ARAF protein kinase activates RAS by antagonizing its binding to RASGAP NF1.

RAF protein kinases are effectors of the GTP-bound form of small guanosine triphosphatase RAS and function by phosphorylating MEK. We showed here that the expression of ARAF activated RAS in a kinase-independent manner. Binding of ARAF to RAS displaced the GTPase-activating protein NF1 and antagonized NF1-mediated inhibition of RAS. This reduced ERK-dependent inhibition of RAS and increased RAS-GTP. By this mechanism, ARAF regulated the duration and consequences of RTK-induced RAS activation and supported the RAS output of RTK-dependent tumor cells. In human lung cancers with EGFR mutation, amplification of ARAF was associated with acquired resistance to EGFR inhibitors, which was overcome by combining EGFR inhibitors with an inhibitor of the protein tyrosine phosphatase SHP2 to enhance inhibition of nucleotide exchange and RAS activation.

Japanese Society of Medical Oncology clinical guidelines: Molecular testing for colorectal cancer treatment, 5th edition.

Molecular testing to determine optimal therapies is essential for managing patients with colorectal cancer (CRC). In October 2022, the Japanese Society of Medical Oncology published the 5th edition of the Molecular Testing Guideline for Colorectal Cancer Treatment. In this guideline, in patients with unresectable CRC, RAS/BRAF V600E mutational and mismatch repair tests are strongly recommended prior to first-line chemotherapy to select optimal first- and second-line therapies. In addition, HER2 testing is strongly recommended because the pertuzumab plus trastuzumab combination is insured after fluoropyrimidine, oxaliplatin, and irinotecan in Japan. Circulating tumor DNA (ctDNA)-based RAS testing is also strongly recommended to assess the indications for the readministration of anti-EGFR antibodies. Both tissue- and ctDNA-based comprehensive genomic profiling tests are strongly recommended to assess the indications for targeted molecular drugs, although they are currently insured in patients with disease progression after receiving standard chemotherapy (or in whom disease progression is expected in the near future). Mutational and mismatch repair testing is strongly recommended for patients with resectable CRC, and RAS/BRAF V600E mutation testing is recommended to estimate the risk of recurrence. Mutational and mismatch repair and BRAF testing are also strongly recommended for screening for Lynch syndrome. Circulating tumor DNA-based minimal residual disease (MRD) testing is strongly recommended for estimating the risk of recurrence based on clinical evidence, although MRD testing was not approved in Japan at the time of the publication of this guideline.

Effects of ABCB1 and ABCG2 polymorphisms on the pharmacokinetics of abemaciclib.

PURPOSE: Adverse events after the use of the CDK4/6 inhibitor abemaciclib are dose-dependent. However, its pharmacokinetics varies among individuals. Abemaciclib is reportedly transported by P-glycoprotein and breast cancer resistance protein. Therefore, we evaluated whether ABCB1 and ABCG2 polymorphisms are pharmacokinetic predictive factors of abemaciclib. METHODS: A total of 45 patients with breast cancer taking abemaciclib (150 mg twice per day) for 2 weeks were evaluated to determine the associations among abemaciclib concentration; adverse events; and ABCB1 1236 T > C, 2677G > T/A, 3435C > T, and ABCG2 421C > A gene polymorphisms. RESULTS: The trough concentration of abemaciclib was significantly higher in the group with grade 2 or greater neutropenia and thrombocytopenia than in those with grades 0 or 1. For ABCB1 2677G > T/A polymorphisms, the concentration of abemaciclib tended to be higher in the homozygous group (TT + AT) than in the wild-type + heterozygous group (GG + GA + GT) (median [range], 222.8 [80.5-295.8] ng/mL vs. 113.5 [23.6-355.2] ng/mL, P = 0.09), Moreover, the ABCB1 2677G > T/A homozygous group had a higher tendency of abemaciclib withdrawal or dose reduction within 4 weeks than the wild-type + heterozygous group (odds ratio, 4.22; 95% confidence interval, 0.86-20.7; P = 0.08). No significant association was observed among abemaciclib concentration; adverse reactions; and ABCB1 1236 T > C, 3435C > T, and ABCG2 421C > A polymorphisms. CONCLUSION: ABCB1 2677G > T/A polymorphism might be a predictor of the pharmacokinetics and tolerability of abemaciclib.

CIRCULATE-Japan: Circulating tumor DNA-guided adaptive platform trials to refine adjuvant therapy for colorectal cancer.

Adjuvant chemotherapy has reduced the risk of tumor recurrence and improved survival in patients with resected colorectal cancer. Potential utility of circulating tumor DNA (ctDNA) prior to and post surgery has been reported across various solid tumors. We initiated a new type of adaptive platform trials to evaluate the clinical benefits of ctDNA analysis and refine precision adjuvant therapy for resectable colorectal cancer, named CIRCULATE-Japan including three clinical trials. The GALAXY study is a prospectively conducted large-scale registry designed to monitor ctDNA for patients with clinical stage II to IV or recurrent colorectal cancer who can undergo complete surgical resection. The VEGA trial is a randomized phase III study designed to test whether postoperative surgery alone is noninferior to the standard therapy with capecitabine plus oxaliplatin for 3 months in patients with high-risk stage II or low-risk stage III colon cancer if ctDNA status is negative at week 4 after curative surgery in the GALAXY study. The ALTAIR trial is a double-blind, phase III study designed to establish the superiority of trifluridine/tipiracil as compared with placebo in patients with resected colorectal cancer who show circulating tumor-positive status in the GALAXY study. Therefore, CIRCULATE-Japan encompasses both "de-escalation" and "escalation" trials for ctDNA-negative and -positive patients, respectively, and helps to answer whether measuring ctDNA postoperatively has prognostic and/or predictive value. Our ctDNA-guided adaptive platform trials will accelerate clinical development toward further precision oncology in the field of adjuvant therapy. Analysis of ctDNA status could be utilized as a predictor of risk stratification for recurrence and to monitor the effectiveness of adjuvant chemotherapy. ctDNA is a promising, noninvasive tumor biomarker that can aid in tumor monitoring throughout disease management.

FMS-like tyrosine kinase 3 (FLT3) amplification in patients with metastatic colorectal cancer.

FMS-like tyrosine kinase 3 (FLT3) plays a key role in hematopoiesis. However, the oncogenic role of FLT3 amplification in patients with metastatic colorectal cancer (mCRC) remains unclear. Here, we aimed to evaluate the characteristics, prognosis, and treatment efficacy of an FLT3 inhibitor (regorafenib) in patients with mCRC with FLT3 amplifications. Tumor tissue samples from 2329 patients were sequenced using NGS in the Nationwide Cancer Genome Screening Project in Japan. The effects of clinicopathological features, co-altered genes, prognosis, and efficacy of regorafenib were investigated. Between April 2015 and June 2018, 85 patients with mCRC with FLT3 amplification were observed. There were no differences in baseline characteristics between patients with or without FLT3 amplification. The frequency of RAS or other gene co-alterations was inversely correlated with the copy number status. Median survival time in patients with FLT3 amplification was significantly shorter compared with those with non-FLT3 amplification. Further investigations of FLT3 amplification as a potential treatment target in mCRC are warranted.

Serum concentration of the CKD4/6 inhibitor abemaciclib, but not of creatinine, strongly predicts hematological adverse events in patients with breast cancer: a preliminary report.

Purpose The CKD4/6 inhibitor abemaciclib is related to adverse events such as hematological toxicity and increase in serum creatinine levels associated with abemaciclib pharmacokinetics. Increase in serum creatinine levels is considered a result of competition with abemaciclib via organic cation transporter 2 and multidrug and toxic compound extrusion. Therefore, we evaluated the association among serum creatinine levels, serum abemaciclib concentrations, and adverse events and whether increase in serum creatinine levels is a useful indicator for predicting the onset of the adverse events of abemaciclib. Methods In total, the data of 12 patients with breast cancer who were treated with abemaciclib (150 mg twice daily) were evaluated to determine the association between increased serum creatinine levels and abemaciclib concentrations and hematological toxicity. Results Grade 3 neutropenia, thrombocytopenia, and anemia were observed at 4 weeks in four (33%), two (17%), and one (8%) patients, respectively. A significant association was observed between steady-state abemaciclib concentrations and the rate of decrease in neutrophil and platelet counts (r = - 0.80, P = 0.003 and r = - 0.70, P = 0.016, respectively). Compared with baseline levels (0.61 [0.53-0.82] mg/mL), serum creatinine levels significantly increased and reached a steady state in at least 2 weeks (0.84 [0.61-1.02] mg/mL, P = 0.01). However, we did not find a significant association between increase in serum creatinine levels and abemaciclib concentrations and hematological toxicity. Conclusions Abemaciclib concentrations are associated with neutropenia and thrombocytopenia. However, increase in serum creatinine levels may not be a useful predictor for estimating abemaciclib pharmacokinetics and hematological toxicity.

The ADAM17 protease promotes tobacco smoke carcinogen-induced lung tumorigenesis.

Lung cancer is the leading cause of cancer-related mortality, with most cases attributed to tobacco smoking, in which nicotine-derived nitrosamine ketone (NNK) is the most potent lung carcinogen. The ADAM17 protease is responsible for the ectodomain shedding of many pro-tumorigenic cytokines, growth factors and receptors, and therefore is an attractive target in cancer. However, the role of ADAM17 in promoting tobacco smoke carcinogen-induced lung carcinogenesis is unknown. The hypomorphic Adam17ex/ex mice-characterized by reduced global ADAM17 expression-were backcrossed onto the NNK-sensitive pseudo-A/J background. CRISPR-driven and inhibitor-based (GW280264X, and ADAM17 prodomain) ADAM17 targeting was employed in the human lung adenocarcinoma cell lines A549 and NCI-H23. Human lung cancer biopsies were also used for analyses. The Adam17ex/ex mice displayed marked protection against NNK-induced lung adenocarcinoma. Specifically, the number and size of lung lesions in NNK-treated pseudo-A/J Adam17ex/ex mice were significantly reduced compared with wild-type littermate controls. This was associated with lower proliferative index throughout the lung epithelium. ADAM17 targeting in A549 and NCI-H23 cells led to reduced proliferative and colony-forming capacities. Notably, among select ADAM17 substrates, ADAM17 deficiency abrogated shedding of the soluble IL-6 receptor (sIL-6R), which coincided with the blockade of sIL-6R-mediated trans-signaling via ERK MAPK cascade. Furthermore, NNK upregulated phosphorylation of p38 MAPK, whose pharmacological inhibition suppressed ADAM17 threonine phosphorylation. Importantly, ADAM17 threonine phosphorylation was significantly upregulated in human lung adenocarcinoma with smoking history compared with their cancer-free controls. Our study identifies the ADAM17/sIL-6R/ERK MAPK axis as a candidate therapeutic strategy against tobacco smoke-associated lung carcinogenesis.

Japanese Society of Medical Oncology Clinical Guidelines: Molecular Testing for Colorectal Cancer Treatment, 4th edition.

Molecular testing to select the appropriate targeted and standard of care therapies is essential for managing patients with colorectal cancer (CRC). The Japanese Society of Medical Oncology previously published clinical guidelines for molecular testing in CRC. In the third edition published in 2018, RAS and BRAF V600E mutations should be tested prior to first-line chemotherapy to assess the benefit of anti-epidermal growth factor receptor (EGFR) antibody therapy in patients with unresectable CRC. Microsatellite instability (MSI) testing was recommended in patients with curatively resected stage II CRC because deficient mismatch repair is associated with low risk of recurrence. MSI testing was also recommended in patients with CRC suspected to be Lynch syndrome. The main aim of this fourth edition is to reflect recent advances in comprehensive genomic profiling (CGP) tests and liquid biopsy. Here, CGP tests performed on tumor tissues are strongly recommended to assess the benefit of molecular targeted drugs in patients with CRC. Circulating tumor DNA (ctDNA)-based CGP tests are also proposed. ctDNA testing is recommended to determine the optimal treatment based on the risk of recurrence for curatively resected CRC and evaluate the suitability and monitor the therapeutic effects of anti-EGFR antibodies in patients with unresectable CRC. While both MSI testing and immunohistochemistry are strongly recommended to determine the indication of immune checkpoint inhibitors in patients with unresectable CRC, next-generation sequencing-based tests are weakly recommended because these tests have not been validated in clinical trials.

Anaplastic lymphoma kinase expression in small-cell lung cancer.

AIMS: Anaplastic lymphoma kinase (ALK) immunohistochemistry has shifted from being a screening tool to being a sole determinant for ALK-targeted therapy. Recent articles have referred to small-cell lung cancer (SCLC) transformation as a resistance mechanism after ALK inhibitor treatments, but few reports have addressed ALK expression in treatment-naive SCLC in a comprehensive manner. Therefore, we examined ALK expression and the mechanisms in treatment-naive SCLCs. METHODS AND RESULTS: We examined ALK expression in a consecutive series of SCLC tumours, and the expression mechanism was analysed regarding gene rearrangement, copy number changes, and point mutations. We also examined whether SCLC with ALK expression can be suppressed by crizotinib treatment in vitro. Immunohistochemical results revealed that ALK was expressed in 16 of 142 (11.3%) SCLCs. The expression was focal and less intense, which is in contrast to strong and uniform expression in adenocarcinoma with ALK rearrangement. Two combined SCLCs showed a positive reaction restricted to the SCLC component. None of the known genetic alterations, including rearrangement, amplification, copy number gain, or point mutations, were associated with ALK expression. A SCLC cell line, SKLC2, which expressed ALK without known genetic alterations, was not inhibited by a practically achievable serum concentration of crizotinib. CONCLUSIONS: Anaplastic lymphoma kinase immunohistochemistry for treatment-naive SCLCs should not be used as a predictive biomarker for ALK inhibitor therapy, because the positive reactions were due to intrinsic expression of normal ALK transcript.

Japanese Society of Medical Oncology Clinical Guidelines: Molecular Testing for Colorectal Cancer Treatment, Third Edition.

The Japanese Society of Medical Oncology (JSMO) previously published 2 editions of the clinical guidelines: "Japanese guidelines for testing of KRAS gene mutation in colorectal cancer" in 2008 and "Japanese Society of Medical Oncology Clinical Guidelines: RAS (KRAS/NRAS) mutation testing in colorectal cancer patients" in 2014. These guidelines have contributed to the proper use of KRAS and RAS mutation testing, respectively. Recently, clinical utility, particularly for colorectal cancer (CRC) patients with BRAF V600E mutation or DNA mismatch-repair (MMR) deficiency, has been established. Therefore, the guideline members decided these genetic alterations should also be involved. The aim of this revision is to properly carry out testing for BRAF V600E mutation and MMR deficiency in addition to RAS mutation. The revised guidelines include the basic requirements for testing for these genetic alterations based on recent scientific evidence. Furthermore, because clinical utility of comprehensive genetic testing using next-generation sequencing and somatic gene testing of analyzing circulating tumor DNA has increasingly evolved with recent advancements in testing technology, we noted the current situation and prospects for these testing technologies and their clinical implementation in the revised guidelines.

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.

Pulmonary carcinosarcoma showing an obvious response to pazopanib: a case report.

BACKGROUND: Pulmonary carcinosarcoma (PCS) is a rare primary lung malignancy and has a poor prognosis among lung tumor histological subtypes. However, an appropriate treatment strategy has not been developed for unresectable PCS. CASE PRESENTATION: A 65-year-old man who was diagnosed with PCS was treated by surgical removal of the primary lung lesion, followed by six cycles of adjuvant chemotherapy with cisplatin plus irinotecan. Following the chemotherapy, he experienced a relapse with brain metastasis, which induced the rapid onset of left leg paralysis. Radical surgical resection and stereotactic radiosurgery to the resection cavity were performed. However, meningeal dissemination and new lung metastases occurred after a year and half. To control these multiple metastatic lesions, the patient was treated with the multiple kinase inhibitor pazopanib. No change was observed in the meningeal dissemination, while the metastatic lung lesions were prominently reduced in size following treatment with pazopanib. Consequently, the patient showed a partial response to pazopanib treatment, although the dose of pazopanib was reduced by half as a result of thrombocytopenia. CONCLUSION: This is the first report of metastatic PCS showing an evident therapeutic response to tumor-targeted therapy. We suggest that pazopanib may be a therapeutic option for patients with metastatic PCS.

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.

[Successful Long-Term Treatment with Intermittent Oxaliplatin Administration for Recurrent Sigmoid Colon Cancer without Developing Neuropathy].

We report a case ofrecurrent sigmoid cancer in which long-term disease control was achieved by intermittent oxaliplatin (L-OHP)administration. A 58-year-old woman underwent first-line chemotherapy with capecitabine and L-OHP(CapeOX) following peritoneal lymph node recurrence of sigmoid cancer. Tumor shrinkage was confirmed by a computed tomography (CT)scan following 4 courses of CapeOX treatment. However, L-OHP administration was discontinued by the 9 course due to peritoneal neuropathy. L-OHP was reintroduced following tumor progression confirmed by CT or elevation of carcinoembryonic antigen levels detected by a blood test. This stop-and-go strategy controlled lymph node recurrence effectively for over 5 years and was not associated with the development ofperitoneal neuropathy. We suggest that the intermittent administration ofL -OHP-containing chemotherapy is an important treatment option for some patients with advanced colorectal cancer, achieving effective long-term disease control with no associated peripheral neuropathy.

Resistance mediated by alternative receptor tyrosine kinases in FGFR1-amplified lung cancer.

Fibroblast growth factor receptor 1 (FGFR1) amplification has been identified in 10-20% of patients with squamous non-small-cell lung cancer. Preclinical models showed promising activity of specific FGFR inhibitors, but early clinical trials showed that only a small fraction of patients with FGFR1-amplified lung cancer responded to FGFR inhibitors. These unsatisfactory results were partly explained by heterogeneous amplicons around the 8p11 genomic region, leading to false-positive amplification results. Furthermore, discrepancies in the gene amplification and protein expression of FGFR1 were also reported. In this study, we identified the roles of alternative receptor tyrosine kinases (RTKs) in FGFR1-amplified lung cancer. These alternative RTKs dominantly activate phosphoinositide 3-kinase-AKT signaling and also mitigate sustained inhibition of mitogen-activated protein kinase signaling by FGFR inhibitors. The rebound activation of extracellular signal-regulated kinase phosphorylation was associated with sensitivity to the drugs. Combinatorial inhibition of alternative RTKs and FGFR1 was required to suppress both AKT and extracellular signal-regulated kinase phosphorylation and to induce key pro-apoptotic proteins BIM and p53 upregulated modulator of apoptosis (PUMA). Furthermore, even in FGFR inhibitor-sensitive NCI-H1581 lung cancer cells, MET-expressing clones were already detectable at a very low frequency before resistance induction. Selection of these pre-existing subclones resulted in FGFR inhibitor resistance because of the activation of AKT and extracellular signal-regulated kinase by MET signaling that was mediated by GRB2 associated binding protein 1 (GAB1). These results suggest that incomplete suppression of key survival signals led to intrinsic and acquired resistance to FGFR inhibitors. Our results may help explain the low clinical response rates to FGFR inhibitors in FGFR1-amplified lung cancer.

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.

Japanese Society of Medical Oncology Clinical Guidelines: RAS (KRAS/NRAS) mutation testing in colorectal cancer patients.

The Japanese guidelines for the testing of KRAS mutations in colorectal cancer have been used for the past 5 years. However, new findings of RAS (KRAS/NRAS) mutations that can further predict the therapeutic effects of anti-epidermal growth factor receptor (EGFR) antibody therapy necessitated a revision of the guidelines. The revised guidelines included the following five basic requirements for RAS mutation testing to highlight a patient group in which anti-EGFR antibody therapy may be ineffective: First, anti-EGFR antibody therapy may not offer survival benefit and/or tumor shrinkage to patients with expanded RAS mutations. Thus, current methods to detect KRAS exon 2 (codons 12 and 13) mutations are insufficient for selecting appropriate candidates for this therapy. Additional testing of extended KRAS/NRAS mutations is recommended. Second, repeated tests are not required for the detection; tissue materials of either primary or metastatic lesions are applicable for RAS mutation testing. Evaluating RAS mutations prior to anti-EGFR antibody therapy is recommended. Third, direct sequencing with manual dissection or allele-specific PCR-based methods is currently applicable for RAS mutation testing. Fourth, thinly sliced sections of formalin-fixed, paraffin-embedded tissue blocks are applicable for RAS mutation testing. One section stained with H&E should be provided to histologically determine whether the tissue contains sufficient amount of tumor cells for testing. Finally, RAS mutation testing must be performed in laboratories with appropriate testing procedures and specimen management practices.

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.

Combined inhibition of KRASG12C and mTORC1 kinase is synergistic in non-small cell lung cancer.

Current KRASG12C (OFF) inhibitors that target inactive GDP-bound KRASG12C cause responses in less than half of patients and these responses are not durable. A class of RASG12C (ON) inhibitors that targets active GTP-bound KRASG12C blocks ERK signaling more potently than the inactive-state inhibitors. Sensitivity to either class of agents is strongly correlated with inhibition of mTORC1 activity. We have previously shown that PI3K/mTOR and ERK-signaling pathways converge on key cellular processes and that inhibition of both pathways is required for inhibition of these processes and for significant antitumor activity. We find here that the combination of a KRASG12C inhibitor with a selective mTORC1 kinase inhibitor causes synergistic inhibition of Cyclin D1 expression and cap-dependent translation. Moreover, BIM upregulation by KRASG12C inhibition and inhibition of MCL-1 expression by the mTORC1 inhibitor are both required to induce significant cell death. In vivo, this combination causes deep, durable tumor regressions and is well tolerated. This study suggests that the ERK and PI3K/mTOR pathways each mitigate the effects of inhibition of the other and that combinatorial inhibition is a potential strategy for treating KRASG12C-dependent lung cancer.