ALBERT

Description: Abstract 1095 Poster Board I-117 Introduction Fanconi anemia (FA) is a rare genetic disorder characterized by bone marrow failure, congenital abnormalities, and an increased risk for cancer and leukemia. Components of the FA-BRCA pathway are thought to function in the repair of DNA interstrand crosslinks (ICLs). Central to this pathway is the monoubiquitylation and chromatin localization of two FA proteins, FANCD2 and FANCI. Recent reports implicate mismatch repair factors in the repair of ICLs and have shown that FANCJ interacts with the MutLαa complex. Here we show that FANCD2 binds several mismatch repair proteins in vivo and that MSH2 is required for the monoubiquitylation and chromatin localization of both FANCD2 and FANCI. Methods Cell lines used: HeLa, human lung carcinoma cell line H1299, FA-A cell line GM6914 and corrected cell line GM6914 + Flag-FANCA, FA-D2 cell line PD20 and corrected cell lines PD20+Flag-FANCD2 and PD20+FANCD2 K561R, human endometrial adenocarcinoma cell line HEC59 (MSH2-deficient) and corrected cell line HEC59+Ch2, and human colon carcinoma cell line HCT116 (MLH1-deficient) and corrected cell line HCT116+Ch3. Cells were treated with the crosslinking agent mitomycin C (MMC). Immunoprecipitation was used to demonstrate the interaction between FANCD2 and MSH2, MLH1, and MSH3. Survival assays were performed by crystal violet staining and extraction. Chromatin loading of FANCD2 and FANCI was determined by cellular fractionation and western blot. Results Through chromatographic purification of FANCD2-containing protein complexes, we identified MSH2 and MLH1 as FANCD2-interacting proteins. Immunoprecipitation using HeLa cell extracts confirmed the interaction between FANCD2, MSH2, MSH3, and MLH1 in vivo. These interactions are all induced upon damage with a DNA crosslinking agent and MSH2 specifically interacts only with the monoubiquitylated form of FANCD2. Additionally, the FANCD2-MSH2 interaction requires ATR, but not ATM, BRCA1, MSH3, or ERCC1/XPF. Human cells lacking MSH2 display increased sensitivity to mitomycin C as compared to their corrected counterparts. FANCD2 and FANCI monoubiquitylation is also greatly diminished in these cells, while cells lacking MLH1 show no effect. Cellular fractionation of MSH2-deficient cells shows that FANCD2 and FANCI are not efficiently loaded onto chromatin after treatment with DNA-damaging agents, while MLH1-deficient cells again show no effect. Interestingly, while knockdown of either MSH2 or FANCD2 in H1299 cells results in increased sensitivity to MMC, double knockdown of both proteins corrects this sensitivity on par with controls. oConclusions These data suggest that mismatch repair proteins play a key role in the activation of the FA-BRCA pathway, likely through recognition of the DNA lesion. Understanding this role could lead to the development of new therapies for the treatment of patients both with FA and cancer. Disclosures No relevant conflicts of interest to declare.

Description: Fanconi anemia (FA) is a rare genetic disorder characterized by bone marrow failure, congenital abnormalities, and an increased risk for cancer and leukemia. Components of the FA-BRCA pathway are thought to function in the repair of DNA interstrand cross-links. Central to this pathway is the monoubiquitylation and chromatin localization of 2 FA proteins, FA complementation group D2 (FANCD2) and FANCI. In the present study, we show that RAD18 binds FANCD2 and is required for efficient monoubiquitylation and chromatin localization of both FANCD2 and FANCI. Human RAD18-knockout cells display increased sensitivity to mitomycin C and a delay in FANCD2 foci formation compared with their wild-type counterparts. In addition, RAD18-knockout cells display a unique lack of FANCD2 and FANCI localization to chromatin in exponentially growing cells. FANCD2 ubiquitylation is normal in cells containing a ubiquitylation-resistant form of proliferating cell nuclear antigen, and chromatin loading of FA core complex proteins appears normal in RAD18-knockout cells. Mutation of the RING domain of RAD18 ablates the interaction with and chromatin loading of FANCD2. These data suggest a key role for the E3 ligase activity of RAD18 in the recruitment of FANCD2 and FANCI to chromatin and the events leading to their ubiquitylation during S phase.

Description: Abstract 3370 Introduction: Fanconi anemia (FA) is a rare genetic disorder characterized by bone marrow failure, an increased risk for cancer and leukemia, and congenital abnormalities. Thirteen FA genes have been identified that when mutated result in hypersensitivity to DNA crosslinking agents. Therefore, components of the FA-BRCA pathway are thought to function in the repair of DNA interstrand crosslinks (ICLs). The monoubiquitylation and chromatin localization of two FA proteins, FANCD2 and FANCI, is considered the hallmark of FA pathway activation. It has been reported that FANCJ interacts with the mismatch repair (MMR) complex MutLα and we have previously shown that FANCD2 binds several mismatch repair proteins in vivo and that MSH2 is required for the monoubiquitylation of FANCD2 and FANCI. Interestingly, mismatch repair deficiency is also associated with leukemia in humans and a defect in hematopoietic repopulation in mouse models, suggesting a possible functional overlap between the FA-BRCA and MMR pathways. Methods: Cell lines used: HeLa, FA-A cell line GM6914 and corrected cell line GM6914 + Flag-FANCA, FA-D2 cell line PD20 and corrected cell lines PD20+Flag-FANCD2 and PD20+FANCD2 K561R, human endometrial adenocarcinoma cell line HEC59 (MSH2-deficient) and corrected cell line HEC59+Ch2, and human colon carcinoma cell line HCT116 (MLH1-deficient) and corrected cell line HCT116+Ch3. Cells were treated with the crosslinking agents mitomycin C (MMC), cisplatin (CDDP), or the alkylating agent temozolomide (TMZ). FANCD2 foci formation was assessed using immunofluorescence. Immunoprecipitation was used to demonstrate interactions between FANCD2, MSH2, and MLH1. Survival assays were performed by crystal violet staining and extraction. Chromosome breakage analysis was performed using metaphase spreads. FANCD2 RNAi flies, spel1-/- flies (MSH2-deficient), and double mutants were treated with diepoxybutane (DEB) and assessed for survival and mutagenicity. Mismatch binding EMSAs were performed using Cy5-labeled matched and mismatched 29-mers. Result: FANCD2 foci formation and chromatin loading is greatly diminished in MSH2-deficient cells, while cells lacking MLH1 show no effect, indicating a requirement for MSH2 in the activation of the FA-BRCA pathway and a possible downstream role for MLH1 in ICL repair. Human and mouse cells lacking MSH2 or human cells lacking MLH1 display increased sensitivity to mitomycin C and cisplatin as compared to their corrected counterparts as well as increased radial formation upon treatment with MMC. Studies in both human cell lines and Drosophila mutants indicate an epistatic relationship between FANCD2 and MSH2 with regards to survival, chromosome breakage, and mutagenicity after treatment with crosslinking agents. MSH2 is required for the interaction between FANCD2 and MLH1, but surprisingly, presence of MLH1 appears to enhance the interaction between FANCD2 and MSH2. Furthermore, the interaction between MSH2 and MLH1 after treatment with MMC is reduced in multiple FA cell lines. FA cell lines also display hypersensitivity to alkylating agents such as temozolomide. These results have led us to examine FA cell lines for a defect in mismatch repair through mismatch binding EMSAs and plasmid-based assays. Conclusion: These data suggest that mismatch repair proteins play a key role in the activation of the FA-BRCA pathway. Conversely, FA proteins appear to be required for interactions between mismatch repair factors and may also be involved in the repair of DNA mismatches, suggesting significant crosstalk between the FA and MMR pathways. Understanding this complex interplay could lead to the development of new therapies for the treatment of patients both with FA and cancer. Disclosures: No relevant conflicts of interest to declare.