genetic hits, follow-up work in each of the 3 additional cell

lines listed used Lipofectamine RNAiMax (Qiagen, Valencia, CA) and Dharmacon SMARTpool

siRNAs for HIF-1α, Aldolase A, AMPK, p300, PCAF, FIH, PLK-1 or the On-Target-Plus non-

targeting pool #4 (OTP4). Total siRNA concentration was kept at 40 nM for single or multiple

siRNA combinations. Knockdown efficiency was determined by Western blotting of cell lysates

96 hours post transfection.

Chemical Compounds

Synthesis of naphthalene-2,6-diyl bis(dihydrogen phosphate) is described in Supplementary

Material and Methods S1.

Western Blotting

Primary antibodies for Western blotting were: HIF-1α (BD Biosciences, San Diego,CA),

Aldolase A (Thermo Scientific, Waltham, Massachusetts), β-actin, p300, phospho-p300 (all from

Santa Cruz Biotechnology Inc.), and AMPK/phosphoAMPK (Cell Signaling Technologies,

Danvers, MA).

Cell Viability and HIF-1α Activity Assays





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Viability of cell populations was quantified photometrically at 475 nm using the XTT Cell

Viability Assay (Biotium, Hayward, CA), according to the manufacturer’s instructions. HIF-1α

activity was measured using a Dual-Glo Luciferase Assay System (Promega, Fitchburg, WI)

according to the manufacturer’s protocol. Relative Luciferase activity (% control) were

calculated to correlate HIF-1 expression with cell viability data for each gene knockdown.

Determination of ATP Concentration

Cellular ATP was measured using an ATP Assay Kit (Abcam, Cambridge, United Kingdom)

according to the manufacturer’s protocol and quantified 96 hours post siRNA-transfection by

both colorimetric (OD 570 nm) and fluorometric (Ex/Em = 535/587 nm) methods,.

Measurement of Cellular Glycolysis

Glycolysis was measured as the rate of extracellular acidification (ECAR) using the Seahorse

Bioscience XF96e platform (Seahorse Bioscience, North Billerica, MA) and the XF Glycolysis

Stress Test Assay according to the manufacturer’s protocol. To measure glycolysis under

hypoxia, a modified hanging drop tissue culture method was used to evaluate 3-dimensional

spheroids of PANC-1 HRE cells transduced with shALDOA constructs. Three days after

seeding cells and 24 hours before measuring glycolysis spheroid shALDOA expression was

induced with 400 ng/ml doxycycline. A final volume of 175 μl of pre-conditioned assay medium

containing 18 spheroids was added to each well of a test plate and incubated at 37ºC in a CO2-

free incubator until the experiment was initiated. Spheroids exhibited a hypoxic core based on

analysis with a fluorescent hypoxia probe LOX-1 (SCIVAX USA, Inc, Woburn, MA) without the

need for hypoxic gassing conditions.

ALDOA kinetic assays.

ALDOA kinetic assays are described in Supplementary Materials and Methods S2.





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Crystallization and structure solution.

Protein crystallization and structure solution are described in Supplementary Materials and

Methods S3, and data collection and refinement statistics in Tables S1 and S2.

Xenografts

Approximately 107 MDA-MB-231 HRE cells, MDA-MB-231 cells harboring shALDOA clones

8.8 and 9.7, and MDA-MB-231 HRE empty vector cells, all in log cell growth, were suspended

each in 0.2 mL PBS and injected subcutaneously into the mammary fat pads of female NOD-

SCID mice. Groups contained five mice. When the tumors reached 250 mm3, chow containing

doxycycline was substituted for control feed (Harlan Laboratories) in test groups. Mice were

euthanized when they became clinically moribund, associated with the metastatic spread of the

MDA-MB-231 tumor to liver and lung (12). Animal studies were approved by SBPMRI’s Animal

Care and Use Committee.

Statistical Analysis

Data are shown as mean ± SD unless indicated otherwise. Student t test assuming two-tailed

distributions was used to calculate statistical significance between groups. Animal survival was

determined by the Kaplan–Meier analysis with P < 0.05 considered statistically significant.

Results:

Glycolytic enzymes regulate HIF-1α activity

To identify genes that regulate HIF activity, we conducted a genome-wide siRNA screen using

MIA PaCa-2 pancreatic cancer cells with a stably integrated 5 x HRE/promoter-luciferase (luc)

HIF reporter to identify genes that when knocked down inhibited HIF activity under hypoxic





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conditions (1% O2). It should be noted that the reporter cannot distinguish between HIF-1 and

HIF-2 activity, although hypoxic MIA PaCa-2 cells express predominantly HIF-1, and HIF-2 has

been reported not to up-regulate glycolysis genes (5). Unexpectedly, the screen identified

several glycolysis-related genes whose knockdown inhibited HIF-1 activity (Table S3). The

expression of glycolysis genes has been reported to be increased by HIF-1 during hypoxia

(5,13). We confirmed this using RNAseq in MIA PaCa-2 cells, and found that 16 glycolysis

genes were up-regulated in hypoxia, 15 of which showed HIF-1 dependence (Table S4).

To test the possibility that a glycolysis–HIF-1 feed-forward loop existed we used a panel of

siRNAs to knockdown 30 glycolysis genes and their isoforms (including pyruvate

dehydrogenase, which is responsible for linking glycolysis to the citric acid cycle in the

mitochondria) modification in mitochondria), and then measured HRE-luciferase activity (a

surrogate of HIF-1 activity) and cell proliferation. Using the same MIA PaCa-2 pancreatic cancer

cells stably transfected with the HRE-luc reporter, we found compelling evidence that glycolytic

enzyme activity is indeed critical for the normal functionality of HIF-1 (Figure 1A). Similar results

were obtained using HRE-luc PANC-1 pancreatic, MDA-MB-231 metastatic breast, and HT-29

colon cancer cell lines (Figure S1, Table S5). The greatest decrease in HRE-luc activity in all

lines, normalized to cell number, was observed when ALDOA was knocked down. Similar

inhibitory effects were observed in the 786-O renal adenocarcinoma cell line that expresses

HIF-2 exclusively, suggesting that regulation of HIF activity by glycolytic enzymes is not limited

to HIF-1 (Figure 1B). Dual knockdown of PGK1 and PGK2 suggest they have additive activities

on HRE-luc inhibition, although this was not accompanied by an additive decrease in cell

proliferation (Figure 1C, and Figure S2). Knockdown of ALDOA, or PGK1 or PGK2 in

combination, inhibited glycolysis in all cell lines (Figure 2A and 2B), which was accompanied by

lower cellular ATP levels (Figure 2C and Figure S3).





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HIF-1α activity is mediated by AMPK activation and p300 inactivation

We first established that, in all tumor cells tested, decreased HIF-1 activity caused by ALDOA

or PGK1 or 2 knockdown occurred without changes in HIF-1 protein levels, and was similar in

normoxia or hypoxia, although a greater overall effect was seen in hypoxia, where HIF-1 levels

are elevated (Figure 3A and Figure S4). In hypoxia the hydroxylation of key HIF-1 proline

residues by oxygen-sensitive dioxygenases is inhibited, thus preventing HIF-1 binding to the

von Hippel-Lindau protein (pVHL) which normaly leads to ubiquitination of HIF-1 and its

proteasomal degradation (4). In order to evaluate possible mechanisms underlying this change

in HIF-1 activity, we co-transfected glycolysis-related siRNAs together with siRNAs targeting

genes that are known to regulate HIF-1 activity. These included AMP-activated protein kinase

(AMPK) (14,15); E1A-associated cellular p300 transcriptional co-activator (p300) (16,17); PCAF

(p300/CBP-associated factor) (18,19); and FIH (Factor Inhibiting HIF-1), which interacts with

HIF-1α and VHL to repress HIF-1 transcriptional activity (20,21). We found that AMPK

knockdown rescued HIF-1 inhibition caused by ALDOA or PGK2 knockdown, but observed little

effect when either p300 or PCAF was knocked down (Figure 3B). FIH knockdown also negated

the effects of ALDOA and PGK1 knockdown on HIF-1 activity, likely due to loss of negative

regulation of HIF-1. Western blotting showed that ALDOA knockdown significantly increased

phosphorylation of AMPK on Thr172, a marker of AMPK activation in response to cellular stress

such as ATP depletion (22,23), in both normoxia and hypoxia (Figure 4A). Further evidence

that AMPK mediates the effects of ALDOA (or PGK1 or PGK2) knockdown on HIF-1 activity was

the rescue of HIF-1 activity by the AMPK inhibitor, dorsomorphin (Figure 4B). AMPK activation

can lead to phosphorylation of p300 at Ser89 that attenuates the interaction of p300 with a

variety of transcription factors in vitro and in vivo, including Hif-1 (24). We observed that

knockdown of ALDOA or PGK2, although not PGK1, resulted in p300 Ser89 phosphorylation in

HT-29 and MiaPaCa-2 cells (Figure 4A). Taken together the results suggest that inhibition of

HIF-1 activity and a concomitant decrease in glycolysis are mediated by AMPK activation





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(possibly in response to low cellular ATP levels), which in turn promotes p300 phosphorylation,

preventing p300 from co-activating HIF-1 transcriptional activity (Figure 4C).

Variable expression of aldolase isoforms suggest compensatory effects on glycolysis

To further understand the basis for variability in the effects of ALDOA knockdown on

proliferation among the 4 HRE luciferase lines, we next tested for possible compensatory

mechanisms when ALDOA is eliminated, by immunoblotting to analyze expression of aldolase B

and C isoforms in normoxia and hypoxia (Figure S5A and 5B). Interestingly, we found that HT-

29 cells (a human colorectal adenocarcinoma cell line with epithelial morphology), which were

the only cells found to be recalcitrant to ALDOA knockdown (Figure S1), were also the only

cells to exhibit significantly higher expression of ALDOC, suggesting a compensatory effect in

the absence of ALDOA at this step of glycolysis.

ALDOA knockdown extends median survival in an in vivo model of metastatic breast

cancer

To validate ALDOA as a potential therapeutic target, and because we have shown that

ALDOA knockdown is acutely toxic to cancer cells in vitro, we expressed a doxycycline-

inducible ALDOA shRNA in MDA-MB-231 breast cancer cells, and then used those cells to

establish an orthotopic model of metastatic breast cancer in female NOD-SCID mice (12). Two

clonal lines that showed complete (clone 8.8) or partial (clone 9.7) ALDOA knockdown,

glycolysis inhibition, and hypoxia response element (HRE) activity inhibition following

doxycycline treatment, were used (Figures 5A and S6). Mice fed doxycycline starting either

one week before implantation of cells or when the primary tumor reached approximately 250

mm3, showed an increased median lifespan. Mice fed doxycycline a week before implantation

showed increases from 37 days in parental, and 41 days in empty vector transfected cells, to 50

and 56 days in two clonal cells lines (p<0.001in both cases); mice treated after tumors were





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established showed increases from 41 days (untreated) to 48 days (p < 0.001 compared to

control) (Figure 5B). Transduction with clone 8.8, which showed complete ALDOA knockdown

associated with glycolysis and HIF-1 inhibition, promoted longer median survival time than did

the partial knockdown clone 9.7. Postmortem analysis of mice indicated marked tumor

metastasis to the lung and liver. While knockdown of ALDOA extended lifespan, doxycycline

given either as pretreatment or when tumors were established had a similar effect. This

suggests that ALDOA knockdown does not affect tumor implantation; rather, that ALDOA

knockdown inhibits metastasis from the primary tumor to the lungs and liver, which occurs late

in the tumor development process, and is the most likely cause of death.

Identification and characterization of a small-molecule allosteric inhibitor of ALDOA,

TDZD-8.

In order to confirm that ALDOA is a target with potential for cancer therapy, we sought a

small-molecule probe inhibitor of human ALDOA. We carried out a chemical library screen for

inhibitors of ALDOA using a novel biochemical assay, which allowed us to identify small

molecule inhibitors that would have interfered with the classic ALDOA assay. We identified the

compound TDZD-8 as an inhibitor with time-dependent inhibition (Figure 6A and 6B). In order

to determine the mechanism of inhibition, we first determined the crystal structure of native

human ALDOA at a resolution of 2.4 Å (see Supplementary Materials and Methods S3 and

Table S1). In the native crystals, the C-terminal tail (“C-tail”; residues 345-363) lies across the

active site, with the C-terminal Tyr363 inserted into the active site (Figure S7B). There is

abundant evidence that the C-tail is highly mobile and that its conformation and dynamics are

critical for catalysis (25). Indeed, we observed a 10-fold reduction in kCAT in an ALDOA construct

truncated before the C-tail (not shown). We next soaked a preformed native crystal in a solution

containing 1 mM TDZD-8, and determined its structure at 2.65 Å resolution (Table S1). We

found unambiguous evidence for TDZD-8 binding covalently to a single site, Cys239, which lies





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on an exposed loop distal to the catalytic site (Figure S7B). Computational docking studies

support our observation that the thiadiazole ring of TDZD-8 can bind to the sulfhydryl group of

Cys239 without the need for significant conformational changes in the protein.

In the crystalline form, however, lattice contacts reduce the mobility of the protein, which may

obscure ligand-binding sites that are available in solution. This may be especially true for

ALDOA, given the known mobility of the C-tail. In the course of our studies, we had co-

crystallized ALDOA bound to the active site substrate-mimetic, ND1, and solved its structure at

2.2 Å resolution (Table S2). The binding of ND1 sterically occludes the C-terminal tyrosine,

causing the entire C-tail to be ejected from its groove and become disordered, a phenomenon

that is typical of this class of inhibitor (Figure S7A). We therefore proceeded to soak preformed

ALDOA-ND1 crystals with TDZD-8, and solved its crystal structure at 2.4 Å resolution (Table

S2). In this case, we observed strong ligand-binding at C239 (as before) as well as at a second

site, C289, which is also distal to the active site but proximal to the C-tail (Figure 6C, 6D, 6E

and Figure S7C and S7D). C289 is partly buried in the native structure, and the binding of

TDZD-8 to C289 induces local conformational changes in loops that would contact the C-tail in

native crystals.

Our data suggest that the reactivity of Cys239 toward TDZD-8 is not significantly influenced by

the absence or presence of the C-tail; by contrast, the reactivity of Cys289 is strongly

influenced, since binding is only observed when the C-tail has been removed. It should

therefore follow that ligand-binding to C289 in solution should perturb the conformation of the C-

tail, and thereby modulate catalytic activity. Indeed, in solution, we observed a near-doubling of

the half-life of inactivation by TDZD-8 (from 120 to 214 min) when Cys289 was mutated to Ala

(Figure 6B). Thus, TDZD-8 appears to act as an allosteric inhibitor, via modulation of the

structure and/or dynamics of the C-tail, mediated principally through modification of Cys289, a

residue that lies within a well-defined, three-dimensional pocket.





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TZDZ-8 inhibits glycolysis, HIF-1 activity, and cancer cell proliferation,

TDZD-8 treatment of MDA-2B-231 breast cancer cells inhibited glycolysis and HIF-1 activity as

well as cancer cell proliferation in a dose dependent manner at low µM concentrations (Figure

6F and S8), and also decreased cellular ATP while increasing phospho-AMPK (Figure 6G and

S9). Treatment of MDA-MB-231 cells with TDZD-8 under hypoxic conditions (1% O2) for 6 h

resulted in an approximately 2-fold reduction of dihydroxy acetone phosphate (DHAP), the

product of the cleavage of fructose-1,6-bisphosphate substrate by ALDOA, and pyruvate, while

levels of the lower-abundance intermediates 3-phosphoglycerate and phosphoenolpyruvate

were not affected (Figure S10).

We therefore used TDZD-8 as a pharmacological probe to see if we could show an antitumor

effect. When administered intraperitoneally daily for 20 days at a dose of 12 mg/kg per day to

mice with MDA-MB-231 orthotopic breast cancer tumors, TDZD-8 caused significant slowing of

tumor growth, by about 60% by day 32 (Figure 6H). Pharmacodynamic studies after a single

dose of TDZD-8 showed a ~50% decrease in tumor lactate levels within 4 h, a 40% decrease in

DHAP, and a slower decrease with daily dosing for 5 days in downstream phosphoglycerate

(Figure 6I). Thus, TDZD-8 itself at low levels, or an active metabolite appears to reach the

tumor in sufficient amounts to inhibit tumor glycolysis, associated with antitumor activity.

Discussion:

We have shown that HIF-1–induced upregulation of glycolytic genes during anaerobic

(hypoxic) glycolysis in cancer cells is itself stimulated by the product of glycolysis, ATP, thereby

completing a glycolysis HIF-1 feed-forward loop that stimulates tumor growth, When anaerobic

glycolysis is inhibited (e.g. via inhibition of ALDOA), ATP levels are reduced, and the feed-

forward loop is broken via activation of the AMP-activated protein kinase-1 (AMPK1), which is

sensitive to the ratio of AMP/ATP in the cell (22,23). Activated AMPK1 inhibits the





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transcriptional activity of HIF-1 by phosphorylating its transcriptional co-activator, the E1A-

associated, cellular p300 (p300), at Ser 89, which blocks formation of the p300–HIF-1 co-

activation complex.

HIF-1 is critical for cancer cell survival and tumor growth in the stressed hypoxic tumor

environment (4,5). However, HIF-1 inhibitors used as single agents for the treatment of human

cancer have not advanced in the clinic. Thus, we took an unbiased approach, utilizing a high-

throughput siRNA synthetic lethal screen, initially to identify genes that might be targets to

inhibit tumor growth, which could also be used in combination with HIF-1 inhibition.

Unexpectedly, several hits in the screen were glycolytic enzymes. While it was known that HIF-

1 increases glycolytic activity in tumors by inducing expression of glycolytic enzymes (5,7,26), it

had not previously been reported that glycolysis increases HIF-1 activity. We found that the

knockdown of 16 of 30 glycolytic enzymes and isoforms were associated with detectable

inhibition of HIF-1 transcriptional activity. Among them, ALDOA and PGK1/2 knockdown

resulted in robust inhibition of HIF-1 activity in all lines tested. Importantly, inhibitors that target

these proteins should have the ability to block two processes critical for tumor growth:

glycolysis, the source of energy and metabolic support (this could be tumor or stroma

glycolysis); and HIF-1, which promotes cancer cell survival and tumor growth through increased

angiogenesis.

We chose ALDOA as a proof-of-principle target for inhibitor development, in part because its

knockdown in cancer cells was associated with greater inhibition of cancer cell proliferation than

PGK1/2 knockdown. This could be because ALDOA has other roles, including “moonlighting’ as

a nuclear protein (27), or that ALDOA is more important as a driver of glycolysis, whereas

PGK1/2 is a driver of tumor angiogenesis, which would not be apparent from our cell-based

studies. ALDOA expression has been reported to be significantly elevated relative to other

glycolytic enzymes in a number of human tumor types (28,29). Another consideration is that





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ALDOA is the major ALDO isoform driving glycolysis in cancer cells, sometimes aided by

ALDOC, whereas PGK1 and PGK2 appear to have redundant activities.

In order to demonstrate the potential of small molecule ALDOA inhibitors for cancer therapy,

we turned to a probe inhibitor of human ALDOA that we discovered through a chemical library

screen, utilizing a novel biochemical assay. The compound, TDZD-8, a 1,2,4-thiadiazole,

showed time-dependent inhibition of ALDOA that suggested a covalent interaction with the

protein. Our crystallographic studies showed that TDZD-8 bound to 2 Cys residues (Cys289 and

Cys239) on the surface of each ALDOA monomer. Although both residues lie distal to the active

site, one of them Cys289, lies in a well-defined pocket, and we found its reactivity toward TDZD-

8 to be strongly influenced by the presence or absence of the C-tail. Thus, we propose that

TDZD-8 binding to Cys289 in solution should allosterically perturb the conformation or flexibility

of the C-tail, thereby inhibiting catalytic activity. The electron density derived by crystallography

is consistent with disulfide bond formation and ring-opening of TDZD-8. Studies using Cys-

directed reagents to inhibit ALDO Cys residues have previously suggested that they are

involved in enzyme activity (30). Of ALDO’s 8 Cys residues, only 4 are accessible in the

absence of denaturing agents, and these include Cys-289 and Cys239. TDZ8-8 has allowed

us, for the first time, to demonstrate crystallographically an allosteric interaction between

ALDOA Cys289 and the catalytic site. Most importantly, although TDZD-8 is a simple chemical

probe without optimized drug like properties, it has nonetheless allowed us to demonstrate an

association between inhibition of glycolysis, HIF-1 activity and the proliferation of cancer cell

lines at low µM concentrations. The compound also exhibited in vivo antitumor against MDA-

MB-231 xenografts in mice and was associated with decreased levels of the glycolytic products

of ALDOA activity in tumors.





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In summary, we have shown that a feed-forward loop in tumors, simultaneously promoting

increased HIF-1 activity and increased glycolysis, offers a target, ALDOA, with which to block

tumor energy/metabolite production pathways and the HIF-1α survival response. Our HIF-1

activity-oriented RNAi screen and subsequent mechanism-based analysis expand our

understanding of known and novel regulators of the HIF-1 transcription factor, and point to a

previously uncharacterized regulation of HIF-1 activity by increased glycolytic enzyme activity.

Acknowledgements

Supported by NIH Grants CA163541, CA188260 (GP) and CCSG grant P30CA030199. The

help of SBPMDI Cancer Center Animal and Genomic Services is gratefully acknowledged.





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ABBREVIATIONS

ALDOA fructose-biphosphatase aldolase A

AMPK1 5-AMP-Activated protein kinase

DHAP dihydroxy acetone phosphate

DMEM Dulbecco’s modified Eagle’s medium

ECAR extracellular acidification rate

FIH factor inhibiting HIF-1

HIF-1 hypoxia inducible factor-1

HIF-2 hypoxia inducible factor-2

HRE hypoxia transcriptional response element

luc luciferase

NDA naphthalene-2,6-diylbis(dihydrogen phosphate)

Ni-NTA nickel nitrilotriacetic acid

p300 E1A binding protein

PDGF platelet derived growth factor

PCAF p300/CBP-associated factor

PGK phosphoglycerate kinase

PLK-1 polo-like kinase

pVHL von Hippel-Lindau protein

TDZD-8 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione

VEGF vascular endothelial growth factor

XTT 2,3-Bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide





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12. Iorns, E., Drews-Elger, K., Ward, T.M., Dean, S., Clarke, J., Berry, D., et al. A new mouse

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21. Mahon, P.C., Hirota, K., Semenza, G.L. FIH-1: a novel protein that interacts with HIF-

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40. Szutowicz, A., Kwiatkowski, J., Angielski, S. Lipogenetic and glycolytic enzyme activities in

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Tumour Biol. 1987; 8: 251-63.

FIGURE LEGENDS:

Figure 1. Changes in glycolytic enzyme expression alters HIF-1 activity. 30 genes

encoding glycolytic enzyme isoforms were selected for validation from a genome-wide siRNA

screen for inhibitors of cellular HIF-1 activity. A, HIF-1 activity was measured using MIA PaCa-2

pancreatic cancer cells harboring a constitutively expressed HRE-luciferase reporter 72 hr after

siRNA reverse transfection and after 24 h hypoxia (1% oxygen). siRNAs were from a different

manufacturer that in the original screen. Values were normalized to MIA PaCa-2 cell viability

under the same conditions as determined using a XTT viability assay and results expressed as

a % of non-treated cells. Closed bars are cell viability and open bars are HRE-luciferase (HIF-

1) activity. Bars represent S.D. of 3 determinations. Scrambled non-targeting (siSCR) and polo

like kinase-1 (PLK1) siRNAs served as a siRNA control and transfection control, respectively. B

Effect of targeted siRNAs for HIF-2α, ALDOA, PGK 1 and PGK2, and a non targeting siRNA

(siSCR) on HRE-luciferase reporter activity in 786-O renal cell carcinoma cells which express

HIF-2 but not HIF-1. The HRE-luciferase reporter responds to both HIF-1 and HIF-2. siRNA

reverse transfection was for 72 hr and hypoxia for 24 hr. Bars are S.D. of 3 determinations.

C, Effect of siRNA knockdown of ALDOA, and PGK1 and PGK2 alone and in combination, 72 hr

after siRNA reverse transfection and 24 hr hypoxia on HIF-1 activity in Panc-1 pancreatic

cancer and MDA-MB-231 triple-negative breast cancer cells harboring HRE-luciferase reporters.

Bars are S.D. of 3 studies. * p = 0.05 compared to siSCR transfected cells.





23

Figure 2. Decreased glycolysis and ATP formation following knockdown of ALDO or

PGK1 and 2. Cellular aerobic glycolysis and glycolytic reserve (defined as excess glycolytic

capacity following consecutive addition of 10 mM glucose, 1.5 µM oligomycin to inhbit

mitochondrial oxidative phosphorylation, and 100 mM 2-deoxyglucose to inhibit glycolysis) was

measured using Seahorse® technology as illustrated in the data key at upper left, in PANC-1

and MIA PaCa-2 pancreatic, MDA-MB-231 metastatic breast and HT-29 colon cancer lines, 72

h after reverse-transfection with siRNA targeting ALDO isoforms. A, Cells transfected with

siRNA targeting ALDO A, B and C, alone and in combination. siSCR non-targeting siRNA

served as a control. ECAR is the extracellular acidification rate measured in mpH/min. B,

Transfection with siRNA targeting PGK1 and PGK2 isoforms alone and in combination. C,

Cellular ATP levels 72 hr after transfection with siRNA targeting HIF-1α, ALDOA, and PGK1 or

2, in PANC-1 and MDA-MB-231 cells. Bars represent S.D. from 3 separate determinations.

Figure 3. Knockdown of ALDOA or PGK1 or 2 inhibits HIF-1 activity without decreasing

HIF-1α protein levels. A, HIF-1 activity measured with a HRE-luciferase reporter in air

(Normoxia) or 1% O2 (Hypoxia) using Panc-1 pancreatic and MDA-MB-231 breast cancer cells

stably transfected with HRE-luciferase reporter 72 h after transfection with siRNA targeting

ALDOA or PGK1 or PGK2, and in hypoxia for the last 24 h. siSCR and HIF-1α siRNAs served

as controls. Bars are S.D. of 3 determinations. HIF-1α protein was measured by Western

blotting. There was no HIF-1 detectable MDA-MB-231 cells in air. B, Dual reverse transfection

of siRNAs targeting ALDOA or PGK, with or without siAMPK (protein kinase, AMP-activated,

alpha 2 catalytic subunit), siP300 (E1A-associated cellular p300 transcriptional co-activator

protein), siPCAF (p300/CBP-associated factor), and siFIH (Factor Inhibiting HIF1). Bars are

S.D. of 3 determinations. The results indicate that AMPK inhibition mediates the effects of loss

of ALDOA, PGK1 or PGK2 on HIF-1 activity, without effect on HIF-1α protein levels.





24

Figure 4. p300 phosphorylation mediates AMPK effects on HIF-1 activity. A, Western blot

showing increased phosphorylation of AMPK (at Thr172) and p300 (at Ser89) following

knockdown of ALDOA, or PGK1 or PGK2 ,72 hr after siRNA transfection and 24 hr in hypoxia in

PANC-1 and MIA PaCa-2. B, HIF-1 activity measured with the HRE-luciferase reporter in MDA-

MB-231 cells relative to untreated cells following knockdown of ALDOA, PGK1, or PGK2 72 hr

after transfection and after 24 hr in hypoxia (filled boxes). Effects are rescued following

treatment with the AMPK inhibitor dorsomorphin at 5 µM, or with siRNA targeting AMPKα * p =

< 0.05 compared to siSCR control. C, Suggested mechanism of glycolytic control of cellular

HIF-1α activity through AMPK/p300 signaling. In normoxic conditions cancer cell aerobic

glycolysis maintains the cellular ATP/ADP ratio at levels sufficient to prevent phosphorylation of

AMPK, thus allowing the unrestricted formation of a p300/HIF1 complex that activates HIF-1.

When cellular ATP formation is decreased under hypoxic conditions, a decreased ratio of

ATP/ADP leads to phosphorylation of AMPK, which in turn phosphorylates p300 preventing its

association HIF-1, thus leading to decreased HIF-1 activity. Since this leads to decreased

synthesis of glycolytic enzymes, a feed-forward inhibition loop of decreased

glycolysis/decreased HIF-1 activity is established despite elevated HIF-1 protein levels due to

HIF-1 stabilization under hypoxic conditions.

Figure 5. Inducible ALDOA knockdown in MDA-MB-231 breast cancer tumors extends

survival of xenografted mice. A, Two lentiviral shALDOA doxycycline-inducible clones of

MDA-MB-231 metastatic breast cancer cells were established (clones 8.8 and 9.7). Both

showed doxycycline-induced ALDOA knockdown together with inhibition of hypoxic glycolysis

measured using Seahorse® technology, and decreased HIF-1α activity under hypoxic





25

conditions measured by HRE-luciferase reporter. B, Groups of 5 female immunodeficient SCID

mice were injected orthotopically in the breast fat pad with 106 MDA-MB-231 parental or vector-

only cells, or with clones 8.8 or 9.7. Animals received dietary doxycycline (625 mg/kg) starting 7

days before injection of cells (pre dox) or when tumors reached approximately 250 mm3 (dox).

When animals became clinically moribund they were euthanized. Median survival of mice with

parental MDA-MB-231 tumors was 37 days and with empty vector cells 41 days. Following

doxycycline treatment mice injected with clone 8.8 or clone 9.7 cells 7 days before cell injection

had a median survival of 56 days and 50 days, respectively (p<0.001 relative to combined

controls groups in both cases). Animals receiving doxycycline treatment when the tumors

reached ~ 250 mm3 had a median survival of 41 and 48 days (p<0.001 compared to combined

control groups in both cases). At the time of euthanization the lungs and liver of the mice

showed extensive metastatic nodules.

Figure 6 Antitumor activity of an ALDOA inhibitor. A, The small molecule TDZD-8 was

discovered as an inhibitor of recombinant human ALDOA activity through a chemical library high

throughput screen. Inhibition of ALDOA was time dependent suggesting that TDZD-8 could be

interacting directly with the protein. B. Time dependent inhibition of expressed ALDOA by

TDZD-8 at different concentrations with a 10 min or a 30 min preincubation. C, Crystallographic

analysis of TDZD-8 binding to ALDOA shown as a ribbon diagram of one monomer of the

tetrameric ALDOA co-crystallized with the active-site inhibitor, ND1, and soaked in 1 mM TDZD-

8. TDZD-8 binds specifically to 2 cysteine residues (239 and 289), as shown. The C-tail is not

visible in this structure, but its conformation derived from the native crystal structure is shown

schematically, colored spectrally with the C-terminus in red. Data collection and refinement

statistics are presented in Tables S3 and S4. D. Surface representation of the pocket occupied

by TDZD-8 bound to Cys289. E. Fit of the Cys289–TDZD-8 covalent adduct into the electron





26

density map (2Fo-Fc), shown as blue chicken-wire. F, Panel showing TDZD-8 inhibition of MDA-

2B-231 breast cancer cell proliferation, medium acidification (lactate formation) measured by

Seahorse® technology, a decreased the ratio of lactate to glucose in the medium, and block of

cellular HIF-1 activity measured with a HRE-luciferase reporter, all at low micromolar IC50

concentrations. G, MDA-MB-231 cells were treated with TDZD-8 or 2-deoxyglucose (2-DG) for 4

hr in normoxia. Left panel: ATP in cell lysates. Values are mean ± S.E.. * p <0.05 compared to

control. Right panel: Western blot showing increased phospho-AMPKα. H, TDZD-8 was

administered at daily doses of 12 mg/kg by intraperitoneal injection daily for 20 days to

immunodeficient SCID mice with MDA-2B -231 tumor xenografts shows significant inhibition of

tumor growth. There were 10 mice per group and bars are S.E. of the mean. * p = < 0.05, and

** p = <0.01 compared to control mice. I , MDA-2B-231 xenografts (~ 250 mm3) were collected

at 1 and 4 hr after a single intraperitoneal dose of TDZD-8 of 12 mg/kg, or 4 hr after the last of 5

daily doses (Q5D), and tumor levels of lactic acid, dihydroxyacetone phosphate (DHAP) and 3-

phospoglycerate (3PG) measured. Values are the mean of 4 mice each and bars are S.E. * p =

< 0.5 compared to non treated control tumors.




0

20

40

60

80

100

120

Cells Only siSCR siHIF2α siALDOA siPGK1 siPGK2

0

20

40

60

80

100

120

siS

CR

siH

IF1α

siA

LD

OA

siP

GK

1

siP

GK

2

siP

GK

1/2

siS

CR

siH

IF1α

siA

LD

OA

siP

GK

1

siP

GK

2

siP

GK

1/2

PANC1 HRE-luc MDAMB231 HRE-luc

A

B

C

FIG 1

PANC-1 MDA-MB-231

*

*

*

*

*

*

* *

*

0

20

40

60

80

100

120

Cells

Only

siS

CR

siH

IF1a

siP

LK

1

siH

K1

siH

K2

siH

K3

siG

P1

siP

FK

FB

1

siP

FK

FB

2

siP

FK

FB

3

siP

FK

FB

4

siP

FK

L

siP

FK

M

siP

FK

P

siA

LD

OA

siA

LD

OB

siA

LD

OC

siT

PI1

siG

AP

DH

siP

GK

1

siP

GK

2

siP

GA

M1

siP

GA

M2

siB

PG

M

siE

NO

1

siE

NO

2

siE

NO

3

siP

KLR

siP

KM

2

siP

DK

1

siP

DK

2

siP

DK

3

siP

DK

4

rela

tive s

igna

l (%

of contr

ol)

MIA PaCa-2 cell proliferation HRE-luciferase

rela

tive s

igna

l (%

of co

ntr

ol)

786-O cell proliferation HRE-luciferase

rela

tive s

igna

l (%

of co

ntr

ol)

cell proliferation HRE-luciferase

*

*

*




FIG 2

A

B

Glycolytic Reserve

Glucose

Injection

Oligomycin

Injection

2-DG

Injection

Glycolysis

Glycolytic

Capacity

Non-glycolytic Acidification

PANC-1 MDA-MB-231

C

0

20

40

60

80

100

120

140

Cells

Only

siS

CR

siH

IF1a

siA

LD

OA

siP

GK

1

siP

GK

2

Re

lati

ve

[A

TP

]

0

20

40

60

80

100

120

140

Cells

Only

siS

CR

siH

IF

siA

LD

OA

siP

GK

1

siP

GK

2

Re

lati

ve

[A

TP

]




0

20

40

60

80

100

120

140

Cel

ls O

nly

siO

TP3

siH

IF1

a

siP

GK

1

siP

GK

2

siA

LDO

A

Cel

ls O

nly

siO

TP3

siH

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a

siP

GK

1

siP

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2

siA

LDO

A

Normoxia Hypoxia

Re

lati

ve

Lu

cif

era

se

Un

its

(R

LU

)

0

20

40

60

80

100

120

140

Cells

Only

siO

TP

3

siH

IF1

a

siP

GK

1

siP

GK

2

siA

LD

OA

Cells

Only

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TP

3

siH

IF1

a

siP

GK

1

siP

GK

2

siA

LD

OA

Normoxia Hypoxia

Re

lati

ve

Lu

cif

era

se

Un

its

(R

LU

)

Cells

Only

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CR

siH

IF1α

siP

GK

1

siP

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2

siA

LD

OA

Cells

Only

siS

CR

siH

IF1α

siP

GK

1

siP

GK

2

siA

LD

OA

Cells

Only

siS

CR

siH

IF1α

siP

GK

1

siP

GK

2

siA

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OA

Cells

Only

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CR

siH

IF1α

siP

GK

1

siP

GK

2

siA

LD

OA

β-Actin

HIF-1α

PANC-1 MDA-MB-231

HIF-1α

β-Actin

FIG 3

A

B

Normoxia Hypoxia Normoxia Hypoxia

0

25

50

75

100

125

150

175

200

225

250

Cells

Only

siS

CR

siH

IF1a

siA

MP

K

siE

P300

siP

CA

F

siF

IH

siA

LD

OA

siA

LD

OA

+ s

iSC

R

siA

LD

OA

+ s

iAM

PK

siA

LD

OA

+ s

iEP

300

siA

LD

OA

+ s

iPC

AF

siA

LD

OA

+ s

iFIH

siP

GK

1

siP

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1 +

siS

CR

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siA

MP

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siP

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1 +

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P300

siP

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siP

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1 +

siF

IH

siP

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2 +

siS

CR

siP

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2 +

siA

MP

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siP

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2 +

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P300

siP

GK

2 +

siP

CA

F

siP

GK

2 +

siF

IH

Re

lati

ve L

uci

fers

e




A

FIG 4

0

20

40

60

80

100

120

140

Ce

lls O

nly

siS

CR

siP

LK

1

siH

IF1α

siA

LD

OA

siP

GK

1

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2

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siA

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+siA

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GK

2+

siA

MP

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HR

E-l

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rase

(p

er

ce

nt co

ntr

ol)

cell proliferation HRE-luciferase

B

* * *

dorsomorphin siAMPKα

C

ATP/ADP AMPK HIF-1α

p300 glycolysis

p

p

Normoxia Hypoxia

β-actin

total AMPK

p-AMPKα(Thr172)

total p300

p-p300(Ser89)

HIF1α

HT

-29 H

RE

β-actin

total AMPK

p-AMPKα(Thr172)

total p300

p-p300(Ser89)

HIF1α

MIA

PaC

a-2

HR

E

siP

GK

2

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1

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siP

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2

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1

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OA

siH

IF-1α

siS

CR

cells

only

cells

only




FIG 5

A

B

perc

ent surv

ival

MDA-MB-231 shALDOA (clones 8.8 and 9.3) + pre dox

MDA-MB-231 parental + pre dox

MDA-MB-231 empty vector + pre dox

30 40 50 60 0

50

100

time (days)

p <.0001

shALDOA clone 8.8

30 40 50 60 0

50

100

time (days)

perc

ent surv

ival

p <.0001

shALDOA clone 9.7

MDA-MB-231 shALDOA (clones 8.8 and 9.3) + dox

HIF1α

ALDOA

β-actin

hypoxia + + +

doxycycline - 24h 48h

shALDOA Clone 9.7

doxycycline - 24h 48h

HIF-1α

ALDOA

β-actin

hypoxia + + +

shALDOA Clone 8.8

cells

cells + 24 hr dox

cells + 48 hr dox




FIG 6

TDZD-8

A

F

0

50

100

150

200

250

300

350

10 15 20 25 30 35

tum

or

volu

me (

mm

3)

days post cell injection

control

TDZD-8 12mg/kg QD ip

* *

**

H I

mM

/mg

0 1 2 3 4 Q5D 0

5

10 Lactate

*

μM

/mg

0 1 2 3 4 0 20 40 60 80

*

Q5D

DHAP

0 1 2 3 4 0

2

4

6

μM

/mg

* *

Q5D

3PG

time (hours)

cell

via

bili

ty (

% c

ontr

ol)

-7 -6 -5 -4 0

25

50

75

100

125

TDZD-8 (M)

IC50 19 μM

TDZD-8 (M)

HR

E –

luc (

% c

ontr

ol)

-7 -6 -5 -4 0

25

50

75

100

125

IC50 9.9 μM

TDZD-8 (M)

lacta

te/g

lucose r

atio)

-7 -6 -5 -4 0

25

50

75

100

125

IC50 9.5 μM

EC

AR

(m

ph/m

in)

TDZD-8 (M) -7 -6 -5 -4

12

16

0

4

8

IC50 22 μM

B

E

D

TDZD-8 (μM)

0 20 40 60 80

AL

DO

A a

ctivity

0

0.2

0.4

0.6

0.8

1.0

1.2

wt 30 min

C289A 10 min

wt 10 min

C289A 30 min

C

p-AMPKα

actin

TDZD-8 (µM) 0 5 10 20

AMPKα

AT

P (

nm

ol/m

g p

rote

in)

TDZD-8

(µM)

2-DG

(mM)

G




Published OnlineFirst June 3, 2016.Cancer Res Geoffrey Grandjean, Petrus R De Jong, Brian P James, et al. adolase A as a therapeutic target

signaling in hypoxic tumors highlightsαglycolysis-HIF1Definition of a novel feed-forward mechanism for

Updated version

10.1158/0008-5472.CAN-16-0401doi:

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