The Clinical Oncolytic Reovirus Formulation Reolysin Synergistically Augments the Anti-Leukemic Activity of Azacitidine

Despite the recent development of new agents for acute myeloid leukemia (AML) therapy, novel approaches are still needed for patients that do not benefit sufficiently from existing regimens. Reolysin (Pelareorep) is a proprietary clinical formulation of the naturally occurring oncolytic reovirus that is non-pathogenic and preferentially replicates in cancer cells, but not in normal tissue. Although Reolysin has been investigated in over 30 adult clinical trials and is very well tolerated when given alone and in combination with chemotherapy, it has never been evaluated for AML therapy. Our major goal was to determine the efficacy and mechanism of action of Reolysin alone and in combination with azacitidine in AML models and primary patient specimens. Reolysin exhibited dose-dependent effects against AML cells with respect to the reduction in leukemia cell viability and induction of apoptosis in all 8 cell lines evaluated. The combination of Reolysin with azacitidine yielded synergistic benefit across all cell lines that was dramatically superior to single agent treatments (p<0.001). The benefit of combination treatment was confirmed in primary AML specimens (N=14 to date). RNASeq-based transcriptome and gene ontology (GO) analyses of the pharmacodynamic effects of each agent and the combination revealed that the Reolysin and azacitidine combination potently altered multiple genes in the immune response pathway (p<0.001). qRT-PCR analyses and ELISA assays confirmed the broad immunomodulatory effects of the Reolysin plus azacitidine combination. Immune priming strategies that can turn "immune cold" cancers to "immune hot" may significantly augment the benefit of many therapeutic approaches. Basic leucine zipper transcription factor ATF-like 2 (BATF2) is a key tumor suppressor that is absent in AML and certain other malignancies. Its deficiency promotes immune escape of cancer cells. BATF2 exhibits anticancer activity through upregulation of interleukin-12 and the activation of CD8+ T cells and therefore represents a novel new target in anticancer treatment with immune checkpoint inhibitors. Our transcriptome analyses identified BATF2 as one of the most significantly upregulated genes in AML cells following treatment with the Reolysin plus azacitidine combination (p<0.0001). Additional mechanistic studies confirmed that BATF2 induction was a key driver of the anti-leukemic effects of combination treatment. Finally, Reolysin (5 x 10 ⁸ TCID50 IV 1x per week), azacitidine (5 mg/kg SC 2x per week), and the combination were administered to mice bearing MOLM-13 FLT3-ITD+ xenografts for 19 days to assess the efficacy and tolerability of each treatment. Combination treatment was well tolerated and antagonized disease progression significantly more effectively than either monotherapy (p<0.01). Additional in vivo studies more rigorously assessing the immunomodulatory effects of Reolysin and azacitidine in immune competent mice are underway. Our collective data demonstrate that Reolysin is a safe and well tolerated agent with potent immunomodulatory effects that synergistically augments the anti-AML effects of azacitidine. A phase I investigator-initiated clinical trial further investigating the safety and preliminary efficacy of this combination in patients with AML is currently being planned.