Inhibition Of c-Met Pathways Research Articles

Enhancing nanoparticle paclitaxel antitumor response through targeted inhibition of c-Met pathway by merestinib in gastric cancer models.

372 Background: Gastric adenocarcinoma (GAC) is the fifth most common malignancy and the third most common cause of cancer-related deaths worldwide. Combination chemotherapy regimens are commonly used to treat GAC, but the median survival time remains less than one year. Nanoparticle albumin-bound paclitaxel ( nab-paclitaxel, NPT), an approved treatment for breast cancer, non-small cell lung cancer and pancreatic cancer, has demonstrated high antitumor activity in previous GAC studies. Many growth factors and their receptors are overexpressed in GAC and have been implicated in its pathophysiology. Aberrant activation of the c-Met pathway has been reported in up to 50% of GAC patients. We hypothesize that merestinib, a small-molecule inhibitor of c-Met, with additional inhibition of Axl and DDR1/2 pathways, will have significant antitumor effects and will enhance nab-paclitaxel chemotherapy response in GAC preclinical models. Methods: GAC subcutaneous xenografts were established by injecting MKN-45 and SNU-1 cells in NOD/SCID mice to study treatment effects on tumor volume, tumor weight, net tumor growth, and mouse body weight. Immunohistochemistry analyses of xenograft tissues were performed using Ki67 and endomucin to examine tumor cell proliferation and microvessel density, respectively. Results: In vitro assays showed that nab-paclitaxel and merestinib decreased cell proliferation in all three GAC-associated cell lines tested, with an additive effect in combination. Reduction in cell proliferation at low doses of nab-paclitaxel (10 nM), merestinib (100 nM), and their combination was 87%, 82%, and 94% (MKN-45, high phospho-c-Met expression), 59%, 50%, and 82% (SNU-1, low phospho-c-Met expression), and 53%, 19%, and 66% in gastric fibroblasts. Immunoblot analysis of merestinib treated MKN-45 cells revealed increased expression of pro-apoptotic proteins and decreased expression of phospho-c-Met, phospho-EGFR, phospho-IGF-1R, phospho-ERK, and phospho-AKT. In gastric fibroblasts, merestinib decreased phospho-ERK and increased pro-apoptotic protein expression. Phospho-c-Met and phospho-EGFR were not detected in SNU-1 immunoblots; however, phospho-ERK, phospho-VEGFR, and apoptotic protein expression increased after treatment. In MKN-45 xenografts, net tumor growth in control, nab-paclitaxel, merestinib, and combination groups was 503 mm3, 115 mm3, 91 mm3, and -9.7 mm3. Immunohistochemistry analysis of tumor cell proliferation and microvessel density corroborated tumor growth study results. Conclusions: These findings suggest that merestinib has strong antitumor activity in GAC and exhibits an additive effect when administered with nab-paclitaxel. These results provide compelling evidence that this therapeutic approach may lead to a clinically relevant combination to improve GAC patients’ survival.

EXTH-21. MECHANISMS OF SYNERGISTIC GROWTH SUPPRESSION BY RADIOTHERAPY AND C-MET INHIBITION IN EXPERIMENTAL GLIOBLASTOMA

Abstract Glioblastoma remains to be one of the most lethal solid cancers and novel therapies are urgently needed. There is increasing interest in the role of the HGF/MET pathway in the response of glioblastoma to radiotherapy. c-MET-mediated radioresistance may be partially induced via proinvasive and DNA damageresponse pathways and HGF may be involved in the regulation of immune responses. Here, we explored the role of the c-MET pathway in response to radiotherapy and investigated potential modes of action that mediate synergistic effects of MET pathway inhibition and irradiation in syngeneic murine glioma models in vitro and in vivo. Murine glioma cells express HGF and c-MET and respond with c-MET phosphorylation upon exposure to exogenous HGF. In vitro, glioma cell viability and proliferation are not affected by pharmacological or genetic c-MET pathway interference, and the c-MET inhibitor tepotinib fails to sensitize glioma cells to irradiation. Conversely, in vivo c-MET inhibition combined with focal radiotherapy synergistically prolongs survival in two syngeneic orthotopic glioma models compared with either treatment alone. Complementary studies demonstrated that synergy was lost when gliomas were established and treated in immunodeficient mice, and importantly, also when c-MET gene expression was disrupted in the tumor. Thus, synergistic suppression of experimental syngeneic glioma growth by irradiation and c-MET inhibition requires at least two mechanisms, expression of c-MET in the tumor and a functional immune system. In summary, our data suggest clinical evaluation of c-MET pathway inhibition in combination with radiotherapy in human glioblastoma.

Crizotinib and PARP inhibitors act synergistically by triggering apoptosis in high-grade serous ovarian cancer.

High-grade serous ovarian cancer (HGSOC) is the predominant and most lethal histological type of epithelial ovarian cancer. During the last few years, several new treatment options with PARP inhibitors have emerged. The FDA has approved the PARP inhibitor olaparib (Lynparza™) as maintenance treatment after first-line platinum-containing chemotherapy and olaparib, niraparib (Zejula™) and rucaparib (Rubraca™) are approved as maintenance therapies in the recurrent, platinum-sensitive setting; nevertheless, development of resistance limits their efficacy. In this study, new combinatorial treatment strategies targeting key signaling pathways were explored to enhance the activity of PARP inhibitors in HGSOC. Carboplatin, olaparib, niraparib, the PI3K inhibitor LY294002 and the c-Met inhibitor crizotinib were used for this investigation. PARP inhibitors and carboplatin alone and in combination caused accumulation of DNA double-strand breaks and G2/M cell cycle arrest. In contrast, crizotinib alone or in combination with PARP inhibitors induced accumulation of cells in sub-G1. Crizotinib together with either of the PARP inhibitors was more strongly synergistic than combinations with a PARP inhibitor and carboplatin or the PI3K inhibitor. Sequential combination of crizotinib and a PARP inhibitor resulted in activation of ATM/CHK2 and inhibition of c-Met pathways, contributing to a decrease in RAD51 levels and induction of caspase-3 dependent apoptotic cell death and suggesting that the combination of crizotinib with a PARP inhibitor may be considered and further explored as a new therapeutic strategy in HGSOC.

Current advances of targeting HGF/c-Met pathway in gastric cancer.

Despite the advances in systemic chemotherapy, gastric adenocarcinoma (GC) remains the third most common cause of cancer-related deaths with poor prognosis. The heterogeneity of GC indicates that novel biomarkers should be established in order to further classify tumors and develop individual targeted therapies. High-quality preclinical and clinical research has demonstrated that growth factor (HGF)-hepatocyte growth factor receptor (c-Met) pathway plays a pivotal role on the growth, survival and invasiveness of GC. In particular, aberrant activation of HGF/c-Met signaling pathway has been associated with poor clinical outcomes, suggesting the therapeutic potential of c-Met. This has stimulated the development and evaluation of a number of c-Met targeted agents in an advance disease setting. In this review, we summarize the current state of the art in the advances on the inhibition of c-Met pathway, with particular emphasis on the clinical testing of c-Met targeted therapeutic agents. Furthermore, we discuss the challenges facing the incorporation of c-Met targeted agents in randomized trials, with the idea that the definition of the appropriate genetic and molecular context for the use of these agents remains the priority.

Have Clinical Trials Properly Assessed c-Met Inhibitors?

The c-Met/HGF pathway is implicated in cancer progression and dissemination. Many inhibitors have been developed to target this pathway. Unfortunately, most trials have failed to demonstrate efficacy. However, clinical trials have not adequately tested the concept of c-Met pathway inhibition due to the lack of appropriate patient selection criteria.

Andrographolide enhanced 5-fluorouracil-induced antitumor effect in colorectal cancer via inhibition of c-MET pathway.

Colorectal cancer (CRC) is the third most common malignant neoplasm worldwide. 5-Fluorouracil (5-Fu) is the most important chemotherapeutic drug used for the treatment of CRC. However, resistance to 5-Fu therapies is a growing concern in CRC clinical practice recently. Andrographolide (Andro) is a main bioactive constituent of the herb Andrographis paniculata, which has various biological effects including anti-inflammation and antitumor activities. In the present study, we investigated the effects of combined Andro with 5-Fu against CRC HCT-116 cells. In vitro studies showed that Andro synergistically enhanced the anti-proliferation effect of 5-Fu on HCT-116 cells due to increased apoptotic cells. Meanwhile, results of the enzyme linked immunosorbent assay indicated that the level of phosphorylated cellular-mesenchymal to epithelial transition factor (p-MET) was decreased by the combination treatment. Further study suggested that Andro promoted the antitumor effect of 5-Fu by down-regulating the level of p-MET. In conclusion, these results confirmed the synergistic antitumor activity of Andro on CRC and provide evidence for possible clinical application of Andro for enhancing the antitumor effect of 5-Fu in CRC treatment.

CyFi: A phase I study exploring the role of cMET pathway inhibition with ficlatuzumab (Fi) combined with high-dose cytarabine (Cy) in patients with high risk relapsed or refractory acute myeloid leukemia (AML).

7040 Background: Pts with AML who are refractory to induction therapy or relapse within 1 year have poor outcomes. Elevated serum HGF level is an adverse prognostic factor in AML (Verstovsek, et al. 2001; Kim, et al. 2005). Pre-clinical models have shown that myeloid blasts produce HGF in an autocrine fashion and pharmacologic blockade of the HGF/c-Met axis sensitizes blasts to cell death (Kentsis, et al. 2012). Methods: We initiated a phase I study to assess the safety and tolerability of Fi combined with Cy in patients with AML who are refractory to 7+3 or have relapsed within 1 year of induction. Fi is given in escalated dosing of 10, 15, or 20 mg/Kg for 4 doses every 2 weeks, starting on day 0, and Cy at a fixed dose of 2g/m2 on days 2-7, using a 3x3 design. PBMCs, BM and serum are collected at defined time points to assess HGF levels and activation of the c-Met pathway. Results: Dose escalation is complete and there were no protocol-defined DLTs identified in 9 evaluable pts. All pts treated to date were refractory to induction. Four had de novo AML; 2 had undifferentiated leukemia; 2 prior MDS; 1 prior MPN. Most frequent grade 3/4 TEAEs were febrile neutropenia (56%), LFT abnormalities (11%), and electrolyte disturbance (11%). There was 1 death (11%) from sepsis and multi-organ failure on day 23, following ANC recovery. Of the 7 evaluable pts, 3 achieved a CR (43%), all in the 2nddose cohort. Two of the 3 CRs are long lasting 11 and 12 months following AlloHCT. All patients had detectable circulating HGF levels at baseline compared to control subjects without AML. HGF levels increased following exposure to Fi by an average of 193%. Baseline HGF levels or change from baseline were not associated with treatment response. Conclusions: Ficlatuzumab can be safely combined with HiDAC in this high-risk AML population and produce durable clinical responses. Circulating HGF levels were detectable at baseline and uniformly increased with treatment suggestive of a feedback response or immune complex stabilization. Dose expansion is ongoing. Clinical trial information: NCT02109627.

In Vivo c-Met Pathway Inhibition Depletes Human Glioma Xenografts of Tumor-Propagating Stem-Like Cells

Solid malignancies contain sphere-forming stem-like cells that are particularly efficient in propagating tumors. Identifying agents that target these cells will advance the development of more effective therapies. Recent converging evidence shows that c-Met expression marks tumor-initiating stem-like cells and that c-Met signaling drives human glioblastoma multiforme (GBM) cell stemness in vitro. However, the degree to which tumor-propagating stem-like cells depend on c-Met signaling in histologically complex cancers remains unknown. We examined the effects of in vivo c-Met pathway inhibitor therapy on tumor-propagating stem-like cells in human GBM xenografts. Animals bearing pre-established tumor xenografts expressing activated c-Met were treated with either neutralizing anti- hepatocyte growth factor (HGF) monoclonal antibody L2G7 or with the c-Met kinase inhibitor PF2341066 (Crizotinib). c-Met pathway inhibition inhibited tumor growth, depleted tumors of sphere-forming cells, and inhibited tumor expression of stem cell markers CD133, Sox2, Nanog, and Musashi. Withdrawing c-Met pathway inhibitor therapy resulted in a substantial rebound in stem cell marker expression concurrent with tumor recurrence. Cells derived from xenografts treated with anti-HGF in vivo were depleted of tumor-propagating potential as determined by in vivo serial dilution tumor-propagating assay. Furthermore, daughter xenografts that did form were 12-fold smaller than controls. These findings show that stem-like tumor-initiating cells are dynamically regulated by c-Met signaling in vivo and that c-Met pathway inhibitors can deplete tumors of their tumor-propagating stem-like cells.

Highlights of This Issue

Suppression of cellular immunity has long been observed in patients with glioblastoma. Sippel and colleagues provide evidence for activation of circulating neutrophils in patients with glioblastoma associated with increased release of the immunosuppressive enzyme Arginase I (ArgI). In vitro targeting of ArgI resulted in restoration of T-cell function in patients with glioblastoma, providing an intriguing new target for clinical intervention designed to restore cellular immune function in this population.Adhesion of circulating tumor cells to the blood vascular endothelium is a pivotal step in metastasis. In this study, Barrow and colleagues revealed that serum levels of several galectin members were greatly increased in colon and breast cancer patients and promoted cancer-endothelial adhesion by interaction with cancer-associated Thomsen-Friedenreich/mucin 1 protein (TF/MUC1). Elevated circulating galectin-2 levels were associated with increased mortality in cancer patients, but this association was suppressed when auto–anti-MUC1 antibodies with specificity for the TF epitope were also present in the circulation. Thus, increased circulation of several galectin members is common in cancer patients and promotes circulating cancer cell metastatic spreading. Targeting the actions of circulating galectins may therefore represent a promising therapeutic approach to reduce metastasis.Accumulating evidence has demonstrated that tumor-initiating cells (T-IC) are resistant to chemotherapy. Zhu and colleagues demonstrated that mir-128 was significantly reduced in breast T-ICs, with the overexpression of its targets Bmi-1 and ABCC5, which regulated the chemotherapeutic resistance. Furthermore, miR-128 reduction in breast cancer tissues was correlated with chemoresistance and poor survival in patients with breast cancer. These findings imply that examination of miR-128 expression is valuable in predicting patient survival and therapeutic response in breast cancer and provide the basis for developing a combined strategy using miR-128 mimics and chemotherapy.Dysregulated c-MET signaling plays important roles in many cancers and represents an attractive therapeutic target. Liu and colleagues discovered a highly selective and potent c-MET kinase inhibitor, INCB28060. INCB28060 effectively inhibits c-MET–dependent signaling and neoplastic activity in various preclinical models. Furthermore, c-MET upregulates epidermal growth factor receptor and HER3 activity through interaction with these receptors and/or by inducing their ligands, and this activity can be reversed by INCB28060. Although numerous c-MET pathway inhibitors are being developed, they may have limitations in their potency, selectivity, pharmacokinetics, safety profile, or mechanisms of action. The properties shown by INCB28060 may enable this agent to overcome these limitations and allow for assessment of the therapeutic potential of c-MET pathway inhibition.

PTEN reconstitution alters glioma responses to c-Met pathway inhibition

Mutations/deletions of the tumor-suppressor phosphatase and tensin homolog PTEN result in PI3K/Akt pathway hyperactivation and potentially alter oncogenic responses to targeted receptor tyrosine kinase inhibitors. We previously showed that hepatocyte growth factor (HGF):c-Met pathway inhibition decreases tumor growth and oncogenic signaling responses in PTEN-null/Met+ gliomas. Here, we use two tet-on PTENwt-inducible glioma cell lines and xenograft models to examine the influence of PTEN on oncogenic signaling responses to HGF:c-Met pathway inhibitors. Reconstitution of PTEN inhibited Akt by more than 80% and inhibited cell growth by approximately 70-75% in both cell lines in vitro. C-Met inhibition alone inhibited in-vitro cell growth by approximately 80-85% and the magnitude of growth inhibition was not altered by combining PTEN reconstitution with c-Met inhibition. Combining PTEN reconstitution with Met inhibition arrested a higher percentage of cells in G(1)/G(0) phase of the cell cycle when compared with either PTEN reconstitution or c-Met inhibition alone. Both PTEN reconstitution alone and inhibiting autocrine HGF:c-Met signaling alone, using anti-HGF mAb, robustly inhibited the growth of subcutaneous and intracranial glioma xenografts. Combining anti-HGF therapy with PTEN reconstitution did not significantly alter the magnitude of xenograft growth inhibition. Semiquantitative immunohistopathological analyses revealed that the inhibition of glioma xenograft angiogenesis and cell proliferation by anti-HGF mAb was greatest in conjunction with PTEN reconstitution. In contrast, xenograft cell apoptosis was greatest in response to anti-HGF therapy alone and PTEN reconstitution abrogated the apoptotic response to anti-HGF therapy. These results provide new insights into how PTEN modulates glioma responses to the inhibition of HGF:c-Met signaling and possibly other receptor tyrosine kinase pathways.

Abstract 3309: Inhibiting c-Met activation depletes glioblastomas of stem-like tumor-initiating cells

Abstract Hepatocyte Growth Factor (HGF) and its tyrosine kinase receptor c-Met are overexpressed in glioblastomas (GBM) and expression levels correlate with poor prognosis. Previously, we showed that anti-HGF therapy using the monoclonal antibody L2G7 inhibits the progression of orthotopic xenografts derived from HGF+/c-Met+ GBM cell lines by inducing apoptosis, and inhibiting tumor cell proliferation and angiogenesis. Increasingly, there is an interest in cancer therapies targeting the stem-like tumor-initiating pool of cells within GBMs (GBM-TICs), as this subset of cells may be particularly resistant to conventional therapies and associated with tumor recurrence. This study examined the effects of c-Met pathway inhibition on the pool of stem-like cells in GBM xenografts. We used the HGF/c-Met dependant U87 cell line as a GBM model system. Wild-type U87 cells were passaged subcutaneously (s.c.) in nude mice for 2-3 weeks. Tumors (>200mm3) were removed and primary tumor-derived cells were cultured in vitro in serum-free stem cell medium containing EGF and FGF to study neurosphere formation, a marker of stem-like cells. We confirmed the expression of stem/progenitor cell markers Sox2, CD133, Nestin, and Musashi in neurosphere-forming cells (U87-NS). Under conditions of forced differentiation, U87-NS cells revealed multipotent capacities and expressed lineage specific differentiation markers such as Neurofilament, BIII-tubulin, O1, and GFAP. Animals bearing intracranial or s.c. xenografts were treated with +/- L2G7 prior to tumor removal and isolation of tumor cells. Overall, L2G7 treated tumors were substantially smaller than control in both intracranial (p<0.0001) and s.c. (p<0.0001) sites. Equal numbers of primary cells derived from control or L2G7 treated s.c. xenografts were either re-transplanted intracranially (10,000 and 1,000 cells per brain), or cultured in stem cell medium containing EGF and FGF to determine the effect of L2G7 on the pool of tumor-initiating and neurosphere-forming stem-like cells, respectively. Cells derived from L2G7 treated xenografts had smaller tumors overall, and demonstrated decreased tumor initiation (50% fewer intracranial tumors at 1000 cells/animal) as compared to the control cells. Similarly, intracranial xenografts were derived from U87-NS cells and treated with +/- L2G7. Cells isolated from L2G7 treated intracranial xenografts were less able to form neurospheres (50% inhibition, p<0.05) than cells derived from control xenografts. Orthotopic and s.c. derived tumors are currently being tested to determine the effects of c-Met inhibition on the expression of stem cell markers in tumor xenografts. In conclusion, we show that treating HGF/c-Met dependant U87 xenografts with the ligand-sequestering antibody L2G7 appears to preferentially deplete tumors of their neurosphere-forming tumor-initiating cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3309. doi:10.1158/1538-7445.AM2011-3309