Victor Olivas

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.

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.

ApoE Suppresses Atherosclerosis by Reducing Lipid Accumulation in Circulating Monocytes and the Expression of Inflammatory Molecules on Monocytes and Vascular Endothelium

Objective—We investigated atheroprotective properties of apolipoprotein (apo) E beyond its ability to lower plasma cholesterol. We hypothesized that apoE reduces atherosclerosis by decreasing lipid accumulation in circulating monocytes and the inflammatory state of monocytes and the vascular endothelium. Methods and Results—We developed mice with spontaneous hyperlipidemia with and without plasma apoE. Hypomorphic apoE mice deficient in low-density lipoprotein receptor (Apoeh/hLdlr−/−) were compared to Apoe−/−Ldlr−/− mice. Despite 4-fold more plasma apoE than WT mice, Apoeh/hLdlr−/− mice displayed similar plasma cholesterol as Apoe−/− Ldlr−/− mice but developed 4-fold less atherosclerotic lesions by 5 months of age. The aortic arch of Apoeh/hLdlr−/− mice showed decreased endothelial expression of ICAM-1, PECAM-1, and JAM-A. In addition, Apoeh/hLdlr−/− mice had less circulating leukocytes and proinflammatory Ly6Chigh monocytes. These monocytes had decreased neutral lipid content and reduced surface expression of ICAM-1, VLA-4, and L-Selectin. Apoeh/hLdlr−/− mice displayed increased levels of apoA1-rich HDL that were potent in promoting cellular cholesterol efflux. Conclusions—Our findings suggest that apoE reduces atherosclerosis in the setting of hyperlipidemia by increasing plasma apoA1-HDL that likely contribute to reduce intracellular lipid accumulation and thereby the activation of circulating leukocytes and the vascular endothelium.

Inactivation of Capicua drives cancer metastasis

Metastasis is the leading cause of death in people with lung cancer, yet the molecular effectors underlying tumor dissemination remain poorly defined. Through the development of an in vivo spontaneous lung cancer metastasis model, we show that the developmentally regulated transcriptional repressor Capicua (CIC) suppresses invasion and metastasis. Inactivation of CIC relieves repression of its effector ETV4, driving ETV4-mediated upregulation of MMP24, which is necessary and sufficient for metastasis. Loss of CIC, or an increase in levels of its effectors ETV4 and MMP24, is a biomarker of tumor progression and worse outcomes in people with lung and/or gastric cancer. Our findings reveal CIC as a conserved metastasis suppressor, highlighting new anti-metastatic strategies that could potentially improve patient outcomes.

Evolution and clinical impact of co-occurring genetic alterations in advanced-stage EGFR-mutant lung cancers

A widespread approach to modern cancer therapy is to identify a single oncogenic driver gene and target its mutant-protein product (for example, EGFR-inhibitor treatment in EGFR-mutant lung cancers). However, genetically driven resistance to targeted therapy limits patient survival. Through genomic analysis of 1,122 EGFR-mutant lung cancer cell-free DNA samples and whole-exome analysis of seven longitudinally collected tumor samples from a patient with EGFR-mutant lung cancer, we identified critical co-occurring oncogenic events present in most advanced-stage EGFR-mutant lung cancers. We defined new pathways limiting EGFR-inhibitor response, including WNT/β-catenin alterations and cell-cycle-gene (CDK4 and CDK6) mutations. Tumor genomic complexity increases with EGFR-inhibitor treatment, and co-occurring alterations in CTNNB1 and PIK3CA exhibit nonredundant functions that cooperatively promote tumor metastasis or limit EGFR-inhibitor response. This study calls for revisiting the prevailing single-gene driver-oncogene view and links clinical outcomes to co-occurring genetic alterations in patients with advanced-stage EGFR-mutant lung cancer.

Apolipoprotein E4 Domain Interaction Accelerates Diet-Induced Atherosclerosis in Hypomorphic Arg-61 Apoe Mice

Objectives—Apolipoprotein (apo) E4 is an established risk factor for atherosclerosis, but the structural components underlying this association remain unclear. ApoE4 is characterized by 2 biophysical properties: domain interaction and molten globule state. Substituting Arg-61 for Thr-61 in mouse apoE introduces domain interaction without molten globule state, allowing us to delineate potential proatherogenic effects of domain interaction in vivo. Methods and Results—We studied atherosclerosis susceptibility of hypomorphic Apoe mice expressing either Thr-61 or Arg-61 apoE (ApoeTh/h or ApoeRh/hmice). On a chow diet, both mouse models were normolipidemic with similar levels of plasma apoE and lipoproteins. However, on a high-cholesterol diet, ApoeRh/h mice displayed increased levels of total plasma cholesterol and very-low-density lipoprotein as well as larger atherosclerotic plaques in the aortic root, arch, and descending aorta compared with ApoeTh/h mice. In addition, evidence of cellular dysfunction was identified in peritoneal ApoeRh/h macrophages which released lower amounts of apoE in culture medium and displayed increased expression of major histocompatibility complex class II molecules. Conclusions—These data indicate that domain interaction mediates proatherogenic effects of apoE4 in part by modulating lipoprotein metabolism and macrophage biology. Pharmaceutical targeting of domain interaction could lead to new treatments for atherosclerosis in apoE4 individuals.

Macrophage-specific apoE gene repair reduces diet-induced hyperlipidemia and atherosclerosis in hypomorphic apoE mice

Background Apolipoprotein (apo) E is best known for its ability to lower plasma cholesterol and protect against atherosclerosis. Although the liver is the major source of plasma apoE, extra-hepatic sources of apoE, including from macrophages, account for up to 10% of plasma apoE levels. This study examined the contribution of macrophage-derived apoE expression levels in diet-induced hyperlipidemia and atherosclerosis. Methodology/Principal Findings Hypomorphic apoE (Apoe h/h) mice expressing wildtype mouse apoE at ∼2–5% of physiological levels in all tissues were derived by gene targeting in embryonic stem cells. Cre-mediated gene repair of the Apoe h/h allele in Apoe h/hLysM-Cre mice raised apoE expression levels by 26 fold in freshly isolated peritoneal macrophages, restoring it to 37% of levels seen in wildtype mice. Chow-fed Apoe h/hLysM-Cre and Apoe h/h mice displayed similar plasma apoE and cholesterol levels (55.53±2.90 mg/dl versus 62.70±2.77 mg/dl, n = 12). When fed a high-cholesterol diet (HCD) for 16 weeks, Apoe h/hLysM-Cre mice displayed a 3-fold increase in plasma apoE and a concomitant 32% decrease in plasma cholesterol when compared to Apoe h/h mice (602.20±22.30 mg/dl versus 888.80±24.99 mg/dl, n = 7). On HCD, Apoe h/hLysM-Cre mice showed increased apoE immunoreactivity in lesional macrophages and liver-associated Kupffer cells but not hepatocytes. In addition, Apoe h/hLysM-Cre mice developed 35% less atherosclerotic lesions in the aortic root than Apoe h/h mice (167×103±16×103 µm2 versus 259×103±56×103 µm2, n = 7). This difference in atherosclerosis lesions size was proportional to the observed reduction in plasma cholesterol. Conclusions/Significance Macrophage-derived apoE raises plasma apoE levels in response to diet-induced hyperlipidemia and by such reduces atherosclerosis proportionally to the extent to which it lowers plasma cholesterol levels.

Inducible Apoe Gene Repair in Hypomorphic ApoE Mice Deficient in the Low-Density Lipoprotein Receptor Promotes Atheroma Stabilization with a Human-Like Lipoprotein Profile

Objective—To study atherosclerosis regression in mice after plasma lipid reduction to moderately elevated apolipoprotein B (apoB)–lipoprotein levels. Approach and Results—Chow-fed hypomorphic Apoe mice deficient in low-density lipoprotein receptor expression (Apoeh/hLdlr–/–Mx1-cre mice) develop hyperlipidemia and atherosclerosis. These mice were studied before and after inducible cre-mediated Apoe gene repair. By 1 week, induced mice displayed a 2-fold reduction in plasma cholesterol and triglyceride levels and a decrease in the non–high-density lipoprotein:high-density lipoprotein-cholesterol ratio from 87%:13% to 60%:40%. This halted atherosclerotic lesion growth and promoted macrophage loss and accumulation of thick collagen fibers for up to 8 weeks. Concomitantly, blood Ly-6Chigh monocytes were decreased by 2-fold but lesional macrophage apoptosis was unchanged. The expression of several genes involved in extracellular matrix remodeling and cell migration was changed in lesional macrophages 1 week after Apoe gene repair. However, mRNA levels of numerous genes involved in cholesterol efflux and inflammation were not significantly changed at this time point. Conclusions—Restoring apoE expression in Apoeh/hLdlr–/–Mx1-cre mice resulted in lesion stabilization in the context of a human-like ratio of non–high-density lipoprotein:high-density lipoprotein-cholesterol. Our data suggest that macrophage loss derived in part from reduced blood Ly-6Chigh monocytes levels and genetic reprogramming of lesional macrophages.