PKN1 is a Novel Therapeutic Target for the Optimal Control of GvHD

Introduction/Rationale: We were the first to demonstrate that targeting JAK1/2 using baricitinib (BARI) and ruxolitinib (RUX) prevents and treats GvHD while enhancing multi-lineage hematopoietic reconstitution and graft-vs-leukemia (GvL) effect in mouse models of allogeneic hematopoietic cell transplantation (allo-HCT). However, unlike BARI, RUX fails to completely prevent or treat GvHD.

Methods: To elucidate the mechanism underlying the superiority of BARI to RUX, we performed both mouse and human kinome analyses. After identifying PKN1 as the best candidate, we have generated T cell-specific PKN1 KO mice which are used as T cell donors in our mouse model of GvHD.

Results: We found that BARI, but not RUX, is a potent inhibitor of both murine and human PKN1. In addition, single-cell RNA sequencing of human PBMCs from patients in a phase 1 trial with BARI also showed downregulation of PKN1-associated genes. Further, mice transplanted with PKN1 KO T cells exhibited significantly reduced GvHD with higher frequencies of Tregs in the spleens, similarly to mice treated with BARI but not RUX. We next investigated if administration of RUX in addition to targeting of PKN1 could prevent GvHD as effectively as BARI. Briefly, we transplanted WT or PKN1 KO T cells along with WT T cell-depleted bone marrow cells (all from B6 mice) into lethally irradiated recipient mice (Balb/c), followed by subcutaneous injection of RUX (400 μg per mouse, 5 times/week). We found that RUX + PKN1 KO T cells demonstrated similar outcomes compared to BARI, showing significantly improved overall survival by reducing GvHD compared to either PKN1 KO T cells or RUX alone.

Conclusion: All of these findings suggest that targeting of PKN1 in addition to JAK1/2 is needed for optimal control of GvHD. Our work provides significant mechanistic insights into GvHD, its prevention and treatment with broad implications for the development and use of PKN1 inhibitors for solid organ transplant rejection and other chronic inflammatory diseases.