SHP2 inhibitors maintain TGFß signalling through SMURF2 inhibition.

Despite the promising antitumor activity of SHP2 inhibitors in RAS-dependent tumours, overall responses have been limited by their narrow therapeutic window. Like with all MAPK pathway inhibitors, this is likely the result of compensatory pathway activation mechanisms. However, the underlying mechanisms of resistance to SHP2 inhibition remain unknown. The E3 ligase SMURF2 limits TGFß activity by ubiquitinating and targeting the TGFß receptor for proteosome degradation. Using a functional RNAi screen targeting all known phosphatases, we identify that the tyrosine phosphatase SHP2 is a critical regulator of TGFß activity. Specifically, SHP2 dephosphorylates two key residues on SMURF2, resulting in activation of the enzyme. Conversely, SHP2 depletion maintains SMURF2 in an inactive state, resulting in the maintenance of TGFß activity. Furthermore, we demonstrate that depleting SHP2 has significant implications on TGFß-mediated migration, senescence, and cell survival. These effects can be overcome through the use of TGFß-targeted therapies. Consequently, our findings provide a rationale for combining SHP2 and TGFß inhibitors to enhance tumour responses leading to improved patient outcomes.

Scribble mis-localization induces adaptive resistance to KRAS G12C inhibitors through feedback activation of MAPK signaling mediated by YAP-induced MRAS.

Tumor cells evade targeted drugs by rewiring their genetic and epigenetic networks. Here, we identified that inhibition of MAPK signaling rapidly induces an epithelial-to-mesenchymal transition program by promoting re-localization of an apical-basal polarity protein, Scribble, in oncogene-addicted lung cancer models. Mis-localization of Scribble suppressed Hippo-YAP signaling, leading to YAP nuclear translocation. Furthermore, we discovered that a RAS superfamily protein MRAS is a direct target of YAP. Treatment with KRAS G12C inhibitors induced MRAS expression, which formed a complex with SHOC2, precipitating feedback activation of MAPK signaling. Abrogation of YAP activation or MRAS induction enhanced the efficacy of KRAS G12C inhibitor treatment in vivo. These results highlight a role for protein localization in the induction of a non-genetic mechanism of resistance to targeted therapies in lung cancer. Furthermore, we demonstrate that induced MRAS expression is a key mechanism of adaptive resistance following KRAS G12C inhibitor treatment.

High-risk neuroblastoma with NF1 loss of function is targetable using SHP2 inhibition.

Reoccurring/high-risk neuroblastoma (NB) tumors have the enrichment of non-RAS/RAF mutations along the mitogen-activated protein kinase (MAPK) signaling pathway, suggesting that activation of MEK/ERK is critical for their survival. However, based on preclinical data, MEK inhibitors are unlikely to be active in NB and have demonstrated dose-limiting toxicities that limit their use. Here, we explore an alternative way to target the MAPK pathway in high-risk NB. We find that NB models are among the most sensitive among over 900 tumor-derived cell lines to the allosteric SHP2 inhibitor SHP099. Sensitivity to SHP099 in NB is greater in models with loss or low expression of the RAS GTPase activation protein (GAP) neurofibromin 1 (NF1). Furthermore, NF1 is lower in advanced and relapsed NB and NF1 loss is enriched in high-risk NB tumors regardless of MYCN status. SHP2 inhibition consistently blocks tumor growth in high-risk NB mouse models, revealing a new drug target in relapsed NB.

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.

Escaping KRAS: Gaining Autonomy and Resistance to KRAS Inhibition in KRAS Mutant Cancers.

Activating mutations in KRAS are present in 25% of human cancers. When mutated, the KRAS protein becomes constitutively active, stimulating various effector pathways and leading to the deregulation of key cellular processes, including the suppression of apoptosis and enhancement of proliferation. Furthermore, mutant KRAS also promotes metabolic deregulation and alterations in the tumor microenvironment. However, some KRAS mutant cancer cells become independent of KRAS for their survival by activating diverse bypass networks that maintain essential survival signaling originally governed by mutant KRAS. The proposed inducers of KRAS independency are the activation of YAP1 and/or RSK-mTOR pathways and co-mutations in SKT11 (LKB1), KEAP1, and NFE2L2 (NRF2) genes. Metabolic reprogramming, such as increased glutaminolysis, is also associated with KRAS autonomy. The presence or absence of KRAS dependency is related to the heterogeneity of KRAS mutant cancers. Epithelial-to-mesenchymal transition (EMT) in tumor cells is also a characteristic phenotype of KRAS independency. Translationally, this loss of dependence is a cause of primary and acquired resistance to mutant KRAS-specific inhibitors. While KRAS-dependent tumors can be treated with mutant KRAS inhibitor monotherapy, for KRAS-independent tumors, we need an improved understanding of activated bypass signaling pathways towards leveraging vulnerabilities, and advancing therapeutic options for this patient subset.

Epithelial-to-Mesenchymal Transition is a Cause of Both Intrinsic and Acquired Resistance to KRAS G12C Inhibitor in KRAS G12C-Mutant Non-Small Cell Lung Cancer.

PURPOSE: KRAS is among the most commonly mutated oncogene in cancer including non-small cell lung cancer (NSCLC). In early clinical trials, inhibitors targeting G12C-mutant KRAS have achieved responses in some patients with NSCLC. Possible intrinsic and acquired resistance mechanisms to KRAS G12C inhibitors are not fully elucidated and will likely become important to identify. EXPERIMENTAL DESIGN: To identify potential resistance mechanisms, we defined the sensitivity of a panel of KRAS G12C-mutant lung cancer cell lines to a KRAS G12C inhibitor, AMG510. Gene set enrichment analyses were performed to identify pathways related to the sensitivity, which was further confirmed biochemically. In addition, we created two cell lines that acquired resistance to AMG510 and the underlying resistance mechanisms were analyzed. RESULTS: KRAS expression and activation were associated with sensitivity to KRAS G12C inhibitor. Induction of epithelial-to-mesenchymal transition (EMT) led to both intrinsic and acquired resistance to KRAS G12C inhibition. In these EMT-induced cells, PI3K remained activated in the presence of KRAS G12C inhibitor and was dominantly regulated by the IGFR-IRS1 pathway. We found SHP2 plays a minimal role in the activation of the PI3K pathway in contrast to its critical role in the activation of the MAPK pathway. The combination of KRAS G12C inhibitor, PI3K inhibitor, and SHP2 inhibitor resulted in tumor regressions in mouse models of acquired resistance to AMG510. CONCLUSIONS: Our findings suggest that EMT is a cause of both intrinsic and acquired resistance by activating the PI3K pathway in the presence of KRAS G12C inhibitor.

MET amplification results in heterogeneous responses to osimertinib in EGFR-mutant lung cancer treated with erlotinib.

The third-generation epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) osimertinib is approved for untreated, or previously EGFR-TKI-treated T790M-positive EGFR-mutated non-small cell lung carcinoma (NSCLC). We investigated the heterogeneity of responses to osimertinib and its underlying mechanisms. A patient with EGFR-L858R-mutated NSCLC was treated with erlotinib. Following treatment, he developed brain and multiple bone metastases and was eventually diagnosed with NSCLC with EGFR-T790M mutation. The responses of various tumor specimens to osimertinib were heterogeneous. We investigated EGFR-T790M and MET amplification using PCR and FISH in autopsy specimens of the cervical spine, lumbar spine, and brain. We established the KNZ osimertinib-resistant (KNZ_OR) tumor cell line with MET amplification using a cervical spine lesion that was intrinsically resistant to osimertinib. We evaluated the effects of MET knockdown and MET inhibitor on KNZ_OR cell sensitivity to osimertinib in vitro and in vivo. Osimertinib-resistant lesions (cervical spine and brain) showed EGFR-L858R and MET amplification, but not EGFR-T790M, whereas osimertinib-sensitive lesions (lumbar spine) showed EGFR-L858R and -T790, but not MET amplification. Osimertinib decreased the association of amplified MET with L858R-mutated EGFR but increased that with human epidermal growth factor receptor 3 in KNZ_OR cells. MET knockdown or MET inhibitor sensitized KNZ_OR cells to osimertinib in vitro, indicating that MET amplification induced osimertinib resistance. Combination with osimertinib plus crizotinib induced tumor shrinkage in the KNZ_OR xenograft model. Hence, MET amplification might induce heterogeneous responses to osimertinib in EGFR-mutated NSCLC. Further investigations on mutated EGFR and amplified MET might lead to the development of effective therapies.

Reduced doses of dabrafenib and trametinib combination therapy for BRAF V600E-mutant non-small cell lung cancer prevent rhabdomyolysis and maintain tumor shrinkage: a case report.

BACKGROUND: A BRAF V600E mutation is found as driver oncogene in patients with non-small cell lung cancer. Although combined treatment with dabrafenib and trametinib is highly effective, the efficacy of reduced doses of the drugs in combination therapy has not yet been reported. CASE PRESENTATION: A Japanese man in his mid-sixties was diagnosed with unresectable lung adenocarcinoma and was unresponsive to cytotoxic chemotherapy and immune checkpoint inhibitors. The BRAF V600E mutation was detected by next generation sequencing, and the patient was subjected to treatment with dabrafenib and trametinib in combination. Although the treatment reduced the tumor size, he experienced myalgia and muscle weakness with elevated serum creatine kinase and was diagnosed with rhabdomyolysis induced by dabrafenib and trametinib. After the patient recovered from rhabdomyolysis, the treatment doses of dabrafenib and trametinib were reduced, which prevented further rhabdomyolysis and maintained tumor shrinkage. CONCLUSION: The reduction of the doses of dabrafenib and trametinib was effective in the treatment of BRAF V600E-mutant NSCLC, and also prevented the incidence of rhabdomyolysis.

Aberrant Methylation of Tumor Suppressive miRNAs in Bile from Patients With Pancreaticobiliary Diseases.

BACKGROUND/AIM: Epigenetic abnormalities in microRNAs (miRNAs) have not been analyzed in samples other than pancreaticobiliary tissues in patients with pancreaticobiliary cancer (PBC). To identify miRNAs specific for PBC, the present study analyzed the methylation of tumor-suppressive miRNAs in bile from patients with pancreaticobiliary diseases. MATERIALS AND METHODS: Bile was collected endoscopically or percutaneously from 52 patients with pancreatic cancer, 26 with biliary tract cancer, and 20 with benign pancreaticobiliary diseases. Sequences encoding 16 tumor-suppressive miRNAs were amplified by polymerase chain reaction and sequenced, and their methylation rates were determined. RESULTS: The methylation rates of miR-1247 and miR-200a were significantly higher in patients with pancreatic cancer, and biliary tract cancer than in those with benign diseases, and the methylation rate of miR-200b was significantly higher in patients with pancreatic cancer than in those with benign diseases. CONCLUSION: Methylation of miR-1247, miR-200a, and miR-200b in bile may be useful for distinguishing PBC from benign diseases.

AXL confers intrinsic resistance to osimertinib and advances the emergence of tolerant cells.

A novel EGFR-tyrosine kinase inhibitor (TKI), osimertinib, has marked efficacy in patients with EGFR-mutated lung cancer. However, some patients show intrinsic resistance and an insufficient response to osimertinib. This study showed that osimertinib stimulated AXL by inhibiting a negative feedback loop. Activated AXL was associated with EGFR and HER3 in maintaining cell survival and inducing the emergence of cells tolerant to osimertinib. AXL inhibition reduced the viability of EGFR-mutated lung cancer cells overexpressing AXL that were exposed to osimertinib. The addition of an AXL inhibitor during either the initial or tolerant phases reduced tumor size and delayed tumor re-growth compared to osimertinib alone. AXL was highly expressed in clinical specimens of EGFR-mutated lung cancers and its high expression was associated with a low response rate to EGFR-TKI. These results indicated pivotal roles for AXL and its inhibition in the intrinsic resistance to osimertinib and the emergence of osimertinib-tolerant cells.

Distinct dependencies on receptor tyrosine kinases in the regulation of MAPK signaling between BRAF V600E and non-V600E mutant lung cancers.

BRAF is one of the most frequently mutated genes across a number of different cancers, with the best-characterized mutation being V600E. Despite the successes of treating BRAF mutant V600E lung cancer with BRAF pathway inhibitors, treatment strategies targeting tumors with non-V600E mutations are yet to be established. We studied cellular signaling differences between lung cancers with different BRAF mutations and determined their sensitivities to BRAF pathway inhibitors. Here, we observed that MEK inhibition induced feedback activation of the receptor tyrosine kinase (RTK) EGFR, and in some cases the RTK FGFR, resulting in transient suppression of ERK phosphorylation in BRAF non-V600E, but not BRAF V600E, mutant cells. Furthermore, we found that both EGFR and FGFR activated the MEK/ERK pathway, despite the presence of BRAF non-V600E mutations with elevated kinase activity. Moreover, in BRAF non-V600E mutants with impaired kinase activities, EGFR had even greater control over the MEK/ERK pathway, essentially contributing completely to the tonic mitogen-activated protein kinase (MAPK) signal. Accordingly, the combination of MEK inhibitor with EGFR inhibitor was effective at shrinking tumors in mouse model of BRAF non-V600E mutant lung cancer. Furthermore, the results were recapitulated with a clinically relevant dual inhibitor of EGFR and RAF, BGB-283. Overall, although BRAF V600E mutant cells are sensitive to BRAF inhibition, non-V600E mutant cancer cells are reliant on RTKs for their MAPK activation and inhibiting both MEK and RTKs are necessary in these cancers. Our findings provide evidence of critical survival signals in BRAF non-V600E mutant cancers, which could pave the way for effective treatment of these cancers.

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.

Epithelial-to-Mesenchymal Transition Defines Feedback Activation of Receptor Tyrosine Kinase Signaling Induced by MEK Inhibition in KRAS-Mutant Lung Cancer.

UNLABELLED: KRAS is frequently mutated in lung cancer. Whereas MAPK is a well-known effector pathway of KRAS, blocking this pathway with clinically available MAPK inhibitors is relatively ineffective. Here, we report that epithelial-to-mesenchymal transition rewires the expression of receptor tyrosine kinases, leading to differential feedback activation of the MAPK pathway following MEK inhibition. In epithelial-like KRAS-mutant lung cancers, this feedback was attributed to ERBB3-mediated activation of MEK and AKT. In contrast, in mesenchymal-like KRAS-mutant lung cancers, FGFR1 was dominantly expressed but suppressed by the negative regulator Sprouty proteins; MEK inhibition led to repression of SPRY4 and subsequent FGFR1-mediated reactivation of MEK and AKT. Therapeutically, the combination of a MEK inhibitor (MEKi) and an FGFR inhibitor (FGFRi) induced cell death in vitro and tumor regressions in vivo These data establish the rationale and a therapeutic approach to treat mesenchymal-like KRAS-mutant lung cancers effectively with clinically available FGFR1 and MAPK inhibitors. SIGNIFICANCE: Adaptive resistance to MEKi is driven by receptor tyrosine kinases specific to the differentiation state of the KRAS-mutant non-small cell lung cancer (NSCLC). In mesenchymal-like KRAS-mutant NSCLC, FGFR1 is highly expressed, and MEK inhibition relieves feedback suppression of FGFR1, resulting in reactivation of ERK; suppression of ERK by MEKi/FGFRi combination results in tumor shrinkage. Cancer Discov; 6(7); 754-69. ©2016 AACR.This article is highlighted in the In This Issue feature, p. 681.

Neurological Symptoms of Sarcoidosis-induced Small Fiber Neuropathy Effectively Relieved with High-dose Steroid Pulse Therapy.

A 59-year-old woman was admitted to our hospital for an evaluation of a 10-day history of progressive pain and hypoesthesia of the right lower back associated with fever and constipation. Sarcoidosis was confirmed on mediastinal lymph node and skin biopsies. Although the neurological symptoms were suspected due to sarcoidosis-induced nerve dysfunction, nerve conduction studies and other routine examinations did not show any abnormalities. The intraepidermal nerve fiber density assessed on a skin biopsy was significantly reduced, suggesting small-fiber neuropathy (SFN). The patient was finally diagnosed with sarcoidosis-induced SFN, and her neurological symptoms were effectively relieved with high-dose steroid therapy.