DNA Damage Response and Mismatch Repair Gene Defects in Advanced and Metastatic Prostate Cancer.

Alterations in DNA damage response (DDR) and related genes are present in up to 25% of advanced prostate cancers (PCa). Most frequently altered genes are involved in the homologous recombination repair, the Fanconi anemia, and the mismatch repair pathways, and their deficiencies lead to a highly heterogeneous spectrum of DDR-deficient phenotypes. More than half of these alterations concern non- BRCA DDR genes. From a therapeutic perspective, poly-ADP-ribose polymerase inhibitors have demonstrated robust clinical efficacy in tumors with BRCA2 and BRCA1 alterations. Mismatch repair-deficient PCa, and a subset of CDK12-deficient PCa, are vulnerable to immune checkpoint inhibitors. Emerging data point to the efficacy of ATR inhibitors in PCa with ATM deficiencies. Still, therapeutic implications are insufficiently clarified for most of the non- BRCA DDR alterations, and no successful targeted treatment options have been established.

PIK3CA hotspot mutations differentially impact responses to MET targeting in MET-driven and non-driven preclinical cancer models.

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.

H2AX promotes replication fork degradation and chemosensitivity in BRCA-deficient tumours.

Histone H2AX plays a key role in DNA damage signalling in the surrounding regions of DNA double-strand breaks (DSBs). In response to DNA damage, H2AX becomes phosphorylated on serine residue 139 (known as γH2AX), resulting in the recruitment of the DNA repair effectors 53BP1 and BRCA1. Here, by studying resistance to poly(ADP-ribose) polymerase (PARP) inhibitors in BRCA1/2-deficient mammary tumours, we identify a function for γH2AX in orchestrating drug-induced replication fork degradation. Mechanistically, γH2AX-driven replication fork degradation is elicited by suppressing CtIP-mediated fork protection. As a result, H2AX loss restores replication fork stability and increases chemoresistance in BRCA1/2-deficient tumour cells without restoring homology-directed DNA repair, as highlighted by the lack of DNA damage-induced RAD51 foci. Furthermore, in the attempt to discover acquired genetic vulnerabilities, we find that ATM but not ATR inhibition overcomes PARP inhibitor (PARPi) resistance in H2AX-deficient tumours by interfering with CtIP-mediated fork protection. In summary, our results demonstrate a role for H2AX in replication fork biology in BRCA-deficient tumours and establish a function of H2AX separable from its classical role in DNA damage signalling and DSB repair.

MND1 and PSMC3IP control PARP inhibitor sensitivity in mitotic cells.

The PSMC3IP-MND1 heterodimer promotes meiotic D loop formation before DNA strand exchange. In genome-scale CRISPR-Cas9 mutagenesis and interference screens in mitotic cells, depletion of PSMC3IP or MND1 causes sensitivity to poly (ADP-Ribose) polymerase inhibitors (PARPi) used in cancer treatment. PSMC3IP or MND1 depletion also causes ionizing radiation sensitivity. These effects are independent of PSMC3IP/MND1's role in mitotic alternative lengthening of telomeres. PSMC3IP- or MND1-depleted cells accumulate toxic RAD51 foci in response to DNA damage, show impaired homology-directed DNA repair, and become PARPi sensitive, even in cells lacking both BRCA1 and TP53BP1. Epistasis between PSMC3IP-MND1 and BRCA1/BRCA2 defects suggest that abrogated D loop formation is the cause of PARPi sensitivity. Wild-type PSMC3IP reverses PARPi sensitivity, whereas a PSMC3IP p.Glu201del mutant associated with D loop defects and ovarian dysgenesis does not. These observations suggest that meiotic proteins such as MND1 and PSMC3IP have a greater role in mitotic DNA repair.

Meiotic Genes and DNA Double Strand Break Repair in Cancer.

Tumor cells show widespread genetic alterations that change the expression of genes driving tumor progression, including genes that maintain genomic integrity. In recent years, it has become clear that tumors frequently reactivate genes whose expression is typically restricted to germ cells. As germ cells have specialized pathways to facilitate the exchange of genetic information between homologous chromosomes, their aberrant regulation influences how cancer cells repair DNA double strand breaks (DSB). This drives genomic instability and affects the response of tumor cells to anticancer therapies. Since meiotic genes are usually transcriptionally repressed in somatic cells of healthy tissues, targeting aberrantly expressed meiotic genes may provide a unique opportunity to specifically kill cancer cells whilst sparing the non-transformed somatic cells. In this review, we highlight meiotic genes that have been reported to affect DSB repair in cancers derived from somatic cells. A better understanding of their mechanistic role in the context of homology-directed DNA repair in somatic cancers may provide useful insights to find novel vulnerabilities that can be targeted.

Deciphering MET-dependent modulation of global cellular responses to DNA damage by quantitative phosphoproteomics.

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.

Targeting the MET Receptor Tyrosine Kinase as a Strategy for Radiosensitization in Locoregionally Advanced Head and Neck Squamous Cell Carcinoma.

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.

Functional Radiogenetic Profiling Implicates ERCC6L2 in Non-homologous End Joining.

Using genome-wide radiogenetic profiling, we functionally dissect vulnerabilities of cancer cells to ionizing radiation (IR). We identify ERCC6L2 as a major determinant of IR response, together with classical DNA damage response genes and members of the recently identified shieldin and CTC1-STN1-TEN1 (CST) complexes. We show that ERCC6L2 contributes to non-homologous end joining (NHEJ), and it may exert this function through interactions with SFPQ. In addition to causing radiosensitivity, ERCC6L2 loss restores DNA end resection and partially rescues homologous recombination (HR) in BRCA1-deficient cells. As a consequence, ERCC6L2 deficiency confers resistance to poly (ADP-ribose) polymerase (PARP) inhibition in tumors deficient for both BRCA1 and p53. Moreover, we show that ERCC6L2 mutations are found in human tumors and correlate with a better overall survival in patients treated with radiotherapy (RT); this finding suggests that ERCC6L2 is a predictive biomarker of RT response.

Mechanisms of PARP inhibitor resistance in cancer and insights into the DNA damage response.

Inhibitors of poly(ADP-ribose) polymerase (PARPi) have entered the clinic for the treatment of patients with cancers that lack homology-directed DNA repair, but drug resistance remains a clinical hurdle. Recent advances in the identification of PARPi resistance mechanisms have yielded a better understanding of DNA end protection and the relevance of endogenous poly(ADP-ribose) glycohydrolase, highlighting new vulnerabilities.

Senescence as biologic endpoint following pharmacological targeting of receptor tyrosine kinases in cancer.

Cellular senescence was first described in 1961 in a seminal study by Hayflick and Moorhead as a limit to the replicative lifespan of somatic cells after serial cultivation. Since then, major advances in our understanding of senescence have been achieved suggesting that this mechanism is activated also by oncogenic stimuli, oxidative stress and DNA damage, giving rise to the concept of premature senescence. Regardless of the initial trigger, numerous experimental observations have been provided to support the notion that both replicative and premature senescence play pivotal roles in early stages of tumorigenesis and in response of tumor cells to anticancer treatments. Moreover, various studies have suggested that the induction of senescence by both chemo- and radiotherapy in a variety of cancer types correlates with treatment outcome. As it is widely accepted that cellular senescence may function as a fundamental barrier of tumor progression, the significance of senescence for clinical interventions that make use of novel molecular targeting-based modalities needs to be well defined. Interestingly, despite numerous studies evaluating efficacies of receptor tyrosine kinases (RTKs) targeting strategies in both preclinical and clinical settings, the relevance of RTKs inhibition-associated senescence in tumors remains less characterized. Here we review the available literature that describes premature senescence as a major mechanism following targeting of RTKs in preclinical as well as in clinical settings. Additionally, we discuss the possible role of diverse RTKs in regulating the induction of senescence following cellular stress and possible implications of this crosstalk in identification of biomarkers of inhibitor-mediated chemo- and radiosensitization approaches.

Targeting the receptor tyrosine kinase RET in combination with aromatase inhibitors in ER positive breast cancer xenografts.

The majority of breast cancers are estrogen receptor positive (ER+). Blockade of estrogen biosynthesis by aromatase inhibitors (AIs) is the first-line endocrine therapy for post-menopausal women with ER+ breast cancers. However, AI resistance remains a major challenge. We have demonstrated previously that increased GDNF/RET signaling in ER+ breast cancers promotes AI resistance. Here we investigated the efficacy of different small molecule RET kinase inhibitors, sunitinib, cabozantinib, NVP-BBT594 and NVP-AST487, and the potential of combining a RET inhibitor with the AI letrozole in ER+ breast cancers. The most effective inhibitor identified, NVP-AST487, suppressed GDNF-stimulated RET downstream signaling and 3D tumor spheroid growth. Ovariectomized mice were inoculated with ER+ aromatase-overexpressing MCF7-AROM1 cells and treated with letrozole, NVP-AST487 or the two drugs in combination. Surprisingly, the three treatment regimens showed similar efficacy in impairing MCF7-AROM1 tumor growth in vivo. However in vitro, NVP-AST487 was superior to letrozole in inhibiting the GDNF-induced motility and tumor spheroid growth of MCF7-AROM1 cells and required in combination with letrozole to inhibit GDNF-induced motility in BT474-AROM3 aromatase expressing cells. These data indicate that inhibiting RET is as effective as the current therapeutic regimen of AI therapy but that a combination treatment may delay cancer cell dissemination and metastasis.

Depletion of FOXM1 via MET Targeting Underlies Establishment of a DNA Damage-Induced Senescence Program in Gastric Cancer.

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.

Targeting of the MET receptor tyrosine kinase by small molecule inhibitors leads to MET accumulation by impairing the receptor downregulation.

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.

The novel ATP-competitive inhibitor of the MET hepatocyte growth factor receptor EMD1214063 displays inhibitory activity against selected MET-mutated variants.

The receptor tyrosine kinase MET is a prime target in clinical oncology due to its aberrant activation and involvement in the pathogenesis of a broad spectrum of malignancies. Similar to other targeted kinases, primary and secondary mutations seem to represent an important resistance mechanism to MET inhibitors. Here, we report the biologic activity of a novel MET inhibitor, EMD1214063, on cells that ectopically express the mutated MET variants M1268T, Y1248H, H1112Y, L1213V, H1112L, V1110I, V1206L, and V1238I. Our results show a dose-dependent decrease in MET autophosphorylation in response to EMD1214063 in five of the eight cell lines (IC50 2-43 nmol/L). Blockade of MET by EMD1214063 was accompanied by a reduced activation of downstream effectors in cells expressing EMD1214063-sensitive mutants. In all sensitive mutant-expressing lines, EMD1214063 altered cell-cycle distribution, primarily with an increase in G1 phase. EMD1214063 strongly influenced MET-driven biologic functions, such as cellular morphology, MET-dependent cell motility, and anchorage-independent growth. To assess the in vivo efficacy of EMD1214063, we used a xenograft tumor model in immunocompromised mice bearing NIH3T3 cells expressing sensitive and resistant MET-mutated variants. Animals were randomized for the treatment with EMD1214063 (50 mg/kg/d) or vehicle only. Remarkably, five days of EMD1214063 treatment resulted in a complete regression of the sensitive H1112L-derived tumors, whereas tumor growth remained unaffected in mice with L1213V tumors and in vehicle-treated animals. Collectively, the current data identifies EMD1214063 as a potent MET small-molecule inhibitor with selective activity towards mutated MET variants.