Abstract 5504: FANCF over expression correlates with selected resistance to topoisomerase II inhibitors independent of other FA/BRCA DNA repair core complex constituents

Abstract

Abstract The Fanconi Anemia (FA)/BRCA DNA repair pathway is integral in repair of the interstrand crosslinks that accumulate during replication and in response to DNA damaging agents. We recently demonstrated that the constitutive over expression of the FA/BRCA pathway contributes to selected melphalan resistance in two isogenic myeloma cell line models.(Yarde et al 2009) These proteins comprise a co-dependent DNA damage response pathway involving thirteen loss of function complementation groups. The key functional event of this pathway is the interdependent mono-ubiquitination (Ub) of FANCD2 and FANCI (ID complex) by the E3 Ub-ligase activity of the FA core complex, a multimer consisting of 8 FA members (FANCA, B, C, E, F, G, L and M). Mono-Ub FANCD2 and FANCI, in turn, facilitate DNA repair by homologus recombination and translesion synthesis. In this study, we screened the 8226/Dox40 and 8226/MR20 cell lines as models of doxorubicin and mitoxantrone resistance for expression of the 12 FA/BRCA pathway members with quantitative PCR (qPCR). QPCR demonstrated a 2.6 (p<0.05) and 1.7 (p<0.05, respectively) fold over expression of FANCF mRNA relative to drug sensitive RPMI8226 cells. Interestingly, elevated FANCF mRNA expression was independent of other core complex constituents (e.g. Dox40: FANCA: 0.90, B: 0.90, C: 0.89, E: 1.1, G 1.1, and L: 1.4-fold RMPI8226 (p>0.05)). To further characterize the FANCF in doxorubicin resistance, we examined mRNA and protein expression of FANCF in RPMI8226, 8226/Dox6 and 8226/Dox40 MM cell lines (representing progressive levels of doxorubicin resistance). FANCF qPCR demonstrated a 2 and 4.7 fold increased in mRNA expression in the 8226/Dox6 and 8226/Dox40 cell lines, respectively (p = 0.103 and p = 0.034). Western blot analysis demonstrated a parallel increase in protein expression. These observations suggest that FANCF may contribute to topoisomerase II inhibitor-mediated DNA double strand break repair, a process that primarily involves non-homologous end joining (NHEJ) independent of the FA/BRCA pathway. To determine if FANCF expression alone may facilitate doxorubicin resistance, pQCXIP-control or pQCXIP-FANCF constructs were expressed in RPMI8226 sensitive MM cells. MTT assays demonstrated a greater than 2 fold resistance to doxorubicin in FANCF over expressing cells at 48 and 96 hours (IC50: 3.26×10−6 and 1.13×10−8M) as compared to control cells (1.33 x10−6 and 5.3×10−9M). These results suggest that FANCF may participate in doxorubicin resistance independently of other FA/BRCA members. To better characterize the role of FANCF and the FA/BRCA pathway in doxorubicin resistance we plan to target FANCF and other FA proteins using lentivirus shRNA and evaluate doxorubicin-mediated FANCD2-Ub, foci formation, DNA damage and repair in sensitive, 8226/Dox6 and Dox40 cells. 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 5504. doi:10.1158/1538-7445.AM2011-5504