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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.

ABSTRACT Inhibiting glycolysis remains an aspirational approach for the treatment of cancer. We recently demonstrated that SF2312, a natural product phosphonate antibiotic, is a potent inhibitor of the glycolytic enzyme Enolase with potential utility for the collateral lethality-based treatment of Enolase-deficient glioblastoma (GBM). However, phosphonates are anionic at physiological pH, limiting cell and tissue permeability. Here, we show that addition of pivaloyloxymethyl (POM) groups to SF2312 (POMSF) dramatically increases potency, leading to inhibition of glycolysis and killing of ENO1 -deleted glioma cells in the low nM range. But the utility of POMSF in vivo is dose-limited by severe hemolytic anemia. A derivative, POMHEX, shows equipotency to POMSF without inducing hemolytic anemia. POMHEX can eradicate intracranial orthotopic ENO1 -deleted tumors, despite sub-optimal pharmacokinetic properties. Taken together, our data provide in vivo proof-of-principal for collateral lethality in precision oncology and showcase POMHEX as a useful molecule for the study of glycolysis in cancer metabolism.

Abstract Glycolysis inhibition is an active area of investigation for the treatment of cancer. However, few compounds have progressed beyond the cell culture stage. We have recently demonstrated that genomic passenger deletion of the glycolytic enzyme Enolase 1 (ENO1) leaves gliomas harboring such deletions solely reliant on ENO2, rendering them exquisitely sensitive to enolase inhibitors Collateral Lethality. However, the tool compound that we employed for these in vitro studies, Phosphonoacetohydroxamate (PhAH), has very poor pharmacological properties and was ineffective in vivo. We recently reported that a structural analogue of PhAH, the natural phosphonate antibiotic SF2312, is a high potency inhibitor of Enolase. While more potent than PhAH, SF2312 remains poorly cell permeable. Here, we generated a Pivaloyloxymethyl (POM) ester pro-drug derivative of SF2312, termed POMSF, which increased the potency in cell based systems by ~50-fold. POMSF is selectively active against ENO1-deleted glioma cells in culture at ~19 nM, versus μM for SF2312. However, POMSF displayed poor aqueous stability. A derivative of POMSF, termed POMHEX, showed greater stability and its active form, HEX, showed 4-fold preference for ENO1 over ENO2. Labeled 13C-glucose tracing shows that POMHEX inhibits glycolysis at the Enolase step in all cell lines tested, but with ~100-fold greater potency in ENO1-deleted lines. POMHEX selectively killed ENO1-deleted glioma cells with an IC50 <30nM, whilst non-deleted cells could readily tolerate μM levels of inhibitor. As such, POMHEX was selected for in vivo experiments. Using an orthotopic intracranial xenografted model where tumor growth and response to therapy are monitored by MRI, we show that POMHEX is capable of eradicating intracranial ENO1-deleted tumors, with mice remaining recurrence-free even after treatment discontinuation. Taken together, these results reinforce that glycolysis is a viable target and provide in vivo proof-of-principal for the concept of using passenger deletions as targetable vulnerabilities in personalized cancer therapy. Citation Format: Yu-Hsi Lin, Nikunj Satani, Naima Hammoudi, Federica Pisaneschi, Paul Leonard, David Maxwell, Zhenghong Peng, Todd Link, Lee IV R. Gilbert, Ananth Bosajou, Duoli Sun, Joe Marszalek, Yuting Sun, John S. McMurray, Pijus K. Mandal, Maria E. Di Francesco, Barbara Czako, Alan Wang, William Bornmann, Ronald A. DePinho, Florian Muller. Pomhex, a cell-permeable high potency enolase inhibitor with utility for collateral lethality treatment of cancer [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr A39.

Despite being crucial for energy generation in most forms of life, few if any microbial antibiotics specifically inhibit glycolysis. To develop a specific inhibitor of the glycolytic enzyme enolase 2 (ENO2) for the treatment of cancers with deletion of ENO1 (encoding enolase 1), we modeled the synthetic tool compound inhibitor phosphonoacetohydroxamate (PhAH) into the active site of human ENO2. A ring-stabilized analog of PhAH, in which the hydroxamic nitrogen is linked to Cα by an ethylene bridge, was predicted to increase binding affinity by stabilizing the inhibitor in a bound conformation. Unexpectedly, a structure-based search revealed that our hypothesized backbone-stabilized PhAH bears strong similarity to SF2312, a phosphonate antibiotic of unknown mode of action produced by the actinomycete Micromonospora, which is active under anaerobic conditions. Here, we present multiple lines of evidence, including a novel X-ray structure, that SF2312 is a highly potent, low-nanomolar inhibitor of enolase.

Abstract Glycolysis inhibition is an active area of investigation in cancer. However, few compounds have progressed beyond the cell culture stage. We have recently demonstrated that genomic passenger deletion of the glycolytic enzyme Enolase 1 (ENO1) leaves gliomas harboring such deletions with less than 10% of normal enzymatic activity, rendering them exquisitely sensitive to enolase inhibitors. However, the tool compound that we employed for these in vitro studies, Phosphonoacetohydroxamate (PhAH), has very poor pharmacological properties and was ineffective in vivo. We performed a SAR studies to increase inhibitor specificity towards ENO2 as well as pro-druging to increase cell permeability. The lead compound generated by these efforts, termed POMHEX, is selectively active against ENO1-deleted glioma cells in culture at ∼35nM (versus μM for PhAH). Using an orthotopic intracranial xenografted model where tumor growth and response to therapy are monitored by MRI, we show that POMHEX is capable of eradicating intracranial ENO1-deleted tumors, with mice remaining recurrence-free even after treatment discontinuation. Taken together, these results reinforce that glycolysis is a viable target and provide in vivo proof-of-principal for the concept of using passenger deletions as targetable vulnerabilities in cancer therapy. Citation Format: Yu-Hsi Lin, Joe Marszalek, Yuting Sun, Naima Hammoudi, Paul Leonard, David Maxwell, Nikunj Satani, Peng Zhang, Todd Link, Gilbert Lee, Maria E. Di Francesco, Barbara Czako, Alan Y. Want, Ronald A. DePinho, Florian L. Muller. Pomhex: a cell-permeable high potency Enolase inhibitor with in vivo anti-neoplastic activity. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C183.

Abstract Background: RecQ helicases are a ubiquitous family of DNA unwinding enzymes involved in the maintenance of chromosome stability. RecQ1 helicase genomic variations have an important prognostic role and represents a therapeutic target in pancreatic cancer [JCO 2006; 24 (11) 1720-1728]. Pubchem data indicated potential RecQ1 activity >600 compounds using a qHTS RecQ1 assay (NIH Chemical Genomics Center). Methods: We applied a set of filters including molecular weight, potency and Hill curve fitting and also removed those interfering with biological assay to identify sixteen compounds for studying RecQ1 inhibition. Two compounds showed strong inhibition of RecQ1 helicase activity at 50 µM and at IC50 < 5 µM, respectively. From a large library of compounds, eight potential inhibitors were synthesized in three simple steps. First, sulfonylation of various amines with 3-Nitrobenzenesulfonyl chloride was performed followed by reduction of the nitro group into an amine. Finally, acylation with variety of acid chlorides resulted in the final library of RecQ1 analogs. These agents were studied for RecQ1 helicase activity assay with the radiolabeled partial duplex substrate in vitro. The RecQ1 inhibitor candidate compounds were investigated in pancreatic cancer cell lines (AsPC-1, BxPC-3, Capan-1, PANC-1, and Mia-Paca2) for anti-tumor efficacy using cell proliferation assay with CellTiter-Glo® luminescence. Combination studies with test compounds and poly (ADP ribose) polymerase (PARP) inhibitor olaparib and topoisomerase Inhibitor SN38 were performed. Results: Though none of the compounds showed the same level of activity of the parent compounds, we were able to demonstrate a modest level of helicase activity with several. Two compounds displayed strong inhibition of pancreatic cancer cell proliferation. Two other compounds displayed synergistic inhibition with SN38 or olaparib of pancreatic cancer cell proliferation and induced DNA damage. Conclusions: Synthesis of RecQ1 inhibitors is feasible and shows potential for anti-tumor efficacy. Note: This abstract was not presented at the meeting. Citation Format: Mingxin Zuo, David Maxwell, Basvoju A. Bhanu Prasad, Zhenghong Peng, William Bornmann, Milind M. Javle. Development of targeted inhibitors against RecQ1 helicase. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5361. doi:10.1158/1538-7445.AM2014-5361

A series of degrasyn-like symmetrical compounds have been designed, synthesized, and screened against B cell malignancy (multiple myeloma, mantle cell lymphoma) cell lines. The lead compounds T5165804 and CP2005 showed higher nanomolar potency against these tumor cells in comparison to degrasyn and inhibited Usp9x activity in vitro and in intact cells. These observations suggest that this new class of compounds holds promise as cancer therapeutic agents.

A virtual library of 54 inositol analog mimics of In(1,4,5)P3 has been docked, scored, and ranked within the binding site of human inositol 1,4,5-trisphosphate 3-kinase A (IP3-3KA). Chemical synthesis of the best scoring structure that also met distance criteria for 3'-OH to -P in Phosphate has been attempted along with the synthesis of (1S,2R,3S,4S)-3-fluoro-2,4-dihydroxycyclohexanecarboxylic acid as an inositol analog, useful for non-invasive visualization and quantitation of IP3-3KA enzymatic activity.

Background: Colorectal cancer (CRC) is the second leading cause of cancer death in the United States. Although the response rate to current systemic therapies is ∼50%, drug resistance develops in nearly all patients leading to 50,000 deaths each year. Overcoming drug resistance involves understanding the mechanisms by which cancer cells adapt to the genotoxic stress. Metabolic changes at levels of mitochondria function, glucose metabolism, and de novo synthesis of fatty acids frequently occurs in malignant cells and impacts tumor development and growth. Our laboratory established in vitro drug-resistant models of CRC cells by chronic exposure of HT29 cells to increasing doses of chemotherapeutic agents (oxaliplatin, 5-FU and SN38) over a period of 4–6 months. We recently reported a metabolic switch to the glycolytic phenotype due to mitochondria defects in the oxaliplatin-resistant CRC cells (HT29-OXR) (Zhou et al, Cancer Research, 2012). In this study, we tested the hypothesis that deregulation of de novo lipogenesis pathways plays an important role in chemoresistance of CRC cells. Methods: A previous study (Bose et al. Br J Ca, 2011) using unbiased proteomic profiling by mass spectroscopy (MS) was used to determine the proteomic signature of defective metabolic pathways in the oxaliplatin-resistant CRC cells (OXR cells). ATP-citrate-lyase (ACLy), the key enzyme of de novo lipogenesis pathway, was examined for protein levels and activation. The lipid content of resistant cells was examined by transmission electron microscope (TEM) and Oil Red staining. Transient knockdown of the ACLy protein by siRNA was used to study re-chemosensitization of the resistant cells. A small molecule inhibitor of ACLy was studied in combination with chemotherapeutic agents in resistant CRC cells for growth inhibitory (MTT assay) and cytotoxic effects (PARP cleavage and Annexin V staining). A cell free assay was used to test the potency of the ACLy inhibitor on ACLy activation. Results: Activated ACLy protein level (phosphorylation on S454) was demonstrated by Western blot analysis in the HT29-OXR and SN38-resistant (HT29-SNR) cells, but not in 5-FU-resistant (HT29-FUR) cells. The OxR cells showed a 2–3 fold increase in lipid droplets numbers (by TEM examination) and fatty acid content (by Oil Red staining) than the parental cells. Furthermore, transient knockdown of the ACLy protein by siRNA demonstrated a return to chemosensitization when cells were treated with oxaliplatin. IC50 values of the ACLy inhibitor for parental HT29 and HT29-OXR, -SNR and —FUR cells were ∼30μM. As a single agent, the ACLy inhibitor blocked phosphorylation of ACLy and induced apoptosis in a concentration-dependent manner in parental HT29 cells, and its resistant derivatives -OXR and -SNR cells. Combination of the ACLy inhibitor at concentrations sufficient to block ACLy phosphorylation with oxaliplatin and SN38 showed enhanced effects on growth inhibition (MTT) and apoptosis induction (PARP cleavage and Annexin V assay) in HT29 cells-OXR and -SNR cells. Conclusions: Chemoresistant CRC cells demonstrated: 1) increased de novo lipogenesis, 2) elevated levels of key lipogenesis enzymes ACLy, 3) dependence on ACLy activity for cell survival under cytotoxic stress. This metabolic switch likely contributes to the chemoresistant phenotype of CRC cells. Targeting an early step of de novo lipogenesis such as blocking ACLy activity may provide a novel strategy to overcome drug-resistance in CRC cells.

Abstract Acquired chemoresistance (ACR) is currently the most important cause of treatment failure and early mortality in DLBCL, arguably the most important but greatest unmet need in lymphoma therapy today. Diffuse Large B cell Lymphoma (DLBCL), the most common human lymphoma, comprises a genetically and clinically diverse group of aggressive B cell non-Hodgkin lymphomas (NHL-B), among a small group of important human cancers increasing in incidence in the US over the last four decades. NHL-B are the fifth most common cancers in the USA (>62,000 new cases/20,000 deaths) expected in 2011. The molecular biologic and genetic basis of the patho-physiology of these important lymphoid tumors is still mostly unresolved. Relapsed/refractory (r/rDLBCL) DLBCL is one of the most difficult scientific challenges and severe unmet therapeutic needs in clinical oncology today. This is due largely to 1.) very drug resistant tumor cells with very poor responses (<20% PR/CR) to current “salvage” therapies; and 2.) non-existent, invalid, or inadequate disease models, that we have recently been able to overcome, with development of valid chemo-refractory DLBCL a unique cell model library, derived from our patients, that now provides the opportunity to delineate critical previously unknown patho-physiologic biomarkers and in vivo molecular model mechanisms, while concurrently providing excellent models for new agent experimental therapeutics. We designed and synthesized novel inhibitors named Turmerax (TMX), when encapsulated with nano-liposomes to overcome solubility/bioavailability issues, is >1000x more effective than curcumin in inhibiting DLBCL growth and survival in vitro, and show no toxicities in normal peripheral blood lymphocytes. Liposomal-TMX (L-TMX) also targets multiple growth and survival signaling pathways, such as NF-kB and AKT, and key cellular regulatory proteins in drug resistance (MDR) in r/r DLBCL. We have also shown in vivo that L-TMX increases in survival time with decreases in lymphoma tumor burden in preliminary studies in SCID/MCL xeno-transplant (XT-SCID) models, without evidence of significant host toxicities. The development of our new agent nano-liposomal Turmerax, has provided a multi-targeted, non-toxic nano-therapeutic small molecule with the critical growth/survival (G/S) targeting specificities similar to natural product therapeutic agents (curcumin) but without the many foibles involving solubility, bioavailability, and poor in vivo potency problems, by providing a well-tolerated, well-delivered (in vivo) effective small molecule therapeutic agent, showing very promising preliminary activities on r/r DLBCL in xeno-transplant human DBLCL models, that appears to be an excellent candidate for future clinical trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A129.

With the goal of developing small molecules as novel regulators of signal transduction and apoptosis, a series of tyrphostin-like compounds were synthesized and screened for their activity against MM-1 (multiple myeloma) cells and other cell lines representing this malignancy. Synthesis was completed in solution-phase initially and then adopted to solid-phase for generating a more diverse set of compounds. A positive correlation was noted between compounds capable of inducing apoptosis and their modulation of protein ubiquitination. Further analysis suggested that ubiquitin modulation occurs through inhibition of cellular deubiquitinase activity. Bulky groups on the sidechain near the α,β-unsaturated ketone caused a complete loss of activity, whereas cyclization on the opposite side was tolerated. Theoretical calculations at the B3LYP/LACV3P(∗∗) level were completed on each molecule, and the resulting molecular orbitals and Fukui reactivity values for C(β) carbon were utilized in developing a model to explain the compound activity.

AG490 is a tyrosine kinase inhibitor with activity against Jak2 and apoptotic activity in specific leukemias. Due to its weak kinase inhibitory activity and poor pharmacology, we conducted a cell-based screen for derivatives with improved Jak2 inhibition and activity in animals. Two hits emerged from an initial small chemical library screen, and more detailed structure–activity relationship studies led to the development of WP1130 with 50-fold greater activity in suppressing Jak2-dependent cytokine signaling than AG490. However, WP1130 did not directly suppress Jak2 kinase activity, but mediated Jak2 ubiquitination resulting in its trafficking through HDAC6 to perinuclear aggresomes without cytokine stimulation or SOCS-1 induction. Jak2 primarily contained K63-linked ubiquitin polymers, and mutation of this lysine blocked Jak2 ubiquitination and mobilization in WP1130-treated cells. Further analysis demonstrated that WP1130, but not AG490, acts as a deubiquitinating enzyme (DUB) inhibitor, possibly through a Michael addition reaction. We conclude that chemical modification of AG490 resulted in development of a DUB inhibitor with activity against a DUB capable of modulating Jak2 ubiquitination, trafficking and signal transduction.

Kit is a membrane-bound tyrosine kinase and receptor for stem cell factor (SCF) with a crucial role in hematopoiesis. Mutations of KIT occur in almost half of patients with core-binding factor leukemias, in which they have been associated with worse outcome. Development of new compounds targeting Kit may therefore hold promise for therapy. We investigated the activity and mechanism of action of APcK110, a novel Kit inhibitor, in the mastocytosis cell line HMC1.2 (KITV560G and KITD816V), acute myeloid leukemia (AML) lines OCIM2 and OCI/AML3 (both wild-type), and primary samples from patients with AML. We show that (a) APcK110 inhibits proliferation of the mastocytosis cell line HMC1.2 and the SCF-responsive cell line OCI/AML3 in a dose-dependent manner; (b) APcK110 is a more potent inhibitor of OCI/AML3 proliferation than the clinically used Kit inhibitors imatinib and dasatinib and at least as potent as cytarabine; (c) APcK110 inhibits the phosphorylation of Kit, Stat3, Stat5, and Akt in a dose-dependent fashion, showing activity of APcK110 on Kit and its downstream signaling pathways; (d) APcK110 induces apoptosis by cleavage of caspase-3 and poly(ADP-ribose) polymerase; and (e) APcK110 inhibits proliferation of primary AML blasts in a clonogenic assay but does not affect proliferation of normal colony-forming cells. Although APcK110 activity may partly depend on cytokine responsiveness (e.g., SCF) and not exclusively KIT mutation status, it remains a potent inhibitor of AML and mastocytosis cell lines and primary AML samples. APcK110 and similar compounds should be evaluated in clinical trials of patients with AML.

Despite the potent anti-proliferative and apoptotic effects of tyrosine kinase inhibitors (imatinib, dasatinib) on mature CML cells these compounds have limited activity on Chronic Myeloid Leukemia (CML) early progenitors (Lin-/CD38−/CD34+). Mechanisms of resistance to these agents in the early progenitor population are poorly understood but are distinct from BCR-ABL mutation induced resistance. Degrasyn (WP1130) was previously shown by our laboratory to inhibit CML cell growth and colony formation. Further structure-activity relationship studies led to the development of a more potent compound, CP2005 (CP). CP displays enhanced activity in suppressing growth and activating apoptosis in various CML and ALL cell lines (IC50 ~1 microM). Here we show that total CD34+ from CML patients are more effectively growth inhibited by CP2005 compared to WP1130. An anti-leukemic effect was also noted in primitive progenitors and was associated with the induction of apoptosis. BaF3 cells expressing an EGFP-tagged BCR-ABL protein treated with CP or WP1130 show that both facilitate translocation of BCR-ABL from the cytoplasm to a detergent insoluble compartment which subsequently leads to degradation of BCR-ABL. We observed that the Akt/mTOR pathway is activated by CP which could be partially suppressed by mTOR or PI3K/Akt inhibition (rapamycin and wortmannin, respectively). CP-induced BCR-ABL translocation was associated with a reduction in the level of tyrosine-phosphorylated Stat3 and Stat5. However, although there is activation of survival signals associated with Akt-signaling 2 microM CP is able to promote significant apoptosis (67%) as a single agent in CML early progenitors. Although imatinib was minimally effective in reducing the early progenitor cell survival, co-incubation of imatinib with CP2005 resulted in near complete induction of apoptosis in early progenitor cells (~ 93%), suggesting that the combined effects of both CP and imatinib on BCR-ABL protein degradation and kinase inhibition markedly impair the survival of CML early progenitors. The combination of BCR-ABL degradation and kinase inhibition may be effective in engaging pro-apoptotic signaling in CML early progenitor cells.

Tumor spheroids more faithfully mimic tumor biology than monolayer cultures and require three-dimensional microscopy. Our goal in this study was to overcome the limitations of signal to noise ratio that have traditionally limited three-dimensional imaging to depths of 100 microm or less. We studied the expression of hypoxia-inducible factor 1alpha (HIF-1alpha), the main regulator of cellular hypoxic response in C6 glioma spheroids. In our spheroids, red fluorescent protein is expressed constitutively and green fluorescent protein is expressed conditionally under control of a HIF-1alpha promoter. In this article, we show a series of optimizations that allowed us to obtain excellent quality confocal microscopy images at imaging depths of up to 320 microm. The combined use of special objectives, glass-bottomed culture dishes, and depth-dependent laser output modulation extended our depth range beyond previously accepted limits. This allowed us to image up to the equator of spheroids of 650 microm diameter, allowing interrogation of HIF-1alpha expression from the spheroid periphery to its hypoxic center.

Enteropathogenic Escherichia coli (EPEC) cause intestinal inflammation, severe diarrhoea and mortality, particularly among children in developing nations. Upon attachment to intestinal epithelial cells, EPEC induces actin-filled membrane protrusions called 'pedestals' and disrupts microvilli to form attaching and effacing (A/E) lesions. EPEC also disrupts epithelial barrier function and causes colitis. Here we have investigated how virulence factors which orchestrate formation of actin pedestals interface with host tyrosine kinases. We show that Tec-family tyrosine kinases localize beneath EPEC and, with Abl-family kinases, comprise a set of redundant host kinases utilized by EPEC to form actin pedestals. We also show that Tir, a virulence factor required for pathogenesis, contains a polyproline region (PPR) that interacts with SH3 domains of redundant kinases, and a phosphorylation site (Y474) that interacts with kinase SH2 domains. These interactions are essential for pedestal formation, and mimic activation of kinases by cellular ligands. Our results suggest that a positive feedback loop exists in which initial phosphorylation of Tir on Y474 by tyrosine kinases causes recruitment of additional redundant kinases via PPR-SH3 interactions and PO(3)-Y474-SH2 interactions, which in turn phosphorylate other Tir molecules as well as proteins that catalyse formation of actin pedestals.