Eugene Lifshits

Preexistence and Clonal Selection of MET Amplification in EGFR Mutant NSCLC

MET amplification activates ERBB3/PI3K/AKT signaling in EGFR mutant lung cancers and causes resistance to EGFR kinase inhibitors. We demonstrate that MET activation by its ligand, HGF, also induces drug resistance, but through GAB1 signaling. Using high-throughput FISH analyses in both cell lines and in patients with lung cancer, we identify subpopulations of cells with MET amplification prior to drug exposure. Surprisingly, HGF accelerates the development of MET amplification both in vitro and in vivo. EGFR kinase inhibitor resistance, due to either MET amplification or autocrine HGF production, was cured in vivo by combined EGFR and MET inhibition. These findings highlight the potential to prospectively identify treatment naive, patients with EGFR-mutant lung cancer who will benefit from initial combination therapy.

EML4-ALK fusion gene and efficacy of an ALK kinase inhibitor in lung cancer

Purpose: The EML4-ALK fusion gene has been detected in ∼7% of Japanese non-small cell lung cancers (NSCLC). We determined the frequency of EML4-ALK in Caucasian NSCLC and in NSCLC cell lines. We also determined whether TAE684, a specific ALK kinase inhibitor, would inhibit the growth of EML4-ALK-containing cell lines in vitro and in vivo. Experimental Design: We screened 305 primary NSCLC [both U.S. (n = 138) and Korean (n = 167) patients] and 83 NSCLC cell lines using reverse transcription-PCR and by exon array analyses. We evaluated the efficacy of TAE684 against NSCLC cell lines in vitro and in vivo. Results: We detected four different variants, including two novel variants, of EML4-ALK using reverse transcription-PCR in 8 of 305 tumors (3%) and 3 of 83 (3.6%) NSCLC cell lines. All EML4-ALK-containing tumors and cell lines were adenocarcinomas. EML4-ALK was detected more frequently in NSCLC patients who were never or light (<10 pack-years) cigarette smokers compared with current/former smokers (6% versus 1%; P = 0.049). TAE684 inhibited the growth of one of three (H3122) EML4-ALK-containing cell lines in vitro and in vivo, inhibited Akt phosphorylation, and caused apoptosis. In another EML4-ALK cell line, DFCI032, TAE684 was ineffective due to coactivation of epidermal growth factor receptor and ERBB2. The combination of TAE684 and CL-387,785 (epidermal growth factor receptor/ERBB2 kinase inhibitor) inhibited growth and Akt phosphorylation and led to apoptosis in the DFCI032 cell line. Conclusions:EML4-ALK is found in the minority of NSCLC. ALK kinase inhibitors alone or in combination may nevertheless be clinically effective treatments for NSCLC patients whose tumors contain EML4-ALK.

PF00299804, an Irreversible Pan-ERBB Inhibitor, Is Effective in Lung Cancer Models with EGFR and ERBB2 Mutations that Are Resistant to Gefitinib

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors gefitinib and erlotinib are effective treatments for a subset of non-small cell lung cancers. In particular, cancers with specific EGFR-activating mutations seem to be the most sensitive to these agents. However, despite their initial response, such cancers almost invariably develop resistance. In 50% of such cancers, a secondary EGFR mutation, T790M, has been identified that renders gefitinib and erlotinib ineffective inhibitors of EGFR kinase activity. Thus, there is a clinical need to develop novel EGFR inhibitors that can effectively inactivate T790M-containing EGFR proteins. In this study, we evaluate the effectiveness of a novel compound, PF00299804, an irreversible pan-ERBB inhibitor. The results from these studies show that PF00299804 is a potent inhibitor of EGFR-activating mutations as well as the EGFR T790M resistance mutation both in vitro and in vivo. Additionally, PF00299804 is a highly effective inhibitor of both the wild-type ERBB2 and the gefitinib-resistant oncogenic ERBB2 mutation identified in lung cancers. These preclinical evaluations support further clinical development of PF00299804 for cancers with mutations and/or amplifications of ERBB family members.

Therapeutic strategies to overcome crizotinib resistance in non-small cell lung cancers harboring the fusion oncogene EML4-ALK

The echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) fusion oncogene represents a molecular target in a small subset of non-small cell lung cancers (NSCLCs). This fusion leads to constitutive ALK activation with potent transforming activity. In a pivotal phase 1 clinical trial, the ALK tyrosine kinase inhibitor (TKI) crizotinib (PF-02341066) demonstrated impressive antitumor activity in the majority of patients with NSCLC harboring ALK fusions. However, despite these remarkable initial responses, cancers eventually develop resistance to crizotinib, usually within 1 y, thereby limiting the potential clinical benefit. To determine how cancers acquire resistance to ALK inhibitors, we established a model of acquired resistance to crizotinib by exposing a highly sensitive EML4-ALK–positive NSCLC cell line to increasing doses of crizotinib until resistance emerged. We found that cells resistant to intermediate doses of crizotinib developed amplification of the EML4-ALK gene. Cells resistant to higher doses (1 μM) also developed a gatekeeper mutation, L1196M, within the kinase domain, rendering EML4-ALK insensitive to crizotinib. This gatekeeper mutation was readily detected using a unique and highly sensitive allele-specific PCR assay. Although crizotinib was ineffectual against EML4-ALK harboring the gatekeeper mutation, we observed that two structurally different ALK inhibitors, NVP-TAE684 and AP26113, were highly active against the resistant cancer cells in vitro and in vivo. Furthermore, these resistant cells remained highly sensitive to the Hsp90 inhibitor 17-AAG. Thus, we have developed a model of acquired resistance to ALK inhibitors and have shown that second-generation ALK TKIs or Hsp90 inhibitors are effective in treating crizotinib-resistant tumors harboring secondary gatekeeper mutations.

Allelic dilution obscures detection of a biologically significant resistance mutation in EGFR -amplified lung cancer

EGFR is frequently mutated and amplified in lung adenocarcinomas sensitive to EGFR inhibitors gefitinib and erlotinib. A secondary mutation, T790M, has been associated with acquired resistance but has not been shown to be sufficient to render EGFR mutant/amplified lung cancers resistant to EGFR inhibitors. We created a model for studying acquired resistance to gefitinib by prolonged exposure of a gefitinib-sensitive lung carcinoma cell line (H3255; EGFR mutated and amplified) to gefitinib in vitro. The resulting resistant cell line acquired a T790M mutation in a small fraction of the amplified alleles that was undetected by direct sequencing and identified only by a highly sensitive HPLC-based technique. In gefitinib-sensitive lung cancer cells with EGFR mutations and amplifications, exogenous introduction of EGFR T790M effectively conferred resistance to gefitinib and continued ErbB-3/PI3K/Akt signaling when in cis to an activating mutation. Moreover, continued activation of PI3K signaling by the PIK3CA oncogenic mutant, p110alpha E545K, was sufficient to abrogate gefitinib-induced apoptosis. These findings suggest that allelic dilution of biologically significant resistance mutations may go undetected by direct sequencing in cancers with amplified oncogenes and that restoration of PI3K activation via either a T790M mutation or other mechanisms can provide resistance to gefitinib.

Activation of ERBB2 Signaling Causes Resistance to the EGFR-Directed Therapeutic Antibody Cetuximab

Several cancers become resistant to cetuximab by activating a bypass signaling pathway and preventing cetuximab inhibition of ERK1/2-stimulated growth. Combating Resistance to an EGF Receptor Inhibitor Many promising anticancer drugs are effective only for a limited time, because the tumor cells develop resistance. Cetuximab, directed against the epidermal growth factor receptor (EGFR), is no exception, and patients with colorectal, head and neck, or non–small cell lung cancer eventually cease to respond to the drug. Yonesaka and colleagues have determined that cetuximab-resistant cancer cells—both in culture and in patients—can up-regulate signaling through the ERBB2 growth factor receptor in several ways, permanently turning on extracellular signal–regulated kinase 1/2 (ERK1/2)–mediated growth, differentiation, and survival. They further show that interference with the ERBB2 pathway restores the ability of cetuximab to control these cancers, pointing to a promising resistance-fighting approach. The authors generated clones of cetuximab-resistant non–small cell lung and colorectal cancer cell lines by exposing the cells to increasing concentration of the drug. In some of these resistant clones, the ERBB2 receptor oncogene was genetically amplified, resulting in activated ERK1/2 signaling. Down-regulation of ERBB2 with a small interfering RNA or antibody restored sensitivity. Other clones did not have amplified ERBB2 genes but did make excess heregulin, an activating ligand for the ERBB2 receptor. Heregulin depletion or ERBB2 inhibition restored cetuximab sensitivity. After replicating these studies in xenografts in mice, the authors also looked for evidence that these resistance-associated alterations pertain to human tumors. In several groups of patients with colorectal cancer, they saw decreased survival or decreased sensitivity to cetuximab in those who exhibited amplified ERBB2 gene or higher heregulin concentrations. The concordance of their cellular data with patient experience improves confidence that concomitant treatment of certain lung, head and neck, or colorectal cancers with cetuximab and an anti-ERBB2 drug may prevent or delay the development of drug resistance. These studies add to other successes for this approach, which has also been used for analysis of other molecular targeted therapies, including EGFR kinase inhibitors. Cetuximab, an antibody directed against the epidermal growth factor receptor, is an effective clinical therapy for patients with colorectal, head and neck, and non–small cell lung cancer, particularly for those with KRAS and BRAF wild-type cancers. Treatment in all patients is limited eventually by the development of acquired resistance, but little is known about the underlying mechanism. Here, we show that activation of ERBB2 signaling in cell lines, either through ERBB2 amplification or through heregulin up-regulation, leads to persistent extracellular signal–regulated kinase 1/2 signaling and consequently to cetuximab resistance. Inhibition of ERBB2 or disruption of ERBB2/ERBB3 heterodimerization restores cetuximab sensitivity in vitro and in vivo. A subset of colorectal cancer patients who exhibit either de novo or acquired resistance to cetuximab-based therapy has ERBB2 amplification or high levels of circulating heregulin. Collectively, these findings identify two distinct resistance mechanisms, both of which promote aberrant ERBB2 signaling, that mediate cetuximab resistance. Moreover, these results suggest that ERBB2 inhibitors, in combination with cetuximab, represent a rational therapeutic strategy that should be assessed in patients with cetuximab-resistant cancers.

Patient-derived models of acquired resistance can identify effective drug combinations for cancer

Targeted cancer therapies have produced substantial clinical responses, but most tumors develop resistance to these drugs. Here, we describe a pharmacogenomic platform that facilitates rapid discovery of drug combinations that can overcome resistance. We established cell culture models derived from biopsy samples of lung cancer patients whose disease had progressed while on treatment with epidermal growth factor receptor (EGFR) or anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors and then subjected these cells to genetic analyses and a pharmacological screen. Multiple effective drug combinations were identified. For example, the combination of ALK and MAPK kinase (MEK) inhibitors was active in an ALK-positive resistant tumor that had developed a MAP2K1 activating mutation, and the combination of EGFR and fibroblast growth factor receptor (FGFR) inhibitors was active in an EGFR mutant resistant cancer with a mutation in FGFR3. Combined ALK and SRC (pp60c-src) inhibition was effective in several ALK-driven patient-derived models, a result not predicted by genetic analysis alone. With further refinements, this strategy could help direct therapeutic choices for individual patients. Secondary chemotherapies can be developed by screening drug-resistant cells from individual cancer patients. Drug resistance, up close and personal Cancer therapies that target specific genetic mutations driving tumor growth have shown promising results in patients; however, the response is often short-lived because the tumors acquire new mutations that render them resistant to these therapies. Complicating matters, the mechanism of resistance can vary from patient to patient. To identify drugs most likely to be effective against resistant tumors, Crystal et al. established cell lines from the tumors of individual patients after resistance occurred and performed a drug screen and genetic analysis on the cultured cells. This strategy successfully identified drug combinations that halted the growth of resistant tumor cells both in culture and in mice. In the future, pharmacological profiling of patient-derived cells could be an efficient way to direct therapeutic choices for individual cancer patients. Science, this issue p. 1480

Differential induction of apoptosis in HER2 and EGFR addicted cancers following PI3K inhibition

Non-small cell lung cancers with activating mutations in the epidermal growth factor receptor (EGFR) are highly responsive to EGFR tyrosine kinase inhibitors (TKIs), such as gefitinib and erlotinib. Such cancers are “addicted” to EGFR, and treatment with a TKI invariably leads to down-regulation of the PI3K-AKT-mTOR and MEK-ERK signaling pathways, resulting in apoptosis. Using a dual PI3K-mTOR inhibitor, NVP-BEZ235, we evaluated whether PI3K-mTOR inhibition alone induced apoptosis in these cancers. In contrast to HER2-amplified breast cancers, we found that PI3K-mTOR inhibition did not promote substantial apoptosis in the EGFR mutant lung cancers. However, blocking both PI3K-mTOR and MEK simultaneously led to apoptosis to similar levels as the EGFR TKIs, suggesting that down-regulation of these pathways may account for much of the apoptosis promoted by EGFR inhibition. In EGFR mutant lung cancers, down-regulation of both intracellular pathways converged on the BH3 family of proteins regulating apoptosis. PI3K inhibition led to down-regulation of Mcl-1, and MEK inhibition led to up-regulation of BIM. In fact, down-regulation of Mcl-1 by siRNA was sufficient to sensitize these cancers to single-agent MEK inhibitors. Surprisingly, an AKT inhibitor did not decrease Mcl-1 levels, and when combined with MEK inhibitors, failed to induce apoptosis. Importantly, we observed that the combination of PI3K-mTOR and MEK inhibitors effectively shrunk tumors in a transgenic and xenograft model of EGFR T790M-L858R cancers. These data indicate simultaneous inhibition of PI3K-mTOR and MEK signaling is an effective strategy for treating EGFR mutant lung cancers, including those with acquired resistance to EGFR TKIs.

Synthetic Lethal Interaction of Combined BCL-XL and MEK Inhibition Promotes Tumor Regressions in KRAS Mutant Cancer Models

KRAS is the most commonly mutated oncogene, yet no effective targeted therapies exist for KRAS mutant cancers. We developed a pooled shRNA-drug screen strategy to identify genes that, when inhibited, cooperate with MEK inhibitors to effectively treat KRAS mutant cancer cells. The anti-apoptotic BH3 family gene BCL-XL emerged as a top hit through this approach. ABT-263 (navitoclax), a chemical inhibitor that blocks the ability of BCL-XL to bind and inhibit pro-apoptotic proteins, in combination with a MEK inhibitor led to dramatic apoptosis in many KRAS mutant cell lines from different tissue types. This combination caused marked in vivo tumor regressions in KRAS mutant xenografts and in a genetically engineered KRAS-driven lung cancer mouse model, supporting combined BCL-XL/MEK inhibition as a potential therapeutic approach for KRAS mutant cancers.

BIM expression in treatment-naïve cancers predicts responsiveness to kinase inhibitors

Cancers with specific genetic mutations are susceptible to selective kinase inhibitors. However, there is a wide spectrum of benefit among cancers harboring the same sensitizing genetic mutations. Herein, we measured apoptotic rates among cell lines sharing the same driver oncogene following treatment with the corresponding kinase inhibitor. There was a wide range of kinase inhibitor-induced apoptosis despite comparable inhibition of the target and associated downstream signaling pathways. Surprisingly, pretreatment RNA levels of the BH3-only pro-apoptotic BIM strongly predicted the capacity of EGFR, HER2, and PI3K inhibitors to induce apoptosis in EGFR-mutant, HER2-amplified, and PIK3CA-mutant cancers, respectively, but BIM levels did not predict responsiveness to standard chemotherapies. Furthermore, BIM RNA levels in EGFR-mutant lung cancer specimens predicted response and duration of clinical benefit from EGFR inhibitors. These findings suggest assessment of BIM levels in treatment-naïve tumor biopsies may indicate the degree of benefit from single-agent kinase inhibitors in multiple oncogene-addiction paradigms.