Haluska, Paul

Abstract Insulin-like growth factor (IGF) signaling has been implicated in the resistance to hormonal therapy in breast cancer. Using a model of postmenopausal, estrogen-dependent breast cancer, we investigated the antitumor effects of the dual IGF-1R/InsR tyrosine kinase inhibitor BMS-754807 alone and in combination with letrozole or tamoxifen. BMS-754807 exhibited antiproliferative effects in vitro that synergized strongly in combination with letrozole or 4-hydroxytamoxifen and fulvestrant. Similarly, combined treatment of BMS-754807 with either tamoxifen or letrozole in vivo elicited tumor regressions not achieved by single-agent therapy. Notably, hormonal therapy enhanced the inhibition of IGF-1R/InsR without major side effects in animals. Microarray expression analysis revealed downregulation of cell-cycle control and survival pathways and upregulation of erbB in response to BMS-754807 plus hormonal therapy, particularly tamoxifen. Overall, these results offer a preclinical proof-of-concept for BMS-754807 as an antitumor agent in combination with hormonal therapies in hormone-sensitive breast cancer. Cooperative cell-cycle arrest, decreased proliferation, and enhanced promotion of apoptosis may contribute to antitumor effects to be gauged in future clinical investigations justified by our findings. Cancer Res; 71(24); 7597–607. ©2011 AACR.

Abstract We have demonstrated that crosstalk between the Hedgehog-GLI-GLI and EGFR signaling pathway exists and contribute to pancreatic tumorigenesis. This interaction can be abrogated by targeting both pathways simultaneously and results in a synergistic antitumor effect in vitro and in vivo in pancreatic tumor models. Based on these results we have undertaken a phase I trial of GDC-0449 (an orally administered Hedgehog pathway inhibitor) and erlotinib (EGFR tyrosine kinase inhibitor to: 1) determine the maximally tolerated dose (MTD) of GDC-0449 combined with erlotinib in unresectable solid tumors; 2) describe the adverse events associated with this treatment combination; 3) describe the responses of this treatment combination; and 4) to assess the effect of the erlotinib and GDC-0449 combination on selected biomarkers in circulating tumor cells (CTCs), tumor biopsies, and FDG-PET in a cohort of patients with metastatic pancreatic cancer patients treated at the MTD. During the dose escalation portion of the trial, eligible patients receive GDC-0449 150 mg p.o. daily and erlotinib 50, 75, 100, or 150 mg daily p.o. at dose levels 1, 2, 3, and 4 respectively. Eligibility criteria include: histologic proof of solid tumors that are unresectable, not amenable to any other standard therapies, or patient refused standard therapy,; ANC ≥> 1500/μL; PLT ≥> 100,000/ μL; Total bilirubin ≤< upper limit of normal (ULN); AST ≤< 3 x ULN; Creatinine ≤< 1.5 x ULN; Hemoglobin ≥> 9.0 g/dL; INR within normal limits (Cohort II [MTD Cohort] only); ECOG performance status (PS) 0, 1 or ≤ 2. For the MTD cohort, only patients with metastatic adenocarcinoma of the pancreas without prior systemic therapy for metastatic disease and tumor amenable to biopsies are eligible. Toxicity assessment is performed at each 28-day treatment cycles and response assessment every second treatment 28 day cycle. 12 patients have been entered-pancreas (3), colorectal (3), neuroendocrine (2), scc anus (2), gallbladder (1), thymoma (1). No dose-limiting toxicity (DLT) was reported at dose level 4. At the MTD, up to 20 patients with previously untreated metastatic pancreatic cancer will be entered to assess the biologic effect of GDC-0449+erlotinib. Pre- and post- treatment biopsies of metastases and FDG-PET will be performed. Weekly collection of CTCs will be undertaken and immunofluorescence microscopy of total & phosphoMAPK, EGFR, AKT, PATCHED1, GLI1, BCL-2, and quantitative PCR (Q-PCR) of PATCHED1, GLI1, and its target genes (BCL-2, BFL-1/A1, 4-1BB) will be performed. In addtion, total & phosphoMAPK, EGFR, and AKT, as well as PATCHED1, GLI1, BCL-2 by immunohistochemistry and Q-PCR of PATCHED1, GLI1, BCL-2, BFL-1/A1, 4-1BB will be analyzed in tumor biopsies. Supported by CA69912, CA136526 and CA102701. 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 1807.

Abstract Preclinical investigations have identified insulin-like growth factor (IGF) signaling as a key mechanism for cancer growth and resistance to clinically useful therapies in multiple tumor types including breast cancer. Thus, agents targeting and blocking IGF signaling have promise in the treatment of solid tumors. To identify possible mechanisms of resistance to blocking the IGF pathway, we generated a cell line that was resistant to the IGF-1R/InsR benzimidazole inhibitors, BMS-554417 and BMS-536924, and compared expression profiles of the parental and resistant cells lines using Affymetrix GeneChip Human Genome U133 arrays. Compared with MCF-7 cells, breast cancer resistance protein (BCRP) expression was increased 9-fold in MCF-7R4, which was confirmed by immunoblotting and was highly statistically significant (P = 7.13E-09). BCRP was also upregulated in an independently derived resistant cell line, MCF-7 924R. MCF-7R4 cells had significantly lower intracellular accumulation of BMS-536924 compared with MCF-7 cells. Expression of BCRP in MCF-7 cells was sufficient to reduce sensitivity to BMS-536924. Furthermore, knockdown of BCRP in MCF-7R4 cells resensitized cells to BMS-536924. Four cell lines selected for resistance to the pyrrolotriazine IGF-1R/InsR inhibitor, BMS-754807, did not have upregulation of BCRP. These data suggest that benzimidazole IGF-1R/InsR inhibitors may select for upregulation and be effluxed by the ATP-binding cassette transporter, BCRP, contributing to resistance. However, pyrrolotriazine IGF-1R/InsR inhibitors do not appear to be affected by this resistance mechanism. Mol Cancer Ther; 10(1); 117–25. ©2011 AACR.

Abstract Resistance to hormonal therapy is a clinically unmet need in breast cancer. IGF signaling has been identified as a major mechanism of resistance to hormonal therapy in breast cancer. As components of the IGF signaling pathway are expressed in most breast cancers, the development of IGF-1R monoclonal antibody (mAb) and tyrosine kinase inhibitors (TKI) are active areas of clinical investigations. A key distinction between the mAb and TKIs are their differences in their ability to inhibit the Insulin Receptor (InsR). While targeting the InsR with TKIs may have a theoretical liability of hyperglycemia, targeting only the IGF-1R may have the theoretical liability of incompletely blocking IGF signaling. As InsR isoform A expression, which can transduce IGF-II-mediated proliferation, is higher in breast cancers compared to normal breast tissue, we investigated whether IGF-1R or IGF-1R/InsR inhibition was sufficient for overcoming resistance to hormonal therapy. To determine the optimal combination strategies for clinical investigations, we tested the hypothesis that IGF signaling inhibition could overcome primary (or de novo/intrinsic) and secondary (or acquired/selected) resistance to hormonal therapy. For these studies, we used either hormone therapy-naïve or hormone therapy-resistant variants of the breast cancer model, MCF-7/AC-1, which has been engineered to stably express full-length human aromatase. We employed and compared a novel, potent dual kinase inhibitor of the IGF-1R and InsR, BMS-754807, which is currently in early clinical investigations, with the IGF-1R antibody mAb391. BMS-754807 has been shown to induce apoptosis more potently than mAb391 in Rh41 human rhabdomyosarcoma cells. In vitro, BMS-754807 demonstrated profound synergy in combination with tamoxifen and letrozole (median effect combination index <0.1). In vivo, BMS-754807 enhanced the anti-tumor activity of tamoxifen and letrozole in hormone-naïve tumors and induced regression of tumors resistant to tamoxifen or letrozole when combined with letrozole. This activity was not observed with mAb therapy, which resulted in greater up-regulation of InsR-A and erbB receptor expression and activation. This suggested a greater susceptibility to resistance pathways with mAb therapy. Dual IGF-1R/InsR blockade alone or in combination was tolerated by the animals and has no significant change in glucose homeostasis. Gene expression profiling experiments to compare the difference between the effects of tamoxifen in combination with BMS-754807 and with mAb revealed alternative pathway signaling is one of the potential mechanisms of resistance.In summary, combined hormonal therapy with BMS-754807 overcomes primary and secondary resistance to tamoxifen and letrozole and was well tolerated. IGF-1R blockade with a mAb alone is insufficient to overcome resistance and induces InsR over-expression. Thus, IGF signaling through either InsR or IGF-1R may be a major mechanism of resistance to hormonal therapy. These data suggest that blockade of IGF-1 and IGF-II from activation of IGF-1R and InsR, with agents such as BMS-754807 have promise in extending the benefits of hormonal therapy in breast cancer. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 402.

Abstract Abstract #2149 Background: Inhibitors of the insulin-like growth factor 1 receptor (IGF-1R) are currently undergoing clinical testing. Preclinical investigations have identified IGF-1 signaling as a key mechanism for breast cancer growth and resistance to clinically useful therapies, including tamoxifen and trastuzumab. Thus, agents targeting IGF-1R have promise in the treatment of breast cancer. Determining mechanisms that can confer resistance to these agents may aid their clinical development.
 Methods: To understand factors may be important in predicting sensitivity to targeting the IGF-1 signaling pathway, we developed a cell line (MCF-7R4) that is resistant to BMS-554417, a small molecule, dual-kinase inhibitor of IGF-1R and insulin receptor (InsR). Compared with the parental MCF-7 cells, MCF-7R4 cells are 40- to 50-fold resistant to BMS-554417 and cross-resistant to the similar compound BMS-536924. The expression profiles of MCF-7R4 and that of MCF-7 were compared using Affymetrix GeneChip Human Genome U133 Plus 2.0 Arrays. Intracellular concentrations of BMS-536924 were examined by reverse phase high performance liquid chromatography. BMS-536924 cellular accumulation in vitro was visualized by fluorescence microscopy using a DAPI filter set. MCF-7 cells stably transfected with either the empty mammalian expression vector pcDNA (MCF-EV) or full length BCRP (MCF-BCRP) were examined for sensitivity to BMS 536924 by MTS assays.
 Results: Compared to MCF-7 cells, BCRP expression was increased 9-fold in MCF-7R4, which was highly statistically significant by t-test (p= 7.13E-09). Little change was observed in other ABC transporter proteins, including ABCB1. No change was observed in IGF-1R or InsR expression. BCRP overexpression in MCF-7R4 cells was confirmed by western blotting. MCF-7R4 cells had significantly lower intracellular accumulation of BMS-536924 compared to MCF-7 cells. Confirming these results, MCF-BCRP cells were significantly less sensitive to the cytoxic effects of BMS-536924 cells than MCF-EV cells.
 Conclusions: BCRP expression was stimulated by prolonged exposure of MCF-7 cells to BMS-554417. Upregulation of BCRP is one of the most significant changes observed in MCF-7R4 cells in comparison to parental cells. BCRP expression decreased cellular exposure to BMS-536924 and was sufficient to confer resistance. These data suggest that BSM-536924 is a substrate for BCRP-mediated efflux. Expression of BCRP may be important in de novo and acquired resistance to benzimidazole –based inhibitors of IGF-1R/InsR. Supported in part by the Mayo Clinic Breast SPORE (CA116201-03), NIH K12 (CA090628-05) and the Mayo Clinic Cancer Center (CA15083). Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2149.

Abstract Heat shock protein 90 (HSP90), which regulates the functions of multiple oncogenic signaling pathways, has emerged as a novel anticancer therapeutic target, and multiple small-molecule HSP90 inhibitors are now in clinical trials. Although the effects of HSP90 inhibitors on oncogenic signaling pathways have been extensively studied, the effects of these agents on tumor suppressor signaling pathways are currently unknown. Here, we have examined how HSP90 inhibitors affect LATS1 and the related protein LATS2, two kinases that relay antiproliferative signals in the Hippo tumor suppressor pathway. Both LATS1 and LATS2 were depleted from cells treated with the HSP90 inhibitors 17-allylamino-17-demethoxygeldanamycin (17-AAG), radicicol, and PU-H71. Moreover, these kinases interacted with HSP90, and LATS1 isolated from 17-AAG–treated cells had reduced catalytic activity, thus showing that the kinase is a bona fide HSP90 client. Importantly, LATS1 signaling was disrupted by 17-AAG in tumor cell lines in vitro and clinical ovarian cancers in vivo as shown by reduced levels of LATS1 and decreased phosphorylation of the LATS substrate YAP, an oncoprotein transcriptional coactivator that regulates genes involved in cell and tissue growth, including the CTGF gene. Consistent with the reduced YAP phosphorylation, there were increased levels of CTGF, a secreted protein that is implicated in tumor proliferation, metastasis, and angiogenesis. Taken together, these results identify LATS1 and LATS2 as novel HSP90 clients and show that HSP90 inhibitors can disrupt the LATS tumor suppressor pathway in human cancer cells. Cancer Res; 70(21); 8642–50. ©2010 AACR.

Abstract Edmonston vaccine strains of measles virus (MV) have shown significant antitumor activity in preclinical models of ovarian cancer. We engineered MV to express the marker peptide carcinoembryonic antigen (MV-CEA virus) to also permit real-time monitoring of viral gene expression in tumors in the clinical setting. Patients with Taxol and platinum-refractory recurrent ovarian cancer and normal CEA levels were eligible for this phase I trial. Twenty-one patients were treated with MV-CEA i.p. every 4 weeks for up to 6 cycles at seven different dose levels (103–109 TCID50). We observed no dose-limiting toxicity, treatment-induced immunosuppression, development of anti-CEA antibodies, increase in anti-MV antibody titers, or virus shedding in urine or saliva. Dose-dependent CEA elevation in peritoneal fluid and serum was observed. Immunohistochemical analysis of patient tumor specimens revealed overexpression of measles receptor CD46 in 13 of 15 patients. Best objective response was dose-dependent disease stabilization in 14 of 21 patients with a median duration of 92.5 days (range, 54–277 days). Five patients had significant decreases in CA-125 levels. Median survival of patients on study was 12.15 months (range, 1.3–38.4 months), comparing favorably to an expected median survival of 6 months in this patient population. Our findings indicate that i.p. administration of MV-CEA is well tolerated and results in dose-dependent biological activity in a cohort of heavily pretreated recurrent ovarian cancer patients. Cancer Res; 70(3); 875–82

Abstract The insulin receptor (IR) and insulin-like growth factor-1 receptor (IGF1R) are receptor tyrosine kinases that participate in mitogenic and antiapoptotic signaling in normal and neoplastic epithelia. In the present study, immunoblotting and reverse transcription-PCR demonstrated expression of IGF1R and IR isoform A in acute myelogenous leukemia (AML) cell lines as well as in >80% of clinical AML isolates. Treatment with insulin enhanced signaling through the Akt and MEK1/2 pathways as well as survival of serum-starved AML cell lines. Conversely, treatment with BMS-536924, a dual IGF1R/IR kinase inhibitor that is undergoing preclinical testing, inhibited constitutive receptor phosphorylation as well as downstream signaling through MEK1/2 and Akt. These changes inhibited proliferation and, in some AML cell lines, induced apoptosis at submicromolar concentrations. Likewise, BMS-536924 inhibited leukemic colony formation in CD34+ clinical AML samples in vitro. Collectively, these results not only indicate that expression of IGF1R and IR isoform A is common in AML but also show that interruption of signaling from these receptors inhibits proliferation in clinical AML isolates. Accordingly, further investigation of IGF1R/IR axis as a potential therapeutic target in AML appears warranted. [Cancer Res 2009;69(19):7635–43]