DNA-damaging Therapies Research Articles

Regulation of the Cell-Intrinsic DNA Damage Response by the Innate Immune Machinery.

Maintenance of genomic integrity is crucial for cell survival. As such, elegant DNA damage response (DDR) systems have evolved to ensure proper repair of DNA double-strand breaks (DSBs) and other lesions that threaten genomic integrity. Towards this end, most therapeutic studies have focused on understanding of the canonical DNA DSB repair pathways to enhance the efficacy of DNA-damaging therapies. While these approaches have been fruitful, there has been relatively limited success to date and potential for significant normal tissue toxicity. With the advent of novel immunotherapies, there has been interest in understanding the interactions of radiation therapy with the innate and adaptive immune responses, with the ultimate goal of enhancing treatment efficacy. While a substantial body of work has demonstrated control of the immune-mediated (extrinsic) responses to DNA-damaging therapies by several innate immune pathways (e.g., cGAS–STING and RIG-I), emerging work demonstrates an underappreciated role of the innate immune machinery in directly regulating tumor cell-intrinsic/cell-autonomous responses to DNA damage.

Identification of copy number variation-driven molecular subtypes informative for prognosis and treatment in pancreatic adenocarcinoma of a Chinese cohort.

BackgroundPancreatic adenocarcinoma (PAAD) is one of the most lethal carcinomas, and the current histopathological classifications are of limited use in clinical decision-making. There is an unmet need to identify new biomarkers for prognosis-informative molecular subtyping and ultimately for precision medicine.MethodsWe profiled genomic alterations for 608 PAAD patients in a Chinese cohort, including somatic mutations, pathogenic germline variants and copy number variations (CNV). Using the CNV information, we performed unsupervised consensus clustering of these patients, differential CNV analysis and functional/pathway enrichment analysis. Cox regression was conducted for progression-free survival analysis, the elastic net algorithm used for prognostic model construction, and rank-based gene set enrichment analysis for exploring tumor microenvironments.FindingsOur data did not support prognostic value of point mutations in either highly mutated genes (such as KRAS, TP53, CDKN2A and SMAD4) or homologous recombination repair genes. Instead, associated with worse prognosis were amplified genes involved in DNA repair and receptor tyrosine kinase (RTK) related signalings. Motivated by this observation, we categorized patients into four molecular subtypes (namely repair-deficient, proliferation-active, repair-proficient and repair-enhanced) that differed in prognosis, and also constructed a prognostic model that can stratify patients with low or high risk of relapse. Finally, we analyzed publicly available datasets, not only reinforcing the prognostic value of our identified genes in DNA repair and RTK related signalings, but also identifying tumor microenvironment correlates with prognostic risks.InterpretationTogether with the evidence from genomic footprint analysis, we suggest that repair-deficient and proliferation-active subtypes are better suited for DNA damage therapies, while immunotherapy is highly recommended for repair-proficient and repair-enhanced subtypes. Our results represent a significant step in molecular subtyping, diagnosis and management for PAAD patients.FundingThis work was supported by the National Natural Science Foundation of China (grant numbers 81470894, 81502695, 81672325, 81871906, 82073326, 82103482 and 32170663), the Shanghai Sailing Program (grant number 20YF1426900), and the Program for Professor of Special Appointment (Eastern Scholar) at Shanghai Institutions of Higher Learning (awarded to H.F.).

The ubiquitin E3 ligase FBXO22 degrades PD-L1 and sensitizes cancer cells to DNA damage

High expression of programmed death-ligand 1 (PD-L1) in cancer cells drives immune-independent, cell-intrinsic functions, leading to resistance to DNA-damaging therapies. We find that high expression of the ubiquitin E3 ligase FBXO22 sensitizes nonsmall cell lung cancer (NSCLC) cells to ionizing radiation (IR) and cisplatin, and that activation of FBXO22 by phosphorylation is necessary for this function. Importantly, FBXO22 activates PD-L1 ubiquitination and degradation, which in turn increases the sensitivity of NSCLC cells to DNA damage. Cyclin-dependent kinase 5 (CDK5), aberrantly active in cancer cells, plays a crucial role in increasing the expression of PD-L1 in medulloblastoma [R. D. Dorand etal, Science 353, 399-403 (2016)]. We show in NSCLC cells that inhibiting CDK5 or reducing its expression increases the level of FBXO22, decreases that of PD-L1, and increases the sensitivity of the cells to DNA damage. We conclude that FBXO22 is a substrate of CDK5, and that inhibiting CDK5 reduces PD-L1 indirectly by increasing FBXO22. Pairing inhibitors of CDK5 with immune checkpoint inhibitors may increase the efficacy of immune checkpoint blockade alone or in combination with DNA-damaging therapies.

CBIO-04. G-QUADRUPLEX STABILIZATION ENHANCES REPLICATION STRESS AND DNA DAMAGE IN ATRX-DEFICIENT HIGH-GRADE GLIOMA

Abstract Loss of function mutations in α-thalassaemia/mental retardation X-linked (ATRX) are a critical molecular hallmark for invariably fatal high-grade glioma (HGG). Mutational inactivation of histone chaperone ATRX leads to accumulations of abnormal DNA secondary structures known as G-quadruplexes (G4s), thereby inducing replication stress and DNA damage. As G4s arise at GC-rich regions (i.e., pericentromeric and telomeric regions), ATRX-deficiency alters genome-wide accessibility of chromatin, leads to transcriptional dysregulation, and induces alternative lengthening of telomeres (ALT). Our goal is to target ATRX deficiency through G4 stabilizers, which represent a class of novel small molecule compounds that selectively bind to and stabilize G4 structures. However, the genomic consequences and efficacy of this therapy for ATRX-deficient HGG are poorly understood. We therefore sought to evaluate the molecular mechanisms that drive selective lethality in patient-derived ATRX-deficient glioma stem cells (GSCs), following G4 stabilization. We found that ATRX-deficient GSCs demonstrate dose-dependent enhanced sensitivity to G4 stabilization, compared to ATRX-intact controls. Cell viability assays confirmed the specificity of our G4 stabilizer in comparison to other commonly used G4 stabilizers. Interestingly, G4 stabilization activated p53-independent apoptosis in ATRX-deficient GSCs. Furthermore, ATRX-deficient GSCs exhibit upregulated expression of both ATR and ATM pathways upon G4 stabilization, indicating enhanced replication stress and DNA damage via double-stranded breaks, respectively. Our preliminary findings suggest that ATR and ATM activation leads to the inhibition of transcription factor NF-κB, which in turn drives apoptosis. Lastly, our data indicate that G4 stabilization perturbs the ALT phenotype in ATRX-deficient GSCs, likely contributing to telomeric dysfunction. Taken together, these findings suggest that G4 stabilizers could synergize with ionizing radiation, the standard of care, as they are both DNA-damaging therapies. Our work defines mechanisms of action and efficacy of a novel therapeutic strategy for ATRX-deficient HGG, with strong implications for other ATRX-deficient cancers.

Therapy-Related Cytopenia of Undetermined Significance (t-CCUS) As a Precursor to Therapy-Related Myeloid Neoplasms (t-MN)

BackgroundClonal cytopenia of undetermined significance (CCUS) is a risk factor for development of myeloid neoplasms (MN) such as myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). Therapy-related myeloid neoplasm (t-MN) is defined as the development of a MN in the context of prior DNA damaging therapies including chemotherapy, radiation, and immunosuppressive therapies. CCUS diagnosed in the setting of prior DNA damaging therapy (t-CCUS) and its outcomes are not known. The aim of this study was to study clinicopathological features and outcome of t-CCUS and compare it with t-MN.MethodsPatients who had received DNA-damaging therapy and subsequently determined to have CCUS or t-MN were identified. The diagnosis of CCUS was made if the patient had non-diagnostic bone marrow biopsy evaluation combined with evidence of pathogenic myeloid somatic genetic alterations using conventional cytogenetics or next generation sequencing (NGS) panel. Pathogenic/likely pathogenic variants (PV) were stratified by DTA (DNMT3A, TET2, and ASXL1) genes vs. others. Event-free survival (EFS) was calculated using interval from t-CCUS to t-MN with death as a competing risk. Overall survival (OS) for t-CCUS and t-MN patients was calculated using interval from t-CCUS or t-MN diagnoses respectively to the last follow up. Statistical analysis was performed using JMP (v14.1, SAS Institute).ResultsWe identified 30 patients with t-CCUS and 259 patients with t-MN. Eleven (37%) t-CCUS patients developed t-MN and were included in both cohorts (Fig. A). Clinical characteristics of t-CCUS patients are shown in Table 1. Briefly, age (median, interquartile range or IQR) at the diagnosis of primary malignancy and t-CCUS was not different between those who developed t-MN and those did not (P=0.09 and 0.378 respectively). There was no difference in hemoglobin, white blood cells, or ANC between the two groups. Patients who developed t-MN had a significantly lower platelet count (68 vs. 130, P<0.01) and higher bone marrow blast percent (2 vs. 0, P=0.04) compared to those who did not.At t-CCUS diagnosis, cytogenetics was abnormal in 11 (37%) patients and none had complex karyotype (CK). Paired cytogenetic analysis at t-CCUS and t-MN was available for 10 patients: 4 had unchanged karyotype at t-MN, whereas 6 had cytogenetic evolution. In contrast, 213 (83%) of 256 t-MN patients with available cytogenetics had abnormalities and 126/256 (49%) had CK. Thus, a significantly higher proportion of patients had CK t-MN compared to t-CCUS (χ 2 26.4, P<0.01).A 42-gene NGS panel identified 36 PV in 21 patients (median 1, range 1-4) at t-CCUS. The most common PV were in TET2 (37.1%), TP53 (11.4%), DNMT3A (8.6%), and ZRSR2 (8.6%) genes. Two patients had paired NGS available at t-CCUS and t-MN: one had BCOR and U2AF1 PV at t-CCUS and acquired an additional NRAS PV at t-MN. The second patient had 2 PV in TP53 at t-CCUS diagnosis, that remained the only PV identified at t-MN. In the t-MN cohort, median number of PV was 2 (range 1-9). The most common PV identified were TP53 (21.5%), TET2 (6.7%), DNMT3A (6.4%), and ASXL1 (6.4%), whereas 4.8% patients had no PV. Three of 22 patients in the t-CCUS cohort had TP53mut, compared to 55 of 157 in the t-MN cohort (χ 2 4, P=0.05).Management strategies for t-CCUS included observation (20), growth factors (4), treatment of primary malignancy (2), immunosuppressives (1), hypomethylating agent followed by allogeneic SCT (1), and others (2). Following t-CCUS, 9 patients progressed to MDS and 2 to AML with a median of 3.5 months (IQR 1.7-43.4). EFS was 43.4 months (IQR 6.8-80.9). The presence of abnormal cytogenetics (Fig. B) and the absence of DTA-mutations (Fig. C) predicted shorter EFS.At last follow up, 12 (40%) deaths were noted. Four (33%) patients died without developing t-MN. Causes of death in the t-CCUS cohort were: t-MN (3), primary malignancy (1), progressive cytopenia without MN (1), infection (1), graft-vs.-host-disease (1), and undetermined (5). t-MN patients had a significantly shorter survival compared to t-CCUS (Fig. D).ConclusionMore than 10% of t-MN patients had a precursor t-CCUS. On the other hand, t-CCUS patients are at a high risk of progression to MN. t-MN is characterized by the acquisition of TP53mut, genomic instability, and a significantly shorter survival compared to t-CCUS. The presence of abnormal cytogenetics and the absence of DTA mutations at t-CCUS predict shorter EFS. [Display omitted] DisclosuresLitzow: Omeros: Other: Advisory Board; AbbVie: Research Funding; Jazz: Other: Advisory Board; Amgen: Research Funding; Astellas: Research Funding; Pluristem: Research Funding; Actinium: Research Funding; Biosight: Other: Data monitoring committee. Patnaik: Kura Oncology: Research Funding; Stemline Therapeutics: Membership on an entity's Board of Directors or advisory committees; Stemline Therapeutics: Membership on an entity's Board of Directors or advisory committees. Al-Kali: Novartis: Research Funding; Astex: Other: Research support to institution.

Targeting DNA2 Overcomes Myeloma Cells' Metabolic Reprogramming in Response to DNA Damage

DNA damage resistance is a major barrier to effective DNA-damaging anticancer therapy in multiple myeloma (MM). To discover novel mechanisms through which MM cells overcome DNA damage, we investigated how MM cells become resistant to antisense therapy targeting ILF2, an important DNA damage regulator in MM (Marchesini et. al., Cancer Cell 2017). We continuously treated JJN3 and KMS11 cells with an ILF2-targeting antisense oligonucleotide (ILF2 ASOs) or control non-targeting antisense oligonucleotide (NT ASOs). Whereas KMS11 cells maintained a high level of DNA damage activation and a significantly increased rate of apoptosis after 3 weeks of ILF2 ASOs treatment, JJN3 cells overcame ILF2 ASO-induced DNA damage activation and became resistant to ILF2 ASOs treatment.To evaluate whether continuous ILF2 ASOs exposure could lead to the selection of MM clones intrinsically resistant to ILF2 ASO-induced DNA damage, we performed single-cell RNA seq (scRNA-seq) analysis of JJN3 cells treated with NT or ILF2 ASOs for 3 weeks. Our analysis divided JJN3 cells into 2 main clusters that were independent of treatment (Fig. 1A), suggesting that persistent exposure to ILF2 ASOs did not induce clonal selection. Differential gene expression analysis of NT ASO- and ILF2 ASO-treated cells in each of these clusters revealed that DNA damage resistant ILF2 ASO-treated cells had significantly upregulated oxidative phosphorylation (OXPHOS), DNA repair signaling, and reactive oxidative species (ROS). Consistent with these results, metabolomic analysis of JJN3 cells after long-term exposure to ILF2-ASOs showed a significant enrichment of tricarboxylic acid cycle (TCA) intermediates (Fig. 1B). ILF2-ASO-resistant MM cells were significantly more sensitive to the OXPHOS inhibitor IACS-010759 than ILF2-ASO-sensitive cells were. These data suggest that MM cells can undergo an adaptive metabolic rewiring to restore energy balance and promote survival in response to DNA damage.We then hypothesized that ILF2-ASO-resistant cells' metabolic reprogramming relies on the repair of DNA damage induced by ILF2 depletion or by the generation of ROS from activated mitochondrial metabolism and that targeting DNA repair proteins involved in these processes overcomes DNA damage resistance. We used a CRISPR/Cas9 library screening strategy to identify DNA repair genes whose loss of function suppresses MM cells' ability to overcome ILF2-ASO-induced DNA damage. Compared with those in NT-ASO-treated cells, DNA2-targeting sgRNAs were significantly depleted after 3 weeks of treatment in ILF2-ASO-treated JJN3 cells but not in ILF2-ASO-treated KMS11 cells. These data suggest that DNA2 is needed to promote resistance to ILF2 depletion. Accordingly, the DNA2 inhibitor NSC105808 (NSC) significantly enhanced ILF2-ASO-induced apoptosis in JJN3 cells. These data gain added significance in light of previous findings that DNA2 is a nuclear and mitochondrial DNA nuclease/helicase that enables cancer cells to counteract the DNA replication stress and mitochondrial oxidative DNA damage induced by DNA-damaging agents. Accordingly, we observed that DNA2 was mainly localized into the mitochondria of MM cells.To dissect the mechanisms of DNA2 inhibition-induced synthetic lethality, we evaluated whether DNA2 activity is essential to maintain activated OXPHOS, which ILF2-ASO-resistant cells require to survive. The quantification of mitochondrial respiratory activity in NT-ASO-and ILF2-ASO-treated MM cells exposed to NSC for 72 hours showed that DNA2 activity inhibition significantly decreased the oxygen consumption rate while increasing ROS production in only ILF2-depleted cells. Transmission electron microscopy analysis showed that NSC-treated ILF2-depleted cells had fragmented mitochondrial cristae structures, whose perturbations affect the OXPHOS system structure and impair cell metabolism. These data suggest that DNA2 is essential to counteract oxidative DNA damage and maintain mitochondrial respiration after MM cells' metabolic reprogramming.In conclusion, our study has revealed a novel mechanism through which MM cells can overcome DNA damage activation. Further studies will clarify whether targeting DNA2 is synthetically lethal in tumors with increased demand of mitochondrial metabolism. [Display omitted] DisclosuresKonopleva: Genentech: Consultancy, Honoraria, Other: grant support, Research Funding; AbbVie: Consultancy, Honoraria, Other: Grant Support, Research Funding; Sanofi: Other: grant support, Research Funding; Ablynx: Other: grant support, Research Funding; Reata Pharmaceuticals: Current holder of stock options in a privately-held company, Patents & Royalties: intellectual property rights; Agios: Other: grant support, Research Funding; Cellectis: Other: grant support; Rafael Pharmaceuticals: Other: grant support, Research Funding; Calithera: Other: grant support, Research Funding; Forty Seven: Other: grant support, Research Funding; Ascentage: Other: grant support, Research Funding; AstraZeneca: Other: grant support, Research Funding; F. Hoffmann-La Roche: Consultancy, Honoraria, Other: grant support; Stemline Therapeutics: Research Funding; Novartis: Other: research funding pending, Patents & Royalties: intellectual property rights; Eli Lilly: Patents & Royalties: intellectual property rights, Research Funding; KisoJi: Research Funding.

Outcome of Therapy-Related Myeloid Neoplasms with Venetoclax-Based Therapy

Outcome of Therapy-Related Myeloid Neoplasms with Venetoclax-Based Therapy

T-MDS Is a Distinct Clinical and Pathological Entity Characterized By Better Survival Compared to t-AML

Introduction: Therapy-related myeloid neoplasm (t-MN) includes acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), and myelodysplastic/myeloproliferative neoplasms (MDS/MPN) that occur as a complication of DNA-damaging therapies. The World Health Organization recommends considering t-MN as a single entity. Whether t-MDS and t-AML have distinct characteristics and outcomes is not known. The aim of this study was to compare clinicopathological characteristics and outcomes of t-MDS and t-AML.Methods: All patients diagnosed with t-MN based on the World Health Organization criteria were identified. Bone marrow biopsies, cytogenetic, and next-generation sequencing (NGS) were obtained at the treating physician's discretion. Pathogenic/likely pathogenic variants (PV) were called based on the standard criteria. Overall survival (OS) was calculated from the time of t-MN diagnosis until the last follow up using Kaplan-Meier analysis using Wilcoxon test. For survival analysis comparing chemotherapy to best supportive care (BSC) only, patients were censored at the time of allogeneic stem cell transplant (SCT). Multivariate analysis was performed using Cox proportional hazard method and corrected using false discovery rate (FDR). Statistical analysis was performed using JMP (v14.1, SAS Institute) and significance was defined as P<0.05.Results: We identified 554 patients, of which 180 (32.4%), 365 (65.8%), and 9 (1.6%) presented as t-AML, t-MDS, and t-MDS/MPN respectively. t-MDS/MPN patients were excluded from further analysis due to a small number. Clinical and laboratory characteristics of the t-AML and t-MDS cohorts is shown in Table 1. t-AML patients were significantly more anemic and thrombocytopenic at presentation. As expected, t-AML had a higher peripheral blood and BM blast count. There was no difference in proportion of patients with chromosomal abnormalities, though a statistically significantly higher proportion of t-MDS patients had chromosome 5 abnormality, 5q deletion, monosomy 5, chromosome 7 abnormality, monosomy 7 compared to t-AML patients. On other hand, 11q23 (mixed lineage leukemia, MLL) rearrangement was more common in t-AML compared to t-MDS (9.3% vs. 2.8%, P=0.005).A higher proportion of t-MDS patients had PV detected by NGS compared to t-AML (92.9% vs. 85.6%, P=0.038). A higher proportion of t-MDS patients had PV in TP53 (37.6% vs. 21.4%, P=0.004) and ASXL1 (23% vs. 11.7%, P=0.016) genes; whereas a higher proportion of t-AML patients had PV in RAS (18.9% vs. 9.1%, P=0.013) and WT1 (8.1% vs. 2.9%, P=0.05) genes.One hundred twenty-eight (35%) of 365 t-MDS patients progressed to t-AML. The presence of any chromosomal abnormality at t-MDS diagnosis predicted a higher risk of transformation to t-AML (χ 2 3.9, P=0.03).t-AML patients had a significantly shorter OS compared to t-MDS (9.2 vs. 19.7 months, P<0.0001, Figure A). This difference persisted when stratified by no disease modifying therapies (BSC only) 2 vs. 17 months (P<0.0001, Figure B), as well as among those who received at least one line of chemotherapy (14 vs. 24.6 months, P<0.001). Finally, a higher proportion of patients with t-AML underwent SCT and there was a trend towards improved survival for t-AML patients (vs. t-MDS 52.9 vs. 20.7 months, P=0.07) from the time of transplant.Multivariate analysis for OS performed to control for all the variables that were different at presentation (except for blast count), showed that t-MDS (as opposed to t-AML) phenotype at diagnosis, and undergoing SCT were independent predictors of improved survival (Table 2).Conclusion: t-MDS and t-AML have distinct clinical, cytogenetic, and genetic features at presentation. In the absence of disease modifying therapies, t-AML is a more aggressive phenotype, consistently associated with a shorter survival. Even after controlling adverse risk features, t-AML phenotype had a shorter survival compared to t-MDS. [Display omitted] DisclosuresLitzow: AbbVie: Research Funding; Astellas: Research Funding; Amgen: Research Funding; Actinium: Research Funding; Pluristem: Research Funding; Jazz: Other: Advisory Board; Omeros: Other: Advisory Board; Biosight: Other: Data monitoring committee. Hiwase: Novartis: Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees.

P-106: Targeting DNA2 overcomes myeloma cells’ metabolic reprogramming in response to DNA damage

<h3>Background</h3> DNA damage resistance is a major barrier to effective DNA-damaging anticancer therapy in multiple myeloma (MM). To discover novel mechanisms through which MM cells overcome DNA damage, we investigated how MM cells become resistant to antisense therapy targeting ILF2, an important DNA damage regulator in 1q21 MM. <h3>Method</h3> We continuously treated MM cells with an ILF2-targeting antisense (ILF2-ASO) or control non-targeting antisense (NT-ASO) for 3 weeks to see if ILF2-ASO exposure leads to the selection of MM clones intrinsically resistant to DNA damage or activates compensatory mechanisms to overcome ILF2 depletion-induced DNA damage. <h3>Results</h3> Single-cell RNA sequencing (scRNA-seq) analysis revealed that DNA damage-resistant ILF2-ASO-treated cells had significantly upregulated oxidative phosphorylation (OXPHOS), DNA repair signaling, and reactive oxidative species (ROS). Metabolomic analysis of MM cells after long-term exposure to ILF2-ASO showed a significant enrichment of tricarboxylic acid cycle (TCA) intermediates. Consistent with these results, ILF2-ASO-resistant MM cells were significantly more sensitive to the OXPHOS inhibitor IACS-010759 than ILF2-ASO-sensitive cells were. These data suggest that MM cells can undergo an adaptive metabolic rewiring to restore energy balance and promote survival in response to DNA damage. We then hypothesized that ILF2-ASO-resistant cells' metabolic reprogramming relies on the repair of DNA damage induced by ILF2 depletion or by the generation of ROS from activated mitochondrial metabolism and that targeting DNA repair proteins involved in these processes overcomes DNA damage resistance. We used a CRISPR/Cas9 screening strategy to identify DNA repair genes whose loss of function suppresses MM cells' ability to overcome ILF2-ASO-induced DNA damage. Compared with those in NT-ASO-treated cells, DNA2-targeting sgRNAs in ILF2-ASO-treated JJN3 cells were significantly depleted after 3 weeks of treatment, suggesting that DNA2 is needed to promote resistance to ILF2 depletion. Accordingly, the DNA2 inhibitor NSC105808 (NSC) significantly enhanced ILF2-ASO-induced apoptosis. To dissect the mechanisms of DNA2 inhibition-induced synthetic lethality, we evaluated whether DNA2 activity is essential to maintain activated OXPHOS, which ILF2-ASO-resistant cells require to survive. The quantification of mitochondrial respiratory activity in NT-ASO- and ILF2-ASO-treated MM cells exposed to NSC for 3 days showed that DNA2 activity inhibition significantly decreased the oxygen consumption rate while increasing ROS production in only ILF2-depleted cells. Transmission electron microscopy analysis showed that NSC-treated ILF2-depleted cells had fragmented mitochondrial cristae structures, whose perturbations affect the OXPHOS system structure and impair cell metabolism. <h3>Conclusion</h3> In conclusion, our study has revealed a novel mechanism through which MM cells counteract oxidative DNA damage and maintain mitochondrial respiration after metabolic reprogramming.

BRCAness as a Biomarker of Susceptibility to PARP Inhibitors in Glioblastoma Multiforme.

Glioblastoma multiforme (GBM) is the most common primary brain cancer. GBMs commonly acquire resistance to standard-of-care therapies. Among the novel means to sensitize GBM to DNA-damaging therapies, a promising strategy is to combine them with inhibitors of the DNA damage repair (DDR) machinery, such as inhibitors for poly(ADP-ribose) polymerase (PARP). PARP inhibitors (PARPis) have already shown efficacy and have received regulatory approval for breast, ovarian, prostate, and pancreatic cancer treatment. In these cancer types, after PARPi administration, patients carrying specific mutations in the breast cancer 1 (BRCA1) and 2 (BRCA2) suppressor genes have shown better response when compared to wild-type carriers. Mutated BRCA genes are infrequent in GBM tumors, but their cells can carry other genetic alterations that lead to the same phenotype collectively referred to as ‘BRCAness’. The most promising biomarkers of BRCAness in GBM are related to isocitrate dehydrogenases 1 and 2 (IDH1/2), epidermal growth factor receptor (EGFR), phosphatase and tensin homolog (PTEN), MYC proto-oncogene, and estrogen receptors beta (ERβ). BRCAness status identified by accurate biomarkers can ultimately predict responsiveness to PARPi therapy, thereby allowing patient selection for personalized treatment. This review discusses potential biomarkers of BRCAness for a ‘precision medicine’ of GBM patients.

Proteins from the DNA Damage Response: Regulation, Dysfunction, and Anticancer Strategies.

Simple SummaryCells respond to genotoxic stress through complex protein pathways called DNA damage response (DDR). These mechanisms ensure the preservation of genomic integrity and activate DNA repair, cell cycle regulation, and, ultimately, programmed cell death. When altered, the DDR protein network leads to several diseases, particularly cancers. In recent years, the vulnerabilities of the DDR network have been successfully exploited to improve cancer treatments using DNA damage strategies and therapies combination.Cells respond to genotoxic stress through a series of complex protein pathways called DNA damage response (DDR). These monitoring mechanisms ensure the maintenance and the transfer of a correct genome to daughter cells through a selection of DNA repair, cell cycle regulation, and programmed cell death processes. Canonical or non-canonical DDRs are highly organized and controlled to play crucial roles in genome stability and diversity. When altered or mutated, the proteins in these complex networks lead to many diseases that share common features, and to tumor formation. In recent years, technological advances have made it possible to benefit from the principles and mechanisms of DDR to target and eliminate cancer cells. These new types of treatments are adapted to the different types of tumor sensitivity and could benefit from a combination of therapies to ensure maximal efficiency.

Abstract 2723: How BRCA1/2 mutations in TNBC affect TME and subsequently immune cell functions

Abstract Breast cancer type 1 and 2 susceptibility proteins (BRCA1/BRCA2) are well known breast cancer genes, mutations in which lead to defective homologous recombination repair (HRR). HR-based DNA repair deficiency (HRD) scores can be used to indicate DNA damage, genomic instability and may predict response to DNA damaging agents in BRCA1/2 mutated tumors. Tumors with a high HRD score caused by complete loss of BRCA1 or BRCA2 function locus-specific LOH are sensitive to DNA damage agents including platinum-based chemotherapy and poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi). To understand the relationship between BRCA1/2 mutations and DNA damage in the tumor microenvironment (TME), we have characterized 107 BRCA1/2 tumors to determine HRD score using whole exome sequencing (WES) and simultaneously measured markers of DNA damage, PARP expression, tumor-infiltrating lymphocytes (TILs) and immune checkpoints to identify potential treatment targets on matching samples (n=47) using highly multiplexed fluorescence microscopy CO-Detection by indEXing (CODEX). We have established and validated a 40-plex breast cancer specific antibody panel consisting of markers to detect DNA damage, TILs and immune checkpoints to deeply profile how the TME is affected by BRCA1/2 mutations using CODEX. Computational image processing of CODEX data was performed to interrogate changes in number, size, morphology, and marker expression in tumor and immune cells. We have characterized BRCA1/2 tumors (n=47) on Tissue Microarrays (TMAs), and we have detected cytotoxic CD8+T and CD107a+NK cells in HRD low (&amp;lt;42) and HRD high groups. Interestingly, their frequency and cytolytic function (measured by Granzyme A and perforin transcript level) appear to be associated with HRD scores. For instance, HRD low groups showed increased cytotoxic CD8+T and CD107a+NK cells whereas HRD high groups revealed decreased cell numbers as well as cytolytic function. We observed that HRD high, LOH positive was associated with increased DNA damage marker expression in tumor cells (H3pSer28, pATM, yH2AX) as well as NK cells (pATM) whereas immune checkpoint protein levels were decreased. We have planned quantitative analysis which allow us to determine the percentage of cell subtypes as well as the spatial compartmentalization of cells to interrogate the tumor microenvironment associated with BRCA1/2 mutations. In conclusion, BRCA1/2 mutated tumors with high HRD score revealed upregulated DNA damage expression in immune cells suggesting that BRCA1/2 mutations can impact HRR in CD8+T cells and CD107a+ NK cells and subsequently affect their ability to produce Granzyme A and Perforin. Our findings will decipher the role of DNA damage in BRCA1/2 mutated tumor cells and immune cell types. Outcomes can potentially predict treatment responses such as DNA damage, PARPi and checkpoint inhibitor therapies in TNBC BRCA1/2 breast cancer. Citation Format: Dana Pueschl, Derek A. Oldrige, Jonathan Belman, Jake S. Shilan, Anupma Nayak, Bradley Wubbenhorst, John Pluta, Robert H. Vonderheide, Michael Feldman, Kara N. Maxwell, E. John Wherry, Susan M. Domchek, Katherine L. Nathanson. How BRCA1/2 mutations in TNBC affect TME and subsequently immune cell functions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2723.

Abstract 2568: Use of kinase inhibitors in Fanconi anemia oral cancercell lines

Abstract Successful Fanconi anemia (FA) treatment allows greater longevity for these patients. However, there is a greatly increased risk of squamous malignancies of the head and neck which are quite deadly in these young patients. There is an unmet need for non DNA damaging therapies for this malignancy which could augment surgery. Our approach has been to examine potential treatments based on known pathophysiology of the disease which would be acceptable for this population so we examined the effects of metformin and pioglitazone, as well as two cell cycle kinase inhibitors, in FA and leukoplakia cell lines. We examined dose-dependent and combination effects on cell proliferation, as judged by MTT assay. We examined metformin, pioglitazone, the polo-like kinase 1 inhibitor, GSK461364, and Wee1 kinase inhibitor, AZD1775.In single agent studies, we observed dose-dependent decreases in cell proliferation with all 4 single agents. We observed decreased cell proliferation in all cell lines at 72 hours at clinically achievable serum levels of 85% to 55% of control levels (P&amp;lt;0.0001). Additionally, we found decreases beyond single agent treatment levels when cells were treated with metformin combined with GSK461364 or AZD1775 and similarly, with combination therapies of pioglitazone and GSK461364 or AZD1775. Our current data demonstrate feasibility and preclinical efficacy of a combined agent approach for FA associated head and neck cancer. The low reported toxicity of these agents clinically allows for all of these agents to potentially move forward clinically, once additional experiments are conducted in mechanistic and animal studies. Citation Format: Walter N. Jungbauer, Mustafa M. Ali, Beverly R. Wuertz, Frank G. Ondrey. Use of kinase inhibitors in Fanconi anemia oral cancercell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2568.

Abstract 2124: Vitamin C enhances H3K36me3 via NSD1 promoter demethylation and sensitizes GBM stem cells to DNA damaging agents

Abstract Primary brain tumors are among the most devastating forms of cancer. Due to profound genetic, epigenetic, and morphologic heterogeneity, GBM represents the most aggressive and lethal form of the disease. GBM exhibits aberrant patterns of DNA methylation, resulting in a highly distorted epigenome, yet the molecular circuits responsible for this epigenetic dysregulation remain unknown. Glioma stem cells (GSCs) are critical modulators of tumor propagation and therapeutic resistance in GBM, and epigenetic deregulation within this cell subpopulation is thought to play fundamental roles in GSC and GBM biology. Understanding and overcoming these tumorigenic epigenetic mechanisms in GSCs is crucial to developing more effective treatment protocols and improving clinical survival. DNA methylation is a reversible process involving the addition of methyl groups, typically, at cytosine residues and demethylation is catalyzed, in part, by the ten-eleven translocation (TET) family of enzymes. These enzymes function as deoxygenases that convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). Multiple studies show negative correlations between 5hmC levels and glioma grade, and loss of 5hmC correlates with poor prognosis of GBM patients. These observations suggest that understanding the mechanisms of 5hmC deregulation in GBM and developing innovative ways to re-establish 5hmC levels in tumors can impact patient outcome. Ascorbate (Vitamin C) is a TET enzyme cofactor that has gained pre-clinical interest as an attractive antitumor agent for GBM due to its ability to regulate redox status, function as potent demethylating agent, and efficiently penetrate the blood-brain barrier. We show through various molecular and biochemical endpoints, that low-dose Vitamin C significantly replenishes 5hmC and decreases stemness in GSCs. We found that, in addition to increasing global levels of 5hmC, low-dose Vitamin C acts synergistically with temozolomide (TMZ) and irradiation to induce cell death in GSCs. We also present a novel mechanism whereby Vitamin C regulates promoter demethylation of the histone methyltransferase NSD1 resulting in an increase in H3K36me3 in GSCs. We propose the increase in H3K36me3 results in euchromatin states that are more susceptible to DNA damage therapies. Our findings strongly indicate that Vitamin C can undo tumor-promoting DNA hypermethylation and sensitize GSCs to conventional radio- and chemotherapeutics and suggest it might serve as an adjunct to standard-of-care GBM treatment. Citation Format: Harmon Khela, Sweta Sudhir, Maria Fagundo-Lugo, Bachchu Lal, John Laterra, Hernando Lopez-Bertoni. Vitamin C enhances H3K36me3 via NSD1 promoter demethylation and sensitizes GBM stem cells to DNA damaging agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2124.

Mutagenic Consequences of Sublethal Cell Death Signaling.

Many human cancers exhibit defects in key DNA damage response elements that can render tumors insensitive to the cell death-promoting properties of DNA-damaging therapies. Using agents that directly induce apoptosis by targeting apoptotic components, rather than relying on DNA damage to indirectly stimulate apoptosis of cancer cells, may overcome classical blocks exploited by cancer cells to evade apoptotic cell death. However, there is increasing evidence that cells surviving sublethal exposure to classical apoptotic signaling may recover with newly acquired genomic changes which may have oncogenic potential, and so could theoretically spur the development of subsequent cancers in cured patients. Encouragingly, cells surviving sublethal necroptotic signaling did not acquire mutations, suggesting that necroptosis-inducing anti-cancer drugs may be less likely to trigger therapy-related cancers. We are yet to develop effective direct inducers of other cell death pathways, and as such, data regarding the consequences of cells surviving sublethal stimulation of those pathways are still emerging. This review details the currently known mutagenic consequences of cells surviving different cell death signaling pathways, with implications for potential oncogenic transformation. Understanding the mechanisms of mutagenesis associated (or not) with various cell death pathways will guide us in the development of future therapeutics to minimize therapy-related side effects associated with DNA damage.

Molecular subtypes of pancreatic adenocarcinoma stratified based on copy number variation profile.

e16232 Background: Pancreatic adenocarcinoma (PAAD) is one of the most lethal carcinomas all over the world, with an estimate of 60,430 new cases and 48,220 deaths in United States according to the cancer statistics of 2021. It is important and urgent to identify molecular subtypes for stratifying PAAD into different groups with respective genomic footprint, appropriate treatment and prognosis status. Methods: We profiled a comprehensive landscape of genomic alterations for 608 PAAD patients in a Chinese cohort, including somatic mutations, pathogenic germline variants and copy number variations (CNVs). 233 out of 608 PAAD had disease-free survival (DFS) information with at least 6-month follow-up. Focusing on this subset of PAAD, we explored prognostic values for several kinds of genomic alterations and finally identified four molecular subtypes based on CNV profile. Results: For point mutations in highly mutated genes ( KRAS, TP53, CDKN2A and SMAD4) or homologous recombination repair (HRR) genes ( ATM, ATR, BARD1, BLM, BRCA1/2, BRIP1, CDK12, CHEK1/2, NBN, PALB2, RAD50/ 51B/ 51C/ 51D/54L and MRE11A), we found no associations between them and prognosis of PAAD patients. Instead, PAAD could be stratified into four molecular subtypes based on CNV profile through unsupervised clustering and CNV score defined by principle component analysis (PCA). According to amplification and deletion pattern of genes in DNA repair and receptor tyrosine kinase related signaling pathways, we named these four subtypes as repair-deficient, proliferation-active, repair-proficient and repair-enhanced ones. Among them, repair-deficient PAAD had the best prognosis with median DFS of 410 days, repair-proficient and proliferation-active one had median DFS of 239 and 197 days, respectively, while repair-enhanced one had the worst prognosis with DFS of 48 days. Genomic footprint analysis showed that both of repair-deficient and proliferation-active subtypes of PAAD possessed higher copy number instability (CNI), while both of repair-proficient and repair-enhanced subtypes possessed higher tumor mutational burden (TMB), implying that the former two subtypes suited for DNA damage therapies (such as platinum-based chemotherapy and PARPi) and the latter two subtypes suited for immunotherapy. Conclusions: We identified four molecular subtypes for PAAD based on CNV profile and found their respective genomic footprint, suited treatment and prognosis status, pacing key steps to precision medicine in PAAD field.

Identification of Copy Number Variation-Driven Molecular Subtypes in Pancreatic Adenocarcinoma of Chinese Cohort

Pancreatic adenocarcinoma (PAAD) is one of the most lethal carcinomas all over the world. The current histopathological classifications for PAAD are of limited use to guide clinical decision-making, and there is an unmet need to identify new biomarkers for molecular subtyping. We profiled a comprehensive landscape of genomic alterations for 608 PAAD patients in a Chinese cohort, including somatic mutations, pathogenic germline variants and copy number variations (CNV). Our data provided no evidence of associations with prognosis based on the information of point mutations in either highly mutated genes (such as KRAS, TP53, CDKN2A and SMAD4) or homologous recombination repair genes. Instead, associated with worse prognosis were amplified genes involved in DNA repair and receptor tyrosine kinase related signaling pathways. Motivated by this observation, we categorized patients into four molecular subtypes (namely repair-deficient, proliferation-active, repair-proficient and repair-enhanced) that differed in prognosis. Together with the evidence from genomic footprint analysis, we suggested that repair-deficient and proliferation-active subtypes were better suited for DNA damage therapies, while immunotherapy was highly recommended for repair-proficient and repair-enhanced subtypes. Collectively, our findings represent a significant step in improving diagnosis and management for PAAD patients. Funding Statement: This work was supported by the National Natural Science Foundation of China (grant numbers 81470894, 81502695, 81672325, 81871906, 82073326) and the Shanghai Sailing Program (grant number 20YF1426900). Declaration of Interests: The authors declare no potential conflicts of interest. Ethics Approval Statement: The overall study protocol was approved by the Medical Ethical Committee of Ruijin Hospital and the research was conducted in accordance with relevant ethical guidelines.

WEE1 Inhibition in Combination With Targeted Agents and Standard Chemotherapy in Preclinical Models of Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal cancer with high incidences of p53 mutations. AZD1775 (adavosertib, previously MK-1775) is a small molecule WEE1 inhibitor that abrogates the G2M checkpoint and can potentially synergize with DNA damaging therapies commonly used in PDAC treatment. The purpose of this study was to identify combination partners for AZD1775, including standard chemotherapy or targeted agents, in PDAC preclinical models. Low powered preliminary screens demonstrated that two of the four PDX models responded better to the combinations of AZD1775 with irinotecan or capecitabine than to either single agent. Following the screens, two full powered PDAC PDX models of differing p53 status were tested with the combinations of AZD1775 and irinotecan or capecitabine. The combinations of AZD1775 and SN38 or 5-FU were also tested on PDAC cell lines. Cellular proliferation was measured using an IncuCyte Live Cell Imager and apoptosis was measured using a Caspase-Glo 3/7 assay. Flow cytometry was conducted to measure alterations in cell cycle distribution. Western blot analysis was used to determine the effects of the drug combinations on downstream effectors. In PDX models with mutated p53 status, there was significant tumor growth inhibition from the combination of AZD1775 with irinotecan or capecitabine (P ≤ 0.03), while PDX models with wild type p53 did not show anti-tumor synergy from the same combinations (P ≥ 0.08). The combination of AZD1775 with SN38 or 5-FU significantly decreased proliferation in all PDAC cell lines, and enhanced apoptosis in multiple cell lines. Cell cycle distribution was disrupted from the combination of AZD1775 with SN38 or 5-FU which was recorded as G2M arrest and decreased G1 phase. AZD1775 inhibited phospho-CDC2 and increased the expression of γH2AX that was either maintained or enhanced after combination with SN38 or 5-FU. The combination of AZD1775 with irinotecan/SN38 or capecitabine/5-FU showed anti-tumor effects in vivo and in vitro in PDAC models. These results support further investigation for these combination strategies to enhance outcomes for PDAC patients.

Effect of prior therapy on tumor mutational burden in NSCLC.

BackgroundHigher tumor mutation burden (TMB) in advanced non-small cell lung cancer (NSCLC) is associated with superior outcomes with checkpoint inhibitor therapy. Tissue samples subject to TMB analysis may be acquired after DNA-damaging therapies such as chemotherapy or radiation. The impact of these therapies on TMB results is unclear. This retrospective analysis explored differences in TMB among treatment-naïve samples and treatment-experienced samples.MethodsNSCLC samples that underwent molecular profiling at a CLIA-certified genomics laboratory (Caris Life Sciences, Phoenix, AZ) and had available treatment and clinical history were identified. TMB was estimated by counting all coding variants (missense, nonsense, frameshift, in-frame InDels) identified by next-generation sequencing. Exceptions were synonymous mutations and any single nucleotide polymorphisms described as germline. History was reviewed under an IRB approved protocol to determine whether patients had received cytotoxic chemotherapy or radiation therapy in the year prior to collection of the tissue subject to TMB analysis. TMB values were compared between cohorts using the Wilcoxon test. Smoking adjusted P values were calculated using the chi-squared test of deviance.ResultsTMB was calculated for 970 annotated tumor specimens. Of these, 155 patients received chemotherapy and/or radiation prior to tissue collection. The median TMB was 8 mut/Mb in both the treatment-naïve and treatment-experienced cohorts. After adjusting for smoking, there was no significant difference in TMB between these cohorts (P=0.22). When analyzed separately, neither prior chemotherapy nor prior radiation therapy influenced TMB. TMB was higher when the specimen source was collected from a metastatic site compared to the primary site.ConclusionsPrior exposure to chemotherapy or radiation therapy was not associated with a significant difference in TMB.

Abstract GS3-05: Classification of triple negative breast cancer (TNBC) by DNA damage immune response (DDIR) signature and homologous recombination deficiency (HRD) status: Analysis of SWOG S9313 adjuvant trial

Abstract Introduction/Aims: DDIR signature, HRD, and stromal tumor infiltrating lymphocytes (sTIL) have each been associated with favorable outcomes in early stage TNBC. We assessed the overlap between these markers and created prognostic categories based on their combined use in a prospective trial of TNBC patients uniformly treated with adjuvant doxorubicin (A) and cyclophosphamide (C) on the SWOG S9313 trial. Methods: SWOG S9313 trial accrued 3,125 women with early stage breast cancer to two alternative dose schedules of AC, with no difference in outcomes between the two arms. 425 centrally determined TNBC cases from S9313 were identified. DDIR signature (score ≥ 0.3681 = DDIR+, Almac Diagnostic Services), HRD status (score ≥ 42 = HRD+, Myriad Genetics), and sTIL were assessed. Gene expression data (Xcel™ array) was subjected to claraT V3.0.0 biological signature analysis (Almac Diagnostic Services), and co-expression cluster analysis was used to identify signatures associated with DDIR and HRD status. The impact of dual classification by DDIR and HRD status (Group 1: DDIR+/HRD+, Group 2: DDIR+/HRD-, Group 3: DDIR-/HRD+, Group 4: DDIR-/HRD-) on disease free survival (DFS) and overall survival (OS) was examined using Cox regression with adjustment for randomized treatment assignment and nodal status. Results: For the 425 patients, median age was 45 years, 33% were node-positive, and 5-year DFS and OS were 74% and 82%, respectively. DDIR and HRD status was available for 89% each, sTIL% was available for 99%, and all three markers were available for 77% (328/425) of patients. 60% were DDIR+ and 65% HRD+. Among DDIR- tumors, 58% were HRD+. sTIL% was associated with DDIR status (P&amp;lt;0.0001) but not with HRD status (P=0.75). The proportion of patients in each group, median sTIL%, and 5-year DFS and OS for each group are outlined in Table 1. DFS and OS were similar for Groups 1, 2, and 3 but significantly lower for Group 4. As expected, cluster analysis showed that immune response signatures dominated Groups 1 and 2 regardless of HRD status. Group 3 tumors were characterized by over-representation of genomic instability signatures, a paucity of immune-related signatures, and low sTIL infiltration. Despite this immune-depleted phenotype, the 5-year OS for Group 3 was similar to that of the immune-enriched DDIR+ groups. Signatures associated with epithelial-mesenchymal transition, mast cell infiltration, and xenobiotic metabolism were over-represented in Group 4. Conclusions: Forty percent of patients with early stage TNBC demonstrate immune-deplete (DDIR-) phenotype, and within this phenotype, more than half demonstrate HRD+ status. HRD+ status within the immune-deplete phenotype predicts for better DFS and OS with adjuvant AC, probably due to underlying genomic instability and increased sensitivity to DNA damaging chemotherapy. Sixty percent of early stage TNBC patients demonstrate an immune-enriched (DDIR+) phenotype, and this phenotype is associated with improved survival with adjuvant AC chemotherapy regardless of HRD status. These findings provide important insights for patient selection and stratification in ongoing and future trials assessing DNA damaging therapy (e.g. PARPi, anthracyclines, platinum agents), immunotherapy, and their combinations in TNBC. Table 1Immune-Enriched GroupsImmune-Deplete GroupsGroup 1 DDIR+/HRD+ N=137 (42%)Group 2 DDIR+/HRD- N=59 (18%)Group 3 DDIR-/HRD+ N=77 (23%)Group 4 DDIR-/HRD- N=55 (17%)5-year DFS82%74%74%56%P=0.001 (Group 1 vs 4); P=0.006 (Group 2 vs 4); P=0.016 (Group 3 vs 4); P=NS (Groups 1 vs 2, 1 vs 3, and 2 vs 3)5-year OS88%86%83%69%P=0.001 (Group 1 vs 4); P=0.003 (Group 2 vs 4); P=0.026 (Group 3 vs 4); P=NS (Group 1 vs 2, 1 vs 3, and 2 vs 3)Median sTIL (%)20%20%5%5%P&amp;lt;0.0001 (Group 1 vs 3); P&amp;lt;0.0001 (Group 2 vs 3); P=NS (Group 1 vs 2, 3 vs 4) Citation Format: Shane R Stecklein, William Barlow, Lajos Pusztai, Kirsten Timms, Richard Kennedy, Sunil Badve, Yesim Gökmen-Polar, Peggy Porter, Hannah Linden, Debu Tripathy, Gabriel N Hortobagyi, Andrew K Godwin, Alastair Thompson, Daniel Hayes, Priyanka Sharma. Classification of triple negative breast cancer (TNBC) by DNA damage immune response (DDIR) signature and homologous recombination deficiency (HRD) status: Analysis of SWOG S9313 adjuvant trial [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr GS3-05.