BRCA1-deficient Cancer Research Articles

Abstract B055: Characterization of highly selective and CNS-penetrant PARP1 inhibitors

Abstract First-generation PARP1/2 inhibitors are FDA-approved for cancers such as breast, ovarian prostate and pancreas and provide significant therapeutic benefit to patients whose tumors harbor BRCA1 or BRCA2 mutations or homologous recombination deficiency (HRD). However, their use is associated with hematological toxicities that narrow their therapeutic index and restrict their application, particularly in combination with standard-of-care chemotherapy. All four FDA-approved PARP1 inhibitors are largely non-selective for the closely related enzyme PARP2, inhibition of which has been shown to drive hematotoxicity. Hence, the development of second-generation molecules highly selective for PARP1 over PARP2 offers a significant opportunity to 1) dramatically enhance therapeutic index, 2) enable additional precision medicine/combination approaches with chemotherapy, radiotherapy, immunotherapy and targeted agents and 3) expand the addressable patient population to those whose tumors harbour additional DDR defects. Utilising X-ray crystallography and structure-based drug design, we describe the characterization of exquisitely selective PARP1 inhibitors. These molecules demonstrate high potency in multiple biochemical and cell-based assays, including viability and colony forming unit read-outs in BRCA-deficient cancer cells, whilst sparing HR-proficient cancer cell lines and primary cells. The molecules are potent PARP1-DNA trappers and exhibit high selectivity across the PARP family and wider secondary pharmacology targets. The molecules potentiate the activity of ATR inhibitors when tested in ATM-deficient cancer cell lines. This chemical series generally exhibits highly desirable physico-chemical and in vitro ADME properties, which translate to high oral bioavailability and low clearance in rodent PK studies. Exemplar molecules also demonstrate high rat CNS penetrance using in vivo microdialysis. Lead molecules yield deep and durable dose-dependent anti-tumor efficacy in a variety of BRCAm xenograft models with responses continuing after cessation of treatment. The compounds demonstrate strong PK/PD relationships which correlate with anti-tumor efficacy using inhibition of tumor PARylation as a PD read-out. Strikingly, the molecules demonstrate compelling anti-tumor efficacy in BRCA-deficient xenograft models that are known to be largely resistant to FDA-approved non-selective PARP1/2 inhibitors. Taken together, these data predict low once daily therapeutic oral dosing based on a highly favorable pre-clinical PK profile and potent in vivo efficacy, with the potential to demonstrate improved efficacy and tolerability compared to marketed PARP inhibitors. In summary, we describe the characterization of next generation potent and selective CNS-penetrant PARP1 inhibitors. These molecules have the potential for an improved safety profile and greater combination potential with standard-of-care and targeted anti-cancer therapies. Citation Format: Phillip M Cowley, Barry E McGuinness, Gillian M Campbell, Alan Wise. Characterization of highly selective and CNS-penetrant PARP1 inhibitors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B055.

TATDN2 resolution of R-loops is required for survival of BRCA1-mutant cancer cells.

BRCA1-deficient cells have increased IRE1 RNase, which degrades multiple microRNAs. Reconstituting expression of one of these, miR-4638-5p, resulted in synthetic lethality in BRCA1-deficient cancer cells. We found that miR-4638-5p represses expression of TATDN2, a poorly characterized member of the TATD nuclease family. We discovered that human TATDN2 has RNA 3' exonuclease and endonuclease activity on double-stranded hairpin RNA structures. Given the cleavage of hairpin RNA by TATDN2, and that BRCA1-deficient cells have difficulty resolving R-loops, we tested whether TATDN2 could resolve R-loops. Using in vitro biochemical reconstitution assays, we found TATDN2 bound to R-loops and degraded the RNA strand but not DNA of multiple forms of R-loops in vitro in a Mg2+-dependent manner. Mutations in amino acids E593 and E705 predicted by Alphafold-2 to chelate an essential Mg2+ cation completely abrogated this R-loop resolution activity. Depleting TATDN2 increased cellular R-loops, DNA damage and chromosomal instability. Loss of TATDN2 resulted in poor replication fork progression in the presence of increased R-loops. Significantly, we found that TATDN2 is essential for survival of BRCA1-deficient cancer cells, but much less so for cognate BRCA1-repleted cancer cells. Thus, we propose that TATDN2 is a novel target for therapy of BRCA1-deficient cancers.

HTS discovery of PARP1-HPF1 complex inhibitors in cancer

PARP1/2 inhibitors (PARPi) are effective clinically used drugs for the treatment of cancers with BRCA deficiencies. PARPi have had limited success and applicability beyond BRCA deficient cancers, and their effect is diminished by resistance mechanisms. The recent discovery of Histone PARylation Factor (HPF1) and the role it plays in the PARylation reaction by forming a shared active site with PARP1 raises the possibility that novel inhibitors that target the PARP1–HPF1 complex can be identified. Herein we describe a simple and cost-effective high-throughput screening (HTS) method aimed at discovering inhibitors of the PARP1–HPF1 complex. Upon HTS validation, we first applied this method to screen a small PARP-focused library of compounds and then scale up our approach using robotic automation to conduct a pilot screen of 10,000 compounds and validating >100 hits. This work demonstrates for the first time the capacity to discover potent inhibitors of the PARP1-HPF1 complex, which may have utility as probes to better understand the DNA damage response and as therapeutics for cancer.

Interferon restores replication fork stability and cell viability in BRCA-defective cells via ISG15

DNA replication and repair defects or genotoxic treatments trigger interferon (IFN)-mediated inflammatory responses. However, whether and how IFN signaling in turn impacts the DNA replication process has remained elusive. Here we show that basal levels of the IFN-stimulated gene 15, ISG15, and its conjugation (ISGylation) are essential to protect nascent DNA from degradation. Moreover, IFNβ treatment restores replication fork stability in BRCA1/2-deficient cells, which strictly depends on topoisomerase-1, and rescues lethality of BRCA2-deficient mouse embryonic stem cells. Although IFNβ activates hundreds of genes, these effects are specifically mediated by ISG15 and ISGylation, as their inactivation suppresses the impact of IFNβ on DNA replication. ISG15 depletion significantly reduces cell proliferation rates in human BRCA1-mutated triple-negative, whereas its upregulation results in increased resistance to the chemotherapeutic drug cisplatin in mouse BRCA2-deficient breast cancer cells, respectively. Accordingly, cells carrying BRCA1/2 defects consistently show increased ISG15 levels, which we propose as an in-built mechanism of drug resistance linked to BRCAness.

A new vulnerability to BET inhibition due to enhanced autophagy in BRCA2 deficient pancreatic cancer

Pancreatic cancer is one of the deadliest diseases in human malignancies. Among total pancreatic cancer patients, ~10% of patients are categorized as familial pancreatic cancer (FPC) patients, carrying germline mutations of the genes involved in DNA repair pathways (e.g., BRCA2). Personalized medicine approaches tailored toward patients’ mutations would improve patients’ outcome. To identify novel vulnerabilities of BRCA2-deficient pancreatic cancer, we generated isogenic Brca2-deficient murine pancreatic cancer cell lines and performed high-throughput drug screens. High-throughput drug screening revealed that Brca2-deficient cells are sensitive to Bromodomain and Extraterminal Motif (BET) inhibitors, suggesting that BET inhibition might be a potential therapeutic approach. We found that BRCA2 deficiency increased autophagic flux, which was further enhanced by BET inhibition in Brca2-deficient pancreatic cancer cells, resulting in autophagy-dependent cell death. Our data suggests that BET inhibition can be a novel therapeutic strategy for BRCA2-deficient pancreatic cancer.

YCH1899, a Highly Effective Phthalazin-1(2H)-one Derivative That Overcomes Resistance to Prior PARP Inhibitors.

Poly(ADP-ribose) polymerase inhibitors (PARPi) have significant efficacy in treating BRCA-deficient cancers, although resistance development remains an unsolved challenge. Herein, a series of phthalazin-1(2H)-one derivatives with excellent enzymatic inhibitory activity were designed and synthesized, and the structure-activity relationship was explored. Compared with olaparib and talazoparib, compound YCH1899 exhibited distinct antiproliferation activity against olaparib- and talazoparib-resistant cells, with IC50 values of 0.89 and 1.13 nM, respectively. Studies of the cellular mechanism revealed that YCH1899 retained sensitivity in drug-resistant cells with BRCA1/2 restoration or 53BP1 loss. Furthermore, YCH1899 had acceptable pharmacokinetic properties in rats and showed prominent dose-dependent antitumor activity in olaparib- and talazoparib-resistant cell-derived xenograft models. Overall, this study suggests that YCH1899 is a new-generation antiresistant PARPi that could provide a valuable direction for addressing drug resistance to existing PARPi drugs.

Long-molecule scars of backup DNA repair in BRCA1- and BRCA2-deficient cancers.

Homologous recombination (HR) deficiency is associated with DNA rearrangements and cytogenetic aberrations1. Paradoxically, the types of DNA rearrangements that are specifically associated with HR-deficient cancers only minimally affect chromosomal structure2. Here, to address this apparent contradiction, we combined genome-graph analysis of short-read whole-genome sequencing (WGS) profiles across thousands of tumours with deep linked-read WGS of 46 BRCA1- or BRCA2-mutant breast cancers. These datarevealeda distinct class of HR-deficiency-enriched rearrangements called reciprocal pairs. Linked-read WGS showed that reciprocal pairs with identical rearrangement orientations gave rise to one of two distinct chromosomal outcomes, distinguishable only with long-molecule data. Whereas one (cis) outcome corresponded to the copying and pasting of a small segment to a distant site, a second (trans) outcome was a quasi-balanced translocation or multi-megabase inversion with substantial (10 kb) duplications at each junction. We propose an HR-independent replication-restart repair mechanism to explain the full spectrum of reciprocalpair outcomes. Linked-read WGS also identified single-strand annealing as a repair pathway that is specific to BRCA2 deficiency in human cancers. Integrating these features in a classifier improved discrimination between BRCA1- and BRCA2-deficient genomes. In conclusion, our data reveal classes of rearrangements that are specific to BRCA1 or BRCA2 deficiency as a source of cytogenetic aberrations in HR-deficient cells.

Functional annotation of variants of the BRCA2 gene via locally haploid human pluripotent stem cells.

Mutations in the BRCA2 gene are associated with sporadic and familial cancer, cause genomic instability and sensitize cancer cells to inhibition by the poly(ADP-ribose) polymerase (PARP). Here we show that human pluripotent stem cells (hPSCs) with one copy of BRCA2 deleted can be used to annotate variants of this gene and to test their sensitivities to PARP inhibition. By using Cas9 to edit the functional BRCA2 allele in the locally haploid hPSCs and in fibroblasts differentiated from them, we characterized essential regions in the gene to identify permissive and loss-of-function mutations. We also used Cas9 to directly test the function of individual amino acids, including amino acids encoded by clinical BRCA2 variants of uncertain significance, and identified alleles that are sensitive to PARP inhibitors used as a standard of care in BRCA2-deficient cancers. Locally haploid human pluripotent stem cells can facilitate detailed structure-function analyses of genes and the rapid functional evaluation of clinically observed mutations.

Selective Killing of BRCA2-Deficient Ovarian Cancer Cells via MRE11 Blockade

The MRE11 nuclease is essential during DNA damage recognition, homologous recombination, and replication. BRCA2 plays important roles during homologous recombination and replication. Here, we show that effecting an MRE11 blockade using a prototypical inhibitor (Mirin) induces synthetic lethality (SL) in BRCA2-deficient ovarian cancer cells, HeLa cells, and 3D spheroids compared to BRCA2-proficient controls. Increased cytotoxicity was associated with double-strand break accumulation, S-phase cell cycle arrest, and increased apoptosis. An in silico analysis revealed Mirin docking onto the active site of MRE11. While Mirin sensitises DT40 MRE11+/− cells to the Top1 poison SN-38, it does not sensitise nuclease-dead MRE11 cells to this compound confirming that Mirin specifically inhibits Mre11 nuclease activity. MRE11 knockdown reduced cell viability in BRCA2-deficient PEO1 cells but not in BRCA2-proficient PEO4 cells. In a Mirin-resistant model, we show the downregulation of 53BP1 and DNA repair upregulation, leading to resistance, including in in vivo xenograft models. In a clinical cohort of human ovarian tumours, low levels of BRCA2 expression with high levels of MRE11 co-expression were linked with worse progression-free survival (PFS) (p = 0.005) and overall survival (OS) (p = 0.001). We conclude that MRE11 is an attractive SL target, and the pharmaceutical development of MRE11 inhibitors for precision oncology therapeutics may be of clinical benefit.

Abstract 6190: Polymerase theta inhibition activates the cGAS-STING pathway and cooperates with immune checkpoint blockade in BRCA-deficient cancers

Abstract Cancers deficient in homologous recombination (HR) repair secondary to mutations in genes such as BRCA1 or BRCA2, are dependent on alternative DNA damage response (DDR) pathways to maintain genomic integrity, rendering them susceptible to synthetic lethal targeting of these pathways. Recently, inhibitors of polymerase theta (POLθ, encoded by POLQ), the critical enzyme in microhomology-mediated end-joining (MMEJ), have been shown to be synthetic lethal with HR repair deficiency (Zhou et al. Nature Cancer 2021). Both HR and MMEJ require nucleolytic DNA end-resection to allow for DSB repair, and we have previously shown that MMEJ acts as a barrier to DNA end-resection at DSBs (Patterson-Fortin et al. Cancer Research 2022). Given the synthetic lethality between HR and MMEJ leads to unrestrained DNA end-resection generating chromosomal abnormalities and the release of nuclear DNA into the cytoplasm, we hypothesized that POLθ inhibition in HR-deficient cancers would activate the cGAS/STING innate immune response pathway and facilitate immunotherapy. To investigate the interactions of POLθ inhibition with the immune microenvironment in HR-deficient cancers, we used human cell lines and genetically modified mouse models representative of BRCA1-deficient triple-negative breast cancer (TNBC) and BRCA2-deficient pancreatic ductal adenocarcinoma (PDAC). Genetic or pharmacological inhibition of POLθ using novobiocin, a first-in-class inhibitor of the POLθ ATPase domain, induced significantly increased cytosolic dsDNA contained in micronuclei. This free DNA was sensed by the cytosolic DNA sensor cyclic GMP-AMP (cGAMP) synthetase (cGAS), increasing synthesis of cGAMP, in HR-deficient tumor cells but not in HR-proficient tumor cells. Increased cGAMP bound to and activated stimulator of interferon genes (STING), triggering phosphorylation of TBK1 and ultimately of IRF3. Activation of the cGAS/STING pathway by POLθ inhibition drove the expression of type I interferon response elements, including PD-L1. Depletion of STING by siRNA or by CRISPR abrogated this pro-inflammatory signaling and abolished the anti-tumor efficacy of novobiocin-mediated POLθ inhibition. Pharmacologic inhibition of POLθ enhanced Granzyme B+ CD8+ T-cell tumor infiltration. Importantly, antibody-mediated depletion of CD8+ T-cell severely compromised the anti-tumor efficacy of novobiocin-mediated POLθ inhibition, whereas anti-tumor activity of POLθ inhibition was augmented with the addition of either anti-PD-1 or anti-CTLA-4 antibodies. These results demonstrate that POLθ inhibition in HR-deficient cancers mediates a pro-inflammatory response in HR-deficient TNBC or PDAC tumor microenvironments, and that immune checkpoint blockade inhibition enhances the therapeutic efficacy of POLθ inhibition. Citation Format: Jeffrey Patterson-Fortin, Heta Jadhav, Constantia Pantelidou, Tin Phan, Carter Grochala, Anita K. Mehta, Jennifer L. Guerriero, Gerburg M. Wulf, Brian M. Wolpin, Ben Z. Stanger, Andrew J. Aguirre, James M. Cleary, Alan D. D'Andrea, Geoffrey I. Shapiro. Polymerase theta inhibition activates the cGAS-STING pathway and cooperates with immune checkpoint blockade in BRCA-deficient cancers [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 6190.

Abstract 1994: Sustained delivery of PARP inhibitor Talazoparib for the treatment of BRCA deficient ovarian cancer

Abstract Ovarian cancer has long been known to be the deadliest cancer associated with the female reproductive system. More than 15% of ovarian cancer patients have a defective BRCA-mediated homologous recombination repair pathway that can be therapeutically targeted with PARP inhibitors, such as Talazoparib (TLZ). The expansion of TLZ clinical approval beyond breast cancer has been hindered due to the highly potent systemic side effects resembling chemotherapeutics. Here we report the development of a novel TLZ-loaded PLGA implant (InCeT-TLZ) that sustainedly releases TLZ over 25 days directly into the peritoneal cavity (i.p.) to treat BRCA-mutated metastatic ovarian cancer (mOC). Methods: InCeT-TLZ was fabricated by dissolving TLZ and PLGA in chloroform followed by extrusion and evaporation. Drug loading and release were confirmed by HPLC. The detailed structure of InCeT-TLZ was checked by scanning electron microscopy. The in vivo therapeutic efficacy of InCeT-TLZ was carried out in a murine Brca2-/-p53R172H/-Pten-/- genetically engineered peritoneally metastatic ovarian cancer model. Treatment started 7 days post tumor inoculation. All mice were monitored by IVIS imaging once a week to confirm and track tumor growth. On day 8, mice with tumors (4-5 mm in diameter) were divided into 4 groups: PBS i.p. injection (50 μl, three times a week), empty implant i.p. implantation (2.5 mm, every 25 days), TLZ i.p. injection (0.33 mg/kg of TLZ by i.p. injection three times a week), and InCeT-TLZ i.p. implantation (2.5 mm, equal to a total amount of 10 i.p. injection doses, every 25 days). Body weight was recorded three times a week as an indicator of treatment tolerance and efficacy. Mice were sacrificed when the body weight increased by 50% of the initial weight. Results and Conclusion: The drug-loaded PLGA implant was developed for the sustained and local delivery of TLZ to treat high-grade serous ovarian cancer with a loading efficiency of 26.75 μg/mm. Implants can be easily placed into the peritoneal cavity of patients or animals with an 18-gauge clinical needle. The extended survival in InCeT-TLZ treated mice compared to the TLZ injection group indicated that the slow release of TLZ maximizes PARP inhibitor therapy in the peritoneal cavity for disseminated cancer treatment. Decrease in ascites formation within the intraperitoneal cavity of the InCeT-TLZ group suggests a promising treatment for ovarian cancer-associated ascites and disease progression. Histopathology following peritoneal InCeT-TLZ implantation showed no toxicity in any surrounding organs at risk. This approach would offer new sustained therapy options for substantially enhancing the therapeutic window, increasing cure rates, reducing mortality and suffering, and offering major enhancements in quality of life for the thousands of women diagnosed with OC yearly. Citation Format: Shicheng Yang, Needa Brown, Abigail L. Campbell, Kushi Aggarwal, Daniela M. Dinulescu, Srinivas Sridhar. Sustained delivery of PARP inhibitor Talazoparib for the treatment of BRCA deficient ovarian 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 1994.

Abstract 1066: PARP inhibitor rucaparib in combination with imipridones ONC201 or ONC212 demonstrates preclinical synergy against BRCA1/2-deficient breast, ovarian, and prostate cancer cells

Abstract BRCA1/2 genes encode proteins that mediate homologous recombination and repair (HRR). BRCA1/2 mutations increase risk of breast, ovarian, prostate and other cancers. BRCA1/2 mutated tumor cells are sensitive to PARP inhibitors (PARPi), that cause DNA replication fork to collapse. Approximately 40% of patients develop PARPi resistance through different mechanisms. One of the mechanisms of PARPi resistance is through P13k/Akt pathway activation. Imipridones are TRAIL-inducing compounds which are PERK-independent activators of the integrated stress response, and dual inhibitors of Akt/ERK. We hypothesized that combining imipridones with PARPi would overcome PARPi resistance due to Akt activation. PARPi also sensitize various solid tumors to recombinant TRAIL and DR5 agonist antibodies thereby further suggesting possible synergies between imipridones and PARPi. Our lab previously showed efficacy of imipridone ONC201 in BRCA-deficient cancer cells and potential synergy with olaparib (2017 AACR annual meeting abstract), without further investigating potential synergistic mechanisms. We explored combination drug synergy of rucaparib (PARP inhibitor) and imipridones (ONC212 and ONC201) in BRCA1/2-deficient breast, ovarian and prostate cancer cell lines. CellTiterGLO viability assays were performed after 72 hours to demonstrate synergistic effects of combination treatment. Western blots were performed to investigate the effect of combination treatment on the Akt pathway, as well as expression of cellular metabolic stress protein ATF4. Cytokine profiling using the Luminex 200 technology was used to study the effect of treatment on the tumor microenvironment. Combination treatment (rucaparib and ONC212, rucaparib and ONC201) in BRCA–deficient cell lines (HCC1937, PEO1, KURAMOCHI, 22RV1, LNCAP) showed synergistic reduction in cell viability. In the HCC 1937 cell line, combination studies of rucaparib-ONC212 and rucaparib-ONC201 showed a synergystic effect, as calculated by Compusyn software, combination indexes below one were observed at concentration of 0.29-37.5 of µM rucaparib with ONC201 at 1.25-5 µM and ONC212 6.25-50 nM with the best combination index of 0.7 for rucaparib-ONC201 combination and 0.31 for rucaparib-ONC212. We similarly observed synergy in other cell lines with combination treatment. Western blot analysis of rucaparib-ONC212 combination showed total Akt protein reduction and an increase in ATF4 consistent with the synergistic effects. Further studies are ongoing to characterize possible mechanisms and effects of PARP inhibitor-imipridone combination treatment on immune-mediated killing. Our findings identify novel PARP inhibitor-imipridone therapy combinations that can be further developed for treatment of BRCA1/2 deficient cancers. Citation Format: Maryam Ghandali, Kelsey E. Huntington, praveen Srinivasan, Don S. Dizon, Stephanie L. Graff, Benedito A. Carneiro, Wafik S. El-Deiry. PARP inhibitor rucaparib in combination with imipridones ONC201 or ONC212 demonstrates preclinical synergy against BRCA1/2-deficient breast, ovarian, and prostate cancer cells [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 1066.

Abstract 6172: Characterization of a novel series of highly selective PARP1 inhibitors

Abstract First-generation PARP1 inhibitors have provided significant therapeutic benefit to patients whose tumors exhibit homologous repair deficiencies, including BRCA mutations, however their use has been associated with hematological toxicities that have restricted their application, particularly in combination with standard-of-care chemotherapy. All four FDA-approved PARP1 inhibitors are largely non-selective for the closely related enzyme PARP2, inhibition of which has been shown to drive hematotoxicity. Hence, the development of second-generation molecules highly selective for PARP1 over PARP2 offers a significant opportunity to 1) dramatically enhance therapeutic index, 2) enable additional precision medicine/combination approaches with chemotherapy, radiotherapy, immunotherapy and targeted agents and 3) expand the addressable patient population to those whose tumors harbor additional DDR defects. Utilizing X-ray crystallography and structure-based drug design, we describe the characterization of a novel chemical series of highly selective PARP1 inhibitors. These molecules demonstrate high potency in multiple biochemical and cell-based assays, including viability and colony forming unit read-outs in BRCA-deficient cancer cells, whilst sparing isogenically-paired BRCA wild-type cells and non-transformed primary cell lines. The molecules are potent PARP1-DNA trappers and exhibit high affinity, high selectivity and prolonged residence time in biophysical surface plasmon resonance (SPR) binding assays. The molecules also demonstrate exceptionally high selectivity for PARP1 over PARP2, and across the mono and polyPARP family, using a cell-based NanoBRET target engagement assay. This chemical series generally exhibits highly desirable physico-chemical and in vitro ADME properties, coupled with an excellent in vitro safety profile, which translate to high oral bioavailability and low clearance in rodent PK studies. Lead molecules yield deep and durable anti-tumor efficacy in a BRCA1m MDA-MB-436 breast cancer xenograft model with responses continuing after cessation of treatment. Percent tumor regression and post-dose tumor control was superior to olaparib at 1/10th of the dose. Importantly, our molecules demonstrate prolonged tumor residence time and a markedly superior tumor:plasma ratio compared to competitor PARP1-selective inhibitors. Taken together, these data predict low therapeutic dosing with the potential to demonstrate improved efficacy and tolerability compared to marketed PARP inhibitors. In summary, we describe the characterization of novel potent and selective PARP1 inhibitors. These molecules demonstrate excellent in vitro ADMET and in vivo PK, coupled with profound anti-tumor efficacy and tumor-targeting properties in a genetically-defined mouse model, supporting their progression into clinical studies. Citation Format: Phillip M. Cowley, Barry E. McGuinness, Gillian M. Campbell, Alan Wise. Characterization of a novel series of highly selective PARP1 inhibitors [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 6172.

RETRACTED ARTICLE: Polymerase θ inhibition activates the cGAS-STING pathway and cooperates with immune checkpoint blockade in models of BRCA-deficient cancer

Recently developed inhibitors of polymerase theta (POLθ) have demonstrated synthetic lethality in BRCA-deficient tumor models. To examine the contribution of the immune microenvironment to antitumor efficacy, we characterized the effects of POLθ inhibition in immunocompetent models of BRCA1-deficient triple-negative breast cancer (TNBC) or BRCA2-deficient pancreatic ductal adenocarcinoma (PDAC). We demonstrate that genetic POLQ depletion or pharmacological POLθ inhibition induces both innate and adaptive immune responses in these models. POLθ inhibition resulted in increased micronuclei, cGAS/STING pathway activation, type I interferon gene expression, CD8+ T cell infiltration and activation, local paracrine activation of dendritic cells and upregulation of PD-L1 expression. Depletion of CD8+ T cells compromised the efficacy of POLθ inhibition, whereas antitumor effects were augmented in combination with anti-PD-1 immunotherapy. Collectively, our findings demonstrate that POLθ inhibition induces immune responses in a cGAS/STING-dependent manner and provide a rationale for combining POLθ inhibition with immune checkpoint blockade for the treatment of HR-deficient cancers.

Abstract P1-13-10: Deconvoluting dynamics of acquired chemoresistance in Triple Negative Breast Cancer tumors

Abstract Background: BRCA deficient Triple negative breast cancers (TNBC) are effectively treated with platinum agents but, upon relapse, resistance is common. A number of genes have been shown to mediate chemoresistance in vitro, but none have been clinically useful due to the heterogeneous mechanisms of in vivo tumor chemo-resilience. Methods: We attacked this problem by recapitulating the generation of in vivo resistance to cisplatin in 50 mice bearing a platinum sensitive TNBC patient derived xenograft (PDX) TM00099, a BRCA1 deficient type 1 tandem duplicator phenotype tumor. Untreated tumors were compared to residual tumors that were sampled after the first cycle of cisplatin, upon recovery, and after the second cycle of drug which generated platinum resistant and sensitive tumors. Our earlier work on TM00099 suggested the existence of two major subspecies, designated A and B, with shifts observed in their proportions post treatment (1). We deconvoluted the bulk tumors into their clonal components to assess the precise numerical fluxes after each treatment cycle to gain insight into the cellular basis of the emergence of in vivo resistance. Results: 55 single cell derived clones isolated from bulk TM00099 tumors were genomically characterized identifying five subclonal populations: B, CCR, and variations of the original A: A25, A33 and A50. SNP analyses indicated that these five subclones comprised the vast majority (~93%) of all TM00099 tumors. Lineage analysis revealed that all A clones were related but distinct from B. CCR however had both A and B SNP markers and relatively higher ploidy indicating that CCR is a fusion of ancestral A and B clones. We found that A50, A33, and CCR were ~1.9X more resistant to cisplatin (mean IC50=4.4 µM) compared to the sensitive clones A25 and B (mean IC50=2.3 µM; p=0.002) in vitro. Although B clones had similar IC50 to A25s, B had improved in vitro survival at higher concentrations of cisplatin suggesting a dormancy-like phenotype: the persistent B cells did not recover within 50 days after in vitro exposure to cisplatin. Using clonal markers in bulk tumors, we found excellent concordance with their in vitro phenotypic analysis: after the first cycle of cisplatin, there was a proportional decline in A25, an enrichment of B, and stable proportions of A50, A33, and CCR. After the second platinum cycle, the emerging resistant tumors were mostly devoid of A25, and had low proportions of B, but highly enriched for A50, A33, CCR and an uncharacterized resistant A clone. The sensitive tumor residuals after the second platinum dose were predominantly comprised of B. Genomic analysis of the clones did not reveal any genetic drivers of resistance. Transciptionally, the sensitive B clones were characterized as mesenchymal or basal-like 1 TNBC subtypes, while the resistant As were categorized as basal-like 2, which have enhanced growth factor signaling and is associated with poorer response to chemotherapy. Correspondingly, the MAPK and stress assosciated NF-κB signaling pathways were augmented in As which were indeed more sensitive to blockade of MEK, EGFR and NF-κB than the platinum sensitive B clone. A25s which are sensitive revertants of the otherwise resistant A group use a mechanism to bypass resistance likely driven by ZNF350 and ZNF93. Conclusions: Our clonal reconstruction of TM00099 showed that acquired resistance can emerge by enrichment of not one but a composite of multiple preexisting resistant clones. The origins and characteristics of these clones are complex and include epigenetically driven resistance (A50 and A33), cell fusion mediated resistance (CCR), reversion to sensitivity (A25), and dormancy despite initial sensitivity (B). These nuances would not be discerned using bulk tumor assessments pointing to single cell genomic analyses as the most precise way to deconvolute the capacity of TNBC tumors to develop resistance.

Deoxycytidine kinase (dCK) inhibition is synthetic lethal with BRCA2 deficiency.

BRCA2 is a well-established cancer driver in several human malignancies. While the remarkable success of PARP inhibitors proved the clinical potential of targeting BRCA deficiencies, the emergence of resistance mechanisms underscores the importance of seeking novel Synthetic Lethal (SL) targets for future drug development efforts. In this work, we performed a BRCA2-centric SL screen with a collection of plant-derived compounds from South America. We identified the steroidal alkaloid Solanocapsine as a selective SL inducer, and we were able to substantially increase its potency by deriving multiple analogs. The use of two complementary chemoproteomic approaches led to the identification of the nucleotide salvage pathway enzyme deoxycytidine kinase (dCK) as Solanocapsine's target responsible for its BRCA2-linked SL induction. Additional confirmatory evidence was obtained by using the highly specific dCK inhibitor (DI-87), which induces SL in multiple BRCA2-deficient and KO contexts. Interestingly, dCK-induced SL is mechanistically different from the one induced by PARP inhibitors. dCK inhibition generates substantially lower levels of DNA damage, and cytotoxic phenotypes are associated exclusively with mitosis, thus suggesting that the fine-tuning of nucleotide supply in mitosis is critical for the survival of BRCA2-deficient cells. Moreover, by using a xenograft model of contralateral tumors, we show that dCK impairment suffices to trigger SL in-vivo. Taken together, our findings unveil dCK as a promising new target for BRCA2-deficient cancers, thus setting the ground for future therapeutic alternatives to PARP inhibitors.

Abstract P4-08-09: Deoxycytidine kinase (dCK) inhibition is synthetic lethal with BRCA2-deficiency

Abstract BRCA2 is a well-established cancer driver in several human malignancies. While the remarkable success of PARP inhibitors proved the clinical potential of targeting BRCA deficiencies, the emergence of resistance mechanisms underscores the importance of seeking novel Synthetic Lethal (SL) targets for future drug development efforts. In this work, we performed a BRCA2-centric SL screen with a collection of plant-derived compounds from South America. We identified the steroidal alkaloid Solanocapsine as a selective SL inducer, and we were able to substantially increase its potency by deriving multiple analogs. The use of two complementary chemoproteomic approaches led to the identification of the nucleotide salvage pathway enzyme deoxycytidine kinase (dCK) as Solanocapsine’s target responsible for its BRCA2-liked SL induction. Additional confirmatory evidence was obtained by using a highly specific dCK inhibitor (DI-87), which induces SL in multiple BRCA2-deficient and KO contexts. Interestingly, dCK-induced SL is mechanistically different from the one induced by PARP inhibitors. dCK inhibition generates substantially lower levels of DNA damage, and cytotoxic phenotypes are associated exclusively with mitosis, thus suggesting that the fine-tuning of nucleotide supply in mitosis is critical for the survival of BRCA2-deficient cells. Moreover, by using a xenograft model of contralateral tumors, we show that dCK impairment suffices to trigger SL in-vivo. Taken together, our findings unveil dCK as a promising new target for BRCA2-deficient cancers, which provides future therapeutic alternatives to PARP inhibitors. Citation Format: María L. Guantay, Cintia A. Garro, Sebastian Siri, María Pansa, Sonja Ghidelli-Disse, Natalia Paviolo, Ana Racca, Viviana Nicotra, Caius Radu, José L. Bocco, Rosana Felice, Israel Gloger, Marcel Muelbaier, Gerard Drewes, Kevin Madauss, Manuela García, Vanesa Gottifredi, Gastón Soria. Deoxycytidine kinase (dCK) inhibition is synthetic lethal with BRCA2-deficiency [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-08-09.

In Silico Investigation of the Molecular Mechanism of PARP1 Inhibition for the Treatment of BRCA-Deficient Cancers.

The protein PARP1, which plays a crucial role in DNA repair processes, is an attractive target for cancer therapy, especially for BRCA-deficient cancers. To overcome the acquired drug resistance of PARP1, PARP1 G-quadruplex (G4) identified in the PARP1-promotor region is gaining increasing attention. Aiming to explore the molecular mechanism of PARP1 inhibition with PARP1 G4 and PARP1 as potential targets, a comparative investigation of the binding characteristics of the newly identified G4 stabilizer MTR-106, which showed modest activity against talazoparib-resistant xenograft models and the FDA-approved PARP1 inhibitor (PARPi) talazoparib, were performed through molecular simulations. Combined analyses revealed that, relative to the groove binding of talazoparib, MTR-106 induced the formation of a sandwich framework through stacking with dT1 and the capping G-pair (dG2 and dG14) of PARP1 G4 to present largely enhanced binding affinity. For the binding with PARP1, although both were located in the catalytic pocket of PARP1, MTR-106 formed more extensive interactions with the surrounding PARP1 residues compared to talazoparib, in line with its increased binding strength. Importantly, vdW interaction was recognized as a decisive factor in the bindings with PARP1 G4 and PARP1. Collectively, these findings demonstrated the ascendancy of MTR-106 over talazoparib at the atomic level and revealed that the dual targeting of PARP1 G4 and PARP1 might be pivotal for PARPi that is capable of overcoming acquired drug resistance, providing valuable information for the design and development of novel drugs.

The SHARON trial: A study of melphalan, BCNU, hydroxocobalamin, ascorbic acid, and autologous stem cell rescue for metastatic pancreatic cancer with an inherited BRCA mutation.

TPS774 Background: SHARON is a phase 1, single-arm trial to assess the safety and preliminary efficacy of 2 cycles of melphalan, BCNU, hydroxocobalamin (vitamin B12b), dose-escalated vitamin C, and autologous stem cells in patients with stage IV pancreatic cancer and an inherited, deleterious BRCA1/2 mutation. Cancers that evolve in patients with a germline BRCA1/ 2 mutation generally have loss or inactivation of the wild type allele, which results in hypersensitivity to melphalan. The melphalan dose-response is log-linear: doubling the melphalan area under the curve (AUC) doubles the log reduction in clonogenic survival. The melphalan AUC that can safely be achieved with melphalan followed by stem cell rescue has the potential to provide major log reductions in cancer cell survival in BRCA-deficient cancers. However, reversion mutations in BRCA can cause resistance to DNA-crosslinking agents such as melphalan. Studies have shown that oxidizing cellular glutathione (GSH) to glutathione disulfide (GSSG) can inhibit DNA repair and hypersensitize wild-type cancer cells to melphalan. In this trial, BCNU, vitamin B12b, and vitamin C are used to increase cancer cell GSSG levels. Methods: Eligibility criteria include stage IV pancreatic cancer, an inherited deleterious BRCA1/2 mutation, life expectancy of at least 6 months, measurable or non-measurable disease, and suitability for apheresis, chemotherapy, and stem cell infusion. Prior treatment with PARP inhibitors is allowed. Stem cells are collected by apheresis. Participants receive 2 cycles of melphalan 100 mg/m2, BCNU 150 mg/m2, vitamin B12b 1.45 g/m2, and vitamin C 3, 6, or 8 g/m2 on Day −2. Stem cell infusion administered on day 0. A 2 + 4 dose-escalation schedule is employed for vitamin C. Adverse reactions summarized by CTCAE 5.0. The primary endpoint is safety, and secondary endpoints are objective response rate (overall and for metastatic lesions) and overall and progression-free survival. Amendment pending to include stage IV, BRCA-related, HER2-negative breast cancer and PALB2 mutations. The trial is enrolling at Memorial Sloan Kettering Cancer Center and Massachusetts General Hospital. Clinical trial information: NCT04150042 .

CX-5461-inspired monofunctional platinum RNA polymerase I selective inhibitors with selective lethality in BRCA1-deficient cancer cells

Two platinum Pol I selective inhibitors P1-Q1 and P1-Q2 target the Pol I transcription machinery, facilitate the formation and stabilization of R-loops, induce severe DNA damage, and display synthetic lethality in BRCA1-deficient A549 cells.