Abstract 2885: Somatic mutation of STAT3 leads to the preferential Th17 differentiation in human naïve CD4-positive cells and favor TCR-mediated proliferation
Authors: Ramona Crescenzo, Valentina Fragliasso, Marcello Gaudiano, Marco Pizzi, Giorgio Inghirami
Journal: Cancer Research (2016)

Abstract Intro: Mature Th17 lymphocytes play a key role in the host defenses against bacteria and fungi although unchecked Th17 activation can lead to autoimmune disorders, inflammation and cancer. The engagement of CD4+ naïve T-cells via IL6 and TGFβ favors a Th17 differentiation program, which requires the presence of IL21 and IL23 to reach a complete maturation and the maintenance of Th17 phenotype. The Th17 differentiation requires multiple lineage defining transcription factors (i.e. RORα, RORγt, and STAT3) and the activation of STAT3 signaling, which regulates a plethora of factors (i.e. RORγt, IL23R), is required for the full execution of the Th17 program. We have recently shown that JAK1/STAT3 activating mutations in Anaplastic Large cell Lymphomas (ALCL) are oncogenic, but it is unknown their protumorigenic contribution in modulating/derailing the host microenviroment and the host responses. Methods: 50 FFPE ALCLs were investigated with pSTAT3, GATA3, RORγ and T-Bet antibodies using a dual color approach. CD4+ CD62L+ CD25- CD45RAhi CD4+ naïve cells of healthy donor were sorted and cultured (7 days) in KBM (IL23 (25ng/ml), IL6 (25ng/ml), IL21 (25ng/ml), IL1β (12,5ng/ml), TGFβ (5ng/ml) + 1% FBS and anti-CD3/CD28 beads (1:50). Forced expressions of WT or mutated Y640F (YF) STAT3 were achieved via lentiviral transduction. STAT3 qRTPCR, luciferase assay, ATPlite, and ELISA were used as readouts on samples collected at day 7 post-transduction. Results: IHC stains showed that pSTAT3+ ALCL preferentially co-expressed nuclear RORγ (p&amp;lt;000.1). Conversely, GATA3+ ALK- ALCL samples were preferentially STAT3-/RORγ- (12/14). Forced expression of STAT3 led to a higher expression of IL23R, RORγt and IL17, compared to controls cells. No statistically differences were seen in cell proliferation or metabolic levels in these conditions. Naïve YF STAT3 CD4 cells, when cultured w/o IL2 (100U/ml) + IL6 + anti-CD3/CD28 beads, were able to efficiently proliferate up to 20 days overcome controls and WT STAT3 cells. Lastly, transfection of HEK293T cells with YF STAT3 in the presence of an IL17 promoter reporter cassette, led to a significant higher luciferase expression levels than control or WT STAT3 transfected cells. Conclusions: Our data demonstrated that the ectopic expression of STAT3 leads to a robust Th17 differentiation of CD4 naïve T-cells and YF STAT3 sustains their growth overtime in the presence of TCR signaling. The preferential expression of RORγ in pSTAT3+ ALCL (ALK+ and mutated STAT3 ALK-ALCL) suggest that the deregulated activation of STAT3 can regulate the plasticity of the neoplastic cells favoring the recruitment of inflammatory host cells within the neoplastic lesions. Additional studies are underway to define the lymphoma-host interactions and the role of JAK TKI in controlling the lymphoma growth and in modulating the pro-tumorigenic inflammatory phenotype of Patient Derived Tumor Xenograft Citation Format: Ramona Crescenzo, Valentina Fragliasso, Marcello Gaudiano, Marco Pizzi, Giorgio Inghirami. Somatic mutation of STAT3 leads to the preferential Th17 differentiation in human naïve CD4-positive cells and favor TCR-mediated proliferation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2885.