Luping Lin

Activation of the AXL kinase causes resistance to EGFR-targeted therapy in lung cancer

Human non–small cell lung cancers (NSCLCs) with activating mutations in EGFR frequently respond to treatment with EGFR-targeted tyrosine kinase inhibitors (TKIs), such as erlotinib, but responses are not durable, as tumors acquire resistance. Secondary mutations in EGFR (such as T790M) or upregulation of the MET kinase are found in over 50% of resistant tumors. Here, we report increased activation of AXL and evidence for epithelial-to-mesenchymal transition (EMT) in multiple in vitro and in vivo EGFR-mutant lung cancer models with acquired resistance to erlotinib in the absence of the EGFR p.Thr790Met alteration or MET activation. Genetic or pharmacological inhibition of AXL restored sensitivity to erlotinib in these tumor models. Increased expression of AXL and, in some cases, of its ligand GAS6 was found in EGFR-mutant lung cancers obtained from individuals with acquired resistance to TKIs. These data identify AXL as a promising therapeutic target whose inhibition could prevent or overcome acquired resistance to EGFR TKIs in individuals with EGFR-mutant lung cancer.

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.

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.

Mechanisms of Resistance to Epidermal Growth Factor Receptor Inhibitors and Novel Therapeutic Strategies to Overcome Resistance in NSCLC Patients.

The epidermal growth factor receptor (EGFR) is a well-characterized oncogene that is frequently activated by somatic kinase domain mutations in non-small cell lung cancer (NSCLC). EGFR TKIs are effective therapies for NSCLC patients whose tumors harbor an EGFR activating mutation. However, EGFR TKI treatment is not curative in patients because of both primary and secondary treatment resistance. Studies over the last decade have identified mechanisms that drive primary and secondary resistance to EGFR TKI treatment. The elucidation of mechanisms of resistance to EGFR TKI treatment provides a basis for the development of therapeutic strategies to overcome resistance and enhance outcomes in NSCLC patients. In this paper, we summarize the mechanisms of resistance to EGFR TKIs that have been identified to date and discusses potential therapeutic strategies to overcome EGFR TKI resistance in NSCLC patients.

Mapping the molecular determinants of BRAF oncogene dependence in human lung cancer

Significance Oncogenic mutations in the BRAF kinase occur in 6–8% of nonsmall cell lung cancers (NSCLCs), but the biological and clinical relevance of these mutations is unclear. We uncovered mechanisms of resistance to BRAF inhibition in NSCLC using an integrated functional chemical genetics approach in human BRAF-mutant NSCLC cells and clinical specimens. Our results provide biological insights into the regulation of BRAF oncogene dependence and identify strategies to optimize outcomes in BRAF-mutant NSCLC patients. Oncogenic mutations in the BRAF kinase occur in 6–8% of nonsmall cell lung cancers (NSCLCs), accounting for more than 90,000 deaths annually worldwide. The biological and clinical relevance of these BRAF mutations in NSCLC is incompletely understood. Here we demonstrate that human NSCLC cells with BRAFV600E, but not other BRAF mutations, initially are sensitive to BRAF-inhibitor treatment. However, these BRAFV600E NSCLC cells rapidly acquire resistance to BRAF inhibition through at least one of two discrete molecular mechanisms: (i) loss of full-length BRAFV600E coupled with expression of an aberrant form of BRAFV600E that retains RAF pathway dependence or (ii) constitutive autocrine EGF receptor (EGFR) signaling driven by c-Jun–mediated EGFR ligand expression. BRAFV600E cells with EGFR-driven resistance are characterized by hyperphosphorylated protein kinase AKT, a biomarker we validated in BRAF inhibitor-resistant NSCLC clinical specimens. These data reveal the multifaceted molecular mechanisms by which NSCLCs establish and regulate BRAF oncogene dependence, provide insights into BRAF–EGFR signaling crosstalk, and uncover mechanism-based strategies to optimize clinical responses to BRAF oncogene inhibition.

The Hippo effector YAP regulates the response of cancer cells to MAPK pathway inhibitors

RAF- and MEK-targeted therapies are approved for patients with BRAFV600E melanoma and under investigation in a several other tumor types, but resistance remains a major challenge. We uncovered yes-associated protein 1 (YAP1) as a mechanism of resistance to RAF-MEK inhibition in BRAF- and RAS-mutant cancers, providing a rationale for co-targeting YAP and RAF-MEK to enhance patient outcomes.

Preclinical efficacy of a RAF inhibitor that evades paradoxical MAPK pathway activation in protein kinase BRAF-mutant lung cancer

Significance Oncogenic mutations in the BRAF protein kinase occur in a large number of patients with lung cancer, the number one cause of cancer mortality worldwide. Despite the relatively high frequency (2–7%) of various oncogenic BRAF alleles in human lung adenocarcinoma (LA), the biological and clinical relevance of these mutations and the optimal therapeutic strategy to improve outcomes remains poorly defined. We report the preclinical efficacy of a next-generation BRAF kinase inhibitor across a spectrum of clinically relevant BRAF-mutant nonsmall cell lung cancer (NSCLC) models. Our results provide biological insight into the regulation of BRAF oncogene dependence and identify strategies to improve outcomes in patients with BRAF-mutant lung cancer. Oncogenic activation of protein kinase BRAF drives tumor growth by promoting mitogen-activated protein kinase (MAPK) pathway signaling. Because oncogenic mutations in BRAF occur in ∼2–7% of lung adenocarcinoma (LA), BRAF-mutant LA is the most frequent cause of BRAF-mutant cancer mortality worldwide. Whereas most tumor types harbor predominantly the BRAFV600E-mutant allele, the spectrum of BRAF mutations in LA includes BRAFV600E (∼60% of cases) and non-V600E mutant alleles (∼40% of cases) such as BRAFG469A and BRAFG466V. The presence of BRAFV600E in LA has prompted clinical trials testing selective BRAF inhibitors such as vemurafenib in BRAFV600E-mutant patients. Despite promising clinical efficacy, both innate and acquired resistance often result from reactivation of MAPK pathway signaling, thus limiting durable responses to the current BRAF inhibitors. Further, the optimal therapeutic strategy to block non-V600E BRAF-mutant LA remains unclear. Here, we report the efficacy of the Raf proto-oncogene serine/threonine protein kinase (RAF) inhibitor, PLX8394, that evades MAPK pathway reactivation in BRAF-mutant LA models. We show that PLX8394 treatment is effective in both BRAFV600E and certain non-V600 LA models, in vitro and in vivo. PLX8394 was effective against treatment-naive BRAF-mutant LAs and those with acquired vemurafenib resistance caused by an alternatively spliced, truncated BRAFV600E that promotes vemurafenib-insensitive MAPK pathway signaling. We further show that acquired PLX8394 resistance occurs via EGFR-mediated RAS-mTOR signaling and is prevented by upfront combination therapy with PLX8394 and either an EGFR or mTOR inhibitor. Our study provides a biological rationale and potential polytherapy strategy to aid the deployment of PLX8394 in lung cancer patients.

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.

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.

Abstract 920: COP1 E3 ligase regulates response to oncogenic MAPK pathway inhibition

Oncogenically activated RAS-MAPK pathway is the driver of several cancers including the majority of non-small cell lung adenocarcinomas (NSCLC). RAS-MAPK pathway is activated in NSCLC tumors via diverse genetic alterations in upstream receptor tyrosine kinases such as EGFR and ALK as well as in RAS, BRAF, MEK and RAS GTPase activating protein (GAP) and tumor suppressor, NF1. Molecular targeting of players of RAS-MAPK pathway can elicit an initial tumor response in many patients. However, most patients show an incomplete response and some fail to respond despite the presence of RAS-MAPK pathway activating genetic lesion in the tumor. Hence, it is important to understand the molecular basis of response and resistance to targeted therapies inhibiting the RAS-MAPK pathway in lung adenocarcinomas in order to improve patient survival. We conducted a genetic screen to identify regulators of response to MAPK pathway inhibition in lung adenocarcinomas. Our genetic screen uncovered the E3 ubiquitin ligase COP1/RFWD2 as a novel modulator of response to inhibition of RAS-MAPK pathway. We found that depletion of COP1 and members of its complex, as well as proteasomal subunits, confers resistance to RAS-MAPK pathway inhibition in human lung adenocarcinoma cells with oncogenically activated RAS-MAPK pathway. Interestingly, oncogenic targets of COP1 include MAPK pathway effectors, presence of which has been shown to support the survival of cancer cells with oncogenically activated MAPK upon inhibition of the RAS-MAPK pathway. Hence, we tested if depletion of COP1 alters the levels of those oncogenic substrates. Excitingly, we observed a substantial impact of COP1 depletion on the levels of certain effectors in the presence of RAS-MAPK inhibitors in genetically diverse NSCLC cells and also in oncogenic-BRAF driven melanoma cells. Our studies suggests that depletion of COP1 confers resistance to MAPK pathway inhibition in RAS-MAPK pathway driven cancers through accumulation of specific MAPK pathway effectors. This work has improved our understanding of the molecular basis of tumor cell resilience during initial treatment as well as of primary treatment resistance. Additionally, we are examining if levels of COP1 could be a biomarker for predicting response to RAS-MAPK pathway inhibitor therapy. We will also determine if the resistance-conferring COP1 substrate could be cotargeted along with RAS-MAPK pathway to improve the patient response in RAS-MAPK pathway driven lung adenocarcinomas and other cancers with low levels of COP1. Citation Format: Manasi K. Mayekar, Luping Lin, Trever G. Bivona. COP1 E3 ligase regulates response to oncogenic MAPK pathway inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 920.