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Ning Xu,1,2 Benjamin Tse,1,2 Lu Yang,1,2 Tiffany CY Tang,1,2 Michelle Haber,1,2 Kenneth Micklethwaite,3– 6 Alla Dolnikov1,2 1Children’s Cancer Institute, University of New South Wales, Sydney, NSW, Australia; 2School of Women’s and Children’s Health, University of New South Wales, Sydney, NSW, Australia; 3Blood Transplant and Cell Therapies Program, Department of Hematology, Westmead Hospital, Sydney, NSW, Australia; 4Sydney Cellular Therapies Laboratory, NSW Health Pathology, Sydney, NSW, Australia; 5Westmead Institute for Medical Research, Sydney, NSW, Australia; 6Sydney Medical School, The University of Sydney, Sydney, NSW, AustraliaCorrespondence: Alla DolnikovChildren’s Cancer Institute, University of New South Wales, PO Box 81, Randwick, NSW, 2031, AustraliaTel +61 449 904 174Email adolnikov@ccia.org.auPurpose: Despite the success of chimeric antigen receptor (CAR) T cells in clinical studies, a significant proportion of responding patients eventually relapsed, with the latter correlating with low CAR T cell expansion and persistence.Methods and Results: Using patient-derived xenograft (PDX) mouse models of CD19+ B cell acute lymphoblastic leukemia (B-ALL), we show that priming leukemia-bearing mice with 5-azacytidine (AZA) enhances CAR T cell therapy. AZA given 1 day prior to CAR T cell infusion delayed leukemia growth and promoted CAR T cell expansion and effector function. Priming leukemia cells with AZA increased CAR T cell/target cell conjugation and target cell killing, promoted CAR T cell divisions and expanded IFNγ+ effector T cells in co-cultures with CD19+ leukemia Nalm-6 and Raji cells. Transcriptome analysis revealed activation of diverse immune pathways in leukemia cells isolated from mice treated with AZA. We propose that epigenetic priming with AZA induces transcriptional changes that sensitize tumor cells to subsequent CAR T cell treatment. Among the candidate genes up-regulated by AZA is TNFSF4 which encodes OX40L, one of the strongest T cell co-stimulatory ligands. OX40L binds OX40, the TNF receptor superfamily member highly specific for activated T cells. TNFSF4 is heterogeneously expressed in a panel of pediatric PDXs, and high TNFSF4 expression correlated with increased CAR T cell numbers identified in co-cultures with individual PDXs. High OX40L expression in Nalm-6 cells increased their susceptibility to CAR T cell killing while OX40L blockade reduced leukemia cell killing.Conclusion: We propose that treatment with AZA activates OX40L/OX40 co-stimulatory signaling in CAR T cells. Our data suggest that the clinical use of AZA before CAR T cells could be considered.Keywords: CAR T cells, leukemia, AZA, patient-derived xenografts, gene expression

Neuroblastoma is a cancer of the sympathetic nervous system that causes up to 15% of cancer-related deaths among children. Among the ~1,000 newly diagnosed cases per year in Europe, more than half are classified as high-risk, with a 5-year survival rate

PurposeTERT gene rearrangement with transcriptional superenhancers leads to TERT overexpression and neuroblastoma. No targeted therapy is available for clinical trials in patients with TERT-rearranged neuroblastoma.Experimental designAnticancer agents exerting the best synergistic anticancer effects with BET bromodomain inhibitors were identified by screening an FDA-approved oncology drug library. The synergistic effects of the BET bromodomain inhibitor OTX015 and the proteasome inhibitor carfilzomib were examined by immunoblot and flow cytometry analysis. The anticancer efficacy of OTX015 and carfilzomib combination therapy was investigated in mice xenografted with TERT-rearranged neuroblastoma cell lines or patient-derived xenograft (PDX) tumor cells, and the role of TERT reduction in the anticancer efficacy was examined through rescue experiments in mice.ResultsThe BET bromodomain protein BRD4 promoted TERT-rearranged neuroblastoma cell proliferation through upregulating TERT expression. Screening of an approved oncology drug library identified the proteasome inhibitor carfilzomib as the agent exerting the best synergistic anticancer effects with BET bromodomain inhibitors including OTX015. OTX015 and carfilzomib synergistically reduced TERT protein expression, induced endoplasmic reticulum stress, and induced TERT-rearranged neuroblastoma cell apoptosis which was blocked by TERT overexpression and endoplasmic reticulum stress antagonists. In mice xenografted with TERT-rearranged neuroblastoma cell lines or PDX tumor cells, OTX015 and carfilzomib synergistically blocked TERT expression, induced tumor cell apoptosis, suppressed tumor progression, and improved mouse survival, which was largely reversed by forced TERT overexpression.ConclusionsOTX015 and carfilzomib combination therapy is likely to be translated into the first clinical trial of a targeted therapy in patients with TERT-rearranged neuroblastoma.