PI3K/AKT Activation Mediates B-Cell Transformation By the “T” Splice-Variant of Fyn Kinase

Abstract

The SRC-family kinase FYN has key roles downstream of growth-factor receptor activation and is implicated in various cancers. Activating FYNutations were reported in angioimmunoblastic T-cell lymphoma (AITL), but its role in transforming lymphocytes is poorly characterized. FYN also may play a role in signaling downstream of the B cell Receptor (BCR) receptor in Diffuse Large B-Cell Lymphoma (DLBCL), and laboratory studies show inhibition of FYN and other SRC family kinases (SFKs) can block BCR-mediated lymphomagenesis. To investigate FYN's role in oncogenic singling, we introduced FYN alleles to IL3-dependent FL5.12 murine pro-B cells and tested for selective advantage upon cytokine withdrawal and ability to transform the cells to cytokine independence. FYN B and T variants result from alternate splicing of exon 7, with B originally detected in brain and T in hematopoietic cells, but both are expressed and may be deregulated associated with disease states in diffuse tissues. We employed wildtype (WT) alleles for both spliceoforms along with mutations detected recurrently in AITL (L174R and R176C), kinase active (Y528H), and kinase dead (K299A). No FYN B alleles showed any selective advantage in the prolymphocyte cells subjected to cytokine withdrawal. In contrast, all FYN T alleles other than kinase dead were strongly enriched through multiple rounds of IL3 withdrawal and rescue. Indeed, complete withdrawal of cytokine resulted in transformation to IL3-independent growth, generating stable cell lines addicted to FYN kinase activity for survival. The SFK inhibitor PP2 was toxic to all FYN-T transformed cells but not to baseline FL5.12 cells growing in cytokine. WT- and L147R-transformed cells were similarly sensitive to PP2, while the other AITL-derived mutant R176C demonstrated reduced sensitivity. Immunoblotting demonstrated PP2-sensitive phosphorylation of AKT and the MTORC1 target 4EBP1, suggesting PI3K/AKT activation as a key survival pathway downstream from transformation by FYN T alleles. Consistently, the AKT inhibitor ipatasertib and PDK1 inhibitor GSK2334470 were highly active against all FYN-transformed lines, and AKT phosphorylation was sensitive to PDK1 inhibition, showing convergence on AKT activation in all transformants. Pan-PI3K inhibition was active against FYN transformants, while the delta isoform-specific inhibitor idelalisib showed only partial activity, suggesting activation of multiple PI3K isoforms downstream of FYN activity. These results suggest that AKT, PI3K, and PDK1 are important targets downstream of FYN, and that the PI3K/AKT pathway is the key survival mechanism in lymphomagenesis mediated by FYN. We show that WT and activating mutants of the FYN T spliceoform specifically, but not FYN B, induce drug-sensitive B lymphocyte malignant transformation by activating PI3K/AKT/MTORC1 signaling. DisclosuresNo relevant conflicts of interest to declare.