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PDF - 35KB, Supplementary table 1 - Fold increase in dead cells upon EMD1214063 treatment as compared to untreated cells in eight studied MET mutated variants.

Radiotherapy (RT) along with surgery is the mainstay of treatment in head and neck squamous cell carcinoma (HNSCC). Radioresistance represents a major source of treatment failure, underlining the urgent necessity to explore and implement effective radiosensitization strategies. The MET receptor widely participates in the acquisition and maintenance of an aggressive phenotype in HNSCC and modulates the DNA damage response following ionizing radiation (IR). Here, we assessed MET expression and mutation status in primary and metastatic lesions within a cohort of patients with advanced HNSCC. Moreover, we investigated the radiosensitization potential of the MET inhibitor tepotinib in a panel of cell lines, in vitro and in vivo, as well as in ex vivo patient-derived organotypic tissue cultures (OTC). MET was highly expressed in 62.4% of primary tumors and in 53.6% of lymph node metastases (LNM), and in 6 of 9 evaluated cell lines. MET expression in primaries and LNMs was significantly associated with decreased disease control in univariate survival analyses. Tepotinib abrogated MET phosphorylation and to distinct extent MET downstream signaling. Pretreatment with tepotinib resulted in variable radiosensitization, enhanced DNA damage, cell death, and G2–M-phase arrest. Combination of tepotinib with IR led to significant radiosensitization in one of two tested in vivo models. OTCs revealed differential patterns of response toward tepotinib, irradiation, and combination of both modalities. The molecular basis of tepotinib-mediated radiosensitization was studied by a CyTOF-based single-cell mass cytometry approach, which uncovered that MET inhibition modulated PI3K activity in cells radiosensitized by tepotinib but not in the resistant ones.

Supplementary Table 1 shows HNSCC patients' characteristics

Supplementary figure 1 provides data concerning establishment of organotypic tissue cultures from freshly-resected HNSCC specimen Supplementary figure 2 shows analyses of MET expression and mutational status in a HNSCC cohort Supplementary figure 3 shows impact of MET inhibition by tepotinib in HNSCC cell lines in vitro Supplementary figure 4 shows effects of combined treatment by tepotinib and ionizing radiation on HNSCC cell lines Supplementary figure 5 shows analyses of OTCs responses to treatment by tepotinib combined with ionizing radiation Supplementary figure 6 shows results of assays studying mechanisms underlying tepotinib-mediated radiosensitization of HNSCC cell lines

Supplementary Methods

Supplementary Table 2 provides odds ratio per protein (CyTOF measurements)

PDF - 36KB, Legends for Supplementary Figure 1 and Supplementary Table 1.

PDF - 2667KB, Supplementary figure 1 - Impact of EMD1214063 treatment on the viability of NIH3T3 MET M1268T and Y1248H variants.

Supplementary figure 1. A, Representative two-parameter flow diagrams (BrdU uptake versus DNA content) of the BrdU proliferation assay; Supplementary figure 2. A, GTL-16 tumor xenograft growth as monitored daily by caliper measurements (METi - days 1-6; IR - day 3); Supplementary figure 3. Total KATO-II cellular protein extracts were subjected to western blotting with a specific antibody against p-p53, p53, p21 or p16 following the indicated treatments; Supplementary figure 4. A, representative images (40x magnification) of SA-βGal activity in MKN-45, SNU-5 and KATO-II cells; Supplementary figure 5. Western blot analysis showing FOXM1 total levels and following MET, MEK or PI3K inhibition; Supplementary figure 6. A, GTL-16 overexpressing FOXM1 cells (clone 1 and 2) and empty vector GTL-16 cells were subjected to western blotting using specific antibodies against FOXM1, BRIP and yH2AX following the indicated treatments.

Table 1. Correlation between MET and FOXM1 expression levels with clinico-pathological features in a cohort of 72 patients with gastric cancer.

Supplementary figures legends

Small proline-rich proteins (SPRRPs) are traditionally known for their function in keratinocyte homeostasis. Recent evidence demonstrates their involvement in additional diverse physiological processes ranging from p53 signaling and direct prevention of DNA damage to bactericidal activities. We highlight these novel, intriguing roles of SPRRPs and discuss them in the context of relevant pathological conditions.

Long noncoding RNAs (lncRNAs) can act as tumour suppressors or oncogenes to repress/promote tumour cell proliferation via RNA-dependent mechanisms. Recently, genome sequencing has identified elevated densities of tumour somatic single nucleotide variants (SNVs) in lncRNA genes. However, this has been attributed to phenotypically-neutral “passenger” processes, and the existence of positively-selected fitness-altering “driver” SNVs acting via lncRNAs has not been addressed. We developed and used ExInAtor2, an improved driver-discovery pipeline, to map pancancer and cancer-specific mutated lncRNAs across an extensive cohort of 2583 primary and 3527 metastatic tumours. The 54 resulting lncRNAs are mostly linked to cancer for the first time. Their significance is supported by a range of clinical and genomic evidence, and display oncogenic potential when experimentally expressed in matched tumour models. Our results revealed a striking SNV hotspot in the iconic NEAT1 oncogene, which was ascribed by previous studies to passenger processes. To directly evaluate the functional significance of NEAT1 SNVs, we used in cellulo mutagenesis to introduce tumour-like mutations in the gene and observed a consequent increase in cell proliferation in both transformed and normal backgrounds. Mechanistic analyses revealed that SNVs alter NEAT1 ribonucleoprotein assembly and boost subnuclear paraspeckles. This is the first experimental evidence that mutated lncRNAs can contribute to the pathological fitness of tumour cells.

Peposertib is a small-molecule inhibitor of DNA-PK, a key regulator of nonhomologous end joining (NHEJ). NHEJ inhibition along with irradiation (IR)-induced DNA double-strand breaks increases antitumor treatment efficacy. Here, we investigate responses of HNSCC models with distinct HPV and p53 status to the treatment with IR, DNA-PK inhibition, and their combination. Three groups of cell lines with various HPV/p53 genotypes (p53-wt/HPV-; p53-mutated/HPV-, and p53-wt/HPV+) were treated by peposertib, 4Gy IR, or a combination of both. In addition to viability and cell cycle assays, caspase 3 activity and senescence-associated β-galactosidase (β-gal) assays were used to evaluate cell fates such as apoptosis and senescence. yH2AX and RAD51 foci immunostainings were implemented to assess the levels of DNA damage and repair. NMRI-nu mice with subcutaneous xenografts of p53-wt/HPV+ and p53-wt/HPV- cell lines were treated with either fractionated 10Gy IR (via the small animal radiation therapy system SmART) alone or in combination with orally distributed peposertib for five consecutive days. Tumors were collected at the end of experiment and the immunostaining targeting Ki67, nuclear DNA fragmentation (TUNEL staining of apoptosis) and β-gal (senescence) was performed. Decreased number of viable cells after IR alone and particularly after combined treatment was observed in most cell lines. Inhibition of NHEJ combined with IR induces an abrogation of proliferation with different cell fates. Whereas HPV+ and p53-mutated cells undergo apoptosis due to a common alteration in the p53 pathways, p53-wt cells are preferentially eliminated through senescence. Elevated yH2AX foci formation after 24h and 48h in the combination treatment group indicates unresolved persistent DNA damage. In vivo, significant effects of IR and of the combination treatment on tumor growth control was observed in p53-wt/HPV+ but not in p53-wt/HPV- xenografts where the tumors relapsed towards end of follow-up. High level of proliferative and apoptotic cells and low level of senescent cells detected in HPV+ compared to HPV- may potentially determine a better treatment response in HPV+ tumors. Diverse pattern of viability and cell cycle distribution upon treatment with IR or combination in cells with dysfunctional p53 compared to cells with p53-wt confirmed the outcome dependence on p53 and HPV status. Peposertib radiosensitizes HNSCC tumors and leads to a better treatment response in dysfunctional p53 cells both in-vitro and in-vivo. Determination of the HPV and p53 status in a particular tumor might be necessary to effectively shape the intervention outcome when combining NHEJ targeting with radiation therapy. The work was supported by the healthcare business of Merck KGaA, Darmstadt, Germany (CrossRef Funder ID: 10.13039/100009945), who provided peposertib free of charge. Citation Format: Liana Hayrapetyan, Selina M. Roth, Lusine Hovhannisyan, Matúš Medo, Daniel M. Aebersold, Yitzhak Zimmer, Michaela Medová. p53-dependent treatment response to DNA-PK inhibition in combination with irradiation in head and neck squamous cell carcinoma (HNSCC) models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 498.

Increasing evidence suggests that interference with growth factor receptor tyrosine kinase (RTK) signaling can affect DNA damage response (DDR) networks, with a consequent impact on cellular responses to DNA‐damaging agents widely used in cancer treatment. In that respect, the MET RTK is deregulated in abundance and/or activity in a variety of human tumors. Using two proteomic techniques, we explored how disrupting MET signaling modulates global cellular phosphorylation response to ionizing radiation (IR). Following an immunoaffinity‐based phosphoproteomic discovery survey, we selected candidate phosphorylation sites for extensive characterization by targeted proteomics focusing on phosphorylation sites in both signaling networks. Several substrates of the DDR were confirmed to be modulated by sequential MET inhibition and IR, or MET inhibition alone. Upon combined treatment, for two substrates, NUMA1 S395 and CHEK1 S345, the gain and loss of phosphorylation, respectively, were recapitulated using invivo tumor models by immunohistochemistry, with possible utility in future translational research. Overall, we have corroborated phosphorylation sites at the intersection between MET and the DDR signaling networks, and suggest that these represent a class of proteins at the interface between oncogene‐driven proliferation and genomic stability., Interference with the MET receptor tyrosine kinase signaling affects the cellular DNA damage response (DDR) to ionizing radiation. Using two proteomic techniques to explore phosphorylation signaling networks, we identified several substrates downstream of MET and the DDR that were modulated by sequential MET inhibition and IR, or MET inhibition alone.

Metastases and tumor recurrence have a major prognostic impact in head and neck squamous cell carcinoma (HNSCC); however, cellular models that comprehensively characterize metastatic and recurrent HNSCC are lacking. To this end, we obtained genomic, transcriptomic, and copy number profiles of the UM-SCC cell line panel, encompassing patient-matched metastatic and recurrent cells. UM-SCC cells recapitulate the most prevalent genomic alterations described in HNSCC, featuring common TP53, PI3K, NOTCH, and Hippo pathway mutations. This analysis identified a novel F977Y kinase domain PIK3CA mutation exclusively present in a recurrent cell line (UM-SCC14B), potentially conferring resistance to PI3K inhibitors. Small proline-rich protein 2A (SPRR2A), a protein involved in epithelial homeostasis and invasion, was one of the most consistently downregulated transcripts in metastatic and recurrent UM-SCC cells. Assessment of SPRR2A protein expression in a clinical cohort of patients with HNSCC confirmed common SPRR2A downregulation in primary tumors (61.9% of cases) and lymph node metastases (31.3%), but not in normal tissue. High expression of SPRR2A in lymph node metastases was, along with nonoropharyngeal location of the primary tumor, an independent prognostic factor for regional disease recurrence after surgery and radiotherapy (HR 2.81; 95% CI, 1.16–6.79; P = 0.02). These results suggest that SPRR2A plays a dual role in invasion and therapeutic resistance in HNSCC, respectively through its downregulation and overexpression. Implications: The current study reveals translationally relevant mechanisms underlying metastasis and recurrence in HNSCC and represents an adjuvant tool for preclinical research in this disease setting. Underlining its discovery potential this approach identified a PIK3CA-resistant mutation as well as SPRR2A as possible theragnostic markers.

The toll-like receptor 5 (TLR5) agonist, CBLB502/Entolimod, is a peptide derived from bacterial flagellin and has been shown to protect against radiation-induced tissue damage in animal models. Here we investigated the protective mechanism of CBLB502 in the liver using models of ischemia-reperfusion injury and concanavalin A (ConA) induced immuno-hepatitis. We report that pretreatment of mice with CBLB502 provoked a concomitant activation of NF-κB and STAT3 signaling in the liver and reduced hepatic damage in both models. To understand the underlying mechanism, we screened for cytokines in the serum of CBLB502 treated animals and detected high levels of IL-22. There was no transcriptional upregulation of IL-22 in the liver, rather it was found in extrahepatic tissues, mainly the colon, mesenteric lymph nodes (MLN), and spleen. RNA-seq analysis on isolated hepatocytes demonstrated that the concomitant activation of NF-κB signaling by CBLB502 and STAT3 signaling by IL-22 produced a synergistic cytoprotective transcriptional signature. In IL-22 knockout mice, the loss of IL-22 resulted in a decrease of hepatic STAT3 activation, a reduction in the cytoprotective signature, and a loss of hepatoprotection following ischemia-reperfusion-induced liver injury. Taken together, these findings suggest that CBLB502 protects the liver by increasing hepatocyte resistance to acute liver injury through the cooperation of TLR5-NF-κB and IL-22-STAT3 signaling pathways.

The DNA-PK holoenzyme is a fundamental element of the DNA damage response machinery (DDR), which is responsible for cellular genomic stability. Consequently, and predictably, over the last decades since its identification and characterization, numerous pre-clinical and clinical studies reported observations correlating aberrant DNA-PK status and activity with cancer onset, progression and responses to therapeutic modalities. Notably, various studies have established in recent years the role of DNA-PK outside the DDR network, corroborating its role as a pleiotropic complex involved in transcriptional programs that operate biologic processes as epithelial to mesenchymal transition (EMT), hypoxia, metabolism, nuclear receptors signaling and inflammatory responses. In particular tumor entities as prostate cancer, immense research efforts assisted mapping and describing the overall signaling networks regulated by DNA-PK that control metastasis and tumor progression. Correspondingly, DNA-PK emerges as an obvious therapeutic target in cancer and data pertaining to various pharmacological approaches have been published, largely in context of combination with DNA-damaging agents (DDAs) that act by inflicting DNA double strand breaks (DSBs). Currently, new generation inhibitors are tested in clinical trials. Several excellent reviews have been published in recent years covering the biology of DNA-PK and its role in cancer. In the current article we are aiming to systematically describe the main findings on DNA-PK signaling in major cancer types, focusing on both preclinical and clinical reports and present a detailed current status of the DNA-PK inhibitors repertoire.

CRISPR-Cas9 deletion (CRISPR-del) is the leading approach for eliminating DNA from mammalian cells and underpins a variety of genome-editing applications. Target DNA, defined by a pair of double strand breaks (DSBs), is removed during non-homologous end-joining (NHEJ). However, the low efficiency of CRISPR-del results in laborious experiments and false negative results. Using an endogenous reporter system, we demonstrate that temporary inhibition of DNA-dependent protein kinase (DNA-PK) – an early step in NHEJ - yields up to 17-fold increase in DNA deletion. This is observed across diverse cell lines, gene delivery methods, commercial inhibitors and guide RNAs, including those that otherwise display negligible activity. Importantly, the method is compatible with pooled functional screens employing lentivirally-delivered guide RNAs. Thus, delaying the kinetics of NHEJ relative to DSB formation is a simple and effective means of enhancing CRISPR-deletion.

MET, the receptor tyrosine kinase (RTK) for hepatocyte growth factor, is a proto-oncogene involved in embryonic development and throughout life in homeostasis and tissue regeneration. Deregulation of MET signaling has been reported in numerous malignancies, prompting great interest in MET targeting for cancer therapy. The present review offers a summary of the biology of MET and its known functions in normal physiology and carcinogenesis, followed by an overview of the most relevant MET-targeting strategies and corresponding clinical trials, highlighting both past setbacks and promising future prospects. By placing their efforts on a more precise stratification strategy through the genetic analysis of tumors, modern trials such as the NCI-MATCH trial could revive the past enthusiasm for MET-targeted therapy.

Tumor metabolism is a thrilling discipline that focuses on mechanisms used by cancer cells to earn crucial building blocks and energy to preserve growth and overcome resistance to various treatment modalities. At the same time, therapies directed specifically against aberrant signalling pathways driven by protein tyrosine kinases (TKs) involved in proliferation, metastasis and growth count for several years to promising anti-cancer approaches. In this respect, small molecule inhibitors are the most widely used clinically relevant means for targeted therapy, with a rising number of approvals for TKs inhibitors. In this review, we discuss recent observations related to TKs-associated metabolism and to metabolic feedback that is initialized as cellular response to particular TK-targeted therapies. These observations provide collective evidence that therapeutic responses are primarily linked to such pathways as regulation of lipid and amino acid metabolism, TCA cycle and glycolysis, advocating therefore the development of further effective targeted therapies against a broader spectrum of TKs to treat patients whose tumors display deregulated signalling driven by these proteins.

MET, the receptor tyrosine kinase (RTK) for hepatocyte growth factor, is a proto-oncogene involved in embryonic development and throughout life in homeostasis and tissue regeneration. Deregulation of MET signaling has been reported in numerous malignancies, prompting great interest in MET targeting for cancer therapy. The present review offers a summary of the biology of MET and its known functions in normal physiology and carcinogenesis, followed by an overview of the most relevant MET-targeting strategies and corresponding clinical trials, highlighting both past setbacks and promising future prospects. By placing their efforts on a more precise stratification strategy through the genetic analysis of tumors, modern trials such as the NCI-MATCH trial could revive the past enthusiasm for MET-targeted therapy.

Lysosomal sequestration of anti-cancer compounds reduces drug availability at intracellular target sites, thereby limiting drug-sensitivity and inducing chemoresistance. For hepatocellular carcinoma (HCC), sorafenib (SF) is the first line systemic treatment, as well as a simultaneous activator of autophagy-induced drug resistance. The purpose of this study is to elucidate how combination therapy with the FDA-approved photosensitizer verteporfin (VP) can potentiate the antitumor effect of SF, overcoming its acquired resistance mechanisms. HCC cell lines and patient-derived in vitro and in vivo preclinical models were used to identify the molecular mechanism of action of VP alone and in combination with SF. We demonstrate that SF is lysosomotropic and increases the total number of lysosomes in HCC cells and patient-derived xenograft model. Contrary to the effect on lysosomal stability by SF, VP is not only sequestered in lysosomes, but induces lysosomal pH alkalinization, lysosomal membrane permeabilization (LMP) and tumor-selective proteotoxicity. In combination, VP-induced LMP potentiates the antitumor effect of SF, further decreasing tumor proliferation and progression in HCC cell lines and patient-derived samples in vitro and in vivo. Our data suggest that combination of lysosome-targeting compounds, such as VP, in combination with already approved chemotherapeutic agents could open a new avenue to overcome chemo-insensitivity caused by passive lysosomal sequestration of anti-cancer drugs in the context of HCC.

Genetic aberrations in the hepatocyte growth factor receptor tyrosine kinase MET induce oncogenic addiction in various types of human cancers, advocating MET as a viable anticancer target. Here, we report that MET signaling plays an important role in conferring a unique metabolic phenotype to cellular models expressing MET-activating mutated variants that are either sensitive or resistant toward MET small molecule inhibitors. MET phosphorylation downregulated by the specific MET inhibitor tepotinib resulted in markedly decreased viability and increased apoptosis in tepotinib-sensitive cells. Moreover, prior to the induction of MET inhibition-dependent cell death, tepotinib also led to an altered metabolic signature, characterized by a prominent reduction of metabolite ions related to amino sugar metabolism, gluconeogenesis, glycine and serine metabolism, and of numerous TCA cycle-related metabolites such as succinate, malate, and citrate. Functionally, a decrease in oxygen consumption rate, a reduced citrate synthase activity, a drop in membrane potential, and an associated misbalanced mitochondrial function were observed exclusively in MET inhibitor-sensitive cells. These data imply that interference with metabolic state can be considered an early indicator of efficient MET inhibition and particular changes reported here could be explored in the future as markers of efficacy of anti-MET therapies.

Radiotherapy (RT) along with surgery is the mainstay of treatment in head and neck squamous cell carcinoma (HNSCC). Radioresistance represents a major source of treatment failure, underlining the urgent necessity to explore and implement effective radiosensitization strategies. The MET receptor widely participates in the acquisition and maintenance of an aggressive phenotype in HNSCC and modulates the DNA damage response following ionizing radiation (IR). Here, we assessed MET expression and mutation status in primary and metastatic lesions within a cohort of patients with advanced HNSCC. Moreover, we investigated the radiosensitization potential of the MET inhibitor tepotinib in a panel of cell lines, in vitro and in vivo, as well as in ex vivo patient-derived organotypic tissue cultures (OTC). MET was highly expressed in 62.4% of primary tumors and in 53.6% of lymph node metastases (LNM), and in 6 of 9 evaluated cell lines. MET expression in primaries and LNMs was significantly associated with decreased disease control in univariate survival analyses. Tepotinib abrogated MET phosphorylation and to distinct extent MET downstream signaling. Pretreatment with tepotinib resulted in variable radiosensitization, enhanced DNA damage, cell death, and G2–M-phase arrest. Combination of tepotinib with IR led to significant radiosensitization in one of two tested in vivo models. OTCs revealed differential patterns of response toward tepotinib, irradiation, and combination of both modalities. The molecular basis of tepotinib-mediated radiosensitization was studied by a CyTOF-based single-cell mass cytometry approach, which uncovered that MET inhibition modulated PI3K activity in cells radiosensitized by tepotinib but not in the resistant ones.

Glioblastoma multiforme (GBM) is the most prevalent primary malignant brain tumor, which has an aggressive phenotype and mostly fatal recurrence after standard therapy. MET, the hepatocyte growth factor (HGF) receptor is a relevant target for GBM treatment as it is expressed in up to 50% of cases and its expression, which may be induced by radiation therapy (RT), can potentially contribute to RT-resistance of GBM stem cells, and tumor recurrence. MET-based chimeric antigen (CAR) T cell therapy is currently being evaluated in several solid tumor working settings. A combination of T cell-based therapies with RT may improve the efficacy of the CAR T cell therapy through by the RT-induced immune activation via release of cytokines and induction of antigen expression, similarly as has been observed when combining RT with immune checkpoint inhibitors. Here we investigated the combination of MET-targeting CAR T cells with RT for GBM treatment, hypothesizing enhanced anti-tumor effects. The current study used a panel of MET-proficient and MET-deficient human GBM and GBM stem-like cell lines. Cells have been irradiated with a single dose of 0, 2, 5, or 10 Gy and RT impact on MET expression has been assessed at various time points after RT administration. Results show MET increase after RT in some cell lines. To test MET-targeting CAR T effect on the GBM cell lines, CAR constructs that are based on HGF-MET binding elements have been introduced into virus-producing cells and used for human T cells transduction to generate MET-targeting CAR T cells. CAR T cells co-cultured with GBM cell lines in vitro have specifically and significantly decreased viability of MET-positive cancer cells. Data resulting from combination of RT and CAR T cells treatment suggest that radiation exhibits an enhancement of CAR T cells anti-tumor killing activity, indicating a synergism between the two modalities. In conclusion, our data are the first to indicate the efficacy of a MET-based CAR T immunotherapy approach in GBM cell lines. The results also demonstrate a basis for the combination of a MET CAR T modality together with RT. The mechanisms for understanding the interaction between RT and the MET CAR T cells are under investigation. Citation Format: Lusine Hovhannisyan, Daniel M. Aebersold, John Maher, Adrian F. Ochsenbein, Carsten Riether, Michaela Medova, Yitzhak Zimmer. Radiation therapy enhances anti-tumor activity of a MET CAR T-based immunotherapy approach for glioblastoma multiforme [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-039.

Double strand breaks (DSBs) are the most lethal DNA lesions as they can result in chromosomal rearrangements or loss of genetic material. To detect and repair DNA, cells have developed the DNA damage response, a cascade of proteins in which the checkpoint kinase 2 (CHK2) is involved. CHK2-deficient cancers, such as breast, prostate or colon, might have a compromised response to ionizing radiation (IR), arresting cell cycle at checkpoints or inducing apoptosis in presence of irreparable damage. To understand how the lack of CHK2 is affecting DNA damage response and repair, the human isogenic HCT116 colorectal cancer cell lines proficient (CHK2 WT) and deficient (CHK2 KO) for CHK2 have been characterized. Cell cycle distribution and γH2AX foci formation were assessed by flow cytometry and immunofluorescence, respectively, to determine the differences in cell cycle checkpoints and DSBs infliction and resolution upon IR. Traffic Light Reporter, complemented by immunofluorescence of RAD51 foci, was employed in both CHK2 WT and KO cell lines to identify the preferred DSB repair pathway. Our results indicate that CHK2-proficient and -deficient cell lines do not exhibit significant differences in cell cycle distribution upon IR. On the other hand, CHK2 KO cells display a significant increase in γH2AX foci per cell compared to the CHK2 WT. Upon 1Gy of IR, cells lacking CHK2 increment the number of foci per cell, indicating that DSBs detection occurs independently of the CHK2 status. Furthermore, the Traffic Light Reporter revealed higher homologous recombination (HR) to non-homologous end-joining (NHEJ) ratio in the CHK2 KO cells as compared to their CHK2 WT counterparts, suggesting that the lack of CHK2 stimulates a shift in the DSBs repair pathway choice. These results contrast with the RAD51 foci formation, which seems to be compromised in the CHK2 KO cells in both treated and untreated conditions as compared to the CHK2-proficient cells. Our results suggest that CHK2 status considerably affects cellular DNA damage response and these differences in execution of DNA repair could be exploited in CHK2-deficient cancers. Citation Format: Carmen Muñoz-Maldonado, Aurélie Quintin, Daniel M. Aebersold, Yitzhak Zimmer, Michaela Medová. DNA damage response and repair characterization in CHK2-deficient cancers [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-026.

Purpose: Deregulated signaling via the MET receptor tyrosine kinase is abundant in gastric tumors, with up to 80% of cases displaying aberrant MET expression. A growing body of evidence suggests MET as a potential target for tumor radiosensitization. Experimental Design: Cellular proliferation and DNA damage-induced senescence were studied in a panel of MET-overexpressing human gastric cancer cell lines as well as in xenograft models after MET inhibition and/or ionizing radiation. Pathways activation and protein expression were assessed by immunoblotting and immunohistochemistry. Tumor tissue microarrays (91 gastric cancer patients) were generated and copy number alteration (178 patients) and gene expression (373 patients) data available at The Cancer Genome Atlas were analyzed to assess the coalterations of MET and FOXM1. Results: MET targeting administered before ionizing radiation instigates DNA damage–induced senescence (∼80%, P < 0.001) rather than cell death. MET inhibition–associated senescence is linked to the blockade of MAPK pathway, correlates with downregulation of FOXM1, and can be abrogated (11.8% vs. 95.3%, P < 0.001) by ectopic expression of FOXM1 in the corresponding gastric tumor cells. Cells with ectopic FOXM1 expression demonstrate considerable (∼20%, P < 0.001) growth advantage despite MET targeting, suggesting a novel clinically relevant resistance mechanism to MET inhibition as the copresence of both MET and FOXM1 protein (33%) and mRNA (30%) overexpression as well as gene amplification (24,7%) are common in patients with gastric cancer. Conclusions: FOXM1, a negative regulator of senescence, has been identified as a key downstream effector and potential clinical biomarker that mediates MET signaling following infliction of DNA damage in gastric tumors. Clin Cancer Res; 22(21); 5322–36. ©2016 AACR.

Various lines of investigation support a signaling interphase shared by various receptor tyrosine kinases and the DNA damage response. However, the underlying driving network nodes and their contribution to the maintenance of DNA integrity remain unknown. We used discovery metabolomics combined with transcriptomics to identify changes in global metabolic pathways that are relevant to DNA repair following MET inhibition (METi). METi was associated with formation of γH2AX foci together with significant alterations in major metabolic circuits. Remarkably, 5′-phosphoribosyl-N-formylglycinamide (FGAR), a de novo purine synthesis pathway metabolite, was decreased in MET-dependent cancer models and a METi-related depletion of dNTPs was observed. Likewise, METi instigated the downregulation of critical purine synthesis enzymes including phosphoribosylglycinamide formyltransferase (GART), which is responsible for FGAR synthesis. Genes encoding these enzymes are regulated through the E2F1 transcription factor, whose levels decreased upon METi in MET-driven cells. To validate those findings, a transient E2F1 overexpression was sufficient to prevent MET targeting-dependent dNTPs depletion and the concomitant METi-associated DNA damage in MET-driven tumor cells. These novel observations support the notion that METi-mediated sensitization to DNA-damaging agents commonly used in cancer therapeutics is largely linked to the depletion of critical metabolites used in DNA repair due to downregulation of key metabolic enzymes whose expression is regulated through MET signaling. This abstract is also being presented as Poster A51. Citation Format: Michaela Poliaková, Matúš Medo, Jonas Koch, SiAna Coggins, Baek Kim, Daniel M. Aebersold, Nicola Zamboni, Michaela Medová, Yitzhak Zimmer. A metabolomics discovery profiling approach identifies an E2F1-associated purine synthesis pathway as a major component of the MET-DNA damage response network [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr PR05.

There is emerging evidence that DNA damage response (DDR) intertwines with signaling pathways downstream of oncogenic receptor tyrosine kinases (RTKs). Two complementary phosphoproteomic approaches revealed global networks and identified and validated core phosphosites that participate in a crosstalk between the MET RTK and the DDR, including a newly discovered phosphosite on MET itself, Ser1016. DNA-PK phosphorylates Ser1016, and this phosphorylation is increased following irradiation. Cell lines as well as animal models expressing Ser1016Ala phosphodeficient receptor exhibit greater sensitivity to irradiation. MET Ser1016Ala mutants fail to undergo proper radiation-induced cell cycle arrest by bypassing the G2 checkpoint with a premature entry into mitosis and abnormal mitotic spindles, leading to a lower proliferation rate of Ser1016-deficient cells. Regulation of MET activity through Ser1016 phosphorylation by a DDR core kinase represents a critical event involved in DNA repair signaling through a growth factor receptor that frequently functions as a tumor driver. Citation Format: Jonas P. Koch, Ariel Bensimon, Selina M. Roth, Matúš Medo, Paola Francica, Eleonora Orlando, Rahel Riedo, Jacopo Gavini, Aurélie Quintin, Astrid A. Glück, Deborah M. Stroka, Andree Blaukat, Daniel M. Aebersold, Yitzhak Zimmer, Ruedi Aebersold, Michaela Medová. Deciphering MET-dependent modulation of global cellular responses to DNA damage by quantitative phosphoproteomics reveals Ser1016 as a novel MET phosphosite that regulates cellular response to irradiation [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr A52.

Deregulated expression of the MET receptor tyrosine kinase has been reported in up to 50% of patients with hepatocellular carcinoma, the most abundant form of liver cancers, and is associated with decreased survival. Consequently, MET is considered as a molecular target in this malignancy, whose progression is highly dependent on extensive angiogenesis. Here we studied the impact of MET small molecule inhibitors on angiogenesis-associated parameters and growth of xenograft liver models consisting of cells expressing MET-mutated variants M1268T and Y1248H, which exhibit constitutive kinase activity. We demonstrate that MET mutations expression is associated with significantly increased production of vascular endothelial growth factor, which is blocked by MET targeting only in cells expressing the M1268T inhibitor-sensitive but not in the Y1248H inhibitor-resistant variant. Decrease in vascular endothelial growth factor production is also associated with reduction of tyrosine phopshorylation of the vascular endothelial growth factor receptor 2 expressed on primary liver sinusoidal endothelial cells and with inhibition of vessel formation. Furthermore, MET inhibition demonstrated an efficient anti-tumor activity and considerable reduction in microvessel density only against the M1268T-derived intrahepatic tumors. Collectively, our data support the role of targeting MET-associated angiogenesis as a major biological determinant for liver tumor growth control.

The MET receptor tyrosine kinase is often deregulated in human cancers and several MET inhibitors are evaluated in clinical trials. Similarly to EGFR, MET signals through the RAS-RAF-ERK/MAPK pathway which plays key roles in cell proliferation and survival. Mutations of genes encoding for RAS proteins, particularly in KRAS, are commonly found in various tumors and are associated with constitutive activation of the MAPK pathway. It was shown for EGFR, that KRAS mutations render upstream EGFR inhibition ineffective in EGFR-positive colorectal cancers. Currently, there are no clinical studies evaluating MET inhibition impairment due to RAS mutations. To test the impact of RAS mutations on MET targeting, we generated tumor cells responsive to the MET inhibitor EMD1214063 that express KRAS G12V, G12D, G13D and HRAS G12V variants. We demonstrate that these MAPK-activating RAS mutations differentially interfere with MET-mediated biological effects of MET inhibition. We report increased residual ERK1/2 phosphorylation indicating that the downstream pathway remains active in presence of MET inhibition. Consequently, RAS variants counteracted MET inhibition-induced morphological changes as well as anti-proliferative and anchorage-independent growth effects. The effect of RAS mutants was reversed when MET inhibition was combined with MEK inhibitors AZD6244 and UO126. In an in vivo mouse xenograft model, MET-driven tumors harboring mutated RAS displayed resistance to MET inhibition. Taken together, our results demonstrate for the first time in details the role of KRAS and HRAS mutations in resistance to MET inhibition and suggest targeting both MET and MEK as an effective strategy when both oncogenic drivers are expressed.

High-throughput molecular profiling approaches have emerged as precious research tools in the field of head and neck translational oncology. Such approaches have identified and/or confirmed the role of several genes or pathways in the acquisition/maintenance of an invasive phenotype and the execution of cellular programs related to cell invasion. Recently published new-generation sequencing studies in head and neck squamous cell carcinoma (HNSCC) have unveiled prominent roles in carcinogenesis and cell invasion of mutations involving NOTCH1 and PI3K-patwhay components. Gene-expression profiling studies combined with systems biology approaches have allowed identifying and gaining further mechanistic understanding into pathways commonly enriched in invasive HNSCC. These pathways include antigen-presenting and leucocyte adhesion molecules, as well as genes involved in cell-extracellular matrix interactions. Here we review the major insights into invasiveness in head and neck cancer provided by high-throughput molecular profiling approaches.

Poor oxygenation is a common hallmark of solid cancers that strongly associates with aggressive tumor progression and treatment resistance. While a hypoxia-inducible factor 1α (HIF-1α)-associated transcriptional overexpression of the hepatocyte growth factor (HGF) receptor tyrosine kinase (RTK) MET has been previously documented, any regulation of the HIF-1α system through MET downstream signaling in hypoxic tumors has not been yet described. By using MET-driven in vitro as well as ex vivo tumor organotypic fresh tissue models we report that MET targeting results in depletion of HIF-1α and its various downstream targets. Mechanistically, we provide evidence that MET regulates HIF-1α levels through a protein translation mechanism that relies on phosphorylation modulation of the eukaryotic initiation factor 4G1 (eIF4G1) on serine 1232 (Ser-1232). Targeted phosphoproteomics data demonstrate a significant drop in eIF4G1 Ser-1232 phosphorylation following MET targeting, which is linked to an increased affinity between eIF4G1 and eIF4E. Since phosphorylation of eIF4G1 on Ser-1232 is largely mediated through mitogen-activated protein kinase (MAPK), we show that expression of a constitutively active K-RAS variant is sufficient to abrogate the inhibitory effect of MET targeting on the HIF-1α pathway with subsequent resistance of tumor cells to MET targeting under hypoxic conditions. Analysis of The Cancer Genome Atlas data demonstrates frequent co-expression of MET, HIF-1α and eIF4G1 in various solid tumors and its impact on disease-free survival of non-small cell lung cancer patients. Clinical relevance of the MET-eIF4G1-HIF-1α pathway is further supported by a co-occurrence of their expression in common tumor regions of individual lung cancer patients.

Background The MET receptor tyrosine kinase represents a promising target in cancer. PIK3CA activating mutations are common in several tumor types and can potentially confer resistance to anti-receptor tyrosine kinase therapy. Methods MET and/or PI3K pathway inhibition was assessed in NIH3T3 cells harboring MET-activating point mutation with or without ectopic expression of PIK3CAE545K and PIK3CAH1047R, as well as in MET-expressing head and neck cancer cells with endogenous PIK3CA mutations. Endpoints included PI3K pathway activation, cell proliferation, colony-forming ability, cell death, wound-healing, and an in vivo model. Results PIK3CAE545K and PIK3CAH1047R confer resistance to MET inhibition in MET-driven models. PIK3CAH1047R was more potent than PIK3CAE545K at inducing resistance in PI3K pathway activation, cell proliferation, colony-forming ability, induction of cell death and wound-healing upon MET inhibition. Resistance to MET inhibition could be synergistically overcome by co-targeting PI3K. Furthermore, combined MET/PI3K inhibition led to enhanced anti-tumor activity in vivo in tumors harboring PIK3CAH1047R. In head and neck cancer cells the combination of MET/PI3K inhibitors led to more-than-additive effects. Conclusions PIK3CA mutations can lead to resistance to MET inhibition, supporting future clinical evaluation of combinations of PI3K and MET inhibitors in common scenarios of malignant neoplasms featuring aberrant MET expression and PIK3CA mutations. Electronic supplementary material The online version of this article (doi:10.1186/s12943-017-0660-5) contains supplementary material, which is available to authorized users.

Idiopathic Pulmonary Fibrosis (IPF) is a progressive, irreversible lung disease with complex pathophysiology. Evidence of endoplasmic reticulum (ER) stress has been reported in alveolar epithelial cells (AEC) in IPF patients. Secreted mediators from bone marrow stem cells (BMSC-cm) have regenerative properties. In this study we investigate the beneficial effects of BMSC-cm on ER stress response in primary AEC and ER stressed A549 cells. We hypothesize that BMSC-cm reduces ER stress. Primary AEC isolated from IPF patients were treated with BMSC-cm. To induce ER stress A549 cells were incubated with Tunicamycin or Thapsigargin and treated with BMSC-cm, or control media. Primary IPF-AEC had high Grp78 and CHOP gene expression, which was lowered after BMSC-cm treatment. Similar results were observed in ER stressed A549 cells. Alveolar epithelial repair increased in presence of BMSC-cm in ER stressed A549 cells. Hepatocyte growth factor (HGF) was detected in biologically relevant levels in BMSC-cm. Neutralization of HGF in BMSC-cm attenuated the beneficial effects of BMSC-cm including synthesis of surfactant protein C (SP-C) in primary AEC, indicating a crucial role of HGF in ER homeostasis and alveolar epithelial repair. Our data suggest that BMSC-cm may be a potential therapeutic option for treating pulmonary fibrosis.

Head and neck cancer constitutes the 6th most common malignancy worldwide and affects the crucial anatomical structures and physiological functions of the upper aerodigestive tract. Classical therapeutic strategies such as surgery and radiotherapy carry substantial toxicity and functional impairment. Moreover, the loco-regional control rates as well as overall survival still need to be improved in subgroups of patients. The scatter-factor/hepatocyte growth factor receptor tyrosine kinase MET is an established effector in the promotion, maintenance and progression of malignant transformation in a wide range of human malignancies, and has been gaining considerable interest in head and neck cancer over the last 15 years. Aberrant MET activation due to overexpression, mutations, tumor-stroma paracrine loops, and cooperative/redundant signaling has been shown to play prominent roles in epithelial-to-mesenchymal transition, angiogenesis, and responses to anti-cancer therapeutic modalities. Accumulating preclinical and translational evidence highly supports the increasing interest of MET as a biomarker for lymph node and distant metastases, as well as a potential marker of stratification for responses to ionizing radiation. The relevance of MET as a therapeutic molecular target in head and neck cancer described in preclinical studies remains largely under-evaluated in clinical trials, and therefore inconclusive. Also in the context of anti-cancer targeted therapy, a large body of preclinical data suggests a central role for MET in treatment resistance towards multiple therapeutic modalities in malignancies of the head and neck region. These findings, as well as the potential use of combination therapies including MET inhibitors in these tumors, need to be further explored.

The MET receptor tyrosine kinase is deregulated primarily via overexpression or point mutations in various human cancers and different strategies for MET inhibition are currently evaluated in clinical trials. We observed by Western blot analysis and by Flow cytometry that MET inhibition by different MET small molecule inhibitors surprisingly increases in a dose-dependent manner total MET levels in treated cells. Mechanistically, this inhibition-related MET accumulation was associated with reduced Tyr1003 phosphorylation and MET physical association with the CBL ubiquitin ligase with concomitant decrease in MET ubiquitination. These data may suggest careful consideration for design of anti-MET clinical protocols.