SOHLH2-RAD54L axis induces radioresistance by promoting homologous recombination repair in non-small cell lung cancer.

A Cell Cycle-Dependent Regulatory Circuit Composed of 53BP1-RIF1 and BRCA1-CtIP Controls DNA Repair Pathway Choice

Glycolytic enzymes are known to play pivotal roles in cancer cell survival, yet their molecular mechanisms remain poorly understood. Phosphoglycerate mutase 1 (PGAM1) is an important glycolytic enzyme that coordinates glycolysis, pentose phosphate pathway, and serine biosynthesis in cancer cells. Herein, we report that PGAM1 is required for homologous recombination (HR) repair of DNA double-strand breaks (DSBs) caused by DNA-damaging agents. Mechanistically, PGAM1 facilitates DSB end resection by regulating the stability of CTBP-interacting protein (CtIP). Knockdown of PGAM1 in cancer cells accelerates CtIP degradation through deprivation of the intracellular deoxyribonucleotide triphosphate pool and associated activation of the p53/p73 pathway. Enzymatic inhibition of PGAM1 decreases CtIP protein levels, impairs HR repair, and hence sensitizes BRCA1/2-proficient breast cancer to poly(ADP-ribose) polymerase (PARP) inhibitors. Together, this study identifies a metabolically dependent function of PGAM1 in promoting HR repair and reveals a potential therapeutic opportunity for PGAM1 inhibitors in combination with PARP inhibitors.

Glycolytic enzymes are known to play pivotal roles in cancer cell survival, yet their molecular mechanisms remain poorly understood. Phosphoglycerate mutase 1 (PGAM1) is an important glycolytic enzyme that coordinates glycolysis, pentose phosphate pathway, and serine biosynthesis in cancer cells. Herein, we report that PGAM1 is required for homologous recombination (HR) repair of DNA double-strand breaks (DSBs) caused by DNA-damaging agents. Mechanistically, PGAM1 facilitates DSB end resection by regulating the stability of CTBP-interacting protein (CtIP). Knockdown of PGAM1 in cancer cells accelerates CtIP degradation through deprivation of the intracellular deoxyribonucleotide triphosphate pool and associated activation of the p53/p73 pathway. Enzymatic inhibition of PGAM1 decreases CtIP protein levels, impairs HR repair, and hence sensitizes BRCA1/2-proficient breast cancer to poly(ADP-ribose) polymerase (PARP) inhibitors. Together, this study identifies a metabolically dependent function of PGAM1 in promoting HR repair and reveals a potential therapeutic opportunity for PGAM1 inhibitors in combination with PARP inhibitors.

PARP inhibitors (PARPi) have exhibited clinical success in inherited breast cancers with mutations in homologous recombination (HR) DNA double strand break (DSB) repair genes BRCA 1/2 by synthetic lethality. Novel PARPi have been shown to function not only by catalytic inhibition of PARP 1/2 activity, but also by “trapping” of PARP to sites of DNA damage. Trapping of PARP is a response generated by PARPi that is independent of the inhibition of PARP’s catalytic activity, and leads to lethal unrepaired DSBs during replication. Importantly, PARP trapping ability of PARPi correlates with its cytotoxic effect. In this study, we investigated whether PARP trapping has distinct effects on regulation of DNA DSB repair in breast cancer cells. Major DSB repair pathways in mammalian cells include HR and non-homologous end joining (c-NHEJ). Alternative NHEJ (ALT-NHEJ) is a recently discovered and highly error-prone pathway which requires DNA resection like HR, and utilizes PARP1. Our hypothesis is that PARP1 catalytic inhibition vs ‘trapping’ leads to distinct outcomes on DSB repair, highlighting inhibitor efficacy. To test our hypothesis, we utilized MCF10A (non-tumorigenic), MDA-MB-231 (TNBC/BRCA+), and SUM149PT (TNBC/BRCA-) breast cancer cell lines with stably integrated GFP-reporter plasmids designed to assess interchromosomal activities of the specific DSB repair pathways. For our analysis, we used varying concentrations of Talazoparib (MDV3800), which is a potent PARP trapping agent when used in combination with DNA damaging agents such as methyl methanesulfonate (MMS). First, we determined optimal conditions to induce PARP trapping in cells without leading to significant toxicity. As observed by others, combination treatments with of MMS/MDV3800 led to a significant increase in PARP trapping relative to treatments with individual drugs. Studies were also performed with a potent inhibitor of coenzyme NAD+ (Daporinad/FK866) that blocks poly-ADP ribosylation activity in combination of MMS. We observed significant PARP trapping with FK866/MMS combination treatment, indicating that PARP depletion under the conditions of DNA damage also leads to potent PARP trapping. Next, we investigated the effects of these inhibitors on DSB repair. Surprisingly, combination MDV3800/MMS lead to an overall decrease in HR and increase in ALT NHEJ activity. These results were observed in both our BRCA+/- lines. Similar results are observed between the repair activities when using FK866/MMS treatments. Overall, our results suggest that PARP inhibitors with potent PARP trapping activity increases ALT NHEJ, which may potentially compensate for lack of HR. Studies are ongoing to determine whether targeting ALT NHEJ may increase cytotoxicity in these cells. Citation Format: Bryan M. Pelkey, Pratik Nagaria, and Feyruz V. Rassool. PARP trapping by PARP inhibitors have distinct effects on HR and ALT NHEJ DSB repair, potentially impacting its therapeutic efficacy in breast cancers [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 2478. doi:10.1158/1538-7445.AM2017-2478

Homologous recombination (HR), which mediates the repair of DNA double-strand breaks (DSB), is crucial for maintaining genomic integrity and enhancing survival in response to chemotherapy and radiotherapy in human cancers. However, the mechanisms of HR repair in treatment resistance for the improvement of cancer therapy remains unclear. Here, we report that the zinc finger protein 830 (ZNF830) promotes HR repair and the survival of cancer cells in response to DNA damage. Mechanistically, ZNF830 directly participates in DNA end resection via interacting with CtIP and regulating CtIP recruitment to DNA damage sites. Moreover, the recruitment of ZNF830 at DNA damage sites is dependent on its phosphorylation at serine 362 by ATR. ZNF830 directly and preferentially binds to double-strand DNA with its 3′ or 5′ overhang through the Zinc finger (Znf) domain, facilitating HR repair and maintaining genome stability. Thus, our study identified a novel function of ZNF830 as a HR repair regulator in DNA end resection, conferring the chemoresistance to genotoxic therapy for cancers those that overexpress ZNF830.

Simple SummaryLung cancer is the most commonly diagnosed cancer worldwide and additionally the most common cause of death from cancer, with non-small cell lung cancers (NSCLC) being the most commonly diagnosed form of the disease. As drug resistance is a key issue halting chemotherapy effectiveness, there is a great need to identify new therapeutic targets. The aims of this study were to investigate the function of the protein, COMMD1, in the repair of DNA double strand breaks and the therapeutic potential of COMMD1 in NSCLC. Here, we demonstrate for the first time how an additional COMMD family member, COMMD1, functions in the repair of DNA double strand breaks and may be relevant as a therapeutic target and prognostic factor in NSCLC. These novel findings highlight the potential of a novel approach to NSCLC therapy, by targeting an overexpressed protein. Lung cancer has the highest incidence and mortality among all cancers, with non-small cell lung cancer (NSCLC) accounting for 85–90% of all lung cancers. Here we investigated the function of COMMD1 in the repair of DNA double strand breaks (DSBs) and as a prognostic and therapeutic target in NSCLC. COMMD1 function in DSB repair was investigated using reporter assays in COMMD1-siRNA-depleted cells. The role of COMMD1 in NSCLC was investigated using bioinformatic analysis, qRT-PCR and immunoblotting of control and NSCLC cells, tissue microarrays, cell viability and cell cycle experiments. DNA repair assays demonstrated that COMMD1 is required for the efficient repair of DSBs and reporter assays showed that COMMD1 functions in both non-homologous-end-joining and homologous recombination. Bioinformatic analysis showed that COMMD1 is upregulated in NSCLC, with high levels of COMMD1 associated with poor patient prognosis. COMMD1 mRNA and protein were upregulated across a panel of NSCLC cell lines and siRNA-mediated depletion of COMMD1 decreased cell proliferation and reduced cell viability of NSCLC, with enhanced death after exposure to DNA damaging-agents. Bioinformatic analyses demonstrated that COMMD1 levels positively correlate with the gene ontology DNA repair gene set enrichment signature in NSCLC. Taken together, COMMD1 functions in DSB repair, is a prognostic maker in NSCLC and is potentially a novel anti-cancer therapeutic target for NSCLC.

Requirement of ATM-Dependent Monoubiquitylation of Histone H2B for Timely Repair of DNA Double-Strand Breaks

DNA non-homologous end joining (NHEJ) and homologous recombination (HR) function to repair DNA double-strand breaks (DSBs) in G2 phase with HR preferentially repairing heterochromatin-associated DSBs (HC-DSBs). Here, we examine the regulation of repair pathway usage at two-ended DSBs in G2. We identify the speed of DSB repair as a major component influencing repair pathway usage showing that DNA damage and chromatin complexity are factors influencing DSB repair rate and pathway choice. Loss of NHEJ proteins also slows DSB repair allowing increased resection. However, expression of an autophosphorylation-defective DNA-PKcs mutant, which binds DSBs but precludes the completion of NHEJ, dramatically reduces DSB end resection at all DSBs. In contrast, loss of HR does not impair repair by NHEJ although CtIP-dependent end resection precludes NHEJ usage. We propose that NHEJ initially attempts to repair DSBs and, if rapid rejoining does not ensue, then resection occurs promoting repair by HR. Finally, we identify novel roles for ATM in regulating DSB end resection; an indirect role in promoting KAP-1-dependent chromatin relaxation and a direct role in phosphorylating and activating CtIP.

Chronic myeloid neoplasms (CMNs) are characterized by excessive expansion of terminally differentiated blood cells arising from myeloid progenitors. This group of hematologic clonal disorders includes the BCR-ABL1-negative myeloproliferative neoplasms (MPNs), myelodysplastic syndromes (MDS) and MPN/MDS disorders with features overlapping both subtypes. CMN patients are at risk of developing acute myeloid leukemia (AML), however curative therapy is lacking with the exception of allogeneic stem cell transplant. Since leukemic transformation portends a dismal treatment outcome and survival, there is a need to identify novel molecular events driving disease progression as a first step in effective targeted therapy. Previously, we achieved complete clearance of leukemic blasts in several CMN patients who had progressed onto AML when treated with poly (ADP-ribose) polymerase (PARP) inhibitor ABT-888, carboplatin and topotecan (McDevitt et al., ASH 2011 and Pratz et al., ASH 2011). PARP inhibitors block single stand break repair which cumulates in an increase in DSBs that cannot be repaired in tumor cells defective in homologous recombination (HR) repair, resulting in the exquisite death response of the malignant clone. In this study, we investigated whether this favorable response can be attributed to defective DNA double-stranded break (DSB) repair resulting from epigenetic silencing of HR genes. To this end, we evaluated the promoter methylation of HR genes and then assessed DNA DSB repair status in CMN patients. We detected BRCA1 promoter hypermethylation in 10% of 99 unique MPN patient samples using quantitative methylation-specific PCR (qMSP) and confirmed decreased BRCA1 transcript expression in several samples. We did not observe DNA promoter methylation of other HR repair genes (Fanconi Anemia genes, Bloom, ATM, ATR, BRCA2). In addition, we characterized the functional DSB repair status in primary mononuclear cells from CMN patients by measuring Rad51 foci formation after irradiation and identified a subset of patients with defective HR repair. Ongoing studies of additional CMN patient samples are underway to further link HR repair gene promoter hypermethylation, transcript expression and functional Rad51 foci formation. These findings will also be compared to karyotype information and in vitro PARP inhibition sensitivity results. By presenting evidence of DNA repair defect that can be linked to epigenetic silencing of HR repair genes in CMN patients, we highlight defective DSB repair as a novel mechanism driving disease progression in myeloid malignancies and also propose examining the DNA promoter methylation status of HR genes as a biomarker to identify patients who will respond favorably to PARP inhibition. Citation Format: Weijie Poh, Robert L. Dilley, Alison R. Moliterno, Keith W. Pratz, Michael A. McDevitt, James G. Herman. BRCA1 promoter methylation and homologous recombination repair status in primary chronic myeloid neoplasms. [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 3536. doi:10.1158/1538-7445.AM2013-3536

Double-Strand Breaks in Heterochromatin Move Outside of a Dynamic HP1a Domain to Complete Recombinational Repair

The importance of chromatin modification, including histone modification and chromatin remodeling, for DNA double-strand break (DSB) repair, as well as transcription and replication, has been elucidated. Phosphorylation of H2AX to γ-H2AX is one of the first responses following DSB detection, and this histone modification is important for the DSB damage response by triggering several events, including the accumulation of DNA damage response-related proteins and subsequent homologous recombination (HR) repair. The roles of other histone modifications such as acetylation, methylation and ubiquitination have also been recently clarified, particularly in the context of HR repair. NBS1 is a multifunctional protein that is involved in various DNA damage responses. Its recently identified binding partner RNF20 is an E3 ubiquitin ligase that facilitates the monoubiquitination of histone H2B, a process that is crucial for recruitment of the chromatin remodeler SNF2h to DSB damage sites. Evidence suggests that SNF2h functions in HR repair, probably through regulation of end-resection. Moreover, several recent reports have indicated that SNF2h can function in HR repair pathways as a histone remodeler and that other known histone remodelers can also participate in DSB damage responses. On the other hand, information about the roles of such chromatin modifications and NBS1 in non-homologous end joining (NHEJ) repair of DSBs and stalled fork-related damage responses is very limited; therefore, these aspects and processes need to be further studied to advance our understanding of the mechanisms and molecular players involved.

Advanced non-small cell lung cancer (NSCLC) is the leading cause of cancer mortality. Despite progress in targeted molecular therapeutics and precision medicine, outcomes in this disease remain poor. Recent evidence suggests that impaired homologous recombination (HR) occurs in a significant subset of NSCLCs and may serve as a predictive biomarker for sensitivity to DNA damaging agents. Poly-ADP ribose polymerase (PARP) and Wee1 inhibition represent two mechanistically distinct approaches to augment the effects of DNA damage. Specifically, the PARP inhibitor olaparib impairs repair of DNA single strand breaks, which during replication lead to the formation of DNA double strand breaks (DSBs), resulting in synthetic lethality in HR deficient tumors. AZD1775 is a Wee1 inhibitor that abrogates the G2 checkpoint and thus removes a safeguard against cell cycle progression with unrepaired DNA damage. Moreover, AZD1775 has been recently reported to exhibit single-agent activity in patients harboring BRCA1/2 mutations. Therefore, we hypothesize that olaparib and AZD1775 would have synergistic effects in a subset of NSCLCs and that HR deficiency could be predictive of tumor response to combination therapy. Utilizing Rad51 focus formation as a marker of HR deficiency, we prospectively selected representative NSCLC cell lines that either did (e.g. Calu6) or did not (e.g. A549) harbor putative defects in HR repair. We treated Calu6 and A549 and other NSCLC cells with AZD1775 and olaparib with varying drug dosing and sequencing to determine the optimal regimen for synergistic effect. Cytotoxicity was determined by CellTiter-Glo cell viability assays and synergy was quantified by calculating the combination index. Additionally, we investigated mechanistic protein markers by Western blot. In response to combined olaparib and AZD1775 treatment, Calu6 cancer cells demonstrated markedly more pronounced synergistic sensitivity (median CI = 0.19) compared to A549 cancer cells (median CI = 0.90). Moreover, a similar trend toward a selective synergistic effect was demonstrated in a panel of 10 additional NSCLC lines. On biochemical analysis, we observed inhibition of p-Cdk1, upregulation of p-Chk1, and upregulation of p-KAP1, suggesting abrogation of the G2/M checkpoint and activation of ATM/ATR repair pathways, all consistent with the mechanistic underpinnings of our hypothesis. Taken together, these results provide early pre-clinical evidence for the rational combination of Wee1 and PARP inhibition in the treatment of advanced NSCLC, and suggest HR deficiency as a predictive marker applicable to NSCLC. Continued mechanistic investigation and further confirmatory studies are warranted to inform the selection of patients who may maximally benefit from such combination treatment. Citation Format: Daniel X. Yang. Synergy between PARP and Wee1 inhibitors suggests homologous recombination repair defect in NSCLC as a mechanistic target for combination therapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3710.

Decision letter: SETD2 is required for DNA double-strand break repair and activation of the p53-mediated checkpoint

While single component E3 ubiquitin ligases have established roles in DNA double-strand break (DSB) repair, the function of multicomponent cullin-RING-ligases (CRL) in DSB repair is only beginning to emerge. FBXW7 is a substrate recognition component of Skp1-Cullin1-F-box E3 ubiquitin ligases previously known only to regulate the proteasomal degradation of substrates such as Cyclin E and MCL1. Given that FBXW7 loss promotes genomic instability, we hypothesized that FBXW7 may have a direct function in DSB repair. To establish the functions of FBXW7 in DSB repair, we first demonstrated the rapid localization of FBXW7 to DSB sites in an ATM-dependent manner. Subsequently, we found that FBXW7 depletion impaired nonhomologous end-joining (NHEJ), but not homologous recombination (HR) repair, resulting in persistent radiation-induced DSBs. Investigation of the molecular mechanisms of FBXW7 in NHEJ revealed that FBXW7 interacts with and promotes K63-linked ubiquitination of XRCC4. This ubiquitination of XRCC4 promotes interaction between the XRCC4/XLF/LIG4 and DNAPK/KU70/KU80 complexes to facilitate NHEJ. To begin to therapeutically leverage this mechanism, strategies to both pharmacologically inhibit FBXW7-CRLs and to exploit FBXW7 mutations occurring in human cancers are being developed. To address the former, pevonedistat (MLN4924), an agent which inhibits CRLs via inhibition of cullin-neddylation, inhibits FBXW7-mediated XRCC4 ubiquitination and NHEJ. This activity leads to increased sensitivity of tumor cells to chemotherapy and radiation and represents a strategy that may be particularly effective in cancers with other DSB repair defects. As another strategy to leverage the mechanisms of FBXW7 in NHEJ, loss-of-function mutations in the WD domain of FBXW7 which occur frequently in human cancers are being explored. These mutations render cells defective in NHEJ and more sensitive to DNA damage. Thus, we hypothesize that inhibition of other DSB repair pathways (e.g. alternative-end-joining) may be an effective therapeutic strategy for FBXW7 mutant cancers. These novel mechanisms of NHEJ regulation as well as their implications in the treatment of human cancers with an emphasis on pancreatic and colorectal cancers will be discussed. This work was supported by NIH grants R01CA163895, P50CA130810, R01CA118762, R01CA156744, and R01CA171277. Citation Format: Meredith Morgan. Exploiting the inhibition of cullin-RING-ligases in DSB repair as a therapeutic strategy [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr IA27.

Akt1 is known to promote non-homologous end-joining (NHEJ)-mediated DNA double-strand break (DSB) repair by stimulation of DNA-PKcs. In the present study, we investigated the effect of Akt1 on homologous recombination (HR)-dependent repair of radiation-induced DSBs in non-small cell lung cancer (NSCLC) cells A549 and H460. Akt1-knockdown (Akt1-KD) significantly reduced Rad51 protein level, Rad51 foci formation and its colocalization with γH2AX foci after irradiation. Moreover, Akt1-KD decreased clonogenicity after treatment with Mitomycin C and HR repair, as tested by an HR-reporter assay. Double knockdown of Akt1 and Rad51 did not lead to a further decrease in HR compared to the single knockdown of Rad51. Consequently, Akt1-KD significantly increased the number of residual DSBs after irradiation partially independent of the kinase activity of DNA-PKcs. Likewise, the number of residual BRCA1 foci, indicating unsuccessful HR events, also significantly increased in the irradiated cells after Akt1-KD. Together, the results of the study indicate that Akt1 seems to be a regulatory component in the HR repair of DSBs in a Rad51-dependent manner. Thus, based on this novel role of Akt1 in HR and the previously described role of Akt1 in NHEJ, we propose that targeting Akt1 could be an effective approach to selectively improve the killing of tumor cells by DSB-inducing cytotoxic agents, such as ionizing radiation.