Identification of an Effective Chemotherapy and DNA Damage Response Inhibitor Combination for Diffuse Large B Cell Lymphoma

Background: Chemotherapy forms the backbone of treatment for Diffuse Large B Cell Lymphoma (DLBCL); however, <∼20% of tumors are chemoresistant. Inhibitors of the DNA damage response (DDR) show promise as chemosensitizers. We therefore set up an in-vitro screen to identify an optimal chemotherapy-DDR inhibitor (DDRi) combination in a panel of DLBCL cell lines. Material and Methods: To achieve this, we harnessed Quadratic Phenotypic Optimization Platform (QPOP), an experimental-analytic method built to identify potent drug combinations. 6 DDRis (to ATR, ATM, CHK1/2, DNA-PK, WEE1, PARP) and 6 routinely used chemotherapy agents were selected for the screen. After identifying the most effective combination using a cell viability assay, we investigated the mechanism of synergy. We observed pathway activation by western blot; apoptosis by Annexin V staining; and cell cycle disruption by flow cytometry with EdU, Propidium Iodide, and phospho-Histone H3 staining. To explore the underlying mechanism of synergy, we performed RNA sequencing. Results: From the combination screen, ATR inhibitor, AZD6738, and chemotherapeutic drug, Gemcitabine (A+G), was the most effective combination across multiple DLBCL cell lines, including Gemcitabine-resistant cell lines. Using two gemcitabine-resistant DLBCL cell lines as models, we investigated the mechanism of A+G synergy. Inhibition of ATR abrogates the G2M checkpoint, causing cells with gemcitabine-induced DNA damage to enter mitosis and die through mitotic catastrophe. We confirmed that the combination reduced cell viability, affected Chk1 phosphorylation, and promoted apoptosis. However, only a small proportion of cells enter mitosis after A+G treatment and cells were primarily arrested in G1 phase with blockade of entry into S phase. Pathway analysis by RNA seq revealed that several cell-cycle and DNA replication-related pathways were suppressed in the combination setting. Interestingly, the transcriptome of A+G treated cells revealed a reversal of a gene expression signature characteristic of dark zone (DZ) biology. The DZ signature reflects a gene expression program associated with B-cells in the DZ of the germinal centre and is enriched in poorly prognostic DLBCL. Conclusions: Taken together, we have identified a chemotherapy-DDRi drug combination, AZD6738 and Gemcitabine, which is effective in killing DLBCL cells in-vitro, including cells that are resistant to Gemcitabine. The mechanism of synergy is not likely to be through mitotic catastrophe, but may potentially involve a cell cycle state reflecting the suppression of a B-cell specific transcriptional program regulating the DZ-LZ transition. Importantly, the reversal of the DZ gene expression signature by the combination indicates its potential utility as a treatment option for these lymphomas that exhibit this poor-prognostic gene signature. Conflict of interest: Advisory Board: ADJ: Consultancy fees from Turbine Ltd, AstraZeneca, Antengene, Janssen MSD and IQVIA Corporate-sponsored Research: ADJ: Research funding from Janssen and AstraZeneca Other Substantive Relationships: ADJ: Travel funding from Perkin Elmer