Uncovering a Novel Functional Interaction Between Adult Hepatic Progenitor Cells, Inflammation and EGFR Signaling During Bile Acids-Induced Injury.

Chronic cholestatic damage is associated to both accumulation of cytotoxic levels of bile acids and expansion of adult hepatic progenitor cells (HPC) as part of the ductular reaction contributing to the regenerative response. Here, we report a bile acid-specific cytotoxic response in mouse HPC, which is partially impaired by EGF signaling. Additionally, we show that EGF synergizes with bile acids to trigger inflammatory signaling and NLRP3 inflammasome activation in HPC. Aiming at understanding the impact of this HPC specific response on the liver microenvironment we run a proteomic analysis of HPC secretome. Data show an enrichment in immune and TGF-ß regulators, ECM components and remodeling proteins in HPC secretome. Consistently, HPC-derived conditioned medium promotes hepatic stellate cell (HSC) activation and macrophage M1-like polarization. Strikingly, EGF and bile acids co-treatment leads to profound changes in the secretome composition, illustrated by an abolishment of HSC activating effect and by promoting macrophage M2-like polarization. Collectively, we provide new specific mechanisms behind HPC regulatory action during cholestatic liver injury, with an active role in cellular interactome and inflammatory response regulation. Moreover, findings prove a key contribution for EGFR signaling jointly with bile acids in HPC-mediated actions.

Epithelial disruption drives mesendoderm differentiation in human pluripotent stem cells by enabling TGF-ß protein sensing.

The processes of primitive streak formation and fate specification in the mammalian epiblast rely on complex interactions between morphogens and tissue organization. Little is known about how these instructive cues functionally interact to regulate gastrulation. We interrogated the interplay between tissue organization and morphogens by using human induced pluripotent stem cells (hiPSCs) downregulated for the morphogen regulator GLYPICAN-4, in which defects in tight junctions result in areas of disrupted epithelial integrity. Remarkably, this phenotype does not affect hiPSC stemness, but impacts on cell fate acquisition. Strikingly, cells within disrupted areas become competent to perceive the gastrulation signals BMP4 and ACTIVIN A, an in vitro surrogate for NODAL, and thus differentiate into mesendoderm. Yet, disruption of epithelial integrity sustains activation of BMP4 and ACTIVIN A downstream effectors and correlates with enhanced hiPSC endoderm/mesoderm differentiation. Altogether, our results disclose epithelial integrity as a key determinant of TGF-ß activity and highlight an additional mechanism guiding morphogen sensing and spatial cell fate change within an epithelium.

MYC and MET cooperatively drive hepatocellular carcinoma with distinct molecular traits and vulnerabilities.

Enhanced activation of the transcription factor MYC and of the receptor tyrosine kinase MET are among the events frequently occurring in hepatocellular carcinoma (HCC). Both genes individually act as drivers of liver cancer initiation and progression. However, their concomitant alteration in HCC has not been explored, nor functionally documented. Here, we analysed databases of five independent human HCC cohorts and found a subset of patients with high levels of MYC and MET (MYChigh/METhigh) characterised by poor prognosis. This clinical observation drove us to explore the functionality of MYC and MET co-occurrence in vivo, combining hydrodynamic tail vein injection for MYC expression in the R26stopMet genetic setting, in which wild-type MET levels are enhanced following the genetic deletion of a stop cassette. Results showed that increased MYC and MET expression in hepatocytes is sufficient to induce liver tumorigenesis even in the absence of pre-existing injuries associated with a chronic disease state. Intriguingly, ectopic MYC in MET tumours increases expression of the Mki67 proliferation marker, and switches them into loss of Afp, Spp1, Gpc3, Epcam accompanied by an increase in Hgma1, Vim, and Hep-Par1 levels. We additionally found a switch in the expression of specific immune checkpoints, with an increase in the Ctla-4 and Lag3 lymphocyte co-inhibitory responses, and in the Icosl co-stimulatory responses of tumour cells. We provide in vitro evidence on the vulnerability of some human HCC cell lines to combined MYC and MET targeting, which are otherwise resistant to single inhibition. Mechanistically, combined blockage of MYC and MET converts a partial cytostatic effect, triggered by individual blockage of MYC or MET, into a cytotoxic effect. Together, these findings highlight a subgroup of HCC characterised by MYChigh/METhigh, and document functional cooperativity between MYC and MET in liver tumorigenesis. Thus, the MYC-R26Met model is a relevant setting for HCC biology, patient classification and treatment.

Enhanced Wild-Type MET Receptor Levels in Mouse Hepatocytes Attenuates Insulin-Mediated Signaling.

Compelling evidence points to the MET receptor tyrosine kinase as a key player during liver development and regeneration. Recently, a role of MET in the pathophysiology of insulin resistance and obesity is emerging. Herein, we aimed to determine whether MET regulates hepatic insulin sensitivity. To achieve this, mice in which the expression of wild-type MET in hepatocytes is slightly enhanced above endogenous levels (Alb-R26Met mice) were analyzed to document glucose homeostasis, energy balance, and insulin signaling in hepatocytes. We found that Alb-R26Met mice exhibited higher body weight and food intake when compared to R26stopMet control mice. Metabolic analyses revealed that Alb-R26Met mice presented age-related glucose and pyruvate intolerance in comparison to R26stopMet controls. Additionally, in Alb-R26Met mice, high MET levels decreased insulin-induced insulin receptor (IR) and AKT phosphorylation compared to control mice. These results were corroborated in vitro by analyzing IR and AKT phosphorylation in primary mouse hepatocytes from Alb-R26Met and R26stopMet mice upon insulin stimulation. Moreover, co-immunoprecipitation assays revealed MET-IR interaction under both basal and insulin stimulation conditions; this effect was enhanced in Alb-R26Met hepatocytes. Altogether, our results indicate that enhanced MET levels alter hepatic glucose homeostasis, which can be an early event for subsequent liver pathologies.

BCL-XL blockage in TNBC models confers vulnerability to inhibition of specific cell cycle regulators.

Cell cycle regulators are frequently altered in Triple-Negative Breast Cancer (TNBC). Emerging agents targeting these signals offer the possibility to design new combinatorial therapies. However, preclinical models that recapitulate TNBC primary resistance and heterogeneity are essential to evaluate the potency of these combined treatments. Methods: Bioinformatic processing of human breast cancer datasets was used to analyse correlations between expression levels of cell cycle regulators and patient survival outcome. The MMTV-R26Met mouse model of TNBC resistance and heterogeneity was employed to analyse expression and targeting vulnerability of cell cycle regulators in the presence of BCL-XL blockage. Robustness of outcomes and selectivity was further explored using a panel of human breast cancer cells. Orthotopic studies in nude mice were applied for preclinical evaluation of efficacy and toxicity. Alterations of protein expression, phosphorylation, and/or cellular localisation were analysed by western blots, reverse phase protein array, and immunocytochemistry. Bioinformatics was performed to highlight drug's mechanisms of action. Results: We report that high expression levels of the BCL2L1 gene encoding BCL-XL and of specific cell cycle regulators correlate with poor survival outcomes of TNBC patients. Blockage of BCL-XL confers vulnerability to drugs targeting CDK1/2/4, but not FOXM1, CDK4/6, Aurora A and Aurora B, to all MMTV-R26Met and human TNBC cell lines tested. Combined blockage of BCL-XL and CDK1/2/4 interfered with tumour growth in vivo. Mechanistically, we show that, co-targeting of BCL-XL and CDK1/2/4 synergistically inhibited cell viability by combinatorial depletion of survival and RTK/AKT signals, and concomitantly restoring FOXO3a tumour suppression actions. This was accompanied by an accumulation of DNA damage and consequently apoptosis. Conclusions: Our studies illustrate the possibility to exploit the vulnerability of TNBC cells to CDK1/2/4 inhibition by targeting BCL-XL. Moreover, they underline that specificity matters in targeting cell cycle regulators for combinatorial anticancer therapies.

The AXL-PYK2-PKCα axis as a nexus of stemness circuits in TNBC.

Cancer stem cells (CSCs) are implicated in tumor initiation, metastasis and drug resistance, and considered as attractive targets for cancer therapy. Here we identified a clinically relevant signaling nexus mediated by AXL receptor, PYK2 and PKCα and show its impact on stemness in TNBC. AXL, PYK2, and PKCα expression correlates with stemness signature in basal-like breast cancer patients, and their depletion in multiple mesenchymal TNBC cell lines markedly reduced the number of mammosphere-forming cells and cells harboring CSCs characteristic markers. Knockdown of PYK2 reduced the levels of AXL, PKCα, FRA1, and PYK2 proteins, and similar trend was obtained upon PKCα depletion. PYK2 depletion decreased AXL transcription through feedback loops mediated by FRA1 and TAZ, whereas PKCα inhibition induced redistribution of AXL to endosomal/lysosomal compartment and enhanced its degradation. PYK2 and PKCα cooperate at a convergence point of multiple stemness-inducing pathways to regulate AXL levels and concomitantly the levels/activation of STAT3, TAZ, FRA1, and SMAD3 as well as the pluripotent transcription factors Nanog and Oct4. Induction of stemness in TNBC sensitized cells to PYK2 and PKCα inhibition suggesting that targeting the AXL-PYK2-PKCα circuit could be an efficient strategy to eliminate CSCs in TNBC.

Modeling Heterogeneity of Triple-Negative Breast Cancer Uncovers a Novel Combinatorial Treatment Overcoming Primary Drug Resistance.

Triple-negative breast cancer (TNBC) is a highly aggressive breast cancer subtype characterized by a remarkable molecular heterogeneity. Currently, there are no effective druggable targets and advanced preclinical models of the human disease. Here, a unique mouse model (MMTV-R26Met mice) of mammary tumors driven by a subtle increase in the expression of the wild-type MET receptor is generated. MMTV-R26Met mice develop spontaneous, exclusive TNBC tumors, recapitulating primary resistance to treatment of patients. Proteomic profiling of MMTV-R26Met tumors and machine learning approach show that the model faithfully recapitulates intertumoral heterogeneity of human TNBC. Further signaling network analysis highlights potential druggable targets, of which cotargeting of WEE1 and BCL-XL synergistically kills TNBC cells and efficiently induces tumor regression. Mechanistically, BCL-XL inhibition exacerbates the dependency of TNBC cells on WEE1 function, leading to Histone H3 and phosphoS33RPA32 upregulation, RRM2 downregulation, cell cycle perturbation, mitotic catastrophe, and apoptosis. This study introduces a unique, powerful mouse model for studying TNBC formation and evolution, its heterogeneity, and for identifying efficient therapeutic targets.

Enhanced differentiation of human induced pluripotent stem cells toward the midbrain dopaminergic neuron lineage through GLYPICAN-4 downregulation.

Enhancing the differentiation potential of human induced pluripotent stem cells (hiPSC) into disease-relevant cell types is instrumental for their widespread application in medicine. Here, we show that hiPSCs downregulated for the signaling modulator GLYPICAN-4 (GPC4) acquire a new biological state characterized by increased hiPSC differentiation capabilities toward ventral midbrain dopaminergic (VMDA) neuron progenitors. This biological trait emerges both in vitro, upon exposing cells to VMDA neuronal differentiation signals, and in vivo, even when transplanting hiPSCs at the extreme conditions of floor-plate stage in rat brains. Moreover, it is compatible with the overall neuronal maturation process toward acquisition of substantia nigra neuron identity. HiPSCs with downregulated GPC4 also retain self-renewal and pluripotency in stemness conditions, in vitro, while losing tumorigenesis in vivo as assessed by flank xenografts. In conclusion, our results highlight GPC4 downregulation as a powerful approach to enhance generation of VMDA neurons. Outcomes may contribute to establish hiPSC lines suitable for translational applications.

ADAMTSL5 is an epigenetically activated gene underlying tumorigenesis and drug resistance in hepatocellular carcinoma.

BACKGROUND & AIMS: The tumour microenvironment shapes tumour growth through cellular communications that include both direct interactions and secreted factors. The aim of this study was to characterize the impact of the secreted glycoprotein ADAMTSL5, whose role in cancer has not been previously investigated, on hepatocellular carcinoma (HCC). METHODS: ADAMTSL5 methylation status was evaluated through bisulfite sequencing, and publicly available data analysis. ADAMTSL5 RNA and protein expression were assessed in mouse models and HCC patient samples and compared to data from published datasets. Functional studies, including association of ADAMTSL5 depletion with responsiveness to clinically relevant drugs, were performed in cellular and in vivo models. Molecular alterations associated with ADAMTSL5 targeting were determined using proteomics, biochemistry, and reverse-transcription quantitative PCR. RESULTS: Methylome analysis revealed hypermethylated gene body CpG islands at the ADAMTSL5 locus in both mouse and human HCC, correlating with higher ADAMTSL5 expression. ADAMTSL5 targeting interfered with tumorigenic properties of HCC cells in vitro and in vivo, whereas ADAMTSL5 overexpression conferred tumorigenicity to pre-tumoural hepatocytes sensitized to transformation by a modest level of MET receptor expression. Mechanistically, ADAMTSL5 abrogation led to a reduction of several oncogenic inputs relevant to HCC, including reduced expression and/or phosphorylation levels of receptor tyrosine kinases MET, EGFR, PDGFRß, IGF1Rß, or FGFR4. This phenotype was associated with significantly increased sensitivity of HCC cells to clinically relevant drugs, namely sorafenib, lenvatinib, and regorafenib. Moreover, ADAMTSL5 depletion drastically increased expression of AXL, accompanied by a sensitization to bemcentinib. CONCLUSIONS: Our results point to a role for ADAMTSL5 in maintaining the function of key oncogenic signalling pathways, suggesting that it may act as a master regulator of tumorigenicity and drug resistance in HCC. LAY SUMMARY: The environment of cancer cells has profound effects on establishment, progression, and response of a tumour to treatment. Herein, we show that ADAMTSL5, a protein secreted by liver cancer cells and overlooked in cancer so far, is increased in this tumour type, is necessary for tumour formation and supports drug resistance. Adamtsl5 removal conferred sensitivity of liver cancer cells to drugs used in current treatment. This suggests ADAMTSL5 as a potential marker in liver cancer as well as a possible drug target.

Synthetic lethal combination targeting BET uncovered intrinsic susceptibility of TNBC to ferroptosis.

Identification of targeted therapies for TNBC is an urgent medical need. Using a drug combination screen reliant on synthetic lethal interactions, we identified clinically relevant combination therapies for different TNBC subtypes. Two drug combinations targeting the BET family were further explored. The first, targeting BET and CXCR2, is specific for mesenchymal TNBC and induces apoptosis, whereas the second, targeting BET and the proteasome, is effective for major TNBC subtypes and triggers ferroptosis. Ferroptosis was induced at low drug doses and was associated with increased cellular iron and decreased glutathione levels, concomitant with reduced levels of GPX4 and key glutathione biosynthesis genes. Further functional studies, analysis of clinical datasets and breast cancer specimens revealed a unique vulnerability of TNBC to ferroptosis inducers, enrichment of ferroptosis gene signature, and differential expression of key proteins that increase labile iron and decrease glutathione levels. This study identified potent combination therapies for TNBC and unveiled ferroptosis as a promising therapeutic strategy.

C3G Is Upregulated in Hepatocarcinoma, Contributing to Tumor Growth and Progression and to HGF/MET Pathway Activation.

The complexity of hepatocellular carcinoma (HCC) challenges the identification of disease-relevant signals. C3G, a guanine nucleotide exchange factor for Rap and other Ras proteins, plays a dual role in cancer acting as either a tumor suppressor or promoter depending on tumor type and stage. The potential relevance of C3G upregulation in HCC patients suggested by database analysis remains unknown. We have explored C3G function in HCC and the underlying mechanisms using public patient data and in vitro and in vivo human and mouse HCC models. We found that C3G is highly expressed in progenitor cells and neonatal hepatocytes, whilst being down-regulated in adult hepatocytes and re-expressed in human HCC patients, mouse HCC models and HCC cell lines. Moreover, high C3G mRNA levels correlate with tumor progression and a lower patient survival rate. C3G expression appears to be tightly modulated within the HCC program, influencing distinct cell biological properties. Hence, high C3G expression levels are necessary for cell tumorigenic properties, as illustrated by reduced colony formation in anchorage-dependent and -independent growth assays induced by permanent C3G silencing using shRNAs. Additionally, we demonstrate that C3G down-regulation interferes with primary HCC tumor formation in xenograft assays, increasing apoptosis and decreasing proliferation. In vitro assays also revealed that C3G down-regulation enhances the pro-migratory, invasive and metastatic properties of HCC cells through an epithelial-mesenchymal switch that favors the acquisition of a more mesenchymal phenotype. Consistently, a low C3G expression in HCC cells correlates with lung metastasis formation in mice. However, the subsequent restoration of C3G levels is associated with metastatic growth. Mechanistically, C3G down-regulation severely impairs HGF/MET signaling activation in HCC cells. Collectively, our results indicate that C3G is a key player in HCC. C3G promotes tumor growth and progression, and the modulation of its levels is essential to ensure distinct biological features of HCC cells throughout the oncogenic program. Furthermore, C3G requirement for HGF/MET signaling full activation provides mechanistic data on how it works, pointing out the relevance of assessing whether high C3G levels could identify HCC responders to MET inhibitors.

Large-Scale Virtual Screening Against the MET Kinase Domain Identifies a New Putative Inhibitor Type.

By using an ensemble-docking strategy, we undertook a large-scale virtual screening campaign in order to identify new putative hits against the MET kinase target. Following a large molecular dynamics sampling of its conformational space, a set of 45 conformers of the kinase was retained as docking targets to take into account the flexibility of the binding site moieties. Our screening funnel started from about 80,000 chemical compounds to be tested in silico for their potential affinities towards the kinase binding site. The top 100 molecules selected-thanks to the molecular docking results-were further analyzed for their interactions, and 25 of the most promising ligands were tested for their ability to inhibit MET activity in cells. F0514-4011 compound was the most efficient and impaired this scattering response to HGF (Hepatocyte Growth Factor) with an IC 50 of 7.2 µ M. Interestingly, careful docking analysis of this molecule with MET suggests a possible conformation halfway between classical type-I and type-II MET inhibitors, with an additional region of interaction. This compound could therefore be an innovative seed to be repositioned from its initial antiviral purpose towards the field of MET inhibitors. Altogether, these results validate our ensemble docking strategy as a cost-effective functional method for drug development.

Tracking Dynamics of Spontaneous Tumors in Mice Using Photon-Counting Computed Tomography.

Computed tomography is a powerful medical imaging modality for longitudinal studies in cancer to follow neoplasia progression and evaluate anticancer therapies. Here, we report the generation of a photon-counting micro-computed tomography (PC-CT) method based on hybrid pixel detectors with enhanced sensitivity and precision of tumor imaging. We then applied PC-CT for longitudinal imaging in a clinically relevant liver cancer model, the Alb-R26Met mice, and found a remarkable heterogeneity in the dynamics for tumors at the initiation phases. Instead, the growth curve of evolving tumors exhibited a comparable exponential growth, with a constant doubling time. Furthermore, longitudinal PC-CT imaging in mice treated with a combination of MEK and BCL-XL inhibitors revealed a drastic tumor regression accompanied by a striking remodeling of macrophages in the tumor microenvironment. Thus, PC-CT is a powerful system to detect cancer initiation and progression, and to monitor its evolution during treatment.

Liver Cancer Gene Discovery Using Gene Targeting, Sleeping Beauty, and CRISPR/Cas9.

Hepatocellular carcinoma (HCC) is a devastating and prevalent cancer with limited treatment options. Technological advances have enabled genetic screens to be employed in HCC model systems to characterize genes regulating tumor initiation and growth. Relative to traditional methods for studying cancer biology, such as candidate gene approaches or expression analysis, genetic screens have several advantages: they are unbiased, with no a priori selection; can directly annotate gene function; and can uncover gene-gene interactions. In HCC, three main types of screens have been conducted and are reviewed here: (1) transposon-based mutagenesis screens, (2) knockdown screens using RNA interference (RNAi) or the CRISPR/Cas9 system, and (3) overexpression screens using CRISPR activation (CRISPRa) or cDNAs. These methods will be valuable in future genetic screens to delineate the mechanisms underlying drug resistance and to identify new treatments for HCC.

Evaluating the landscape of gene cooperativity with receptor tyrosine kinases in liver tumorigenesis using transposon-mediated mutagenesis.

BACKGROUND & AIMS: The variety of alterations found in hepatocellular carcinoma (HCC) makes the identification of functionally relevant genes and their combinatorial actions in tumorigenesis challenging. Deregulation of receptor tyrosine kinases (RTKs) is frequent in HCC, yet little is known about the molecular events that cooperate with RTKs and whether these cooperative events play an active role at the root of liver tumorigenesis. METHODS: A forward genetic screen was performed using Sleeping Beauty transposon insertional mutagenesis to accelerate liver tumour formation in a genetic context in which subtly increased MET RTK levels predispose mice to tumorigenesis. Systematic sequencing of tumours identified common transposon insertion sites, thus uncovering putative RTK cooperators for liver cancer. Bioinformatic analyses were applied to transposon outcomes and human HCC datasets. In vitro and in vivo (through xenografts) functional screens were performed to assess the relevance of distinct cooperative modes to the tumorigenic properties conferred by RTKs. RESULTS: We identified 275 genes, most of which are altered in patients with HCC. Unexpectedly, these genes are not restricted to a small set of pathway/cellular processes, but cover a large spectrum of cellular functions, including signalling, metabolism, chromatin remodelling, mRNA degradation, proteasome, ubiquitination, cell cycle regulation, and chromatid segregation. We validated 15 tumour suppressor candidates, as shRNA-mediated targeting confers tumorigenicity to RTK-sensitized cells, but not to cells with basal RTK levels. This demonstrates that the context of enhanced RTK levels is essential for their action in tumour initiation. CONCLUSION: Our study identifies unanticipated genetic interactions underlying gene cooperativity with RTKs in HCC. Moreover, these results show how subtly increased levels of wild-type RTKs provide a tumour permissive cellular environment allowing a large spectrum of deregulated mechanisms to initiate liver cancer. LAY SUMMARY: Receptor tyrosine kinases (RTKs) are among signals frequently deregulated in patients with hepatocellular carcinoma and their deregulation confers essential biological properties to cancer cells. We have applied a genetic method to randomly mutate large numbers of genes in the context of a mouse model with increased RTK levels, predisposed to develop liver cancer. We identified mechanisms that accelerate tumour formation in cooperation with enhanced RTK levels. The wide array of cellular functions among these cooperators illustrates an extraordinary capability of RTKs to render the liver more vulnerable to additional alterations, by priming cells for tumour initiation.

Publisher Correction: Hypermethylation of gene body CpG islands predicts high dosage of functional oncogenes in liver cancer.

In the original version of this Article, the sixth sentence of the abstract incorrectly read 'Most of the genes upregulated and with hypermethylated CGIs in the Alb-R26Met HCC model undergo the same change.', and should have read 'Most of the genes upregulated and with hypermethylated CGIs in the Alb-R26Met HCC model undergo the same change in a large proportion of HCC patients.'. This has been corrected in both the PDF and HTML versions of the Article.

Hypermethylation of gene body CpG islands predicts high dosage of functional oncogenes in liver cancer.

Epigenetic modifications such as aberrant DNA methylation reshape the gene expression repertoire in cancer. Here, we used a clinically relevant hepatocellular carcinoma (HCC) mouse model (Alb-R26Met) to explore the impact of DNA methylation on transcriptional switches associated with tumorigenesis. We identified a striking enrichment in genes simultaneously hypermethylated in CpG islands (CGIs) and overexpressed. These hypermethylated CGIs are located either in the 5'-UTR or in the gene body region. Remarkably, such CGI hypermethylation accompanied by gene upregulation also occurs in 56% of HCC patients, which belong to the "HCC proliferative-progenitor" subclass. Most of the genes upregulated and with hypermethylated CGIs in the Alb-R26Met HCC model undergo the same change. Among reprogrammed genes, several are well-known oncogenes. For others not previously linked to cancer, we demonstrate here their action together as an "oncogene module". Thus, hypermethylation of gene body CGIs is predictive of elevated oncogene levels in cancer, offering a novel stratification strategy and perspectives to normalise cancer gene dosages.

Genomic Status of MET Potentiates Sensitivity to MET and MEK Inhibition in NF1-Related Malignant Peripheral Nerve Sheath Tumors.

Malignant peripheral nerve sheath tumors (MPNST) are highly resistant sarcomas that occur in up to 13% of individuals with neurofibromatosis type I (NF1). Genomic analysis of longitudinally collected tumor samples in a case of MPNST disease progression revealed early hemizygous microdeletions in NF1 and TP53, with progressive amplifications of MET, HGF, and EGFR To examine the role of MET in MPNST progression, we developed mice with enhanced MET expression and Nf1 ablation (Nf1fl/ko;lox-stop-loxMETtg/+;Plp-creERTtg/+ ; referred to as NF1-MET). NF1-MET mice express a robust MPNST phenotype in the absence of additional mutations. A comparison of NF1-MET MPNSTs with MPNSTs derived from Nf1ko/+;p53R172H;Plp-creERTtg/+ (NF1-P53) and Nf1ko/+;Plp-creERTtg/+ (NF1) mice revealed unique Met, Ras, and PI3K signaling patterns. NF1-MET MPNSTs were uniformly sensitive to the highly selective MET inhibitor, capmatinib, whereas a heterogeneous response to MET inhibition was observed in NF1-P53 and NF1 MPNSTs. Combination therapy of capmatinib and the MEK inhibitor trametinib resulted in reduced response variability, enhanced suppression of tumor growth, and suppressed RAS/ERK and PI3K/AKT signaling. These results highlight the influence of concurrent genomic alterations on RAS effector signaling and therapy response to tyrosine kinase inhibitors. Moreover, these findings expand our current understanding of the role of MET signaling in MPNST progression and identify a potential therapeutic niche for NF1-related MPNSTs.Significance: Longitudinal genomic analysis reveals a positive selection for MET and HGF copy number gain early in malignant peripheral nerve sheath tumor progression. Cancer Res; 78(13); 3672-87. ©2018 AACR.