Defective Homologous Recombination Research Articles

Abstract ED12-1: Pol-theta inhibitors in HRD tumors

Abstract Deficiency of homologous recombination (HR)-mediated DNA repair occurs through genetic or epigenetic inactivation of the BRCA1 and BRCA2 (BRCA1/2) genes, and it plays a role in the initiation and progression of many tumor types. HR-deficiency also provides unique opportunities for targeted therapy, as exemplified by the extreme sensitivity of BRCA1/2-mutated tumors to poly (ADP-ribose) polymerase inhibitors (PARPi). In the last decade, several PARP inhibitors have been approved for clinical use. At first, PARPi were solely used in combination with chemotherapeutics, and platinum sensitivity was used as a surrogate marker of HR-deficiency for enrolling patients for treatment. The promising clinical response of patients with germline BRCA1/2-mutations prompted the use of PARPi for patients with somatic BRCA1/2 mutations as well. In addition, it also opened the possibility for extended use of PARPi for the treatment of various types of ovarian, breast, pancreatic and prostate tumors with HR defects. PARPi efficacy is currently being evaluated in different clinical settings such as first line chemotherapy, neoadjuvant therapy, and combination therapy with chemo- or immuno-therapies. Despite initial sensitivity to PARPi, resistance to these drugs is emerging as the major obstacle to its clinical effectiveness in patients with HR-deficient tumors. PARPi resistance can result from several independent mechanisms, often leading to the restoration of Homologous Recombination and/or Replication Fork stabilization. In addition, resistance to PARPi often correlates with platinum resistance, which remains the backbone therapy for most BRCA1/2-mutated tumors. The absence of alternative therapeutic options for patients with tumors with innate or acquired resistance underlines the urgency to develop additional therapeutics. While several mechanisms of PARPi resistance have been described, an effective method for overcoming such resistance is still lacking. DNA polymerase theta (POLθ or POLQ) recently emerged as a new promising drug target for the treatment of HR-deficient tumors. POLθ expression is particularly high in subtypes of breast and ovarian tumors with defects in HR, where it mediates backup DNA double-strand breaks (DSBs) repair, thus compensating for the loss of HR. As a result, POLθ is synthetic lethal with HR, and POLθ inhibition in HR-deficient tumors induces cell death. In addition, POLθ inhibition synergizes with PARPi in the killing of HR-deficient tumors. Synthetic lethality between HR-deficiency and POLθ inhibition hinges on several functions of POLθ. The enzyme maintains genomic stability and prevents tumorigenesis. It is a crucial enzyme in the mutagenic microhomology-mediated end joining (MMEJ) repair of DSBs, a pathway that plays critical role in genomic stability. Inhibiting PARP1, a key enzyme in MMEJ, prevents POLθ recruitment to sites of laser micro-irradiation. Since POLθ inhibition and PARPi have additive effects on HR-deficient cells, these data suggest that POLθ also functions outside the PARP-mediated MMEJ pathway and in pathways that are critical to the survival of HR-deficient cells. POLθ is a large protein that contains 3 domains and is structurally and functionally distinct from other polymerases. The N-term contains a helicase-like ATPase domain that can unwind several types of DNA structures, while the central domain binds RAD51, displaces RPA proteins from resected DSBs, and antagonizes HR repair in an ATP-hydrolysis dependent manner. Finally, the C-term contains a nuclease domain which trims DNA ends and a polymerase domain that fills in nucleotides during MMEJ. Both the ATPase and polymerase domains are required for MMEJ repair. POLθ has several functions that preserve genomic stability, and POLθ-mediated MMEJ repair can compensate for the loss of HR. It remains unclear which of the many functions of POLθ underlies the synthetic lethality with HR. Nevertheless, POLθ exhibits unique features of druggability, providing a strong rationale for developing POLθ inhibitors. We have recently performed a small-molecule screen for inhibitors of POLθ ATPase activity and identified the antibiotic novobiocin (NVB) as a specific and potent inhibitor of human POLθ. NVB binds to purified POLθ protein, prevents its recruitment to DNA damage, and inhibits MMEJ repair. Importantly, we have shown that NVB selectively kills HR-deficient (BRCA1- and BRCA2-deficient) cells over wild-type cells and potentiates the cytotoxic effect of PARPi in HR-deficient tumor cells in vitro and in vivo. Moreover, NVB kills HR-deficient, PARPi-resistant tumor cells. These results suggest that NVB can be used alone or in combination with PARPi for treating HR-deficient tumors, even after they have acquired PARPi resistance. Accordingly, clinical trials have now been initiated for the use of NVB in the management of these tumors. Citation Format: Alan d’Andrea. Pol-theta inhibitors in HRD tumors [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 ED12-1.

Abstract P3-05-33: High expression of MiR-99b in breast cancer is associated with cell proliferation signaling and worse patient survivals in breast cancer

Abstract Background: MicroRNA (miR) is single stranded RNA which regulates the gene expression epigenetically by inhibiting the mRNA translation as well as promoting mRNA degradation. MiR-99b is known as a regulator of mechanistic target of rapamycin (mTOR) signaling and was reported as both onco-miR that promote cell proliferation and tumor suppressor-miR in multiple cancers. We hypothesized that there is a complex interaction between miR-99b and cancer signaling pathways as well as tumor microenvironment, which may influence outcomes. Methods: We studied the clinical relevance of miR-99b expression by performing in silico analyses of 1,961 breast cancer patients using two independent large cohorts; METABRIC and TCGA. Results: We found that high miR-99b breast cancer enriched MTORC1 signaling gene set (normalized enrichment score (NES)>1.50 in both cohorts), but not epithelial mesenchymal transition, NF-kB, nor TGF-β signaling gene sets (all false discovery rate (FDR)>0.40). High miR-99b breast cancer was significantly associated with high rates of mutation scores; silent- and non-silent-mutation rate, fraction altered, single-nucleotide variant neoantigens, as well as intratumor heterogeneity and homologous recombination defects. MiR-99b high tumors also enriched several cell proliferation-related gene sets; E2F targets, G2M checkpoint, and Mitotic spindle signaling, and was significantly associated with pathological grade, but not with subtype nor AJCC stage. High miR-99b breast cancer was significantly associated with low fraction of several stromal cells, including adipocytes cells, keratinocytes cells, and lymphatic endothelial cells in tumor microenvironment (all p< 0.001). On the other hand, miR-99b expression was not associated with immune function nor immune cell infiltration in breast cancer, except for dendritic cells (p=0.006 and 0.020, respectively). Finally, breast cancer with high miR-99b expression was significantly associated with worse overall survival (OS) (hazard ratio (HR)=1.23 (p< 0.001) and 1.26 (p=0.027), respectively) and disease-specific survival (DSS) (HR=1.28 (p< 0.001) and 1.39 (p=0.022), respectively), particularly DSS for ER-positive/HER2-negative breast cancer (HR=1.29 (p< 0.001) and 1.82 (p=0.017), respectively), consistently in two cohorts. In conclusion, we found that high miR-99b expressing breast cancer was significantly associated with not only MTORC1 but also cell proliferation and worse patient outcomes particularly in ER-positive/HER2-negative breast cancer. Citation Format: Masanori Oshi, Yoshihisa Tokumaru, Nobuhiko Sugito, Mahato Sasamoto, Rongrong Wu, Li Yan, Akimitsu Yamada, Takashi Ishikawa, Itaru Endo, Kazuaki Takabe. High expression of MiR-99b in breast cancer is associated with cell proliferation signaling and worse patient survivals in breast cancer [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 P3-05-33.

Abstract P4-01-06: LP-184, an acylfulvene class small molecule therapeutic, is synthetically lethal in HR deficient and PARP inhibitor resistant triple negative breast cancer

Abstract Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer accounting for approximately 15% to 20% of all newly diagnosed breast cancer cases. TNBCs are defined by lack of expression of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER-2).Due to its special molecular phenotype, it is not sensitive to endocrine therapy or targeted therapy. Therefore, chemotherapy such as anthracyclines or taxanes is the main systemic treatment, but the efficacy of conventional postoperative adjuvant chemoradiotherapy is poor. ~35% of TNBC tumors show abnormalities in the HR pathway, making them sensitive to poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi) and DNA-damaging agents. Although more than 40% BRCA1/2-deficient patients fail to respond to PARPi and a substantial proportion of patients acquire PARPi resistance over time. TNBCs heterogeneous nature, poor prognosis and limited treatment options presents the urgent need of development of novel agents targeting tumor specific alterations. Lantern Pharma is advancing LP-184, an acylfulvene-derived prodrug that is specifically activated in tumors that overexpress the oxidoreductase enzyme Prostaglandin Reductase 1 (PTGR1), for the treatment of solid tumor indications including TNBC. LP-184 has multiple mechanisms of action (MOA). While It is synthetically lethal in tumors harboring DNA damage repair defects including Homologous Recombination (HR) deficiencies, it can also interrupt transcription. LP-184 efficacy was tested in a panel of breast cancer cell lines and patient derived TNBC xenografts models, both sensitive as well as resistant to PARP inhibitors and anthracyclines. LP-184 demonstrated nanomolar potency in six NCI-60 breast cancer cell lines (median IC50 = 327 nM). Subcutaneous patient-derived TNBC xenograft mouse models were used to determine tumor volume responses to LP-184 treatment in vivo. Xenograft tumors were derived from 10 treatment-naive HR deficient (HRD score > 50) primary TNBC patients with known BRCA1/2 loss of heterozygosity (LOH), 7 of which subsequently progressed on PARP inhibitor Olaparib. LP-184 (4mg/kg i.v., (q2d x 5 then 7 days off)x2) led to complete and durable regression in all 10 TNBC HRD-PDX models tested as compared to control (p < 0.0001). A tumor growth inhibition range of 107-141% was achieved across all the 10 models. For LP-184, T/C at the control group end day was 0% in 10/10 models, whereas across the same models for Olaparib, T/C was 0% in 2/10 models and ranged from 15 - 90% in 8/10 models. Terminal body weight change was only a transient weight loss < 4% with LP-184 treatment across all models. LP-184 exhibited superior potency than olaparib in TNBC PDX models that carry HRD mutations including PARP resistant models and was well tolerated in mice. As acylfulvene-induced damage is primarily repaired by transcription-coupled nucleotide-excision repair (TC-NER) and HR pathways, response to LP-184 is influenced by tumor DNA damage repair pathway status. Recent data highlight an important role of super enhancer driven core transcription regulatory circuits in the pathogenesis of TNBCs. Our results support the superior efficacy of LP-184 in TNBCs, likely linked to the multiple MOAs, and establish LP-184 as a promising new agent for future clinical testing in TNBC patients. We finally propose that LP-184 may be broadly efficacious in solid tumors with HR and/or TC-NER pathway defects, such as pancreatic, prostate, ovarian and bladder cancers. Citation Format: Aditya Kulkarni, Neha Biyani, Laura Brullé-Soumaré, Stefano Cairo, Panna Sharma, Kishor Bhatia. LP-184, an acylfulvene class small molecule therapeutic, is synthetically lethal in HR deficient and PARP inhibitor resistant triple negative breast cancer [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 P4-01-06.

Abstract P2-23-13: Highly proliferative estrogen receptor-positive breast cancer is associated with better response to neoadjuvant chemotherapy but worse clinical outcome

Abstract Introduction: Highly proliferative cancers are known to respond better to neoadjuvant chemotherapy (NAC), and pathological complete response (pCR) is a surrogate for survival benefit in breast cancer. Ki67 is the most commonly used cell proliferation marker; however, conventional immunostaining is subjected to variability by the assessor and the institution including the antibody used. To this end, we used the Ki67 gene expression to identify highly proliferative breast cancer and investigated whether it is associated with better response to NAC and survival by intrinsic subtype. Materials and Methods: Total of 6623 breast cancer patients from 11 independent cohorts with full transcriptome and clinical data were analyzed. The top 20% of Ki67 gene expression in each cohort were defined as highly proliferative group based on previous studies using immuno-staining. Results: Highly proliferative breast cancer was associated with high proliferation score, and all five cell proliferation-related gene sets in the Hallmark collection; E2F Targets, G2M Checkpoint, Mitotic Spindle, and Myc Targets v1 and v2 were enriched by gene set enrichment analysis in multiple cohorts regardless of subtype. MKI67 gene expression increased with higher grade in both ER-positive breast cancer and TNBC in multiple cohorts, but no clear association with staging was observed. Highly proliferative ER-positive breast cancer was significantly associated with worse disease-free, disease-specific, and overall survival consistently in METABRIC and GSE96058 cohorts, and showed a trend toward worse overall survival in TCGA; however, this was not the case in TNBC. Further, we found that highly proliferative tumor was significantly associated with better pathologic complete response (pCR) rate in six of the eight neoadjuvant chemotherapy cohorts of ER-positive breast cancer, but only two for TNBC. We also found that highly proliferative ER-positive breast cancer was associated with abundant silent and non-silent mutations, increased intra-tumoral heterogeneity, and homologous recombination defect scores when compared to lower proliferation groups, and this was not seen in TNBC in TCGA cohort. Although DNA repair gene set was enriched in the highly proliferative ER-positive breast cancer, it was also associated with higher neoantigen activity, higher infiltration of tumor infiltrating lymphocytes including Th1 cells, Th2 cells, regulatory T cells, and M1 macrophages, as well as with enrichment of Interferon-gamma response gene set consistently in 3 large cohorts. In contrast, several stromal cell fractions were decreased in this group. Conclusions: Highly proliferative ER-positive breast cancer is significantly associated with pCR after NAC with increased tumor infiltrating lymphocytes, but is not a surrogate for survival, which are not the case in TNBC. Citation Format: Rongrong Wu, Yoshihisa Tokumaru, Mariko Asaoka, Masanori Oshi, Li yan, Takashi Ishikawa, Kazuaki Takabe. Highly proliferative estrogen receptor-positive breast cancer is associated with better response to neoadjuvant chemotherapy but worse clinical outcome [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 P2-23-13.

Abstract PD11-12: PD11-12 DORA: A Phase II, Multicenter, International, Non-Comparator Study of Olaparib (O) +/- Durvalumab (D) as a chemotherapy-free maintenance strategy in Platinum tReated Advanced Triple-Negative Breast Cancer (aTNBC)

Abstract Introduction: A significant proportion of aTNBC patients carry homologous recombination defects associated with platinum sensitivity. Olaparib is an approved PARP inhibitor (PARPi) for germline BRCA (gBRCA) associated early and metastatic breast cancer as well as maintenance therapy in platinum-sensitive ovarian cancer irrespective of gBRCA status. PARPi enhances immune response via cGAS/STING activation and is synergistic with anti-PD-1 blockade in preclinical models without overlapping toxicities. Here, the efficacy of maintenance olaparib (O) +/- durvalumab (D) in aTNBC patients following clinical benefit from platinum chemotherapy is investigated (NCT03167619). Methods: Eligible pts had aTNBC with investigator-assessed clinical benefit (SD, PR, CR) after a minimum of 3 q3-weekly or 6 q1-weekly cycles of platinum-based chemotherapy in the 1st or 2nd line treatment setting. Patients were randomized 1:1 to receive O 300 mg BID daily or O 300mg BID daily + D 1.5g IV q4 wks. The study was a non-comparator trial; randomization aimed to reduce bias. Tumors were evaluated by RECIST1.1 at baseline and q8 wks. Known gBRCA carriers were limited to 10. The primary endpoint was progression-free survival (PFS). Secondary endpoints were disease control rate (DCR), clinical benefit rate (CBR), and overall survival (OS). Results: From 2/4/2019-12/24/2020, 45 pts were randomized (23 pts in O arm; 22 in O+D arm). 82.2% received platinum as 1st line therapy and 82% received a platinum-doublet. As of data cutoff (6/30/2021), median follow-up of 9.8m (7.2-15.1), the median PFS was 3.95m (p= 0.0023; 95% CI 2.55-6.13) with O monotherapy. The median PFS was 6.1 mos (p= <.0001; 95% CI 3.68-10.11) in the O+D arm. CBR (CR, PR or SD ≥ 24 wks) was 39.1% (19.7%-61.5%) and 36.4% (17.2%-59.3%) in the O and O+D arms, respectively. DCR was 52.2% (30.6%, 73.2%) and 68.2% (45.1%, 86.1%) in the O and O + D arms, respectively. Currently, 7 pts (15.6%) remain on study treatment, only 2 have gBRCA alterations. No new safety signals were reported. Correlative analysis including germline/somatic BRCA, HRR genes, BRCA methylation, TMB and PDL-1 in association with clinical outcomes will be presented. Conclusions: A subset of non-gBRCA altered aTNBC pts who derived clinical benefit from platinum-based chemotherapy had a durable disease control with a chemotherapy-free maintenance strategy of olaparib +/- durvalumab. Citation Format: Sarah L. Sammons, Tira J. Tan, Young Hyunk Im, Tiffany A. Traina, Carey Anders, Ezekiel Renzulli, Sung-Bae Kim, Rebecca Dent. PD11-12 DORA: A Phase II, Multicenter, International, Non-Comparator Study of Olaparib (O) +/- Durvalumab (D) as a chemotherapy-free maintenance strategy in Platinum tReated Advanced Triple-Negative Breast Cancer (aTNBC) [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 PD11-12.

Homologous Recombination Defects and Mutations in DNA Damage Response (DDR) Genes Besides BRCA1 and BRCA2 as Breast Cancer Biomarkers for PARP Inhibitors and Other DDR Targeting Therapies.

Homologous recombination repair (HRR) is the cellular mechanism for error-free repair of double strand DNA (dsDNA) breaks. Cancer cells with mutations in both alleles of genes encoding for proteins involved in HRR, such as BRCA1 and BRCA2, have defects in the repair process. As a result, these cells repair dsDNA breaks with alternative mechanisms, such as non-homologous end joining. In breast cancers with germline mutations in BRCA1 and BRCA2 genes, HRR defects result in sensitivity to PARP inhibitors, drugs that interfere with the function of PARP enzyme and promote trapping of the enzyme on DNA and stalling of the process of repairing single strand breaks. HRR defects also lead to sensitivity to DNA damaging chemotherapy due to the inability of cells to repair chemotherapy induced DNA lesions. Besides germline mutations in BRCA1 and BRCA2, somatic mutations in these genes or germline and somatic mutations or other genetic and epigenetic alterations of other genes involved in homologous recombination (HR) may produce HRR defects leading to sensitivity to PARP inhibitors. However, studies are less conclusive, a fact that may relate to the common lack of bi-allelic loss of function in these cases, as opposed to cancers with germline BRCA1 or BRCA2 defects that usually acquire bi-allelic loss of function. In addition, there is heterogeneity between the different HRR genes and the severity of the resulting HRR defects, as measured by HR defect assays. This review article examines the landscape of HRR gene mutations in breast cancer and the possible therapeutic implications of HRR defects other than germline BRCA1 and BRCA2 mutations for targeted therapies. Identification of a wider range of breast cancers with HRR defects may expand the subset of patients that derive benefit from PARP inhibitors and other DDR-targeting drugs in the clinic.

Small-Molecule-Mediated Stabilization of PP2A Modulates the Homologous Recombination Pathway and Potentiates DNA Damage-Induced Cell Death.

High-grade serous carcinoma (HGSC) is the most common and lethal ovarian cancer subtype. PARP inhibitors (PARPi) have become the mainstay of HGSC-targeted therapy, given that these tumors are driven by a high degree of genomic instability (GI) and homologous recombination (HR) defects. Nonetheless, approximately 30% of patients initially respond to treatment, ultimately relapsing with resistant disease. Thus, despite recent advances in drug development and an increased understanding of genetic alterations driving HGSC progression, mortality has not declined, highlighting the need for novel therapies. Using a small-molecule activator of protein phosphatase 2A (PP2A; SMAP-061), we investigated the mechanism by which PP2A stabilization induces apoptosis in patient-derived HGSC cells and xenograft (PDX) models alone or in combination with PARPi. We uncovered that PP2A genes essential for cellular transformation (B56α, B56γ, and PR72) and basal phosphatase activity (PP2A-A and -C) are heterozygously lost in the majority of HGSC. Moreover, loss of these PP2A genes correlates with worse overall patient survival. We show that SMAP-061-induced stabilization of PP2A inhibits the HR output by targeting RAD51, leading to chronic accumulation of DNA damage and ultimately apoptosis. Furthermore, combination of SMAP-061 and PARPi leads to enhanced apoptosis in both HR-proficient and HR-deficient HGSC cells and PDX models. Our studies identify PP2A as a novel regulator of HR and indicate PP2A modulators as a therapeutic therapy for HGSC. In summary, our findings further emphasize the potential of PP2A modulators to overcome PARPi insensitivity, given that targeting RAD51 presents benefits in overcoming PARPi resistance driven by BRCA1/2 mutation reversions.

BMAL1 collaborates with CLOCK to directly promote DNA double-strand break repair and tumor chemoresistance

Accumulating evidence indicates a correlation between circadian dysfunction and genomic instability. However, whether the circadian machinery directly regulates DNA damage repair, especially in double-strand breaks (DSBs), remains poorly understood. Here, we report that in response to DSBs, BMAL1 is activated by ATM-mediated phosphorylation at S183. Phosphorylated BMAL1 is then localized to DNA damage sites, where it facilitates acetylase CLOCK to load in the chromatin, regulating the acetylation of histone H4 (H4Ac) at DSB sites. In this way, the BMAL1-CLOCK-H4Ac axis promotes the DNA end-resection to generate single-stranded DNA (ssDNA) and the subsequent homologous recombination (HR). BMAL1 deficient cells display defective HR, accumulation of unrepaired DSBs and genome instability. Accordingly, depletion of BMAL1 significantly enhances the sensitivity of adrenocortical carcinoma (ACC) to DNA damage-based therapy in vitro and in vivo. These findings uncover non-canonical function of BMAL1 and CLOCK in HR-mediated DSB repair, which may have an implication in cancer therapeutics.

The clinical utility of integrative genomics in childhood cancer extends beyond targetable mutations

We conducted integrative somatic–germline analyses by deeply sequencing 864 cancer-associated genes, complete genomes and transcriptomes for 300 mostly previously treated children and adolescents/young adults with cancer of poor prognosis or with rare tumors enrolled in the SickKids Cancer Sequencing (KiCS) program. Clinically actionable variants were identified in 56% of patients. Improved diagnostic accuracy led to modified management in a subset. Therapeutically targetable variants (54% of patients) were of unanticipated timing and type, with over 20% derived from the germline. Corroborating mutational signatures (SBS3/BRCAness) in patients with germline homologous recombination defects demonstrates the potential utility of PARP inhibitors. Mutational burden was significantly elevated in 9% of patients. Sequential sampling identified changes in therapeutically targetable drivers in over one-third of patients, suggesting benefit from rebiopsy for genomic analysis at the time of relapse. Comprehensive cancer genomic profiling is useful at multiple points in the care trajectory for children and adolescents/young adults with cancer, supporting its integration into early clinical management.

Identification of necroptosis subtypes and development of necroptosis-related risk score model for in ovarian cancer.

Background: ith the ongoing development of targeted therapy, non-apoptotic cell death, including necroptosis, has become a popular topic in the field of prevention and treatment. The purpose of this study was to explore the effect of necroptosis-related genes (NRGs) on the classification of ovarian cancer (OV) subtypes and to develop a necroptosis-related risk score (NRRS) classification system. Methods: 74 NRGs were obtained from the published studies, and univariate COX regression analysis was carried out between them and OV survival. Consensus clustering analysis was performed on OV samples according to the expression of NRGs related to prognosis. Furthermore, the NRRS model was developed by combining Weighted Gene Co-Expression Network Analysis (WGCNA) with least absolute shrinkage and selection operator (Lasso)-penalized Cox regression and multivariate Cox regression analysis. And the decision tree model was constructed based on the principle of random forest screening factors principle. Results: According to the post-related NRGs, OV was divided into two necroptosis subtypes. Compared with Cluster 1 (C1), the overall survival (OS) of Cluster 2 (C2) was significantly shorter, stromal score and immune score, the infiltration level of tumor associated immune cells and the expression of 20 immune checkpoints were significantly higher. WGCNA identified the blue module most related to necroptosis subtype, and 12 genes in the module were used to construct NRRS. NRRS was an independent prognostic variable of OV. The OS of samples with lower NRRS was significantly longer, and tumor mutation burden and homologous recombination defect were more obvious. Conclusion: This study showed that necroptosis plays an important role in the classification, prognosis, immune infiltration and biological characteristics of OV subtypes. The evaluation of tumor necroptosis may provide a new perspective for OV treatment.

Abstract B065: Polymerase theta inhibition activates the cGAS-STING signaling pathway and elicits an immune response in homologous recombination-deficient pancreatic adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy that harbors mutations in homologous recombination (HR) repair genes in up to 25% of cases. Defects in HR impart to tumor cells a specific vulnerability to poly-ADP ribose polymerase inhibitors and platinum-based chemotherapy. However, not all patients respond, and some who initially respond develop resistance and progress. Thus, new therapies that are effective against HR-deficient PDAC are needed. Here, we show that inactivation of the HR pathway in PDAC is associated with overexpression of polymerase theta (Polθ, or POLQ), a key enzyme that regulates the alternative non-homologous end-joining (alt-NHEJ) pathway of double-strand break (DSB) DNA repair. Using both human and murine HR-deficient PDAC models, we find that POLQ knockdown is synthetically lethal with mutations in HR genes (BRCA1, BRCA2, and PALB2) as well as the DNA damage repair gene ATM. Further, we present the first findings that POLQ knockdown enhances cytosolic micronucleus formation and activates cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) signaling. In an in vivo murine model, POLQ inhibition also leads to STING-dependent recruitment of immune cells and enhanced CD8+ T cell infiltration in the BRCA2-deficient tumor microenvironment (TME). Through its role in the alt-NHEJ pathway, we thus find that POLQ is critical for DSB repair in HR-deficient PDAC, and its inhibition presents a novel synthetic lethal approach to suppress tumor growth while simultaneously stimulating an immune response in the PDAC TME. Citation Format: Grace Oh, Annie Wang, Lidong Wang, Jiufeng Li, Gregor Werba, Daniel Weissinger, Ende Zhao, Surajit Dhara, Rosmel Hernandez, Amanda Ackermann, Despoina Kalfakakou, Emily A. Kawaler, Talia Golan, Theodore H. Welling, Agnel Sfeir, Diane M. Simeone. Polymerase theta inhibition activates the cGAS-STING signaling pathway and elicits an immune response in homologous recombination-deficient pancreatic adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr B065.

Therapeutic Targeting of Spliceosome Mutant Myeloid Neoplasms Via PARP1 Inhibition

Somatic mutations in splicing factors (SF) SF3B1, SRSF2, U2AF1 are observed across a spectrum of clonal myeloid disorders including myelodysplastic syndromes (MDS). Previous studies identified increased R-loops in SF-mutant cells. Abnormal R-loop accumulation is associated with increased genomic instability and preferential sensitivity to ATR kinase inhibitor. Motivated by these observations, we set out to identify novel therapeutic targets in SF-mutant myeloid malignancies. Using MLLAF9 Srsf2WT and Srsf2P95Hcells, we performed a focused drug screen using inhibitors targeting different DNA damage response pathways and drugs that inhibit DNA replication or DNA metabolic processes. Unexpectedly, Srsf2P95H cells displayed increased sensitivity to inhibitors targeting PARP1/2 (Fig. 1A-C). Olaparib (PARPi) treatment induced higher apoptosis in Srsf2P95Hcells than in Srsf2WTcells (Fig. 1D). To ensure this phenomenon extends beyond Srsf2 mutation, we generated MLLAF9 leukemia on U2af1WT and U2af1S34Fbackgrounds. PARPi treatment in U2af1S34F cells resulted in reduced cell viability and increased apoptosis compared to U2af1WT cells (Fig. 1E-G). Next, we genetically inactivated Parp1 in Srsf2WT and Srsf2P95H cells. The absence of Parp1 did not affect the growth of Srsf2WT cells (Fig. 1H). In contrast, Parp1 deletion significantly reduced proliferation of Srsf2P95H cells (Fig. 1I).Finally, to confirm this phenotype in vivo, we transplanted established MLLAF9 Srsf2WTor Srsf2P95H leukemia into syngeneic mice. Eleven days post-transplant, mice received either vehicle or PARPi. Mice transplanted with Srsf2WT cells showed no survival advantage when treated with PARPi (Fig. 1J). In contrast, PARPi treatment significantly prolonged survival of mice transplanted with Srsf2P95H cells compared to vehicle-treated mice (Fig. 1K). To examine whether PARPi sensitivity is conserved in humans, we treated K562 cells expressing either SRSF2P95H or SF3B1K700E mutation. SRSF2P95H and SF3B1K700E cells showed increased sensitivity to olaparib and rucaparib (Fig. 1L-M). This observation was further validated in a panel of primary AML blasts carrying SRSF2 and U2AF1 mutations(Fig. 1N). Taken together, our results identify PARP1 as a novel therapeutic target in SF-mutant myeloid neoplasms. PARP inhibitors preferentially kill cells with defective homologous recombination (HR)-mediated DNA repair. We asked if the observed PARPi sensitivity in SF-mutant cells was due to defects in HR. To measure HR activity, we performed a CRISPR-Cas9/mClover assay. The assay measures HR-dependent insertion of an mClover-containing donor cassette into Cas9-mediated double-stranded breaks in the LMNA gene, which results in the expression of a green fluorescent Lamin A/C protein. There was no significant difference in the relative percentage of mClover positivity in SRSF2P95H or U2AF1S34F cells compared to control (Fig. 1O), suggesting that these cells are HR-proficient. Interestingly, SF3B1K700E cells showed significantly reduced HR efficiency, consistent with a published study. We next investigated cellular PARP1 activity in SRSF2- and U2AF1-mutant cells by monitoring Poly-ADP-ribosylated chain (PAR) levels. SRSF2P95H cells had elevated PAR level compared to SRSF2WT cells (Fig. 1P). Since PARP1 is known to associate with R-loops, we wanted to test whether increased PARP1 activity and PARPi sensitivity are R-loop dependent. We generated SRSF2-mutant cells that inducibly express RNaseH1, an enzyme that specifically cleaves the RNA moiety within the RNA:DNA hybrids. Induction of RNaseH1 reduced PAR level and PARPi sensitivity in SRSF2P95H cells, suggesting that SRSF2-mutant cells elicit a PARP1 response to promote survival in an R-loop-dependent manner (Fig. 1Q). Lastly, since SF-mutant cells are also sensitive to ATRi, we tested if combining PARPi and ATRi can further sensitize SF-mutant cells. Combined PARPi + ATRi treatment induced synergistic killing of Srsf2- and U2af1-mutant cells compared to wildtype cells in cell viability and colony forming assays (Fig. 1R-S). In summary, this study provides a pre-clinical rationale for therapeutic targeting of PARP1 in SF-mutant leukemia. Moreover, PARP and ATR inhibitor combination could emerge as a new therapeutic strategy in this genetically distinct disease subtype. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Homologous Recombination Deficiency (HRD) in Hematological Malignancies

Introduction: The inability of a cell to repair the double-stranded breaks because of a defective homologous recombination repair (HRR) pathway results in Homologous Recombination Deficiency (HRD). The defective DNA repair leads to genomic instability and tumorigenesis, which has a characteristic scar predictive of the HRD phenotype. The presence of HRD has been found to make tumors sensitive to ICL-inducing platinum-based therapies and poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) inhibitors (PARPi). However, there has been limited investigation of HRD phenotype in hematological malignancies, and herein, we aim to evaluate HRD phenotype using Optical genome mapping (OGM) technology in comparison to large gene next-generation sequencing (NGS) panel. Methods: This retrospective study included the analysis of 60 hematological malignancy samples that were evaluated with a 523-gene next-generation sequencing (NGS) panel and OGM. The HRD scores were calculated using a combination of three HRD signatures that included loss of heterozygosity (LOH), telomeric allelic imbalance (TAI), and large-scale transitions (LST). Results: In the 60 hematological malignancy cases analyzed with OGM and 523-gene NGS panel, the HRD scores were 11 (±9.8) with OGM compared to 2.4 (±3.8) with 523-gene NGS panel. OGM detected 67% additional structural variants that resulted from HRD in these cases. Conclusion: This study highlights the HRD scars that are missed by current technologies used for accessing HRD phenotype and presents OGM as an alternative tool with high resolution and sensitivity to accurately assess the HRD phenotype. We recommend the use of OGM for HRD phenotype evaluation as the most sensitive method, that could potentially lead to the differentiation of tumors with or without HRD phenotype in the ongoing clinical trials. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Clinicogenomic characteristics and synthetic lethal implications of germline homologous recombination-deficient hepatocellular carcinoma.

We performed an in-depth examination of pathogenic germline variants (PGVs) and somatic variants in DNA damage response (DDR) genes in hepatocellular carcinoma (HCC) to explore their clinical and genomic impacts. We used a merged whole-exome or RNA sequencing data set derived from in-house ( n = 230) and The Cancer Genome Atlas ( n = 362) databases of multiethnic HCC samples. We also evaluated synthetic lethal approaches targeting mutations in homologous recombination (HR) genes using HCC cells selected from five genomic databases of cancer cell lines. A total of 110 PGVs in DDR pathways in 96 patients were selected. Of the PGV carriers, 44 were HR-altered and found to be independently associated with poorer disease-free survival after hepatectomy. The most frequently altered HR gene in both germline and somatic tissues was POLQ , and this variant was detected in 22.7% (10/44) and 23.8% (5/21) of all the corresponding carriers, respectively. PGVs in HR were significantly associated with upregulation of proliferation and replication-related genes and familial risk of HCC. Samples harboring PGVs in HR with loss of heterozygosity were most strongly correlated with the genomic footprints of deficient HR, such as mutation burden and denovoSig2 (analogous to Catalogue of Somatic Mutations in Cancer [COSMIC] 3), and poor outcome. Pharmacologic experiments with HCC cells defective in BRCA2 or POLQ suggested that tumors with this phenotype are synthetic lethal with poly(ADP-ribose) polymerase inhibitors. Our findings suggest that germline HR defects in HCC tend to confer a poor prognosis and result in distinctive genomic scarring. Tests of the clinical benefits of HR-directed treatments in the affected patients are needed.

Lack of impact of the ALDH2 rs671 variant on breast cancer development in Japanese BRCA1/2-mutation carriers.

The aldehyde degrading function of the ALDH2 enzyme is impaired by Glu504Lys polymorphisms (rs671, termed A allele), which causes alcohol flushing in east Asians, and elevates the risk of esophageal cancer among habitual drinkers. Recent studies suggested that the ALDH2 variant may lead to higher levels of DNA damage caused by endogenously generated aldehydes. This can be a threat to genome stability and/or cell viability in a synthetic manner in DNA repair-defective settings such as Fanconi anemia (FA). FA is an inherited bone marrow failure syndrome caused by defects in any one of so far identified 22 FANC genes including hereditary breast and ovarian cancer (HBOC) genes BRCA1 and BRCA2. We have previously reported that the progression of FA phenotypes is accelerated with the ALDH2 rs671 genotype. Individuals with HBOC are heterozygously mutated in either BRCA1 or BRCA2, and the cancer-initiating cells in these patients usually undergo loss of the wild-type BRCA1/2 allele, leading to homologous recombination defects. Therefore, we hypothesized that the ALDH2 genotypes may impact breast cancer development in BRCA1/2 mutant carriers. We genotyped ALDH2 in 103 HBOC patients recruited from multiple cancer centers in Japan. However, we were not able to detect any significant differences in clinical stages, histopathological classification, or age at clinical diagnosis across the ALDH2 genotypes. Unlike the effects in hematopoietic cells of FA, our current data suggest that there is no impact of the loss of ALDH2 function in cancer initiation and development in breast epithelium of HBOC patients.

Definition of a Novel Cuproptosis-Relevant lncRNA Signature for Uncovering Distinct Survival, Genomic Alterations, and Treatment Implications in Lung Adenocarcinoma.

Objective Cuproptosis is a newly discovered copper-independent cell death modality, and limited evidence suggests the critical implications in human cancers. Nonetheless, the clinical impacts of cuproptosis-relevant lncRNAs in lung adenocarcinoma (LUAD) remain largely ill-defined. The present study was aimed at defining a cuproptosis-relevant lncRNA signature for LUAD and discuss the clinical utility. Methods We collected transcriptome expression profiling, clinical information, somatic mutation, and copy number variations from TCGA-LUAD cohort retrospectively. The genetic alterations of cuproptosis genes were systematically assessed across LUAD, and cuproptosis-relevant lncRNAs were screened for defining a LASSO prognostic model. Genomic alterations, immunological and stemness features, and therapeutic sensitivity were studied with a series of computational approaches. Results Cuproptosis genes displayed aberrant expression and widespread genomic alterations across LUAD, potentially modulated by m6A/m5C/m1A RNA modification mechanisms. We defined a cuproptosis-relevant lncRNA signature, with a reliable efficacy in predicting clinical outcomes. High-risk subset displayed higher somatic mutations, CNVs, TMB, SNV neoantigens, aneuploidy score, CTA score, homologous recombination defects, and intratumor heterogeneity, cytolytic activity, CD8+ T effector, and antigen processing machinery, proving that this subset might benefit from immunotherapy. Increased stemness indexes and activity of oncogenic pathways might contribute to undesirable prognostic outcomes for high-risk subset. Additionally, high-risk patients generally exhibited higher response to chemotherapeutic agents (cisplatin, etc.). We also predicted several small molecule compounds (GSK461364, KX2-391, etc.) for treating this subset. Conclusion Accordingly, this cuproptosis-relevant lncRNA signature offers an efficient approach to identify and characterize diverse prognosis, genomic alterations, and treatment outcomes in LUAD, thus potentially assisting personalized therapy.

Abstract IA029: The origins of leiomyosarcoma: implications for prognosis and defining new therapeutic options

Abstract Introduction: Leiomyosarcomas (LMS) are genetically heterogenous tumors differentiating along smooth muscle lines. Currently, LMS treatment is not informed by molecular subtyping and is associated with highly variable survival. While disease site continues to dictate clinical management, the contribution of genetic factors to LMS subtype, origins, and timing are unknown. Here we analyze 70 genomes and 130 transcriptomes of LMS, including multiple tumor regions and paired metastases, with the goal of defining the molecular origins of LMS and using both computational analysis and functional genomics to define new therapeutic arenas for metastatic disease. Results: Molecular profiling highlight the very early origins of LMS. We uncover three specific subtypes of LMS that likely develop from distinct lineages of smooth muscle cells. Of these, dedifferentiated LMS with high immune infiltration and tumors primarily of gynecological origin harbor genomic dystrophin deletions and/or loss of dystrophin expression, acquire the highest burden of genomic mutation, and are associated with worse survival. Homologous recombination defects lead to genome-wide mutational signatures, and a corresponding sensitivity to PARP trappers and other DNA damage response inhibitors, suggesting a promising therapeutic strategy for LMS. Finally, by phylogenetic reconstruction, we present evidence that clones seeding lethal metastases arise decades prior to LMS diagnosis. Conclusions: In summary, LMS, the catch all-label for any primary malignant smooth muscle neoplasm, encompasses at least three distinct entities. We report that the utility of DNA damage response inhibitors is promising and warrants further exploration. Finally, the molecular evolution and timing of LMS provide a broad window for early intervention and risk stratification for these patients. Citation Format: Rebecca A. Gladdy. The origins of leiomyosarcoma: implications for prognosis and defining new therapeutic options [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr IA029.

570P Hypomethylating agents synergistically combine with PARP inhibitors in ovarian cancer

Poly (ADP-Ribose) polymerase inhibitors (PARPi’s) are currently used for first and second line maintenance therapy of ovarian cancer (OC) in patients whose tumors harbor defects in homologous recombination. Unfortunately, the rapid emergence of resistance leads to a short duration of response, and thus preventing PARPi resistance and extending the spectrum of patients who would benefit from PARP inhibition are urgent needs. While multiple drug classes have demonstrated synergy when combined with PARPi’s preclinically, head-to-head comparisons which would facilitate clinical prioritization of the most promising combinations are missing.

Prognostic and Immunological Significance of FUNDC1 in Hepatocellular Carcinoma: A Study on TCGA Mining.

Background Hepatocellular carcinoma (HCC) is an inflammation-related malignancy influenced by the immune microenvironment, such as immune tolerance and evasion. HFUN14 domain-with protein 1 (FUNDC1) is a necessary mitochondrial outer membrane protein, functioning as a receptor for hypoxia-caused mitophagy, which is related to human immunity. The relationship between HCC and FUNDC1 in terms of prognosis and immunology was demonstrated in the current investigation. Even so, the function of FUNDC1 in liver cancer is yet unknown. Methods The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) datasets were utilized for examining if FUNDC1 expression is associated with clinicopathological characteristics and prognosis. Genetic changes (mutation), DNA methylation, and their relationship with patient prognosis were identified by cBioPortal and MethSurv. Utilizing the Tumor Immune Estimation Resource (TIMER), immune checkpoints, infiltration, and immune cell biomarkers were analyzed. Utilizing the STRING database, the network of protein-protein interactions was created. Using Gene Set Enrichment Analysis, the FUNDC1 biological roles were determined (GSEA). Results FUNDC1 elevation was significantly linked with gender (p < 0.001), tumor stage (p = 0.01349), tumor grade (p < 0.001), and alpha-fetoprotein (AFP) (p < 0.001) levels in HCC. It was illustrated by ROC curve analysis that FUNDC1 had a significant diagnostic and prognostic value. The FUNDC1 genetic change rate was 0.6%. Four out of 6 DNA methylation CpG sites were associated with the HCC prognosis. FUNDC1 is associated strongly with immune cell infiltration in HCC. Moreover, FUNDC1 was positively related to immune checkpoints such as mutant-allele tumor heterogeneity (MATH) (p < 0.001), ploidy (p < 0.05), homologous recombination defect (HRD) (p < 0.001), and loss of heterozygosity (LOH). GSEA revealed significant FUNDC1 enrichment in the cell cycle, hedgehog, and MAPK signaling pathways. Conclusion FUNDC1 is a mitophagy regulator that could be a therapeutic, prognostic, and putative diagnostic biomarker for HCC.

PRECISION ONCOLOGY- BASIC TO BEDSIDE

Basic science technologies and platforms have unraveled the molecular basis of carcinogenesis, both genetic and epigenetic events. Few of the events which are repeatedly observed and are actionable, gave birth to the science “Precision Oncology”. The evolution of molecular testing has made a major stride in the recent times, and has empowered personalized cancer medicine over the conventional blanket treatment. Among the important diagnostic technologies, Next Generation Sequencing (NGS) based in-vitro diagnostics have contributed substantially to comprehensive genomic profiling (CGP) in clinical practice that is meant for the detection of substitutions, insertions and deletions (indels), copy number alterations, and gene rearrangements, as well as genomic signatures including Microsatellite Instability (MSI), Loss of Heterozygosity (LOH), Homologous Recombination Defect (HRD Score), Tumor Mutation Burden (TMB). CGP has come into clinical practice with various guidelines endorsing its clinical utility in various indications, both tumor specific and tumor agnostic. Lung cancer is an excellent example where the role of NGS based comprehensive genomic profiling (CGP) has been recommended upfront either on tissue or blood (Liquid Biopsy). In ovarian cancer, calculation like LOH or HRD score, based on the tissue NGS, has widen the eligibility of PARP inhibitor drugs. Further, NTRK fusions, MSI and TMB biomarkers are universal supporting tumor agnostic approach. CGP is a tremendous diagnostic application but brings challenges especially interpretation which are affected by intra/inter tumor heterogeneity, identification of multiple driver mutations, multiple targets and treatment options, cross talk of various mutations and genomic signatures, and various signaling pathways, variable variant allele frequency (VAF), cut-offs for various scores like TMB, LOH, HRD. All these challenges are currently overcome by Molecular Tumor Boards, where basic science understanding in conjunct with clinical experience, has made a huge difference. Thus, offering true precision oncology with unique treatment options for individual patient.&nbsp;