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How Do DNA repair defective tumors become resistant to chemotherapy?

Chemoresistance remains a major problem: Cisplatin has been used for over 30 years for the treatment of ovarian cancer. Within six months following treatment, one quarter of patients develop platinum-resistant cancer and only a third of ovarian cancer patients live past five years. This makes ovarian cancer the fifth-leading cause of cancer death among women in the United States. While genome studies are making great strides in uncovering the genetic determinants that underlie tumorigenesis, the genetic determinants of therapeutic resistance are still largely unknown.

Sensitivity to platinum agents is, in part, determined by the integrity of DNA repair. In particular, platinum sensitivity is enhanced in breast and ovarian tumors that derive from inherited mutations in DNA repair genes, BRCA1 and BRCA2 (BRCA). The sensitivity derives from the fact that BRCA mutant tumors are defective in DNA repair by homologous recombination (HR), which contributes to processing of DNA interstrand crosslinks (ICLs) induced by platinum agents such as cisplatin The loss of DNA repair in BRCA- tumors also sensitizes them to inhibitors of PARP (PARPi). The promise of these therapies is limited, however, by the development of resistance.

About half the time chemoresistance in BRCA1- and BRCA2-mutant cancers is due to restoredDNA repair. One mechanism by which repair is restored is through secondary mutations in the BRCA genes that reinstate the function of the BRCA gene in DNA repair 1-4.  When DNA repair is not restored, however the mechanism of chemoresistance was a mystery.

A new mechanism of chemoresistance in BRCA2 mutant cancer. Recently, we found another way resistance occurs. To identify the genetic determinants of cisplatin sensitivity, we performed a survival-based, loss of function RNAi screen in the cisplatin-sensitive BRCA2 mutant PEO1 ovarian cancer cell line.  Of the ~100 gene targets identified in the screen whose loss promotes cisplatin resistance, we initially focused on CHD4, for several reasons. Multiple unrelated shRNAs targeting CHD4 conferred reistance and re-expression of a CHD4 restored cisplatin sensitivity. Moreover, CHD4 was implicated as a tumor suppressor in several female malignancies. While CHD4 is best known for its function in the nucleosome-remodeling (NuRD) complex, depletion of other NuRD members did not confer resistance. Thus, we considered NuRD complex independent functions  of CHD4. Interestingly, CHD4 functions in the DDR similar to BRCA1 and BRCA2. CHD4 has an active role in recruiting DNA repair factors to sites of lesions and actively promotes DNA repair. As such, CHD4 depletion activates a DNA damage response, reduces proliferation, and enhances sensitivity to DNA damaging agents.  While CHD4 depletion has negative effects on the DDR, this was avoided in BRCA2 mutant cells in which proliferation and cisplatin resistance was enhanced. In addition to enhancing cisplatin resistance and correcting cell cycle progression defects, CHD4 depletion dramatically reduces cisplatin induced chromosomal aberrations 5.

Why does CHD4 loss rescue BRCA2-mutant cancer? We considered that cisplatin resistance along with reduced chromosomal aberrations could reflect that CHD4 depletion restored DNA repair. However, we found that DNA repair was not restored as measured by analysis of RAD51 function (chromatin localization and focal formation) as well as in standard DR-GFP reporter assay. Instead, we found that PCNA monoubiquitination was enhanced, a modification that provides a landing pad for mutagenic translesion synthesis (TLS) polymerases. Consistent with a bias towards a mutagenic bypass mechanism, the frequency of mutagenesis is elevated both in untreated and cisplatin treated BRCA2 mutant cells in which CHD4 is depleted. Moreover, RAD18, the ligase that monoubiquitinates PCNA is more focal and RAD18 depletion substantially re-sensitizes BRCA2-mutant cells with low CHD4 to cisplatin, but had little effect on cells with functional BRCA2. Collectively, these findings suggest that CHD4 loss primes BRCA2-mutant cells for DNA damage tolerance.

Effective Therapy for BRCA cancers. Strikingly, analysis of CHD4 mRNA specifically in BRCA2-mutant ovarian cancers revealed that low CHD4 expression significantly correlated with shorter progression free and overall survival. These results suggest that CHD4 mRNA expression levels could serve as a valuable predictor of response to chemotherapy in ovarian and potentially other cancers carrying a BRCA2 mutation. CHD4 loss also conferred enhanced resistance to inhibitors of PARP.  Thus, current efforts include identifying novel agents that either enhance the efficacy of current therapies or work effectively as monotherapy to kill otherwise chemoresistant cancer. We are also addressing how CHD4 loss confers cisplatin resistance through tolerance pathways and if loss of other genes found in our initial screen will confer cisplatin resistance by a mechanism similar to loss of CHD4.  Finally, because CHD4 loss uniquely rescues a BRCA2 mutant background and BRCA2-associated cancers typically retain an N-terminal protein, we are determining if this N-terminal mutant protein retains a function important for cisplatin resistance and is therefore a worthy target.

Routes of chemo resistance in BRCA2 mutant cancer cells.

Figure 1: Routes of chemo resistance in BRCA2 mutant cancer cells.

1        Sakai, W. et al. Secondary mutations as a mechanism of cisplatin resistance in BRCA2-mutated cancers. Nature 451, 1116-1120, doi:nature06633 [pii]10.1038/nature06633 (2008).

2        Edwards, S. L. et al. Resistance to therapy caused by intragenic deletion in BRCA2. Nature 451, 1111-1115 (2008).

3        Sakai, W. et al. Functional restoration of BRCA2 protein by secondary BRCA2 mutations in BRCA2-mutated ovarian carcinoma. Cancer Res 69, 6381-6386, doi:0008-5472.CAN-09-1178 [pii]10.1158/0008-5472.CAN-09-1178 (2009).

4        Norquist, B. et al. Secondary somatic mutations restoring BRCA1/2 predict chemotherapy resistance in hereditary ovarian carcinomas. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 29, 3008-3015, doi:10.1200/JCO.2010.34.2980 (2011).

5        Guillemette, S. et al. Resistance to therapy in BRCA2 mutant cells due to loss of the nucleosome remodeling factor CHD4. Genes Dev 29, 489-494, doi:10.1101/gad.256214.114 (2015).