Abstract 4438: Promoter hypermethylation status of Fanconi Anemia (FA) pathway genes FANCF, FANCL and FANCS in non-small cell lung cancer (NSCLC)

The Fanconi Anemia (FA) pathway is essential for human cells to maintain integrity following DNA damage. This pathway is involved in repairing double stranded DNA breaks. Cancers with defective FA pathway are expected to be more sensitive to cross-link based therapy, or to treatments in which additional repair mechanisms are targeted. The FA pathway contains 15 genes, and some of the members have been implicated in susceptibility to a number of cancers by genetic or epigenetic alterations. The methylation of CpG islands in the FANCF gene is thought to plays a role in the occurrence of ovarian cancer. We have recently reported the detection of up to 20% of NSCLC to be FA functionally inactive (lack of FANCD2 foci formation in the nucleus of proliferating cells by triple stain immunofluorescence, FATSI negative). Since epigenetic inactivation can be one of the mechanisms for FA functional deficiency in these tumors, we evaluated a series of NSCLC samples for FANCF methylation. Human lung tumor tissue samples were obtained from The Tissue Procurement Shared Resources of the Ohio State University Comprehensive Cancer Center after IRB approval. FA pathway status was evaluated by FATSI. Genomic DNA and total RNA samples were isolated from frozen lung tumor and matching non-tumor tissues. The methylation status of the FANCF gene promoter was evaluated using methylation-specific PCR (MS-PCR). FANCF gene expression was evaluated by NanoString assays. We screened total of 40 NSCL tumors by the FATSI assay, and the ratio of FANCD2 foci negative tumor was 20% (8/40). Squamous cell carcinoma, adenocarcinoma and large cell carcinoma histologies were all represented in the samples. FANCF promoter methylation was present in 2 of 8 FATSI negative tumors (1 adenocarcinoma and 1 large cell carcinoma) based on MS-PCR analysis, and absent in 8 tested FATSI positive tumors. NanoString analysis was performed in 5 FATSI positive and 5 FATSI negative tumors which the latter including the two tumors with FANCF methylation identified with the MS-PCR. FANCF mRNA level was 1.8-fold lower in tumors with promoter methylation as comparing to matched non-tumor tissues. One of the two tumors containing FANCF methylation was also analyzed with RNAseq, and the results showed a 1.6 fold reduction in FANCF mRNA in the tumor as comparing to matched non-tumor tissue. However the FANCF mRNA level was similar between tumor and non-tumor (tumor/non-tumor = 1.04) in the samples without methylation. The above observation suggests that epigenetic alterations can be the base for FA functional deficiency in NSCLC patients. These findings may have clinical implications, since these tumors may be more sensitive to cross-link based therapy. However, an important caveat is that these changes may not be stable and could revert during treatment. Evaluation of pre and post treatment samples in FATSI negative patients undergoing therapy would be necessary to support this hypothesis. Citation Format: Wenrui Duan, Tyler Rees, Kevin Vu, Brittany Barnwell, Li Gao, Arjun Kalvala, xin wu, Gregory A. Otterson, Miguel A. Villalona-Calero. Promoter hypermethylation and gene expression of FANCF in non-small cell lung cancer (NSCLC). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4246. doi:10.1158/1538-7445.AM2013-4246

The Fanconi Anemia (FA) pathway is essential for human cells to maintain integrity following DNA damage. This pathway is involved in the endogenous repair of double stranded DNA breaks and homologous recombination as well as repair of DNA cross-linking caused by exogenous agents. Cancers with defective FA pathway are expected to be more sensitive to cross-link based therapy, or to treatments in which additional repair mechanisms are targeted. We have recently reported the detection of 22% of NSCLC to be FA functionally inactive by Fanconi Anemia Triple Staining Immunofluorescence (FATSI) test. Studies have shown involvement of certain micro RNA (miRNA) as regulatory elements in the development of lung cancer. We set out to evaluate potential involvement of miRNAs in the regulation of the Fanconi Anemia (FA) pathway. Using Nanostring counter miRNA array we screened 734 different miRNA expression in two FA defective lung cancer cells and matched control cells along with two FA pathway deficient lung tumors and matched non-tumor lung tissue samples. Selected miRNA expression were validated with real-time PCR analysis. miRNA target gene expression was analyzed through AmpliSeq RNA gene expression analysis. Among 734 different miRNAs, a cluster of microRNAs were found to be up-regulated including an important cancer related micro RNA, miR-200C. Nanostring data showed that miR-200C was increased 7.5 fold on average in the FA defective lung cancer cells as compared to control cell. An average of 22 fold increase in miR-200C was detected in the FA defective lung tumor tissues comparing to matching non-tumor tissues. AmpliSeq analysis showed the ZEB1(zinc finger E-box binding homeobox 1) mRNA expression was down regulated in10 out 10 lung tumors (100%) comparing to non-tumor tissues, and 9 out of 10 samples (90%) showed reduction in ZEB2 expression. MiRNA-200C has been reported as a negative regulator of epithelial-mesenchymal transition (EMT) and inhibiting cell migration and invasion by promoting the upregulation of E-cadherin through targeting ZEB1 and ZEB2 transcription factors. Our findings indicate that the FA pathway regulates downstream genes through regulation of miRNAs in lung cancer. MiR-200C appears to be one of the most important FA downstream regulators in lung cancer. Validation with a larger sample size will be needed to confirm our findings. Citation Format: Wenrui Duan, Shirley Tang, Li Gao, Kathleen Dotts, Andrew Fink, Arjun Kalvala, Brittany Aguila, Miguel A. Villalona-Calero. Micro RNA-200C is one of the important Fanconi Anemia (FA) pathway downstream regulators in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1361.

Fanconi anemia (FA) is a cancer susceptibility syndrome characterized by sensitivity to DNA-damaging agents. The FA proteins (FANCs) are implicated in DNA repair, although the precise mechanisms by which FANCs process DNA lesions are not fully understood. An epistatic relationship between the FA pathway and translesion synthesis (TLS, a post-replication DNA repair mechanism) has been suggested, but the basis for cross-talk between the FA and TLS pathways is poorly understood. We show here that ectopic overexpression of the E3 ubiquitin ligase Rad18 (a central regulator of TLS) induces DNA damage-independent mono-ubiquitination of proliferating cell nuclear antigen (PCNA) (a known Rad18 substrate) and FANCD2. Conversely, DNA damage-induced mono-ubiquitination of both PCNA and FANCD2 is attenuated in Rad18-deficient cells, demonstrating that Rad18 contributes to activation of the FA pathway. WT Rad18 but not an E3 ubiquitin ligase-deficient Rad18 C28F mutant fully complements both PCNA ubiquitination and FANCD2 activation in Rad18-depleted cells. Rad18-induced mono-ubiquitination of FANCD2 is not observed in FA core complex-deficient cells, demonstrating that Rad18 E3 ligase activity alone is insufficient for FANCD2 ubiquitylation. Instead, Rad18 promotes FA core complex-dependent FANCD2 ubiquitination in a manner that is secondary to PCNA mono-ubiquitination. Taken together, these results demonstrate a novel Rad18-dependent mechanism that couples activation of the FA pathway with TLS.

The poly ADP-ribose polymerase (PARP) inhibitors are novel agents being developed with the hope of inhibiting base excision repair, a process that is of prime importance for tumors to survive genotoxic insult. BRCA gene homozygous deficiency has been identified as a potential predictor of response to PARP inhibitors. BRCA is involved in homologous recombination (HR), an example of double-strand break repair. Thus, inhibiting tumors which have lost one DNA repair pathway by targeting a second DNA repair pathway represents groundbreaking therapeutic strategy. BRCA is just one of many genes that collaborate in the same repair pathway, which has been named the Fanconi Anemia (FA) pathway. The FA pathway is a major mechanism of homologous recombination DNA repair in response to genotoxic insults. Deficiencies in FA pathway have been reported as a predictor of cisplatin and mitomycin C (MMC) sensitivity in cancer cells. Given that this pathway plays essential roles in response to the DNA damage, cancers with defective FA pathway are expected to be more sensitive to DNA cross-link based therapy, or to treatments in which additional repair mechanisms are targeted. In order to preclinically evaluate the effect of PARP inhibition in lung cancer cells with spontaneously defective FA pathway, we utilized RNAi technology to create FANCD2 knockdown cancer cells. Lung cancer cells H1299 and A549 were transduced with FANCD2-specific shRNA-expressing and puromycin-resistant lentiviral particles, or control shRNA lentiviral particles to create stably transduced cells. Successful FANCD2 knockdown was confirmed by the Western blot by reduction in the FANCD2 protein. Cell viability was evaluated with MTT (Dimethylthiazolyl-2-5-diphenyltetrazolium bromide) analysis. The FA defective H1299 and A549 lung cancer cells and the controls (transected with empty vectors) were treated with the PARP inhibitor ABT888 at a dose of 5µM. Then the treated cells were harvested at 24, 48 and 72 hours post treatment. MTT cell viability analysis showed that ABT888 alone was cytotoxic to the FA deficient lung cancer cells (H1299D2-down and A549D2-down) 72 hours post treatment. In both cell types, the FA defective lung cancer cells had less viable cells compared to controls 72 hours post treatment. Recent clinical data confirmed the PARP inhibitors could be used not only as chemosensitizers but as well as single agents to selective kill tumors with BRCA-deficient breast cancers. Disruption of FA cascade has been reported in solid tumors. Given that the FA pathway plays an essential role in response to therapy-induced DNA interstrand cross-links, it is very plausible that cancers with defective FA pathway are more sensitive to cross-link based therapy and that treatments like PARP inhibitors in which an additional repair mechanism is targeted. A clinical trial to validate this hypothesis is under way. 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 5500. doi:10.1158/1538-7445.AM2011-5500

Bcl-2 is a central apoptotic inhibitor, and overexpression is associated with tumor progression and treatment resistance in cancers. Overexpression has been reported in up to 80% of small cell lung cancers (SCLC). ABT-263 (Nativoclax) is a potent and selective inhibitor of Bcl-2 and Bcl-xL, disrupting their interactions with pro-death proteins leading to the initiation of apoptosis within 2 hours post exposure. However a recent phase II study of single-agent ABT-263 showed low rate of response to single-agent treatment in advanced and recurrent SCLC. Thus, pre-selection of patients most likely to derive benefit from BCL-2 inhibitors will be needed for further development of these agents in SCLC. The Fanconi Anemia (FA) pathway is a major mechanism of homologous recombination DNA repair in response to genotoxic insults. The repair abnormalities resulting from deficiencies in FA pathway potentially select for the persistence of prosurvival pathways. We hypothesized that cancers with defective FA pathway would be more sensitive to not only DNA interstrand crosslinking based therapy, but also to treatments in which prosurvival pathways are targeted, like BCL-2 inhibition. We utilized RNAi technology to create FANCD2 knockdown SCLC cancer cells. H719 cells were transduced with FANCD2-specific shRNA-expressing and puromycin-resistant lentiviral particles or control shRNA lentiviral particles to create stably transduced cells. Successful FANCD2 knockdown was confirmed by Western blot by reduction in the FANCD2 protein. Cell viability was evaluated with MTT (Dimethylthiazolyl-2-5-diphenyltetrazolium bromide) analysis, and apoptosis was evaluated with Western immunoblot PARP cleavage assay. The FA defective H719 small cell lung cancer cells and the control cells (transfected with empty vectors) were treated with ABT-263 at a dose of 2μM. The treated cells were then harvested at 6, 24 and 48 hours post treatment. MTT cell viability analysis showed that ABT-263 alone was cytotoxic to the FA deficient lung cancer cells with less viable cells comparing to controls 6-48 hours post treatment. In addition, Western immunoblot analysis with anti-PARP [poly (ADP-ribose) polymerase] antibody showed PARP cleavage was increased in the FA defective H719 cells as compared to control cells 6 hours post ABT-263. Disruption of FA cascade has been reported in solid tumors. Recently we have developed a FA triple-staining immunofluorescence (FATSI) method to detect FANCD2 foci formation, which is capable of evaluating the functionality of the whole pathway using formalin fixed paraffin embedded (FFPE) tumor samples and have identified up to 15% of small cell lung cancer tumor samples to be functionally deficient. Based on our preliminary studies with the H719 cells, we propose that SCLCs with defective FA pathway would be more sensitive to BCL-2 inhibitors compared to those retaining an intact repair function. Citation Format: Li Gao, Wenrui Duan, Brittany Barnwell, Arjun Kalvala, Gregory A. Otterson, Miguel A. Villalona-Calero. Sensitivity of small cell lung cancer cells with defective Fanconi Anemia (FA) pathway to BCL2 inhibitors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4365. doi:10.1158/1538-7445.AM2013-4365

Upregulation of Fanconi Anemia DNA Repair Genes in Melanoma Compared with Non-Melanoma Skin Cancer

The Fanconi Anemia (FA) pathway is essential for human cells to maintain integrity following DNA damage. This pathway is involved in the endogenous repair of double stranded DNA breaks and homologous recombination as well as repair of DNA cross-linking caused by exogenous agents. Cancers with defective FA pathway are expected to be more sensitive to cross-link based therapy, or to treatments in which additional repair mechanisms are targeted. We have recently reported the detection of up to 20% of NSCLC to be FA functionally inactive (lack of FANCD2 foci formation in the nucleus of proliferating cells by triple stain immunofluorescence, FATSI negative). MicroRNAs (miRs, miRNA) are small, 19-25 nucleotide, single stranded, non-coding RNA molecules that have regulatory roles in humans by targeting mRNAs via cleaved or translational repression which can affect the output of protein coding genes and cause various changes in developmental and physiological processes including cancer. It has been published that some miRNAs are tumor suppressors, and others are oncogenes. However, microRNAs and the FA pathway in relationship to lung cancer progression remain unknown. In order to elucidate the role of FA related miRs in human lung cancer development or treatment, we analyzed miR expression in lung cancer cells and patient lung tumors. Several cell lines were generated using RNAi technology with FANCD2 deficiency and confirmed via reduction in FANCD2 protein expression. Using Nanostring counter technology, microRNA array analysis was performed on FANCD2 foci positive and foci negative tumors as well as matching normal lung tissues. A cluster of microRNAs (mir-431, 377, 376c, 200c, 26a, 135a, and let-7e) were found to be up-regulated, and miR-494,205, 630, 145,671 were found to be down-regulated in the foci negative tumors samples compared to matching non-tumor lung tissue. The same group of miRs were upregulated and its corresponding group were down-regulated in the foci negative tumors as compared to foci positive tumors. These findings indicate that the FA pathway may regulate downstream genes through regulation of microRNAs in lung cancer. In order to validate this hypothesis, specific miRs were be analyzed with Real-Time Quantitative PCR via foci positive and foci negative samples (cell and patient tissue) in hopes of finding significant and consistent regulation. This study contributes to further understanding in miRNA and cancer pathology that may be applicable to futuristic lung cancer treatment. Citation Format: Shirley Tang, Li Gao, Kathleen Kathleen, Andrew Fink, Arjun Kalvala, Brittany Aguila, Gregory Otterson, Miguel Villalona-Calero, Wenrui Duan. micoRNA in FA defective tumor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4442. doi:10.1158/1538-7445.AM2017-4442

BRCA 1/2 dysfunction sensitizes cells to inhibition of PARP enzymatic activity, due to the persistence of DNA breaks normally repaired by homologous recombination (HR), thus cancer pts carriers of germ line BRCA deleterious mutations have been targeted for treatment with PARP inhibitors. An example of HR repair is the FA pathway where BRCA 1/2 collaborate with several other genes resulting in FancI and FancD2 activation and nucleus repair foci formation. We have developed a triple stain immunofluorescence based method to evaluate FA pathway functionality (foci formation) (FancD2/DAPI/Ki67) (FATSI) in paraffin embedded tumor tissues, and hypothesized that among patients with sporadic tumors, a significant proportion with tumoral dysfunctional FA pathway can be identified. A two steps clinical trial (NCT01017640) was designed to 1- screen solid tumor patients for absence of FA tumor foci formation, and 2- evaluate safety and recommended doses in these pts of the PARPi veliparib, either alone or in combination with chemotherapy (MMC). 607 pts were consented, 532 had their tumor tissue screened, 142 (27%) had absent foci. All major solid tumors were represented in FA dysfunction, notably 36 of 105 (34%) colorectal and 40 of 135 (30%) breast cancers. 52 pts received 192 cycles of veliparib, 29 pts as monotherapy in escalating dose cohorts (starting at 50 mg PO BID continuously), and 23 following MMC 10 mg-sqm every 4 weeks (40mg-sqm cumulative cap) at 50 mg BID at increased duration cohorts (1, 2 and 3 wks) every 4 wks (100 and 200 mg BID for 3 wks in subsequent cohorts). Recommended dose levels are: veliparib 300 mg BID as monotherapy (2DLT/3 pts at 400 BID:G3 fatigue) and MMC/veliparib 10mg-sqm/200 mg BID three weeks duration. Analysis for germ line mutations (BRCA1 and 2 sequencing and for the 5 most common BRCA1 large rearrangements) in PBMC among 50 pts showed 7 pts (3 breast, 1 ampullary, 2 ovarian) with BRCA abnormalities: 6 known deleterious mutations and 1 polymorphism. Five RECIST criteria responses occurred (1CR, 4 PR) (1 lung, 2 breast, 1 ovarian, 1 endometrial), all in the combination arm. Two breast cancer pts (39 and 13 cycles) and 1 ovarian (6 cycles) on monotherapy, plus 1 breast (15 cycles), 1 colon (7 cycles) on the combination, had prolonged stability. rH2AX (n=49) and PAR (n=17) analysis in PBMC suggests veliparib dose dependency (blunting of rH2AX induction, lower PAR level and slower recovery for higher doses). The trial was amended to enroll an expanded cohort of 10 FATSI negative colorectal patients who will undergo fresh biopsy for whole exome sequencing (RNAseq) and FA/DNA repair gene panel analysis prior to treatment at the recommended dose in the combination arm, with the goal of identifying the specific genetic defect mediating the FA dysfunction, as well as partner mutations to be targeted in subsequent trials. Supported by R01CA152101 & N01CM62207 Citation Format: Miguel A. Villalona-Calero, Wenrui Duan, Weiqiang Zhao, Konstantin Shilo, Jiuping Ji, Jennifer Thurmond, Adam Norris, Jeffrey Rose, Rachel Layman, John Marshall, Tanios Bekaii-Saab, Alice Chen. Phase I trial of veliparib or mitomycin (MMC) + veliparib in patients with sporadic solid tumors screened for somatic deficiency in the Fanconi Anemia (FA) pathway. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-139. doi:10.1158/1538-7445.AM2013-LB-139

The Fanconi Anemia (FA) pathway is a major mechanism of homologous recombination DNA repair in response to genotoxic insults. Lack of formation of FANCD2 foci has been reported as a predictor of cisplatin and mitomycin C (MMC) sensitivity in cancer cells. Given that this pathway plays essential roles in response to the DNA damage, cancers with defective FA pathway are expected to be more sensitive to DNA cross-link based therapy, or to treatments in which additional repair mechanisms are targeted. We have developed a test to identify tumors with defective FA pathway using paraffin embedded tissues. Paraffin embedded lung primary cancer specimens were subjected to immunofluorescence microscopy (IF) analysis to evaluate the status of FancD2 subnuclear foci formation, Ki67 protein expression and DAPI staining of nuclei simultaneously in order to eliminate false negative tumor specimens due to absence of tumor cells or low proliferation. Additional validation using FA defective PD220 and PD20 cell-lines as well as fresh tumor tissues was done; IF analysis and Western blotting analysis of FDACD2 protein mono-ubiqutination were performed. The ratio of FANCD2 foci negative tumor was 20% among 20 paraffin embedded non-small cell lung cancer samples analyzed. Histological characteristics of the defective tumors include large cell carcinoma and adenocarcinoma. The proposed immunofluorecence staining method evaluates functional status of FA pathway using paraffin embedded tissues even after long term storage. Given the clinical availability of agents targeting alternative mechanisms of repair, the status of FA pathway may be an important biomarker for personalized therapeutic treatment in patients with lung cancers. A clinical trial to validate this hypothesis is under way. 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 3750.

There is a growing appreciation that defects in homologous recombination repair may increase sensitivity of tumors to certain DNA-damaging agents, plausibly through a synthetic lethal interaction. The Fanconi Anemia (FA) pathway is a major mechanism of homologous recombination (HR) DNA repair. Deficiencies in FA pathway have been reported as a predictor of cisplatin, mitomycin C (MMC) or PARP inhibitor sensitivity in cancer cells. The novel PARP inhibitor BMN-673 has emerged from preclinical studies as a best in class PARP trapping agent. BMN-673 has also demonstrated single-agent cytotoxicity in BRCA mutant cells, and activity in cancer patients with BRCA germ line deficiency. To evaluate novel targeted agents in the background of FA deficiency we utilized RNAi technology to generate several lung cancer cell lines with FANCD2 deficiency. Successful FANCD2 knockdown was confirmed by reduction in the FANCD2 protein. Cell viability was evaluated with MTT assay. We treated the FA defective H1299D2-down and A549D2-down non-small cell lung cancer cells and their FA competent counterparts H1299E and A549E (empty vector controls) with the PARP inhibitors veliparib (ABT-888, 5μM) and BMN673 (0.5μM), as well as with the CHK1 inhibitor Arry-575 at a dose of 0.5μM. We also treated the BCL2 expressing small cell lung cancer cells H719D2-down, H792D2-down and their controls H719E and H792E with the BCL2/XL inhibitor navitoclax (ABT263) at a dose of 2μM. The treated cells were harvested at 24, 48 and 72 hours (h) post treatment. Cell viability analysis showed that H1299D2-down cells had 80% of viable cells compared to 100% viable cells in H1299E controls 72h post treatment with veliparib. The A549D2-down cells had 68% viable cells compared to 83% viable cells in the A549E cells 72 h post veliparib treatment. FA defective cells were also more sensitive to treatment of BMN-673 (25% for H1299D2-down vs 62% for H1299E; 29% for A549D2-down vs 46% for A549E) 72 h post BMN-673 treatment at dose of 0.5μM. BMN-673 was more potent compared to veliparib. FA defective cells were also more sensitive to the treatment of CHK1 or BCL2/XL inhibition. H1299D2-down cells had 38% of viable cells comparing to 60% viable cells in the H1299E cells post treatment of CHK1 inhibitor Arry-575 at a dose of 0.5 μM. In addition, MTT analysis showed that BCL2/XL inhibitor navitoclax was more cytotoxic to the H719D2-down (51%) as compared to H719E (85%) 48 h post treatment. Similarly, the H792D2-down cells were more sensitive to the treatment of navitoclax (58% viable cell) as compared to H792E cells (86% viable cell) 48 h post treatment at dose of 2μM. Given that FA pathway plays essential roles in response to DNA damage, our results suggest that a subset of lung cancer patients are likely to be more susceptible to treatments in which additional pathways (e.g PARP, CHK1 and BCL2/XL) are targeted. Clinical trials to evaluate this therapeutic concept are needed. Citation Format: Li Gao, Wenrui Duan, Kathleen Dotts, Arjun Kalvala, Brittany Aguila, Gregory A. Otterson, Miguel A. Villalona-Calero. Inhibition of pro-survival pathways in lung cancer cells with functional defects in the Fanconi Anemia pathway. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 559. doi:10.1158/1538-7445.AM2015-559

10509 Loss of the fanconi anemia (FA) pathway function has been described in a number of sporadic tumor types including breast, ovarian, pancreatic, head and neck and hematological malignancies. Functionally, the FA pathway responds to stalled DNA replication following DNA damage. Given the importance of the FA pathway in the response to DNA damage, we hypothesized that cells deficient in this pathway may become hyper-dependent on alternative DNA damage response pathways in order to respond to endogenous genotoxic stress such as occurs during metabolism. Therefore, targeting these alternative pathways could offer therapeutic strategies in FA pathway deficient tumors. To identify new therapeutic targets we treated FA pathway competent and deficient cells with a DNA damage response siRNA library, that individually knocked out 230 genes. We identified a number of gene targets that were specifically toxic to FA pathway deficient cells, amongst which was the DNA damage response kinase Ataxia Telangiectasia Mutated (ATM). To test the requirement for ATM in FA pathway deficient cells, we interbred Fancg ± Atm± mice. Consistent with the siRNA screen result, Fancg-/- Atm-/- mice were non viable and Fancg± Atm-/- and Fancg-/- Atm ± progeny were less frequent that would have been expected. Several human cell lines with FA gene mutations were observed to have constitutive activation of ATM which was markedly reduced on correction with the appropriate wild-type FA gene. Interestingly, FA pathway deficient cells, including the FANCC mutant and FANCG mutant pancreatic cancer cell lines, were selectively sensitive to monotherapy with the ATM inhibitor KU55933, as measured by dose inhibition and colony count assays. FA pathway deficient cells also demonstrated an increased level of chromosomal breakage, cell cycle arrest and apoptosis following KU55933 treatment when compared to FA pathway corrected cells. We conclude that FA pathway deficient cells have an increased requirement for ATM activation in order to respond to sporadic DNA damage. This offers the possibility that monotherapy with ATM inhibitors could be a therapeutic strategy for tumors that are deficient for the FA pathway. No significant financial relationships to disclose.

Some of the restarting events of stalled replication forks lead to sister chromatid exchange (SCE) as a result of homologous recombination (HR) repair with crossing over. The rate of SCE is elevated by the loss of BLM helicase or by a defect in translesion synthesis (TLS). We found that spontaneous SCE levels were elevated approximately 2-fold in chicken DT40 cells deficient in Fanconi anemia (FA) gene FANCC. To investigate the mechanism of the elevated SCE, we deleted FANCC in cells lacking Rad51 paralog XRCC3, TLS factor RAD18, or BLM. The increased SCE in fancc cells required Xrcc3, whereas the fancc/rad18 double mutant exhibited higher SCE than either single mutant. Unexpectedly, SCE in the fancc/blm mutant was similar to that in blm cells, indicating functional linkage between FANCC and BLM. Furthermore, MMC-induced formation of GFP-BLM nuclear foci was severely compromised in both human and chicken fancc or fancd2 cells. Our cell survival data suggest that the FA proteins serve to facilitate HR, but not global TLS, during crosslink repair.

The Fanconi anemia (FA) pathway is essential for the repair of DNA interstrand cross-links. At the heart of this pathway is the monoubiquitination of the FANCI-FANCD2 (ID) complex by the multiprotein “core complex” containing the E3 ubiquitin ligase FANCL. Vertebrate organisms have the eight-protein core complex, whereas invertebrates apparently do not. We report here the structure of the central domain of human FANCL in comparison with the recently solved Drosophila melanogaster FANCL. Our data represent the first structural detail into the catalytic core of the human system and reveal that the central fold of FANCL is conserved between species. However, there are macromolecular differences between the FANCL proteins that may account for the apparent distinctions in core complex requirements between the vertebrate and invertebrate FA pathways. In addition, we characterize the binding of human FANCL with its partners, Ube2t, FANCD2, and FANCI. Mutational analysis reveals which residues are required for substrate binding, and we also show the domain required for E2 binding.

Purpose: To characterize the prevalence of germline pathogenic or likely pathogenic (PLP) variants in Fanconi Anemia (FA) pathway genes among patients with invasive breast cancer (IBC) and evaluate opportunities for clinical actionability through germline and somatic analysis. Background: Disruption of the FA DNA repair pathway can impair homologous recombination (HR). While high-penetrance PLP variants in BRCA1, BRCA2, and PALB2 are established breast cancer susceptibility genes and contribute to HR deficiency (HRD), the clinical consequences of other FA germline PLP variants remain unclear. Using the updated cohort from the City of Hope INSPIRE Study, we assessed the prevalence and functional impact of germline PLP variants in 17 FA pathway genes. HRD, mutational signatures, and loss of heterozygosity were used to evaluate effects on HR integrity. These findings may have implications for HRD-related precision therapeutic intervention and clinical trial eligibility. Methods: This retrospective study included consented patients with histologically confirmed IBC enrolled in the INSPIRE Study as of January 14, 2025. Germline sequencing of 155 cancer predisposition genes was performed; patients with multiple FA variants were classified according to the FA gene of the highest penetrance. A cohort subset underwent paired tumor-normal whole exome sequencing (>400x tumor, >180x normal), including HRD scoring (scarHRD), LOH determination, and SBS3 assessment (SigProfiler). Clinical data were obtained from cancer registries and medical records. Clinical trial eligibility criteria were evaluated using ClinicalTrials.gov. Results: Of 8,242 patients with IBC, 7,982 underwent germline sequencing; 592 (7.4%) had a PLP variant in an FA gene, including 197 (33.3%) in non-high-penetrance (non-HP) genes. Among 1,833 patients with paired tumor-normal sequencing, 145 (7.9%) had an FA PLP variant, of which 53 (2.89%) were in non-HP genes. HRD scores were ≥42 in 45.3% of non-HP carriers (Table 1). 24 of 53 patients (45.3%) with a non-HP FA germline PLP variant had HRD-positive (≥42) tumors. HRD-positivity is more likely among patients with germline PLP variants in non-HP FA pathway genes compared to those without a variant in any FA gene (RR 1.51, 95% CI 1.12 to 2.05; p < 0.0077). Stage data were available for 1,548 patients; 128 (8.3%) were stage IV at diagnosis, including 7/128 (5.5%) with non-HP FA gene PLP variants. Conclusion: Germline PLPs in non-HP FA genes are present in a meaningful subset of patients with IBC and may be functionally relevant. These findings support the value of expanded germline testing and trial eligibility criteria that encompass the broader FA pathway and suggest that non-HP FA gene variants may serve as biomarkers for identifying patients likely to benefit from HRD-targeted therapies. Citation Format: L. Naghi, K. G. Roth, S. Lindsey, A. Seuylemezian, J. Bonner, L. Kruper, S. B. Gruber, J. Mortimer, K. McDonnell. Lower Penetrance Germline Fanconi Anemia Variants in Breast Cancer: Opportunities for Expanded Genetic Testing and Therapeutic Targeting in the Updated INSPIRE Study Cohort [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS4-01-27.

Fanconi Anemia (FA) pathway resolves DNA interstrand cross links (ICL). The FA pathway was initially recognized in vertebrates, but was later confirmed in other animals and speculated in fungi. FA proteins FANCM, FANCL and FANCJ are present in Saccharomyces cerevisiae but, their mechanism of interaction to resolve ICL is still unclear. Unlike Dikarya, early diverging fungi (EDF) possess more traits shared with animals. We traced the evolutionary history of the FA pathway across Opisthokonta. We scanned complete proteomes for FA-related homologs to establish their taxonomic distribution and analyzed their phylogenetic trees. We checked transcription profiles of FA genes to test if they respond to environmental conditions and their genomic localizations for potential co-localization. We identified fungal homologs of the activation and ID complexes, 5 out of 8 core proteins, all of the endonucleases, and deubiquitination proteins. All fungi lack FANCC, FANCF and FANCG proteins responsible for post-replication repair and chromosome stability in animals. The observed taxonomic distribution can be attributed to a gradual degradation of the FA pathway from EDF to Dikarya. One of the key differences is that EDF have the ID complex recruiting endonucleases to the site of ICL. Moreover, 21 out of 32 identified FA genes are upregulated in response to different growth conditions. Several FA genes are co-localized in fungal genomes which also could facilitate co-expression. Our results indicate that a minimal FA pathway might still be functional in Mucoromycota with a gradual loss of components in Dikarya ancestors.