PARP1 Enzymatic Activity Research Articles

Nuclear localization of APLF facilitates breast cancer metastasis

Most breast cancer deaths result from metastases. We previously reported that DNA repair factor and histone chaperone Aprataxin PNK-like Factor (APLF) is involved in EMT-associated metastasis of triple negative breast cancer (TNBC) cells. However, non-metastatic cells also expressed APLF, the implications of which in disease advancement remain uncertain. Here, we demonstrate that the metastatic prognosis of breast cancer cells may be determined by the cellular localization of APLF. Using TNBC patient samples and cell lines, we discovered that APLF was localized in the nucleus and cytoplasm, whereas other subtypes of breast cancer had cytosolic or perinuclear localization. To investigate metastatic properties in vitro and in vivo, we modeled APLF differential localization by stably producing APLF-tagged nuclear localization signal (NLS) in the luminal subtype MCF7 cells in the absence of putative APLF NLS. Nuclear APLF in non-metastatic MCF7 cells demonstrated pronounced migration, invasion and metastatic potential. We obtained the mechanistic insight from molecular studies that PARP1 could facilitate the transport of APLF from the cytosol to the nucleus, assisting in the metastasis of TNBC cells linked with EMT. Inhibition of PARP1 enzymatic activity with olaparib abrogated the nuclear expression of APLF with loss in expression of genes associated with EMT. Thus, our findings reveal that cellular localization of APLF may predict the risk of breast cancer to metastasize and hence could be exploited to determine the disease progression. We anticipate that the inhibition of cytosolic PARP1-APLF interaction may potentially aid in the prevention of breast cancer metastasis in TNBC patients.

Abstract 4534: HSK40495, a highly selective PARP1 inhibitor with improved hematopoietic safety for the treatment of HRD cancers

Abstract It is well known that several PARP inhibitors have been approved for tumors with homologous recombination repair deficiencies (HRD), such as BRCA mutations. However, the common side effect of these inhibitors is hematologic toxicity, which limits their therapeutic index and combination potential with first-line chemotherapies. One possible reason is that currently approved PARP inhibitors do not specifically target PARP1, and might also affect other PARP family members. Recent studies have shown that the synthetic lethality caused by PARP trapping with HRD is mainly dependent on PARP1 inhibition, but not other PARP family members. Accordingly, we developed a selective PARP1 inhibitor that effectively inhibits tumor growth with less hematologic toxicity. HSK40495 is a potent PARP1 inhibitor with highly selectivity of PARP2. HSK40495 inhibited PARP1 enzymatic activity with a single digit nanomolar potency and with greater than 5000-fold selectivity against PARP2 in the protein binding assay. In addition, HSK40495 exhibited potent anti-proliferation activity in both DLD1(BRCA KO) cell and MDA-MB-436 cell, while had minimal effects against BRCA-WT DLD1 cell up to 30 µM in an anti-proliferation assay. In a subcutaneous MDA-MB-436 xenograft tumor model, HSK40495 administered orally once daily achieved excellent tumor growth inhibition of 100% at 3mg/kg. HSK40495 demonstrated significant PARylation inhibition in tumor tissues. Importantly, in CD34+ human bone marrow progenitor cell, HSK40495 showed improved cytotoxicity when compare to first-generation PARP inhibitor, Talazoparib. Meanwhile, in a lineage-specific hematotoxicity assay, HSK40495 exhibited improved cytotoxic safety in the process of differentiation of myeloid, erythroid and megakaryocyte compared with Talazoparib treatment. In summary, HSK40495, a highly selective PARP1inhibitor over other PARP family members, exhibits excellent pharmacological and safety properties, and also has the favorable pharmacokinetics profile in preclinical studies. All these findings suggest that HSK40495 has the great potential to be a promising PARP1 inhibitor for further development in the patients with HRD tumors. Citation Format: Fan Li, Xiaoli Gou, Lei Chen, Qingyuan Meng, Yao Li, Hongjiao Dong, Pingming Tang, Ju Wang, Pangke Yan. HSK40495, a highly selective PARP1 inhibitor with improved hematopoietic safety for the treatment of HRD cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4534.

Transcription–replication conflicts underlie sensitivity to PARP inhibitors

An important advance in cancer therapy has been the development of poly(ADP-ribose) polymerase (PARP) inhibitors for the treatment of homologous recombination (HR)-deficient cancers1–6. PARP inhibitors trap PARPs on DNA. The trapped PARPs are thought to block replisome progression, leading to formation of DNA double-strand breaks that require HR for repair7. Here we show that PARP1 functions together with TIMELESS and TIPIN to protect the replisome in early S phase from transcription–replication conflicts. Furthermore, the synthetic lethality of PARP inhibitors with HR deficiency is due to an inability to repair DNA damage caused by transcription–replication conflicts, rather than by trapped PARPs. Along these lines, inhibiting transcription elongation in early S phase rendered HR-deficient cells resistant to PARP inhibitors and depleting PARP1 by small-interfering RNA was synthetic lethal with HR deficiency. Thus, inhibiting PARP1 enzymatic activity may suffice for treatment efficacy in HR-deficient settings.

Abstract C034: Understanding the role of PARP activity in prostate cancer

Abstract Prostate cancer (PCa) is the second most fatal cancer in men in the United States. African American men have an incidence and death rate two times higher than that of European American men. The androgen receptor (AR) is the primary pro-oncogenic transcription factor responsible for PCa emergence and progression. AR directed therapies including androgen deprivation (ADT), direct receptor antagonism and inhibition of androgen synthesis are the current standard of care for advanced PCa. Adaptive resistance to anti-androgen therapy can occur resulting in castration-resistant prostate cancer (mCRPC). mCRPC remains universally fatal with no curative treatment options, which highlights the need for new treatment regimes. Poly (ADP-ribose) polymerase 1 (PARP-1) inhibitors are approved to treat mCRPC tumors that have homologous recombination (HR) mutations. PARP-1 is an enzyme that recruits DNA damage repair enzymes. PARP-1 enzymatic activity is elevated in mCRPC patient tumors compared to primary tumors. PARP1 enzymatic activity has been implicated in driving pro-survival mechanisms through recruitment of DNA-damage repair enzymes and regulation of oncogenic transcription factors, including AR. Although increased PARP activity has been associated with driving cancer cell survival, the mechanisms and consequences of elevated PARP activity in PCa remains an area to be further explored. The biologically relevant doses of five PARPi were established in regular and steroid depleted growth conditions in CRPC models. PARP enzymatic activity was assessed by immunoblotting for changes in PARlyation, revealing that PARP protein remains stable, while enzymatic activity is attenuated upon challenge with PARPi. Transcriptomic analysis will assess changes in gene expression programs that are associated with inhibition of PARP enzymatic activity as it relates to key oncogenic pathways in PCa. It will be imperative to gain a deeper understanding of how PARP-1 manipulation modulates pro-oncogenic transcriptional networks to better inform clinical response. Patient derived explants (PDEs) from patients with distinct racial backgrounds will be used to examine differential PARPi responses as it relates to race. This will serve to unravel the mechanisms attributed to the increased lethality of PCa observed in African American populations. Collectively, these studies and future studies for this project will further establish the impact of PARP on PCa gene expression programs and provide resources for future studies exploring PARP in PCa. Further, they may help elucidate mechanisms to address PCa racial disparities. Citation Format: Moriah Cunningham, Latese Evans, Salome Tchotorlishvili, Jasibel Vasquez Gonzalez, Candice Bizzaro, Tessa Mulders, Matthew Schiewer. Understanding the role of PARP activity in prostate cancer [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr C034.

Abstract C062: Defining the interplay between PARP and the RB/E2F axis in prostate cancer

Abstract Prostate cancer (PCa) is the second leading cause of cancer-related death in American men. Non-Hispanic Black men are 1.8 times more likely to develop PCa and 2.3 times more likely to die from PCa. Tumor suppressor retinoblastoma (RB1) is frequently deleted in PCa. RB represses the transcriptional activity of E2Fs and the phosphorylation of RB by CDK4/6 results in dissociation of the complex and a subsequent increase in E2F transcriptional activity, leading to cell cycle progression. RB loss is associated with tumor aggressiveness occurring in 10-15% of castration-resistant prostate cancer (CRPC) tumors and corresponds to a decrease in overall survival. RB depletion leads to increased expression of DNA repair proteins due to elevated transcriptional activity of E2F1. PARP-1 is an enzyme that plays a role in DNA repair. Elevated PARP-1 enzymatic activity is associated with PCa progression and correlates with poor clinical outcomes in CRPC. E2F1 transcriptional activity is supported by PARP enzymatic activity, however the consequences of the functional interaction between the RB/E2F axis and PARP remains an open line of inquiry. Defining the interplay between PARP and RB/E2F is likely to contribute to the identification of novel treatment strategies and/or biomarker response. Canonically, E2F1-3 are transcription factors that positively regulate gene expression programs associated with pro-proliferative signals. Prior research by other groups has shown that E2F target genes expression is elevated in tumors excised from Black men compared to Asian and White men. There was an increase in germline RAD51 mutations observed. RAD51 is an E2F target gene involved in DDR, therefore attenuating PARP enzymatic activity may be a potential mode of targeting this axis. These data suggest that there may be differential DNA damage response (DDR) in African American men driven by aberrations in the RB/E2F signaling axis, however the mechanism is yet to be fully explored. Analysis of publicly available patient datasets were performed and demonstrate that RB1 deletions is more frequent than RB1/BRCA2 co-deletion in metastatic PCa. Isogenic models of RB1 knockdown were used to evaluate the impact of RB loss on PARP enzymatic activity. PARP-1 mRNA and PARP-1 protein are elevated upon RB1 depletion with a concomitant increase in PARP activity. Growth curve assays were conducted to examine the biological response to PARP inhibition in the isogenic models. In hormone-depleted conditions, results suggest a differential response between RB depleted models compared to the control when challenged with PARP inhibition. Furthermore, an IC50 dose of Olaparib elicits differential PARP enzymatic response in the RB1 depleted model. RB1 loss is associated with an enrichment in E2F1 binding at the PARP-1 locus. Future studies have been designed to determine the impact of the RB/E2F pathway alterations on downstream consequences PARP enzymatic function and establish the contribution of PARP and the RB/E2F axis on racial disparity in PCa. Citation Format: Latese Evans. Defining the interplay between PARP and the RB/E2F axis in prostate cancer [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr C062.

Abstract B063: Interplay between PARP and the RB/E2F axis in prostate cancer

Abstract Retinoblastoma (RB) protein is a tumor suppressor that represses the transcriptional activity of E2Fs by forming an RB-E2F repressor complex. The phosphorylation of RB by CDK4/6 results in dissociation of the complex and subsequent E2F transcriptional activity, leading to cell cycle progression. RB loss occurs in 10-15% of castration-resistant prostate cancer (CRPC) which is associated tumor aggressiveness and poor clinical outcomes. RB loss is also associated differential treatment response to an array of treatment modalities, including hormone therapy and DNA damage inducing chemotherapy. RB depletion leads to enhanced expression of DNA repair proteins due to increased transcriptional activity of E2F1. PARP-1 is an enzyme that plays a role in multiple nuclear processes including DNA repair, transcriptional regulation, and chromatin dynamics. Increased PARP-1 activity is associated with and correlates to poor clinical outcomes in CRPC. E2F1 transcriptional activity is supported by PARP enzymatic activity, but the consequences of the functional interaction between the RB/E2F axis and PARP remains an open line of inquiry, which may yield novel treatment strategies and/or biomarkers of response. Isogenic models of RB1 knockdown were used to evaluate the impact of RB loss on PARP enzymatic activity. PARP-1 protein increases upon RB1 depletion with a concomitant increase in PARP activity. Growth curve assays were conducted to examine the biological response to PARP inhibition in the isogenic models. Results suggest a differential response between control and RB1 depleted cells when treated with Olaparib. RB1depleted cells also exhibit differential PARP activity in response to treatment with the IC50 dose of Olaparib. Future studies have been designed to evaluate the impact of RB alteration and manipulation of PARP activity on biological and molecular processes that are governed by RB/E2F and PARP. Citation Format: Latese Evans, Moriah Cunningham, Jasibel Vasquez Gonzalez, Matthew Schiewer. Interplay between PARP and the RB/E2F axis in prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B063.

Abstract 1696: Systematic identification of pathways associated with antibody drug conjugate sensitivity in breast cancer

Abstract Antibody drug conjugates (ADC) are emerging as paradigm-changing precision therapeutics. Sacituzumab Govitecan (SG), which combines a Topoisomerase I (TOP1) inhibitor payload (SN38) with hRS7, an antibody targeting the tumor-selective antigen TROP2, is proving to be a successful cancer therapeutic for some highly refractory cancers including triple negative breast cancer (TNBC). However, mechanisms and biomarkers of sensitivity and resistance to SG remain poorly understood. Prior clinical trials have identified positive correlations between TROP2 expression and better outcome for metastatic TNBC patients receiving SG. Additionally, patient-derived specimen analyses by our group identified mutations in both TROP2 and TOP1 in post-progression metastatic lesions, suggesting multifaceted resistance mechanisms. To systematically interrogate ADC sensitivity mechanisms, we utilized genome-wide CRISPR-Cas9 knockout screening to identify novel regulators mediating SG sensitivity in human TNBC cells. Interactome analysis of top SG sensitizing hits showed clustered pathways of interest, including DNA repair/replication, mTORC1/metabolism, and endosome trafficking/sorting. The top druggable hit sensitizing to SG was PARP1, encoding the poly (ADP-ribose) polymerase 1 that is a key enzyme required for single-strand break repair pathways and for DNA replication fork stability. Conversely, the very top SG resistance-inducing hit in the genome was PARG, encoding the glycohydrolase that opposes PARP enzymatic activity. Furthermore, the synergistic lethality between TOP1 and PARP1 was confirmed in experiments combining SG with PARP inhibitor treatment of TNBC cell lines. In addition, to identify SG/ADC specific genes and pathways, we performed a secondary round of CRISPR screens using a custom library comprising top hits discovered in the primary screen, treating cells in parallel with either SG or cytotoxic payload SN38 alone. Pathway analysis of genes selectively modulating sensitivity to SG but not SN38 revealed novel mediators involved in TROP2 turnover and recycling. We then performed validation and mechanistic studies to elucidate how specific genetic permutations regulate antibody delivery and sensitivity to SG. In conclusion, through a systematic approach using CRISPR screening, we identified several novel resistance and sensitizing pathways that are implicated in ADC delivery and target protein turnover. We are currently prioritizing the druggable genes and pathways in preparation for proof-of-concept studies combining ADCs with select targeted therapeutics to achieve greater tumor killing efficacy. Citation Format: Bogang Wu, Sheng Sun, Nayana Thimmiah, Aiko Nagayama, James Coates, Win Thant, David Li, John Doench, Aditya Bardia, Leif Ellisen. Systematic identification of pathways associated with antibody drug conjugate sensitivity in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1696.

Analysis of the PARP1, ADP-Ribosylation, and TRIP12 Triad With Markers of Patient Outcome in Human Breast Cancer

PARP inhibitors (PARPi) are increasingly used in breast cancer therapy, including high-grade triple-negative breast cancer (TNBC) treatment. Varying treatment responses and PARPi resistance with relapse currently pose limitations to the efficacy of PARPi therapy. The pathobiological reasons why individual patients respond differently to PARPi are poorly understood. In this study, we analyzed expression of PARP1, the main target of PARPi, in normal breast tissue, breast cancer, and its precursor lesions using human breast cancer tissue microarrays covering a total of 824 patients, including more than 100 TNBC cases. In parallel, we analyzed nuclear adenosine diphosphate (ADP)-ribosylation as a marker of PARP1 activity and TRIP12, an antagonist of PARPi-induced PARP1 trapping. Although we found PARP1 expression to be generally increased in invasive breast cancer, PARP1 protein levels and nuclear ADP-ribosylation were lower in higher tumor grade and TNBC samples than non-TNBCs. Cancers with low levels of PARP1 and low levels of nuclear ADP-ribosylation were associated with significantly reduced overall survival. This effect was even more pronounced in cases with high levels of TRIP12. These results indicate that PARP1-dependent DNA repair capacity may be compromised in aggressive breast cancers, potentially fueling enhanced accumulation of mutations. Moreover, the results revealed a subset of breast cancers with low PARP1, low nuclear ADP-ribosylation, and high TRIP12 levels, which may compromise their response to PARPi, suggesting a combination of markers for PARP1 abundance, enzymatic activity, and trapping capabilities might aid patient stratification for PARPi therapy.

DDDR-01. AZD9574 IS A NOVEL, BRAIN PENETRANT PARP-1 SELECTIVE INHIBITOR WITH ACTIVITY IN AN INTRACRANIAL XENOGRAFT MODEL OF TRIPLE NEGATIVE BREAST CARCINOMA WITH HOMOLOGOUS RECOMBINATION REPAIR DEFICIENCY

Abstract The Poly (ADP-ribose) polymerase (PARP) family has numerous essential functions in cellular processes such as transcription, chromatin remodelling, DNA damage response and repair as well as apoptosis. PARP inhibition blocks base excision repair and results in conversion of SSBs to DNA double-strand break (DSBs), the most deleterious form of DNA damage. DSBs can be repaired by homologous recombination repair (HRR) or non-homologous end joining (NHEJ). The physiological importance of HRR is underscored by the observation of genomic instability in HRR-deficient (HRD+) cells and, importantly, the association of cancer predisposition and developmental defects with mutations in HRR genes. PARP1 and PARP2 are required for SSB repair, while PARP1 is also involved in the repair of DNA double-strand breaks (DSBs) and replication fork damage. AZD9574 is a novel brain penetrant PARP1 inhibitor that acts by selectively inhibiting and trapping PARP1 at the sites of SSBs. While AZD9574 inhibited PARP1 enzymatic activity in all tested cell lines irrespective of the HRR status (IC50 range between 0.3 – 2 nM), colony formation assay in isogenic cell lines pairs confirmed higher potency and selectivity towards HRD+ models. In vivo, AZD9574 demonstrated dose-dependent efficacy in a BRCA1 mutant MDA-MB-436 subcutaneous xenograft model. Anti-tumour effects of AZD9574 were manifested by significant growth regressions that were durable after treatment withdrawal. An intracranial xenograft model of breast cancer brain metastases was developed to assess the efficacy of AZD9574 in the context of blood-brain barrier penetrance. Treatment of animals with established intracranial lesions showed sustained tumour growth suppression resulting in a significantly extended survival of tumour-bearing mice. Collectively, we believe that our data support the development of AZD9574 as a potential therapy for patients with HRD+ breast cancer whose disease has spread to the brain.This abstract was previously presented at AACR 2022 (Hamerlik et al, AACR 2022, Abs #3880)

The C-Terminal Domain of Y-Box Binding Protein 1 Exhibits Structure-Specific Binding to Poly(ADP-Ribose), Which Regulates PARP1 Activity.

Y-box-binding protein 1 (YB-1) is a multifunctional protein involved in the regulation of gene expression. Recent studies showed that in addition to its role in the RNA and DNA metabolism, YB-1 is involved in the regulation of PARP1 activity, which catalyzes poly(ADP-ribose) [PAR] synthesis under genotoxic stress through auto-poly(ADP-ribosyl)ation or protein trans-poly(ADP-ribosyl)ation. Nonetheless, the exact mechanism by which YB-1 regulates PAR synthesis remains to be determined. YB-1 contains a disordered Ala/Pro-rich N-terminal domain, a cold shock domain, and an intrinsically disordered C-terminal domain (CTD) carrying four clusters of positively charged amino acid residues. Here, we examined the functional role of the disordered CTD of YB-1 in PAR binding and in the regulation of PARP1-driven PAR synthesis in vitro. We demonstrated that the rate of PARP1-dependent synthesis of PAR is higher in the presence of YB-1 and is tightly controlled by the interaction between YB-1 CTD and PAR. Moreover, YB-1 acts as an effective cofactor in the PAR synthesis catalyzed by the PARP1 point mutants that generate various PAR polymeric structures, namely, short hypo- or hyperbranched polymers. We showed that either a decrease in chain length or an increase in branching frequency of PAR affect its binding affinity for YB-1 and YB-1–mediated stimulation of PARP1 enzymatic activity. These results provide important insight into the mechanism underlying the regulation of PARP1 activity by PAR-binding proteins containing disordered regions with clusters of positively charged amino acid residues, suggesting that YB-1 CTD-like domains may be considered PAR “readers” just as other known PAR-binding modules.

Abstract 2609: AZD9574 is a novel, brain penetrant PARP-1 selective inhibitor with activity in an orthotopic, intracranial xenograft model with aberrant DNA repair

Abstract The Poly (ADP-ribose) polymerase (PARP) family has numerous essential functions in cellular processes such as transcription, chromatin remodelling, DNA damage response and repair as well as apoptosis. PARP inhibition blocks base excision repair and results in conversion of SSBs to DNA double-strand break (DSBs). DSBs are the most deleterious form of DNA damage that can be generated by exogenous DNA damaging agents or endogenous replication stress. DSBs can be repaired by homologous recombination repair (HRR) or non-homologous end joining (NHEJ). The physiological importance of HRR is underscored by the observation of genomic instability in HRR-deficient (HRD+) cells and, importantly, the association of cancer predisposition and developmental defects with mutations in HRR genes. PARP1 and PARP2 are required for SSB repair, while PARP1 is also involved in the repair of DNA double-strand breaks (DSBs) and replication fork damage. Recent reports suggest the PARP1 inhibition is sufficient to elicit an anti-proliferative effect and that PARP2 is essential for the survival of hematopoietic stem and progenitor cells in animal models.AZD9574 is a novel brain penetrant PARP1 inhibitor that acts by selectively inhibiting and trapping PARP1 at the sites of SSBs. AZD9574 exhibited >8000-fold selectivity for PARP1 compared to PARP2 and other members of the PARP family (PARP2, PARP3, PARP5a and PARP6) in biochemical assays. While AZD9574 inhibited PARP1 enzymatic activity in all tested cell lines irrespective of the HRR status (IC50 range between 0.3 - 2 nM), colony formation assay in isogenic cell lines pairs confirmed higher potency and selectivity towards HRD+ models (DLD1 BRCA2-/-; SKOV-3 BRCA2-/- and SKOV-3 PALB2-/-). For example, AZD9574 IC50 in the BRCA2-/- DLD1 cells was 1.38 nM compared to IC50 > 40 µM BRCA2wt cells, which corresponds to a ~20,000-fold greater efficacy in the BRCA2-/-cells (ratio of AZD9574 IC50 in BRCA2wt divided by BRCA2-/-) compared to the wild type parental line. In vivo, AZD9574 demonstrated dose-dependent efficacy in a BRCA1 mutant MDA-MB-436 subcutaneous xenograft model. Anti-tumour effects of AZD9574 were manifested by pronounced growth regressions that were durable after treatment withdrawal. An intracranial xenograft model of breast cancer brain metastases was developed to assess the efficacy of AZD9574 in the context of blood-brain barrier penetrance. Treatment of animals with established intracranial lesions using a dose of 3 mg/kg AZD9574 showed sustained tumour growth suppression resulting in a significantly extended survival of tumour-bearing mice. Collectively, we believe that our data support the development of AZD9574 as a potential therapy for patients with HRD+ breast cancer whose disease has spread to the brain. Citation Format: Kunzah Jamal, Anna Staniszewska, Jacob Gordon, Shenghua Wen, Frank McGrath, Gregory Dowdell, Dominic Kabbabe, Giuditta illuzzi, Matthew Griffin, Barry R. Davies, Petra Hamerlik. AZD9574 is a novel, brain penetrant PARP-1 selective inhibitor with activity in an orthotopic, intracranial xenograft model with aberrant DNA repair [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2609.

Abstract 1792: FGFR3 mediated PARP1 tyrosine 158 phosphorylation promotes PARP inhibitor resistance

Abstract Poly(ADP-ribose) polymerase (PARP) inhibitors (PARPi), which induce DNA damage by inhibiting PARP1 enzymatic activity and trapping PARP on the damaged DNA, are used to eliminate BRCA1/2-mutated (BRCAm) cancer. However, clinical observations suggest that BRCAm tumors develop PARPi resistance. Current strategies to overcome PARPi resistance include impeding multiple DNA repair pathways to induce excessive DNA damage. Here, we propose a novel strategy targeting oncogenic receptor tyrosine kinases to enhance PARP trapping. By developing triple-negative breast cancer (TNBC) cells with acquired talazoparib resistance, we observed a high prevalence of activated fibroblast growth factor receptor 3 (FGFR3) kinase in these cells through kinase antibody array analysis. Mass spectrometry analysis and in vitro kinase assay suggested that FGFR3 phosphorylated PARP1 at tyrosine residues 158 and 176. Biochemistry studies suggested that only PARP1 tyrosine 158 phosphorylation contributes to PARPi resistance in the cells we developed. We then developed a monoclonal antibody against tyrosine 158 phosphorylated PARP1, and found that high-level PARP1 tyrosine 158 phosphorylation positively correlated with PARPi resistance in breast cancer patient-derived xenograft models. We further demonstrated that the combination of FGFR inhibitor and PARPi delayed DNA repair with prolonged PARP trapping. Moreover, synergy between PARPi and FGFR inhibition was observed in multiple TNBC cell lines with PARPi resistance in vitro. The combination of PARPi and FGFR inhibitor also showed synergism in vivo, and treatment with the combination of PARPi and FGFR inhibitor was tolerated in mouse models. These findings reveal that PARP1 tyrosine 158 phosphorylation facilitates resolving of the PARPi-induced PARP-trapping, and that the tyrosine 158 phosphorylated PARP1 may be an effective biomarker to indicate FGFR3 mediated PARPi resistance. Citation Format: MeiKuang Chen, Yuan Gao, Weiya Xia, Yu-Han Wang, Jennifer K. Litton, Yu-Yi Chu, Funda Meric-Bernstam, Helen Piwnica-Worms, Banu Arun, Jordi Rodon Ahnert, Yongkun Wei, Wei-Chao Chang, Hung-Ling Wang, Coya Tapia, Constance T. Albarracin, Shao-Chun Wang, Ying-Nai Wang, Gabriel N. Hortobagyi, Chunru Lin, Liuqing Yang, Dihua Yu, Mien-Chie Hung. FGFR3 mediated PARP1 tyrosine 158 phosphorylation promotes PARP inhibitor resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1792.

DNA Methylation Malleability and Dysregulation in Cancer Progression: Understanding the Role of PARP1.

Mammalian genomic DNA methylation represents a key epigenetic modification and its dynamic regulation that fine-tunes the gene expression of multiple pathways during development. It maintains the gene expression of one generation of cells; particularly, the mitotic inheritance of gene-expression patterns makes it the key governing mechanism of epigenetic change to the next generation of cells. Convincing evidence from recent discoveries suggests that the dynamic regulation of DNA methylation is accomplished by the enzymatic action of TET dioxygenase, which oxidizes the methyl group of cytosine and activates transcription. As a result of aberrant DNA modifications, genes are improperly activated or inhibited in the inappropriate cellular context, contributing to a plethora of inheritable diseases, including cancer. We outline recent advancements in understanding how DNA modifications contribute to tumor suppressor gene silencing or oncogenic-gene stimulation, as well as dysregulation of DNA methylation in cancer progression. In addition, we emphasize the function of PARP1 enzymatic activity or inhibition in the maintenance of DNA methylation dysregulation. In the context of cancer remediation, the impact of DNA methylation and PARP1 pharmacological inhibitors, and their relevance as a combination therapy are highlighted.

PARP-DNA trapping ability of PARP inhibitors jeopardizes astrocyte viability: Implications for CNS disease therapeutics

There is emerging interest in the role of poly(ADP-ribose) polymerase-1 (PARP-1) in neurodegeneration and potential of its therapeutic targeting in neurodegenerative disorders. New generations of PARP inhibitors exhibit polypharmacological properties; they do not only block enzymatic activity with lower doses, but also alter how PARP-1 interacts with DNA. While these new inhibitors have proven useful in cancer therapy due to their ability to kill cancer cell, their use in neurodegenerative disorders has an opposite goal: cell protection. We hypothesize that newer generation PARP-1 inhibitors jeopardize the viability of dividing CNS cells by promoting DNA damage upon the PARP-DNA interaction. Using enriched murine astrocyte cultures, our study evaluates the effects of a variety of drugs known to inhibit PARP; talazoparib, olaparib, PJ34 and minocycline. Despite similar PARP enzymatic inhibiting activities, we show here that these drugs result in varied cell viability. Talazoparib and olaparib reduce astrocyte growth in a dose-dependent manner, while astrocytes remain unaffected by PJ34 and minocycline. Similarly, PJ34 and minocycline do not jeopardize DNA integrity, while treatment with talazoparib and olaparib promote DNA damage. These two drugs impact astrocytes similarly in basal conditions and upon nitrosative stress, a pathological condition typical for neurodegeneration. Mechanistic assessment revealed that talazoparib and olaparib promote PARP trapping onto DNA in a dose-dependent manner, while PJ34 and minocycline do not induce PARP-DNA trapping. This study provides unique insight into the selective use of PARP inhibitors to treat neurodegenerative disorders whereby inhibition of PARP enzymatic activity must occur without deleteriously trapping PARP onto DNA.

Abstract IA23: Marker-guided targeted therapy, PARP inhibitors, and novel post-translational modification of PD-L1

Abstract Poly (ADP-ribose) polymerase (PARP) inhibitors have emerged as promising therapeutics for many diseases, including cancer, in clinical trials. Three PARP inhibitors have been approved by the FDA to treat ovarian cancer with (olaparib and rucaparib) or without BRCA mutations (niraparib). BRCA1 and BRCA2 play essential roles in repairing DNA double-strand breaks, and a deficiency of BRCA proteins sensitizes cancer cells to PARP inhibition. My group recently demonstrated that receptor tyrosine kinase c-Met associates with and phosphorylates PARP1 at Tyr907. Phosphorylation of PARP1 Tyr907 increases PARP1 enzymatic activity and reduces binding to a PARP inhibitor, thereby rendering cancer cells resistant to PARP inhibition. Combining c-Met and PARP1 inhibitors synergized to suppress growth of breast cancer cells in vitro and xenograft tumor models. Similar synergistic effects were observed in a lung cancer xenograft tumor model. These results suggest that PARP1 pTyr907 abundance may predict tumor resistance to PARP inhibitors, and that treatment with a combination of c-Met and PARP inhibitors may benefit patients bearing tumors with high c-Met expression who do not respond to PARP inhibition alone (Nature Medicine 2016;22:194). We are working with clinicians at MD Anderson to initiate marker-guided clinical trials using combinational therapy of PARP and c-MET inhibitors. Extracellular interaction between programmed death ligand-1 (PD-L1) and programmed cell death protein-1 (PD-1) leads to tumor-associated immune escape. We further explored whether a cross-talk exists between PARP inhibition and PD-L1/PD-1 immune checkpoint axis and determined whether blockade of PD-L1/PD-1 potentiates PARP inhibitor (PARPi) in tumor suppression. PARP inhibitor (PARPi) treatment upregulates tumor cell PD-L1 expression, which attenuates PARPi efficacy via cancer-associated immunosuppression. The blockade of PD-L1 can restore the attenuated antitumor immunity and potentiate PARPi in tumor suppression (Clinical Cancer Res 2017;23:3711). My group also conducted a highly translational study, which identified a link between the ubiquitination and glycosylation pathways that regulate the immunosuppressive activity of PD-L1 (Nature Communications 2016;7:12632). We showed that glycosylation of PD-L1 is required for its protein stability and interaction with PD-1. In addition, we demonstrated that metformin-activated AMPK kinase downregulates PD-L1 through phosphorylation of Ser-195, which alters glycosylation of PD-L1 and functionally mimics anti-PD-L1 to block PD-L1/PD-1 interaction. It exhibited a highly potent synergistic effect of combination therapy of metformin, a drug that has been used to treat patients with diabetes and anti-CTLA4. The therapeutic efficacy could reach to survival rate of 50-70% in different syngeneic mouse models, which were treated by this combination therapy (Molecular Cell 2018;71:606). We also demonstrated that epithelial-mesenchymal transition (EMT) enhances PD-L1 in cancer stem-like cells (CSCs) by the EMT/β-catenin/STT3-PD-L1 signaling axis. Etoposide, a commonly used anticancer chemotherapy drug, is able to suppress this signaling axis, resulting in downregulation of PD-L1 to sensitize cancer cells to anti-Tim 3 therapy (Nature Communications 2018;9:1908). These findings provide potential therapeutic strategies to enhance cancer immune therapy efficacy by targeting PD-L1 stabilization to combat multiple cancer types. We identified TNFα as a major factor triggering cancer cell immunosuppression against T-cell surveillance via stabilization of programmed cell death-ligand 1 (PD-L1) (Cancer Cell 2016;30:925). To this end, in collaboration with StCube Pharmaceuticals Inc., we have developed monoclonal antibodies against glycosylation-specific PD-L1. Impressive therapeutic effect was observed through antibody-drug-conjugate approach (Cancer Cell 2018;33:187). Most recently, we unraveled an active defense system of PD-L1, previously known to only associate with passive defense activity. We discovered that cancer cells that express PD-L1 can secrete exosomes containing PD-L1 (exosome-PD-L1). The exosome-PD-L1 is able to directly interact with PD-1 to inhibit T-cell activation as well as T-cell killing activity (Cell Res 2018;28:862). Our new discoveries provide a new means to enhance therapeutic efficacy of immune checkpoint therapy. Citation Format: Mien-Chie Hung. Marker-guided targeted therapy, PARP inhibitors, and novel post-translational modification of PD-L1 [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr IA23.

Abstract NT-098: WINDOW OF OPPORTUNITY TRIAL: ASSESSING THE ADAPTIVE RESPONSE OF HGSOC TO PARPI FOR INFORMED COMBINATION THERAPIES

Abstract At least 50% of high grade serous ovarian cancer (HGSOC) patients display defects in the components of the homologous recombination (HR) DNA repair pathway, which is the only high fidelity DNA repair mechanism for double strand breaks (DSB). This provides an exciting opportunity for targeted therapies using drugs such as poly (ADP-ribose) polymerase inhibitors (PARPi). PARP is an important component of the base excision repair and alternative non-homologous recombination DNA repair pathways. Therefore, inhibition of PARP enzymatic activity induces DNA damage and leads to synthetic lethality in HR defective tumors. Several clinical trials with PARPi have shown an improvement of progression-free survival in patients but have only shown a limited effect on overall survival. As for many other targeted therapies, the cancer cells rapidly adapt to the stress induced by the drug and develop resistance. Resistance can either be genomic or adaptive. In adaptive resistance the cellular networks are rewired in the absence of genomic changes to mediate resistance. To overcome this resistance, several groups, including ours, suggested the use of PARPi-based combination therapies. Several clinical trials are currently testing PARPi combinations with drugs targeting the immune checkpoint, the DNA damage checkpoint, the PI3K pathway, and the MAPK pathway. Although several of these combinations are promising, the main challenge remains in finding the right drug combination for the right patient since each tumor has the potential to adapt differently to PARPi. We thus implemented a window of opportunity trial to determine whether it is possible to identify different adaptive responses to PARPi by comparing pre and post-treatment tumor samples from multiple sites in the peritoneal cavity. We used reversed phase protein array (RPPA) analysis to measure the expression of over 300 proteins in each tumor samples and compared pathways activity in pre and post treatment samples from individual patients. We also compared the adaptive response detected in cancer patients with the one observed in cell lines. Base on pathway scores, we developed an approach to predict which combination with PARPi would be most likely to benefit a specific patient. Overall, our results indicate that the adaptive response to PARPi treatment can be detected early during treatment and that individual patients utilize different pathways to adapt to the stress induced by the drug. This study reinforces the need and opportunity for informed combination therapies to improve outcomes in this devastating disease. Citation Format: Marilyne Labrie, Tae-Beom Kim, Zhenlin Ju, Sanghoon Lee, Yiling Lu, Ken Chen, Gordon B. Mills and Shannon N. Westin. WINDOW OF OPPORTUNITY TRIAL: ASSESSING THE ADAPTIVE RESPONSE OF HGSOC TO PARPI FOR INFORMED COMBINATION THERAPIES [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr NT-098.

Downregulation of Human DAB2IP Gene Expression in Renal Cell Carcinoma Results in Resistance to Ionizing Radiation.

Renal cell carcinoma (RCC) is known to be highly radioresistant but the mechanisms associated with radioresistance have remained elusive. We found DOC-2/DAB2 interactive protein (DAB2IP) frequently downregulated in RCC, is associated with radioresistance. In this study, we investigated the underlying mechanism regulating radioresistance by DAB2IP and developed appropriate treatment. Several RCC lines with or without DAB2IP expression were irradiated with ionizing radiation (IR) for determining their radiosensitivities based on colony formation assay. To investigate the underlying regulatory mechanism of DAB2IP, immunoprecipitation-mass spectrometry was performed to identify DAB2IP-interactive proteins. PARP-1 expression and enzymatic activity were determined using qRT-PCR, Western blot analysis, and ELISA. In vivo ubiquitination assay was used to test PARP-1 degradation. Furthermore, in vivo mice xenograft model and patient-derived xenograft (PDX) model were used to determine the effect of combination therapy to sensitizing tumors to IR. We notice that DAB2IP-deficient RCC cells acquire IR-resistance. Mechanistically, DAB2IP can form a complex with PARP-1 and E3 ligases that is responsible for degrading PARP-1. Indeed, elevated PARP-1 levels are associated with the IR resistance in RCC cells. Furthermore, PARP-1 inhibitor can enhance the IR response of either RCC xenograft model or PDX model. In this study, we unveil that loss of DAB2IP resulted in elevated PARP-1 protein is associated with IR-resistance in RCC. These results provide a new targeting strategy to improve the efficacy of radiotherapy of RCC.

Positron-Emission Tomographic Imaging of a Fluorine 18–Radiolabeled Poly(ADP-Ribose) Polymerase 1 Inhibitor Monitors the Therapeutic Efficacy of Talazoparib in SCLC Patient–Derived Xenografts

IntroductionInhibitors of poly-(ADP)-ribose polymerase (PARP) are promising therapeutics for SCLC. We tested whether PARP inhibitor (PARPi) target engagement as measured by a fluorine 18–radiolabeled PARPi ([18F]PARPi) has the potential to predict drug efficacy in vivo. MethodsTumor growth inhibition during daily talazoparib treatment was evaluated in mice engrafted with SCLC patient-derived xenografts to evaluate talazoparib efficacy at multiple doses. Mice were intravenously injected with [18F]PARPi radiotracer at multiple timepoints after single doses of oral talazoparib to quantitatively assess the extent to which talazoparib could reduce tumor radiotracer uptake and positron-emission tomographic (PET)/computer tomographic activity. Tumors were harvested and tumor poly-(ADP) ribose level was measured by enzyme-linked immunosorbent assay. ResultsA dose range of talazoparib with differential therapeutic efficacy was established, with significant delay in time to reach 1000 mm3 for tumors treated with 0.3 mg/kg (p = 0.02) but not 0.1 mg/kg talazoparib. On PET/computed tomography with [18F]PARPi, reduction in [18F]PARPi uptake after talazoparib dosing was consistent with talazoparib clearance, with reduction in PET activity attenuating over 24 hours. Talazoparib target engagement, measured by maximum tumor PET uptake, increased in a dose-dependent manner (3.9% versus 2.1% injected dose/g for 0.1 and 0.3 mg/kg at 3 hours post-talazoparib, p = 0.003) and correlated with PARP enzymatic activity among individual tumors as measured by total tumor poly-(ADP) ribose (p = 0.04, R = 0.62 at 1 hour post-talazoparib). ConclusionsPET imaging using [18F]PARPi has the potential to be a powerful tool in treatment monitoring by assessing PARPi target engagement in real-time.

Poly-ADP Ribosyl Polymerase 1 (PARP1) Regulates Influenza A Virus Polymerase.

Influenza A viruses (IAV) are evolutionarily successful pathogens, capable of infecting a number of avian and mammalian species and responsible for pandemic and seasonal epidemic disease in humans. To infect new species, IAV typically must overcome a number of species barriers to entry, replication, and egress, even while virus replication is counteracted by antiviral host factors and innate immune mechanisms. A number of host factors have been found to regulate the replication of IAV by interacting with the viral RNA-dependent RNA polymerase (RdRP). The host factor PARP1, a poly-ADP ribosyl polymerase, was required for optimal functions of human, swine, and avian influenza RdRP in human 293T cells. In IAV infection, PARP1 was required for efficient synthesis of viral nucleoprotein (NP) in human lung A549 cells. Intriguingly, pharmacological inhibition of PARP1 enzymatic activity (PARylation) by 4-amino-1,8-naphthalimide led to a 4-fold increase in RdRP activity, and a 2.3-fold increase in virus titer. Exogenous expression of the natural PARylation inhibitor PARG also enhanced RdRP activity. These data suggest a virus-host interaction dynamic where PARP1 protein itself is required, but cellular PARylation has a distinct suppressive modality, on influenza A viral polymerase activity in human cells.

PARP1 Inhibitors Eliminated Imatinib-Refractory Chronic Myeloid Leukemia Cells in Bone Marrow Microenvironment Conditions

Bone marrow microenvironment (BMM) also defined as a stem cell niche provides a major obstacle for current anti-leukemia treatment modalities including tyrosine kinase inhibitors (TKIs) such as imatinib. We recently reported that chronic myeloid leukemia (CML) stem cells are sensitive to PARP inhibitor (PARPi)-triggered synthetic lethality in conditions mimicking peripheral blood (normoxia, no stromal cells). We reported that PARPi olaparib and talazoparib eliminate imatinib-refractory CML cells in the conditions mimicking BMM (hypoxia, stromal cells present). The effect, however, is not as robust as that observed in normoxia implicating BMM-specific protective impact. Therefore, more efficient targeting of PARP is required to enhance synthetic lethal effect against CML in BMM.Most PARPi's have been designed to compete with NAD for a binding site on the PARP1 molecule. This strategy resulted in the discovery of nucleotide-like PARPi's that not only target PARP, but, unfortunately, other enzymatic pathways involving NAD and other nucleotides as co-factors. Using such inhibitors affects multiple NAD/nucleotide-dependent enzymatic pathways, which results in secondary toxic effects proceeding from the inactivation of other pathways, while the efficiency against the PARP pathway is diminished. Thus, the challenge is to design inhibitors based on other activities of PARP1. We reported that DSBs-dependent interaction of PARP1 with histone 4 (H4) resulted in activation of PARP1 enzymatic activity and stimulation of Alt-NHEJ.Using high-throughput screen we identified non-NAD-like inhibitors, which interfered with H4-mediated activation of PARP1 and were effective against several types of tumors including BRCA1-deficient CML cells. Here we show that combination of two structurally different PARPi's, NAD-like olaparib or talazoparib and non-NAD-like 5F02 resulted in more abundant elimination of CML cells and reduced toxicity to normal counterparts. Finally, the combination exerted synergistic effect in humanized immunodeficient mice bearing primary CML xenografts. Altogether, non-NAD-like PARP1i synergistically enhanced synthetic lethal effect of NAD-like PARPi in tumor cells without increasing the cytotoxic effect in normal cells.While synthetic lethality mediated by NAD-like PARPi is usually associated with enhanced accumulation of potentially lethal DSBs, non-NAD-like PARP1i 5F02 did not induce DSBs when used as a single agent and also in combination with olaparib. Thus, the mechanistic aspect of the synergistic effect of NAD-like and non-NAD-like PARPis needs to be elucidated. Nevertheless, our data suggest that combining NAD-like and allosteric non-NAD-like PARPi's may represent a viable therapeutic strategy for enhancing CML response to PARP inhibition and reducing the resistance that inevitably results from treatment with NAD-like PARPi's alone. DisclosuresValent:Novartis: Honoraria; Incyte: Honoraria; Pfizer: Honoraria.