Naima Hammoudi

Mitochondrial dysfunction in some triple-negative breast cancer cell lines: role of mTOR pathway and therapeutic potential.

IntroductionTriple-negative breast cancer (TNBC) is a subtype of highly malignant breast cancer with poor prognosis. TNBC is not amenable to endocrine therapy and often exhibit resistance to current chemotherapeutic agents, therefore, further understanding of the biological properties of these cancer cells and development of effective therapeutic approaches are urgently needed.MethodsWe first investigated the metabolic alterations in TNBC cells in comparison with other subtypes of breast cancer cells using molecular and metabolic analyses. We further demonstrated that targeting these alterations using specific inhibitors and siRNA approach could render TNBC cells more sensitive to cell death compared to other breast cancer subtypes.ResultsWe found that TNBC cells compared to estrogen receptor (ER) positive cells possess special metabolic characteristics manifested by high glucose uptake, increased lactate production, and low mitochondrial respiration which is correlated with attenuation of mTOR pathway and decreased expression of p70S6K. Re-expression of p70S6K in TNBC cells reverses their glycolytic phenotype to an active oxidative phosphorylation (OXPHOS) state, while knockdown of p70S6K in ER positive cells leads to suppression of mitochondrial OXPHOS. Furthermore, lower OXPHOS activity in TNBC cells renders them highly dependent on glycolysis and the inhibition of glycolysis is highly effective in targeting TNBC cells despite their resistance to other anticancer agents.ConclusionsOur study shows that TNBC cells have profound metabolic alterations characterized by decreased mitochondrial respiration and increased glycolysis. Due to their impaired mitochondrial function, TNBC cells are highly sensitive to glycolytic inhibition, suggesting that such metabolic intervention may be an effective therapeutic strategy for this subtype of breast cancer cells.

Metabolic activation of mitochondria in glioma stem cells promotes cancer development through a reactive oxygen species-mediated mechanism

IntroductionCancer stem cells (CSCs) possess characteristics associated with normal stem cells, specifically the abilities to renew themselves and to give rise to all cell types (differentiation). It is assumed that induction of differentiation in CSCs would reduce their ability to form tumors. What triggers CSC differentiation and the role of “differentiation” in tumorigenesis remain elusive.MethodsGlioma stem cell (GSC) lines and subcutaneous as well as orthotopic xenografts established from fresh surgical specimens of glioblastoma multiforme were used.ResultsExposure of GSCs to serum activates mitochondrial respiration and causes an increase in mitochondrial reactive oxygen species (ROS) as well as oxidative stress responses, leading to the appearance of differentiation morphology and a deceased expression of CSC markers. Chemical perturbation of the mitochondrial electron transport chain causes ROS increase and further downregulation of stem cell markers, while antioxidant N-acetyl-cysteine reduces ROS and suppresses the differentiation of GSCs. Surprisingly, the serum-induced differentiated GSCs exhibit greater ability to form tumor in both orthotopic and subcutaneous xenograft models, which can be suppressed by N-acetyl-cysteine. Mitochondrial ROS from the serum-stimulated cells triggered the activation of nuclear factor-kappa-B (NFκB) pathway, which is a potential mechanism for the promotion of tumorigenesis.ConclusionThis study suggests that ROS generated from active mitochondrial respiration in the presence of serum is critical in CSCs activation, which promotes tumor development in vivo.

SF2312 is a natural phosphonate inhibitor of enolase

Despite being critical 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 for the treatment of cancers with deletion of Enolase 1, we modeled the synthetic tool compound inhibitor, Phosphonoacetohydroxamate (PhAH) into the active site of human ENO2. A ring-stabilized analogue of PhAH, with the hydroxamic nitrogen linked to the alpha-carbon 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 back-bone-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 nM inhibitor of Enolase.

ENOblock does not inhibit the activity of the glycolytic enzyme enolase

Inhibition of glycolysis is of great potential for the treatment of cancer. However, inhibitors of glycolytic enzymes with favorable pharmacological profiles have not been forthcoming. Due to the nature of their active sites, most high-affinity transition-state analogue inhibitors of glycolysis enzymes are highly polar with poor cell permeability. A recent publication reported a novel, non-active site inhibitor of the glycolytic enzyme Enolase, termed ENOblock (N-[2-[2-2-aminoethoxy)ethoxy]ethyl]4-4-cyclohexylmethyl)amino]6-4-fluorophenyl)methyl]amino]1,3,5-triazin-2-yl]amino]benzeneacetamide). This would present a major advance, as this is heterocyclic and fully cell permeable molecule. Here, we present evidence that ENOblock does not inhibit Enolase enzymatic activity in vitro as measured by three different assays, including a novel 31P NMR based method which avoids complications associated with optical interferences in the UV range. Indeed, we note that due to strong UV absorbance, ENOblock interferes with the direct spectrophotometric detection of the product of Enolase, phosphoenolpyruvate. Unlike established Enolase inhibitors, ENOblock does not show selective toxicity to ENO1-deleted glioma cells in culture. While our data do not dispute the biological effects previously attributed to ENOblock, they indicate that such effects must be caused by mechanisms other than direct inhibition of Enolase enzymatic activity.

Alterations of mitochondrial biogenesis in chronic lymphocytic leukemia cells with loss of p53

Deletion of chromosome 17p with a loss of p53 is an unfavorable cytogenetic change in chronic lymphocytic leukemia (CLL) with poor clinical outcome. Since p53 affects mitochondrial function and integrity, we examined possible mitochondrial changes in CLL mice with TCL1-Tg/p53-/- and TCL1-Tg/p53+/+ genotypes and in primary leukemia cells from CLL patients with or without 17p-deletion. Although the expression of mitochondrial COX1, ND2, and ND6 decreased in p53-/-CLL cells, there was an increase in mitochondrial biogenesis as evidenced by higher mitochondrial mass and mtDNA copy number associated with an elevated expression of TFAM and PGC-1α. Surprisingly, the overall mitochondrial respiratory activity and maximum reserved capacity increased in p53-/- CLL cells. Our study suggests that leukemia cells lacking p53 seem able to maintain respiratory function by compensatory increase in mitochondrial biogenesis.

Eradication of ENO1-deleted Glioblastoma through Collateral Lethality

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 A39: Pomhex, a cell-permeable high potency enolase inhibitor with utility for collateral lethality treatment of cancer

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 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.

Abstract 4242: Rhodamine esters as fluorescent tumor painting agents for glioblastoma

Proceedings: AACR 107th Annual Meeting 2016; April 16-20, 2016; New Orleans, LA GBM remains one of the most difficult cancers to treat. Despite surgery, radiation and chemotherapy, tumors invariably recur. There is a strong correlation between extend of resection and subsequent time to recurrence and ultimate patient survival. A key challenge for the neurosurgeon is to minimize removal normal brain tissue whilst being as aggressive as possible with regards of removal of tumor tissue. Defining tumor boundary during surgery remains challenging. Fluorescent dyes such as 5- Aminolevulinic acid, have been studied as aids for tumor delineation but have failed to gain widespread acceptance in clinical practice. It has long been known that a substantial number of cancers, including Glioblastoma, exhibit a hyperpolarization of the plasma and mitochondrial membrane potential. This hyperpolarization is manifest by increased retention of lipophilic cationic dyes (Nernstian probes), such as the SPECT-CT probes 99Tc-Sestamibi and 99Tc-Tetrofosmin in both glioma cells in culture and primary tumors. We synthesized a series of ester derivatives of Rhodamine B with high fluorescence quantum yield in the red spectrum and a very favorable biological safety profile. Utilizing a series of intracranial xenografted glioblastoma pre-clinical mouse models, we show that a single I.V. injection of 1 mg/kg fluoroethylrhodamine ester (RhoFe) results in dramatic selective fluorescence in tumor but not surrounding normal brain tissue. This was observed even in tumors with minimal breakdown of the blood brain barrier, as determined by T1-contrast enhancing agents. RhoFe-tumor-selective fluorescence can remain visible up to 72 hours after injection. Similar results were obtained with Tetramethylrhodamine ester (TMRE) and rhodamine 123 (Rho123), but unlike RhoFe, TMRE proved highly toxic, while Rho123 fluorescence was both weaker and in the green spectrum, accompanied by higher endogenous auto-fluorescence. While well tolerated in vivo, RhoFe shows strong photoxicity in cell culture, suggesting potential as a photodynamic therapy agent. Taken together, these data suggest that RhoFe may be a promising tumor painting agent, with potential utility in fluorescence guided surgery as well as photodynamic therapy. Citation Format: Yu-Hsi Lin, Steven Millward, Seth Gammon, Nikunj Satani, Naima Hammoudi, Joshua Philip Gray, Lindsay E. Kelderhouse, Argentina Ornelas, Haley Schroeder, Florian L. Muller. Rhodamine esters as fluorescent tumor painting agents for glioblastoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4242.

Abstract 3837: Passenger deletion of ENO1 as a collateral lethality target in cancer

Large scale genomic characterization efforts such as TCGA have painted an unprecedentedly detailed picture of the genetic alterations that underlie tumorigenesis. Yet, the majority of genetic alterations are passenger rather than driver events and are considered unactionable. We have previously proposed that passenger or collateral deletions could serve as pharmacologically targetable vulnerabilities Collateral Lethality, in case passenger genes are homozygously deleted and member of a paralogous gene family carrying out an essential housekeeping function. We have presented proof-of-principal, whereby passenger deletion of the glycolytic gene Enolase 1 (ENO1) as part of the 1p36-tumor suppressor locus, renders glioma cells harboring such deletions highly sensitive to ablation of its redundant paralogue, ENO2. While our original analysis identified ENO1-homozygous deletions in Glioblastoma (GBM), recent bioinformatics analyzes, backed by immunohistochemistry, show that ENO1-homozygous deletions also occur in Hepatocellular Carcinoma and Cholangiocarcinoma. In GBM, multisector analysis of primary and recurrent tumors, indicate that ENO1 deletion is an early event which is homogenously distributed through the primary tumor and persist during recurrence. To pharmacologically exploit ENO1-deletion, we have pursued two approaches. First, we have synthesized cell-permeable prodrug derivatives of the natural Enolase inhibitor SF2312. The lead compound, POMHEX, shows potent killing of ENO1-deleted glioma cells in the low nM range while ENO1-restored isogenic or normal cells can tolerate μM doses. POMHEX has a short half-life yet can eradicate intracranial xenografted ENO1-deleted tumors, provided extensive breakdown of the blood-brain barrier. Our second approach to targeting ENO1-deletion consisted of chemical biology screening of drug libraries for the ability to kill ENO1-deleted but not isogenic rescued cells. We find that ENO1-deleted cells show a dramatic sensitization to inhibitors of the mitochondrial electron transport chain. These include tool compounds such as rotenone as well as compounds not previously associated with mitochondria, such as Mubritinib and an experimental anti-neoplastic agent previously described as a HIF1-inhibitor, now known to inhibit mitochondrial Complex I. The latter agent shows potent activity against ENO1-deleted intracranial xenografts. The likely cause for this sensitivity is the inability of ENO1-deleted cells to compensatory upregulate glycolysis in response mitochondrial inhibition, the typical response of ENO1-intact glioma cells and normal cells. Together, these data indicate that passenger deletion of ENO1 is an encouraging drug-target and provide support for collateral lethality as a viable therapeutic strategy, which, given the large number of passenger deletions in the cancer genome, may be broadly applicable. Citation Format: Yu-Hsi Lin, Nikunj Satani, Naima Hammoudi, Joe Marszalek, Yuting Sun, Marina Protopopova, Maria E. Di Francesco, Barbara Czako, Alan Wang, Ronald A. DePinho, Florian L. Muller. Passenger deletion of ENO1 as a collateral lethality target in cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3837.

Abstract C183: Pomhex: a cell-permeable high potency Enolase inhibitor with in vivo anti-neoplastic activity

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.