Inhibiting glycolysis remains an aspirational approach for the treatment of cancer. We have previously identified a subset of cancers harbouring homozygous deletion of the glycolytic enzyme enolase (ENO1) that have exceptional sensitivity to inhibition of its redundant paralogue, ENO2, through a therapeutic strategy known as collateral lethality. Here, we show that a small-molecule enolase inhibitor, POMHEX, can selectively kill ENO1-deleted glioma cells at low-nanomolar concentrations and eradicate intracranial orthotopic ENO1-deleted tumours in mice at doses well-tolerated in non-human primates. Our data provide an in vivo proof of principle of the power of collateral lethality in precision oncology and demonstrate the utility of POMHEX for glycolysis inhibition with potential use across a range of therapeutic settings.
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There is no restriction in any data presented in this work. The data that support the findings of this study are available from the corresponding author upon request. The POMHEX NCI-60 Human Tumor Cell Lines Screen data are attached as Supplementary Note 1. The metabolomic data, and Figures and Extended Data Figures source data have been deposited in Figshare (https://doi.org/10.6084/m9.figshare.12931676.v1). The DICOM source MRI scans are stored on a secure sever at MDA and are available on request. X-ray structures of ENO2 bound with HEX (PDB 5IDZ) have been deposited in the Protein Data Bank. Source data are provided with this paper.
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We thank S. Millward, S. Gammon, F. Lang, D. Pwinica-Worms, J. De Groot, P. Cserjesi, C. Halbrook, C. Lyssiotis and J. Huse for critical comments and suggestions. We thank H. Butterfield, L. Jacobs, A. Portal, P. Shresta, M. Washington and P. Suriyamongkol for technical assistance. We thank J. Holton and G. Meigs for their assistance with X-ray diffraction data collection. We thank K. Kalurachchi and J. McMurray for assistance with NMR. The UT M.D. Anderson Cancer Center NMR Core Facility is supported in part by the NCI Cancer Center Support Grant (CA16672). We thank M. Yuan and S. Breitkopf for help with the mass-spectrometry experiments. We thank Z. Jia for technical support with NHP experiments at CRL. We thank J. Long for help with statistical analysis and for critical reading. We thank K. Michel, C. Kingsley, V. Tran and the rest of the SAIF team for expert assistance with MRI imaging, and G. Rao, V. Henry, J. Gumin and F. Lang for GSC’s and intracranial cell implantations. The Pharmaceutical Chemistry Facility and the Small Animal Imaging Facility at The UT M.D. Anderson Cancer Center were supported by the NIH/NCI Cancer Center Support Grant under award number P30CA016672. Financial support was provided by NIH CDP SPORE P50CA127001-07 (F.L.M.), NIH SPORE 2P50CA127001-11A1 (F.L.M.), ACS RSG-15-145-01-CDD (F.L.M.), NCCN YIA170032 (F.L.M. Young Investigator Award), The Andrew Sabin Family Foundation (F.L.M. Fellow award), The Dr. Marnie Rose Foundation (F.L.M.), The Uncle Kory Foundation (F.L.M.) and The University of Texas MD Anderson Cancer Center Institutional Research Grant (F.L.M.), CPRIT RP140612 (R.A.D.) and NIH R01 CA225955 (R.A.D). S.K. was supported in part by MD Anderson Cancer Center CPRIT Research Training Program Grant RP170067. This work is dedicated to the memory of Dr. John Stuart McMurray, an expert in phosphonate prodrug synthesis, without whom this would not have been possible.
F.L.M. and R.A.D. are inventors on a patent covering the concept of targeting ENO1-deleted tumours with inhibitors of ENO2 (US patent 9,452,182 B2). F.L.M., R.A.D., D.M., W.B., Y.-H.L, N.S., Z.P., B.C. and F.P. are inventors on a patent application for the use of enolase inhibitors for the treatment of ENO1-deleted tumours (US patent 10,363,261 B2). V.C.Y, C-D.P and FLM are inventors on a patent application describing the synthesis and utility of novel prodrug inhibitors of enolase US 62/797,315 (filed 27 January 2019) R.A.D. is founder, director and/or advisor of Tvardi Therapeutics, Asylia Therapeutics and Stellanova Therapeutics, and the focus of these companies is not related to the content of this manuscript.
Peer review information Primary Handling Editor: George Caputa.
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a, Non pro-drugged phosphonate Enolase inhibitors were administered to mice by tail vein IV injection at the times indicated (white arrows) to determine effects on hematocrit (fraction of RBC / total plasma volume). Each trace represents an individual mouse treated and sampled repeatedly over 10 days, with two animals per drug or vehicle (green). Phosphoacetohydroxamate (PhAH; purple) caused a rapid and significant drop in hematocrit (and visible jaundice), which is then restored to initial levels after discontinuing treatment. PhAH is a pan-Enolase inhibitor with slightly greater potency against ENO1 over ENO2. As ENO1 is the sole isoform expressed in RBCs, anemia caused by PhAH is most likely due to on-target inhibitory activity against ENO1. b, Representative capillary tube after centrifugation from the hematocrit experiment described in a. The ratio of RBC fraction to total blood volume is decreased in response to PhAH treatment; pale yellow plasma in the PhAH sample indicates hemolysis. c,d, POMSF is a POM-pro-drug version of SF2312 that was generated for in vivo testing. Mice were IV tail vein injected with either vehicle (DMSO), POMSF, or POMHEX dissolved in PBS at the indicated doses. Capillary tubes were used to measure percent hematocrit. c, Hematocrit was determined as a function of time in response to Enolase inhibitors over a 10-day treatment course. Mice were administered either DMSO (green), POMSF (blue), or POMHEX (red) for 5 days before discontinuing treatment. Compared to the DMSO control and POMHEX, administration of POMSF results in decreased percent hematocrit, which is then restored to initial levels after discontinuing treatment. Significant differences by non-paired t-test with Bonferroni correction are indicated in d. e, Representative capillary tubes from mice treated with DMSO vehicle (left), POMSF (middle), and POMHEX (right). Note the decrease hematocrit (red bracket) as fraction of total blood volume (black bracket) from POMSF treatment; pale orange plasma indicates hemolysis.
a, b, Enolase activity (1/v, y-axis) was measured spectrophotometrically in vitro using an NADH-coupled assay1,2, with ENO1 and ENO2 activity plotted as a function of substrate concentration (x-axis, 1/S: 2-phosphoglycerate in mM; HEX in nM). Data were plotted as Lineweaver-Burke with x-axis showing inverse of substrate versus inverse activity (1/V). Each dot represents on independent biochemical rate determination. Slopes (Km-apparent/Vmax) were re-plotted as a function of inhibitor concentration in Fig. 1c. c. Enolase activity in lysates from ENO1 and ENO2 overexpressing D423 cells, measured as a function of HEX concentration; 0.5 mM 2-PG substrate concentration.
Extended Data Fig. 3 The Gli56 ENO1-homozygously deleted glioma cell line is sensitive to POMHEX and is pyruvate auxotrophic.
Sensitivity to POMHEX was determined in additional ENO1-homozygous deleted cell line, Gli56 (dark red) and its isogenic ENO1-rescued control3,4. a, Absence of ENO1 protein confirmed by western blot in the Gli56 cell line; note levels of ENO2 are lower than in D423 (ENO1-/-), which is confirmed by mRNA. b, c, RNA-seq mRNA expression of ENO1 and ENO2 in cell lines differing in ENO1-deletion status, confirming lower ENO2-expression in Gli56 compared to D423 cell. Each point represent one biological replicate, means are shown, n number is indicated for each cell line. d, Gli56 (dark red) is selectively sensitive to POMHEX compared to its isogenic ENO1-rescued control3 (blue). Another Gli56 rescued cell line (purple), in which ENO1 and a neighboring deleted gene, NMNAT1, are re-expressed (generated for a separate collateral lethality project), also shows resistance to POMHEX. Cell density was visualized by crystal violet after 14 days of growth; fresh media (DMEM, contains 1.25 mM pyruvate) containing Enolase inhibitor was provided every 4 days (N = 4 biological replicates +/- S.EM.). In contrast to the D423 cell line, Gli56 cannot be grown in pyruvate-free DMEM media, likely due to lower level of residual ENO2 expression. e, f, Gli56 ENO1-deleted, but not Gli56 ENO1-rescued cells, can grow in pyruvate-free DMEM. g, h, D423 ENO1-deleted cells can grow in pyruvate-free DMEM media, but slower than D423 ENO1-rescued or LN319 (ENO1-WT) cells. N = 8 biological replicates, mean +/- S.E.M. P value by 2-way ANOVA are indicated.
Extended Data Fig. 4 Relative sensitivities of glioma cell lines to the pro-drug POMHEX versus the active Enolase inhibitor HEX.
Sensitivity of a panel of glioma cell lines with with varying ENO1 statuses to the Enolase inhibitors POMHEX and HEX. IC50 values were calculated based on terminal cell density measured by crystal violet. D502 and U343 are ENO1-heterozygous deleted cell lines (~50% total Enolase3,4). Consistent with our previous reports for pan-Enolase inhibitors3,4, ENO1-homozygous deletion confers the greatest sensitivity to HEX, while ENO1-heterozygous deletion status shows intermediate sensitivity. While sensitivity to HEX is dependent on ENO1-deletion status, the sensitivity to POMHEX like depends not only on ENO1-deletion status but also expression of pro-drug bioactivation enzymes that transform POMHEX into active HEX enolase inhibitor (carboxylesterases, CES and phosphodiesterases, PDE). On average, the potency of POMHEX is ~75-fold greater than HEX though with substantial variation across cell lines. D502 is considerably more sensitive to POMHEX than U343 (IC50 82 vs 559 nM), yet U343 is more sensitive to HEX than D502 (IC50 19,723 nM vs 28,756 nM). This can be explained by higher levels of expression of pro-drug activating enzymes in the D502 glioma cell line result in greater sensitivity to POMHEX as compared to U343. Identification of the specific genes responsible, and their expression could be used for patient stratification, expanding the utility of Enolase inhibitors beyond those with ENO1-homozygous deletions.
Extended Data Fig. 5 ENO1-deletion status predicts sensitivity to Enolase inhibitors in glioma sphere- forming cells (GSC).
GSCs and omics data were kindly shared by the Lang lab9,10. a, Copy number variation at the 1p36 locus identifies GSC296 (red) as ENO1-homozygous deleted and GSC231, 268, 275, 289 as heterozygous deleted (pink). Dark blue regions represent bi-allelic (homozygous) deletion, while light blue regions correspond to mono-allelic loss. b, Confirmed lack of ENO1 expression by western blot in GSC296 (red), with corresponding levels of ENO2 shown. The western blot analysis for GSC296 was performed at least 3 times with similar outcomes. c, RNA-seq mRNA expression of ENO1 (bottom) and ENO2 (top) in ENO1-homozygous deleted and heterozygous deleted GSCs, confirming lack of ENO1 expression. Each bar represents one biological replicate. d,e, Sensitivity of GSCs with varying ENO1-deletion status to POMHEX. Dissociated GSCs were seeded in 96-well round bottom-low attachment plates in duplicate and allowed to form spheroids. Spheroids were treated with a serial dilution of POMHEX for 1 week; media was changed every 3 days with fresh drug. Viability was visualized with 5 nM TMRE (red). TMRE was quantified by ImageJ and expressed as a function of vehicle controls. Each dot represents one biological replicate. GSC296 (ENO1-/-) was distinctly sensitive to POMHEX. ENO1-heterozygous deleted GSCs showed intermediate sensitivity. ENO1-WT GSCs with the lowest residual ENO2 expression showed the greatest sensitivity amongst ENO1-WT GSCs. Experiments with GSC296 were reproduced independently once.
All indicated cells were grown in RPMI and treated with a serial dilution of POMHEX for 5 days. Cell density was then quantified by crystal violet after 5 days of growth and expressed relative to control (n = 2 biological replicates). a, A panel of hepatocellular carcinoma cell lines (SNU-423, SNU-398, SK-HEP-1; grey) and highly differentiated, hepatoblastoma line (HepG2 C3A; blue) were treated with POMHEX; D423 (red) was tested as a point of reference. The IC50 of POMHEX for most HCC cell lines is about 750 nM (grey), which is slightly lower than that for ENO1-WT glioma cell lines. However, the highly differentiated cell line, HepG2 C3A, was essentially insensitive with an IC50 >10,000 nM. This resistance likely derives from the dependence of hepatocytes on OxPhos, with minimal requirements for glycolysis-derived ATP (excess ATP allows for glycolysis reversal, gluconeogenesis) and concurs with our data in NHP, which indicate no hepatotoxicity. b, HEK293 kidney cells (grey) are immortalized, non-transformed kidney cells. Sensitivity to POMHEX was comparable to that observed in ENO1-WT cancer cell lines. c, Non-immortalized human astrocytes (grey) showed IC50 of >2,500 nM to POMHEX, which is also comparable to ENO1-WT glioma cell lines and far higher than that for ENO1-deleted D423 cells.
Hyperactivation of HIF by loss of VHL tumor suppressor gene has been reported to sensitize cells to glycolysis inhibition12. a, Proliferation of renal clear cell carcinoma cell lines that lack functional VHL (786-O and RCC4, red) and isogenic rescued cell lines re-expressing VHL (blue) was followed by Incucyte live cell imager (x-axis, time; y-cell confluence) in response to POMHEX treatment. Each box represents one biological replicates. VHL-deleted parental lines showed 4- to 8-fold greater sensitivity than isogenic rescued VHL lines. b, Cell density quantified by crystal violet for the same cell lines corroborates Incucyte live imaging data (N = 4 biological replicates, with +/- S.E.M.). IC50 for POMHEX in the RCC4 line is ~250 nM and 1,000 nM in the isogenic rescued line. TCA-cycle deficiency and defective oxidative phosphorylation by loss of function of Fumarase (FH) increase reliance on glycolysis and sensitize to glucose deprivation11. c, Likewise, the FH-null renal carcinoma cell line, UOK262, (red) shows dramatically higher sensitivity to the Enolase inhibitor POMHEX than isogenic FH-rescued cell line control (blue). Cell density in response to POMHEX treatment was quantified by crystal violet (N = 4 biological replicates, with +/- S.E.M.).
Extended Data Fig. 8 The synthetic cell permeable pyruvate pro-metabolite, methyl 2-oxopropanoate, significantly attenuates toxicity of POMHEX.
ENO1-deleted (D423, red), ENO1-isogenically rescued (D423 ENO1, blue), and ENO1-WT (LN319, grey) cells were treated with POMHEX at the doses indicated (x-axis) in media (DMEM) free of pyruvate with either (a) 2.5 mM methyl 2-oxopropanoate “methyl pyruvate,” (b) 5 mM lactate, or (c) 5 mM acetate (d). Cell density after 5 days exposure was determined by crystal violet staining and expressed relative to non-drug contain controls (n = 4 biological replicates as indicated, +/ S.E.M.). The IC50 for pyruvate-free media is indicated by a dashed line, for comparison. Exogenous methyl pyruvate attenuates sensitivity to Enolase inhibitors especially in ENO1-homozygously deleted cells (IC50 shifted from ~20 nM to ~150 nM), but supplementation with lactate or acetate had minimal effects. (e) Methyl pyruvate is a cell-permeable, synthetic pro-metabolite of pyruvate that can passively diffuse into the cell without requiring monocarboxylate transporter (SLC16) activity. It can then be hydrolyzed by intracellular carboxylesterases to release pyruvate. While lactate and acetate could serve a similar purpose, the present data (c, d) suggest that the rate of acetyl-CoA production by these carbon sources is insufficient to compensate for loss of ATP and pyruvate by POMHEX-mediated inhibition of glycolysis.
Extended Data Fig. 9 ShRNA knockdown of ENO2 recapitulates metabolic disruptions caused by small-molecule ENO2 inhibitors.
a, Knockdown of ENO2 by doxycycline-inducible shRNA in D423 ENO1-deleted and ENO1-rescued cells with the same constructs and cell lines used previously3. Induction of shENO2 results in ~70% decrease in ENO2-protein levels (ratios of band densities ENO2 to TPI, loading control, are indicated). The western blot analysis was performed once with each line representing a single biological replicate. Polar metabolites were profiled at specific times after induction of shENO2 with doxycycline (times indicated in x-axis, hours) in both conditioned media (extracellular) and cell pellet (intracellular), with cells passaged after 120 h. b, Schematic showing the Enolase reaction in the context of central carbon metabolism and associated pathways, with metabolites altered selectively in D423 ENO1-deleted cells in response to ENO2-knockdown. c-e, Accumulation of metabolites upstream of Enolase in response to ENO2-knockdown in ENO1-deleted but not ENO1-rescued cells recapitulates observation with Enolase inhibitors (Fig. 4). Glycerate (c, d) was the most significantly altered metabolite in polar metabolomic profile of conditioned media in ENO1-deleted but not rescued cells. Intracellular glycerate levels (c) recapitulate this trend and mirror the levels of 3-PG (e), from which it is hydrolyzed. f-j, TCA-cycle metabolites were selectively decreased in response to ENO2-knockdown, recapitulating effects of small molecule Enolase inhibition (Fig. 4). Similar to observations with small molecule Enolase inhibition, hexosamine biosynthesis pathway (HBP) metabolites increase in response to ENO2 knockdown. k-l, Each bar represents one biological replicate. P values from T-test of the log of the normalized values of each biological replicate are indicated.
The PK properties of phosphonate containing drugs have been well-studied in diverse model species as well as human patients and exhibit remarkable similarity, despite extensive chemical diversity. The very similar PK properties of these diverse drugs, derives from the fact their physiochemistry and pharmacokinetics are predominantly dictated by the negatively charged phosphonate group, conferring water solubility and limited protein binding. This water solubility means that drugs distribute with water and are cleared by a predominantly renal mechanism. Renal excretion is passive glomerular filtration and active tubular secretion, which can be modulated by drugs like Probenecid64. Rodent species rapidly eliminate phosphonate drugs, with >95% of injected drug excreted unchanged within 1 hr. In non-human primates, half-lives are typically 10-times longer, and in human patients even more so. This copious body of PK data provides a guide towards predictions of how HEX might behave in human patients. The comparison with fosmidomycin is especially informative as it is nearly identical, except for 1 C-C bond, to fosmidomycin. The elimination half-life of HEX in rodents and NHP falls very much in line with other phosphonate drugs and allows reasonable confidence in PK prediction in other models (canines) and in human patients. N/A = data not found in the literature.
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Lin, YH., Satani, N., Hammoudi, N. et al. An enolase inhibitor for the targeted treatment of ENO1-deleted cancers. Nat Metab 2, 1413–1426 (2020). https://doi.org/10.1038/s42255-020-00313-3
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