Abstract 4146: Calcineurin represses IL-12 secretion in acute lymphoblastic leukemia

Abstract

Abstract Acute lymphoblastic leukemia (ALL) remains a leading cause of disease-related deaths in children. Although immuno-therapeutics have become a powerful strategy for the treatment of ALL, the mechanisms by which the leukemia cells avoid recognition and clearance by the immune system have not been clearly defined. Previous studies by our group defined leukemia-cell calcineurin (Cn) as a critical mediator of immune evasion in an aggressive, clinically relevant, murine model of BCR-ABL1+ B-ALL, as knocking it down in leukemia cells extended survival in immunocompetent, but not B and/or T cell-deficient mice (P=0.001). To identify downstream mediators of Cn-dependent immune evasion, we performed a cytokine array that demonstrated increased levels in chemokines and cytokines in the supernatant of Cn-deficient leukemia compared to the control. To validate this finding, ELISA was performed to quantify cytokine production and secretion. IL-12 secretion in the supernatant of Cn-deficient leukemia was significantly increased compared to the control. Ex vivo analyses demonstrated that T cell stimulation with the supernatant of Cn-deficient leukemia cells led to CD8+ T cell activation and CD4+ T cell differentiation into Th1 cells, as determined by measuring intracellular levels of IFN-γ and TNF-α. Furthermore, we found that the enhanced T cell activation by factors secreted from Cn-deficient leukemia was ablated by IL-12 neutralizing antibodies. Notably, analyses of publicly available gene expression profiles demonstrate that children with ALL in which either IL-12A or IL-12B is expressed at high levels have better outcomes, suggesting that IL-12 has clinical relevance. Importantly, treatment of mice with recombinant IL-12 (rIL-12) dramatically reduced the leukemia burden and prolonged survival in WT mice with parental leukemia as compared to vehicle treated WT controls (P=0.002). Successfully treated mice survived a second challenge with leukemia, suggesting the establishment of immunologic memory in these mice. In addition, rIL-12 treated Rag1-/- mice with leukemia had a similar reduction in leukemia burden, and transient prolongation of survival as compared to vehicle treated Rag1-/- mice (P=0.003). Given that Rag1-/- recipients lack mature B and T-cells, these results suggest that IL-12 responsive innate immune cells, such as NK cells, also play a major role in the effective suppression of leukemia cells. To begin to address this hypothesis, WT, Rag1-/-, IL2rg-/-, and double knockout (DKO) mice lacking B, T and NK cells (Rag1-/-/IL2rg-/-) were engrafted with calcineurin-deficient leukemia. A trend was observed in which DKO mice showed the highest rate of leukemia progression compared to the other groups. Overall, these data suggest that Cn-mediated inhibition of IL-12 secretion contributes to evasion of both innate and adaptive immunity by leukemia cells, and that novel strategies for therapeutic IL-12 delivery are needed. Citation Format: Rae Hunter, Jairo Fonseca, Jodi Dougan, Lori A. Gardner, Jennifer Rabe, Manali Rupji, Bhakti Dwivedi, Curtis J. Henry, Christopher C. Porter. Calcineurin represses IL-12 secretion in acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4146.