Abstract Deficiency in the homologous recombination repair (HRR) pathway, which is key for high-fidelity DNA double-strand break (DSB) repair, is prevalent across various cancers (including ovarian, breast, pancreatic, and prostate). It frequently arises from loss-of-function mutations in BRCA1, BRCA2, or other HRR-associated genes. PARP inhibitors (PARPi) have transformed the treatment landscape and survival outcomes in patients with HRR-deficient tumors. However, despite the remarkable efficacy of PARPi, clinical benefit is variable, and a significant number of patients develop resistance. Combinations of PARPi with targeted agents blocking the cellular DNA damage response have been pursued to improve efficacy of PARPi treatments, but enhanced myelosuppression has limited dosing and, consequently, efficacy of these approaches. DNA polymerase theta (Polθ; POLQ) is a key component of microhomology-mediated end joining (MMEJ) DSB repair pathway. Under HRR-deficient conditions, cellular DSB repair becomes increasingly dependent on POLQ-driven MMEJ. We report the identification and optimization of a novel chemotype of POLQ inhibitors, culminating in the discovery of the clinical candidate AZD4956. AZD4956 is a potent and selective inhibitor of the polymerase domain of POLQ (IC50 <10 nM) in biochemical assays. AZD4956 suppresses cellular MMEJ activity with single digit nanomolar potency (IC50 3.95 nM) and shows potent antiproliferative activities (IC50 values between 3-10 nM) and induction of genomic instability (assessed by micronuclei quantification) in a wide range of HRR defective cell lines, with no measurable activity in HRR proficient genetic backgrounds. AZD4956 further enhances genomic instability and antiproliferative activity induced by the PARP1-selective inhibitor, saruparib, in multiple HRR defective cell lines, with no activity of the combination in HRR proficient settings. Additionally, combination of AZD4956 and saruparib does not exacerbate hematotoxicity of saruparib treatment in a 3D-bone marrow microphysiological system. Oral daily treatment of AZD4956 combined with saruparib drives sustained regression of the HRR deficient BRCA2-/- DLD-1 xenograft model, superior to the respective activities of the two agents as monotherapies. Increased efficacy of the combination can be pharmacodynamically tracked by increased accumulation of micronuclei in circulating red blood cells. The preclinical profile, spanning efficacy, safety, DMPK and physicochemical properties, supports clinical development of AZD4956 as a combination partner with saruparib in patients with HRR deficient cancers. Clinical evaluation of AZD4956 is ongoing in a first-in-human, open-label, multicenter, phase 1/2a study in patients with HRR deficient solid tumors (PARTHENON study) in combination with saruparib. Citation Format: Bernard Barlaam, Josep V. Forment, Lee Mulderrig, de Renty Christelle, Harriet Southgate, Adina Hughes, Derek Barratt, Nina Akrap, Marcello Maresca, Oliver Turner, Dougles Ferguson, Andy Pike, Ross Hill, Lenka Oplustil O’Connor, Kainat Khan, Sonja Gill, Stefan Kavanagh, Susan Critchlow, Sabina Cosulich, David Wilson. Discovery of AZD4956, a potent and selective inhibitor of DNA polymerase theta, a clinical candidate for treatment of HRR-deficient tumors in combination with saruparib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 5745.

Abstract Introduction: Loss-of-function mutations in the tumour suppressors BRCA1/BRCA2 inactivate the homologous recombination repair (HRR) pathway, driving genomic instability and cancer progression but conferring sensitivity to DNA double-strand break–inducing agents such as platinum salts and poly(ADP-ribose) polymerase inhibitors (PARPi). BRCA1/BRCA2 mutations are frequent in ovarian, breast, pancreatic, and prostate cancers, underpinning approvals of PARPi in these settings. Despite strong activity in HRR-defective tumours, PARPi responses vary and resistance emerges. Attempts to boost efficacy by combining PARPi with other DNA damage response inhibitors have been limited by enhanced myelosuppression, constraining dose intensity and benefit. Results: AZD4956 is a novel, potent, and selective inhibitor of the polymerase activity of DNA polymerase theta (Polθ), a key effector of microhomology-mediated end joining—a repair pathway critical when HRR is compromised. Combining AZD4956 with the PARP1-selective inhibitor saruparib improves efficacy versus either agent alone across HRR-defective cellular backgrounds, with no activity in HRR-proficient settings. Enhanced efficacy correlates with increased genomic instability: in HRR-deficient cells, the combination produces a ∼4-fold rise in chromosomal aberrations. AZD4956 also potentiates other DNA-damaging agents (e.g., cisplatin, TOP1 inhibitors) specifically in HRR-deficient lines.AZD4956 shows single-agent in vitro activity in the low-nanomolar range in cell lines with PALB2, BRCA2, and RAD51C mutations, like PARPi, but no single-agent activity in BRCA1-mutant cells. In vivo, AZD4956 monotherapy yields modest tumour growth inhibition (TGI ∼50–80%) in some BRCA2 and PALB2 mutant models. By contrast, AZD4956 (≥10 mg/kg BID) combined with the maximal efficacious mouse dose of saruparib (1 mg/kg QD) consistently outperforms either monotherapy. Increased efficacy aligns with pharmacodynamic modulation: the combination drives an average ∼2-fold increase in micronuclei in red blood cells versus monotherapy. Combination activity is observed in both BRCA1- and BRCA2-mutant patient-derived xenografts from diverse tissues (breast, prostate) and is restricted to HRR-deficient models where PARPi alone confers some TGI. Notably, maximal combination benefit requires the maximal efficacious saruparib dose. Conclusions: Preclinical pharmacology supports AZD4956 selectivity and its potential to amplify antitumour activity when combined with PARPi in HRR-defective cancers. AZD4956 is being evaluated in PARTHENON, a first-in-human, open-label, multicentre, phase 1/2a study of AZD4956 plus saruparib in patients with HRR-deficient solid tumours. Citation Format: Josep V. Forment, Lee Mulderrig, Christelle de Renty, Harriet Southgate, Gemma Jones, Martina Gesu, Rebecca Sargeant, Daniel Sutton, Lenka Oplustil O'Connor, Susan Critchlow, Sabina Cosulich. AZD4956, a potent and selective inhibitor of DNA polymerase theta, enhances the activity of DNA-damaging agents in HRR defective cellular backgrounds and improves efficacy of the new generation PARP1-selective inhibitor, saruparib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 230.

Abstract Introduction: This study aimed to identify genes that sensitize NSCLC cells to AZD3470, an MTA-cooperative PRMT5 inhibitor that targets MTAP-deficient tumors while sparing normal tissues. Early clinical data for this class of drugs as monotherapy has shown objective response rates (ORRs) of approximately 20% across multiple tumor indications. Encouragingly, the duration of response and disease control rates are substantial, suggesting that PRMT5 inhibitors may play a significant role in disease stabilization. While these findings are promising, they also highlight the need to further improve clinical efficacy. Therefore, we aimed to identify genetic alterations—beyond MTAP deficiency—that contribute to sensitivity to PRMT5 inhibition. Experimental procedures: To identify genes associated with sensitivity to AZD3470 we performed a genome-wide CRISPR screen across six NSCLC cell lines. Hit validation was conducted using two NSCLC CRISPR/Cas9 generated isogenic pairs. RNAseq, cell cycle analysis, apoptosis assay and γH2AX staining were used to provide mechanistic insight for AZD3470 sensitization. Finally, findings were validated in 29 NSCLC PDX models. Results: The genome-wide CRISPR screen identified CAAP1 loss as one of the top sensitizers to AZD3470 treatment. The CAAP1 (Caspase activity and apoptosis inhibitor 1) gene is located on chromosome 9p21, in close proximity to MTAP, and was found to be co-deleted with MTAP in approximately 20% of tumors. CRISPR screen results were further validated using HCC15 and NCI-H838 CAAP1 KO/WT isogenic pairs in long term proliferation assays, where CAAP1 null cells showed increased sensitivity to AZD3470 compared to CAAP1 WT cells. In addition, cell cycle analysis of the NCI-H838 and HCC15 CAAP1 KO cells after 96h of treatment with 1µM AZD3470 showed a higher percentage of cells in G2/M phase compared to CAAP1 WT cells. Moreover, CAAP1 deficiency potentiated both DNA damage and apoptosis induced by AZD3470. RNAseq analysis of HCC15 CAAP1 KO/WT isogenic cell line pairs revealed significant number of differentially spliced events in CAAP1 KO versus CAAP1 WT cells that increased with AZD3470 dose. Notably, the most abundant changes were detected in the transcripts with skipped exons that were enriched for pathways such as DNA repair or cell cycle consistent with the increased DNA damage and cell cycle alterations detected in CAAP1 null cells treated with AZD3470. Finally, our findings were confirmed using PDX models, where 71% of NSCLC PDXs in which AZD3470 induced regression were CAAP1 null. Conclusions: In summary, CAAP1 deletion in MTAP-null tumors increases sensitivity to AZD3470, both in vivo and in vitro, by potentiating AZD3470 induced cell cycle alterations and DNA damage, potentially through the regulation of alternative splicing. Citation Format: Jelena Urosevic, Angelos Papadopoulos, Shaun Moore, Ted Hong, Valentina Quarantotti, Harriet Southgate, Maximilian Wechsung, Lukasz Magiera, Daniel Barrell, Grainne Gernon, Elissavet Kentepozidou, Laura Rosenberg, Anisha Solanki, James T. Lynch, Stephen Fawell, Ho Man Chan, Susan Critchlow, Emma Dean. CAAP1 loss uncovers vulnerability of MTAP-deficient NSCLC to PRMT5 inhibition with AZD3470 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4489.

Abstract Background: PRMT5 is an epigenetic enzyme that catalyzes symmetric di-methylation of arginine (SDMA) of multiple substrates that regulate biological processes including RNA splicing and cell cycle. The role of PRMT5 in controlling chromatin accessibility has been investigated in some cancer contexts, but its role in non-small cell lung cancer (NSCLC) global chromatin regulation is not known. AstraZeneca has developed an MTA-cooperative PRMT5 inhibitor (AZD3470) that selectively inhibits PRMT5 in MTAP-null tumors and currently is in Phase I clinical trial (NCT06130553, NCT06130553). Here we applied integrative dynamic MultiOMICs analysis upon AZD3470 treatment in six MTAP-null NSCLC cancer cell lines to inform on PRMT5’s function in maintaining tumor cell growth and survival. Methods: Utilizing an integrative approach, our study explored the effects of AZD3470 on human MTAP-deleted NSCLC cells. MultiOMICs analysis including SDMA post-translational modification (PTM) scans, ATAC-seq, RNA-seq, and mass spectrometry proteomics were employed to delineate the immediate effects and downstream phenotypes of AZD3470 PRMT5 inhibition. Results: AZD3470 induced significant SDMA modification of splicing factors consistent with known roles for PRMT5. PRMT5 inhibition by AZD3470 led to changes in global chromatin accessibility in NSCLC cells. Specific changes in genes involved in the cell cycle pathway were observed including CDK6. Integrative analysis of transcriptomics and proteomics data consistently showed alterations in cell cycle, DNA damage repair and splicing related pathways upon treatment, consistent with the established biological roles of PRMT5. Alternative splicing analysis showed that AZD3470 treatment induced a unique retained intron within the ATM gene and resulted in reduced ATM protein levels. Conclusion: Here we identify AZD3470-induced changes to protein post-translational modification, chromatin accessibility, gene expression and RNA splicing in MTAP-deficient NSCLC cancer cell lines. Different area of MultiOMICs data together pointed to changes in cell cycle and DNA repair pathways. Further, we identified alternative splicing of ATM as a consequence of AZD3470 PRMT5i treatment resulting in significant reduction of ATM protein expression. Keywords: AZD3470, NSCLC, PRMT5 Inhibition, Chromatin Accessibility, Alternative Splicing, DNA Repair, ATM Gene. Citation Format: Ted Hong, Steven Criscione, Andrew Jarnuczak, Jelena Urosevic, Stephanie Ashenden, Ghaith Hamza, Praveen Kumar, Nevena Cvetesic, Lauren Bradshaw, Shaun Moore, Daniel Karl, Tianhui Zhang, Abel Sousa, Jonathan Cairns, Anthony Iannetta, Andrew Zhang, Eric Miele, John Reicha, Chris Chambers, Ramy Elgendy, Julia Lindgren, Daniel Jachimowicz, Maryam Clausen, Graham Belfield, Tony Cheung, Michael Grondine, James T. Lynch, Ho Man Chan, Susan Critchlow, Emma Dean. Integrative dynamic MultiOMICs analysis identifies cell cycle and DNA damage response phenotypes as downstream consequences of using the PRMT5 inhibitor AZD3470 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB008.

Background: PRMT5 is an epigenetic enzyme that catalyzes symmetric dimethylation of arginine substrates (SDMA), regulating multiple cell processes. The PRMT5-MTAP collateral vulnerability describes the accumulation of the metabolite methylthioadenosine (MTA) in tumor cells (as a result of the deletion of the MTAP gene), which acts as a natural endogenous partial inhibitor of PRMT5. This provides an opportunity to selectively inhibit PRMT5 in MTAP deleted tumors, which occurs in ~15% of all cancers. First generation PRMT5 inhibitors, which cannot discriminate between MTAP-proficient normal cells and MTAP-deficient tumor cells, have a narrow therapeutic index which may limit clinical efficacy due to on-target toxicity. MTAP-selective PRMT5 inhibitors spare MTAP-proficient normal cells, improving therapeutic index and clinical efficacy. We have developed a novel MTAP-selective PRMT5 inhibitor, AZ-PRMT5i-1, that selectively targets MTAP-deficient tumors and spares MTAP-proficient tissue. Methods: In vitro activity of AZ-PRMT5i-1 was profiled using six MTAP isogenic cell lines, as well as in a panel of 300 cancer cell lines. In vivo efficacy was assessed using three MTAP deleted models and hematological toxicity was addressed using a 3D human bone marrow model. Results: Using a MTAP CRISPR KO isogenic pair we demonstrate potent inhibition of SDMA in MTAP-null tumors with a 54-fold margin over the MTAP wildtype (WT) counterpart. MTAP differential activity was maintained in proliferation assays across a number of MTAP isogenic cell lines and in a panel of 300 cancer cell lines. AZ-PRMT5i-1 demonstrates strong dose dependent efficacy across MTAP deleted xenograft and PDX models of gastric and lung origin, where greater than 80% of tumor growth inhibition was detected, with no apparent toxicity. In addition, corresponding dose-dependent inhibition of SDMA is observed in the treated tumors. In an in vitro 28-day 3D human bone marrow model, AZ-PRMT5i-1 has reduced toxicity in erythroid and megakaryocyte cell lineages, compared to a first generation PRMT5 inhibitor. Conclusion: Overall, these studies demonstrate that AZ-PRMT5i-1 is a potent PRMT5 inhibitor demonstrating MTAP selectivity and anti-tumor activity in in vitro and in vivo pre-clinical models. Citation Format: James T. Lynch, Shaun Moore, Ivan Del Barco Barrantes, Lauren Bradshaw, Chris Chambers, Ted Hong, Sophie Cooke, Jelena Urosevic, Mercedes Vazquez-Chantada, James M. Smith, Anna Cronin, Benedicte Recolin, Sonja Gill, Susan Critchlow, Ho Man Chan, Emma Dean. AZ-PRMT5i-1: A potent MTAP-selective PRMT5 inhibitor with pharmacodynamic and monotherapy anti-tumor activity in MTAP-deleted tumours [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 6272.

Drug combinations are highly desirable in oncology as they can improve therapeutic response and overcome drug resistance. However, responses can be context-specific, meaning there is a real need for large-scale studies to fully explore the drug combination response landscape. We present a screen of 109 drug combinations in over 750 diverse cell lines covering 41 hematological and solid tumor types. Each combination was screened in a full 7 × 7 matrix format, representing over 2.3 million individual data points, the largest such study to date. We took a novel approach to assess combination benefit. Growth inhibition and highest single agent (HSA) and Bliss excess combination heatmaps were generated for all >68,000 combination-cell line pairs, and each was scored for high activity (combination Emax>0.5) and combination benefit/synergy (HSA>0.1). Out of 4578 combination-cancer type pairs 489 met the filtering criteria. We prioritized combinations based on their efficacy and cancer type selectivity to minimize risk of potential tissue toxicity. We identified several known combination-cancer type pairs (e.g. MCL1 inhibitor AZD5991 plus BCL2 inhibitor venetoclax in AML) as well as novel indications for combinations already known to be active in a different cancer type (e.g. AZD5991 plus AKT inhibitor capivasertib in endometrial cancer or AURKB inhibitor AZD2811 plus venetoclax in DLBCL). We selected 4 combinations based on clinical need and validated them further in vitro and in vivo (selumetinib plus venetoclax or AZD5991, AZD2811 plus venetoclax, and capivasertib plus AZD5991). To understand how molecular context affects drug response, we also used GDSC tools ANOVA to perform over 5.4 million statistical tests to identify statistically significant associations between drug response metrics and multi-omics features including mutations, CNAs, gene expression and methylation. Associations were identified with five response metrics, covering both single agent and combination responses. We looked for significant associations across 21 different subgroups of cell lines and 6 molecular ‘baskets’ (TP53, KRAS, MLL2/KMT2D, PTEN, PIK3CA, and BRAF). We identified 11,611 biomarkers which met the significance criteria (p⇐0.001, FDR⇐10%, and both positive and negative Glass deltas >=1). Moreover, we used biomarkers of single-agent response to identify any combination Emax or Bliss associated biomarkers that could not be explained by the action of either drug. This resulted in identification of 1,631 ‘emergent’ biomarkers. In summary, our screen and pipeline have been designed to optimize preclinical interpretation with a focus on actionability, particularly by our unique approach, and to provide a valuable resource for exploration by the wider research community. As a result of this screen, we identified and validated novel combination-tumor types in multiple cancer types. Citation Format: Azadeh Bashi, Elizabeth A. Coker, Krishna Bulusu, Marta Milo, Claire Crafter, James Lynch, David Jenkins, Howard Lightfoot, Brandon Willis, Courtney Andersen, Omid Tavana, Kevin Mongeon, Jacob Gordon, Paul Smith, Simon Barry, Ultan McDermott, Lisa Drew, Susan Critchlow, Mathew J. Garnett, Jerome Mettetal. Large scale pan cancer drug combination screening to identify effective and actionable combinations and biomarker hypothesis. [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 5321.

Background: PRMT5 is type II arginine methyltransferase that catalyses symmetric dimethylation of arginine residues (SDMA) and plays an important role in cancer biology. By methylating a number of substrates, PRMT5 can regulate important processes such as DNA repair, RNA splicing, and cellular proliferation. In addition, PRMT5 is overexpressed in various cancer types and has been identified as a candidate for therapeutic intervention through the development of small molecules that inhibit PRMT5 methyltransferase activity. However, first generation PRMT5 inhibitors have shown limited clinical benefit mainly due to development of on-target toxicity, primarily in the bone marrow. AstraZeneca has developed a second generation PRMT5 inhibitor (AZ-PRMT5i), that selectively inhibits PRMT5 in MTAP deficient tumours while sparing MTAP proficient normal cells. MTAP (methylthioadenosine phosphorylase) is a metabolic enzyme involved in methionine salvage pathway. MTAP deficiency results in accumulation of the metabolite methylothioadenosine (MTA) in tumor cells that induces partial inhibition of PRMT5, rendering these tumors sensitive to PRMT5 inhibition. Homozygous deletion of the MTAP gene, that results in the loss of MTAP protein, has been found in approximately 15% of advanced solid tumors. Here, for the first time, we describe epigenetic silencing of the MTAP gene in Hodgkin's Lymphoma (HL) cell lines. This silencing results in the loss of MTAP protein expression thus increasing sensitivity to PRMT5 inhibition. Importantly, MTAP protein loss was also observed in primary cHL samples, opening a novel opportunity for the treatment of HL. Methods: Bioinformatic data mining of the Cancer Cell Line Encyclopedia (CCLE) dataset has been used to overlay levels of expression of MTAP mRNA with MTAP copy number and MTAP promoter DNA methylation. MTAP protein expression and activity of AZ-PRMT5i were assessed in 5 HL cell lines in vitro; efficacy of AZ-PRMT5i and levels of target engagement were tested in vivo in the L540 xenograft model. MTAP expression levels were determined by IHC analysis using 55 primary samples from cHL patients. Results: Using an unbiased analysis of the CCLE dataset, we have identified a subset of cell lines that in the absence of MTAP genetic loss were showing low levels of MTAP mRNA expression, equivalent to levels of MTAP mRNA in MTAP homozygous deleted cell lines. Interestingly, this was predominantly the case in HL cell lines where it was detected in 67% of cases (4 out of 6 cell lines). MTAP methylation profile analysis of HL cell lines revealed elevated MTAP DNA promoter methylation in cell lines with low MTAP mRNA expression. In addition, MTAP protein was not detected by Western Blot. Accordingly, lack of MTAP protein expression resulted in increased sensitivity of these cells to AZ-PRMT5i compared to a HL cell line that expressed MTAP protein. Utilizing RNA-seq for transcriptional profiling of AZ-PRMT5i on a MTAP-deficient HL cell line has identified robust changes in genes that regulate the cell cycle, DNA replication and TP53 pathways thus confirming on target activity. In addition, AZ-PRMT5i demonstrated strong dose dependent efficacy in a MTAP silenced L540 xenograft model where greater than 90% of tumour growth inhibition was detected, with no significant body weight loss. Corresponding dose-dependent inhibition of SDMA is observed in the treated tumours. Finally, IHC analysis of MTAP expression in 55 primary cHL samples has demonstrated that 46 of 55 cHL samples (84%) had absent nuclear MTAP expression. Conclusion: Here we are presenting a novel finding showing MTAP protein expression loss in the majority of tested primary cHL samples, which could provide a collateral vulnerability and novel therapeutic opportunity to 2 nd generation PRMT5 inhibitors as demonstrated in our pre-clinical models.

Abstract Background: PRMT5 is an epigenetic enzyme that catalyzes symmetric dimethylation of arginine substrates (SDMA), regulating multiple cell processes. The PRMT5-MTAP collateral vulnerability describes the accumulation of the metabolite methylthioadenosine (MTA) in tumor cells (as a result of the deletion of the MTAP gene), which acts as a natural endogenous partial inhibitor of PRMT5. This provides an opportunity to selectively inhibit PRMT5 in MTAP deleted tumors, which occurs in ~15% of all cancers. First generation PRMT5 inhibitors, which cannot discriminate between MTAP-proficient normal cells and MTAP-deficient tumor cells, have a narrow therapeutic index which may limit clinical efficacy due to on-target toxicity. MTAP-selective PRMT5 inhibitors spare MTAP-proficient normal cells, improving therapeutic index and clinical efficacy. We have developed a novel MTAP-selective PRMT5 inhibitor, AZ-PRMT5i-1, that selectively targets MTAP-deficient tumors and spares MTAP-proficient tissue. Methods: In vitro activity of AZ-PRMT5i-1 was profiled using six MTAP isogenic cell lines, as well as in a panel of 300 cancer cell lines. In vivo efficacy was assessed using three MTAP deleted models and hematological toxicity was addressed using a 3D human bone marrow model. Results: Using a MTAP CRISPR KO isogenic pair we demonstrate potent inhibition of SDMA in MTAP-null tumors with a 54-fold margin over the MTAP wildtype (WT) counterpart. MTAP differential activity was maintained in proliferation assays across a number of MTAP isogenic cell lines and in a panel of 300 cancer cell lines. AZ-PRMT5i-1 demonstrates strong dose dependent efficacy across MTAP deleted xenograft and PDX models of gastric and lung origin, where greater than 80% of tumor growth inhibition was detected, with no apparent toxicity. In addition, corresponding dose-dependent inhibition of SDMA is observed in the treated tumors. In an in vitro 28-day 3D human bone marrow model, AZ-PRMT5i-1 has reduced toxicity in erythroid and megakaryocyte cell lineages, compared to a first generation PRMT5 inhibitor. Conclusion: Overall, these studies demonstrate that AZ-PRMT5i-1 is a potent PRMT5 inhibitor demonstrating MTAP selectivity and anti-tumor activity in in vitro and in vivo pre-clinical models. Citation Format: James T. Lynch, Shaun Moore, Ivan Del Barco Barrantes, Lauren Bradshaw, Chris Chambers, Ted Hong, Sophie Cooke, Jelena Urosevic, Mercedes Vazquez-Chantada, James M. Smith, Anna Cronin, Benedicte Recolin, Sonja Gill, Susan Critchlow, Ho Man Chan, Emma Dean. AZ-PRMT5i-1: A potent MTAP-selective PRMT5 inhibitor with pharmacodynamic and monotherapy anti-tumor activity in MTAP-deleted tumours [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 6272.

Cancer cells reprogram their metabolism to support growth and invasion. While previous work has highlighted how single altered reactions and pathways can drive tumorigenesis, it remains unclear how individual changes propagate at the network level and eventually determine global metabolic activity. To characterize the metabolic lifestyle of cancer cells across pathways and genotypes, we profiled the intracellular metabolome of 180 pan‐cancer cell lines grown in identical conditions. For each cell line, we estimated activity for 49 pathways spanning the entirety of the metabolic network. Upon clustering, we discovered a convergence into only two major metabolic types. These were functionally confirmed by 13C‐flux analysis, lipidomics, and analysis of sensitivity to perturbations. They revealed that the major differences in cancers are associated with lipid, TCA cycle, and carbohydrate metabolism. Thorough integration of these types with multiomics highlighted little association with genetic alterations but a strong association with markers of epithelial–mesenchymal transition. Our analysis indicates that in absence of variations imposed by the microenvironment, cancer cells adopt distinct metabolic programs which serve as vulnerabilities for therapy.

Supplementary Figure from Identification of a Molecularly-Defined Subset of Breast and Ovarian Cancer Models that Respond to WEE1 or ATR Inhibition, Overcoming PARP Inhibitor Resistance

ABSTRACTThe unravelling of the complexity of cellular metabolism is in its infancy. Cancer-associated genetic alterations may result in changes to cellular metabolism that aid in understanding phenotypic changes, reveal detectable metabolic signatures, or elucidate vulnerabilities to particular drugs. To understand cancer-associated metabolic transformation we performed untargeted metabolite analysis of 173 different cancer cell lines from 11 different tissues under constant conditions for 1099 different species using liquid chromatography-mass spectrometry (LC-MS). We correlate known cancer-associated mutations and gene expression programs with metabolic signatures, generating novel associations of known metabolic pathways with known cancer drivers. We show that metabolic activity correlates with drug sensitivity and use metabolic activity to predict drug response and synergy. Finally, we study the metabolic heterogeneity of cancer mutations across tissues, and find that genes exhibit a range of context specific, and more general metabolic control.

Microphysiological in vitro systems are platforms for preclinical evaluation of drug effects and significant advances have been made in recent years. However, existing microfluidic devices are not yet able to deliver compounds to cell models in a way that reproduces the real physiological drug exposure. Here, we introduce a novel tumour-on-chip microfluidic system that mimics the pharmacokinetic profile of compounds on 3D tumour spheroids to evaluate their response to the treatments. We used this platform to test the response of SW620 colorectal cancer spheroids to irinotecan (SN38) alone and in combination with the ATM inhibitor AZD0156, using concentrations mimicking mouse plasma exposure profiles of both agents. We explored spheroid volume and viability as a measure of cancer cells response and changes in mechanistically relevant pharmacodynamic biomarkers (γH2AX, cleaved-caspase 3 and Ki67). We demonstrate here that our microfluidic tumour-on-chip platform can successfully predict the efficacy from in vivo studies and therefore represents an innovative tool to guide drug dose and schedules for optimal efficacy and pharmacodynamic assessment, while reducing the need for animal studies.

A fundamental feature of cancer cells is genomic heterogeneity. It is a main driver of phenotypic differences, including the response to drugs, and therefore a key factor in therapy selection. Motivated by the increasing role attributed to metabolic reprogramming in tumor development, we wondered how genomic heterogeneity affects metabolic phenotype. To this end, we profiled the intracellular metabolome of 180 cancer cell lines grown in similar conditions to exclude environmental factors. For each cell line, we estimate activity for 49 pathways across the whole metabolic network. Upon clustering of activity data, we found a convergence into only two major metabolic types. These were further characterized by 13C-flux analysis, lipidomics, and analysis of sensitivity to perturbations. These experiments revealed differences in lipid, mitochondrial, and carbohydrate metabolism between the two major types. Finally, a thorough integration of our metabolic data with multiple omics data revealed a strong association with markers of epithelial-mesenchymal transition (EMT). Our analysis indicates that in absence of variations imposed by the microenvironment, the metabolism of cancer cell lines falls into only two major classes despite genetic heterogeneity.

Replication stress (RS) is a hallmark of cancer and has the potential to be exploited by selective DNA Damage Response inhibitors. During replication stress, the DNA polymerase is uncoupled from the replisome helicase activity resulting in extended regions of single strand DNA that lead to the initiation of a replication stress response (RSR). Two key regulators of the RSR are the ATR and WEE1 kinases. Here, we aimed to identify patient selection biomarkers for WEE1 and ATR inhibitors (WEE1i, ATRi) from a cohort of 37 patient-derived tumor xenografts (PDX) from ovarian or triple negative breast cancer, 20 of which harbored BRCA1/BRCA2deleterious mutations. The antitumor response of the WEE1i AZD1775 and the ATRi AZD6837 was evaluated, and PDX tumors were characterized by exome sequencing, metabolomics, western blot and immunohistochemistry. AZD1775 treatment response in PDXs, measured as tumor regressions (≤-30% change in tumor volume), was observed in 28% of models tested. Exome sequencing identified an association between treatment response and concomitant alterations in RB1 and STK11 (LKB1), whose functional relevance was validated in an in vitro model. Immunoblots showed that WEE1i treatment resulted in downregulation of the ribonucleotide reductase subunit RRM2 and induction of an S-phase DNA damage response (DDR) in vivo. Metabolomics identified a profound change in the purine and pyrimidine synthesis pathways in WEE1i-sensitive PDXs and these changes appear functionally relevant since PDX ex vivo data demonstrate rescue of the WEE1i effects after addition of nucleosides. Another important association with WEE1i sensitivity was increased cyclin E expression. Together, these data suggest that early entry into S phase and an imbalance of origin firing to available dNTPs represent drivers of sensitivity to the WEE1i. Interestingly by contrast, ATRi-sensitive PDXs (representing 10% of the models) were characterized primarily by those tumors harboring ATM alterations, suggesting that synthetic lethality with this major DDR pathway is the primary mechanism of sensitivity to the ATR inhibitor in ovarian and triple negative breast cancer. This study therefore highlights differences between the drivers of sensitivity to inhibitors of these two important RSR proteins.Citation Format: Violeta Serra, Cristina Cruz, Zhongwu Lai, Marta Castroviejo-Bermejo, Marta Palafox, Urszula M. Polanska, Gemma N. Jones, Anderson Wang, Filippos Michopoulos, Rachel Brough, Brian Dougherty, Elaine Cadogan, Susan Critchlow, Alejandra Bruna, J. Carl Barrett, Cristina Saura, Christopher J. Lord, Carlos Caldas, Joaquin Arribas, Judith Balmaña, Mark J. O’Connor. Biomarkers for inhibitors of the replication stress response proteins WEE1 and ATR in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3503.

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Compounds targeting phosphatidylinositol-3-kinase/mammalian target of rapamycin (PI3K/mTOR) signaling are being investigated in multiple clinical settings, but drug resistance may reduce their benefit. Compound rechallenge after drug holidays can overcome such resistance, yet little is known about the impact of drug holidays on cell biochemistry. We found that PI3K inhibitor (PI3Ki)-resistant cells cultured in the absence of PI3Ki developed a proliferative defect, increased oxygen consumption and accumulated reactive oxygen species (ROS), leading to lactate production through hypoxia-inducible factor-1α. This metabolic imbalance was reversed by mammalian target of rapamycin complex 1 (mTORC1) inhibitors. Interestingly, neither AKT nor c-MYC was involved in mediating the metabolic phenotype, despite the latter contributing to resistant cells’ proliferation. These data suggest that an AKT-independent PI3K/mTORC1 axis operates in these cells. The excessive ROS hampered cell division, and the metabolic phenotype made resistant cells more sensitive to hydrogen peroxide and nutrient starvation. Thus, the proliferative defect of PI3Ki-resistant cells during drug holidays is caused by defective metabolic adaptation to chronic PI3K/mTOR pathway inhibition. This metabolic imbalance may open the therapeutic window for challenge with metabolic drugs during drug holidays.