Matthew O'Connor

A novel, potent, and selective insulin-like growth factor-I receptor kinase inhibitor blocks insulin-like growth factor-I receptor signaling in vitro and inhibits insulin-like growth factor-I receptor–dependent tumor growth in vivo

Insulin-like growth factor-I receptor (IGF-IR) and its ligands, IGF-I and IGF-II, are up-regulated in a variety of human cancers. In tumors, such as colorectal, non–small cell lung, ovarian, and pediatric cancers, which may drive their own growth and survival through autocrine IGF-II expression, the role of IGF-IR is especially critical. Here, we present a novel small-molecule IGF-IR kinase inhibitor, cis-3-[3-(4-methyl-piperazin-l-yl)-cyclobutyl]-1-(2-phenyl-quinolin-7-yl)-imidazo[1,5-a]pyrazin-8-ylamine (PQIP), which displayed a cellular IC50 of 19 nmol/L for inhibition of ligand-dependent autophosphorylation of human IGF-IR with 14-fold cellular selectivity relative to the human insulin receptor. PQIP showed minimal activity against a panel of 32 other protein kinases. It also abolished the ligand-induced activation of downstream phosphorylated AKT and phosphorylated extracellular signal-regulated kinase 1/2 in both IGF-IR transfectant cells and a GEO human colorectal cancer cell line. Analysis of GEO cells revealed a significant level of both phosphorylated IGF-IR and IGF-II expression. Furthermore, inactivation of IGF-II in conditioned GEO culture medium by a neutralizing antibody diminished IGF-IR activation, indicating the presence of a functional IGF-II/IGF-IR autocrine loop in GEO cells. Once daily oral dosing of PQIP induced robust antitumor efficacy in GEO xenografts. The antitumor efficacy correlated with the degree and duration of inhibition of tumor IGF-IR phosphorylation in vivo by this compound. Moreover, when mice were treated for 3 days with a dose of PQIP that maximally inhibited tumor growth, only minor changes in blood glucose were observed. Thus, PQIP represents a potent and selective IGF-IR kinase inhibitor that is especially efficacious in an IGF-II–driven human tumor model. [Mol Cancer Ther 2007;6(8):2158–67]

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.

Abstract 2403: Proteomic profiling of signaling pathways in LKB1 deficient non-small cell lung cancers (NSCLC) identifies novel therapeutic targets including IGF1R pathway.

Background: LKB1 is a tumor suppressor gene that is lost in ∼35% of non-small cell lung cancers (NSCLC), half of which carry concurrent KRAS mutations. When functioning normally, LKB1 plays a critical role in energy sensing and regulates the PI3K/mTOR pathway. We have previously shown that LKB1 loss is associated with greater resistance to certain targeted drugs. Here, we investigate signaling pathways dysregulated in the setting of LKB1 loss, with a particular focus on potential novel therapeutic targets. Methods: Expression of >140 total and phospho-proteins were measured in 109 NSCLC cell lines by reverse phase protein array (RPPA). Differences in protein expression between LKB1 deficient versus intact cell lines were determined by t-test, with correction for multiple testing. IC 50 s for targeted therapies were determined by MTS assay in cell lines with and without LKB1. For the drug combination assay, nine NSCLC cell lines with and without LKB1 were treated with the IGFR inhibitor OSI-906 plus serial dilutions of the dual mTOR inhibitor OSI-027. Proliferation was measured after 72 hours. Bliss algorithm was used to calculate synergy and fractional inhibition for each plate independently. Further validation was performed using NSCLC cells stably overexpressing LKB1. Results: Loss of LKB1 via mutation or deletion was strongly correlated with LKB1 protein expression (p Conclusions: These studies identify new potential targets in LKB1 deficient NSCLC, which represents a significant portion of lung cancer patients. Specifically, the combination of IGFR and mTOR inhibition demonstrated synergy, supporting further investigation as a therapeutic approach for this molecularly defined subset of NSCLC patients. Citation Format: Lauren Averett Byers, Maria Angelica Cortez, Chao Yang, Jing Wang, Lixia Diao, You Hong Fan, Luc Girard, Adi Gazdar, Ignacio Wistuba, John D. Minna, Matthew O9Connor, Sharon Barr, John V. Heymach. Proteomic profiling of signaling pathways in LKB1 deficient non-small cell lung cancers (NSCLC) identifies novel therapeutic targets including IGF1R pathway. [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 2403. doi:10.1158/1538-7445.AM2013-2403

Abstract A2: Selective inhibition of mTORC1/mTORC2 sensitizes prostate cancer cells to the effects of AR or IGF-1R/IR antagonists, providing an approach to overcome intrinsic mechanisms of resistance

Prostate cancer is a complex disease primarily characterized by dependence on androgen receptor (AR) signaling. Androgen deprivation therapy is efficacious; however, prostate tumors which initially respond to castration or androgen antagonists eventually progress. These castration-resistant tumors may develop hypersensitivity to low levels of androgens and AR overexpression or may adapt to rely on alternate signaling pathways such as the PI3K/mTOR axis. Deregulation of the PI3K/mTOR axis is a feature of prostate cancer, as evidenced by the fact that 40% of primary and 70% of metastatic prostate tumors exhibit loss of the tumor suppressor PTEN. Recent publications have shown that crosstalk between the AR and PTEN/PI3K/mTOR pathways plays a role in prostate cancer development and progression (1,2). Building upon these data, we evaluated the effects of ASP7486 (OSI-027), a selective inhibitor of mTORC1/mTORC2 as monotherapy and combined with bicalutamide, an androgen receptor antagonist. ASP7486 inhibited proliferation across a panel of prostate cancer cell lines, including those refractory to bicalutamide, supporting the hypothesis that tumors which have acquired androgen independence may rely upon mTOR signaling for survival. ASP7486 but not rapamycin, an allosteric mTORC1 inhibitor, induced apoptosis, implying that mTORC2 signaling is an important regulator of survival in prostate cancer. ASP7486, but not rapamycin, significantly upregulated AR expression which may serve to sensitize cells to the effect of an antiandrogen while limiting sensitivity to mTOR inhibition as a monotherapy. Consistent with this, the combination of ASP7486 and bicalutamide synergistically inhibited proliferation in vitro. In prostate tumors which have acquired resistance to androgen antagonists, activation of alternate RTKs may provide a survival mechanism in the absence of functional AR. PI3K pathway alterations and increased expression of IFG-1R are observed in castrate-resistant tumors (3,4). Treatment of prostate cancer cells with ASP7486 leads to increased phosphorylation of multiple RTKs, including IGF-1R and IR. We reasoned that ASP7486-mediated activation of IGF-1R and IR would sensitize these cells to the effects of OSI-906, a selective IGF-1R/IR inhibitor. The combination of ASP7486 and OSI-906, a selective inhibitor of IGF-1R/IR, synergistically inhibited proliferation and induced apoptosis. The combination of the two drugs provided greater inhibition of key signaling effectors than either monotherapy. These effects are not limited to prostate cancer. We have observed ASP7486-induced upregulation of RTK expression and phosphorylation in multiple tumor types, and the combination of ASP7486 and OSI-906 synergistically inhibited proliferation in the majority of cell lines tested. Together these data demonstrate that combinations of ASP7486 with targeted inhibitors can attenuate crosstalk between signaling networks and provide synergistic efficacy in vitro. The importance of AR and IGF-1R/PI3K/mTOR signaling in prostate cancer provides a rationale for targeting the disease and its intrinsic mechanisms of resistance with inhibitors of these pathways or in combination.

Abstract 4463: OSI-027, a dual mTORC1/mTORC2 inhibitor, induces autophagy in cancer cells

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Inhibitors of mTORC1 have proven clinical activity in renal cell carcinoma (RCC), indicating that mTOR is an important signaling hub in this tumor type. mTOR exists in two functionally distinct protein complexes: mTORC1 and mTORC2. mTORC1 regulates multiple cellular functions including protein translation and tumor metabolism While this complex binds rapamycin some aspects of mTORC1 function are rapamycin insensitive. mTORC2 controls distinct cellular functions including survival and is not directly inhibited by rapalogs. In order to evaluate the relative contribution of mTORC1 and mTORC2 to the survival of RCC cells, we compared the effects of rapamycin, an allosteric inhibitor of mTORC1, to OSI-027, a selective catalytic-site inhibitor of mTORC1/mTORC2. In RCC cell lines, OSI-027 provided greater maximal inhibition of proliferation than rapamycin, with reduction of cell viability to less than baseline in 5 of 12 cell lines, however caspase 3/7-dependent apoptosis was observed only in one cell line. We identified a subset of RCC cell lines which were sensitive to OSI-027 but not rapamycin, and another set which was relatively sensitive to both. Comparison of gene expression profiles of these groups identified MAP1LC3, encoding LC3, a critical effector of autophagy, as significantly upregulated in cell lines which were preferentially sensitive to OSI-027. We therefore sought to further define the role of autophagy in response to OSI-027 treatment. Autophagy is a catabolic process by which cells consume proteins and organelles to promote survival under adverse conditions, and which can result in cell death. Both mTORC1 and mTORC2 have been shown to negatively regulate autophagy, and inhibition of mTOR has been shown to induce formation of autophagic vesicles. Treatment in vitro with OSI-027 resulted in the conversion of LC3 to the lipidated form LC3-II as well as accumulation of LC3 in autophagosomes, robust indicators of autophagy. Increased autophagosome content occurred in a dose-dependent manner and is inversely proportional to viable cell number although induction of apoptosis was not observed, implying that in these cells OSI-027-induced autophagy leads to complete growth arrest or apoptosis-independent cell death. OSI-027-mediated induction of autophagy occurred in the majority of RCC cell lines tested, whereas little or no autophagy resulted from rapamycin treatment. In xenograft tumors sensitive to OSI-027 but not rapamycin, we analyzed induction of LC3-II and apoptosis in tumor lysates. Consistent with our observations in vitro, we observed that treatment with OSI-027 but not rapamycin resulted in induction of autophagy in vivo, while neither compound induced apoptosis. Together these data indicate that autophagy may play a key role in mediating OSI-027 but not rapamycin efficacy and supports further exploration of the utility of dual mTORC1/mTORC2 inhibitors for the treatment of renal cell carcinoma. 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 4463. doi:10.1158/1538-7445.AM2011-4463

Abstract 5063: Preclinical evaluation of an EMT multi-gene biomarker index for predicting response to inhibitors of EGFR and IGF1R

In oncology, the goal of personalized medicine is to improve patient outcome by tailoring therapy to the biochemical signaling within the individual9s tumor. This requires identifying effective predictors of response that can be measured in biopsy material. High expression of the epithelial marker E-cadherin is associated with increased sensitivity in cultured cells and an improved survival benefit in response to erlotinib in patients. Downregulation of E-cadherin is a critical event in epithelial to mesenchymal transition (EMT) and is associated with decreased sensitivity to erlotinib. In order to determine if a more complete characterization of the EMT state would better predict sensitivity to erlotinib as well as other epithelial-targeting drugs, we developed an 88-gene signature that can be used to calculate a numerical index value which represents the EMT state of cells or tumors. In a panel of human tumor cell lines, the EMT index predicted erlotinib sensitivity correctly in 21 out of 24 lines. The index also predicted sensitivity to the IGF1R inhibitor OSI-906. While the EMT index marginally improved sensitivity prediction in vitro compared to E-cadherin, the numerical index allowed for a comparison of relative EMT states that was not possible with E-cadherin alone. Using a microarray database of human tumor sections, we computed EMT index values across 8 solid tumor types in order to compare their relative EMT states. Breast and lung tumors had a fairly even distribution between epithelial and mesenchymal tumors, while colon tumors were more epithelial, and kidney tumors were more mesenchymal. When comparing EMT index values to E-cadherin mRNA levels, some tumor types showed good agreement between E-cadherin and the EMT index (colon, kidney, lung) while others showed less agreement (breast, pancreas). We next compared our EMT signature to other published signatures of EMT and EGFR inhibitor response. Interestingly, most signatures were equally predictive of erlotinib sensitivity in vitro, even though less than 20% of the genes overlaped between any two signatures. We also analyzed signatures developed for individual characteristics of EMT such as invasion or stem cells. These did not correlate with erlotinib sensitivity, suggesting the protection from current therapeutics is not controlled solely by these specific aspects of EMT. Finally, we characterized sub-types of lung cancer for EMT state to determine if the EMT classification might correlate with established response rates to erlotinib. Adenocarcinomas, which tend to respond well, had more epithelial index values while squamous cell carcinomas, which tend to be less sensitive, had more mesenchymal values. This suggests that more epithelial index values might predict for erlotinib response in patients, potentially providing clinicians with a tool to more effectively treat patients based on the EMT status of their tumors. 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 5063. doi:10.1158/1538-7445.AM2011-5063

Abstract 1463: Pharmacological impact of epithelial-mesenchymal plasticity in the H358 lung tumor model

The phenotypic changes associated with epithelial to mesenchymal transition (EMT) give tumor cells at least two attributes that increase metastatic efficiency. First, mesenchymal cells have lost cell-cell contacts and are more invasive and can therefore escape the primary tumor, whereas epithelial cells remain anchored by cell-cell contacts in the primary tumor. Second, mesenchymal cells in vitro are less sensitive than epithelial cells to agents that inhibit the EGFR pathway. This appears to translate into the clinic, since patients with tumors that express mesenchymal markers do not respond to EGFR targeting therapeutics as well as patients with more epithelial tumors. To determine whether we can control sensitivity to EGFR inhibitors by manipulating a cell9s epithelial/mesenchymal status, we examined the role of EMT and its reversibility in tumor cell sensitivity to the EGFR inhibitor erlotinib. We studied in vitro models of EMT driven by Transforming Growth Factor β (TGFβ), Hepatocyte Growth Factor + Oncostatin M (HGF+OSM), Snail or Zeb1 in the H358 non-small cell lung carcinoma model. EMT driven by TGFβ or HGF+OSM caused a significant decrease in sensitivity to erlotinib, while EMT driven by induced expression of Snail or Zeb1 was much less effective, suggesting a change in drug sensitivity requires more input than these canonical EMT transcription factors. Once mesenchymal tumor cells have localized to a metastatic site, it is thought the tumor cells must undergo a mesenchymal to epithelial transition (MET) in order to form a cohesive tumor. We examined the reversibility of EMT in our models and the corresponding erlotinib sensitivity, both by withdrawal of ligand and by pharmacological inhibition of the signaling pathways downstream of the drivers. Using these approaches, we found EMT driven by HGF+OSM was reversible as evidenced by morphology and marker changes, and this reversion correlated with an increase in erlotinib sensitivity, comparable to the parental H358 cells. EMT driven by TGFβ was partially reversed by both ligand withdrawal and pharmacological inhibition of the receptor, and the extent of reversion in erlotinib sensitivity correlated with the extent of marker reversion. These results demonstrate that the extent of reversibility of EMT is dependent on the driver. Furthermore, it may be possible to drive some erlotinib-insensitive tumors back to a more erlotinib-sensitive state with pharmacological agents, thus improving patient response. 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 1463.