Selective JAK2 Pathway Inhibitor, CHZ868, Ameriorates Anti-Leukemic Functionaliry in CD19 CAR-T Cells

Introduction: A subset of B cell acute lymphoblastic leukemia (B-ALL) cases remains treatment-resistant, pointing to the need for a deeper understanding of their molecular biology and the development of optimized therapies. Although tyrosine kinase inhibitors (TKIs) based on the unique gene expression profile of each B-ALL case have been developed to address this problem, the genetic background of the tumor and the acquisition of resistance through genetic mutations has resulted in inconsistent TKI therapeutic outcomes. Conversely, CD19 chimeric antigen receptor T (CAR-T) cell therapy, which involves engineering T cells to express a chimeric antigen receptor that specifically binds to CD19, has shown strong and sustained antitumor effects against CD19-positive B cell malignancies. However, despite its efficacy, CAR-T cell therapy is associated with significant challenges, such as the potential for immune evasion, leading to diminished antitumor effects. Combining molecular targeted therapies with CAR-T cell therapy represents a novel strategy for amplifying the antitumor efficacy of immunotherapy. However, the concurrent use of Janus kinase (JAK) 1/2 inhibitors, such as ruxolitinib, has been reported to reduce CAR-T cell potency by inhibiting the JAK1-dependent T cell activation pathway. Unlike JAK1 and JAK3, which participate in common gamma receptor-dependent cytokine signaling that is crucial for T cell-mediated immunity, selective JAK2 inhibitors are predicted to have minimal impact on CAR-T cell activity. Our research explored the anti-tumor activity of a combination of a selective type II JAK2 inhibitor, CHZ868, and CD19 CAR-T cells.

Methods: B-ALL and CD19 CAR-T cells were co-cultured with 100 nM of each JAK inhibitor at an appropriate E:T ratio to confirm the effect of each JAK inhibitor on the cytotoxicity of CD19 CAR-T cells. Sequential killing assays were performed by adding fresh tumor cells to CAR-T cells co-cultured with tumor cells every 4 days with or without JAK inhibitors. CAR-T cell cytotoxicity was assessed by flow cytometry, and tumor cell survival was determined by measuring CD19 expression and was defined as the exclusion of the absence of CD3 expression. High throughput RNA sequencing was performed to investigate the gene expression profiles of CAR-T cells in the presence of CHZ868 to investigate the molecular mechanisms underlying CAR-T cell function and persistence. To observe the effect of CHZ868 on the antitumor efficacy of CAR-T in vivo, an in vivo stress test was performed, in which the CAR T-cells dose was lowered to determine the functional limits of different CAR-T cell populations. A total of 5 x 10⁵ Firefly luciferase-labeled CD19+ tumor cells (REH) were injected into the tail vein of NSG mice, and the mice were then treated with 1 x 10⁵ of CD19 CAR-T or control CAR-T cells, on day 7 post tumor cell injection. Concurrently, CHZ868 (30 mg/kg/day) or vehicle control (DMSO) was administered orally over a three-week period.

Results: Consistent with previous studies, ruxolitinib completely blocked the cytotoxic effects of CD19 CAR-T cells on JAK2 wild-type REH cells. By contrast, in the presence of fedratinib and CHZ868, CD19 CAR-T cells maintained their anti-tumor function even after multiple rounds of tumor rechallenge, including with JAK2 wild-type REH cells. Furthermore, CHZ868 had a greater synergistic effect on CD19 CAR-T cells than fedratinib.

Transcriptome analysis revealed that 116 genes were more highly expressed and 211 genes were more lowly expressed in the CHZ868 group than in the control group. Gene Set Enrichment Analysis (GSEA) revealed decreased expression of activation-related genes, such as MYC and mTOR, and increased expression of genes related to inactive memory T cells in the CHZ868 group. The expression of ribosome-related genes, an indicator of proliferative ability in early differentiation stages, was upregulated in the CHZ868 group.

In the in vivo stress test, the CD19 CAR-T cell group showed a modest anti-leukemic effect, which was enhanced when CD19 CAR-T was combined with CHZ868, as evidenced by the results of live tumor imaging and the prolonged survival of the mice.

Conclusion: Our findings suggest that selective inhibition of the JAK2 pathway may potentiate CAR-T cell therapy and offer a viable treatment strategy for the treatment of patients with resistant B-ALL, regardless of the JAK2 mutation status of their tumor cells.

Nakazawa:A-SEEDS Co., Ltd. with equity: Membership on an entity's Board of Directors or advisory committees. Yagyu:A-SEEDS Co., Ltd. with equity: Membership on an entity's Board of Directors or advisory committees.