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

Program: Oral and Poster Abstracts
Type: Oral
Session: 651. Multiple Myeloma and Plasma Cell Dyscrasias: Basic and Translational: Multiple Myleoma Circulating Tumor Cells, Novel Mechanisms, and Immune Interactions
Hematology Disease Topics & Pathways:
Research, Biological therapies, Antibody Therapy, apoptosis, Translational Research, Plasma Cell Disorders, Chimeric Antigen Receptor (CAR)-T Cell Therapies, Combination therapy, Checkpoint Inhibitor, Diseases, immune mechanism, cell expansion, Therapies, Immunotherapy, immunology, Lymphoid Malignancies, Biological Processes, Technology and Procedures, profiling, Study Population, pathogenesis, Animal model, omics technologies
Monday, December 11, 2023: 4:00 PM

Yubin Kang, MD1, Xiaobei Wang, PhD1*, Jian Wu, MD, PhD1*, Shaima Jabbar, PhD1*, Chongming Tom Jiang, PhD2*, Xiling Shen, PhD2* and Besim Ogretmen, PhD3*

1Division of Hematologic Malignancies and Cellular Therapy, Duke University, Durham, NC
2Terasaki Institute, Los Angeles, CA
3Departments of Biochemistry and Molecular Biology and Hollings Cancer Center, Medical University of South Carolina, Charleston, SC

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.

Disclosures: Kang: OrPro Therapeutics: Other: Patent application.

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