Investigating Tumor Suppressors in the DNA Damage Response: Caretakers of the Genome and Biomarkers to Predict Therapeutic Response: A Dissertation

Our genome is constantly challenged by sources that cause DNA damage. To repair DNA damage and maintain genomic stability eukaryotes have evolved a complex network of pathways termed the DNA damage response (DDR). The DDR consists of signal transduction pathways that sense DNA damage and mediate tightly coordinated reactions to halt the cell cycle and repair DNA with a collection of different enzymes. In this manner, the DDR protects the genome by preventing the accumulation of mutations and DNA aberrations that promote cellular transformation and cancer development. Loss of function mutations in DDR genes and genomic instability occur frequently in many tumor types and underlie numerous cancer-prone hereditary syndromes such as Fanconi Anemia (FA). My thesis research applies candidate-based and unbiased experimental approaches to investigate the role of several tumor suppressor genes (TSGs) in the DDR. My dissertation will first describe a novel function for the breast and ovarian cancer tumor suppressor and FA-associated gene FANCJ in the DDR to ultraviolet (UV) irradiation. In response to UV irradiation FANCJ supports checkpoint induction, the arrest of DNA synthesis, and suppresses UV induced point mutations. Suggesting that FANCJ could suppress UV induced cancers, in sequenced melanomas from multiple databases I found somatic mutations in FANCJ previously associated with breast/ovarian cancer and FA syndrome. The second part of my dissertation will describe an RNA interference screen to identify genes modulating cellular sensitivity to the chemotherapeutic drug cisplatin. The hereditary breast/ovarian cancer tumor suppressor BRCA2 is essential for DNA repair, thus BRCA2 mutant ovarian cancer cells are initially sensitive to cisplatin chemotherapy that induces DNA damage. However, drug resistance develops and remains a major problem in the clinic. My screen identified the chromatin remodeling factor CHD4 as a potent modulator of cisplatin sensitivity and predictor of response to chemotherapy in BRCA2 mutant cancers. Taken together, my investigations highlight the important contribution of the DDR and the role they play in tumorigenesis and predicting therapeutic response

Resistance to therapy in BRCA2 mutant cells due to loss of the nucleosome remodeling factor CHD4

Hereditary cancers derive from gene defects that often compromise DNA repair. Thus, BRCA-associated cancers are sensitive to DNA-damaging agents such as cisplatin. The efficacy of cisplatin is limited, however, by the development of resistance. One cisplatin resistance mechanism is restoration of homologous recombination (HR), which can result from BRCA reversion mutations. However, in BRCA2 mutant cancers, cisplatin resistance can occur independently of restored HR by a mechanism that remains unknown. Here we performed a genome-wide shRNA screen and found that loss of the nucleosome remodeling factor CHD4 confers cisplatin resistance. Restoration of cisplatin resistance is independent of HR but correlates with restored cell cycle progression, reduced chromosomal aberrations, and enhanced DNA damage tolerance. Suggesting clinical relevance, cisplatin-resistant clones lacking genetic reversion of BRCA2 show de novo loss of CHD4 expression in vitro. Moreover, BRCA2 mutant ovarian cancers with reduced CHD4 expression significantly correlate with shorter progression-free survival and shorter overall survival. Collectively, our findings indicate that CHD4 modulates therapeutic response in BRCA2 mutant cancer cells

FANCJ/BACH1 Acetylation at Lysine 1249 Regulates the DNA Damage Response

BRCA1 promotes DNA repair through interactions with multiple proteins, including CtIP and FANCJ (also known as BRIP1/BACH1). While CtIP facilitates DNA end resection when de-acetylated, the function of FANCJ in repair processing is less well defined. Here, we report that FANCJ is also acetylated. Preventing FANCJ acetylation at lysine 1249 does not interfere with the ability of cells to survive DNA interstrand crosslinks (ICLs). However, resistance is achieved with reduced reliance on recombination. Mechanistically, FANCJ acetylation facilitates DNA end processing required for repair and checkpoint signaling. This conclusion was based on the finding that FANCJ and its acetylation were required for robust RPA foci formation, RPA phosphorylation, and Rad51 foci formation in response to camptothecin (CPT). Furthermore, both preventing and mimicking FANCJ acetylation at lysine 1249 disrupts FANCJ function in checkpoint maintenance. Thus, we propose that the dynamic regulation of FANCJ acetylation is critical for robust DNA damage response, recombination-based processing, and ultimately checkpoint maintenance

FANCJ Localization by Mismatch Repair Is Vital to Maintain Genomic Integrity after UV Irradiation

Nucleotide excision repair (NER) is critical for the repair of DNA lesions induced by UV radiation, but its contribution in replicating cells is less clear. Here, we show that dual incision by NER endonucleases, including XPF and XPG, promotes the S-phase accumulation of the BRCA1 and Fanconi anemia-associated DNA helicase FANCJ to sites of UV-induced damage. FANCJ promotes replication protein A phosphorylation and the arrest of DNA synthesis following UV irradiation. Interaction defective mutants of FANCJ reveal that BRCA1 binding is not required for FANCJ localization, whereas interaction with the mismatch repair (MMR) protein MLH1 is essential. Correspondingly, we find that FANCJ, its direct interaction with MLH1, and the MMR protein MSH2 function in a common pathway in response to UV irradiation. FANCJ-deficient cells are not sensitive to killing by UV irradiation, yet we find that DNA mutations are significantly enhanced. Thus, we considered that FANCJ deficiency could be associated with skin cancer. Along these lines, in melanoma we found several somatic mutations in FANCJ, some of which were previously identified in hereditary breast cancer and Fanconi anemia. Given that, mutations in XPF can also lead to Fanconi anemia, we propose collaborations between Fanconi anemia, NER, and MMR are necessary to initiate checkpoint activation in replicating human cells to limit genomic instability.clos

FANCJ acetylation mutants are functional.

<p>A. The acetylation mutants are expressed in FA-J cells. FA-J cells were complemented with vector, FANCJ^WT, FANCJ^K1249R, or FANCJ^K1249Q. The FA-J cell lines were collected and analyzed or B. lysates were immunoprecipitated with FANCJ antibodies and immunoblot was performed with the indicated antibodies. C. The acetylation mutants localize in nuclear foci of FA-J cells. The FA-J cell lines were seeded onto 6-well plates and incubated overnight. The cells were treated with 1 mM HU and 24 h later immunoflourescence was performed with the indicated antibodies. D. The FA-J cell lines have similar cell cycle profiles. The FA-J cells lines were collected and analyzed by FACS to determine the percentage of cells with 2N and 4N DNA content. E. Expression of acetylation mutants restores MMC resistance. The FA-J cell lines were seed onto 6 well plates and incubated overnight. The cells were either left untreated or treated with increasing doses of MMC. Cells were counted 8 days later and percent survival was calculated. Data represent mean percent ± s.d. of survival from three independent experiments. F. Expression of acetylation mutants restores G2/M checkpoint exit. The FA-J cell lines were untreated or treated with 0.25 µg/ml melphalan, collected at the indicated times, and analyzed by FACS to determine the percentage of cells in G2/M. Data represent mean percent ± s.d. of survival from three independent experiments.</p

FANCJ acetylation is induced after DNA damage.

<p>A. Endogenous FANCJ is acetylated in response DNA damage. MCF7 and HeLa cells were left untreated (UT) or treated with zeocin (6.25 µg/ml for 1 h), MMC (250 nM for 1 h), UV (30 J/m²), MMS (300 µg/ml for 4 h), HU (1 mM for 24 h), or CPT (1 µM for 1 h). Cell lysates were collected at distinct times post damage (zeocin 24 h, MMC 24 h or as indicated, UV 6 h, CPT 24 h, MMS 4 h, and HU 24 h) and analyzed for expression and/or acetylation following immunoprecipitation with the indicated antibodies. B. Exogenous FANCJ is acetylated on lysine 1249 in response to DNA damage. Myc-tagged FANCJ wild-type or mutant species and CBP were co-transfected into 293T cells and left untreated (UT) or treated with zeocin (12.5 µg/ml for 1 h or C. CPT (1 µM for 1 h). Cells were processed at different time points post DNA damage and analyzed for expression and/or acetylation following immunoprecipitation with the indicated antibodies.</p

FANCJ^K1249R or FANCJ^K1249Q promotes polη- or Rad54-dependent repair, respectively.

<p>A. FA-J cells expressing acetylation mutants have a distinct response and reliance on repair or tolerance factors for DNA damage survival. The FA-J cell lines were transfected with siRNA against Luc, Polη, or Rad54. The cells were treated with indicated doses of zeocin, UV, or as in B., with MMC and the percent survival was calculated 8 days later. Data represent mean percent ± s.d. of survival from three independent experiments. C. Cells were collected and analyzed for expression with the indicated antibodies.</p

FANCJ and its acetylation at 1249 promote an RPA response.

<p>A. Deficiency in FANCJ or its acetylation impairs the CPT-induced RPA focus formation. The FA-J cell lines were seeded onto 6-well plates, incubated overnight, left untreated or treated with CPT 1 h and immunoflourescence was performed with the indicated antibodies. The percent of cells with RPA foci was quantified and graphed. Data represent mean ± s.d. from three independent experiments. B. FANCJ and its acetylation promote RPA phosphorylation at 1 h post-CPT. The complemented FA-J cell lines were either left untreated or treated for 1 h with the indicated dose of CPT and analyzed 1 h post-treatment. Cell lysates were collected, lysed, and analyzed with the indicated antibodies. C. FANCJ acetylation promotes RPA phosphorylation at times greater than 1 h post-CPT. Same as above but collected at the time points indicated.</p