A functional sgRNA-CRISPR screening method for generating murine RET and NTRK1 rearranged oncogenes.

CRISPR/Cas9 gene editing represents a powerful tool for investigating fusion oncogenes in cancer biology. Successful experiments require that sgRNAs correctly associate with their target sequence and initiate double stranded breaks which are subsequently repaired by endogenous DNA repair systems yielding fusion chromosomes. Simple tests to ensure sgRNAs are functional are not generally available and often require single cell cloning to identify successful CRISPR-editing events. Here, we describe a novel method relying on acquisition of IL3-independence in Ba/F3 cells to identify sgRNA pairs that generate oncogenic gene rearrangements of the Ret and Ntrk1 tyrosine kinases. The rearrangements were confirmed with PCR, RT-PCR and sequencing and Ba/F3 cells harboring Ret or Ntrk1 rearrangements acquired sensitivity to RET and TRK inhibitors, respectively. Adenoviruses encoding Cas9 and sgRNA pairs inducing the Kif5b-Ret and Trim24-Ret rearrangements were intratracheally instilled into mice and yielded lung adenocarcinomas. A cell line (TR.1) established from a Trim24-Ret positive tumor exhibited high in vitro sensitivity to the RET inhibitors LOXO-292 and BLU-667 and orthotopic TR.1 cell-derived tumors underwent marked shrinkage upon LOXO-292 treatment. Thus, the method offers an efficient means to validate sgRNAs that successfully target their intended loci for the generation of novel, syngeneic murine oncogene-driven tumor models.

A Rapid, Functional sgRNA Screening Method for Generating Murine RET and NTRK1 Fusion Oncogenes.

CRISPR/Cas9 gene editing technology is an indispensable and powerful tool in the field of cancer biology. To conduct successful CRISPR-based experiments, it is crucial that sgRNAs generate their designed alterations. Here, we describe a simple and efficient sgRNA screening method for validating sgRNAs that generate oncogenic gene rearrangements. We used IL3-independence in Ba/F3 cells as an assay to identify sgRNA pairs that generate fusion oncogenes involving the Ret and Ntrk1 tyrosine kinases. We confirmed these rearrangements with PCR or RT-PCR as well as sequencing. Ba/F3 cells harboring Ret or Ntrk1 rearrangements acquired sensitivity to RET and TRK inhibitors, respectively. Adenoviruses encoding Cas9 and sgRNAs that catalyze the Kif5b-Ret and Trim24-Ret rearrangements were intratracheally instilled into mice and yielded lung adenocarcinomas. A cell line (TR.1) was established from a Trim24-Ret positive tumor that exhibited high in vitro sensitivity to RET-specific TKIs. Moreover, orthotopic transplantation of TR.1 cells into the left lung yielded well-defined tumors that shrank in response to LOXO-292 treatment. The method offers an efficient means to validate sgRNAs that successfully target their intended loci for the generation of novel murine oncogene-driven tumor models.

Evaluation of KRASG12C inhibitor responses in novel murine KRASG12C lung cancer cell line models.

Introduction: The KRAS(G12C) mutation is the most common genetic mutation in North American lung adenocarcinoma patients. Recently, direct inhibitors of the KRASG12C protein have been developed and demonstrate clinical response rates of 37-43%. Importantly, these agents fail to generate durable therapeutic responses with median progression-free survival of ~6.5 months. Methods: To provide models for further preclinical improvement of these inhibitors, we generated three novel murine KRASG12C-driven lung cancer cell lines. The co-occurring NRASQ61L mutation in KRASG12C-positive LLC cells was deleted and the KRASG12V allele in CMT167 cells was edited to KRASG12C with CRISPR/Cas9 methods. Also, a novel murine KRASG12C line, mKRC.1, was established from a tumor generated in a genetically-engineered mouse model. Results: The three lines exhibit similar in vitro sensitivities to KRASG12C inhibitors (MRTX-1257, MRTX-849, AMG-510), but distinct in vivo responses to MRTX-849 ranging from progressive growth with orthotopic LLC-NRAS KO tumors to modest shrinkage with mKRC.1 tumors. All three cell lines exhibited synergistic in vitro growth inhibition with combinations of MRTX-1257 and the SHP2/PTPN11 inhibitor, RMC-4550. Moreover, treatment with a MRTX-849/RMC-4550 combination yielded transient tumor shrinkage in orthotopic LLC-NRAS KO tumors propagated in syngeneic mice and durable shrinkage of mKRC.1 tumors. Notably, single-agent MRTX-849 activity in mKRC.1 tumors and the combination response in LLC-NRAS KO tumors was lost when the experiments were performed in athymic nu/nu mice, supporting a growing literature demonstrating a role for adaptive immunity in the response to this class of drugs. Discussion: These new models of murine KRASG12C mutant lung cancer should prove valuable for identifying improved therapeutic combination strategies with KRASG12C inhibitors.

Durable responses to alectinib in murine models of EML4-ALK lung cancer requires adaptive immunity.

Lung cancers bearing oncogenic EML4-ALK fusions respond to targeted tyrosine kinase inhibitors (TKIs; e.g., alectinib), with variation in the degree of shrinkage and duration of treatment (DOT). However, factors that control this response are not well understood. While the contribution of the immune system in mediating the response to immunotherapy has been extensively investigated, less is known regarding the contribution of immunity to TKI therapeutic responses. We previously demonstrated a positive association of a TKI-induced interferon gamma (IFNγ) transcriptional response with DOT in EGFR-mutant lung cancers. Herein, we used three murine models of EML4-ALK lung cancer to test the role for host immunity in the alectinib therapeutic response. The cell lines (EA1, EA2, EA3) were propagated orthotopically in the lungs of immunocompetent and immunodeficient mice and treated with alectinib. Tumor volumes were serially measured by µCT and immune cell content was measured by flow cytometry and multispectral immunofluorescence. Transcriptional responses to alectinib were assessed by RNAseq and secreted chemokines were measured by ELISA. All cell lines were similarly sensitive to alectinib in vitro and as orthotopic tumors in immunocompetent mice, exhibited durable shrinkage. However, in immunodeficient mice, all tumor models rapidly progressed on TKI therapy. In immunocompetent mice, EA2 tumors exhibited a complete response, whereas EA1 and EA3 tumors retained residual disease that rapidly progressed upon termination of TKI treatment. Prior to treatment, EA2 tumors had greater numbers of CD8+ T cells and fewer neutrophils compared to EA1 tumors. Also, RNAseq of cancer cells recovered from untreated tumors revealed elevated levels of CXCL9 and 10 in EA2 tumors, and higher levels of CXCL1 and 2 in EA1 tumors. Analysis of pre-treatment patient biopsies from ALK+ tumors revealed an association of neutrophil content with shorter time to progression. Combined, these data support a role for adaptive immunity in durability of TKI responses and demonstrate that the immune cell composition of the tumor microenvironment is predictive of response to alectinib therapy.

Novel EGFR-mutant mouse models of lung adenocarcinoma reveal adaptive immunity requirement for durable osimertinib response.

Lung cancers bearing oncogenically-mutated EGFR represent a significant fraction of lung adenocarcinomas (LUADs) for which EGFR-targeting tyrosine kinase inhibitors (TKIs) provide a highly effective therapeutic approach. However, these lung cancers eventually acquire resistance and undergo progression within a characteristically broad treatment duration range. Our previous study of EGFR mutant lung cancer patient biopsies highlighted the positive association of a TKI-induced interferon γ transcriptional response with increased time to treatment progression. To test the hypothesis that host immunity contributes to the TKI response, we developed novel genetically-engineered mouse models of EGFR mutant lung cancer bearing exon 19 deletions (del19) or the L860R missense mutation. Both oncogenic EGFR mouse models developed multifocal LUADs from which transplantable cancer cell lines sensitive to the EGFR-specific TKIs, gefitinib and osimertinib, were derived. When propagated orthotopically in the left lungs of syngeneic C57BL/6 mice, deep and durable shrinkage of the cell line-derived tumors was observed in response to daily treatment with osimertinib. By contrast, orthotopic tumors propagated in immune deficient nu/nu or Rag1-/- mice exhibited modest tumor shrinkage followed by rapid progression on continuous osimertinib treatment. Importantly, osimertinib treatment significantly increased intratumoral T cell content and decreased neutrophil content relative to diluent treatment. The findings provide strong evidence supporting the requirement for adaptive immunity in the durable therapeutic control of EGFR mutant lung cancer.

EphB4 and ephrinB2 act in opposition in the head and neck tumor microenvironment.

Differential outcomes of EphB4-ephrinB2 signaling offers formidable challenge for the development of cancer therapeutics. Here, we interrogate the effects of targeting EphB4 and ephrinB2 in head and neck squamous cell carcinoma (HNSCC) and within its microenvironment using genetically engineered mice, recombinant constructs, pharmacologic agonists and antagonists. We observe that manipulating the EphB4 intracellular domain on cancer cells accelerates tumor growth and angiogenesis. EphB4 cancer cell loss also triggers compensatory upregulation of EphA4 and T regulatory cells (Tregs) influx and their targeting results in reversal of accelerated tumor growth mediated by EphB4 knockdown. EphrinB2 knockout on cancer cells and vasculature, on the other hand, results in maximal tumor reduction and vascular normalization. We report that EphB4 agonism provides no additional anti-tumoral benefit in the absence of ephrinB2. These results identify ephrinB2 as a tumor promoter and its receptor, EphB4, as a tumor suppressor in HNSCC, presenting opportunities for rational drug design.

Upregulation of complement proteins in lung cancer cells mediates tumor progression.

Introduction: In vivo, cancer cells respond to signals from the tumor microenvironment resulting in changes in expression of proteins that promote tumor progression and suppress anti-tumor immunity. This study employed an orthotopic immunocompetent model of lung cancer to define pathways that are altered in cancer cells recovered from tumors compared to cells grown in culture. Methods: Studies used four murine cell lines implanted into the lungs of syngeneic mice. Cancer cells were recovered using FACS, and transcriptional changes compared to cells grown in culture were determined by RNA-seq. Results: Changes in interferon response, antigen presentation and cytokine signaling were observed in all tumors. In addition, we observed induction of the complement pathway. We previously demonstrated that activation of complement is critical for tumor progression in this model. Complement can play both a pro-tumorigenic role through production of anaphylatoxins, and an anti-tumorigenic role by promoting complement-mediated cell killing of cancer cells. While complement proteins are produced by the liver, expression of complement proteins by cancer cells has been described. Silencing cancer cell-specific C3 inhibited tumor growth In vivo. We hypothesized that induction of complement regulatory proteins was critical for blocking the anti-tumor effects of complement activation. Silencing complement regulatory proteins also inhibited tumor growth, with different regulatory proteins acting in a cell-specific manner. Discussion: Based on these data we propose that localized induction of complement in cancer cells is a common feature of lung tumors that promotes tumor progression, with induction of complement regulatory proteins protecting cells from complement mediated-cell killing.

TP53 Null Mutations Identify Lung Cancer Cell Lines with Highest Sensitivity to the Nontaxane Microtubule Inhibitor Eribulin.

The nontaxane microtubule inhibitor eribulin is an approved therapeutic for metastatic breast cancer and liposarcoma. Eribulin was previously tested in unselected patients with lung cancer and yielded a modest objective response rate of ∼5%-12%. Because lung cancers represent diverse histologies and driving oncogenic mutations, we postulated that eribulin may exhibit properties of a precision oncology agent with a previously undefined specificity for a molecularly distinct subset of lung cancers. Herein, we screened a panel of 44 non-small cell and small-cell lung cancer cell lines for in vitro growth sensitivity to eribulin. The results revealed a greater than 15,000-fold range in eribulin sensitivity (IC50 = 0.005-89 nM) among the cell lines that was not correlated with their sensitivity to the taxane-based inhibitor paclitaxel. The quartile of cell lines exhibiting the lowest eribulin IC50 values was not enriched for specific histologies, epithelial-mesenchymal differentiation, or specific oncogene drivers but was significantly enriched for nonsense/frameshift TP53 mutations and low-TP53 mRNA but not missense TP53 mutations. By comparison, the mutation status of cyclin-dependent kinase inhibitor 2A, STK11, and KEAP1 was not associated with eribulin sensitivity. Finally, the highest eribulin IC50 quartile (>1 nM) exhibited significantly elevated mRNA expression of the drug pump, ATP binding cassette B1, defined resistance mechanism to eribulin, and paclitaxel. The findings support further investigations into basic mechanisms by which complete lack of TP53 function regulates anticancer activity of eribulin and the potential utility of TP53 null phenotypes distinct from TP53 missense mutations as a biomarker of response in patients with lung cancer. SIGNIFICANCE STATEMENT: Distinct from precision oncology agents that are matched to cancers bearing oncogenically activated versions of their targets, microtubule inhibitors, such as eribulin, are deployed in an unselected manner. The results in this study demonstrate that lung cancer cell lines exhibiting the highest sensitivity to eribulin bear TP53 null phenotypes, supporting a rationale to consider the status of this tumor suppressor in the clinical setting.

A tyrosine kinase inhibitor-induced interferon response positively associates with clinical response in EGFR-mutant lung cancer.

Tyrosine kinase inhibitors (TKIs) targeting EGFR-mutant lung cancers promote a range of tumor regression responses to yield variable residual disease, a likely incubator for acquired resistance. Herein, rapid transcriptional responses induced by TKIs early in treatment that associate with the range of patient responses were explored. RNAseq was performed on EGFR mutant cell lines treated in vitro with osimertinib and on tumor biopsies of eight EGFR mutant lung cancer patients before and after 2 weeks of TKI treatment. Data were evaluated for gene expression programs altered upon TKI treatment. Chemokine and cytokine expression were measured by ELISA and quantitative RT-PCR. IκB Kinase (IKK) and JAK-STAT pathway dependence was tested with pharmacologic and molecular inhibitors. Tumor sections were stained for the T-cell marker CD3. Osimertinib stimulated dynamic, yet wide-ranging interferon (IFN) program regulation in EGFR mutant cell lines. IL6 and CXCL10 induction varied markedly among the EGFR mutant cell lines and was sensitive to IKK and JAK-STAT inhibitors. Analysis of matched patient biopsy pairs revealed marked, yet varied enrichment of IFN transcriptional programs, effector immune cell signatures and T-cell content in treated tumors that positively correlated with time to progression in the patients. EGFR-specific TKIs induce wide-ranging IFN response program activation originating within the cancer cell. The strong association of IFN program induction and duration of clinical response indicates that the TKI-induced IFN program instructs variable recruitment and participation of immune cells in the overall therapeutic response.

Role of epidermal growth factor receptor inhibitor-induced interferon pathway signaling in the head and neck squamous cell carcinoma therapeutic response.

BACKGROUND: Epidermal growth factor receptor (EGFR) is frequently amplified or overexpressed in head and neck squamous cell carcinoma (HNSCC) and is a clinically validated target for the therapeutic antibody, cetuximab, in the management of this cancer. The degree of response to EGFR inhibitors measured by tumor shrinkage varies widely among HNSCC patients, and the biological mechanisms that underlie therapeutic heterogeneity amongst HNSCC patients remain ill-defined. METHODS: EGFR-dependent human and murine HNSCC cell lines were treated with the EGFR/ERBB inhibitors, gefitinib and AZD8931, and submitted to RNAseq, GSEA, and qRT-PCR. Conditioned media was analyzed by ELISA and Luminex assays. Murine HNSCC tumors were stained for T cell markers by immunofluorescence. Primary HSNCC patient specimens treated with single agent cetuximab were stained with Vectra multispectral immunofluorescence. RESULTS: The transcriptional reprogramming response to EGFR/ERBB-specific TKIs was measured in a panel of EGFR-dependent human HNSCC cell lines and interferon (IFN) α and γ responses identified as top-ranked TKI-induced pathways. Despite similar drug sensitivity, responses among 7 cell lines varied quantitatively and qualitatively, especially regarding the induced chemokine and cytokine profiles. Of note, the anti-tumorigenic chemokine, CXCL10, and the pro-tumorigenic factor, IL6, exhibited wide-ranging and non-overlapping induction. Similarly, AZD8931 exerted potent growth inhibition, IFNα/IFNγ pathway activation, and CXCL10 induction in murine B4B8 HNSCC cells. AZD8931 treatment of immune-competent mice bearing orthotopic B4B8 tumors increased CD8 + T cell content and the therapeutic response was abrogated in nu/nu mice relative to BALB/c mice. Finally, Vectra 3.0 analysis of HNSCC patient tumors prior to and after 3-4 weeks of single agent cetuximab treatment revealed increased CD8 + T cell content in specimens from patients exhibiting a therapeutic response relative to non-responders. CONCLUSIONS: The findings reveal heterogeneous, tumor cell-intrinsic, EGFR/ERBB inhibitor-induced IFN pathway activation in HNSCC and suggest that individual tumor responses to oncogene-targeted agents are a sum of direct growth inhibitory effects and variably-induced participation of host immune cells.

Cancer cell-intrinsic expression of MHC II in lung cancer cell lines is actively restricted by MEK/ERK signaling and epigenetic mechanisms.

BACKGROUND: Programmed death 1/programmed death ligand 1 (PD-1/PD-L1) targeted immunotherapy affords clinical benefit in ~20% of unselected patients with lung cancer. The factor(s) that determine whether a tumor responds or fails to respond to immunotherapy remains an active area of investigation. We have previously defined divergent responsiveness of two KRAS-mutant cell lines to PD-1/PD-L1 blockade using an orthotopic, immunocompetent mouse model. Responsiveness to PD-1/PD-L1 checkpoint blockade correlates with an interferon gamma (IFNγ)-inducible gene signature and major histocompatibility complex class II (MHC II) expression by cancer cells. In the current study, we aim to identify therapeutic targets that can be manipulated in order to enhance cancer-cell-specific MHC II expression. METHODS: Responsiveness to IFNγ and induction of MHC II expression was assessed after various treatment conditions in mouse and human non-small cell lung cancer (NSCLC) cell lines using mass cytometric and flow cytometric analysis. RESULTS: Single-cell analysis using mass and flow cytometry demonstrated that IFNγ consistently induced PD-L1 and MHC class I (MHC I) across multiple murine and human NSCLC cell lines. In contrast, MHC II showed highly variable induction following IFNγ treatment both between lines and within lines. In mouse models of NSCLC, MHC II induction was inversely correlated with basal levels of phosphorylated extracellular signal-regulated kinase (ERK) 1/2, suggesting potential mitogen-activated protein (MAP) kinase-dependent antagonism of MHC II expression. To test this, cell lines were subjected to varying levels of stimulation with IFNγ, and assessed for MHC II expression in the presence or absence of mitogen-activated protein kinase kinase (MEK) inhibitors. IFNγ treatment in the presence of MEK inhibitors significantly enhanced MHC II induction across multiple lung cancer lines, with minimal impact on expression of either PD-L1 or MHC I. Inhibition of histone deacetylases (HDACs) also enhanced MHC II expression to a more modest extent. Combined MEK and HDAC inhibition led to greater MHC II expression than either treatment alone. CONCLUSIONS: These studies emphasize the active inhibitory role that epigenetic and ERK signaling cascades have in restricting cancer cell-intrinsic MHC II expression in NSCLC, and suggest that combinatorial blockade of these pathways may engender new responsiveness to checkpoint therapies.

Cancer Cell-Intrinsic Expression of MHC Class II Regulates the Immune Microenvironment and Response to Anti-PD-1 Therapy in Lung Adenocarcinoma.

MHC class II (MHCII) expression is usually restricted to APC but can be expressed by cancer cells. We examined the effect of cancer cell-specific MHCII (csMHCII) expression in lung adenocarcinoma on T cell recruitment to tumors and response to anti-PD-1 therapy using two orthotopic immunocompetent murine models of non-small cell lung cancer: CMT167 (CMT) and Lewis lung carcinoma (LLC). We previously showed that CMT167 tumors are eradicated by anti-PD1 therapy, whereas LLC tumors are resistant. RNA sequencing analysis of cancer cells recovered from tumors revealed that csMHCII correlated with response to anti-PD1 therapy, with immunotherapy-sensitive CMT167 cells being csMHCII positive, whereas resistant LLC cells were csMHCII negative. To test the functional effects of csMHCII, MHCII expression was altered on the cancer cells through loss- and gain-of-function of CIITA, a master regulator of the MHCII pathway. Loss of CIITA in CMT167 decreased csMHCII and converted tumors from anti-PD-1 sensitive to anti-PD-1 resistant. This was associated with lower levels of Th1 cytokines, decreased T cell infiltration, increased B cell numbers, and decreased macrophage recruitment. Conversely, overexpression of CIITA in LLC cells resulted in csMHCII in vitro and in vivo. Enforced expression of CIITA increased T cell infiltration and sensitized tumors to anti-PD-1 therapy. csMHCII expression was also examined in a subset of surgically resected human lung adenocarcinomas by multispectral imaging, which provided a survival benefit and positively correlated with T cell infiltration. These studies demonstrate a functional role for csMHCII in regulating T cell infiltration and sensitivity to anti-PD-1.

Translating mesothelioma molecular genomics and dependencies into precision oncology-based therapies.

Malignant pleural mesothelioma (MPM) is a rare, yet lethal asbestos-induced cancer and despite marked efforts to reduce occupational exposure, the incidence has not yet significantly declined. Since 2003, combined treatment with a platinum-based agent and pemetrexed has been the first-line therapy and no effective or approved second-line treatments have emerged. The seemingly slow advance in developing new MPM treatments does not appear to be related to a low level of clinical and pre-clinical research activity. Rather, we suggest that a key hurdle in successfully translating basic discovery into novel MPM therapeutics is the underlying assumption that as a rare cancer, it will also be molecularly and genetically homogeneous. In fact, lung adenocarcinoma and melanoma only benefitted from precision oncology upon full appreciation of the high degree of molecular heterogeneity inherent in these cancers, especially regarding the diversity of oncogenic drivers. Herein, we consider the recent explosion of molecular and genetic information that has become available regarding MPM and suggest ways in which the unfolding landscape may guide identification of novel therapeutic vulnerabilities within subsets of MPM that can be targeted in a manner consistent with the tenets of precision oncology.

Functional RNAi Screens Define Distinct Protein Kinase Vulnerabilities in EGFR-Dependent HNSCC Cell Lines.

The inhibitory epidermal growth factor receptor (EGFR) antibody, cetuximab, is an approved therapy for head and neck squamous cell carcinoma (HNSCC). Despite tumor response observed in some HNSCC patients, cetuximab alone or combined with radio- or chemotherapy fails to yield long-term control or cures. We hypothesize that a flexible receptor tyrosine kinase coactivation signaling network supports HNSCC survival in the setting of EGFR blockade, and that drugs disrupting this network will provide superior tumor control when combined with EGFR inhibitors. In this work, we submitted EGFR-dependent HNSCC cell lines to RNA interference-based functional genomics screens to identify, in an unbiased fashion, essential protein kinases for growth and survival as well as synthetic lethal targets for combined inhibition with EGFR antagonists. Mechanistic target of rapamycin kinase (MTOR) and erythroblastosis oncogene B (ERBB)3 were identified as high-ranking essential kinase hits in the HNSCC cell lines. MTOR dependency was confirmed by distinct short hairpin RNAs (shRNAs) and high sensitivity of the cell lines to AZD8055, whereas ERBB3 dependency was validated by shRNA-mediated silencing. Furthermore, a synthetic lethal kinome shRNA screen with a pan-ERBB inhibitor, AZD8931, identified multiple components of the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase pathway, consistent with ERK reactivation and/or incomplete ERK pathway inhibition in response to EGFR inhibitor monotherapy. As validation, distinct mitogen-activated protein kinase kinase (MEK) inhibitors yielded synergistic growth inhibition when combined with the EGFR inhibitors, gefitinib and AZD8931. The findings identify ERBB3 and MTOR as important pharmacological vulnerabilities in HNSCC and support combining MEK and EGFR inhibitors to enhance clinical efficacy in HNSCC. SIGNIFICANCE STATEMENT: Many cancers are driven by nonmutated receptor tyrosine kinase coactivation networks that defy full inhibition with single targeted drugs. This study identifies erythroblastosis oncogene B (ERBB)3 as an essential protein kinase in epidermal growth factor receptor-dependent head and neck squamous cell cancer (HNSCC) cell lines and a synthetic lethal interaction with the extracellular signal-regulated kinase mitogen-activated protein kinase pathway that provides a rationale for combining pan-ERBB and mitogen-activated protein kinase inhibitors as a therapeutic approach in subsets of HNSCC.

Subcellular Localization and Activity of the Mitogen-Activated Protein Kinase Kinase 7 (MKK7) γ Isoform are Regulated through Binding to the Phosphatase Calcineurin.

Calcineurin (CaN) phosphatase signaling is regulated by targeting CaN to substrates, inhibitors, and scaffold proteins containing docking motifs with the consensus sequence of PxIxIT. Here, we identify the docking of CaN to the γ isoform of MKK7, a component of the c-Jun N-terminal kinase (JNK) pathway. Because of alternative splicing of a single exon within the N-terminal domain, MKK7γ encodes a unique PxIxIT motif (PIIVIT) that is not present in MKK7α or ß We found that MKK7γ bound directly to CaN through this PIIVIT motif in vitro, immunoprecipitated with CaN from cell extracts, and exhibited fluorescence resonance energy transfer (FRET) with CaN in the cytoplasm but not in the nucleus of living cells. In contrast, MKK7α and ß exhibited no direct binding or FRET with CaN and were localized more in the nucleus than the cytoplasm. Furthermore, the inhibition of CaN phosphatase activity increased the basal phosphorylation of MKK7γ but not MKK7ß Deletion of the MKK7γ PIIVIT motif eliminated FRET with CaN and promoted MKK7γ redistribution to the nucleus; however, the inhibition of CaN activity did not alter MKK7γ localization, indicating that MKK7γ cytoplasmic retention by CaN is phosphatase activity independent. Finally, the inhibition of CaN phosphatase activity in vascular smooth muscle cells, which express MKK7γ mRNA, enhances JNK activation. Overall, we conclude that the MKK7γ-specific PxIxIT motif promotes high-affinity CaN binding that could promote novel cross talk between CaN and JNK signaling by limiting MKK7γ phosphorylation and restricting its localization to the cytoplasm.

Preselection of Lung Cancer Cases Using FGFR1 mRNA and Gene Copy Number for Treatment With Ponatinib.

INTRODUCTION: Preclinically, high epidermal growth factor receptor 1 (FGFR1) messenger RNA (FGFR1-MRNA) and FGFR1 amplification (FGFR1-AMP) predicted sensitivity to fibroblast growth factor receptor inhibitors in non-small-cell lung cancer and small-cell lung cancer cell lines. KRAS mutations did not preclude sensitivity. PATIENTS AND METHODS: Metastatic EGFR- and ALK-negative lung cancers were screened for FGFR1-MRNA by in-situ hybridization (ISH) and FGFR1-AMP by silver in-situ hybridization (SISH). Patients with positive findings were offered ponatinib, a multi-kinase inhibitor of FGFR1-4. Differences in overall survival (OS) between cohorts were assessed by the log-rank test. Association of FGFR1 positivity with clinicopathologic features were assessed by Fisher exact test and Kruskal-Wallis rank sum test. RESULTS: A total of 171 cases were prescreened: 9 (7.3%) of 123 SISH+; 53 (42.1%) of 126 ISH+; and 6 cases concordantly positive for SISH and ISH. SISH+ cases had fewer coincident KRAS mutations (P = .03) than SISH- cases, and ISH+ cases had worse OS (P = .020) than ISH- cases. Data distributions suggested a distinct higher positivity cut point for FGFR1 ISH (≥ 20%), occurring in 29 (23%) of 126 cases, was associated with small-cell lung cancer histology (P = .022), soft tissue metastases (P = .050) and shorter OS (P = .031). Four patients received ponatinib on study: All ISH+ by the initial cut point, 2 of 4 by higher cut point, 1 of 4 SISH+. Tolerability was poor. The best response for the 2 higher ISH cases was stable disease and progressive disease for the 2 lower ISH cases. CONCLUSION: Elevated FGFR1-MRNA is more common than FGFR1-AMP and associated with worse OS. Higher FGFR1 mRNA expression may be associated with a specific phenotype and is worthy of further exploration. Ponatinib's poor tolerance suggests further fibroblast growth factor receptor exploration in ISH+ cases should utilize more selective FGFR1 inhibitors.

A miRNA Panel Predicts Sensitivity of FGFR Inhibitor in Lung Cancer Cell Lines.

PURPOSE: To test whether a microRNA (miRNA) panel may serve as an alternative biomarker of fibroblast growth factor receptor (FGFR) tyrosine kinase inhibitor sensitivity in lung cancer. METHODS: Histologically diverse lung cancer cell lines were submitted to assays for ponatinib and AZD4547 sensitivity. miRNAs, FGFR1 messenger RNA, gene copy number, and protein expression were detected by real-time quantitative PCR, fluorescence in-situ hybridization, and immunoblotting in 34 lung cancer cell lines. RESULTS: Among 34 cell lines, 14 exhibited ponatinib sensitivity and 20 exhibited AZD4547 sensitivity (drug concentration causing 50% inhibition values < 100 nmol/L). A total of 39 of the 377-miRNA set were initially identified from the 4 paired ponatinib-sensitive or -insensitive cell lines to have at least an 8-fold differential expression and then were detected in all the 34 cell lines. A predictive panel of 3 miRNAs (let-7c, miRNA155, and miRNA218) was developed that had an area under the curve (AUC) of 0.886 with a sensitivity of 71.4% and specificity of 77.3% to predict response to ponatinib. The miRNA panel performed similar to FGFR1 protein expression (AUC = 0.864) and messenger RNA expression (AUC = 0.939), and better than FGFR1 amplification (AUC = 0.696). Furthermore, we validated this panel using data for sensitivity to AZD4547 in the cell line cohort with an AUC of 0.931 and a sensitivity of 73.3% and specificity of 76.2%, respectively. CONCLUSION: The developed miRNA panel (let-7c, miRNA155, and miRNA218) may be useful in predicting response to FGFR tyrosine kinase inhibitors, either ponatinib or AZD4547 in lung cancer cell lines, and warrants further validation in the clinical setting.

ALK is a critical regulator of the MYC-signaling axis in ALK positive lung cancer.

A subset of lung cancers is dependent on the anaplastic lymphoma kinase (ALK) oncogene for survival, a mechanism that is exploited by the use of the ALK inhibitor crizotinib. Despite exceptional initial tumor responses to ALK inhibition by crizotinib, durable clinical response is limited and the emergence of drug resistance occurs. Furthermore, intrinsic resistance is frequently observed, where patients fail to respond initially to ALK-inhibitor therapy. These events demonstrate the underlying complexity of a molecularly-defined oncogene-driven cancer and highlights the need to identify compensating survival pathways. Using a loss-of-function whole genome short-hairpin (shRNA) screen, we identified MYCBP as a determinant of response to crizotinib, implicating the MYC signaling axis in resistance to crizotinib-treated ALK+ NSCLC. Further analysis reveals that ALK regulates transcriptional expression of MYC and activates c-MYC transactivation of c-MYC target genes. Inhibition of MYC by RNAi or small molecules sensitizes ALK+ cells to crizotinib. Taken together, our findings demonstrate a dual oncogene mechanism, where ALK positively regulates the MYC signaling axis, providing an additional oncogene target whose inhibition may prevent or overcome resistance.

Mechanisms of rapid cancer cell reprogramming initiated by targeted receptor tyrosine kinase inhibitors and inherent therapeutic vulnerabilities.

Receptor tyrosine kinase (RTK) pathways serve as frequent oncogene drivers in solid cancers and small molecule and antibody-based inhibitors have been developed as targeted therapeutics for many of these oncogenic RTKs. In general, these drugs, when delivered as single agents in a manner consistent with the principles of precision medicine, induce tumor shrinkage but rarely complete tumor elimination. Moreover, acquired resistance of treated tumors is nearly invariant such that monotherapy strategies with targeted RTK drugs fail to provide long-term control or cures. The mechanisms mediating acquired resistance in tumors at progression treated with RTK inhibitors are relatively well defined compared to the molecular and cellular understanding of the cancer cells that persist early on therapy. We and others propose that these persisting cancer cells, termed "residual disease", provide the reservoir from which acquired resistance eventually emerges. Herein, we will review the literature that describes rapid reprogramming induced upon inhibition of oncogenic RTKs in cancer cells as a mechanism by which cancer cells persist to yield residual disease and consider strategies for disrupting these intrinsic responses for future therapeutic gain.