NPM-ALK Upregulates Jab1/Csn5 through STAT3 Activation in Anaplastic Large Cell Lymphoma: A Novel Function of NPM-ALK That Contributes to PD1/PD-L1 Immune Checkpoint Regulation

Introduction: ALK+ anaplastic large cell lymphoma (ALCL) frequently carries the t(2;5) resulting in overexpression of NPM-ALK oncoprotein, which activates many oncogenic pathways. The Jab1 (c-Jun activation domain-binding protein-1), initially discovered as a c-Jun co-activator, represents the fifth component of an evolutionary highly conserved 8-subunit protein complex, named COP9 signalosome (CSN). The Jab1/Csn5 gene operates as an oncogene in cancer through multiple mechanisms including cell cycle control via the CDK inhibitor p27. Recent evidence suggests that Jab1/Csn5 is also involved in immune checkpoint regulation through PD-L1 stabilization by inhibition of PD-L1 proteasomal degradation. Recently, we reported that the protein levels of Jab1/Csn5 are the highest in ALCL as compared to other peripheral T-cell lymphomas (PTCL) and significantly correlate with PD-L1 levels in PTCLs (Drakos et al, HemaSphere 2019; (3):p596, EHA abstract). In this study, we hypothesized that NPM-ALK may regulate Jab1/Csn5 expression and thus contributes to cell proliferation as well as PD-L1/PD1 immune checkpoint regulation. Methods: The in vitro system included 4 ALK+ (Karpas 299, DEL, SUPM2, SUDHL1) and 2 ALK- (Mac1, Mac2a) ALCL cell lines as well as murine Ba/F3 parental and Ba/F3 clones stably transfected with NPM-ALK (Ba/F3-NPM-ALK), EEF1G (Ba/F3-EEF1G-ALK) or control plasmid (Ba/F3-MIG). Transient transfections with Jab1/CSN si-RNAs and STAT3 si-RNAs were also performed in ALCL cells. In addition, ALCL cells were treated with ALK (Crizotonib) or STAT3 (XIII) inhibitors. Two animal models were used in the study: 1) in the ex vivo model, Karpas-299 clones stably transfected with Jab1/CSN5 shRNA constructs were generated and injected in both thighs of SCID-beige immunocompromised mice. The Jab1/CSN5 shRNA and the control mice were followed for tumor development and their tumors were measured and analysed by immunohistochemistry. 2) in the patient derived xenograft (PDX) model of ALK+ ALCL, the mice were treated with the ALK inhibitor Ceritinib or control vehicle and were monitored for changes in tumor characteristics over two weeks. Tumor specimens were taken at early time points (24, 48, and 72 hrs) following treatment in order to obtain viable tumor cells for protein analysis. Results: Jab1/Csn5 was substantially upregulated in the Ba/F3-NPM-ALK and Ba/F3-EEF1G-ALK stable clones as compared to paternal or control Ba/F3-MIG clones. Jab1/Csn5 upregulation was associa