Abstract
Introduction: Dark zone-like diffuse large B-cell lymphoma (DZ-DLBCL) represents a distinct germinal center B-cell (GCB) subtype, characterized by transcriptional programs reminiscent of physiological germinal center dark zones. These tumors have poor prognosis and resistance to both chemotherapy and novel immunotherapies. In our recent study (Cancila et al., JCI 2025), we identified that ataxia telangiectasia and Rad3-related (ATR) kinase activity suppresses T-cell infiltration in both physiological DZs and DZ-DLBCLs. Pharmacologic ATR inhibition reprogrammed the DZ gene expression landscape toward a light zone (LZ)-like state, increasing T-cell association and susceptibility to T-cell mediated killing. We hypothesized that enhancing ATR inhibition-induced replication stress could overcome chemoresistance while reprogramming DZ-DLBCLs toward immune-accessible light zone (LZ)-like phenotypes.
Methods and Results: Through drug screening of various chemotherapeutics combined with replication stress inhibitors, we identified a potent synergy between the nucleoside analog gemcitabine and ATR inhibition specifically in DZ-DLBCL cell lines. While gemcitabine + ATR inhibitor combinations have been explored in solid tumors, their potential in hematologic malignancies has not been explored. Gemcitabine displayed limited cytotoxicity in DZ-DLBCL cell lines as a single agent. However, its combination with ATR inhibition resulted in robust synergistic cell death both in vitro across 14 cell lines (mean ZIP score : 75) and in vivo. In NSG mouse models, combination treatment resulted in a 50% mean reduction in tumor volume, compared to a 92% and 141% change in single agent of GEM or ATR inhibition alone, respectively. Unexpectedly, this synergy was not solely driven by mitotic catastrophe (as has been reported in solid cancers), with the majority of cell death occurring within late G1 and S-phase of the cell cycle.
Importantly, RNA sequencing revealed that dual therapy with ATR inhibition and gemcitabine strongly reversed the DZ transcriptional signature and induced LZ-associated programs, more than that by either drug alone. This was associated with enhanced replication stress, activation of non-canonical NF-κB signaling and suppression of key oncogenic regulators, including MYC and the DZ master transcriptional repressor BCL6- leading to a multifactorial activation of apoptosis and eventual cell death. A CRISPR screen comparing control and combination-treated cells identified NF-κB pathway genes as essential for cell death induced by the combined therapy. Chromatin profiling by H3K27ac ChIP-seq revealed reduced enhancer activity at BCL6 and increased acetylation at immune-permissive loci such as NFKB2, suggesting that epigenetic remodeling reinforces the transcriptional switch induced by the exacerbated replication stress under conditions of combination therapy of DZ DLBCL cells with a nucleoside analog and an ATR inhibitor.
As we have shown the DZ gene expression signature to be an ATR regulated process limiting T-cell infiltration (Cancila et al., JCI 2025), we then tested the hypothesis that reversal of the DZ signature by combined nucleoside analog + ATR inhibitor treatment could also improve T-cell directed therapy in DZ-DLBCL models. T cell–engager cytotoxicity assays using Glofitamab and autologous PBMCs co-cultured with DZ-DLBCL cells showed enhanced levels of cell kill in combination with gemcitabine and ATR inhibition.
Conclusion: Together, these findings suggest that supraphysiological levels of replication stress induced by the combination of gemcitabine and ATR inhibition synergize to reprogram the DZ transcriptional architecture and enable immune recognition. Together, these findings position ATR inhibition plus gemcitabine as a rational therapeutic strategy for DZ-DLBCL, simultaneously disrupting DZ oncogenic circuitry and priming tumor cells for immune attack. These findings provide immediate clinical translation potential through modification of the STARGLO protocol, replacing oxaliplatin with ATR inhibition to enhance Glofitamab-Gemcitabine efficacy in relapsed DLBCL, particularly DZ-like subtypes.
