Romel Somwar

Acquired Resistance of Lung Adenocarcinomas to Gefitinib or Erlotinib Is Associated with a Second Mutation in the EGFR Kinase Domain

Background Lung adenocarcinomas from patients who respond to the tyrosine kinase inhibitors gefitinib (Iressa) or erlotinib (Tarceva) usually harbor somatic gain-of-function mutations in exons encoding the kinase domain of the epidermal growth factor receptor (EGFR). Despite initial responses, patients eventually progress by unknown mechanisms of “acquired” resistance. Methods and Findings We show that in two of five patients with acquired resistance to gefitinib or erlotinib, progressing tumors contain, in addition to a primary drug-sensitive mutation in EGFR, a secondary mutation in exon 20, which leads to substitution of methionine for threonine at position 790 (T790M) in the kinase domain. Tumor cells from a sixth patient with a drug-sensitive EGFR mutation whose tumor progressed on adjuvant gefitinib after complete resection also contained the T790M mutation. This mutation was not detected in untreated tumor samples. Moreover, no tumors with acquired resistance had KRAS mutations, which have been associated with primary resistance to these drugs. Biochemical analyses of transfected cells and growth inhibition studies with lung cancer cell lines demonstrate that the T790M mutation confers resistance to EGFR mutants usually sensitive to either gefitinib or erlotinib. Interestingly, a mutation analogous to T790M has been observed in other kinases with acquired resistance to another kinase inhibitor, imatinib (Gleevec). Conclusion In patients with tumors bearing gefitinib- or erlotinib-sensitive EGFR mutations, resistant subclones containing an additional EGFR mutation emerge in the presence of drug. This observation should help guide the search for more effective therapy against a specific subset of lung cancers.

Novel D761Y and Common Secondary T790M Mutations in Epidermal Growth Factor Receptor–Mutant Lung Adenocarcinomas with Acquired Resistance to Kinase Inhibitors

Purpose: In patients whose lung adenocarcinomas harbor epidermal growth factor receptor (EGFR) tyrosine kinase domain mutations, acquired resistance to the tyrosine kinase inhibitors (TKI) gefitinib (Iressa) and erlotinib (Tarceva) has been associated with a second-site EGFR mutation, which leads to substitution of methionine for threonine at position 790 (T790M). We aimed to elucidate the frequency and nature of secondary EGFR mutations in patients with acquired resistance to TKI monotherapy. Experimental Design: Tumor cells from patients with acquired resistance were examined for secondary EGFR kinase domain mutations by molecular analyses. Results: Eight of 16 patients (50% observed rate; 95% confidence interval, 25-75%) had tumor cells with second-site EGFR mutations. Seven mutations were T790M and one was a novel D761Y mutation found in a brain metastasis. When combined with a drug-sensitive L858R mutation, the D761Y mutation modestly reduced the sensitivity of mutant EGFR to TKIs in both surrogate kinase and cell viability assays. In an autopsy case, the T790M mutation was found in multiple visceral metastases but not in a brain lesion. Conclusions: The T790M mutation is common in patients with acquired resistance. The limited spectrum of TKI-resistant mutations in EGFR, which binds to erlotinib in the active conformation, contrasts with a wider range of second-site mutations seen with acquired resistance to imatinib, which binds to ABL and KIT, respectively, in closed conformations. Collectively, our data suggest that the type and nature of kinase inhibitor resistance mutations may be influenced by both anatomic site and mode of binding to the kinase target.

Optimization of dosing for EGFR-mutant non-small cell lung cancer with evolutionary cancer modeling.

Predictive models of EGFR-mutant tumor behavior point to alternative drug dosing strategies to prevent and treat acquired resistance. Harnessing Evolution to Improve Lung Cancer Therapy Like any organism under severe evolutionary pressure, a few select members of a cancer cell population acquire molecular changes that strengthen the clan’s chances of survival. Therapeutic drugs exert a powerful selective force on characteristically compliant cancer cells, as the common recurrence of drug-resistant cancers testifies. To learn how to better fight the potent forces of evolution, Chmielecki et al. examined the behavior of non–small cell lung cancer (NSCLC) before and after the cells acquire resistance to targeted therapy, which inevitably they do. The growth characteristics of these cells were consistent with patient tumor behavior, enabling construction of a mathematical model that predicted alternative therapeutic strategies to delay the development of drug-resistant cancer cells. The authors made paired isogenic cell lines that were sensitive and resistant to afatinib and erlotinib—drugs used to treat NSCLC that are directed against the epidermal growth factor receptor (EGFR) tyrosine kinase, which is activated in a subset of NSCLCs. To the authors’ surprise, the drug-resistant cells grew more slowly than their sensitive counterparts, and resistance was not maintained in the absence of selection. Multiple clinical observations corroborated these findings. For example, patients with resistant tumors showed a slow course of disease progression, and patients with acquired resistance have re-responded to tyrosine kinase inhibitor (TKI) therapy after a drug holiday. The authors then constructed an evolutionary mathematical model of tumor behavior based on the differential growth rates of TKI-sensitive and TKI-resistant cells in heterogeneous tumor cell populations. Understanding the growth dynamics of how tumors behave allowed the authors to calculate what would happen under different treatment regimes. Their models predicted that continuous administration of a low-dose EGFR TKI combined with high-dose pulses of an EGFR TKI should delay the onset of resistance. Subsequent cellular studies bore out this prediction. Modeling also indicated the wisdom of prolonging treatment with the EGFR TKI after the development of resistance to prevent fast overgrowth by the sensitive cells, a result also born out in vitro and in vivo. Ultimate proof will have to come from patients. Clinical trials based on these alternative dosing strategies will be the true test of the utility of evolutionary mathematical modeling in designing cancer treatments. If they prove beneficial, individual models based on the characteristics of diverse cancer cell types could offer clues for designing optimal treatment strategies. Non–small cell lung cancers (NSCLCs) that harbor mutations within the epidermal growth factor receptor (EGFR) gene are sensitive to the tyrosine kinase inhibitors (TKIs) gefitinib and erlotinib. Unfortunately, all patients treated with these drugs will acquire resistance, most commonly as a result of a secondary mutation within EGFR (T790M). Because both drugs were developed to target wild-type EGFR, we hypothesized that current dosing schedules were not optimized for mutant EGFR or to prevent resistance. To investigate this further, we developed isogenic TKI-sensitive and TKI-resistant pairs of cell lines that mimic the behavior of human tumors. We determined that the drug-sensitive and drug-resistant EGFR-mutant cells exhibited differential growth kinetics, with the drug-resistant cells showing slower growth. We incorporated these data into evolutionary mathematical cancer models with constraints derived from clinical data sets. This modeling predicted alternative therapeutic strategies that could prolong the clinical benefit of TKIs against EGFR-mutant NSCLCs by delaying the development of resistance.

Induction of BIM is essential for apoptosis triggered by EGFR kinase inhibitors in mutant EGFR-dependent lung adenocarcinomas.

Background Mutations in the epidermal growth factor receptor (EGFR) gene are associated with increased sensitivity of lung cancers to kinase inhibitors like erlotinib. Mechanisms of cell death that occur after kinase inhibition in these oncogene-dependent tumors have not been well delineated. We sought to improve understanding of this process in order to provide insight into mechanisms of sensitivity and/or resistance to tyrosine kinase inhibitors and to uncover new targets for therapy. Methods and Findings Using a panel of human lung cancer cell lines that harbor EGFR mutations and a variety of biochemical, molecular, and cellular techniques, we show that EGFR kinase inhibition in drug-sensitive cells provokes apoptosis via the intrinsic pathway of caspase activation. The process requires induction of the proapoptotic BH3-only BCL2 family member BIM (i.e., BCL2-like 11, or BCL2L11); erlotinib dramatically induces BIM levels in sensitive but not in resistant cell lines, and knockdown of BIM expression by RNA interference virtually eliminates drug-induced cell killing in vitro. BIM status is regulated at both transcriptional and posttranscriptional levels and is influenced by the extracellular signal-regulated kinase (ERK) signaling cascade downstream of EGFR. Consistent with these findings, lung tumors and xenografts from mice bearing mutant EGFR-dependent lung adenocarcinomas display increased concentrations of Bim after erlotinib treatment. Moreover, an inhibitor of antiapoptotic proteins, ABT-737, enhances erlotinib-induced cell death in vitro. Conclusions In drug-sensitive EGFR mutant lung cancer cells, induction of BIM is essential for apoptosis triggered by EGFR kinase inhibitors. This finding implies that the intrinsic pathway of caspase activation may influence sensitivity and/or resistance of EGFR mutant lung tumor cells to EGFR kinase inhibition. Manipulation of the intrinsic pathway could be a therapeutic strategy to enhance further the clinical outcomes of patients with EGFR mutant lung tumors.

An integrated genomic analysis of lung cancer reveals loss of DUSP4 in EGFR-mutant tumors

To address the biological heterogeneity of lung cancer, we studied 199 lung adenocarcinomas by integrating genome-wide data on copy number alterations and gene expression with full annotation for major known somatic mutations in this cancer. This showed non-random patterns of copy number alterations significantly linked to EGFR and KRAS mutation status and to distinct clinical outcomes, and led to the discovery of a striking association of EGFR mutations with underexpression of DUSP4, a gene within a broad region of frequent single-copy loss on 8p. DUSP4 is involved in negative feedback control of EGFR signaling, and we provide functional validation for its role as a growth suppressor in EGFR-mutant lung adenocarcinoma. DUSP4 loss also associates with p16/CDKN2A deletion and defines a distinct clinical subset of lung cancer patients. Another novel observation is that of a reciprocal relationship between EGFR and LKB1 mutations. These results highlight the power of integrated genomics to identify candidate driver genes within recurrent broad regions of copy number alteration and to delineate distinct oncogenetic pathways in genetically complex common epithelial cancers.

Cabozantinib in patients with advanced RET-rearranged non-small-cell lung cancer: an open-label, single-centre, phase 2, single-arm trial

SUMMARY Background RET rearrangements are found in 1–2% of non-small cell lung cancers. Cabozantinib is a multikinase RET inhibitor that produced a 10% response rate in unselected patients with lung cancers. To evaluate the activity of cabozantinib in patients with RET-rearranged lung cancers, we conducted a prospective phase 2 trial in this molecular subgroup. Methods We enrolled patients in this open-label, Simon two-stage, phase 2 trial if they met the following criteria: metastatic or unresectable lung cancer harboring a RET rearrangement, Karnofsky performance status of >70%, and measurable disease. Cabozantinib was administered at 60 mg daily. The primary objective was to determine the overall response rate (RECIST v1·1). This analysis was performed in an intent to treat fashion in patients who received at least one dose of cabozantinib and underwent imaging performed at baseline and at least one protocol-specified follow up time point. The secondary objectives were to determine progression-free survival, overall survival, and toxicity. The accrual of RET-rearranged lung cancer patients to this protocol has been completed. This study was registered with ClinicalTrials.gov, number NCT01639508. Findings Twenty six patients with RET-rearranged lung adenocarcinomas were treated with cabozantinib. KIF5B-RET was the predominant fusion type identified in 16 (62%) patients. The study met its primary endpoint with confirmed partial responses observed in seven of 25 response-evaluable patients (overall response rate 28% [95% CI 12–49%]). The most common grade 3 treatment-related adverse events were asymptomatic lipase elevation in four patients (15%), increased alanine aminotransferase in two patients (8%), increased aspartate aminotransferase in two patients (8%), thrombocytopenia in two patients (8%), and hypophosphatemia in two patients (8%). No drug-related deaths were observed. Nineteen patients (73%) required dose reduction due to drug-related adverse events. Interpretation The observed activity of cabozantinib in patients with RET-rearranged lung cancers defines RET rearrangements as actionable drivers in patients with lung cancers. An improved understanding of tumor biology and novel therapeutic approaches will be required to improve outcomes with RET-directed targeted therapy.BACKGROUND RET rearrangements are found in 1-2% of non-small-cell lung cancers. Cabozantinib is a multikinase inhibitor with activity against RET that produced a 10% overall response in unselected patients with lung cancers. To assess the activity of cabozantinib in patients with RET-rearranged lung cancers, we did a prospective phase 2 trial in this molecular subgroup. METHODS We enrolled patients in this open-label, Simon two-stage, single-centre, phase 2, single-arm trial in the USA if they met the following criteria: metastatic or unresectable lung cancer harbouring a RET rearrangement, Karnofsky performance status higher than 70, and measurable disease. Patients were given 60 mg of cabozantinib orally per day. The primary objective was to determine the overall response (Response Criteria Evaluation in Solid Tumors version 1.1) in assessable patients; those who received at least one dose of cabozantinib, and had been given CT imaging at baseline and at least one protocol-specified follow-up timepoint. We did safety analyses in the modified intention-to-treat population who received at least one dose of cabozantinib. The accrual of patients with RET-rearranged lung cancer to this protocol has been completed but the trial is still ongoing because several patients remain on active treatment. This study was registered with ClinicalTrials.gov, number NCT01639508. FINDINGS Between July 13, 2012, and April 30, 2016, 26 patients with RET-rearranged lung adenocarcinomas were enrolled and given cabozantinib; 25 patients were assessable for a response. KIF5B-RET was the predominant fusion type identified in 16 (62%) patients. The study met its primary endpoint, with confirmed partial responses seen in seven of 25 response-assessable patients (overall response 28%, 95% CI 12-49). Of the 26 patients given cabozantinib, the most common grade 3 treatment-related adverse events were lipase elevation in four (15%) patients, increased alanine aminotransferase in two (8%) patients, increased aspartate aminotransferase in two (8%) patients, decreased platelet count in two (8%) patients, and hypophosphataemia in two (8%) patients. No drug-related deaths were recorded but 16 (62%) patients died during the course of follow-up. 19 (73%) patients required dose reductions due to drug-related adverse events. INTERPRETATION The reported activity of cabozantinib in patients with RET-rearranged lung cancers defines RET rearrangements as actionable drivers in patients with lung cancers. An improved understanding of tumour biology and novel therapeutic approaches will be needed to improve outcomes with RET-directed targeted treatment. FUNDING Exelixis, National Institutes of Health and National Cancer Institute Cancer Center Support Grant P30 CA008748.

Superoxide dismutase 1 (SOD1) is a target for a small molecule identified in a screen for inhibitors of the growth of lung adenocarcinoma cell lines

We previously described four small molecules that reduced the growth of lung adenocarcinoma cell lines with either epidermal growth factor receptor (EGFR) or KRAS mutations in a high-throughout chemical screen. By combining affinity proteomics and gene expression analysis, we now propose superoxide dismutase 1 (SOD1) as the most likely target of one of these small molecules, referred to as lung cancer screen 1 (LCS-1). siRNAs against SOD1 slowed the growth of LCS-1 sensitive cell lines; conversely, expression of a SOD1 cDNA increased proliferation of H358 cells and reduced sensitivity of these cells to LCS-1. In addition, SOD1 enzymatic activity was inhibited in vitro by LCS-1 and two closely related analogs. These results suggest that SOD1 is an LCS-1–binding protein that may act in concert with mutant proteins, such as EGFR and KRAS, to promote cell growth, providing a therapeutic target for compounds like LCS-1.

A Novel Crizotinib-Resistant Solvent-Front Mutation Responsive to Cabozantinib Therapy in a Patient with ROS1-Rearranged Lung Cancer

Purpose: Rearranged ROS1 is a crizotinib-sensitive oncogenic driver in lung cancer. The development of acquired resistance, however, poses a serious clinical challenge. Consequently, experimental and clinical validation of resistance mechanisms and potential second-line therapies is essential. Experimental Design: We report the discovery of a novel, solvent-front ROS1D2033N mutation in a patient with CD74-ROS1–rearranged lung adenocarcinoma and acquired resistance to crizotinib. Crizotinib resistance of CD74-ROS1D2033N was functionally evaluated using cell-based assays and structural modeling. Results: In biochemical and cell-based assays, the CD74-ROS1D2033N mutant demonstrated significantly decreased sensitivity to crizotinib. Molecular dynamics simulation revealed compromised crizotinib binding due to drastic changes in the electrostatic interaction between the D2033 residue and crizotinib and reorientation of neighboring residues. In contrast, cabozantinib binding was unaffected by the D2033N substitution, and inhibitory potency against the mutant was retained. Notably, cabozantinib treatment resulted in a rapid clinical and near-complete radiographic response in this patient. Conclusions: These results provide the first example of successful therapeutic intervention with targeted therapy to overcome crizotinib resistance in a ROS1-rearranged cancer. Clin Cancer Res; 22(10); 2351–8. ©2015 AACR.

Single-cell analysis of insulin-regulated fatty acid uptake in adipocytes

Increased body fat correlates with the enlargement of average fat cell size and reduced adipose tissue insulin sensitivity. It is currently unclear whether adipocytes, as they accumulate more triglycerides and grow in size, gradually become less insulin sensitive or whether obesity-related factors independently cause both the enlargement of adipocyte size and reduced adipose tissue insulin sensitivity. In the first instance, large and small adipocytes in the same tissue would exhibit differences in insulin sensitivity, whereas, in the second instance, adipocyte size per se would not necessarily correlate with insulin response. To analyze the effect of adipocyte size on insulin sensitivity, we employed a new single-cell imaging assay that resolves fatty acid uptake and insulin response in single adipocytes in subcutaneous adipose tissue explants. Here, we report that subcutaneous adipocytes are heterogeneous in size and intrinsic insulin sensitivity. Whereas smaller adipocytes respond to insulin by increasing lipid uptake, adipocytes with cell diameters larger than 80-100 microm are insulin resistant. We propose that, when cell size approaches a critical boundary, adipocytes lose insulin-dependent fatty acid transport. This negative feedback mechanism may protect adipocytes from lipid overload and restrict further expansion of adipose tissue, which leads to obesity and metabolic complications.

Protein interaction discovery using parallel analysis of translated ORFs (PLATO)

Identifying physical interactions between proteins and other molecules is a critical aspect of biological analysis. Here we describe PLATO, an in vitro method for mapping such interactions by affinity enrichment of a library of full-length open reading frames displayed on ribosomes, followed by massively parallel analysis using DNA sequencing. We demonstrate the broad utility of the method for human proteins by identifying known and previously unidentified interacting partners of LYN kinase, patient autoantibodies, and the small-molecules gefitinib and dasatinib.