Ribose Polymerase Research Articles

Immune correlates of outcome in patients with leiomyosarcoma (LMS) treated with durvalumab plus olaparib or cediranib: Transcriptome analysis from the DAPPER study.

11567 Background: Immune checkpoint blockade (ICB) as monotherapy has not shown clinical benefit in non-inflamed (cold) tumors such as LMS. Combining ICB with angiogenesis, or poly-ADP ribose polymerase (PARP) inhibitors may increase tumor immunogenicity by altering the immune cell composition of the tumor microenvironment (TME). In the DAPPER trial [NCT03851614], advanced LMS pts were randomized to receive ICB (Durvalumab 1500mg IV q4w) with either angiogenesis- (Cediranib 20mg qd PO on 5 days/week) or PARP inhibition (Olaparib 300mg bid PO) until unacceptable toxicity or disease progression (Ayodele et al, J Clin Oncol, 2021). Here, we present the results of transcriptomic and immune biomarker analyses of patients (pts) in the whole cohort. Methods: Radiologic responses were assessed using RECISTv1.1 and survival analysis performed by Kaplan-Meier method. Transcriptome analysis by RNAseq was performed on fresh tumor biopsies obtained from all pts at screening. Relative fractions of 22 immune cell subsets in the TME were inferred from gene-expression profiles using CIBERSORT v1.06. Gene set variation analysis (GVSA) to identify transcriptomic signatures associated with progression-free (PFS) or overall survival (OS) benefit was performed using GSVA R package v1.42. For comparison with an independent cohort, GVSA was also performed using transcriptome data from LMS pts (n = 104) in the cancer genome atlas (TCGA) dataset. Results: Among 28 response-evaluable pts, 1 (3.6%) had partial response; 10 (35.7%) had stable disease (SD); and 17 (60.7%) had progressive disease. Median PFS and OS were 2.8 months (95% CI, 2.8 – 5.4) and 14.6 months (95% CI 10.7 – NR), respectively. RNAseq data of adequate quality was available for 17 pts. Using the median CIBERSORT score as cut-off, pts with high M1 macrophage levels at baseline had significantly longer OS (p = 0.0019). High M1/M2 macrophage ratio score was also associated with longer OS (p = 0.05). GVSA identified 7 immune- and angiogenesis-related signatures that were associated with significantly longer OS. After false discovery rate correction, 1 signature comprising genes reflective of high overall B-cell activity remained significant. No transcriptomic signatures were associated with OS in the TCGA LMS cohort where pts did not receive ICB. Conclusions: Transcriptomic analysis shows that macrophage presence in the TME is associated with longer OS. Association of the B-cell activity signature with longer OS in LMS pts on the DAPPER trial, but not in the ICB treatment-naïve TCGA cohort suggests that high B-cell activity may identify pts who are more likely to have favorable outcomes with ICB and supports previous reports in sarcoma (Petitprez et al, Nature, 2020). Our results support ongoing further evaluation and integration of these biomarkers in LMS. Clinical trial information: NCT03851614 .

Harnessing olaparib, palbociclib, and endocrine therapy: A phase I/II trial of olaparib, palbociclib and fulvestrant in patients with BRCA mutation-associated, hormone receptor-positive, human epidermal growth factor receptor 2 (HER2)-negative metastatic breast cancer (HOPE).

TPS1126 Background: Hormone receptor-positive (HR+) metastatic breast cancer (MBC) in patients (pts) with germline or somatic BRCA1 or BRCA2 (g/s BRCA1/2) mutations has dual drivers: hormone receptor signaling and homologous recombination deficiency. The PALOMA-3 trial showed superiority of fulvestrant (F) with palbociclib (P) compared to F alone. The OlympiAD trial showed the poly-ADP ribose polymerase inhibitor (PARPi) olaparib (O) to be superior to chemotherapy in BRCA1/2-associated MBC. However, treatment resistance inevitably develops; targeting dual therapeutic drivers concurrently may delay or circumvent resistance. O and P have overlapping hematologic toxicity and the safety of combined O, P and F is unknown. The tolerability of O + P is particularly relevant given the emergence of potentially less myelosuppressive PARPi. Methods: HOPE (NCT03685331) is a phase I/II trial to evaluate safety and efficacy of O, P and F in pts with g/s BRCA1/2-associated, HR+ MBC. Eligible pts have an ECOG performance status 0-1, measurable/evaluable breast cancer, any/no prior endocrine therapy, and 0-2 lines of chemotherapy for MBC. Prior platinum is allowed for curative intent if completed at least 12 months prior to MBC diagnosis, or for MBC if no progression during therapy. Prior PARPi and CDK4/6i are permitted on phase I, and are permitted during phase II if there was no progression on them. Treatment (28-day cycles) consists of: O orally twice daily continuously; F 500mg intramuscularly on day 1 of each cycle plus load; and P orally once daily on days 1-21. Phase I begins with a 28-day safety run-in of O and F alone. O and P doses are based on dose level (DL): DL 0 (starting level), O 300mg, P 75mg; DL 1, O 300mg, P 100mg; DL 2, O 300mg, P 125mg; DL-1, O 250mg, P 75mg and DL-2, O 200mg, P 75mg. For phase II, O and P will be dosed at MTD. Pts have tissue collection at baseline and peripheral blood cfDNA analysis at baseline, at progression, and at all scan timepoints (every 3 cycles). Phase I primary endpoint is MTD. A 3+3 dose escalation design is used with a 30% rate of dose limiting toxicity (DLT) deemed acceptable, and 6 patients treated at a dose for it to be declared MTD. A minimum of 2 and a maximum of 18 patients will be enrolled on the phase I. Phase II primary endpoint is PFS using Kaplan-Meier methods and secondary efficacy endpoints are objective response rate and 24-week clinical benefit rate. Phase II will evaluate 54 subjects to provide 80% power to detect an increase in PFS from 7 months with O monotherapy to 10 months. Exploratory objectives include examination of baseline tissue for PARPi predictive biomarkers and measures of tumor immunogenicity as well as serial serum evaluation for reversion mutations. Enrollment is ongoing. Clinical trial information: NCT03685331 .

POLQ inhibition elicits an immune response in homologous recombination-deficient pancreatic adenocarcinoma via cGAS/STING signaling.

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy that harbors mutations in homologous recombination-repair (HR-repair) proteins in 20%-25% of cases. Defects in HR impart a specific vulnerability to poly ADP ribose polymerase inhibitors and platinum-containing chemotherapy in tumor cells. However, not all patients who receive these therapies respond, and many who initially respond ultimately develop resistance. Inactivation of the HR pathway is associated with the overexpression of polymerase theta (Polθ, or POLQ). This key enzyme regulates the microhomology-mediated end-joining (MMEJ) pathway of double-strand break (DSB) repair. Using human and murine HR-deficient PDAC models, we found that POLQ knockdown is synthetically lethal in combination with mutations in HR genes such as BRCA1 and BRCA2 and the DNA damage repair gene ATM. Further, POLQ knockdown enhances cytosolic micronuclei formation and activates signaling of cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING), leading to enhanced infiltration of activated CD8+ T cells in BRCA2-deficient PDAC tumors in vivo. Overall, POLQ, a key mediator in the MMEJ pathway, is critical for DSB repair in BRCA2-deficient PDAC. Its inhibition represents a synthetic lethal approach to blocking tumor growth while concurrently activating the cGAS-STING signaling pathway to enhance tumor immune infiltration, highlighting what we believe to be a new role for POLQ in the tumor immune environment.

Nipavect: Phase II study of niraparib and panitumumab in advanced RAS WT colorectal cancer.

3579 Background: Panitumumab (Pmab) is a recombinant monoclonal antibody that binds specifically to the human epidermal growth factor receptor (EGFR) and it is indicated for the treatment of metastatic colorectal carcinoma (mCRC). However, the objective response rates (ORR) for the single agent are historically about 8-11%. EGFR inhibition induces synthetic lethality with poly ADP ribose polymerase inhibitors (PARPi) such as Niraparib, by attenuating DNA repair pathways. Combining PARP and EGFR inhibition has the potential to confer synergistic benefit, while also ameliorating resistance mechanism to EGFRi. We conducted this study to define the safety and efficacy of the combination of Niraparib and Pmab in RAS wildtype (WT) mCRC. Methods: Patients (pts) with RAS WT mCRC who have progressed on at least one line of systemic chemotherapy were eligible for the trial. Other selected inclusion criteria were ≥18 years of age, ECOG PS 0-1 and measurable disease per RECIST 1.1. Pmab was administered at 6 mg/kg IV on days 1 & 15 of each 28-day cycle, while Niraparib was taken orally at 200mg or 300mg (based on body weight and platelet count) daily throughout the cycle. The primary endpoint was clinical benefit rate (CBR) defined as complete (CR) + partial (PR) response + stable disease (SD) per RECIST v1.1. Multiple secondary endpoints included safety/tolerability, ORR, progression-free survival (PFS), overall survival (OS), and duration of response (DoR). Results: A safety run-in cohort of 6 pts was initially enrolled. Following a preplanned safety analysis showing an acceptable toxicity profile, an additional 19 pts were enrolled. Of the total 25 enrolled pts, 24 were evaluable for response. Male - 50%; Whites/African Americans/Asians – 65.2%/21.7%/13%; median age – 58.5yrs. The majority had left sided tumor (92%) and the median line of prior therapy was 2 (range 1-4). CBR was 83.3% (0 CR, 6 PR, 14 SD) and ORR was 25%. mPFS – 5.6mos (95% CI: 3.7, 6.9); mOS – 20.9mos (95% CI: 9.2, NR). Six-month PFS and OS rates were 48.4% and 100% respectively. At a median follow up time of 7.5mos (95% CI: 4.2, 10.4), 3 patients remained on treatment and DoR was yet to be determined. Muco-cutaneous adverse events (AEs) of any grade included rash (58.4%), oral mucositis (20.8%), dry skin (16.7%) and paronychia (4.2%). Most common (grade > 2) treatment related AEs (TRAEs) were anemia (G3; 12.5%), dermatitis, oral mucositis, hypertension and neutropenia (G3; 4.2% each). There were no grade 4-5 TRAEs. Conclusions: The combination of Pmab and Niraparib had an acceptable safety profile, and showed considerable antitumor activity in pts with advanced RAS WT mCRC compared to historical rates. Additional biomarker analyses such as survival correlation with immune cell infiltration in paired skin biopsies and HER2/BRAF mutational status are ongoing. Funding and product (Niraparib) for the study were provided by GSK (NCT03983993). Clinical trial information: NCT03983993 .

Polymerase θ inhibition steps on the cGAS pedal.

Deficiencies in homologous recombination (HR) repair lead to an accumulation of DNA damage and can predispose individuals to cancer. Polymerase theta (Pol θ, encoded by POLQ) is overexpressed by HR-deficient cancers and promotes cancer cell survival by mediating error-prone double-stranded break (DSB) repair and facilitating resistance against poly-ADP ribose polymerase inhibitor treatment. In this issue of the JCI, Oh, Wang, et al. report on the impact of Pol θ inhibition on activation of antitumor immunity. The authors used pancreatic ductal adenocarcinoma (PDAC) cell and mouse models characterized by HR-associated gene alterations and POLQ overexpression. POLQ knockdown showed synthetic lethality in combination with gene mutations involving DNA repair, including BRCA1, BRCA2, and ATM. Notably, Pol θ deficiency or inhibition suppressed tumor growth, increased the accumulation of unrepaired DNA damage, and enhanced T cell infiltration via the cGAS/STING pathway. These findings suggest a broader scope for Pol θ inhibition in HR-deficient cancers.

First trial of chemotherapy de-escalation in ovarian cancer (OC): A phase III randomized, open label study of niraparib maintenance after carboplatin and paclitaxel in patients with optimally debulked advanced (homologous recombination deficient) HRD high-grade ovarian cancer in first line therapy (N-PLUS/NOGGO-ov53/ENGOT-ov62).

TPS5620 Background: OC is the fifth most common cause of death from cancer in women. More than 70% of the patients are diagnosed with advanced disease and less than 40% of women with OC are cured. Currently, the standard therapy in HRD OC is surgical debulking followed by 6 cycles of chemotherapy with subsequent maintenance treatment with poly ADP ribose polymerase (PARP) inhibitors. Within the last decades, no focus was set on the benefits of fewer cycles of chemotherapy, even though there is evidence for comparable efficacy and less toxicity in comparison to more cycles. Chemotherapies are usually associated with side effects, which can be severe and even lead to therapy discontinuation. The data available about the needed number of chemotherapy cycles is weak, especially in the context of PARP inhibition. Furthermore, patients with stage III/IV with optimal cytoreduction are underrepresented in previous studies. In the N-PLUS trial, we hypothesize that recurrence-free survival (RFS) in patients receiving 3 cycles of chemotherapy followed by maintenance with niraparib is not inferior to 6 cycles of chemotherapy followed by niraparib in advanced HRD high-grade OC patients with no visible disease following primary tumor debulking. Methods: In this multicenter, randomized, open-label study 650 patients with advanced HRD high-grade OC with no residual tumor mass following primary tumor debulking will be enrolled. Patients will be randomized 1:1 to receive either 3 cycles carboplatin + paclitaxel and maintenance therapy with niraparib (Arm A) or 6 cycles carboplatin + paclitaxel and maintenance therapy with niraparib (Arm B) for a maximum of 36 months. Randomization will be performed according to the results of the NGS analysis and stratified either to BRCAm/LOH-independent or BRCAwt/LOHhigh, FIGO stage III vs. IV, and participating countries. The primary endpoint will be RFS, secondary endpoints will be OS, TFST, TWIST, PFS2, Quality of Life and safety assessments. Clinical trial information: NCT05460000 .

Association of KMT2C loss-of-function mutations in circulating tumor DNA and prolonged response to the combination of PARPi and PI3Ki.

e13003 Background: The emergence of somatic KMT2C mutations has been associated with resistance to various treatments, including chemotherapy and endocrine therapy in patients with estrogen receptor-positive breast cancers. The impact of these mutations in patients treated with poly-ADP ribose polymerase inhibitors (PARPi) and phosphatidylinositol-3-kinase inhibitors (PI3Ki) is unknown. Methods: Circulating tumor DNA (ctDNA) was obtained from plasma and peripheral blood at different time points from 38 patients prior to treatment with the combination of the PARPi olaparib with the alpha-specific PI3Ki alpelisib (NCT01623349). CtDNA was also collected immediately before the first cycle and at the time of disease progression. CtDNA underwent deep targeted sequencing (tumor-normal pairs, 397 genes (2Mb) at 25,000x somatic and 10,000x germline) at the Broad Institute of MIT and Harvard. Variants that were exonic, non-synonymous, and predicted to lead to loss-of-function were considered mutations. Cohorts were stratified according to the presence or absence of the genomic alteration, and progression-free survival was compared with the Kaplan-Meier method and log-rank test. A Cox regression was performed using age, tumor type, germline BRCA1/2 mutation status, performance status, and number of previous lines of treatment as covariates. Results: The KMT2C gene was one of the 3 genes most frequently mutated in our cohort, affecting 5 patients (15%) after the exclusion of those with variants with non-deleterious alterations. The other 2 commonly mutated genes were TP53 (76%) and PIK3CA (15%). In the comparison of patients based on these most common mutations, we identified that patients with somatic KMT2C mutations (n=5) have longer mPFS than those that were wild type (n=28): mPFS 386 vs. 151 days, log-rank p = 0.025. Cox proportional hazards model accounting for age, tumor type, germline BRCA1/2 mutation status, performance status, and number of previous lines revealed a hazard ratio of 0.16; p = 0.007. All somatic KMT2C mutations were proximal to the SET domain critical for methyltransferase activity. Upon progression, the KMT2C-mutated clones were suppressed to below the detection level of mutation calling algorithms. Analysis of the raw files revealed that the clones were suppressed but not eliminated. Conclusions: While the emergence of KMT2C has been reported to be associated with cancer progression and resistance to different therapies, patients with deleterious KMT2C mutations in the ctDNA may benefit from therapy with PARPi and PI3Ki.

A four-tier classification system for studying homologous recombination repair gene reversion mutations as a mechanism of resistance to PARP inhibitors and platinum chemotherapy.

3140 Background: Pathogenic mutations in homologous recombination repair (HRR) genes are highly associated with the oncogenesis of multiple cancers and represent favorable biomarkers for platinum (Pt)-based chemotherapy and poly ADP ribose polymerase inhibitors (PARPi). While the development of drug resistance by restoring protein function via secondary mutation is commonly observed, a thorough analysis of the landscape of HRR gene reversion mutation is lacking. Methods: A real-world database with ~13,000 pan-cancer patients who underwent targeted next-generation sequencing and contained a primary germline or somatic pathogenic mutation of 25 pre-defined HRR genes, including ATM, ATR, BARD1, BLM, BRCA1, BRCA2, BRIP1, CDK12, CHEK1, CHEK2, ERCC3, FANCA, FANCC, FANCD2, FANCI, FANCL, MRE11A, NBN, PALB2, RAD51, RAD51C, RAD51B, RAD51D, RAD54L, and WRN, was used to identify HRR secondary mutations, which were further classified into four tiers by mutation type and intended reversion mechanism. Patient’s clinical data and treatment history were retrospectively reviewed. Results: The potential mechanism of HRR gene reversion was categorized into four tiers, including Tier 1 (single nucleotide variation, SNV), Tier 2 (frameshift), Tier 3 (deletion), and Tier 4 of other mutation types that were not further discussed in this study. A total of 42 patients were identified with Tier 1/2/3 secondary HRR gene mutations in the presence of a primary pathogenic germline (N = 32) or somatic (N = 10) mutation. BRCA1 (15/42, 35.7%) and BRCA2 (23/42, 54.8%) were most frequently observed in this cohort with significant enrichment of exon 11 aberrations of both genes. The other HRR genes identified of Tier 1/2/3 reversion mutations included PALB2 (N = 3, Tier 3) and RAD51D (N = 1, Tier 1). More specifically, a majority of BRCA1 reversion mutations (8/15) were Tier 1 SNV, while the most prevalent reversion mechanism for BRCA2 was Tier 3 deletion, which accounted for 60.9% (14/23) of all BRCA2 cases with a median deleted length of 1164 bp (range: 153 – 6745 bp). Furthermore, we reported the treatment history data of three cancer patients (pancreatic, prostate, and lung, respectively) demonstrated the acquisition of BRCA2 reversion mutations (Tier 2 and Tier 3) at post-Pt/Olaparib in the presence of a primary germline BRCA2 pathogenic mutation, strongly suggestive of the role of reversion mutations in driving resistance to Olaparib or Pt-chemotherapy. Conclusions: Owing to a large-scale real-world dataset, we retrospectively investigated the landscape of the reversion mechanism of HRR genes and established a four-tier classification system for secondary HRR mutations in the presence of primary germline/somatic pathogenic HRR mutations. This study is of great clinical value in studying mechanisms of drug resistance to Pt-chemotherapy and PARPi.

The CD73 immune checkpoint promotes tumor cell metabolic fitness.

CD73 is an ectonucleotidase overexpressed on tumor cells that suppresses anti-tumor immunity. Accordingly, several CD73 inhibitors are currently being evaluated in the clinic, including in large randomized clinical trials. Yet, the tumor cell-intrinsic impact of CD73 remain largely uncharacterized. Using metabolomics, we discovered that CD73 significantly enhances tumor cell mitochondrial respiration and aspartate biosynthesis. Importantly, rescuing aspartate biosynthesis was sufficient to restore proliferation of CD73-deficient tumors in immune deficient mice. Seahorse analysis of a large panel of mouse and human tumor cells demonstrated that CD73 enhanced oxidative phosphorylation (OXPHOS) and glycolytic reserve. Targeting CD73 decreased tumor cell metabolic fitness, increased genomic instability and suppressed poly ADP ribose polymerase (PARP) activity. Our study thus uncovered an important immune-independent function for CD73 in promoting tumor cell metabolism, and provides the rationale for previously unforeseen combination therapies incorporating CD73 inhibition.

Investigation of synthetic lethality gene pairs in the DNA damage response pathway in response to immunotherapy in pan-cancer.

2567 Background: The concept of synthetic lethality (SL) refers to the phenomenon where the simultaneous loss of function of two genes leads to cell death, while the loss of function of either gene alone has no effect. PARP inhibitors target poly ADP ribose polymerase (PARP) enzymes, which are involved in DNA repair, are the successful cases of utilizing synthetic lethality-based therapy to treat cancers with DNA damage response (DDR) deficiency. The prognosis for cancer patients already has a combination of synthetic lethal mutations especially in DDR pathway can vary and it is not yet fully understood how these mutations may impact the response to immunotherapy. Methods: SL gene combinations in the DDR pathway were screened out from SynLethDB database (http://histone.sce.ntu.edu.sg/SynLethDB/). The immunotherapy cohort (Samstein 2019, n=1661) and non-immunotherapy cohort (Zehir 2017, n=10336) with solid tumor were used to Kaplan-Meier analyze the correlation between SL gene combinations and prognosis. TMB scores were compared between DDR co-mutation group and non DDR co-mutation group. For investigating the associations between immune infiltrates and genetic features, TIMER 2.0 database ( http://timer.cistrome.org/ ) was conducted to estimation of immune infiltration levels on DDR mutation genes in pan-cancer. Results: 430 pairs of DDR mutation combinations were used to subgroup stratification, the DDR co-mutation group was associated with worse prognosis in non-immunotherapy cohort (HR=1.305, p<0.0001) but better in immunotherapy cohort (HR=0.741,p<0.0061). Interestingly, the TMB score in the DDR co-mutation group was significantly higher than non DDR co-mutation group (mean 50.03 vs 8.868, p<0.0001) in immunotherapy cohort. The high-frequency mutation combinations (>10%) were ATM-POLE(14.40%), ATM-ATR (13.60%), ATR-BRCA2 (13.60%), ATM-BRCA2 (12.00%) and BRCA2-RECQL4 (10.40%), respectively. Comparing common immune component with statistically significant under different DDR mutant genes, the ATM-POLE co-mutation tumor tended to have more CD4+ T cell infiltration while the ATM-ATR co-mutation tumor appeared to have more myeloid dendritic cell infiltration. Conclusions: The combinations of DDR SL mutation tumor might result to worse prognosis whereas these patients might benefit from immunotherapy. Our results will help to shed light on the complex interplay for the combinations of SL mutation in DDR pathway between cancer cells and the immune system and provide new insights into the development of effective cancer treatments.

The landscape of BRCA1 and BRCA2 large rearrangements in an international cohort of over 20 000 ovarian tumors identified using next-generation sequencing.

Approximately half of ovarian tumors have defects within the homologous recombination repair pathway. Tumors carrying pathogenic variants (PVs) in BRCA1/BRCA2 are more likely to respond to poly-ADP ribose polymerase (PARP) inhibitor treatment. Large rearrangements (LRs) are a challenging class of variants to identify and characterize in tumor specimens and may therefore be underreported. This study describes the prevalence of pathogenic BRCA1/BRCA2 LRs in ovarian tumors and discusses the importance of their identification using a comprehensive testing strategy. Sequencing and LR analyses of BRCA1/BRCA2 were conducted in 20 692 ovarian tumors received between March 18, 2016 and February 14, 2023 for MyChoice CDx testing. MyChoice CDx uses NGS dosage analysis to detect LRs in BRCA1/BRCA2 genes using dense tiling throughout the coding regions and limited flanking regions. Of the 2217 PVs detected, 6.3% (N = 140) were LRs. Overall, 0.67% of tumors analyzed carried a pathogenic LR. The majority of detected LRs were deletions (89.3%), followed by complex LRs (5.7%), duplications (4.3%), and retroelement insertions (0.7%). Notably, 25% of detected LRs encompassed a single or partial single exon. This study identified 84 unique LRs, 2 samples each carried 2 unique LRs in the same gene. We identified 17 LRs that occurred in multiple samples, some of which were specific to certain ancestries. Several cases presented here illustrate the intricacies involved in characterizing LRs, particularly when multiple events occur within the same gene. Over 6% of PVs detected in the ovarian tumors analyzed were LRs. It is imperative for laboratories to utilize testing methodologies that will accurately detect LRs at a single exon resolution to optimize the identification of patients who may benefit from PARP inhibitor treatment.

Targeting Poly(ADP)ribose polymerase in BCR/ABL1-positive cells

BCR/ABL1 causes dysregulated cell proliferation and is responsible for chronic myelogenous leukemia (CML) and Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph1-ALL). In addition to the deregulatory effects of its kinase activity on cell proliferation, BCR/ABL1 induces genomic instability by downregulating BRCA1. PARP inhibitors (PARPi) effectively induce cell death in BRCA-defective cells. Therefore, PARPi are expected to inhibit growth of CML and Ph1-ALL cells showing downregulated expression of BRCA1. Here, we show that PARPi effectively induced cell death in BCR/ABL1 positive cells and suppressed colony forming activity. Prevention of BCR/ABL1-mediated leukemogenesis by PARP inhibition was tested in two in vivo models: wild-type mice that had undergone hematopoietic cell transplantation with BCR/ABL1-transduced cells, and a genetic model constructed by crossing Parp1 knockout mice with BCR/ABL1 transgenic mice. The results showed that a PARPi, olaparib, attenuates BCR/ABL1-mediated leukemogenesis. One possible mechanism underlying PARPi-dependent inhibition of leukemogenesis is increased interferon signaling via activation of the cGAS/STING pathway. This is compatible with the use of interferon as a first-line therapy for CML. Because tyrosine kinase inhibitor (TKI) monotherapy does not completely eradicate leukemic cells in all patients, combined use of PARPi and a TKI is an attractive option that may eradicate CML stem cells.

Roles of the PARP Inhibitor in BRCA1 and BRCA2 Pathogenic Mutated Metastatic Prostate Cancer: Direct Functions and Modification of the Tumor Microenvironment.

Cancer cells frequently exhibit defects in DNA damage repair (DDR), leading to genomic instability. Mutations in DDR genes or epigenetic alterations leading to the downregulation of DDR genes can result in increased dependency on other DDR pathways. Therefore, DDR pathways could be a treatment target for various cancers. In fact, polyadenosine diphosphatase ribose polymerase (PARP) inhibitors, such as olaparib (Lynparza®), have shown remarkable therapeutic efficacy against BRCA1/2-mutant cancers through synthetic lethality. Recent genomic analytical advancements have revealed that BRCA1/BRCA2 pathogenic variants are the most frequent mutations among DDR genes in prostate cancer. Currently, the PROfound randomized controlled trial is investigating the efficacy of a PARP inhibitor, olaparib (Lynparza®), in patients with metastatic castration-resistant prostate cancer (mCRPC). The efficacy of the drug is promising, especially in patients with BRCA1/BRCA2 pathogenic variants, even if they are in the advanced stage of the disease. However, olaparib (Lynparza®) is not effective in all BRCA1/2 mutant prostate cancer patients and inactivation of DDR genes elicits genomic instability, leading to alterations in multiple genes, which eventually leads to drug resistance. In this review, we summarize PARP inhibitors' basic and clinical mechanisms of action against prostate cancer cells and discuss their effects on the tumor microenvironment.

A Pronectin™ AXL-targeted first-in-class bispecific T cell engager (pAXLxCD3ε) for ovarian cancer

BackgroundPronectins™ are a new class of fibronectin-3-domain 14th-derived (14Fn3) antibody mimics that can be engineered as bispecific T cell engager (BTCE) to redirect immune effector cells against cancer. We describe here the in vitro and in vivo activity of a Pronectin™ AXL-targeted first-in-class bispecific T cell engager (pAXLxCD3ε) against Epithelial Ovarian Cancer (EOC).MethodspAXLxCD3ε T-cell mediated cytotoxicity was evaluated by flow cytometry and bioluminescence. pAXLxCD3ε mediated T-cell infiltration, activation and proliferation were assessed by immunofluorescence microscopy and by flow cytometry. Activity of pAXLxCD3ε was also investigated in combination with poly-ADP ribose polymerase inhibitors (PARPi). In vivo antitumor activity of pAXLxCD3ε was evaluated in immunocompromised (NSG) mice bearing intraperitoneal or subcutaneous EOC xenografts and immunologically reconstituted with human peripheral blood mononuclear cells (PBMC).ResultspAXLxCD3ε induced dose-dependent cytotoxicity by activation of T lymphocytes against EOC cells, regardless of their histologic origin. The addition of PARPi to cell cultures enhanced pAXLxCD3ε cytotoxicity. Importantly, in vivo, pAXLxCD3ε was highly effective against EOC xenografts in two different NSG mouse models, by inhibiting the growth of tumor cells in ascites and subcutaneous xenografts. This effect translated into a significantly prolonged survival of treated animals.ConclusionpAXLxCD3ε is an active therapeutics against EOC cells providing a rational for its development as a novel agent in this still incurable disease. The preclinical validation of a first-in-class agent opens the way to the development of a new 14Fn3-based scaffold platform for the generation of innovative immune therapeutics against cancer.

PARP inhibitors in metastatic prostate cancer.

Poly-ADP ribose polymerase inhibitors (PARPi) are an emerging therapeutic option for the treatment of prostate cancer. Their primary mechanism of action is via induction of synthetic lethality in cells with underlying deficiencies in homologous recombination repair (HRR). In men with metastatic castrate-resistant prostate cancer (mCRPC) and select HRR pathway alterations, PARPi treatment has been shown to induce objective tumor responses as well as improve progression free and overall survival. Presently, there are two PARPi, olaparib and rucaparib, that are FDA approved in the treatment of mCRPC. Ongoing research is focused on identifying which HRR alterations are best suited to predict response to PARPi so that these therapies can be most effectively utilized in the clinic. While resistance to PARPi remains a concern, combination therapies may represent a mechanism to overcome or delay resistance.

MODERN APPROACHES TO THE SYSTEMIC TREATMENT OF RECURRENT OVARIAN CANCER

Introduction: Introduction: Recurrent ovarian cancer is one of the most challenging issues in medical oncology. Being the fifth in the cancer mortality list among women, ovarian cancer is the reason of more deaths than any other cancer of the female reproductive system. A meta-analysis was conducted to investigate the optimal treatment options for recurrent platinum-sensitive and platinum-resistant ovarian cancer. Methods: Data for writing this article was collected from the publications in English, Russian and Italian from 2002 to 2023. PubMed and Web of Science were searched for the relevant articles. Survival rates, particularly overall survival (OS), progression-free survival (PFS), and adverse events (AEs) of the chemotherapy regimens were discussed. We analyzed the literature data comparing the effectiveness and safety of chemotherapy, antiangiogenic therapy, the use of poly-ADP ribose polymerase (PARP) inhibitors, and checkpoint inhibitors in the treatment of recurrent ovarian cancer. Results: An overview of the literature devoted to modern approaches to the systemic treatment of recurrent ovarian cancer is given in the article. General principles of classification and treatment of recurrent ovarian cancer are analyzed. The existing regimens of chemotherapy in combination with targeted therapy are given separately for various types of ovarian cancer relapses. Chemotherapy with doublet platinum compounds (carboplatin or cisplatin) continues to be the standard of care in platinum-sensitive relapsed ovarian cancer with or without targeted therapy. Treatment with poly-ADP ribose polymerase inhibitors, vascular endothelial growth factor inhibitors, immunotherapy with checkpoint inhibitors, and antibody-drug conjugate for patients with folate receptor alpha-positive, can be considered in platinumresistant epithelial ovarian cancer. Conclusion: In summary, we can conclude that despite the success of the primary treatment of ovarian cancer, the majority of patients with a widespread tumor process develop a relapse of the disease over the next two years, which is the cause of death of these patients. The development of effective treatment regimens for recurrent ovarian/fallopian tube/primary peritoneal cancer remains particularly acute and important in gynecological oncology and requires further study.

One fixed dosing of a poly(adenosine diphosphate ribose) polymerase inhibitor does not fit all

Optimization of poly(adenosine diphosphate ribose) polymerase inhibitor dosing is important for maximizing the side effect and safety profile without attenuating the clinical benefit. Individualized dosing of niraparib as first‐line maintenance therapy for newly diagnosed ovarian cancer decreases adverse events while maintaining efficacy and should be considered for patients receiving niraparib in this setting.

Abstract LB164: Entinostat restores sensitivity to olaparib in two in vitro models of PARPi resistant ovarian cancer cells

Abstract The use of Poly ADP ribose polymerase inhibitors (PARPi) has revolutionized the treatment of homologous recombination (HR) deficient ovarian cancer tumors. A subset of these tumors exhibit genetic and acquired resistance to PARPi treatment. We have previously shown histone deacetylase inhibitors (HDACi) resensitizes ovarian cancer cells to PARPi. The objective of this study is to examine the effectiveness of combination HDACi and PARPi treatment in two in vitro models of mouse ovarian cancer cells. To investigate these effects, we used the following three ID8 murine ovarian cancer epithelial cell lines: TP53− / − (HR deficient), TP53− / −/BRCA2− / − (HR proficient), and an olaparib resistant line TP53− / −/BRCA2− / −-OR (ID8_OR). We also created ovarian cancer organoids from these three lines. In short, 8-week old female C57BL/6 mice were injected with 7 million untreated cells in PBS. Following the formation of ascites, mice were sacrificed and organoids were derived from both ascites and tumors. For viability assays, cells were pretreated with entinostat (Ent, 0.25uM) for 24h, followed by 72h (2D cells) or 7 days (organoids) of Ent (0-2uM), olaparib (Ola, 0-40uM), or in combination. Cell proliferation and viability was assessed using MTS and ATP-based assays. For immunofluorescence and western blotting analysis, cells were treated with 0.5uM Ent, 10uM Ola or the combination.TP53 adherent cells and organoids treated with 0.5uM Ent + 10uM of Ola significantly reduced cell proliferation when compared to Ola alone (p= 0.0028, 0.0021 Two-Way ANOVA). When compared to Ola and Ent alone, TP53/BRCA2 adherent cells treated with 0.125uM of Ent + 2.5uM of Ola also reduced cell proliferation (p=0.0011 and p=0.0072, 0.0845). Lastly, ID8_OR adherent cells and organoids treated with 0.125uM of Ent + 2.5μM of Ola significantly reduced cell proliferation when compared to Ola alone (p=0.0018, 0.0264).Immunofluorescences analysis revealed, when treated with Ola and Ent combination, adherent TP53 (p= 0.002, n.s), TP53/BRCA2 (p=0.0033, 0.0315), and ID8_OR (n.s) cells displayed increased expression of DNA damage marker gH2AX when compared to Control and Ola alone. We also observed decreased expression of RAD51 in cells treated with combination when compared to control and Ola alone, TP53 (p= <0.0001, 0.0023) and ID8_OR (n.s). In our organoids, we found an increase in gH2AX across all three cell lines when treated with Ola+Ent compared to control and Ola alone: TP53 (p= <0.0001, 0.0057), TP53/BRCA2 (n.s), and ID8_OR (p= 0.0116, 0.0151). Similarly, we found a decrease in RAD51 when cells were treated with Ola+Ent compared to control and Ola alone: TP53 (p= n.s, 0.0418) and ID8_OR (n.s). Lastly, preliminary western blot analysis of the organoids, revealed a decreases in proliferation marker PCNA in both adherent cells and organoids when comparing the control to combination treatment. In conclusion, the combination of Ola and Ent treatment restores responsiveness of two in vitro models of genetic and acquired PARPi resistance through increasing DNA damage and reducing DNA repair and proliferation. Citation Format: Bisiayo E. Fashemi, Lilian N. van Biljon1, Tyler Woodard, Vijayalaxmi Gupta, Mary M. Mullen, Benjamin Bitler, Dineo Khabele. Entinostat restores sensitivity to olaparib in two in vitro models of PARPi resistant ovarian cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB164.

Abstract 4977: Development of small molecule inhibitors of protein interaction with glycosaminoglycans

Abstract Glycosaminoglycans (GAGs), such as heparan sulfate and chondroitin sulfate, are an important component of the tumor microenvironment (TME). These carbohydrates are attached to proteoglycans at the surface of tumor cells and play a vital role in malignant transformation. Heparan sulfate (HS) glycosaminoglycans have been demonstrated to play a pleiotropic role in regulating the activity of important cellular receptors and downstream signaling pathways, and are implicated in cancer cell proliferation, angiogenesis, invasion, and metastasis. Heparinase, an endoglycosidase that specifically cleaves heparan sulfate side chains of heparan sulfate proteoglycans is expressed in several sarcoma subtypes. We measured heparinase activity in several cell lines and found that A673, CHP100, CHLA9 and RD-ES Ewing’s sarcoma cell lines have significantly higher levels of heparinase activity compared to the heparinase-deficient CHO-K1 psgD-677 cell line. Interference with heparinase or heparin sulfate function(s) may represent a therapeutic approach in sarcoma. To this end, we tested these cell lines by measuring cell growth using a cell viability assay, in the presence or absence of GTC3300P, a small molecule inhibitor of protein interaction with glycosaminoglycan. We demonstrate that the Ewing’s sarcoma cell lines exhibit potent and significant inhibition in cell growth in vitro, in the presence of drug. Western blot analysis shows induction of cleaved Poly-ADP Ribose Polymerase (PARP), yH2AX, as well as cleaved caspase-3, known markers of apoptosis and down regulation of mTORC1 targets such as phospho-S6 (p-S6), in Ewing’s sarcoma. Apoptosis was confirmed by the induction of sub-G1 population in Ewing’s sarcoma by cell cycle analysis. Previous reports have indicated that heparinase activity is strongly implicated in cell migration and invasion and is associated with tumor metastasis, angiogenesis and inflammation. This was confirmed by LC-MS/MS proteomics and bioinformatics (gene ontology and gene set enrichment) analysis of these multiple cell lines, which identified alterations in numerous biomarkers in these pathways, including multiple ribosomal protein subunits, spliceosomes components, and complement coagulation cascade. We further conducted a cell migration assay and demonstrate that GTC3300 significantly inhibits cell migration in Ewing’s sarcoma cell lines, but not in heparinase-deficient psg677 cells. Taken together our data strongly suggests that targeting protein interaction with glycosaminoglycans including heparan sulfate in Ewing’s sarcoma represents a novel approach and merits further investigation Citation Format: Tahir N. Sheikh, Elgilda Musi, Alex J. Rai, Gary K. Schwartz. Development of small molecule inhibitors of protein interaction with glycosaminoglycans. [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 4977.

Advances in the application of immune checkpoint inhibitors in gynecological tumors.

Immune checkpoints are regulatory molecules that suppress immune effector cells, and are essential for maintaining tolerance, preventing autoimmune reactions, and minimizing tissue damage by controlling the duration and intensity of the immune responses. However, immune checkpoints are frequently upregulated during cancer and dampen the anti-tumor immune responses. Immune checkpoint inhibitors (ICIs) have been effective against multiple tumors, and have improved patients' survival outcomes. Recent clinical trials have also reported promising therapeutic effects of ICIs in some gynecological cancers. To review the current research and future directions in the treatment of gynecological malignancies, including ovarian, cervical and endometrial cancers, using ICIs. Currently, cervical and ovarian cancers are the only gynecological tumors that are treated by immunotherapeutic approaches. In addition, ICIs, chimeric antigen receptor (CAR)- and T cell receptor (TCR)-engineered T cells targeting endometrial tumors, especially those originating in the vulva and fallopian tubes, are under development. Nevertheless, the molecular mechanism underlying the effects of ICIs, especially in combination with chemotherapy, radiation therapy, anti-angiogenesis drugs and poly ADP ribose polymerase inhibitors (PARPi), needs to be elucidated. Furthermore, novel predictive biomarkers have to be identified in order to increase the therapeutic efficacy of ICIs while reducing adverse reactions.