Biological and Clinical Impact of JAK2/mTOR Blockade in Gvhd Prevention: Preclinical and Phase I Trial Results

Background: Distinct from broadly acting graft-versus-host disease (GVHD) prophylaxis, JAK2 inhibition suppresses alloreactive T cells, while sparing regulatory T cells (Tregs) and graft-versus-leukemia (GVL). Early IL-6 activity via JAK2 and phosphorylated STAT3 in CD4⁺ T cells is associated with acute GVHD onset. In mice, we show combined JAK2/mTOR blockade synergistically prevents xenogeneic GVHD. In this first-in-human phase I/II GVHD prevention trial we combine pacritinib, a JAK2 inhibitor, with sirolimus to concurrently reduce T-cell costimulation via mTOR and IL-6 activity. With phase I complete, we demonstrate that dual JAK2/mTOR inhibition is safe, suppresses pathogenic Th1 and Th17 cells, spares Tregs and key GVL effector cells, and exhibits preliminary activity in preventing GVHD. The primary aim of phase I was to identify the lowest biologically active dose of pacritinib (defined as < 35% of CD4⁺ pSTAT3⁺ T cells at day +21) that is safe when combined with sirolimus-based immune suppression. The preliminary activity of JAK2/mTOR inhibition in GVHD prevention was also investigated.

Materials and Methods: This single-arm phase I/II trial (NCT02891603) tested the safety of pacritinib when administered with sirolimus plus low-dose tacrolimus (PAC/SIR/TAC) after allogeneic hematopoietic cell transplantation (alloHCT). A 3+3 dose escalation design was followed, including dose level 1 (PAC 100mg daily), level 2 (PAC 100mg twice daily), and level 3 (PAC 200mg twice daily). Clinical safety, pharmacodynamic assessments, and pharmacokinetic (PK) studies were followed during the study. Acute GVHD was scored through day +100. Patient characteristics are described in Table 1 (n=12). Allowed donor types were HLA-A, -B, -C, and -DRB1 matched-related or unrelated donors. Adequate vital organ function and Karnofsky performance status (KPS ≥ 80%) were required.

Results: Dose level 2, PAC 100mg twice a day, was the lowest biologically active and safe dose, and thus the recommended phase II dose. Blood samples acquired at day +21 showed that PAC 100mg twice a day reduced the mean frequency and geometric MFI of CD4⁺ pSTAT3⁺ T cells (Figure 1A, B). Consistent with suppressed pSTAT3, PAC 100mg twice a day decreased pathogenic Th1 and Th17 cells (Figure 1C, D). pSTAT5 is critical for Tregs and effectors of GVL. PAC 100mg twice a day favored STAT5 phosphorylation in CD4⁺ T cells, preserved Tregs and increased the ratio of Tregs to pathogenic T helper cells, and supported CD3⁺ T cell and NK cell effectors (Figure 1E-J). Patients treated on dose level 2 of PAC exhibited a robust increase in Th2 cells at day +21 (29.5% v 4.87% level 1 or 4.5% baseline, P<0.001 ANOVA). Additionally, neutrophil and platelet engraftment occurred without delay (Figure 1K, L). A single dose limiting toxicity was observed in dose level I only, and consisted of angioedema possibly related to PAC. CMV reactivation or disease were not observed among patients treated at dose level 2, with only a single case of CMV reactivation among dose level 1 (8 of 12 recipients were CMV seropositive). A single patient treated on dose level 2 developed grade 4 acute GVHD and died, after prematurely discontinuing TAC for acute kidney injury and electively stopping PAC. A patient died of relapsed disease in dose level 1.

To test the efficacy of dual JAK2/mTOR inhibition in vivo, NSG mice were transplanted with human peripheral blood mononuclear cells (PBMCs) and treated with either vehicle, PAC, STAT3 inhibitor S3I-201, SIR, PAC/SIR, or S3I/SIR. The combination of JAK2 or downstream STAT3 inhibition plus SIR significantly reduced xenogeneic GVHD in mice (Figure 1M) and maintained donor anti-tumor activity by CD8⁺ T cells (data not shown). Further, dual JAK2 or STAT3 inhibition with mTOR blockade significantly increased the induction of Tregs in mice transplanted with Treg-depleted human PBMCs (62.3% PAC/SIR or 74% S3I/SIR v 29.9-38% with vehicle or inhibitors alone, P<0.01 ANOVA).

Conclusions: We demonstrate that PAC/SIR/TAC (RP2D: PAC 100mg twice a day) is safe and effectively reduces IL-6 signal transduction, pathogenic Th1 and Th17 cells, and preserves Tregs and effectors necessary for GVL and antiviral immunity. Preliminarily, adding pacritinib limits acute GVHD, preserves donor CMV immunity, and permits timely engraftment. The efficacy of PAC/SIR/TAC will be tested in our ongoing phase II GVHD prevention trial.