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Merestinib is an oral multi-kinase inhibitor targeting a limited number of oncokinases including MET, AXL, RON and MKNK1/2. Here, we report that merestinib inhibits neurotrophic receptor tyrosine kinases NTRK1/2/3 which are oncogenic drivers in tumors bearing NTRK fusion resulting from chromosomal rearrangements. Merestinib is shown to be a type II NTRK1 kinase inhibitor as determined by x-ray crystallography. In KM-12 cells harboring TPM3-NTRK1 fusion, merestinib exhibits potent p-NTRK1 inhibition in vitro by western blot and elicits an anti-proliferative response in two- and three-dimensional growth. Merestinib treatment demonstrated profound tumor growth inhibition in in vivo cancer models harboring either a TPM3-NTRK1 or an ETV6-NTRK3 gene fusion. To recapitulate resistance observed from type I NTRK kinase inhibitors entrectinib and larotrectinib, we generated NIH-3T3 cells exogenously expressing TPM3-NTRK1 wild-type, or acquired mutations G595R and G667C in vitro and in vivo. Merestinib blocks tumor growth of both wild-type and mutant G667C TPM3-NTRK1 expressing NIH-3T3 cell-derived tumors. These preclinical data support the clinical evaluation of merestinib, a type II NTRK kinase inhibitor (NCT02920996), both in treatment naïve patients and in patients progressed on type I NTRK kinase inhibitors with acquired secondary G667C mutation in NTRK fusion bearing tumors.

Vascular endothelial growth factor receptor 2 (VEGFR2) is an attractive therapeutic target in solid malignancies due to its central role in tumor angiogenesis. Ramucirumab (Cyramza®, LY3009806) is a human monoclonal antibody specific for VEGFR2 approved for several adult indications and currently in a phase 1 clinical trial for pediatric patients with solid tumors (NCT02564198). Here, we evaluated ramucirumab in vitro and the anti-murine VEGFR2 antibody DC101 in vivo with or without chemotherapy across a range of pediatric cancer models. Ramucirumab abrogated in vitro endothelial cord formation driven by cancer cell lines representing multiple pediatric histologies; this response was independent of the origin of the tumor cell-line. Several pediatric cancer mouse models responded to single agent DC101-mediated VEGFR2 inhibition with tumor growth delay. Preclinical stable disease and partial xenograft regressions were observed in mouse models of Ewing's sarcoma, synovial sarcoma, neuroblastoma, and desmoplastic small round cell tumor treated with DC101 and cytotoxic chemotherapy. In contrast, DC101 treatment in osteosarcoma models had limited efficacy alone or in combination with chemotherapeutics. Our data indicate differential efficacy of targeting the VEGFR2 pathway in pediatric models and support the continued evaluation of VEGFR2 inhibition in combination with cytotoxic chemotherapy in multiple pediatric indications.

Interaction of numerous signaling pathways in endothelial and mesangial cells results in exquisite control of the process of physiological angiogenesis, with a central role played by vascular endothelial growth factor receptor 2 (VEGFR-2) and its cognate ligands. However, deregulated angiogenesis participates in numerous pathological processes. Excessive activation of VEGFR-2 has been found to mediate tissue-damaging vascular changes as well as the induction of blood vessel expansion to support the growth of solid tumors. Consequently, therapeutic intervention aimed at inhibiting the VEGFR-2 pathway has become a mainstay of treatment in cancer and retinal diseases. In this review, we introduce the concepts of physiological and pathological angiogenesis, the crucial role played by the VEGFR-2 pathway in these processes, and the various inhibitors of its activity that have entered the clinical practice. We primarily focus on the development of ramucirumab, the antagonist monoclonal antibody (mAb) that inhibits VEGFR-2 and has recently been approved for use in patients with gastric, colorectal, and lung cancers. We examine in-depth the pre-clinical studies using DC101, the mAb to mouse VEGFR-2, which provided a conceptual foundation for the role of VEGFR-2 in physiological and pathological angiogenesis. Finally, we discuss further clinical development of ramucirumab and the future of targeting the VEGF pathway for the treatment of cancer.

Treatment of metastatic gastric cancer typically involves chemotherapy and monoclonal antibodies targeting HER2 (ERBB2) and VEGFR2 (KDR). However, reliable methods to identify patients who would benefit most from a combination of treatment modalities targeting the tumor stroma, including new immunotherapy approaches, are still lacking. Therefore, we integrated a mouse model of stromal activation and gastric cancer genomic information to identify gene expression signatures that may inform treatment strategies. We generated a mouse model in which VEGF-A is expressed via adenovirus, enabling a stromal response marked by immune infiltration and angiogenesis at the injection site, and identified distinct stromal gene expression signatures. With these data, we designed multiplexed IHC assays that were applied to human primary gastric tumors and classified each tumor to a dominant stromal phenotype representative of the vascular and immune diversity found in gastric cancer. We also refined the stromal gene signatures and explored their relation to the dominant patient phenotypes identified by recent large-scale studies of gastric cancer genomics (The Cancer Genome Atlas and Asian Cancer Research Group), revealing four distinct stromal phenotypes. Collectively, these findings suggest that a genomics-based systems approach focused on the tumor stroma can be used to discover putative predictive biomarkers of treatment response, especially to antiangiogenesis agents and immunotherapy, thus offering an opportunity to improve patient stratification. Cancer Res; 76(9); 2573–86. ©2016 AACR.

Antiangiogenesis–based cancer therapies, specifically those targeting the VEGF-A/VEGFR2 pathway, have been approved for subsets of solid tumors. However, these therapies result in an increase in hematologic adverse events. We surmised that both the bone marrow vasculature and VEGF receptor–positive hematopoietic cells could be impacted by VEGF pathway–targeted therapies. We used a mouse model of spontaneous breast cancer to decipher the mechanism by which VEGF pathway inhibition alters hematopoiesis. Tumor-bearing animals, while exhibiting increased angiogenesis at the primary tumor site, showed signs of shrinkage in the sinusoidal bone marrow vasculature accompanied by an increase in the hematopoietic stem cell–containing Lin-cKit+Sca1+ (LKS) progenitor population. Therapeutic intervention by targeting VEGF-A, VEGFR2, and VEGFR3 inhibited tumor growth, consistent with observed alterations in the primary tumor vascular bed. These treatments also displayed systemic effects, including reversal of the tumor-induced shrinkage of sinusoidal vessels and altered population balance of hematopoietic stem cells in the bone marrow, manifested by the restoration of sinusoidal vessel morphology and hematopoietic homeostasis. These data indicate that tumor cells exert an aberrant systemic effect on the bone marrow microenvironment and VEGF-A/VEGFR targeting restores bone marrow function. Cancer Res; 76(3); 517–24. ©2015 AACR.

Inhibition of VEGFR signaling is an effective treatment for renal cell carcinoma, but resistance continues to be a major problem. Recently, the sphingosine phosphate (S1P) signaling pathway has been implicated in tumor growth, angiogenesis, and resistance to antiangiogenic therapy. S1P is a bioactive lipid that serves an essential role in developmental and pathologic angiogenesis via activation of the S1P receptor 1 (S1P1). S1P1 signaling counteracts VEGF signaling and is required for vascular stabilization. We used in vivo and in vitro angiogenesis models including a postnatal retinal angiogenesis model and a renal cell carcinoma murine tumor model to test whether simultaneous inhibition of S1P1 and VEGF leads to improved angiogenic inhibition. Here, we show that inhibition of S1P signaling reduces the endothelial cell barrier and leads to excessive angiogenic sprouting. Simultaneous inhibition of S1P and VEGF signaling further disrupts the tumor vascular beds, decreases tumor volume, and increases tumor cell death compared with monotherapies. These studies suggest that inhibition of angiogenesis at two stages of the multistep process may maximize the effects of antiangiogenic therapy. Together, these data suggest that combination of S1P1 and VEGFR-targeted therapy may be a useful therapeutic strategy for the treatment of renal cell carcinoma and other tumor types.

Blood vessels are crucial components for normal tissue development and homeostasis, so it is not surprising that endothelial dysfunction and dysregulation results in a variety of different pathophysiological conditions. The large number of vascular-related disorders and the emergence of angiogenesis as a major hallmark of cancer has led to significant interest in the development of drugs that target the vasculature. While several in vivo models exist to study developmental and pathological states of blood vessels, few in vitro assays have been developed that capture the significant complexity of the vascular microenvironment. Here, we describe a high content endothelial colony forming cells (ECFC)/adipose-derived stem cell (ADSC) coculture assay that captures many elements of in vivo vascular biology and is ideal for in vitro screening of compounds for pro- or anti-angiogenic activities.

Purpose: Checkpoint kinase 1 (CHK1) is a key regulator of the DNA damage response and a mediator of replication stress through modulation of replication fork licensing and activation of S and G2–M cell-cycle checkpoints. We evaluated prexasertib (LY2606368), a small-molecule CHK1 inhibitor currently in clinical testing, in multiple preclinical models of pediatric cancer. Following an initial assessment of prexasertib activity, this study focused on the preclinical models of neuroblastoma. Experimental Design: We evaluated the antiproliferative activity of prexasertib in a panel of cancer cell lines; neuroblastoma cell lines were among the most sensitive. Subsequent Western blot and immunofluorescence analyses measured DNA damage and DNA repair protein activation. Prexasertib was investigated in several cell line–derived xenograft mouse models of neuroblastoma. Results: Within 24 hours, single-agent prexasertib promoted γH2AX–positive double-strand DNA breaks and phosphorylation of DNA damage sensors ATM and DNA–PKcs, leading to neuroblastoma cell death. Knockdown of CHK1 and/or CHK2 by siRNA verified that the double-strand DNA breaks and cell death elicited by prexasertib were due to specific CHK1 inhibition. Neuroblastoma xenografts rapidly regressed following prexasertib administration, independent of starting tumor volume. Decreased Ki67 and increased immunostaining of endothelial and pericyte markers were observed in xenografts after only 6 days of exposure to prexasertib, potentially indicating a swift reduction in tumor volume and/or a direct effect on tumor vasculature. Conclusions: Overall, these data demonstrate that prexasertib is a specific inhibitor of CHK1 in neuroblastoma and leads to DNA damage and cell death in preclinical models of this devastating pediatric malignancy. Clin Cancer Res; 23(15); 4354–63. ©2017 AACR.

Background Antiangiogenic agents have established efficacy in the treatment of metastatic colorectal cancer. We investigated whether bevacizumab could improve disease-free survival in the adjuvant setting after resection of the primary tumour. Methods For the open-label, randomised, controlled QUASAR 2 trial, which was done at 170 hospitals in seven countries, we recruited patients aged 18 years or older with WHO performance status scores of 0 or 1 who had undergone potentially curative surgery for histologically proven stage III or high-risk stage II colorectal cancer. Patients were randomly assigned (1:1) to receive eight 3-week cycles of oral capecitabine alone (1250 mg/m2 twice daily for 14 days followed by a break for 7 days) or the same regimen of oral capecitabine plus 16 cycles of 7·5 mg/kg bevacizumab by intravenous infusion over 90 min on day 1 of each cycle. Randomisation was done by a computer-generated schedule with use of minimisation with a random element stratified by age, disease stage, tumour site, and country. The study was open label and no-one was masked to treatment assignment. The primary endpoint was 3-year disease-free survival, assessed in the intention-to-treat population. Toxic effects were assessed in patients who received at least one dose of randomised treatment. This trial is registered with the ISRCTN registry, number ISRCTN45133151. Findings Between April 25, 2005, and Oct 12, 2010, 1952 eligible patients were enrolled, of whom 1941 had assessable data (968 in the capecitabine alone group and 973 in the capecitabine and bevacizumab group). Median follow-up was 4·92 years (IQR 4·00–5·16). Disease-free survival at 3 years did not differ between the groups (75·4%, 95% CI 72·5–78·0 in the capecitabine and bevacizumab group vs 78·4%, 75·7–80·9 in the capecitabine alone group; hazard ratio 1·06, 95% CI 0·89–1·25, p=0·54). The most common grade 3–4 adverse events were hand–foot syndrome (201 [21%] of 963 in the capecitabine alone group vs 257 [27%] of 959 in the capecitabine and bevacizumab group) and diarrhoea (102 [11%] vs 104 [11%]), and, with the addition of bevacizumab, expected increases were recorded in all-grade hypertension (320 [33%] vs 75 [8%]), proteinuria (197 [21%] vs 49 [5%]), and wound healing problems (30 [3%] vs 17 [2%]). 571 serious adverse events were reported (221 with capecitabine alone and 350 with capecitabine and bevacizumab). Most of these were gastrointestinal (n=245) or cardiovascular (n=169). 23 deaths within 6 months of randomisation were classified as being related to treatment, eight in the capecitabine alone group and 15 in the capecitabine and bevacizumab group. Interpretation The addition of bevacizumab to capecitabine in the adjuvant setting for colorectal cancer yielded no benefit in the treatment of an unselected population and should not be used.

CHK1 is a serine/threonine protein kinase essential for S-phase and G2/M cell cycle checkpoint regulation following DNA damage. Targeted inhibition of CHK1 in several tumor types increases DNA damage and replication stress, culminating in cell death through mitotic catastrophe. Recent studies have identified CHK1 as a therapeutic target in several pediatric tumor types. We evaluated the antitumor efficacy of LY2606368 mesylate monohydrate (“LY”), a checkpoint kinase 1 (CHK1)/CHK2 inhibitor currently in early phase clinical trials for adult solid cancers, in a panel of pediatric tumor cell lines and mouse models of embryonal tumors and pediatric sarcoma. In vitro effects of LY were assessed via Cell Titer Glo, immunoblotting, and cell cycle analysis by flow cytometry. For in vivo studies, mice bearing cell-derived (CDX) or patient-derived xenografts (PDX) of several pediatric tumor types were treated with four weekly cycles of 10 mg/kg LY BID for 3 consecutive days, followed by a 4 day dosing holiday. Tumor volume and body weight were measured 2x weekly. Xenograft tumor health following LY, chemotherapy, or combination treatment was evaluated by fluorescent immunohistochemistry (IHC) for a panel of markers for cell proliferation (Ki67), apoptosis (TUNEL), and angiogenesis (CD31, smooth muscle actin [SMA], MECA32). Single digit nanomolar sensitivity to LY was observed in the majority of pediatric cancer cell lines evaluated in vitro. A more detailed analysis of LY-treated neuroblastoma and pediatric sarcoma cell lines showed increased DNA damage, CHK1 phosphorylation, and MAPK pathway activation. Significant single agent LY activity was observed in mouse models of neuroblastoma and pediatric sarcoma, but not in models of hepatoblastoma or retinoblastoma. Acquired resistance to LY was observed in the ST162 and SJCRH30 models of alveolar rhabdomyosarcoma. Interestingly, more stroma was observed following LY single agent treatment as measured by CD31, SMA, and MECA32 IHC staining; co-treatment with chemotherapy reduced the amount of SMA expressing-cells. Overall, our data demonstrate that LY is highly effective as a single agent in murine in vivo models of human neuroblastoma and several pediatric sarcoma subtypes. Current studies include further evaluation of the LY mechanism of action; investigation into the mechanism of intrinsic and acquired resistance; and identification of possible biomarkers for LY sensitivity. Citation Format: Caitlin D. May, Richard Beckmann, Wayne Blosser, Michele Dowless, Alle VanWye, Teresa Burke, Gerard Oakley, Jennifer Stephens, Julie Stewart, Beverly Falcon, Louis Stancato. Targeting checkpoint kinase 1 (CHK1) with the small molecule inhibitor LY2606368 mesylate monohydrate in models of high-risk pediatric cancer yields significant antitumor effects. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2458.