JAK2 activation promotes tumorigenesis in ALK-negative anaplastic large cell lymphoma via regulating oncogenic STAT1-PVT1 lncRNA axis
Authors: Kang Le, Linda E. Wellik, Matthew J. Maurer, Ellen D. McPhail, Thomas E. Witzig, Mamta Gupta
Journal: Blood Cancer Journal (2021)

Anaplastic large cell lymphoma (ALCL), a subgroup of peripheral T-cell lymphoma (PTCL), is characterized by high expression of CD30 with varying genetics and clinical outcome 1 .Studies confirm that the 5 years overall survival rates for ALK-positive and ALK-negative ALCL at 80% and 49%, respectively 2,3 .Inferior clinical outcome in patients with ALK-negative ALCL as compared to ALK-positive ALCL highlights the need for new druggable targets for this unique subgroup of ALCL.Prior studies have demonstrated upregulated JAK kinases and their downstream STAT-signaling proteins in various subtypes of PTCLs 4-8 ; however, studies delineating the role of JAK isoforms in ALK-negative ALCL are lacking.The overall goal of this study was to assess role of JAK kinases in the ALK-negative ALCL subset and to evaluate the potential impact on disease prognosis and therapeutic advances.We performed immunohistochemistry on ALK-negative tumors (n = 12) using phospho-specific antibodies against JAK1, JAK2, and JAK3.Using 30% cut-off, ALK-negative ALCL tumors were negative for p-JAK1;10% positive for p-JAK3, while 33.3% showed elevated p-JAK2.Consistent with the p-JAK2 immunostaining seen in ALK-negative ALCL, robust tyrosine phosphorylation of JAK2 was detected in Mac-2A and FE-PD cells (Fig. 1A;S1A,B).These results suggest that JAK2 activation is exclusively associated with the ALK-negative ALCL subtype, and may have an important role in the disease pathogenesis.Pharmacologic inhibition of JAK2 specific inhibitor fedratinib, resulted in a dose-dependent decrease in cell proliferation and survival in Mac-2A and FE-PD cells in vitro.Similarly, shRNA-mediated knockdown of JAK2 significantly (p < 0.01) decreased the growth of Mac-2A cells.Furthermore, inhibition of JAK2 abated colony formation capabilities of the FE-PD and Mac-2A cells (Fig. 1B-D; S1C).Taken together, these results suggest that ALK-negative ALCL cells have an exquisite sensitivity to JAK2 inhibition.In order to find out the downstream mediators of JAK2 kinase in the ALK-negative ALCL, effects of fedratinib or JAK2 siRNA were assessed on the STATs signaling.Treatment with fedratinib decreased the phosphorylation of STAT1 and STAT3 in Mac-2A and FE-PD cells.Similarly, JAK2 knockdown by siRNA also decreased phosphorylation of STAT1 and STAT3 in Mac-2A cells (Fig. 1E; S2A).Interestingly, fludarabine (a STAT1-inhibitor) but not stattic (a-STAT3 inhibitor) had a significant (p < 0.01) inhibitory effect on cell proliferation and survival of Mac2A and FE-PD cells (Fig. 1F; S2B).STAT1 depletion by STAT1-shRNA significantly (p < 0.01) inhibited the proliferation of FE-PD cells (Fig. S2C,D).The CFU assay also demonstrated that the STAT1 inhibition with fludarabine or STAT1 shRNA significantly inhibited the colony-forming ability of ALK-negative ALCL cells (Fig. S2E).These data suggest that STAT1 is an important downstream mediator of oncogenic effects of JAK2 in the ALK-negative ALCL cells.We and others have previously shown that c-Myc (referred as Myc hereafter) plays an important role in the lymphoma subtypes and acts as an important downstream effector to JAK/STAT pathway [9][10][11] .In line with this notion, inhibition of JAK2 with fedratinib decreased Myc at protein level whereas mRNA level or distribution pattern between cytoplasm and nucleus remained unaltered in both Mac-2A and FE-PD cell lines.A rapid