Maria Emilia Di Francesco

Genetic Events That Limit the Efficacy of MEK and RTK Inhibitor Therapies in a Mouse Model of KRAS-Driven Pancreatic Cancer

Mutated KRAS (KRAS*) is a fundamental driver in the majority of pancreatic ductal adenocarcinomas (PDAC). Using an inducible mouse model of KRAS*-driven PDAC, we compared KRAS* genetic extinction with pharmacologic inhibition of MEK1 in tumor spheres and in vivo. KRAS* ablation blocked proliferation and induced apoptosis, whereas MEK1 inhibition exerted cytostatic effects. Proteomic analysis evidenced that MEK1 inhibition was accompanied by a sustained activation of the PI3K-AKT-MTOR pathway and by the activation of AXL, PDGFRa, and HER1-2 receptor tyrosine kinases (RTK) expressed in a large proportion of human PDAC samples analyzed. Although single inhibition of each RTK alone or plus MEK1 inhibitors was ineffective, a combination of inhibitors targeting all three coactivated RTKs and MEK1 was needed to inhibit proliferation and induce apoptosis in both mouse and human low-passage PDAC cultures. Importantly, constitutive AKT activation, which may mimic the fraction of AKT2-amplified PDAC, was able to bypass the induction of apoptosis caused by KRAS* ablation, highlighting a potential inherent resistance mechanism that may inform the clinical application of MEK inhibitor therapy. This study suggests that combinatorial-targeted therapies for pancreatic cancer must be informed by the activation state of each putative driver in a given treatment context. In addition, our work may offer explanative and predictive power in understanding why inhibitors of EGFR signaling fail in PDAC treatment and how drug resistance mechanisms may arise in strategies to directly target KRAS.

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.

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 3016: Identification of protein arginine methyltransferase 1 as novel epigenetic vulnerability in KRAS/p53 mutant PDAC primary patient models

Pancreatic ductal adenocarcinoma (PDAC) is a rapidly progressing disease associated with less than 10% 5-year survival rate. Various treatment regimens failed to improve survival of PDAC patients, thus a critical need exists to identify druggable targets essential for tumor maintenance. We developed a powerful in vivo platform that enables the identification of new molecular drivers in the PDAC context where activating mutation of KRAS gene and loss of p53 dominate the genetic landscape. Through an in vivo loss of function screen performed in KRAS/p53 mutant PDAC primary patient models, we identify protein arginine methyltransferase 1 (PRMT1) as top scoring hit. This novel dependency in PDAC was subsequently validated in multiple PDAC models using both shRNA mediated as well as CRISPR base genetic inhibition and we demonstrated that PRMT1 knockdown induces a significant growth inhibition in vitro. Methylation of arginine 3 on histone H4 (H4R3me2a) as well as global arginine methylation was also evaluated and showed a dramatic reduction upon PRMT1 knockdown, correlating observed phenotype with target engagement. To further confirm a role for PRMT1 in tumor maintenance, we developed inducible PRMT1 knockdown in a primary patient model and showed a dramatic tumor growth inhibition (TGI) in vivo upon PRMT1 knockdown. PRMT1 is the primary enzyme responsible for arginine asymmetric demethylation, however other members of the Type I family are also involved in this process and we evaluated the role of protein arginine methyltransferase 4 (PRMT4) and 6 (PRMT6) in our workhorse model. Surprisingly, no significant phenotypic response was observed upon genetic inhibition of PRMT4 or PRMT6 suggesting no redundancy between different PRMT type I and a unique dependency on PRMT1. To strengthen and complement the genetic validation, we leveraged a PRMT Type I inhibitor and confirmed in vitro results as well as in vivo efficacy at tolerated doses (xenograft vs allograft). Key models have been prioritized in order to inform on PRMT1 dependency and to refine responder population. Our research has identified and validated for the first time an arginine methyltransferase as a novel genetic vulnerability in PDAC and strongly suggest PRMT1 as a new therapeutic opportunity in PDAC cancers. Citation Format: Virginia Giuliani, Bhavatarini Vangamudi, Erika Suzuki, Meredith Miller, Chiu-Yi Liu, Alessandro Carugo, Christopher Bristow, Guang Gao, Jing Han, Yuting Sun, Ningping Feng, Edward Chang, Joseph Marszalek, Jeffrey Kovacs, Maria Emilia Di Francesco, Carlo Toniatti, Timothy Heffernan, Philip Jones, Giulio Draetta. Identification of protein arginine methyltransferase 1 as novel epigenetic vulnerability in KRAS/p53 mutant PDAC primary patient models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3016. doi:10.1158/1538-7445.AM2017-3016

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 PL07-01: Altered metabolism in leukemic microenvironment

Interactions of leukemia cells and their bone marrow (BM) microenvironment are known to play a key role in the survival and growth of leukemic cells. It has been postulated that specific niches provide a sanctuary where subpopulations of leukemic cells evade chemotherapy-induced death and acquire a drug-resistant phenotype. Understanding the cellular and molecular biology of the leukemia stem cell (LSC) niche and of microenvironment/leukemia interactions may provide new targets that allow destruction of LSCs without adversely affecting normal stem cell self-renewal. Key emerging therapeutic targets include chemokine receptors such as CXCR4 and hypoxia-related proteins, as well as the metabolic abnormalities of the leukemia-associated stroma. We have recently reported that CXCR4 inhibition causes leukemia cell dislodgement from CXCL12-producing marrow niches, reduced proliferation and induction of differentiation of AML cells in an in vivo model of AML, translating into pronounced anti-leukemia effects. Studies in murine leukemia models using the hypoxia probe pimonidazole demonstrated extensive areas of hypoxia within leukemic, but not healthy normal, bone marrow. Time-course analysis of bone marrow spaces within calvaria and femurs by multiphoton intravital microscopy (MP-IVM) demonstrated lodging of p190-Bcr/Abl tdTomato cells in close proximity to blood vessels, followed by accumulation of leukemia cells localized within the sinusoidal and marrow spaces resulting in the demise of the animals within 3 weeks. In this model, pimonidazole detected hypoxic areas despite abundant vascular supply in the marrow cavities. In vivo magnetic resonance imaging with hyperpolarized pyruvate showed higher pyruvate-lactate conversion (high glycolytic flux) in leukemic marrows. These findings were supported by significant pimonidazole uptake by the diseased bone marrow in patients with acute leukemia, causing stabilization of HIF-1α in 55% (76/138) of primary AML patients and of its target CA9. Paradoxically, AML cells become highly dependent on mitochondrial oxidative phosphorylation (OXPHOS) for their survival, and inhibition of OXPHOS with the novel small molecule complex I inhibitor IACS-10759 inhibits oxygen consumption, eliminates hypoxia in vivo and inhibits AML growth. These findings suggest that altered tumor metabolism underlies the hypoxia observed in leukemias. We postulate that the altered tumor microenvironment within the hypoxic niche cells will influence leukemia development and response to therapy. Hence, targeting key metabolic alterations should be considered in the armamentarium of anti-AML therapies. IACS-10759 is presently completing IND enabling safety/toxicity studies with first in human studies targeting relapsed/refractory AML planned for 2016. Citation Format: Marina Y. Konopleva, Tomasz Zal, Niki M. Zacharias Millward, Byoung-Sik Cho, Karine Harutyunyan, Anna Zal, Hong Mu, Sergej Konoplev, Juliana Benito, Juliana Velez, Carlos Bueso-Ramso, Jennifer Molina, Pratip K. Bhattacharya, Maria Emilia Di Francesco, Joseph Marszalek, Michael Andreeff. Altered metabolism in leukemic microenvironment. [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 PL07-01.

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.

Abstract 976: Metabolic eradication of treatment resistant cancer stem cells in pancreatic tumors: A clonal tracking-based platform for identifying the best personalized treatment

Pancreatic ductal adenocarcinoma (PDAC) continues to have a poor prognosis despite new drugs advancing to the clinic. We recently characterized a population of cells able to survive the genetic and pharmacologic extinction of oncogenic pathways and demonstrated that the surviving cells (SC) are tumor stem cells (CSC) able to remain in a quiescent state for months before relapsing. In-depth transcriptomics and metabolomics analyses revealed SCs to exhibit different metabolic features compared to the bulk of tumor cells. Specifically, SC relied on mitochondrial respiration (OXPHOS) and displayed impaired glycolytic capacity. In accord with their decreased dependence on glycolysis these SC were highly sensitive to OXPHOS inhibitors, which prevented tumor recurrence. Notably, preliminary results in patients suggest that SC resistant to conventional chemoradiation also have similar features. We analyzed specimens after neoadjuvant treatment and observed dormant cells positive for CSC markers and characterized by an increased mitochondrial mass. Evaluation of TMA of hundreds of treated tumors revealed that high mitochondrial content is a common feature of SC. Furthermore, functional metabolic characterization of human cells resistant to gemcitabine revealed that SC have a severely impaired glycolytic reserve, closely resembling mouse SC. Based on these findings, we posit that OXPHOS inhibitors may be an effective adjuvant therapy to eradicate resistant cells in patients. To validate the efficacy of OXPHOS inhibition, we developed a new platform to study tumor evolution in response to treatments based on clonal tracking. Lentivirus-based systems have been extensively used as a tool to investigate the clonal dynamics, but they have been limited by lack of sensitivity and the impossibility of tracking identical clones in different animals. Here, using a new version of the barcoded technology coupled with deep-sequencing, we track hundreds of thousands of clones at the single-cell level. We generated patient-derived xenograft animal cohorts in which tumors were clonal replicas of each other (each tumor is maintained by the same clones of all other tumors), representing a unique tool to evaluate responses to treatments. More importantly, resistant tumor clones generated in vivo can be isolated and fully characterized and compared to pre-treatment clones to identify new mechanisms of resistance. Our integrated analysis paves the way for new therapeutic strategies for patients to eradicate treatment-resistant CSC by specifically targeting their unique metabolism. In addition, our clonal tracking-based platform monitoring the efficacy of different treatments in eradicating resistant clones represents an unprecedented tool for exploring treatment responses at the single-cell level, which will help guide the development of personalized treatments. Citation Format: Denise Corti, Alessandro Carugo, Seth Sahil, Matteo Marchesini, Piergiorgio Pettazzoni, Luigi Nezi, Tessa Green, Joseph R Marszalek, Maria Emilia Di Francesco, Timothy P Heffernan, Giulio F Draetta, Andrea Viale. Metabolic eradication of treatment resistant cancer stem cells in pancreatic tumors: A clonal tracking-based platform for identifying the best personalized treatment. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 976. doi:10.1158/1538-7445.AM2015-976