Christine Heske

IGF-1R Inhibition Activates a YES/SFK Bypass Resistance Pathway: Rational Basis for Co-Targeting IGF-1R and Yes/SFK Kinase in Rhabdomyosarcoma

The insulin-like growth factor 1 receptor (IGF-1R) has surfaced as a significant target in multiple solid cancers due to its fundamental roles in pro-survival and anti-apoptotic signaling. However, development of resistance to IGF-1R blockade represents a significant hindrance and limits treatment efficacy in the clinic. In this study, we identified acquired resistance to IGF-1R blockade with R1507, an antibody against IGF-1R, and with BMS-754807, a small molecular inhibitor of IGF-1R/insulin receptor (IR). We showed that treatment with an IGF-IR antibody, R1507, or an IR/IGF-IR kinase inhibitor, BMS-754807, was associated with increased activation of YES/SRC family tyrosine kinase (SFK) in rhabdomyosarcoma (RMS). Combining anti–IGF-1R agents with SFK inhibitors resulted in blockade of IGF-1R inhibition–induced activation of YES/SFK and displayed advantageous antitumor activity in vitro and in vivo. Our data provide evidence that IGF-1R blockade results in activation of the YES/SRC family kinase bypass resistance pathway in vitro and in vivo. This may be of particular clinical relevance since both Yes and IGF components are overexpressed in RMS. Increased YES/SFK activation might serve as a clinical biomarker for predicting tumor resistance to IGF-1R inhibition. Dual inhibition of IGF-1R and SFK may have a broader and enhanced clinical benefit for patients with RMS.

STA-8666, a novel HSP90 inhibitor/SN-38 drug conjugate, causes complete tumor regression in preclinical mouse models of pediatric sarcoma

Long-term survival in patients with metastatic, relapsed, or recurrent Ewing sarcoma and rhabdomyosarcoma is dismal. Irinotecan, a topoisomerase 1 inhibitor, has activity in these sarcomas, but due to poor bioavailability of its active metabolite (SN-38) has had limited clinical efficacy. In this study we have evaluated the efficacy and toxicity of STA-8666, a novel drug conjugate which uses an HSP90 inhibitor to facilitate intracellular, tumor-targeted delivery of the topoisomerase 1 inhibitor SN-38, thus preferentially delivering and concentrating SN-38 within tumor tissue. We present in vivo evidence from mouse xenograft models that STA-8666 results in more persistent inhibition of topoisomerase 1 and prolonged DNA damage compared to irinotecan. This translates into superior antitumor efficacy and survival in multiple aggressive models of both diseases in mouse xenografts, as well as in an irinotecan-resistant model of pediatric osteosarcoma, demonstrated by dramatic tumor shrinkage, durable remission and prolonged complete regressions following short-term treatment, compared to conventional irinotecan. Gene expression analysis performed on xenograft tumors treated with either irinotecan or STA-8666 showed that STA-8666 affected expression of DNA damage and repair genes more robustly than irinotecan. These results suggest that STA-8666 may be a promising new agent for patients with pediatric-type sarcoma.

Matrix screen identifies synergistic combination of PARP inhibitors and nicotinamide phosphoribosyltransferase (NAMPT) inhibitors in Ewing sarcoma

Purpose: Although many cancers are showing remarkable responses to targeted therapies, pediatric sarcomas, including Ewing sarcoma, remain recalcitrant. To broaden the therapeutic landscape, we explored the in vitro response of Ewing sarcoma cell lines against a large collection of investigational and approved drugs to identify candidate combinations. Experimental Design: Drugs displaying activity as single agents were evaluated in combinatorial (matrix) format to identify highly active, synergistic drug combinations, and combinations were subsequently validated in multiple cell lines using various agents from each class. Comprehensive metabolomic and proteomic profiling was performed to better understand the mechanism underlying the synergy. Xenograft experiments were performed to determine efficacy and in vivo mechanism. Results: Several promising candidates emerged, including the combination of small-molecule PARP and nicotinamide phosphoribosyltransferase (NAMPT) inhibitors, a rational combination as NAMPTis block the rate-limiting enzyme in the production of nicotinamide adenine dinucleotide (NAD+), a necessary substrate of PARP. Mechanistic drivers of the synergistic cell killing phenotype of these combined drugs included depletion of NMN and NAD+, diminished PAR activity, increased DNA damage, and apoptosis. Combination PARPis and NAMPTis in vivo resulted in tumor regression, delayed disease progression, and increased survival. Conclusions: These studies highlight the potential of these drugs as a possible therapeutic option in treating patients with Ewing sarcoma. Clin Cancer Res; 23(23); 7301–11. ©2017 AACR.

The Role of PDGFR-β Activation in Acquired Resistance to IGF-1R Blockade in Preclinical Models of Rhabdomyosarcoma

To determine what alternative pathways may act as mechanisms of bypass resistance to type 1 insulin-like growth factor receptor (IGF-1R) blockade in rhabdomyosarcoma (RMS), we compared expression of receptor tyrosine kinase activity in a number of IGF-1R antibody-resistant and -sensitive RMS cell lines. We found that platelet-derived growth factor receptor β (PDGFR-β) activity was upregulated in three xenograft-derived IGF-1R antibody-resistant cell lines that arose from a highly sensitive fusion-positive RMS cell line (Rh41). Furthermore, we identified four additional fusion-negative RMS cell lines that similarly upregulated PDGFR-β activity when selected for IGF-1R antibody resistance in vitro. In the seven cell lines described, we observed enhanced growth inhibition when cells were treated with dual IGF-1R and PDGFR-β inhibition in vitro. In vivo studies have confirmed the enhanced effect of targeting IGF-1R and PDGFR-β in several mouse xenograft models of fusion-negative RMS. These findings suggest that PDGFR-β acts as a bypass resistance pathway to IGF-1R inhibition in a subset of RMS. Therapy co-targeting these receptors may be a promising new strategy in RMS care.

Abstract B14: Hsp90-inhibitor drug conjugate STA-12-8666 demonstrates complete tumor regression in preclinical models of pediatric sarcoma

Long-term survival in patients with metastatic relapsed or recurrent Ewing sarcoma (ES) and rhabdomyosarcoma (RMS) is dismal. Encouraging responses to irinotecan, a topoisomerase 1 (Top1) inhibitor, have been seen in these patients; however, limitations in irinotecan bioavailability, including low conversion rate to the active metabolite (SN-38) and high excretion rate of the inactive form, hinder efficacy. HSP90 is widely expressed in cancer cells, and HSP90 inhibitors (HSP90i) have favorable pharmacokinetics for anticancer use, as they remain in tumors for longer periods of time and at higher steady-state levels compared to normal tissue. This property makes them ideal intracellular delivery vehicles for chemotherapeutic drugs, allowing for high tumor exposure and low systemic toxicity. STA-12-8666 (Synta Pharmaceuticals) is an HSP90i drug conjugate (HDC) consisting of a weak HSP90i attached to SN-38 through a cleavable chemical linker. The purpose of this study was to test this HDC in xenograft models of pediatric sarcoma and to investigate its mechanism of action. To test therapeutic efficacy of this HDC, female SCID mice underwent orthotopic injection of ES or RMS cells from established cell lines or PDX tissue. When tumors reached a desired size, mice were randomized and then treated weekly with HDC, vehicle, ganetespib (a highly potent HSP90i), high dose irinotecan, protracted dose irinotecan, or irinotecan plus ganetespib. Tumors were measured twice per week, and mice were weighed weekly to determine drug tolerability. Tumors were harvested at midpoints and at study endpoint for biology studies. Activity of pharmacodynamic (PD) markers was investigated in tumor tissue. In xenograft models of ES and RMS, treatment with HDC produced superior antitumor efficacy compared to the other arms. When initial treatment began in mice with palpable tumors (between 100 and 500 mm3 (ES) or 50 and 90 mm3 (RMS)), all tumors underwent complete regression after 2 doses of HDC, with total tumor eradication in all ES mice and several RMS mice. In the RMS group, 6/8 mice relapsed by 23 weeks, and all 6 of those responded to retreatment with HDC. When initial treatment was delayed until tumors reached between 800 mm3 and 1000 mm3, complete regressions were again achieved in ES after 2 doses and RMS after 4 doses. Compared with high dose weekly irinotecan, which also induced tumor regression, mice treated with HDC had longer and more persistent remissions. A dose response effect was seen in HDC with cures noted in mice with ES at the 100- and 150-mg/kg doses and longest remissions noted in RMS in the 150-mg/kg group. Tolerability of the HDC was excellent with no toxicity-related deaths or weight loss in any treated mice. Studies using PDX models are ongoing and will be reported. Activity of γH2AX in tumor samples was explored as a PD marker of Top1 inhibitor activity. Mice bearing ES were treated with a single dose of vehicle, irinotecan, or HDC and one mouse per group was sacrificed at serial intervals between 6 hours and 10 days post-treatment. Expression of γH2AX in irinotecan mice began to wane between days 1 and 3, whereas in HDC mice, it was still detectable at day 7, suggesting that HDC results in more persistent inhibition of topoisomerase 1 compared to irinotecan. To look at the potential role of HSP90 inhibition in this HDC, HSP70 activity was investigated as a marker of HSP90 inhibition in samples from the dose finding experiment. In ES, no HSP70 was detected in samples from mice treated with irinotecan or HDC, suggesting the primary mechanism of action is via SN-38. In RMS, HSP70 was slightly induced in mice treated with higher doses of the HDC, perhaps contributing to the higher relapse rate in this model. Preclinical data suggest that this HDC may be a promising anticancer agent for ES and RMS patients. Citation Format: Christine M. Heske, Arnulfo Mendoza, Choh Yeung, David A. Proia, Len Neckers, Lee J. Helman. Hsp90-inhibitor drug conjugate STA-12-8666 demonstrates complete tumor regression in preclinical models of pediatric sarcoma. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr B14.

Abstract IA33: Combination targeted therapy in pediatric sarcomas

Our group, and many others have documented a crucial role for IGFIR signaling in several pediatric sarcomas. These studies, along with the development of fully human IGFIR antibodies led to clinical testing of these blocking antibodies in rhabdomyosarcoma, Ewing9s sarcoma, and osteosarcoma, along with a variety of other sarcomas. In general these single agent Phase 2 studies showed objective responses rates below 20%. Furthermore, even in responding patients, the duration of response is short-lived, typically less than 18 weeks. Thus the majority of patients do not have long term benefit from IGFIR blockade, indicating the presence of innate or acquired resistance to this therapeutic approach. We have been using xenografts of human rhabdomyosarcoma cell lines RD(embryonal) and RH30 (alveolar) to model both innate and acquired resistance to IGFIR blockade. Our xenograft models of rhabdomyosarcoma predicted short duration responses that mimicked the clinical observations, and we have been attempting to use these models to better understand how various signaling pathways in addition to IGFIR may interact and indeed be used as “by-pass” resistance pathways. Our hope is that this knowledge will lead to rational combination targeted therapy that we assume will be necessary for maximal clinical benefit. I will discuss one example of an additional signaling pathway that was activated upon IGFIR blockade, and how this information led to combination targeted therapy that holds therapeutic promise. We recently demonstrated that Src-Family Kinase (SFK) YES is highly expressed and functional in rhabdomyosarcomas. Since SFK signaling has been previously linked to IGF signaling in other tumor types, we evaluated the effect of IGFIR on YES activation. YES is rapidly activated upon IGFIR blockade using either a monoclonal antibody directed against the IGFIR or an IGFIR kinase inhibitor, suggesting that YES activation could play a role in resistance to IGFIR blockade. We next tested dual SFK and IGFIR inhibition and found that combined SFK and IGFIR inhibition led to more potent inhibition of both RD and RH30 rhabdomyosarcoma cell lines and perhaps most importantly led to increased apoptosis. Based on these promising in vitro effects, we evaluated the combination of IGFIR Ab, R1507 plus the SFK inhibitor, dasatinib, in our RD and RH30 xenografts. While both R1507 and dasatinib led to tumor growth inhibition as single agents, mice developed resistance to both single agents within 70-90 days of treatment. In contrast, the mice treated with the combination of R1507 and dasatinib did not develop resistance after 90 days and also showed enhanced tumor growth inhibition. This example highlights the need to better understand how signaling pathways interact so that one may predict consequences of therapeutic intervention using targeted agents and develop combinations that will undoubtedly be necessary to achieve the maximal clinical benefit of agents designed to inhibit specific signaling pathways. Citation Format: Lee J. Helman, Fernanda Arnaldez, Christine Heske, Xiaolin Wan, Choh Yeung. Combination targeted therapy in pediatric sarcomas. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr IA33.