Inhibition of Sphingosine Kinase 2 Enhances Immunotherapy in Mouse Model of Multiple Myeloma

Introduction: Sphingolipid metabolism, in particular the ceramide:sphigosine 1-phophate (S1P) rheostat, is increasingly recognized as a key pathway in cancer biology, inflammation and immune responses. Sphingosine kinase (SK) 1 and 2 catalyze the formation of S1P. We previously reported that SK2 but not SK1 was overexpressed in multiple myeloma (MM) cells. Treatment with opaganib, an SK2-specific inhibitor, downregulated the expression of c-Myc and Mcl-1, resulting in apoptosis of MM cells. A phase I clinical trial of opaganib in patients with relapsed/refractory MM demonstrated the safety and evidence of anti-myeloma activity of SK2 inhibition. However, the roles of SK2 in host's anti-tumor immunity are unclear. We herein determined the effects of SK2 on the functions of T cells and myeloid derived suppressor cells (MDSCs), the underlying mechanisms, and potential clinical application of SK2 inhibition in enhancing immunotherapy.

Methods: For tumor models, transplantable mouse VK*Myc myeloma cells, CT-2A gliomas cells, B16F10 melanoma cells, or TRAMP C2 prostate cancer cells were implanted into SK2 ^-/- knockout (KO), SK1 ^-/- KO or WT mice. Multi-color flow cytometry was used to measure various immune cells. Anti-CD8 antibody (clone YST-169.4) and anti-Gr-1 antibody (clone RB6-8C5) were used to deplete CD8 T cells and MDSCs in vivo, respectively. Adaptive transfer of enriched CD8 T cells and bone marrow derived MDSCs was performed. Cytokines and chemokines were measured by multiplex assay. Transcriptome sequencing and omics analyses were performed on mouse primary CD8 T cells and MDSCs. CRISPR/Cas9 genetic knockout experiments were used to validate genes identified from the sequencing results. Mouse BCMA CAR T model was established using syngeneic VK*Myc myeloma cells and anti-mouse BCMA CAR T cells. Additionally, the combinatorial effects of opaganib and 4-1BB agonist were examined in VK* Myc myeloma mouse model.

Results: When we injected VK*Myc myeloma cells IV into SK1 ^-/- KO, SK2 ^-/- KO or WT mice, none of SK2 ^-/- KO mice developed myeloma or died from myeloma whereas 85-90% of WT recipient mice and all SK1 ^-/- KO mice developed myeloma and died. Similarly, tumor development was significantly attenuated in SK2 ^-/- KO mice when CT-2A gliomas cells (intracranial injection), B16F10 melanoma cells (IV) or TRAMP C2 prostate cancer cells (IV) were implanted, demonstrating broad anti-tumor effects of SK2 deletion. We found that the homing of myeloma cells was not affected in SK2 ^-/- KO mice. SK2 ^-/- KO recipient mice showed significantly increased number of CD8 T cells and decreased number of MDSCs. Depletion of CD8 T cells using CD8 antibody rendered SK2 ^-/- KO mice susceptible to myeloma development and administration of anti-Gr-1 antibody increased CD8 T cells and made SK2 WT mice resistant to myeloma development. Adaptive transfer of SK2 ^-/- KO CD8 T cells suppressed myeloma development, indicative of a critical role of SK2 in T cell regulation. Cytokine profiling revealed significantly increased level of IFNγ and IL-12 and suppressed level of IL-6, GM-CSF, CCL2, TNFα, and IL-10 in SK2 ^-/- KO recipient mice. CD8 T cells isolated from SK2 ^-/- KO mice were more proliferative in response to CD3/CD28 antibody stimulation, had higher expression of CD69, Granzyme B and IFNγ but lower level of PD-1, LAG-3, TIGIT and TIM-3, and were more cytotoxic against myeloma cells. Differential gene expression analyses showed up-regulation of IL-36gamma and kallikrein 1-related peptidase b22 (KLK1B22) in SK2 ^-/- KO CD8 T cells. Knockout of IL-36gamma or KLK1B22 with CRISPR/Cas9 reversed the effects of SK2 deletion on T cell activation and exhaustion, demonstrating the important role of IL-36gamma and KLK1B22 in SK2 mediated T cell regulation. Compared to T cells from WT mice, T cells from SK2 ^-/- KO mice exhibited enhanced anti-myeloma activities in our VK*Myc myeloma anti-BCMA CAR T mouse model. Finally, treatment of mice with opaganib increased the number of T cells and decreased the number of MDSCs and showed synergistic anti-myeloma activities when combined with 4-1BB agonist in VK*Myc myeloma model.

Conclusions: Our study demonstrated that inhibition of SK2 enhances anti-tumor immunity by promoting CD8 T cell activation likely through IL-36gamma and KLK1B22. These studies provide rationale for clinical trials investigating the combination of opaganib with CAR T therapy or other immunotherapy in cancer treatment.