Karen A. Reeves

The Mechanisms of Differential Sensitivity to an Insulin-like Growth Factor-1 Receptor Inhibitor (BMS-536924) and Rationale for Combining with EGFR/HER2 Inhibitors

Overexpression and enhanced activity of insulin-like growth factor-I receptor (IGF-IR) in diverse tumor types make it an attractive target for cancer therapy. BMS-536924 is a potent small molecule inhibitor of IGF-IR, which shows antitumor activity in multiple tumor models, including sarcoma. To facilitate the development of IGF-IR inhibitors as cancer therapy, identification of biomarkers for selecting patients most likely to derive clinical benefit is needed. To do so, 28 sarcoma and neuroblastoma cell lines were screened for in vitro response to BMS-536924 to identify sensitive and resistant cell lines. Notably, Ewings sarcoma, rhabdomyosarcoma, and neuroblastoma are more responsive to BMS-536924, suggesting these specific subtypes may represent potential targeted patient subpopulations for the IGF-IR inhibitor. Gene expression and protein profiling were performed on these cell lines, and candidate biomarkers correlating with intrinsic and/or acquired resistance to BMS-536924 were identified. IGF-I, IGF-II, and IGF-IR were highly expressed in sensitive cell lines, whereas IGFBP-3 and IGFBP-6 were highly expressed in resistant lines. Overexpression of epidermal growth factor receptor (EGFR) and its ligands in resistant cell lines may represent one possible resistance mechanism by the adaptation of IGF-IR-independent growth using alternative signaling pathways. Based on cross-talk between IGF-IR and EGFR pathways, combination studies to target both pathways were performed, and enhanced inhibitory activities were observed. These results provide a strategy for testing combinations of IGF-IR inhibitors with other targeted therapies in clinical studies to achieve improved patient outcomes. Further exploration of mechanisms for intrinsic and acquired drug resistance by these preclinical studies may lead to more rationally designed drugs that target multiple pathways for enhanced antitumor efficacy.

Differential Mechanisms of Acquired Resistance to Insulin-like Growth Factor-I Receptor Antibody Therapy or to a Small-Molecule Inhibitor, BMS-754807, in a Human Rhabdomyosarcoma Model

Agents targeting insulin-like growth factor-I receptor (IGF-IR), including antibodies and small-molecule inhibitors, are currently in clinical development for the treatment of cancers including sarcoma. However, development of resistance is a common phenomenon resulting in failures of anticancer therapies. In light of this problem, we developed two resistant models from the rhabdomyosarcoma cell line Rh41: Rh41-807R, with acquired resistance to BMS-754807, a small-molecule dual-kinase inhibitor targeting IGF-IR and insulin receptor (IR), and Rh41-MAB391R, with resistance to MAB391, an IGF-IR-blocking antibody. In addition, tumor xenograft models were established from Rh41 and Rh41-807R cell lines. Gene expression and DNA copy number analyses of these models revealed shared as well as unique acquired resistance mechanisms for the two types of IGF-IR inhibitors. Each resistant model used different signaling pathways as a mechanism for proliferation. Platelet-derived growth factor receptor α (PDGFRα) was amplified, overexpressed, and constitutively activated in Rh41-807R cells and tumors. Knockdown of PDGFRα by small interfering RNA in Rh41-807R resensitized the cells to BMS-754807. Synergistic activities were observed when BMS-754807 was combined with PDGFRα inhibitors in the Rh41-807R model in vitro. In contrast, AXL expression was highly elevated in Rh41-MAB391R but downregulated in Rh41-807R. Notably, BMS-754807 was active in Rh41-MAB391R cells and able to overcome resistance to MAB391, but MAB391 was not active in Rh41-807R cells, suggesting potentially broader clinical activity of BMS-754807. This is the first study to define and compare acquired resistance mechanisms for IGF-IR-targeted therapies. It provides insights into the differential acquired resistance mechanisms for IGF-IR/IR small-molecule inhibitor versus anti-IGF-IR antibody.

Dual IGF-1R/InsR Inhibitor BMS-754807 Synergizes with Hormonal Agents in Treatment of Estrogen-Dependent Breast Cancer

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.

Oncogenic NRAS has multiple effects on the malignant phenotype of human melanoma cells cultured in vitro

Activating mutations in the NRAS gene, which occur predominantly in codon 61 (Q61R, Q61K) are among the most common genetic events in malignant melanoma. NRAS protein with oncogenic codon 61 mutations may therefore be good therapeutic targets. In the present study, we used gene expression profiling as a method for global characterization of gene expression alterations that resulted from treatment of melanoma cells with siRNA specifically targeting NRASQ61R. Sixteen probe sets representing 15 unique genes were identified whose expression was significantly altered by siRNA against NRASQ61R in 2 melanoma cell lines. The genes with altered expression are involved in several functions, including modulation of cell growth, invasion and migration. The results suggest that downregulation of cyclin E2 and cyclin D1 and also upregulation of the negative cell‐cycle regulator HBP1 in NRASQ61R knockdown cells contribute to the inhibition of cell proliferation. Furthermore, suppression of oncogenic NRAS results in reduced migration and invasion, which is accompanied by downregulation of EphA2 (a receptor tyrosine kinase), uPAR (urokinase receptor) and cytoskeleton proteins such as leupaxin, paxillin and vinculin. These studies support the concept that suppression of oncogenic NRAS by siRNA can induce growth arrest and inhibit invasion of human melanoma cells by modulating the levels of these gene products.

Insulin Receptor (IR) Pathway Hyperactivity in IGF-IR Null Cells and Suppression of Downstream Growth Signaling Using the Dual IGF-IR/IR Inhibitor, BMS-754807

The biology of IGF-IR/IR signaling was studied in normal mouse embryonic fibroblasts (MEFs) that were either wild type (wt), heterozygous (het), or null for the IGF-IR. The ability of IGF-I, IGF-II, or insulin to stimulate serum-starved MEFs was characterized by gene expression profiling and biochemical analyses for activation of downstream signals. Each genotypic group of MEFs exhibited distinct patterns of expression both while resting and in response to stimulation. The insulin receptor (IR) pathway in IGF-IR null MEFs was hypersensitive to insulin ligand stimulation resulting in greater AKT phosphorylation than in wt or het MEFs stimulated with the same ligand. Interestingly, the IR pathway hypersensitivity in IGF-IR null MEFs occurred with no observed changes in the levels of IR isoforms A or B. A new small molecule IGF-IR inhibitor (BMS-754807), having equipotent activity against both IGF-IR and IR, proved effective in suppressing both AKT and ERK phosphorylation from both the IGF-IR and IR pathways by all three ligands tested in wt, het, and null MEFs. The use of a dual IGF-IR/IR inhibitor addresses concerns about the use of growth inhibiting therapies directed against the IGF-IR receptor in certain cancers. Lastly, comparison of the antiproliferative effects (IC(50)s) of various compounds in wt vs. null MEFs demonstrates that genetically characterized MEFs provide a simple and inexpensive tool with which to define compounds as having mostly on-target or off-target IGF-IR activities because off-target compounds affect both wt and null MEFs equally.

IRS2 Copy Number Gain, KRAS and BRAF Mutation Status as Predictive Biomarkers for Response to the IGF-1R/IR Inhibitor BMS-754807 in Colorectal Cancer Cell Lines

Insulin-like growth factor receptor 1 (IGF-1R)–targeting therapies are currently at an important crossroad given the low clinical response rates seen in unselected patients. Predictive biomarkers for patient selection are critical for improving clinical benefit. Coupling in vitro sensitivity testing of BMS-754807, a dual IGF-1R/IR inhibitor, with genomic interrogations in 60 human colorectal cancer cell lines, we identified biomarkers correlated with response to BMS-754807. The results showed that cell lines with BRAFV600E or KRASG13D mutation were resistant, whereas cell lines with wild-type of both KRAS and BRAF were particularly sensitive to BMS-754807 if they have either higher RNA expression levels of IR-A or lower levels of IGFBP6. In addition, the cell lines with KRAS mutations, those with either insulin receptor substrate 2 (IRS2) copy number gain (CNG) or higher IGF-1R expression levels, were more sensitive to the drug. Furthermore, cell lines with IRS2 CNG had higher levels of ligand-stimulated activation of IGF-1R and AKT, suggesting that these cell lines with IGF-IR signaling pathways more actively coupled to AKT signaling are more responsive to IGF-1R/IR inhibition. IRS2 siRNA knockdown reduced IRS2 protein expression levels and decreased sensitivity to BMS-754807, providing evidence for the functional involvement of IRS2 in mediating the drug response. The prevalence of IRS2 CNG in colorectal cancer tumors as measured by qPCR-CNV is approximately 35%. In summary, we identified IRS2 CNG, IGF-1R, IR-A, and IGFBP6 RNA expression levels, and KRAS and BRAF mutational status as candidate predictive biomarkers for response to BMS-754807. This work proposed clinical development opportunities for BMS-754807 in colorectal cancer with patient selection to improve clinical benefit. Mol Cancer Ther; 14(2); 620–30. ©2014 AACR.

Abstract 363: Therapeutic targeting of insulin receptor-A with a dual IGF-1R/IR inhibitor: Overcoming a potential escape mechanism from IGF-1R-specific signaling inhibition in insulin receptor-A transfected cancer cells

Type I Insulin-like Growth Factor Receptor (IGF-1R) is a tyrosine kinase receptor that is activated by binding to IGF-1 and IGF-II ligands; IGF-1R has been shown to play a role in cancer development and progression and therapies targeting IGF-1R have resulted in clinical benefit in cancer patients. Insulin Receptor (IR) which is closely related to IGF-1R, is expressed in normal tissues and tumors, and is expressed as two isoforms, IR-A and IR-B. IR-A exhibits mitogenic activity and is preferentially found in fetal tissue and cancer cells, whereas IR-B regulates glucose metabolism in response to insulin. Both IR isoforms bind insulin, and IR-A also binds IGF-II. Several cancer cell types that express IR-A also overexpress IGF-II, suggesting a possible autocrine loop that enhances tumor survival. Since IGF-1R antibodies bind specifically to IGF-1R and not to IR, IR signaling could represent a potential escape mechanism to antibody treatment through the IR-A pathway. BMS-754807, a small molecule inhibitor with dual activity against IGF-1R and IR, recently entered clinical development. It is anticipated that BMS-754807 can prevent or overcome therapeutic resistance resulting from increased IR-activated signaling. The goal of the presented studies was to demonstrate whether dual IGF-1R/IR inhibition by BMS-754807 may prevent IR-mediated resistance to IGF-1R inhibition. To test this hypothesis, a human rhabdomyosarcoma cell line, Rh41 expressing IGF-1R, but little IR, was engineered to express either IR-A (Rh41-IR-A) or IR-B (Rh41-IR-B). IR expression was confirmed by PCR analysis of specific IR isoforms. Rh41-IR-A cells showed phosphorylation of IR after stimulation with IGF-II and/or insulin. MAB391, a neutralizing monoclonal antibody to IGF-1R, inhibited the proliferation of parental Rh41 and Rh41-IR-B cells in vitro, but Rh41-IR-A cells exhibited no sensitivity to the IGF-1R mAb. Combination treatment with both IGF-1R and IR antibodies in Rh41-IR-A cells were strongly synergistic, suggesting dual receptor blocking is required for enhanced anti-tumor efficacy. Inhibition of IGF-1R and IR with BMS-754807 showed the same inhibitory activity in Rh41-IR-A cells as was observed in the parental Rh41 cells. Therefore, dual IGF-1R/IR inhibition by BMS-754807 treatment may overcome the ability of cancer cells to utilize the IR-A pathway and provide an advantage over IGF-1R specific antibodies in the treatment of cancer. 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 363.

Abstract A153: Mechanisms of acquired resistance to an IGF‐1R inhibitor in multiple models

Background: The IGF‐1R pathway plays a major role in cancer cell proliferation, survival and resistance to anti‐cancer therapies in many human malignancies. Targeting IGF‐1R represents a promising strategy in the development of novel anti‐cancer therapeutics. BMS‐754807, a small molecule inhibitor targeting both IGF‐1R/IR, demonstrated anti‐tumor activity pre‐clinically in multiple tumor types, and is currently in clinical development. A general concern in the clinic with antitumor agents is the development of resistance. In light of this problem, we have developed and characterized cell line models with acquired resistance to BMS‐754807, and identified potential mechanisms and biomarkers for the resistance. Methods: Five cell lines of different tumor types, MCF7, Rh41, Rh1, Geo and SW480 (breast, sarcomas and colon), were induced to develop acquired resistance to BMS‐754807 (range from 5‐ to 38‐fold increased resistance) by stepwise exposure to increasing concentrations of the drug for extended periods. Analyses of in vitro and in vivo drug response, gene expression profiles, signaling pathways and gene copy numbers were performed to characterize the resistant models and the corresponding sensitive parental cells. Results: Cell line specific as well as shared molecular alterations were observed in the different resistant cells using genomic approaches to define mechanisms of resistance to BMS‐754807. The resistant models were also tested against multiple IGF‐1R inhibitors and showed cross‐resistance suggesting common mechanisms of resistance to IGF‐1R inhibition. This presentation will focus on results from the MCF7‐807R model. MCF‐807R cells showed a 5‐fold decrease in IGF‐1R and > 200‐fold increase in IGFBP3 RNA expression levels, as well as overexpression of other growth receptors, such as EGFR, with > 70‐fold increase in both protein and RNA levels compared to the sensitive parental cells. MCF7‐807R and the parental MCF7 cells had differential response to inhibitors of IGF1‐R and EGFR by preferential inhibition of pAKT and pERK. When tested in vitro, MCF7‐807R cells became more sensitive than the parental to multiple EGFR inhibitors presumably due to overexpression of EGFR and growth‐dependence on EGFR; an in vivo xenograft study showed dose‐dependent tumor growth inhibition with cetuximab treatment, whereas the growth of MCF7 parental tumor xenografts was not inhibited by cetuximab. Conclusion: EGFR plays a critical role in resistance to IGF‐1R inhibition; resistant cells and tumors utilized the EGFR pathway to escape growth inhibition. Crosstalk between IGF‐1R and EGFR can confer acquired resistance to IGF‐1R inhibition through compensatory mechanisms by the enhanced activity of the reciprocal EGFR pathway. Dual EGFR and IGFR inhibition may prevent or reverse resistance to IGFR inhibitors offering a promising strategy for exploration in clinical studies to yield greater anticancer activity. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A153.