BRCA1-deficient Breast Cancer Research Articles

DNA double-strand break-capturing nuclear envelope tubules drive DNA repair.

Current models suggest that DNA double-strand breaks (DSBs) can move to the nuclear periphery for repair. It is unclear to what extent human DSBs display such repositioning. Here we show that the human nuclear envelope localizes to DSBs in a manner depending on DNA damage response (DDR) kinases and cytoplasmic microtubules acetylated by α-tubulin acetyltransferase-1 (ATAT1). These factors collaborate with the linker of nucleoskeleton and cytoskeleton complex (LINC), nuclear pore complex (NPC) protein NUP153, nuclear lamina and kinesins KIF5B and KIF13B to generate DSB-capturing nuclear envelope tubules (dsbNETs). dsbNETs are partly supported by nuclear actin filaments and the circadian factor PER1 and reversed by kinesin KIFC3. Although dsbNETs promote repair and survival, they are also co-opted during poly(ADP-ribose) polymerase (PARP) inhibition to restrain BRCA1-deficient breast cancer cells and are hyper-induced in cells expressing the aging-linked lamin A mutant progerin. In summary, our results advance understanding of nuclear structure-function relationships, uncover a nuclear-cytoplasmic DDR and identify dsbNETs as critical factors in genome organization and stability.

Abstract 7130: Dysregulated DNA repaired elicited by type 1 PRMT inhibitor in clear cell renal cell carcinoma enables development of novel combination therapies

Abstract Clear cell renal cell carcinoma (ccRCC) is the most prevalent form of kidney cancer, notorious for its resistance to treatment and dismal prognosis. Advanced or metastatic cases on average display an 8% 5-year survival rate. Given the considerable number of diagnoses in advanced stages, it is evident that we require innovative therapeutic approaches. Our lab has developed a methodology to establish cell lines from tumors and adjacent normal tissues sourced from patients. Consequently, we've amassed a collection of cell lines, including those from patient-derived ccRCC and normal kidney cells. Using these ccRCC models, we demonstrated that the type I protein arginine methyltransferase inhibitor, MS023, elicits a dysregulated DNA repair phenotype. RNA-Seq analysis of ccRCC cells treated with MS023 unveiled a marked downregulation of genes associated with the homologous recombination (HR) pathway for double-strand break repair and the Fanconi Anemia (FA) pathway for interstrand crosslink (ICL) repair. In a follow-up investigation using paired normal and cancerous cell lines from the same patient, we identified resistance to MS023 in normal kidney cells that is not mirrored in the corresponding ccRCC cell line. This hints at a potential therapeutic window and provides a strong rationale for clinical pursuit. Poly-ADP ribose polymerase (PARP) inhibitors are known to yield a synergistic effect when employed in HR-deficient diseases, such as BRCA-deficient breast cancers. Since MS023 induces an HR-deficient phenotype, we hypothesize that combining it with Talazoparib, a PARP inhibitor, will produce a similar synergistic effect. Additionally, mitomycin C (MMC) is a clinically approved compound that induces ICLs. As MS023 impedes ICL repair by downregulating crucial ICL-repairing genes, combining these two drugs should likewise generate a synergistic effect. Through a drug screen across two of our ccRCC cell lines, we discovered that the cells exhibit sensitivity to various PARP inhibitors. Employing synergy checkerboard assays and the highest single-agent model of synergy, we have found that the combination of MS023 and Talazoparib, as well as the combination of MS023 and MMC, demonstrate synergy, significantly reducing cellular viability at lower doses of the combination treatments compared to mono-treatment conditions at their respective doses. Preliminary in vivo studies using patient-derived xenograft models have revealed that both MS023 and Talazoparib are well-tolerated in mice, both when administered individually and in combination. Our current knowledge base indicates that MS023 holds promise for clinical investigation, both as a standalone treatment and in combination with Talazoparib or MMC. These results present an exciting opportunity to shed new light on the challenging therapeutic landscape faced by ccRCC patients today. Citation Format: Karen Arevalo, Joseph Walton, Jalna Meens, Angel Sau Ni Ng, Laurie Ailles. Dysregulated DNA repaired elicited by type 1 PRMT inhibitor in clear cell renal cell carcinoma enables development of novel combination therapies [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 7130.

Aryl hydrocarbon receptor suppresses STING-mediated type I IFN expression in triple-negative breast cancer

Triple-negative breast cancer (TNBC) is one of the most aggressive types of cancer. Despite decades of intense investigation, treatment options remain limited, and rapid recurrence with distant metastases remains a significant challenge. Cancer cell-intrinsic production of cytokines such as type I interferons (IFN-I) is a known potent modulator of response to therapy in many cancers, including TNBC, and can influence therapeutic outcome. Here, we report that, in TNBC systems, the aryl hydrocarbon receptor (AhR) suppresses IFN-I expression via inhibition of STImulator of Interferon Genes (STING), a key mediator of interferon production. Intratumoral STING activity is essential in mediating the efficacy of PARP inhibitors (PARPi) which are used in the treatment of cancers harboring BRCA1 deficiency. We find that, in TNBC cells, PARPi treatment activates AhR in a BRCA1 deficiency-dependent manner, thus suggesting the presence of a negative feedback loop aimed at modulating PARPi efficacy. Importantly, our results indicate that the combined inhibition of PARP and AhR is superior in elevating IFN-I expression as compared to PARPi-alone. Thus, AhR inhibition may allow for enhanced IFN-I production upon PARPi in BRCA1-deficient breast cancers, most of which are of TNBC origin, and may represent a therapeutically viable strategy to enhance PARPi efficacy.

Abstract 1187 A novel activity of the RAD52 protein that is essential for the viability of BRCA-deficient breast and ovarian cancer cells

Abstract 1187 A novel activity of the RAD52 protein that is essential for the viability of BRCA-deficient breast and ovarian cancer cells

Abstract B037: Effect of cross-sex estrogen therapy on olaparib treatment in mice

Abstract Background: Transfeminine individuals—assigned male at birth—pursue gender-affirming estrogen therapy to treat their gender dysphoria. When a transmasculine individual is diagnosed with breast cancer, there are no clinical guidelines on how to manage gender-affirming estrogen therapy during breast cancer treatment. There are limited studies on the effects of estrogen therapy during breast cancer treatment. In this study, we used a BRCA-deficient breast cancer mouse model to investigate the effect of estrogen on olaparib treatment outcomes. Method: We established a cross-sex hormone model of femininity using 60 µg of weekly subcutaneous estrogen and implanted viable fragments of female K14-Cre Brca1f/fp53f/f murine tumors into healthy recipient mice (n=184): female controls, male controls, male castrated controls, males receiving estrogen, and castrated males receiving estrogen. When the tumors reached 5 mm, mice receiving estrogen were first randomized to stop or continue estrogen. Then, mice were further randomized in every arm to be treated with olaparib or vehicle. Mice were euthanized when the tumor reached 20 mm. Only olaparib treated arms are reported here, and all comparisons are with female controls. Results: Female control mice receiving olaparib have the longest median tumor-specific survival times (n=17; 103 days). Male controls (n=16) had significantly shorter survival than female controls (75 versus 103 days; p=0.031). Survival times for males that stopped estrogen (n=10; 39 days), males that continued estrogen (n=10; 50 days), and castrated males that stopped estrogen (n=19; 58 days) were significantly shorter than females (all p<0.01). Survival for male castrated mice that continued estrogen (n=10) was also shorter than females, but did not achieve significance (68 days; p=0.09). All mice that did not receive olaparib had a median survival of 14 days, validating the stability of our tumor model. Conclusion: When the same tumor is treated with olaparib, females had better outcomes then males, regardless of castration status or whether the male mice stopped or continued estrogen during olaparib treatment. Our work indicates that the gender at birth, rather than the hormonal milieu, affects outcomes for hormone-independent BRCA1-related breast cancer. Molecular analysis of the tumors is underway to understand the hormonal effects on the tumors and interaction with olaparib treatment. Citation Format: Yujing J. Heng, Lin Wang, Abhishek Thavamani, Erica S. Massicott, Vanessa C. Bret-Mounet, Gabrielle M. Baker, John G. Clohessy, Gerburg M. Wulf. Effect of cross-sex estrogen therapy on olaparib treatment in mice [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 B037.

Mechanism of substrate hydrolysis by the human nucleotide pool sanitiser DNPH1

Poly(ADP-ribose) polymerase (PARP) inhibitors are used in the clinic to treat BRCA-deficient breast, ovarian and prostate cancers. As their efficacy is potentiated by loss of the nucleotide salvage factor DNPH1 there is considerable interest in the development of highly specific small molecule DNPH1 inhibitors. Here, we present X-ray crystal structures of dimeric DNPH1 bound to its substrate hydroxymethyl deoxyuridine monophosphate (hmdUMP). Direct interaction with the hydroxymethyl group is important for substrate positioning, while conserved residues surrounding the base facilitate target discrimination. Glycosidic bond cleavage is driven by a conserved catalytic triad and proceeds via a two-step mechanism involving formation and subsequent disruption of a covalent glycosyl-enzyme intermediate. Mutation of a previously uncharacterised yet conserved glutamate traps the intermediate in the active site, demonstrating its role in the hydrolytic step. These observations define the enzyme’s catalytic site and mechanism of hydrolysis, and provide important insights for inhibitor discovery.

βhCG mediates immune suppression through upregulation of CD11b+ Gr1+ myeloid derived suppressor cells, CD206+ M2 macrophages, and CD4+ FOXP3+ regulatory T-cells in BRCA1 deficient breast cancers.

BRCA1 mutation is reported in about 70% of all triple negative breast cancers (TNBC), while BRCA1 defect due to promoter hypermethylation is seen in about 30%-60% of sporadic breast cancers. Although PARP inhibitors and platinum-based chemotherapy are used to treat these cancers, more efficient therapeutic approaches are required to overcome the resistance to treatment. Our previous findings have reported elevated βhCG expression but not αhCG in BRCA1 deficient breast cancers. As βhCG causes immune suppression in pregnancy, this study explored the immunomodulatory effect of βhCG in BRCA1mutated/deficient TNBC. We observed that Th1, Th2, and Th17 cytokines are upregulated in the presence of βhCG in BRCA1 defective cancers. In NOD-SCID and syngeneic mouse models, βhCG increases the frequency of Myeloid-derived suppressor cells in tumour tissues and contributes to macrophage reprogramming from antitumor M1 to pro-tumour M2 phenotype. βhCG reduces the CD4+ T-cell infiltration while increasing the density of CD4+ CD25+ FOXP3+ regulatory T-cell in BRCA1 deficient tumour tissues. In contrast, xenograft tumours with βhCG knocked down TNBC cells did not show these immune suppressive effects. We have also shown that βhCG upregulates pro-tumorigenic markers arginase1(Arg1), inducible nitric oxide synthase, PD-L1/PD-1, and NFκB in BRCA1 defective tumours. Thus, for the first time, this study proves that βhCG suppresses the host antitumor immune response and contributes to tumour progression in BRCA1 deficient tumours. This study will help develop new immunotherapeutic approaches for treating BRCA1 defective TNBC by regulating βhCG.

Transmembrane nuclease NUMEN/ENDOD1 regulates DNA repair pathway choice at the nuclear periphery.

Proper repair of DNA damage lesions is essential to maintaining genome integrity and preventing the development of human diseases, including cancer. Increasing evidence suggests the importance of the nuclear envelope in the spatial regulation of DNA repair, although the mechanisms of such regulatory processes remain poorly defined. Through a genome-wide synthetic viability screen for PARP-inhibitor resistance using an inducible CRISPR-Cas9 platform and BRCA1-deficient breast cancer cells, we identified a transmembrane nuclease (renamed NUMEN) that could facilitate compartmentalized and non-homologous end joining-dependent repair of double-stranded DNA breaks at the nuclear periphery. Collectively, our data demonstrate that NUMEN generates short 5' overhangs through its endonuclease and 3'→5' exonuclease activities, promotes the repair of DNA lesions-including heterochromatic lamina-associated domain breaks as well as deprotected telomeres-and functions as a downstream effector of DNA-dependent protein kinase catalytic subunit. These findings underline the role of NUMEN as a key player in DNA repair pathway choice and genome-stability maintenance, and have implications for ongoing research into the development and treatment of genome instability disorders.

MicroRNA-155-5p, Reduced by Curcumin-Re-Expressed Hypermethylated BRCA1, Is a Molecular Biomarker for Cancer Risk in BRCA1-methylation Carriers.

Constitutional BRCA1-methylation is a cancer risk factor for breast (BC) and ovarian (OC) cancer. MiR-155, regulated by BRCA1, is a multifunctional microRNA that plays a crucial role in the immune system. The present study assessed the modulation of miR-155-5p expression in peripheral white blood cells (WBCs) of BC and OC patients and cancer-free (CF) BRCA1-methylation female carriers. Additionally, we investigated the potential of curcumin to suppress miR-155-5p in BRCA1-deficient breast cancer cell lines. MiR-155-5p expression was measured using a stem-loop RT-qPCR method. Gene expression levels were determined using qRT-PCR and immunoblotting. MiR-155-5p was more highly expressed in the BRCA1-hypermethylated HCC-38 and UACC-3199 BC cell lines than in the BRCA1-mutated (HCC-1937) and WT BRCA1 (MDA-MB-321) cell lines. Curcumin suppressed miR-155-5p in the HCC-38 cells but not in the HCC-1937 cells via the re-expression of BRCA1. Elevated levels of miR-155-5p were detected in patients with non-aggressive and localized breast tumors and in patients with late-stage aggressive ovarian tumors, as well as in CF BRCA1-methylation carriers. Notably, IL2RG levels were reduced in the OC and CF groups but not in the BC group. Together, our findings suggest opposing effects of WBC miR-155-5p, according to the cell and cancer type. In addition, the results point to miR-155-5p as a candidate biomarker of cancer risk among CF-BRCA1-methylation carriers.

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.

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.

Homologous recombination deficiency prediction using low-pass whole genome sequencing in breast cancer

Homologous recombination repair deficiency (HRD) results in a defect in DNA repair and is a frequent driver of tumorigenesis. Poly(ADP-ribose) polymerase inhibitors (PARPi) or platinum-based therapies have increased theraputic effectiveness when treating HRD positive cancers. For breast cancer and ovairan cancer HRD companion diagnostic tests are commonly used. However, the currently used HRD tests are based on high-depth genome sequencing or hybridization-based capture sequencing, which are technically complex and costly. In this study, we modified an existing method named shallowHRD, which uses low-pass whole genome sequencing (WGS) for HRD detection, and estimated the performance of the modified shallowHRD pipeline. Our shallowHRD pipeline achieved an AUC of 0.997 in simulated low-pass WGS data, with a sensitivity of 0.981 and a specificity of 0.964; and achieved a higher HRD risk score in clinical BRCA-deficient breast cancer samples (p = 5.5 × 10−5, compared with BRCA-intact breast cancer samples). We also estimated the limit of detection the shallowHRD pipeline could accurately predict HRD status with a minimum sequencing depth of 0.1 ×, a tumor purity of > 20%, and an input DNA amount of 1 ng. Our study demostrates using low-pass sequencing, HRD status can be determined with high accuracy using a simple approach with greatly reduced cost.

Determination of BRCAness Phenotype in Breast Tumors for the Appointment of Neoadjuvant Chemotherapy Based on Platinum and Taxanes

The concept of BRCAness was developed because of similarities between sporadic and hereditary breast cancer. BRCAness defines the pathogenesis and treatment sensitivity of many types of cancer, as well as the presence of a defect in the homologous recombination repair of tumor cells simulating the loss of BRCA1 or BRCA2, as in the presence of germline mutations. The question of treatment effectiveness for BRCA-like tumors is controversial and open. Thus, the aim of this work was to study the effectiveness of neoadjuvant chemotherapy (NAC) in BRCA-deficient breast cancer patients without germline mutations. The study involved 130 patients with breast cancer in stages IIA-IIIB. The treatment regimen included neoadjuvant chemotherapy, surgery, and adjuvant chemotherapy. The materials used were tumor samples from before and after chemotherapy. DNA and RNA were isolated from the tumor material. RNA was used to assess the expression level of BRCA1, while DNA was used for methyl-sensitive PCR. A microarray analysis was performed on high-density DNA chips from an Affymetrix CytoScanTM HD Array to assess DNA copy number aberration (CNA status) and loss of heterozygosity. A statistical analysis was performed using the Statistica 8.0 application package. It was noted that the existence of copy number aberrations in genes was statistically significantly associated with tumor treatment response and disease prognosis. Patients with partial regression had a statistically significantly higher amount of deletion than patients without an objective response (5/25 patients; 16%), as shown in the general sample of patients (52.9% versus 27.1%, respectively) at p = 0.0001 and in patients treated with anthracycline-containing regimen (p = 0.0001). In addition, it was shown that patients with BRCA1 deletion had higher rates of metastatic-free survival (log rank test, p = 0.009). BRCAness patients had a higher rate of 5-year metastatic survival, but not of treatment efficacy. The prospective study showed the positive effect of assessing the BRCAness phenotype of a tumor before treatment and of prescribing personalized NAC regimens. The objective response rate was statistically significantly more often observed in the group of patients with personalized chemotherapy (85.0% (34/40 patients) versus 62.3% (56/90 patients); p = 0.007). Despite the controversial effectiveness of BRCA-like tumor treatment, our data showed high predictive and prognostic significance of the BRCAness phenotype for the personalization of platinum and taxane regimens.

STING agonism enhances anti-tumor immune responses and therapeutic efficacy of PARP inhibition in BRCA-associated breast cancer

Poly (ADP-ribose) polymerase (PARP) inhibitors exert their efficacy via synthetic lethal effects and by inducing cGAS/STING-mediated immune responses. We demonstrate that compared to monotherapies, combined PARP inhibition and STING agonism results in increased STING pathway activation, greater cytotoxic T-cell recruitment and enhanced dendritic cell activation in BRCA1-deficient breast cancer models. The combination markedly improved anti-tumor efficacy in vivo, with evidence of complete tumor clearance, prolongation of survival and induction of immunologic memory.

Revisiting PARP2 and PARP1 trapping through quantitative live-cell imaging.

Poly (ADP-ribose) polymerase-1 (PARP1) and 2 (PARP2) are two DNA damage-induced poly (ADP-ribose) (PAR) polymerases in cells and are the targets of PARP inhibitors used for cancer therapy. Strand breaks recruit and activate PARP1 and 2, which rapidly generate PAR from NAD+. PAR promotes the recruitment of other repair factors, relaxes chromatin, and has a role in DNA repair, transcription regulation, and RNA biology. Four PARP1/2 dual inhibitors are currently used to treat BRCA-deficient breast, ovarian, prostate, and pancreatic cancers. In addition to blocking the enzymatic activity of PARP1 and 2, clinical PARP inhibitors extend the appearance of PARP1 and PARP2 on chromatin after damage, termed trapping. Loss of PARP1 confers resistance to PARP inhibitors, suggesting an essential role of trapping in cancer therapy. Yet, whether the persistent PARP1 and 2 foci at the DNA damage sites are caused by the retention of the same molecules or by the continual exchange of different molecules remains unknown. Here, we discuss recent results from quantitative live-cell imaging studies focusing on PARP1 and PARP2's distinct DNA substrate specificities and modes of recruitment and trapping with implications for cancer therapy and on-target toxicities of PARP inhibitors.

Combined inhibition of EZH2 and ATM is synthetic lethal in BRCA1-deficient breast cancer

BackgroundThe majority of BRCA1-mutant breast cancers are characterized by a triple-negative phenotype and a basal-like molecular subtype, associated with aggressive clinical behavior. Current treatment options are limited, highlighting the need for the development of novel targeted therapies for this tumor subtype.MethodsOur group previously showed that EZH2 is functionally relevant in BRCA1-deficient breast tumors and blocking EZH2 enzymatic activity could be a potent treatment strategy. To validate the role of EZH2 as a therapeutic target and to identify new synergistic drug combinations, we performed a high-throughput drug combination screen in various cell lines derived from BRCA1-deficient and -proficient mouse mammary tumors.ResultsWe identified the combined inhibition of EZH2 and the proximal DNA damage response kinase ATM as a novel synthetic lethality-based therapy for the treatment of BRCA1-deficient breast tumors. We show that the combined treatment with the EZH2 inhibitor GSK126 and the ATM inhibitor AZD1390 led to reduced colony formation, increased genotoxic stress, and apoptosis-mediated cell death in BRCA1-deficient mammary tumor cells in vitro. These findings were corroborated by in vivo experiments showing that simultaneous inhibition of EZH2 and ATM significantly increased anti-tumor activity in mice bearing BRCA1-deficient mammary tumors.ConclusionTaken together, we identified a synthetic lethal interaction between EZH2 and ATM and propose this synergistic interaction as a novel molecular combination for the treatment of BRCA1-mutant breast cancer.

Abstract 1288: STING agonism enhances anti-tumor immune responses and therapeutic efficacy of PARP inhibition in BRCA-associated breast cancer

Abstract Poly (ADP-ribose) polymerase (PARP) inhibitors exert their efficacy by inducing synthetic lethal effects, as well as cGAS/STING-mediated immune responses in BRCA- and other homologous recombination repair-deficient cancer cells. Here, we investigated whether the immunologic and therapeutic effects of PARP inhibition in BRCA-deficient breast cancer models could be augmented by synthetic cyclic dinucleotide agonists of STING. In in vitro experiments, combined olaparib-mediated PARP inhibition and ADU-S100-mediated STING agonism induced a greater degree of STING pathway activation and proinflammatory cytokine production compared to monotherapies in BRCA1-deficient human MDA-MB-436 cells and mouse triple-negative breast cancer cell lines derived from a genetically engineered model of BRCA-deficient breast cancer. In vivo experiments were conducted in mice bearing completely established tumors derived from the genetically engineered mouse model expanded syngeneically. In this model, the combination also induced an improved immune response compared to either monotherapy alone, evidenced by a greater degree of cytotoxic T cell recruitment and activation, and enhanced dendritic cell activation and antigen presentation. Nanostring mRNA analysis indicated that combinatorial effects were the result of augmented interferon signaling and antigen processing, as well as heightened leukocyte and dendritic cell functions. The combination markedly improved anti-tumor efficacy in vivo compared to monotherapy treatment, with evidence of complete tumor clearance and prolongation of survival. Finally, in mice treated with the combination, tumors could not be re-established upon rechallenge, indicating the induction of immunologic memory. These results support the development of combined PARP inhibition and STING agonism in BRCA-associated breast cancer. Citation Format: Heta Jadhav, Constantia Pantelidou, Aditi Kothari, Renyan Liu, Gerburg Wulf, Jennifer L. Guerriero, Geoffrey I. Shapiro. STING agonism enhances anti-tumor immune responses and therapeutic efficacy of PARP inhibition in BRCA-associated breast cancer [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 1288.

Abstract 1133: Novobiocin-mediated polymerase theta inhibition induces cGAS/STING pathway activation and T-cell infiltration in BRCA-associated cancers

Abstract Recently developed inhibitors of polymerase theta have demonstrated synthetic lethality in pre-clinical tumor models of BRCA-associated cancers. Given that other DNA damage repair inhibitor therapies, including PARP inhibitors, induce changes in the tumor immune microenvironment, we asked whether polymerase theta inhibition would also alter the tumor microenvironment in BRCA-associated cancers. We have demonstrated that novobiocin, a first-in-class inhibitor of the polymerase theta ATPase domain, induces both innate and adaptive immune responses through activation of the cGAS/STING pathway in BRCA-deficient tumor cells. Novobiocin-mediated polymerase theta inhibition generates micronuclei, activating the cGAS/STING pathway in both BRCA1-deficient MDA-MB-436 triple-negative breast cancer and BRCA2-deficient CAPAN1 pancreatic cancer models in vitro. Activation of the STING pathway upregulated the expression of interferon response genes, which was abrogated upon siRNA-mediated STING depletion. As an adaptive response, novobiocin also increased expression of PD-L1 in BRCA-deficient tumor cells. These effects were not observed in isogenic cell lines in which wild-type BRCA expression was corrected by add-back. Using a genetically engineered syngeneic mouse model of BRCA1-deficient breast cancer, we confirmed novobiocin-mediated activation of the cGAS/STING pathway in vivo, resulting in the induction of pro-inflammatory signaling and ultimately robust CD8+ T-cell infiltration and activation. Taken together, our findings demonstrate that novobiocin-mediated polymerase theta inhibition induces local and systemic immune responses in a cGAS/STING-dependent manner and provide a rationale for combining polymerase theta inhibition with immunotherapies for the treatment of BRCA-associated cancers. Citation Format: Jeffrey Patterson-Fortin, Heta Jadhav, Tin Phan, Alan D'Andrea, Geoffrey I. Shapiro. Novobiocin-mediated polymerase theta inhibition induces cGAS/STING pathway activation and T-cell infiltration in BRCA-associated cancers [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 1133.

OGG1 Inhibition Triggers Synthetic Lethality and Enhances The Effect of PARP Inhibitor Olaparib in BRCA1-Deficient TNBC Cells.

BackgroundPARP1 plays a critical role in the base excision repair (BER) pathway, and PARP1 inhibition leads to specific cell death, through a synthetic lethal interaction, in the context of BRCA1/2 deficiency. To date, up to five different PARP inhibitors (PARPi), have been approved, nevertheless, the acquisition of resistance to PARPi is common and there is increasing interest in enhancing responses and expand their use to other tumour types.MethodsWe hypothesized that other BER members could be additional synthetic lethal partners with mutated BRCA genes. To test this, we decided to evaluate the glycosylase OGG1 as a potential candidate, by treating BRCA1 proficient and deficient breast cancer cells with PARPi olaparib and the OGG1 inhibitor TH5478.ResultsKnocking out BRCA1 in triple-negative breast cancer cell lines causes hypersensitivity to the OGG1 inhibitor TH5487. Besides, TH5487 enhances the sensitivity to the PARP inhibitor olaparib, especially in the context of BRCA1 deficiency, reflecting an additive interaction.DiscussionThese results provide the first evidence that OGG1 inhibition is a promising new synthetic lethality strategy in BRCA1-deficient cells, and could lead to a new framework for the treatment of hereditary breast and ovarian cancer.

47P Immune tumor microenvironment and markers of apoptosis in breast cancer in carriers of hereditary mutations in the BRCA1 gene

Tumor immune microenvironment is currently recognized as an important participant in carcinogenesis, influencing the behavior of tumor cells and treatment efficacy. It is suggested that defects in BRCA1/2 genes contribute to a high mutation load and high immunogenicity, which modulates immune microenvironment. At the same time, it was shown that BRCA1/2-associated breast cancer tumors do not belong to the category of immunoactive ones. In addition, there is evidence of the additional evaluation of TP53 mutation in these tumors and disruption of the cell death process, which can also be a factor of resistance to therapy. 40 patients with breast cancer with BRCA1 mutations 185delAG, 4153delA, 5382insC, 3819DELGTAA, 3875delGTCT, 300T>G, 2080delA and BRCA2 (6174delT)) determined by real-time polymerase chain reaction (PCR) were included. Immunohistochemical study was performed using monoclonal antibodies to Ki67, p53, CD8, CD4, CD68, CD163, BCL2. In the presented study patients with BRCA1-associated breast cancer, had high low tumor CD4/CD8 ratio in 50% of cases, which characterizes an immunosuppressive microenvironment. Mutation BRCA1 5382insC is associated with high level of CD4+ T-lymphocytes (p˂0.05), G3 is associated with a low CD4/CD8 ratio (p=0.056) and a low levels of CD68+M1, CD163+M2-macrophages (p=0,022, p=0.002); T1 correlates with high levels of CD8+ TILs (p=0.038) and high levels of CD163 (p=0.033). High Ki-67 is associated with a lack of BCL2 expression (p=0.04). Negative expression of BCL2 occurred in 52.5% of cases. A high level of p53 expression is described as the main type of expression in these tumors, suggesting a combination of TB53 and BRCA1 mutations and a violation of the mechanism of cell death in these patients. The immunosuppressive type of microenvironment prevails in the tumors of breast cancer patients with hereditary mutations in the BRCA1 gene, where a violation of the mechanism of cell death in BRCA1-deficient breast cancer was revealed. The main directions of future therapy of these tumors may be modification of the immune microenvironment and activation of the mechanisms of cell death.