Functional investigation into NK cells identifies salt-inducible kinases as novel therapeutic targets against NK malignancies

Abstract Background: Natural Killer (NK) cells are innate lymphoid cells that play critical roles in anti-tumor and anti-viral immunity. However, hyperproliferation and overactivation of NK cells may lead to autoimmunity and NK cell malignancies. Extranodal NK-cell lymphoma (ENKL) is a rare but aggressive non-Hodgkin lymphoma with a 5-year overall survival (OS) in 25% patients. Also, Advanced ENKL or Aggressive NK Leukemia (ANKL) has poor prognosis with a 2-year OS in 38% and 18% patients, respectively. Although occurring worldwide, the disease is prevalent in Asian and South American population and is strongly associated with Epstein-Barr Virus (EBV) infection. We previously demonstrated that Salt-Inducible Kinase inhibitors (SIKi), identified via a 4000-drug all-human-material screen, potentiate T cell activity and improve survival in an ovarian cancer model. Surprisingly, we further found that SIKi reduces NK cell numbers in the tumor. We aimed to investigate the hitherto unexplored roles of SIKs in NK cells and their possible implication in NK malignancies. Approach and Results: Using conditional knockout mice models, we found that NK-specific deletion of Sik2 and Sik3 obliterates NK cells in peripheral blood, spleen and bone marrow (8-10 fold reduction). Sik2 Sik3 deletion led to increased experimental lung metastasis of B16F10 melanoma, correlating with the low NK cell count. Sik2 Sik3 knockout impaired IFNγ production and reduced mitotic potential (Ki67) in NK cells. We took a pharmaceutical approach to show that a low dose (1 μM) of SIKi (YKL-05-099, SK-124) significantly reduces IFNγ (-47%), TNFα (-20%) and Granzyme B (-48%) production from cytokine-stimulated mouse and human NK cells. Transcriptomic analysis revealed that SIKi causes striking reduction in gene transcription and protein translation in NK cells and downregulates proliferation signatures. In vitro SIK inhibition or in vivo Sik2 Sik3 deletion led to downregulation of STAT5 signaling and increase in hallmarks of apoptosis in NK cells. Also, SIK inhibition reduced the cell surface expression of cytokine receptors that are essential for NK cell activation. Hence, Sik2 and Sik3 are critical to maintain normal NK cell biology including viability, proliferation and function. Interestingly, Sik2 Sik3 knockout suppressed signatures of NK-cell malignancies. By analyzing published transcriptomic datasets, we found that expressions of SIK genes are dysregulated in more aggressive form of NK malignancy with STAT5B-N642H gain-of-function (GOF). Also, STAT5B GOF mutations in clinical NK malignancy leads to upregulation of MYC and E2F target pathways. SIK deletion downregulated transcripts of STAT5 signaling, and MYC and E2F targets, indicating functional overlap with STAT5 signaling. We found that SIK inhibitors downregulate the proliferation and viability of multiple human NK lymphoma/leukemia cell lines of patient origin, such as NK92MI, IMC-1-STAT5BN642H and KHYG-1-STAT5BN642H, in vitro and reduce cytokine production and cytotoxicity signatures such as Perforin 1. Also, SIK inhibitors downregulated STAT5 activation (pSTAT5A/B) in NK-lymphoma cell lines. Combination of SIKi with a potent STAT5 inhibitor (AC-4-130) further decreased proliferation and cytotoxicity signatures of human NK lymphoma cells. To test the in vivo efficacy, we generated multiple xenograft models of human NK-cell malignancies. We used both aggressive NK leukemia model (intravenous transplant) and extranodal primary tumor model (subcutaneous injection) to test the drug efficacy. SIKi monotherapy at a non-toxic dose (10 mg/Kg every alternate day) significantly improved survival in NK92MI and KHYG-1-STAT5BN642H NK-leukemia models. Moreover, SIKi remarkably suppressed subcutaneous growth of human SNK-6 NK lymphoma cells. Hence, SIKi impedes NK-cell lymphoma growth in xenograft models. Conclusion and future: We identified a critical role of SIK in normal NK cell development and function. Also, SIKi is efficacious in controlling clinically relevant NK malignancies in vivo. STAT5 signaling emerges as a top downstream target of SIK. Hence, SIKi may potentially synergize with JAK/STAT5 inhibitors to further improve clinical outcome. Analysis of the effect of SIKi on the immune landscape of NK malignancies using clinical tissue specimens (ex vivo analysis) or immunocompetent preclinical models (in vivo) may further identify its role as an immunotherapeutic and potential synergy with anti-PD1 therapy.