Maryland Franklin

OSI-930: A Novel Selective Inhibitor of Kit and Kinase Insert Domain Receptor Tyrosine Kinases with Antitumor Activity in Mouse Xenograft Models

OSI-930 is a novel inhibitor of the receptor tyrosine kinases Kit and kinase insert domain receptor (KDR), which is currently being evaluated in clinical studies. OSI-930 selectively inhibits Kit and KDR with similar potency in intact cells and also inhibits these targets in vivo following oral dosing. We have investigated the relationships between the potency observed in cell-based assays in vitro, the plasma exposure levels achieved following oral dosing, the time course of target inhibition in vivo, and antitumor activity of OSI-930 in tumor xenograft models. In the mutant Kit-expressing HMC-1 xenograft model, prolonged inhibition of Kit was achieved at oral doses between 10 and 50 mg/kg and this dose range was associated with antitumor activity. Similarly, prolonged inhibition of wild-type Kit in the NCI-H526 xenograft model was observed at oral doses of 100 to 200 mg/kg, which was the dose level associated with significant antitumor activity in this model as well as in the majority of other xenograft models tested. The data suggest that antitumor activity of OSI-930 in mouse xenograft models is observed at dose levels that maintain a significant level of inhibition of the molecular targets of OSI-930 for a prolonged period. Furthermore, pharmacokinetic evaluation of the plasma exposure levels of OSI-930 at these effective dose levels provides an estimate of the target plasma concentrations that may be required to achieve prolonged inhibition of Kit and KDR in humans and which would therefore be expected to yield a therapeutic benefit in future clinical evaluations of OSI-930.

Novel 6-aminofuro[3,2-c]pyridines as potent, orally efficacious inhibitors of cMET and RON kinases

A series of novel 6-aminofuro[3,2-c]pyridines as kinase inhibitors is described, most notably, OSI-296 (6). We discuss our exploration of structure-activity relationships and optimization leading to OSI-296 and disclose its pharmacological activity against cMET and RON in cellular assays. OSI-296 is a potent and selective inhibitor of cMET and RON kinases that shows in vivo efficacy in tumor xenografts models upon oral dosing and is well tolerated.

Abstract 5194: Focal radiation enhances paclitaxel therapy in a mouse model of triple negative breast cancer

Triple negative breast cancer (TNBC) accounts for 15-20% of all breast cancers in the US. In general, patients have poorer prognosis and therapeutic intervention is more challenging due to insensitivity to hormonal and anti-HER2 therapies. Therapeutic options include chemotherapy and/or radiation (RT) treatment. In this work we utilized HCC70, a human TNBC cell line, grown subcutaneously in NSG mice. We investigated whether the combination of focal RT and paclitaxel would improve anti-tumor activity over paclitaxel alone. RT therapy has typically been delivered in low doses over long periods of time. More recently higher fractions of RT are being given in shorter time intervals. Utilizing the Small Animal Radiation Research Platform (SARRP; Xstrahl, Suwanee, GA) we compared a low dose (2.5Gy) fractionated regimen to a higher dose but less frequent regimen (8Gy, QDx3) either as monotherapy or in combination with paclitaxel (15mg/kg weekly). The total cumulative dose of radiation was 24-25Gy in both radiation regimens. Paclitaxel alone produced moderate activity with a 10 day tumor growth delay and no tumor regressions. Treatment with low dose RT resulted in tumor stasis for ≥68 days, but there were no tumor regressions or tumor free survivors (TFS). Treatment with 8Gy, produced a 75% incidence of confirmed partial regressions (PR) and a 25% incidence of complete regressions (CR) with no TFS. In this group we observed slightly more body weight loss (BWL), more frequent clinical signs, and more frequent need for food and water supplementation than with the fractionated RT regimen. Mean BWL was ~10% with a nadir around 8-10 days post first RT dose. BWL was recovered over the next 1-2 week period. The combination of low dose focal RT with paclitaxel produced activity similar to that with high dose radiation (50% PR, 40% CR, and 1 TFS). Treatment with paclitaxel plus 8Gy RT was highly efficacious, producing an 80% incidence of CRs, all of which remained tumor free at study day 108. The remaining mouse experienced tumor stasis in excess of 63 days. Thus, a higher dose of focal RT given in shorter intervals in combination with systemic paclitaxel resulted in the most efficacious therapeutic strategy but at the cost of increased BWL and clinical signs. Citation Format: Maryland Franklin, Thomas Dailey, Wilbur Leopold. Focal radiation enhances paclitaxel therapy in a mouse model of triple negative breast cancer [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 5194. doi:10.1158/1538-7445.AM2017-5194

Abstract 2619: Combination of ECP1014 and anti-PD-L1 reduces tumor growth in the CT26 murine colon carcinoma model of a cold tumor

Objective: The objective of this study was to evaluate the effect of ECP1014, a new selective COX2 inhibitor, in combination with a PDL1 inhibitor on tumor growth in a CT26 murine colon carcinoma model. Colon cancer has been described as a “cold tumor” and antiPDL1monotherapy has shown limited or no benefit. In addition, many colon cancer cell lines produce high levels of PGE2, which may in turn dampen the immune system. The hypothesis was that combining ECP1014, which we have shown potently decreases PGE2 in CT26, with a checkpoint inhibitor would increase immune response to the tumor, turning it “hot” and producing superior tumor control versus either agent alone. Methods: Balb/C mice were implanted with CT26 cells and randomly assigned to 7 treatment groups; vehicle (G1), 10mg/kg rat IgG2b control (G2), 10mg/kg antiPDL1 (G3), 10mg/kg ECP1014 (G4), 10mg/kg ECP1014+10mg/kg rat IgG2b (G5), 10mg/kg ECP1014+10mg/kg antiPDL1 (G6), 1mg/kg ECP1014+10mg/kg antiPDL1(G7). ECP1014 was given daily by oral gavage; rat IgG2b and antiPDL1 (clone 10F.9G2) were administered IP. Treatment was started on day 3 post implant and mice were followed for 16 days. Tumor measurements were taken every 2 to 3 days, and a nonparametric approach was taken to assess the pairwise differences among groups using day 16 tumor volumes. In addition, a responder analysis was used. Tumor infiltrating lymphocytes (TIL) were also assessed by flow cytometry on day 16. Results: All animals developed tumors. G4 to 7 demonstrated statistically significantly (p Summary: ECP1014 alone was superior to antiPDL1 in slowing tumor growth. However, the combination of ECP1014+antiPDL1showed the greatest tumor response and growth suppression and markedly increased CD8+ Tcells infiltrating into the tumor vs. single agent anti-PDL1 treatment. Citation Format: Bobby W. Sandage, John J. Talley, Eduardo J. Martinez, Maryland R. Franklin, Mary Anne Meade, Dan Saims, Matt Thayer, Scott Wise, David Draper, Wilber Leopold. Combination of ECP1014 and anti-PD-L1 reduces tumor growth in the CT26 murine colon carcinoma model of a cold tumor [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 2619. doi:10.1158/1538-7445.AM2017-2619

Abstract 4915: Evaluation of the immune response following treatment with anti-CTLA-4 antibody, radiation therapy or the combination in a murine model of breast cancer

While breast cancers are considered poorly immunogenic, several approaches utilizing immunotherapies are being undertaken in the clinic to evaluate their potential for improving outcomes. Radiation therapy (RT) is a highly utilized clinical treatment modality in breast cancer, is known to modify the tumor microenvironment and has been shown to potentially synergize with immunotherapies. To evaluate this potential synergy preclinically, we utilized the 4T1 murine mammary carcinoma model to investigate the immune response following treatment with either the 9H10 clone of an anti-CTLA-4 antibody (9H10), RT (8Gy, QDx3) or the combination of the two. Tumors were established in the mammary fat pad and treatments initiated when tumors reached a mean volume of 63-72mm3. We found that both monotherapies resulted in a period of stable disease followed by tumor regrowth. The combination did not demonstrate added benefit over either single agent treatment. As the 4T1 model is known to be highly metastatic, with tumors seen in lung, liver and lymph nodes, we closely examined each mouse at the time of euthanasia for evidence of metastatic disease. None of the treatments effectively reduced metastasis. To attempt to gain a better mechanistic understanding around the lack of added benefit of the combination, we profiled different immune cell subsets in the tumor by flow cytometry. We found that within the tumor infiltrating lymphocytes (TILs), 9H10 antibody and RT treatment resulted in an increased percentage of CD4+ T cells. Conversely a decreased percentage of CD8+ T cells was observed. However, the greatest effect exerted by 9H10 and RT was on the myeloid derived suppressive cell (MDSC) population. Although RT slightly decreased the percentage of MDSC within tumors and 9H10 had no effect, the two treatments synergized to reduce the number of tumor-infiltrating MDSC. Interestingly, a similar synergistic effect was observed when we analyzed the expression of the proliferation marker Ki-67 within CD8+ T cells. We also found that RT, and to a lesser extent 9H10, increased the percentage of CD45+ cells expressing the T cell inhibitory receptors PD1, PD-L1, and CTLA-4. No additive effect on the expression of these receptors was observed with combined therapy. Taken together, our results suggest that anti-CTLA-4 treatment combined with RT triggers both pro- and anti-tumor signaling pathways thus providing a possible explanation for the marginal anti-tumor responses we observed with these therapies in this disease model. Furthermore, this model could be very beneficial to evaluate other agents either in combination with 9H10 or RT or as a triple combination with both 9H10 and RT. Citation Format: Maryland R. Franklin, Matt Thayer, David Draper, Dan Saims, Scott Wise. Evaluation of the immune response following treatment with anti-CTLA-4 antibody, radiation therapy or the combination in a murine model of breast cancer. [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 4915.

Abstract 2915: Discovery and characterization of OSI-296, a dual inhibitor of cMET and RON kinases

cMET and RON are receptor tyrosine kinases of the MET proto-oncogene family that are activated by their respective ligands HGF and MSP. Signaling through the cMET/HGF system can be deregulated in cancer by HGF-dependent autocrine activation, gene amplification, and/or the presence of activating mutations, among others, while for RON, constitutively active variants generated by alternative splicing or methylation-dependent promoter usage [short-form RON (sfRON)] have been identified. Approaches to abrogate aberrant cMET and RON signaling that have led to agents in clinical trials include inhibiting their kinase function with small molecules. We report here the discovery and characterization of OSI-296, a dual inhibitor of cMET and RON. The compound exhibited selectivity in a panel of 96 kinases with potent activity against cMET, including common Y1230 mutants, and RON. OSI-296 blocked cMET autophosphorylation in MKN45 cells, resulting in dose-dependent inhibition of downstream ERK, AKT, and STAT3 phosphorylation. It also showed potent cellular activity in ELISA-format sfRON and caRON cell mechanistic assays that we developed, resulting in dose-dependent inhibition of downstream ERK and AKT phosphorylation. OSI-296 showed a PK profile in rodents suitable for oral dosing with >70% bioavailability. In multiple xenografts models (cMET: MKN45, SNU-5, U87MG; RON: caRON), significant tumor growth inhibition was observed upon oral dosing with regression at higher doses. OSI-296 was very well tolerated with little body weight loss and no adverse effects even at the highest tested dose of 300 mg/kg p.o. qdx14. Solid PK/PD/TGI correlations have been established wherein >90% inhibition of cMET or RON phosphorylation sustained over 24 h by OSI-296 translated to 100% TGI. In summary, OSI-296 was shown to be a well tolerated, dual inhibitor of cMET and RON with in vivo activity in mouse xenografts models for both targets upon oral dosing. 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 2915. doi:1538-7445.AM2012-2915

Abstract 4160: Evaluating epithelial-to-mesenchymal transition (EMT) in the EL1-luc/EL1-SV40 T-antigen transgenic mouse model of pancreatic cancer

Epithelial-to-mesenchymal transition (EMT) is a biological function important in normal cellular processes such as embryonic development and wound healing. In cancer it is thought that the tumor cell machinery can re-activate these normal pathways resulting in more aggressive and invasive tumors. The loss of E-cadherin and the gain of vimentin are hallmarks which identify the process of EMT and have been shown to correlate with poor prognosis in multiple solid tumor types. While many preclinical models are utilized to evaluate mechanisms of tumorigenesis few in vivo models evaluating parameters of EMT have been described. The EL1-luc/EL1-SV40 T-antigen transgenic mouse represents a model of pancreatic cancer whereby mice develop tissue specific, spontaneous and bioluminescent pancreatic tumors. To evaluate whether EMT occurs in the EL1-luc/EL1-SV40 T-antigen model in vivo, we collected primary pancreatic tissue from male mice between 10 and 21 weeks of age. The tissue was formalin fixed, paraffin embedded and then utilized for histopathological endpoints such as Hemotoxylin and Eosin staining as well as immunohistochemistry for markers known to be involved in EMT such as E-cadherin and vimentin. We found the tumors to express both markers and become very heterogeneous over time. In early tumors E-cadherin expression is membrane localized and very high. Over time there are areas of the tumors that have reduced or lost E-cadherin expression. Vimentin expression was highly variable but when present tended to be highly expressed. In many of the later stage tumors there was substantial heterogeneity reflected by the appearance of multiple cell types within a tumor. We utilized the Aperio (Aperio Technologies, Vista, CA) slide scanner and software system to evaluate serial sections of tumor samples and found that in some sections of the tumor E-cadherin is present and vimentin is absent, whereas in other areas of the tumor vimentin is present in the absence of E-cadherin. Additionally, we identified areas of the tumor that seem to be expressing both markers which suggests that the EL1-luc/EL1-SV40 T-antigen transgenic mouse may recapitulate many aspects of EMT observed in vivo, thus offering a model system to study the signaling and molecular changes necessary for this process during cancer progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4160.