The BCR-Associated Tyrosine Kinase SYK Is Linked to the Activation of AKT in Mantle Cell Lymphoma.
Authors: Rudelius Martina , Sebasigari Denise , Tabe Yoko , Davies-Hill Theresa , Fend Falko , Leticia Quintanilla-Martinez , Elaine Jaffe S. , Stefania Pittaluga , Mark Raffeld 
Published: April 2020

Abstract
Mantle cell lymphoma (MCL) is comprised of two major clinical-pathologic subtypes, the more common typical MCL and the blastoid variant (bv-MCL). We recently demonstrated that the PI3K/AKT pathway is preferentially activated in bv-MCL. In all bv-MCL cases, activated phosphorylated AKT (p-AKT) expression was accompanied by the phosphorylation of downstream targets, and pharmacological inhibition of the pathway abrogated or reduced the phosphorylation of AKT and its targets and resulted in cell cycle arrest and apoptosis. Activation of the pathway was neither the result of mutations in the p110 PI3K catalytic subunit, nor the loss of the negative regulator PTEN, in the majority of cases. Since the B-cell receptor (BCR) signaling pathway has been shown to be a major activator of the AKT pathway via SYK/PI3K interactions, the goal of the current study was to gain insight into its possible role in MCL. Initial studies revealed activated SYK in 4 MCL cell lines studied, and immunoprecipitation studies showed a linkage between SYK, the adaptor protein BCAP, and the p85 regulatory subunit of PI3K. To analyze a possible causal connection between SYK and AKT activation, we used both a classical inhibitor approach [piceatannol and SYK-II (Calbiochem)] and a targeted gene knockdown approach employing a lentiviral mirRNA delivery system in two cell lines (Z138C and Granta 519). Functional knock-down and downstream effects were analyzed by western blotting, assessment of PIP3 levels, MTT-test, and flow-cytometry. Using the two approaches, we could block the activation of PI3K/AKT pathway and phosphorylation of AKT downstream targets in the two MCL cell lines, indicating the importance of the B-cell receptor associated SYK kinase in activation of the PI3K/AKT pathway. Activation of BCR signaling components, including SYK, PLCγ2, and LYN, were then assessed by western blotting in 28 primary MCL cases (12 typical and 16 blastoid). Phosphorylated (active) SYK and PLCγ2 (a direct SYK target) were detected in all 28 primary cases (12 blastoid and 16 typical). Surprisingly, there were no significant differences between the two groups, despite the difference in their AKT status. However, there was a distinct difference in the activation state of LYN, as assessed by its phosphorylation at Tyr 396, which was correlated with AKT activation. While Tyr 396 was phosphorylated in the majority of typical MCL, the converse was true for the bv-MCL and cell lines, suggesting that LYN was inactive in the latter. Although LYN has been shown to have both positive and negative roles in BCR activation, the net effect of loss of LYN activity is hyperactivation of B-cell signaling. Given the consistent effect of SYK inhibition on the activity of the PI3K/AKT pathway in the cell lines, we hypothesize that loss of a negative regulator downstream of SYK, possibly LYN, is additionally required to allow activation of the PI3K/AKT pathway in MCL.