Therapy-induced APOBEC3A drives evolution of persistent cancer cells.

Acquired drug resistance to anticancer targeted therapies remains an unsolved clinical problem. Although many drivers of acquired drug resistance have been identified1-4, the underlying molecular mechanisms shaping tumour evolution during treatment are incompletely understood. Genomic profiling of patient tumours has implicated apolipoprotein B messenger RNA editing catalytic polypeptide-like (APOBEC) cytidine deaminases in tumour evolution; however, their role during therapy and the development of acquired drug resistance is undefined. Here we report that lung cancer targeted therapies commonly used in the clinic can induce cytidine deaminase APOBEC3A (A3A), leading to sustained mutagenesis in drug-tolerant cancer cells persisting during therapy. Therapy-induced A3A promotes the formation of double-strand DNA breaks, increasing genomic instability in drug-tolerant persisters. Deletion of A3A reduces APOBEC mutations and structural variations in persister cells and delays the development of drug resistance. APOBEC mutational signatures are enriched in tumours from patients with lung cancer who progressed after extended responses to targeted therapies. This study shows that induction of A3A in response to targeted therapies drives evolution of drug-tolerant persister cells, suggesting that suppression of A3A expression or activity may represent a potential therapeutic strategy in the prevention or delay of acquired resistance to lung cancer targeted therapy.

Dependency of a therapy-resistant state of cancer cells on a lipid peroxidase pathway.

Plasticity of the cell state has been proposed to drive resistance to multiple classes of cancer therapies, thereby limiting their effectiveness. A high-mesenchymal cell state observed in human tumours and cancer cell lines has been associated with resistance to multiple treatment modalities across diverse cancer lineages, but the mechanistic underpinning for this state has remained incompletely understood. Here we molecularly characterize this therapy-resistant high-mesenchymal cell state in human cancer cell lines and organoids and show that it depends on a druggable lipid-peroxidase pathway that protects against ferroptosis, a non-apoptotic form of cell death induced by the build-up of toxic lipid peroxides. We show that this cell state is characterized by activity of enzymes that promote the synthesis of polyunsaturated lipids. These lipids are the substrates for lipid peroxidation by lipoxygenase enzymes. This lipid metabolism creates a dependency on pathways converging on the phospholipid glutathione peroxidase (GPX4), a selenocysteine-containing enzyme that dissipates lipid peroxides and thereby prevents the iron-mediated reactions of peroxides that induce ferroptotic cell death. Dependency on GPX4 was found to exist across diverse therapy-resistant states characterized by high expression of ZEB1, including epithelial-mesenchymal transition in epithelial-derived carcinomas, TGFß-mediated therapy-resistance in melanoma, treatment-induced neuroendocrine transdifferentiation in prostate cancer, and sarcomas, which are fixed in a mesenchymal state owing to their cells of origin. We identify vulnerability to ferroptic cell death induced by inhibition of a lipid peroxidase pathway as a feature of therapy-resistant cancer cells across diverse mesenchymal cell-state contexts.

A combinatorial strategy for treating KRAS-mutant lung cancer.

Therapeutic targeting of KRAS-mutant lung adenocarcinoma represents a major goal of clinical oncology. KRAS itself has proved difficult to inhibit, and the effectiveness of agents that target key KRAS effectors has been thwarted by activation of compensatory or parallel pathways that limit their efficacy as single agents. Here we take a systematic approach towards identifying combination targets for trametinib, a MEK inhibitor approved by the US Food and Drug Administration, which acts downstream of KRAS to suppress signalling through the mitogen-activated protein kinase (MAPK) cascade. Informed by a short-hairpin RNA screen, we show that trametinib provokes a compensatory response involving the fibroblast growth factor receptor 1 (FGFR1) that leads to signalling rebound and adaptive drug resistance. As a consequence, genetic or pharmacological inhibition of FGFR1 in combination with trametinib enhances tumour cell death in vitro and in vivo. This compensatory response shows distinct specificities: it is dominated by FGFR1 in KRAS-mutant lung and pancreatic cancer cells, but is not activated or involves other mechanisms in KRAS wild-type lung and KRAS-mutant colon cancer cells. Importantly, KRAS-mutant lung cancer cells and patients' tumours treated with trametinib show an increase in FRS2 phosphorylation, a biomarker of FGFR activation; this increase is abolished by FGFR1 inhibition and correlates with sensitivity to trametinib and FGFR inhibitor combinations. These results demonstrate that FGFR1 can mediate adaptive resistance to trametinib and validate a combinatorial approach for treating KRAS-mutant lung cancer.

Resensitization to Crizotinib by the Lorlatinib ALK Resistance Mutation L1198F.

In a patient who had metastatic anaplastic lymphoma kinase (ALK)-rearranged lung cancer, resistance to crizotinib developed because of a mutation in the ALK kinase domain. This mutation is predicted to result in a substitution of cysteine by tyrosine at amino acid residue 1156 (C1156Y). Her tumor did not respond to a second-generation ALK inhibitor, but it did respond to lorlatinib (PF-06463922), a third-generation inhibitor. When her tumor relapsed, sequencing of the resistant tumor revealed an ALK L1198F mutation in addition to the C1156Y mutation. The L1198F substitution confers resistance to lorlatinib through steric interference with drug binding. However, L1198F paradoxically enhances binding to crizotinib, negating the effect of C1156Y and resensitizing resistant cancers to crizotinib. The patient received crizotinib again, and her cancer-related symptoms and liver failure resolved. (Funded by Pfizer and others; ClinicalTrials.gov number, NCT01970865.).

A murine lung cancer co-clinical trial identifies genetic modifiers of therapeutic response.

Targeted therapies have demonstrated efficacy against specific subsets of molecularly defined cancers. Although most patients with lung cancer are stratified according to a single oncogenic driver, cancers harbouring identical activating genetic mutations show large variations in their responses to the same targeted therapy. The biology underlying this heterogeneity is not well understood, and the impact of co-existing genetic mutations, especially the loss of tumour suppressors, has not been fully explored. Here we use genetically engineered mouse models to conduct a 'co-clinical' trial that mirrors an ongoing human clinical trial in patients with KRAS-mutant lung cancers. This trial aims to determine if the MEK inhibitor selumetinib (AZD6244) increases the efficacy of docetaxel, a standard of care chemotherapy. Our studies demonstrate that concomitant loss of either p53 (also known as Tp53) or Lkb1 (also known as Stk11), two clinically relevant tumour suppressors, markedly impaired the response of Kras-mutant cancers to docetaxel monotherapy. We observed that the addition of selumetinib provided substantial benefit for mice with lung cancer caused by Kras and Kras and p53 mutations, but mice with Kras and Lkb1 mutations had primary resistance to this combination therapy. Pharmacodynamic studies, including positron-emission tomography (PET) and computed tomography (CT), identified biological markers in mice and patients that provide a rationale for the differential efficacy of these therapies in the different genotypes. These co-clinical results identify predictive genetic biomarkers that should be validated by interrogating samples from patients enrolled on the concurrent clinical trial. These studies also highlight the rationale for synchronous co-clinical trials, not only to anticipate the results of ongoing human clinical trials, but also to generate clinically relevant hypotheses that can inform the analysis and design of human studies.

Systematic identification of genomic markers of drug sensitivity in cancer cells.

Clinical responses to anticancer therapies are often restricted to a subset of patients. In some cases, mutated cancer genes are potent biomarkers for responses to targeted agents. Here, to uncover new biomarkers of sensitivity and resistance to cancer therapeutics, we screened a panel of several hundred cancer cell lines--which represent much of the tissue-type and genetic diversity of human cancers--with 130 drugs under clinical and preclinical investigation. In aggregate, we found that mutated cancer genes were associated with cellular response to most currently available cancer drugs. Classic oncogene addiction paradigms were modified by additional tissue-specific or expression biomarkers, and some frequently mutated genes were associated with sensitivity to a broad range of therapeutic agents. Unexpected relationships were revealed, including the marked sensitivity of Ewing's sarcoma cells harbouring the EWS (also known as EWSR1)-FLI1 gene translocation to poly(ADP-ribose) polymerase (PARP) inhibitors. By linking drug activity to the functional complexity of cancer genomes, systematic pharmacogenomic profiling in cancer cell lines provides a powerful biomarker discovery platform to guide rational cancer therapeutic strategies.

Improved tumor vascularization after anti-VEGF therapy with carboplatin and nab-paclitaxel associates with survival in lung cancer.

Addition of anti-VEGF antibody therapy to standard chemotherapies has improved survival and is an accepted standard of care for advanced non-small cell lung cancer (NSCLC). However, the mechanisms by which anti-VEGF therapy increases survival remain unclear. We evaluated dynamic CT-based vascular parameters and plasma cytokines after bevacizumab alone and after bevacizumab plus chemotherapy with carboplatin and nab-paclitaxel in advanced NSCLC patients to explore potential biomarkers of treatment response and resistance to this regimen. Thirty-six patients were enrolled in this study. The primary end point was 6-mo progression-free survival rate, which was 74% (95% CI: 57, 97). This regimen has a promising overall response rate of 36% and median time to progression of 8.5 (6.0, 38.7) mo and overall survival of 12.2 (9.6, 44.1) mo. We found that anti-VEGF therapy led to a sustained increase in plasma PlGF, a potential pharmacodynamic marker. We also found that higher levels of soluble VEGFR1 measured before starting bevacizumab with chemotherapy were associated with worse survival, supporting its potential role as biomarker of treatment resistance. Our imaging biomarker studies indicate that bevacizumab-based treatment-while reducing blood flow, volume, and permeability in the overall population-may be associated with improved survival in patients with improved tumor vasculature and blood perfusion after treatment. This hypothesis-generating study supports the notion that excessively decreasing vascular permeability and pruning/rarefaction after bevacizumab therapy may negatively impact the outcome of combination therapy in NSCLC patients. This hypothesis warrants further dose-titration studies of bevacizumab to examine the dose effect on tumor vasculature and treatment efficacy.

Assessment of ABT-263 activity across a cancer cell line collection leads to a potent combination therapy for small-cell lung cancer.

BH3 mimetics such as ABT-263 induce apoptosis in a subset of cancer models. However, these drugs have shown limited clinical efficacy as single agents in small-cell lung cancer (SCLC) and other solid tumor malignancies, and rational combination strategies remain underexplored. To develop a novel therapeutic approach, we examined the efficacy of ABT-263 across >500 cancer cell lines, including 311 for which we had matched expression data for select genes. We found that high expression of the proapoptotic gene Bcl2-interacting mediator of cell death (BIM) predicts sensitivity to ABT-263. In particular, SCLC cell lines possessed greater BIM transcript levels than most other solid tumors and are among the most sensitive to ABT-263. However, a subset of relatively resistant SCLC cell lines has concomitant high expression of the antiapoptotic myeloid cell leukemia 1 (MCL-1). Whereas ABT-263 released BIM from complexes with BCL-2 and BCL-XL, high expression of MCL-1 sequestered BIM released from BCL-2 and BCL-XL, thereby abrogating apoptosis. We found that SCLCs were sensitized to ABT-263 via TORC1/2 inhibition, which led to reduced MCL-1 protein levels, thereby facilitating BIM-mediated apoptosis. AZD8055 and ABT-263 together induced marked apoptosis in vitro, as well as tumor regressions in multiple SCLC xenograft models. In a Tp53; Rb1 deletion genetically engineered mouse model of SCLC, the combination of ABT-263 and AZD8055 significantly repressed tumor growth and induced tumor regressions compared with either drug alone. Furthermore, in a SCLC patient-derived xenograft model that was resistant to ABT-263 alone, the addition of AZD8055 induced potent tumor regression. Therefore, addition of a TORC1/2 inhibitor offers a therapeutic strategy to markedly improve ABT-263 activity in SCLC.

Ceritinib in ALK-rearranged non-small-cell lung cancer.

BACKGROUND: Non-small-cell lung cancer (NSCLC) harboring the anaplastic lymphoma kinase gene (ALK) rearrangement is sensitive to the ALK inhibitor crizotinib, but resistance invariably develops. Ceritinib (LDK378) is a new ALK inhibitor that has shown greater antitumor potency than crizotinib in preclinical studies. METHODS: In this phase 1 study, we administered oral ceritinib in doses of 50 to 750 mg once daily to patients with advanced cancers harboring genetic alterations in ALK. In an expansion phase of the study, patients received the maximum tolerated dose. Patients were assessed to determine the safety, pharmacokinetic properties, and antitumor activity of ceritinib. Tumor biopsies were performed before ceritinib treatment to identify resistance mutations in ALK in a group of patients with NSCLC who had had disease progression during treatment with crizotinib. RESULTS: A total of 59 patients were enrolled in the dose-escalation phase. The maximum tolerated dose of ceritinib was 750 mg once daily; dose-limiting toxic events included diarrhea, vomiting, dehydration, elevated aminotransferase levels, and hypophosphatemia. This phase was followed by an expansion phase, in which an additional 71 patients were treated, for a total of 130 patients overall. Among 114 patients with NSCLC who received at least 400 mg of ceritinib per day, the overall response rate was 58% (95% confidence interval [CI], 48 to 67). Among 80 patients who had received crizotinib previously, the response rate was 56% (95% CI, 45 to 67). Responses were observed in patients with various resistance mutations in ALK and in patients without detectable mutations. Among patients with NSCLC who received at least 400 mg of ceritinib per day, the median progression-free survival was 7.0 months (95% CI, 5.6 to 9.5). CONCLUSIONS: Ceritinib was highly active in patients with advanced, ALK-rearranged NSCLC, including those who had had disease progression during crizotinib treatment, regardless of the presence of resistance mutations in ALK. (Funded by Novartis Pharmaceuticals and others; ClinicalTrials.gov number, NCT01283516.).

MET Exon 14 Skipping in Non-Small Cell Lung Cancer.

BACKGROUND: Non-small cell lung cancers (NSCLCs) harboring specific genetic alterations can be highly sensitive to targeted therapies. MATERIALS AND METHODS: We performed a targeted rearrangement assay on 54 NSCLCs across all stages that were from patients who were never smokers and did not have driver mutations. Because MET exon 14 skipping was the most frequent alteration found, we surveyed the results for MET exon 14 skipping at Massachusetts General Hospital (MGH) since the inclusion of this alteration into our current molecular profiling panel. RESULTS: In a cohort of 54 never-smokers with lung cancers that were wild-type for known driver mutations, MET exon 14 skipping was the most frequently recurring alteration, occurring in 10 cancers (19%). Clinical testing at MGH via our next-generation sequencing (NGS) and NGS-rearrangement panels showed an additional 16 cases of MET exon 14 skipping, for an overall estimated frequency of 5.6%. A clinical case of a patient with MET exon 14 skipping treated with the MET inhibitor crizotinib is also described. CONCLUSION: MET exon 14 skipping is a targetable gene alteration found in NSCLC. Patients with these alterations may respond well to MET inhibition. IMPLICATIONS FOR PRACTICE: MET exon 14 skipping occurs with an approximately 5% frequency in NSCLC and is seen in both squamous and adenocarcinoma histology. Patients whose cancers have MET exon 14 skipping can respond well to MET inhibitors. Molecular testing for MET exon 14 skipping should be performed on all lung cancers because this is a targetable alteration.

Acquired resistance to crizotinib from a mutation in CD74-ROS1.

Crizotinib, an inhibitor of anaplastic lymphoma kinase (ALK), has also recently shown efficacy in the treatment of lung cancers with ROS1 translocations. Resistance to crizotinib developed in a patient with metastatic lung adenocarcinoma harboring a CD74-ROS1 rearrangement who had initially shown a dramatic response to treatment. We performed a biopsy of a resistant tumor and identified an acquired mutation leading to a glycine-to-arginine substitution at codon 2032 in the ROS1 kinase domain. Although this mutation does not lie at the gatekeeper residue, it confers resistance to ROS1 kinase inhibition through steric interference with drug binding. The same resistance mutation was observed at all the metastatic sites that were examined at autopsy, suggesting that this mutation was an early event in the clonal evolution of resistance. (Funded by Pfizer and others; ClinicalTrials.gov number, NCT00585195.).

Inconsistency and features of single nucleotide variants detected in whole exome sequencing versus transcriptome sequencing: A case study in lung cancer.

Whole exome sequencing (WES) and RNA sequencing (RNA-Seq) are two main platforms used for next-generation sequencing (NGS). While WES is primarily for DNA variant discovery and RNA-Seq is mainly for measurement of gene expression, both can be used for detection of genetic variants, especially single nucleotide variants (SNVs). How consistently variants can be detected from WES and RNA-Seq has not been systematically evaluated. In this study, we examined the technical and biological inconsistencies in SNV detection using WES and RNA-Seq data from 27 pairs of tumor and matched normal samples. We analyzed SNVs in three categories: WES unique - those only detected in WES, RNA-Seq unique - those only detected in RNA-Seq, and shared - those detected in both. We found a small overlap (average ∼14%) between the SNVs called in WES and RNA-Seq. The WES unique SNVs were mainly due to low coverage, low expression, or their location on the non-transcribed strand in RNA-Seq data, while the RNA-Seq unique SNVs were primarily due to their location out of the WES-capture boundary regions (accounting ∼71%), as well as low coverage of the regions, low coverage of the mutant alleles or RNA-editing. The shared SNVs had high locus-specific coverage in both WES and RNA-Seq and high gene expression levels. Additionally, WES unique and RNA-Seq unique SNVs showed different nucleotide substitution patterns, e.g., ∼55% of RNA-Seq unique variants were A:T→G:C, a hallmark of RNA editing. This study provides an important evaluation on the inconsistencies of somatic SNVs called in WES and RNA-Seq data.

PI3K regulates MEK/ERK signaling in breast cancer via the Rac-GEF, P-Rex1.

The PI3K pathway is genetically altered in excess of 70% of breast cancers, largely through PIK3CA mutation and HER2 amplification. Preclinical studies have suggested that these subsets of breast cancers are particularly sensitive to PI3K inhibitors; however, the reasons for this heightened sensitivity are mainly unknown. We investigated the signaling effects of PI3K inhibition in PIK3CA mutant and HER2 amplified breast cancers using PI3K inhibitors currently in clinical trials. Unexpectedly, we found that in PIK3CA mutant and HER2 amplified breast cancers sensitive to PI3K inhibitors, PI3K inhibition led to a rapid suppression of Rac1/p21-activated kinase (PAK)/protein kinase C-RAF (C-RAF)/ protein kinase MEK (MEK)/ERK signaling that did not involve RAS. Furthermore, PI3K inhibition led to an ERK-dependent up-regulation of the proapoptotic protein, BIM, followed by induction of apoptosis. Expression of a constitutively active form of Rac1 in these breast cancer models blocked PI3Ki-induced down-regulation of ERK phosphorylation, apoptosis, and mitigated PI3K inhibitor sensitivity in vivo. In contrast, protein kinase AKT inhibitors failed to block MEK/ERK signaling, did not up-regulate BIM, and failed to induce apoptosis. Finally, we identified phosphatidylinositol 3,4,5-trisphosphate-dependent Rac exchanger 1 (P-Rex1) as the PI(3,4,5)P3-dependent guanine exchange factor for Rac1 responsible for regulation of the Rac1/C-RAF/MEK/ERK pathway in these cells. The expression level of P-Rex1 correlates with sensitivity to PI3K inhibitors in these breast cancer cell lines. Thus, PI3K inhibitors have enhanced activity in PIK3CA mutant and HER2 amplified breast cancers in which PI3K inhibition down-regulates both the AKT and Rac1/ERK pathways. In addition, P-Rex1 may serve as a biomarker to predict response to single-agent PI3K inhibitors within this subset of breast cancers.

Transformation from non-small-cell lung cancer to small-cell lung cancer: molecular drivers and cells of origin.

Lung cancer is the most common cause of cancer deaths worldwide. The two broad histological subtypes of lung cancer are small-cell lung cancer (SCLC), which is the cause of 15% of cases, and non-small-cell lung cancer (NSCLC), which accounts for 85% of cases and includes adenocarcinoma, squamous-cell carcinoma, and large-cell carcinoma. Although NSCLC and SCLC are commonly thought to be different diseases owing to their distinct biology and genomic abnormalities, the idea that these malignant disorders might share common cells of origin has been gaining support. This idea has been supported by the unexpected findings that a subset of NSCLCs with mutated EGFR return as SCLC when resistance to EGFR tyrosine kinase inhibitors develops. Additionally, other case reports have described the coexistence of NSCLC and SCLC, further challenging the commonly accepted view of their distinct lineages. Here, we summarise the published clinical observations and biology underlying tumours with combined SCLC and NSCLC histology and cancers that transform from adenocarcinoma to SCLC. We also discuss pre-clinical studies pointing to common potential cells of origin, and speculate how the distinct paths of differentiation are determined by the genomics of each disease.

Bronchial and peripheral murine lung carcinomas induced by T790M-L858R mutant EGFR respond to HKI-272 and rapamycin combination therapy.

The EGFR T790M mutation has been identified in tumors from lung cancer patients that eventually develop resistance to erlotinib. In this study, we generated a mouse model with doxycycline-inducible expression of a mutant EGFR containing both L858R, an erlotinib-sensitizing mutation, and the T790M resistance mutation (EGFR TL). Expression of EGFR TL led to development of peripheral adenocarcinomas with bronchioloalveolar features in alveoli as well as papillary adenocarcinomas in bronchioles. Treatment with an irreversible EGFR tyrosine kinase inhibitor (TKI), HKI-272, shrunk only peripheral tumors but not bronchial tumors. However, the combination of HKI-272 and rapamycin resulted in significant regression of both types of lung tumors. This combination therapy may potentially benefit lung cancer patients with the EGFR T790M mutation.

Combined targeting of HER2 and VEGFR2 for effective treatment of HER2-amplified breast cancer brain metastases.

Brain metastases are a serious obstacle in the treatment of patients with human epidermal growth factor receptor-2 (HER2)-amplified breast cancer. Although extracranial disease is controlled with HER2 inhibitors in the majority of patients, brain metastases often develop. Because these brain metastases do not respond to therapy, they are frequently the reason for treatment failure. We developed a mouse model of HER2-amplified breast cancer brain metastasis using an orthotopic xenograft of BT474 cells. As seen in patients, the HER2 inhibitors trastuzumab and lapatinib controlled tumor progression in the breast but failed to contain tumor growth in the brain. We observed that the combination of a HER2 inhibitor with an anti-VEGF receptor-2 (VEGFR2) antibody significantly slows tumor growth in the brain, resulting in a striking survival benefit. This benefit appears largely due to an enhanced antiangiogenic effect: Combination therapy reduced both the total and functional microvascular density in the brain xenografts. In addition, the combination therapy led to a marked increase in necrosis of the brain lesions. Moreover, we observed even better antitumor activity after combining both trastuzumab and lapatinib with the anti-VEGFR2 antibody. This triple-drug combination prolonged the median overall survival fivefold compared with the control-treated group and twofold compared with either two-drug regimen. These findings support the clinical development of this three-drug regimen for the treatment of HER2-amplified breast cancer brain metastases.

Lung cancers with acquired resistance to EGFR inhibitors occasionally harbor BRAF gene mutations but lack mutations in KRAS, NRAS, or MEK1.

Acquired resistance to EGF receptor (EGFR) tyrosine kinase inhibitors (TKIs) is inevitable in metastatic EGFR-mutant lung cancers. Here, we modeled disease progression using EGFR-mutant human tumor cell lines. Although five of six models displayed alterations already found in humans, one harbored an unexpected secondary NRAS Q61K mutation; resistant cells were sensitive to concurrent EGFR and MEK inhibition but to neither alone. Prompted by this finding and because RAS/RAF/MEK mutations are known mediators of acquired resistance in other solid tumors (colon cancers, gastrointestinal stromal tumors, and melanomas) responsive to targeted therapies, we analyzed the frequency of secondary KRAS/NRAS/BRAF/MEK1 gene mutations in the largest collection to date of lung cancers with acquired resistance to EGFR TKIs. No recurrent NRAS, KRAS, or MEK1 mutations were found in 212, 195, or 146 patient samples, respectively, but 2 of 195 (1%) were found to have mutations in BRAF (G469A and V600E). Ectopic expression of mutant NRAS or BRAF in drug-sensitive EGFR-mutant cells conferred resistance to EGFR TKIs that was overcome by addition of a MEK inhibitor. Collectively, these positive and negative results provide deeper insight into mechanisms of acquired resistance to EGFR TKIs in lung cancer and inform ongoing clinical trials designed to overcome resistance. In the context of emerging knowledge about mechanisms of acquired resistance to targeted therapies in various cancers, our data highlight the notion that, even though solid tumors share common signaling cascades, mediators of acquired resistance must be elucidated for each disease separately in the context of treatment.

Not just gRASping at flaws: finding vulnerabilities to develop novel therapies for treating KRAS mutant cancers.

Mutations in Kirsten rat-sarcoma (KRAS) are well appreciated to be major drivers of human cancers through dysregulation of multiple growth and survival pathways. Similar to many other non-kinase oncogenes and tumor suppressors, efforts to directly target KRAS pharmaceutically have not yet materialized. As a result, there is broad interest in an alternative approach to develop therapies that induce synthetic lethality in cancers with mutant KRAS, therefore exposing the particular vulnerabilities of these cancers. Fueling these efforts is our increased understanding into the biology driving KRAS mutant cancers, in particular the important pathways that mutant KRAS governs to promote survival. In this mini-review, we summarize the latest approaches to treat KRAS mutant cancers and the rationale behind them.

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.