Evangelos Pazarentzos

The Hippo effector YAP promotes resistance to RAF- and MEK-targeted cancer therapies

Resistance to RAF- and MEK-targeted therapy is a major clinical challenge. RAF and MEK inhibitors are initially but only transiently effective in some but not all patients with BRAF gene mutation and are largely ineffective in those with RAS gene mutation because of resistance. Through a genetic screen in BRAF-mutant tumor cells, we show that the Hippo pathway effector YAP (encoded by YAP1) acts as a parallel survival input to promote resistance to RAF and MEK inhibitor therapy. Combined YAP and RAF or MEK inhibition was synthetically lethal not only in several BRAF-mutant tumor types but also in RAS-mutant tumors. Increased YAP in tumors harboring BRAF V600E was a biomarker of worse initial response to RAF and MEK inhibition in patients, establishing the clinical relevance of our findings. Our data identify YAP as a new mechanism of resistance to RAF- and MEK-targeted therapy. The findings unveil the synthetic lethality of combined suppression of YAP and RAF or MEK as a promising strategy to enhance treatment response and patient survival.

AXL Mediates Resistance to PI3Kα Inhibition by Activating the EGFR/PKC/mTOR Axis in Head and Neck and Esophageal Squamous Cell Carcinomas

Phosphoinositide-3-kinase (PI3K)-α inhibitors have shown clinical activity in squamous cell carcinomas (SCCs) of head and neck (H&N) bearing PIK3CA mutations or amplification. Studying models of therapeutic resistance, we have observed that SCC cells that become refractory to PI3Kα inhibition maintain PI3K-independent activation of the mammalian target of rapamycin (mTOR). This persistent mTOR activation is mediated by the tyrosine kinase receptor AXL. AXL is overexpressed in resistant tumors from both laboratory models and patients treated with the PI3Kα inhibitor BYL719. AXL dimerizes with and phosphorylates epidermal growth factor receptor (EGFR), resulting in activation of phospholipase Cγ (PLCγ)-protein kinase C (PKC), which, in turn, activates mTOR. Combined treatment with PI3Kα and either EGFR, AXL, or PKC inhibitors reverts this resistance.

RAS-MAPK dependence underlies a rational polytherapy strategy in EML4-ALK–positive lung cancer

One strategy for combating cancer-drug resistance is to deploy rational polytherapy up front that suppresses the survival and emergence of resistant tumor cells. Here we demonstrate in models of lung adenocarcinoma harboring the oncogenic fusion of ALK and EML4 that the GTPase RAS–mitogen-activated protein kinase (MAPK) pathway, but not other known ALK effectors, is required for tumor-cell survival. EML4-ALK activated RAS-MAPK signaling by engaging all three major RAS isoforms through the HELP domain of EML4. Reactivation of the MAPK pathway via either a gain in the number of copies of the gene encoding wild-type K-RAS (KRASWT) or decreased expression of the MAPK phosphatase DUSP6 promoted resistance to ALK inhibitors in vitro, and each was associated with resistance to ALK inhibitors in individuals with EML4-ALK–positive lung adenocarcinoma. Upfront inhibition of both ALK and the kinase MEK enhanced both the magnitude and duration of the initial response in preclinical models of EML4-ALK lung adenocarcinoma. Our findings identify RAS-MAPK dependence as a hallmark of EML4-ALK lung adenocarcinoma and provide a rationale for the upfront inhibition of both ALK and MEK to forestall resistance and improve patient outcomes.

Adaptive stress signaling in targeted cancer therapy resistance

The identification of specific genetic alterations that drive the initiation and progression of cancer and the development of targeted drugs that act against these driver alterations has revolutionized the treatment of many human cancers. Although substantial progress has been achieved with the use of such targeted cancer therapies, resistance remains a major challenge that limits the overall clinical impact. Hence, despite progress, new strategies are needed to enhance response and eliminate resistance to targeted cancer therapies in order to achieve durable or curative responses in patients. To date, efforts to characterize mechanisms of resistance have primarily focused on molecular events that mediate primary or secondary resistance in patients. Less is known about the initial molecular response and adaptation that may occur in tumor cells early upon exposure to a targeted agent. Although understudied, emerging evidence indicates that the early adaptive changes by which tumor cells respond to the stress of a targeted therapy may be crucial for tumo r cell survival during treatment and the development of resistance. Here we review recent data illuminating the molecular architecture underlying adaptive stress signaling in tumor cells. We highlight how leveraging this knowledge could catalyze novel strategies to minimize or eliminate targeted therapy resistance, thereby unleashing the full potential of targeted therapies to transform many cancers from lethal to chronic or curable conditions.

Dose-dependent Neuroprotection of VEGF165 in Huntington's Disease Striatum

Huntingtons disease (HD) is a devastating neurodegenerative disorder caused by abnormal polyglutamine expansion in the huntingtin protein (Exp-Htt). Currently, there are no effective treatments for HD. We used bidirectional lentiviral transfer vectors to generate in vitro and in vivo models of HD and to test the therapeutic potential of vascular endothelial growth factor 165 (VEGF₁₆₅). Lentiviral-mediated expression of Exp-Htt caused cell death and aggregate formation in human neuroblastoma SH-SY5Y and rat primary striatal cultures. Lentiviral-mediated VEGF₁₆₅ expression was found to be neuroprotective in both of these models. Unilateral stereotaxic vector delivery of Exp-Htt vector in adult rat striatum led to progressive inclusion formation and striatal neuron loss at 10 weeks post-transduction. Coinjection of a lower dose VEGF₁₆₅ significantly attenuated DARPP-32(+) neuronal loss, enhanced NeuN staining and reduced Exp-Htt aggregation. A tenfold higher dose VEGF₁₆₅ led to overt neuronal toxicity marked by tissue damage, neovascularization, extensive astrogliosis, vascular leakage, chronic inflammation and distal neuronal loss. No overt behavioral phenotype was observed in these animals. Expression of VEGF₁₆₅ at this higher dose in the brain of wild-type rats led to early mortality with global neuronal loss. This report raises important safety concerns about unregulated VEGF₁₆₅ CNS applications.Huntingtons disease (HD) is a devastating neurodegenerative disorder caused by abnormal polyglutamine expansion in the huntingtin protein (Exp-Htt). Currently, there are no effective treatments for HD. We used bidirectional lentiviral transfer vectors to generate in vitro and in vivo models of HD and to test the therapeutic potential of vascular endothelial growth factor 165 (VEGF165). Lentiviral-mediated expression of Exp-Htt caused cell death and aggregate formation in human neuroblastoma SH-SY5Y and rat primary striatal cultures. Lentiviral-mediated VEGF165 expression was found to be neuroprotective in both of these models. Unilateral stereotaxic vector delivery of Exp-Htt vector in adult rat striatum led to progressive inclusion formation and striatal neuron loss at 10 weeks post-transduction. Coinjection of a lower dose VEGF165 significantly attenuated DARPP-32+ neuronal loss, enhanced NeuN staining and reduced Exp-Htt aggregation. A tenfold higher dose VEGF165 led to overt neuronal toxicity marked by tissue damage, neovascularization, extensive astrogliosis, vascular leakage, chronic inflammation and distal neuronal loss. No overt behavioral phenotype was observed in these animals. Expression of VEGF165 at this higher dose in the brain of wild-type rats led to early mortality with global neuronal loss. This report raises important safety concerns about unregulated VEGF165 CNS applications.

Anticancer Gene Transfer for Cancer Gene Therapy

Gene therapy vectors are among the treatments currently used to treat malignant tumors. Gene therapy vectors use a specific therapeutic transgene that causes death in cancer cells. In early attempts at gene therapy, therapeutic transgenes were driven by non-specific vectors which induced toxicity to normal cells in addition to the cancer cells. Recently, novel cancer specific viral vectors have been developed that target cancer cells leaving normal cells unharmed. Here we review such cancer specific gene therapy systems currently used in the treatment of cancer and discuss the major challenges and future directions in this field.

Novel computational method for predicting polytherapy switching strategies to overcome tumor heterogeneity and evolution

The success of targeted cancer therapy is limited by drug resistance that can result from tumor genetic heterogeneity. The current approach to address resistance typically involves initiating a new treatment after clinical/radiographic disease progression, ultimately resulting in futility in most patients. Towards a potential alternative solution, we developed a novel computational framework that uses human cancer profiling data to systematically identify dynamic, pre-emptive, and sometimes non-intuitive treatment strategies that can better control tumors in real-time. By studying lung adenocarcinoma clinical specimens and preclinical models, our computational analyses revealed that the best anti-cancer strategies addressed existing resistant subpopulations as they emerged dynamically during treatment. In some cases, the best computed treatment strategy used unconventional therapy switching while the bulk tumor was responding, a prediction we confirmed in vitro. The new framework presented here could guide the principled implementation of dynamic molecular monitoring and treatment strategies to improve cancer control.

Abstract 1713: PDK1 and hexokinase 2 are downstream effectors of PTEN loss and regulate response to targeted therapies in multiple tumor types

Recent advances in molecular profiling of many human tumor types has enabled the development and clinical use of molecularly-targeted therapies in patients. Many tumors exhibit inactivation of PTEN which is predicted to sensitize tumor cells to PI3 kinase or AKT inhibitor therapy. However, clinical responses to PI3 kinase or AKT inhibitors are variable and not curative. Furthermore, these responses in patients do not correlate uniformly with PTEN inactivation. Therefore, we investigated whether PTEN inactivation could lead to dependence on signaling pathways and components that function independently of PI3K or AKT and that could serve as novel therapeutic targets in PTEN deficient tumors. Using multiple human tumor models including lung adenocarcinoma, lung squamous, renal cell carcinoma, and prostate adenocarcinoma that express or lack PTEN and Gene Set Enrichment Analysis, we identified alterations in numerous metabolic regulatory genes in response to PTEN inactivation, including hexokinase 2 (HK2) and pyruvate dehydrogenase kinase 1 (PDK1). We found that in PTEN-deficient tumor cells HK2 is upregulated and selectively localizes to the mitochondria, where it enhances glycolytic flux and inhibits apoptosis. Furthermore, we found that PDK1 is also upregulated upon PTEN inactivation and suppresses oxidative phosphorylation, upregulating glycolysis and the production of lactate. Thus, we uncovered a novel role for HK2 and PDK1 in the metabolic reprogramming that occurs as a consequence of PTEN inactivation in tumor cells. Furthermore, we found that overexpression of either HK2 or PDK1 confers resistance to targeted therapies against PI3K and other oncogenic drivers more broadly. Conversely, genetic or pharmacological suppression of HK2 or PDK1 enhances response to targeted therapy in multiple tumor cell types. Together, our data identify HK2 and PDK1 as novel molecular biomarkers and promising therapeutic targets in multiple PTEN deficient tumor types. Citation Format: Evangelos Pazarentzos, Trever G. Bivona. PDK1 and hexokinase 2 are downstream effectors of PTEN loss and regulate response to targeted therapies in multiple tumor types. [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 1713. doi:10.1158/1538-7445.AM2014-1713

Abstract 1836: Erlotinib induces NF-kappa B dependence that promotes EGFR tyrosine kinase inhibitor resistance in lung adenocarcinoma

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The vast majority of patients with lung adenocarcinomas harboring activating mutation in EGFR respond to EGFR tyrosine inhibitors (TKI) (i.e. erlotinib). However, the magnitude of tumor regression is variable and responses are short-lived with a median duration of 9-12 months. We recently identified activation of the NF-κB (Nuclear Factor kappa-light-chain-enhancer of activated B cells) signaling pathway as a key mediator of de novo resistance to EGFR TKI therapy in cell lines and tumor xenograft models of lung adenocarcinoma. However, the role of NF-κB activation in mediating EGFR TKI acquired resistance in lung adenocarcinoma is not well defined. We have found that erlotinib treatment of EGFR TKI sensitive lung adenocarcinoma cell lines promotes the rapid phosphorylation and degradation of IκB (inhibitor of NF-κB) and subsequent activation of the NF-κB subunit RelA. RelA activation is accompanied by transcriptional activation of downstream NF-κB target genes, including IL6. Prolonged exposure of EGFR TKI sensitive lung adenocarcinoma cell lines to erlotinib leads to EGFR TKI acquired resistance that is accompanied by sustained NF-κB activation and IL6 expression. EGFR TKI acquired resistance can be overcome by treating cells with erlotinib in combination with PBS-1086 (rel∼MD, Inc.), a direct Rel inhibitor. Furthermore, concomitant treatment of EGFR TKI sensitive lung adenocarcinoma cells with erlotinib + PBS-1086 prevents the development of EGFR TKI acquired resistance. Together, these results demonstrate the molecular basis for the synthetic lethality of combined EGFR and NF-κB inhibition and provide mechanism-based rationale for polytherapies against both EGFR and NF-κB to enhance response in lung adenocarcinoma patients. Broadly, our findings provide novel insights into the biological and clinical consequences of the context-specific and dynamic functional interplay between EGFR and NF-κB signaling. Citation Format: Collin M. Blakely, Evangelos Pazarentzos, Saurabh Asthana, Victor Olivas, Irena Tan, Timothy Fouts, Jeffrey Meshulam, Trever G. Bivona. Erlotinib induces NF-kappa B dependence that promotes EGFR tyrosine kinase inhibitor resistance in lung adenocarcinoma. [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 1836. doi:10.1158/1538-7445.AM2014-1836

Synthetic essentiality of metabolic regulator PDHK1 in PTEN-deficient cells and cancers

PTEN is a tumor suppressor that is often inactivated in cancer and possesses both lipid and protein phosphatase activities. We report the metabolic regulator PDHK1 (pyruvate dehydrogenase kinase1) is a synthetic-essential gene in PTEN-deficient cancer and normal cells. The predominant mechanism of PDHK1 regulation and dependency is the PTEN protein phosphatase dephosphorylates NFκ;B activating protein (NKAP) and limits NFκB activation to suppress expression of PDHK1, a NFκB target gene. Loss of the PTEN protein phosphatase upregulates PDHK1 to drive aerobic glycolysis and induce PDHK1 cellular dependence. PTEN-deficient human tumors harbor increased PDHK1, which is a biomarker of decreased patient survival, establishing clinical relevance. This study uncovers a PTEN-regulated signaling pathway and reveals PDHK1 as a potential target in PTEN-deficient cancers. SIGNIFICANCE The tumor suppressor PTEN is widely inactivated in cancers and tumor syndromes. PTEN antagonizes PI3K/AKT signaling via its lipid phosphatase activity. The modest success of PI3K/AKT inhibition in PTEN-deficient cancer patients provides rationale for identifying other vulnerabilities in PTEN-deficient cancers to improve clinical outcomes. We show that PTEN-deficient cells are uniquely sensitive to PDHK1 inhibition. PTEN and PDHK1 co-suppression reduced colony formation and induced cell death in vitro and tumor regression in vivo. PDHK1 levels were high in PTEN-deficient patient tumors and associated with inferior patient survival, establishing clinical relevance. Our study identifies a PTEN-regulated signaling pathway linking the PTEN protein phosphatase to the metabolic regulator PDHK1 and provides a mechanistic basis for PDHK1 targeting in PTEN-deficient cancers.