Abstract 1741: Inhibition of type 1 insulin-like growth factor receptor (IGF-1R) influences processing of replication-associated DNA double-strand breaks (DSBs) and induces schedule-dependent sensitization of human melanoma to temozolomide (TMZ)
Authors: Ramcharan Roger N. , Aleksic Tamara , Gao Shan , Tanner Jordan , Darvill Nicholas , Bridges Esther , Asher Ruth , Watson Amanda J. , Margison Geoffrey P. , Repapi Emmanouela , Li Ji-Liang , Middleton Mark R. , Macaulay Valentine M. 
Published: May 2015

Abstract
IGF-1R is frequently up-regulated in cancers including melanoma, and is implicated in mediating resistance to chemotherapy and targeted agents via regulation of apoptosis. Here, we investigated responses to temozolomide (TMZ), a methylating agent that generates toxic O6-methylguanine (O6meG) adducts, which are removed by O6-methylguanine-DNA methyltransferase (MGMT). Persistent O6meG adducts trigger futile cycles of mismatch repair, ultimately leading to formation of replication-associated DSBs. In a first approach to test links between IGF-1R and response to TMZ, we used a panel of 10 human melanoma cell lines in which we characterized MGMT expression, activation and expression of IGF axis components, and growth inhibitory effects of TMZ. We found that TMZ GI50 values correlated with MGMT levels (r=0.79, p=0.009), and in 7 MGMT-proficient cell lines, with phospho-IGF-1R (r=0.81, p=0.038) suggesting a link between TMZ resistance and IGF-1R activation. In a second approach we used IGF-1R tyrosine kinase inhibitors (TKIs) AZ12253801 and linsitinib (OSI-906), which is currently undergoing clinical evaluation, to assess effects of blocking IGF signaling. These IGF-1R TKIs were capable of sensitizing wild-type and mutant BRAF melanoma cells to TMZ; lack of correlation with apoptosis induction suggested that other factors contributed to this effect. IGF-1R inhibition did not influence MGMT expression or activity, was not epistatic with MGMT inhibition, and TMZ-sensitized MGMT-null cells, suggesting that chemo-sensitization was MGMT-independent. We found that IGF-1R TKI sensitized melanoma cells to inhibition of poly(ADP ribose) polymerase (PARP), and influenced processing of TMZ-induced DSBs, with increased RPA focus formation within 24hr of TMZ application, and persistence of RAD51 foci at 72hr. These data suggest functional impairment of homologous recombination (HR)-mediated DSB repair. Compared with simultaneous or prior IGF-1R TKI, TMZ sensitization was greater when IGF-1R TKI followed TMZ, consistent with a model in which IGF-1R inhibitor pre-treatment reduces DSB yield. In mice bearing melanoma xenografts with a clinically relevant (V600E BRAF, wild-type p53) genotype, this sequential (TMZ→linsitinib) combination treatment was tolerable, and linsitinib achieved peak plasma levels comparable to clinical Cmax values. TMZ and linsitinib caused minor inhibition of tumor growth (gradient reduction of 13% and 25% respectively), while the combination caused supra-additive growth delay (72%) that was significantly different from control (p&lt;0.01), TMZ (p&lt;0.01) and linsitinib (p&lt;0.05) groups. In summary, IGF-1R inhibition influences HR repair of TMZ-induced DSBs and sensitizes melanomas to TMZ using treatment schedules relevant to clinical practice.
Citation Format: Roger N. Ramcharan, Tamara Aleksic, Shan Gao, Jordan Tanner, Nicholas Darvill, Esther Bridges, Ruth Asher, Amanda J. Watson, Geoffrey P. Margison, Emmanouela Repapi, Ji-Liang Li, Mark R. Middleton, Valentine M. Macaulay. Inhibition of type 1 insulin-like growth factor receptor (IGF-1R) influences processing of replication-associated DNA double-strand breaks (DSBs) and induces schedule-dependent sensitization of human melanoma to temozolomide (TMZ). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1741. doi:10.1158/1538-7445.AM2014-1741