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2782 ATR Inhibition and Replication Stress Converge on a BCL6-Mediated Reversal of the Dark Zone Signature in DLBCL

Program: Oral and Poster Abstracts
Session: 605. Molecular Pharmacology and Drug Resistance: Lymphoid Neoplasms: Poster II
Hematology Disease Topics & Pathways:
Research, Combination therapy, Apoptosis, Translational Research, CHIP, Assays, Treatment Considerations, Biological Processes, Molecular biology, Technology and Procedures
Sunday, December 8, 2024, 6:00 PM-8:00 PM

Allison Chan, PhD1*, Akshaya Anbuselvan1*, Patrick Jaynes1*, Nayana Prabhu2*, Yuan Ju3*, Jane Jia Hui Lee3*, Tze King Tan1*, Jasmine Goh1*, Marco Foiani1,4,5*, Takaomi Sanda, MD, PhD6, Wai Leong Tam1,3,7,8*, Mark J O'Connor9*, Edward K Chow10* and Anand Devaprasath D. Jeyasekharan, MBBS, MRCP, PhD1,11,12,13*

1Cancer Science Institute of Singapore, National University of Singapore, Singapore, Singapore
2A*STAR Institute of Molecular and Cell Biology, Singapore, Singapore
3Laboratory of Translational Cancer Biology, Genome Institute of Singapore, Singapore, Singapore
4IFOM, The FIRC Institute of Molecular Oncology, Milan, Italy
5University of Milan, Department of Oncology and Hematology-Oncology, Milan, Italy
6Department of Hematology and Oncology, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan
7Department of Biochemistry, National University of Singapore, Singapore, Singapore
8School of Biological Science, Nanyang Technological University, Singapore, Singapore
9Oncology R&D, AstraZeneca, Cambridge, United Kingdom
10KYAN Therapeutics, Singapore, Singapore
11NUS Centre for Cancer Research, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore
12Department of Haematology-Oncology, National University Cancer Institute, Singapore, National University Health System, Singapore, Singapore
13Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore

Chemotherapy forms the backbone of treatment for Diffuse Large B Cell Lymphoma (DLBCL); however, approximately 20% of tumors are chemoresistant. Inhibitors of the DNA damage response (DDR) sensitize various tumor types to chemotherapy and show pre-clinical activity in lymphoma. DLBCL with replication stress and genomic instability are particularly sensitive to ATR and WEE1 inhibitors, making DDR inhibition a promising therapeutic avenue to enhance chemosensitivity.

We therefore conducted an in-vitro screen to identify optimal chemotherapy-DDR inhibitor (DDRi) combinations in a panel of eight DLBCL cell lines. This screen utilized the Quadratic Phenotypic Optimization Platform (QPOP), an experimental-analytic method designed to identify potent drug combinations. Six DDRis targeting various pathways (ATR, ATM, CHK1/2, DNA-PK, WEE1, PARP) were screened along with six routinely used chemotherapy agents. The ATR inhibitor AZD6738 and replication stress-inducing chemotherapeutic gemcitabine (A+G) emerged as the most effective combination, inducing synergistic cell death via apoptosis across fourteen DLBCL cell lines, including gemcitabine-resistant lines. This efficacy was further demonstrated in vivo, with the A+G combination significantly reducing tumor growth in NSG mouse xenograft models.

Contrary to the expected mechanism of the A+G combination causing mitotic catastrophe, flow cytometry revealed that only a small proportion of cells entered mitosis. RNA sequencing of A+G treated cell lines revealed expected suppression of cell-cycle and DNA replication-related pathways. Interestingly, the combination also strongly reversed a poor-prognostic gene expression signature characteristic of dark zone (DZ) biology in the DZ-like and gemcitabine-resistant cell lines HT and SUDHL4. This was accompanied by changes in a variety of chromatin regulation pathways. To further investigate the mechanism underlying this transcriptional shift, we performed chromatin immunoprecipitation sequencing (ChIP-seq) for the enhancer marker H3K27ac. This revealed that A+G treated cells showed a significant loss of super-enhancer marks for BCL6, a master regulatory transcription repressor in DLBCL. BCL6 is a proto-oncogene that represses genes involved in cell cycle arrest and apoptosis, allowing lymphoma cells to survive and proliferate, and its expression was significantly downregulated at the RNA level in A+G treated cells. Additionally, mass-spectrometry cellular thermal shift assays (MS-CETSA) demonstrated that gemcitabine initiated an early destabilization of BCL6 protein, which was sustained by ATR inhibition. However, only the A+G combination led to sustained loss of BCL6 protein levels, explaining improved cell kill with the reversal of the DZ signature. We hypothesize that the replication stress induced by gemcitabine treatment leads to activation of pathways that cause degradation of BCL6, which is then reinforced by transcriptional changes induced by ATR inhibition.

In conclusion, we have identified a chemotherapy-DDRi combination, ATR inhibition and gemcitabine, that is highly effective in killing chemoresistant DLBCL cells in vitro and in vivo. The mechanism of synergy involves suppression of a BCL6-regulated transcriptional program driving dark zone biology. Gemcitabine is a routinely used chemotherapeutic for second-line treatment of DLBCL, and neither it nor AZD6738 shows lymphodepletion in humans. As DLBCL with DZ-like signatures show poor outcomes on standard chemo-immunotherapy, our finding that ATR inhibition with gemcitabine reverses DZ signatures suggests the possibility of this being a non-lymphodepleting genotoxic backbone for combinations with T-cell engaging bispecific antibodies.

Disclosures: Sanda: Kyowa Kirin: Honoraria; Amgen: Honoraria; Astellas: Honoraria. O'Connor: AstraZeneca: Current Employment. Chow: Kyan Technologies: Current Employment, Current equity holder in private company. Jeyasekharan: BeiGene: Consultancy; Roche: Consultancy; Gilead Sciences: Consultancy; AstraZeneca: Consultancy, Research Funding; Antengene Corp: Consultancy; MSD: Consultancy; IQVIA: Consultancy; Janssen: Consultancy, Research Funding; Turbine: Consultancy.

*signifies non-member of ASH