Jennifer R. Stephens

Therapeutic Inhibition of MAP Kinase Interacting Kinase Blocks Eukaryotic Initiation Factor 4E Phosphorylation and Suppresses Outgrowth of Experimental Lung Metastases

Activation of the translation initiation factor 4E (eIF4E) promotes malignant transformation and metastasis. Signaling through the AKT-mTOR pathway activates eIF4E by phosphorylating the inhibitory 4E binding proteins (4E-BP). This liberates eIF4E and allows binding to eIF4G. eIF4E can then be phosphorylated at serine 209 by the MAPK-interacting kinases (Mnk), which also interact with eIF4G. Although dispensable for normal development, Mnk function and eIF4E phosphorylation promote cellular proliferation and survival and are critical for malignant transformation. Accordingly, Mnk inhibition may serve as an attractive cancer therapy. We now report the identification of a potent, selective and orally bioavailable Mnk inhibitor that effectively blocks 4E phosphorylation both in vitro and in vivo. In cultured cancer cell lines, Mnk inhibitor treatment induces apoptosis and suppresses proliferation and soft agar colonization. Importantly, a single, orally administered dose of this Mnk inhibitor substantially suppresses eIF4E phosphorylation for at least 4 hours in human xenograft tumor tissue and mouse liver tissue. Moreover, oral dosing with the Mnk inhibitor significantly suppresses outgrowth of experimental B16 melanoma pulmonary metastases as well as growth of subcutaneous HCT116 colon carcinoma xenograft tumors, without affecting body weight. These findings offer the first description of a novel, orally bioavailable MNK inhibitor and the first preclinical proof-of-concept that MNK inhibition may provide a tractable cancer therapeutic approach.

LY2875358, a Neutralizing and Internalizing Anti-MET Bivalent Antibody, Inhibits HGF-Dependent and HGF-Independent MET Activation and Tumor Growth

Purpose: MET, the receptor for hepatocyte growth factor (HGF), has been implicated in driving tumor proliferation and metastasis. High MET expression is correlated with poor prognosis in multiple cancers. Activation of MET can be induced either by HGF-independent mechanisms such as gene amplification, specific genetic mutations, and transcriptional upregulation or by HGF-dependent autocrine or paracrine mechanisms. Experimental Design/Results: Here, we report on LY2875358, a novel humanized bivalent anti-MET antibody that has high neutralization and internalization activities, resulting in inhibition of both HGF-dependent and HGF-independent MET pathway activation and tumor growth. In contrast to other bivalent MET antibodies, LY2875358 exhibits no functional agonist activity and does not stimulate biologic activities such as cell proliferation, scattering, invasion, tubulogenesis, or apoptosis protection in various HGF-responsive cells and no evidence of inducing proliferation in vivo in a monkey toxicity study. LY2875358 blocks HGF binding to MET and HGF-induced MET phosphorylation and cell proliferation. In contrast to the humanized one-armed 5D5 anti-MET antibody, LY2875358 induces internalization and degradation of MET that inhibits cell proliferation and tumor growth in models where MET is constitutively activated. Moreover, LY2875358 has potent antitumor activity in both HGF-dependent and HGF-independent (MET-amplified) xenograft tumor models. Together, these findings indicate that the mechanism of action of LY2875358 is different from that of the one-armed MET antibody. Conclusions: LY2875358 may provide a promising therapeutic strategy for patients whose tumors are driven by both HGF-dependent and HGF-independent MET activation. LY2875358 is currently being investigated in multiple clinical studies. Clin Cancer Res; 20(23); 6059–70. ©2014 AACR.

The Checkpoint Kinase 1 Inhibitor Prexasertib Induces Regression of Preclinical Models of Human Neuroblastoma

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.

Olaratumab Exerts Antitumor Activity in Preclinical Models of Pediatric Bone and Soft Tissue Tumors through Inhibition of Platelet-Derived Growth Factor Receptor α

Purpose: Platelet-derived growth factor receptor α (PDGFRα) is implicated in several adult and pediatric malignancies, where activated signaling in tumor cells and/or cells within the microenvironment drive tumorigenesis and disease progression. Olaratumab (LY3012207/IMC-3G3) is a human mAb that exclusively binds to PDGFRα and recently received accelerated FDA approval and conditional EMA approval for treatment of advanced adult sarcoma patients in combination with doxorubicin. In this study, we investigated olaratumab in preclinical models of pediatric bone and soft tissue tumors. Experimental Design: PDGFRα expression was evaluated by qPCR and Western blot analysis. Olaratumab was investigated in in vitro cell proliferation and invasion assays using pediatric osteosarcoma and rhabdoid tumor cell lines. In vivo activity of olaratumab was assessed in preclinical mouse models of pediatric osteosarcoma and malignant rhabdoid tumor. Results: In vitro olaratumab treatment of osteosarcoma and rhabdoid tumor cell lines reduced proliferation and inhibited invasion driven by individual platelet-derived growth factors (PDGFs) or serum. Furthermore, olaratumab delayed primary tumor growth in mouse models of pediatric osteosarcoma and malignant rhabdoid tumor, and this activity was enhanced by combination with either doxorubicin or cisplatin. Conclusions: Overall, these data indicate that olaratumab, alone and in combination with standard of care, blocks the growth of some preclinical PDGFRα-expressing pediatric bone and soft tissue tumor models. Clin Cancer Res; 24(4); 847–57. ©2017 AACR.

Abstract 2738: c-Met antibody LY2875358 (LA480) has pre-clinical enhanced efficacy with gastric cancer standard-of-care in vitro and in vivo

cMet is a member of the receptor tyrosine kinase family and is the receptor for hepatocyte growth factor (HGF). cMet has been implicated in the initiation and progression of cancer due to the range of activities that cMet stimulates including proliferation, migration, morphogenesis, and survival. Inappropriate activation of c-Met can be induced by ligand-independent mechanisms such as gene amplification, specific genetic mutations, and transcriptional upregulation, or by ligand-dependent autocrine or paracrine mechanisms. Indeed, amplification of the c-Met gene, with consequent protein overexpression and constitutive kinase activation, has been reported in a number of human cancers, including gastric, esophageal and non-small-cell lung carcinomas. It has been reported that ∼10-20% of gastric tumors have increased copy numbers of the MET gene and overexpression of c-Met significantly correlates with poor prognosis in gastric cancer. c-Met antibody LY2875358 treatment reduces proliferation of gastric cancer cell lines with ligand-independent activation of c-Met resulting from gene amplification. The ability of LY2875358 to internalize and deplete cell surface c-Met is implicated in its activity against ligand-independent driven gastric cell lines. Here, we demonstrate that the pre-clinical combination of c-Met antibody LY2875358 with gastric cancer standard-of-care treatment has better efficacy than either treatment alone, both in vitro and in vivo. These data suggest that LY2875358 in combination with standard-of-care may be a promising treatment for gastric cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2738. doi:1538-7445.AM2012-2738

Abstract 2734: c-Met antibody LY2875358 (LA480) shows differential antitumor effects in non-small cell lung cancer

c-Met is a member of the receptor tyrosine kinase family and is the receptor for hepatocyte growth factor (HGF). c-Met is involved in many mechanisms of cancer proliferation and metastasis. Inappropriate activation of c-Met can be induced by ligand-independent mechanisms such as gene amplification, specific genetic mutations, and transcriptional up-regulation, or by ligand-dependent autocrine or paracrine mechanisms. Lung cancer is the leading cause of cancer death worldwide. Despite the successful development of EGFR- or EML4-ALK-targetd therapies, treatment options remain limited for patients with advanced lung cancer, making the identification of new therapeutic targets essential. c-Met expression was reported in 41-72% non-small cell lung cancer (NSCLC), amplification of c-Met occurs in 5-10 % of patients, and c-Met mutations have been detected in 8-13% of patients. We have developed a bi-valent c-Met antibody LY2875358 (LA480), which blocks ligand-dependent and ligand-independent c-Met activations. It is currently in clinical development. Here, we have demonstrated that LY2875358 alone or in combination with standard-of-care (SOC) affected cell proliferation, migration and signal transduction in NSCLC cells with c-Met gene amplification, mutations and overexpression. In vitro, LY2875358 induces wild type and mutant c-Met internalization and degradation. In vivo, LY2875358 alone shows a marked antitumor activity in Met amplification NSCLC xenograft models. The combination of LY2875358 with SOC chemotherapeutics treatments has better efficacy than either treatment alone, both in vitro and in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2734. doi:1538-7445.AM2012-2734

Abstract 5465: LY2875358, a bivalent MET antibody with anti-tumor activity through blocking HGF as well as inducing degradation of MET, differentiates from a one-armed 5D5 MET antibody.

MET is involved in many mechanisms of cancer proliferation and metastasis. Inappropriate activation of MET can be induced by HGF-independent mechanisms such as gene amplification, specific genetic mutations, and transcriptional up-regulation, or by HGF-dependent autocrine or paracrine mechanisms. LY2875358 is a novel humanized bivalent MET antibody currently in phase I clinical testing (trial NCT01287546). LY2875358 has high neutralization and internalization activities against MET for inhibiting HGF-dependent and HGF-independent MET pathway activation and tumor growth. In HGF-dependent MET activation, LY2875358 blocks HGF binding to MET, HGF-induced MET phosphorylation and tumor growth both in cell culture and in mouse xenograft models, resembling activities of a humanized one-armed 5D5 MET antibody (monovalent antibody similar to Onartuzumab). In tumors with HGF-independent MET activation through MET gene amplification, LY2875358 induces internalization and degradation of MET, which results in decreased pMET and total MET, inhibition of cell proliferation and tumor growth in MKN45 and SNU5 gastric tumor lines and EBC-1 and H1993 NSCLC tumor lines. Moreover, LY2875358 enhances antitumor activity in combination with cisplatin or 5-FU in vitro and in vivo in MET amplified tumor cells. However, under the same ligand-independent conditions, the one-armed 5D5 antibody did not have anti-tumor activities. When HGF is added to tumor cells with high MET gene amplification, LY2875358 decreases cell proliferation, while the one-armed 5D5 antibody does not. In contrast to other bivalent MET antibodies, LY2875358 has no or otherwise negligible agonist activity and does not stimulate biological activities such as cell proliferation, scattering, invasion, tubulogenesis, apoptosis protection or angiogenesis in various HGF responsive cells. These findings indicate that LY2875358 has a different mechanism of action from the humanized one-armed 5D5 MET antibody. LY2875358 may be a promising therapy for treatment of patients whose tumors are driven by HGF-dependent and HGF-independent MET activation. Citation Format: Wei Zeng, Victoria Peek, Mark Wortinger, Jonathan Tetreault, Jinqi Xia, Jennifer Stephens, Kelly Credille, Darryl Ballard, Trish Brown-Augsburger, Jirong Lu, Chi-Kin Chow, Peter Vaillancourt, Ying Tang, Sau-Chi B. Yan, Ling Liu. LY2875358, a bivalent MET antibody with anti-tumor activity through blocking HGF as well as inducing degradation of MET, differentiates from a one-armed 5D5 MET antibody. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5465. doi:10.1158/1538-7445.AM2013-5465

Merestinib (LY2801653) inhibits neurotrophic receptor kinase (NTRK) and suppresses growth of NTRK fusion bearing tumors

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.

MET-targeting antibody (emibetuzumab) and kinase inhibitor (merestinib) as single agent or in combination in a cancer model bearing MET exon 14 skipping

SummaryPurpose Approximately 3% of lung cancer bears mutations leading to MET exon 14 skipping, an oncogenic driver which is further evidenced by case reports of patient response to MET kinase inhibitor treatment. Approximately 15% of tumors harboring MET exon14 skipping have concurrent MET amplification. Experimental Design Merestinib is a type II MET kinase inhibitor. Emibetuzumab, a bivalent anti-MET antibody, internalizes MET receptor. Each single agent and the combination were evaluated in the Hs746t gastric cancer line bearing MET exon14 skipping and MET amplification. Results Merestinib inhibited Hs746t cell proliferation (IC50=34 nM) and totally eliminated pMET at 100nM. Emibetuzumab showed little anti-proliferative activity against Hs746t cells (IC50>100nM), did not reduce pMET, and slightly reduced cell surface MET. In the Hs746t xenograft model, dose dependent differences in durability of response were seen with merestinib including durable tumor regression (91.8%) at 12 mg/kg qd. Emibetuzumab treatment (10mg/kg qw) provided transient tumor regression (37.7%), but tumors re-grew while on treatment. Concurrent combination of merestinib (6 mg/kg qd) and emibetuzumab resulted in 85% tumor regression, while a sequential combination (initiating merestinib first) resulted in longer duration of treatment response. Conclusions Data in this study support a clinical evaluation of merestinib in patients with MET exon 14 skipping (NCT02920996). As a type II MET kinase inhibitor, merestinib may provide a therapeutic option to treatment naïve patients or to patients who progress on type I MET inhibitor treatment. Data also support clinical evaluation of the sequential combination of merestinib with emibetuzumab when patients progress on single agent merestinib.

Abstract 583: The CDK4/6 inhibitor abemaciclib induces synergistic immune activation and antitumor efficacy in combination with PD-L1 blockade

Targeting cyclin dependent kinases 4 and 6 (CDK4/6) with inhibitors such as abemaciclib has shown promise in early and late phase clinical trials in both breast cancer and NSCLC. While there is evidence that patients benefit from single-agent abemaciclib, combination strategies leveraging this compound together with immunotherapy are of interest for the treatment of these and other cancers. Consequently, it is important to understand if and how a cell cycle inhibitor can be combined with immunotherapy. However, because most preclinical studies have been performed using xenograft tumors in immune-compromised mice, the potential immunomodulatory effects of abemaciclib have not been adequately ascertained. To investigate the immune combinatorial potential of abemaciclib, we studied the effects of treatment alone and in combination with checkpoint immunotherapy in a murine syngeneic tumor model sensitive to abemaciclib using immuno-competent mice. Abemaciclib monotherapy of established murine CT26 tumors, which harbor KRAS G12C mutation and CDKN2A deletion, caused a dose-dependent delay in tumor growth. Surprisingly, gene expression analysis showed that treatment was associated with an increase in intra-tumor immune inflammation without major alteration in immune subset frequencies. Testing of various dosing regimens in this preclinical model found that monotherapy abemaciclib pretreatment followed by combination with anti-PD-L1 antibody therapy, induced an enhanced anti-tumor response compared to abemaciclib and anti-PD-L1 monotherapies. Optimal combination therapy exhibited superior anti-tumor efficacy, resulting in complete tumor regression (CR) in 50-60% of mice in a setting where anti-PD-L1 monotherapy showed little or no efficacy (0% CRs). Mice which maintained CRs after cessation of combination therapy were able to resist later CT26 rechallenge, demonstrating that abemaciclib in combination with anti-PD-L1 enabled the generation of an immunologic memory. Examination of intra-tumor gene expression during treatment found that combination therapy further amplified the immune/T cell activation signature compared to both monotherapies. Intra-tumoral suppression of cell cycle genes, which are indicative of inhibition of CDK4/6, was also greater during the combination therapy, suggesting that the effects anti-PD-L1 therapy may augment the cell cycle arrest induced by abemaciclib. Although it was uncertain if agents that inhibit cell proliferation could be combined with immunotherapy, these preclinical results demonstrate that it is possible to combine CDK4/6 inhibition by abemaciclib with checkpoint immunotherapy to improve tumor efficacy. The synergistic responses observed in terms of tumor efficacy, immune activation, and cell cycle control provides support for the clinical investigation of this combination. Citation Format: Jack Dempsey, Lysiane Huber, Amelie Forest, Jennifer R. Stephens, Thompson N. Doman, Jason Manro, Andrew Capen, Robert S. Flack, Gregory P. Donoho, Sean Buchanan, Alfonso De Dios, Kyla Driscoll, Michael Kalos, Ruslan Novosiadly, Richard P. Beckmann, David A. Schaer. The CDK4/6 inhibitor abemaciclib induces synergistic immune activation and antitumor efficacy in combination with PD-L1 blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 583. doi:10.1158/1538-7445.AM2017-583