Therapeutic Repurposing of Brincidofovir in Non-Hodgkin Lymphoma - Potential Synergy with Immune Checkpoint Blockade

Introduction: Brincidofovir (BCV) is a novel nucleoside phosphonate analogue with unique dual anti-viral and anti-tumor properties. Notably, BCV elicits potent anti-lymphoproliferative activity, and has demonstrated potential immunogenic effects in pre-clinical studies on T-cell and NK/T-cell lymphoma (T/NK-NHL) cell-line models. Methods: Activity of BCV was evaluated in forty-four cell-line models, including T/NK-NHL (n=25) and B-cell lymphoma (BCL, n=19), as well as their respective NOD/SCID mice xenograft models (MYC/BCL2-rearranged diffuse large B-cell lymphoma, TP63-rearranged anaplastic large cell lymphoma, JAK3-mutant NK/T-cell lymphoma and peripheral T-cell lymphoma, not otherwise specified; n=1 each). Potential in vivo immunogenic effects were examined in a syngeneic EL4-C57BL/6 murine lymphoma model. Results: BCV demonstrated potent inhibitory effects in vitro across the majority of cell-lines regardless of EBV positivity, with IC50 values within clinically-achievable human plasma concentrations (2 µg/ml) in 17/25 (68.0%) T/NK-NHL and in 13/19 (68.4%) BCL. In vivo treatment via intraperitoneal BCV (IP, 40mg/kg, 2X per week) significantly inhibited tumor growth in all xenograft models when compared to vehicle control. Notably, RNAseq analysis demonstrated BCV downregulated MYC and MYC-target pathways in T/NK-NHL models. Further mechanistic studies showed that BCV evoked S-phase cell cycle arrest, replication stress, DNA damage and apoptosis, while also triggering STING pathway-mediated interferon responses, PD-L1 expression and immunogenic cell death. In EL4 murine lymphoma, BCV similarly inhibited cell viability in a dose-dependent manner (IC50 516 ng/ml) and increased PD-L1 expression on flow cytometry. In the syngeneic EL4-C57BL/6 model, BCV alone or in combination with anti-PD1 (IP 200 µg 1X per week) significantly inhibited tumor growth at 8 days post-treatment, compared to isotype or anti-PD1 treatment alone. No significant difference in tumor volume or weight was observed with the combination of BCV and anti-PD1, compared with BCV alone. Intriguingly, at this early time point, histologic examination suggested a significantly heavier immune infiltration only in the BCV+anti-PD1 combination group, which was corroborated by the highest scores for adaptive immune response, cytokines/chemokines & receptors, cytotoxic cells, dendritic cells, NK CD56dim cells and neutrophils (NanoString nCounter, Mouse Immunology Panel). Gene expression of CCL2, CCL12, CXCL9, GZMB, CHIL3 and CTLA4 were also highest in the combination treatment group. Conclusions: Taken together, these results demonstrate a novel role of BCV in the treatment of lymphoma. The immunogenic effects of BCV suggests potential for combination with PD1/PDL1 and CTLA4 checkpoint immunotherapy.