JAK 1/2 Inhibition Preserves Hematopoietic Progenitor and Stem Cells, Prevents Aplasia, Inhibits Pro-Inflammatory Cytokines, and Prolongs Survival in Murine Immune Bone Marrow Failure

Background: Immune aplastic anemia (AA) is clinically characterized by marked pancytopenia due to cytotoxic T-lymphocyte mediated hematopoietic stem and progenitor cell (HSPC) destruction. Though hematopoietic stem cell transplant and immunosuppressive therapies are effective, both are onerous to administer and a fully oral regimen is desirable. Ruxolitinib (RUX) is a Janus Kinase (JAK) 1/2 inhibitor which suppresses cytotoxic T cell activation and inhibits production of inflammatory cytokines; RUX is FDA-approved for myeloproliferative neoplasms and graft versus host disease.

Methods: RUX was administered as a previously validated food additive (Rux-chow) in an immune bone marrow failure (BMF) murine model (major histocompatibility complex mismatched C57BL/6 (B6)ÞCByB6F1 lymph node (LN) cell infusion following sublethal total body irradiation (TBI)). Animals were fed Rux-chow starting at two, four, and six days after BMF induction (D+2, D+4, D+6) until day 28; a control group ate chow without RUX for the duration. Survival was assessed for 56 (D+2) or 70 (D+4, D+6) days. Blood counts, T-cells, apoptosis, and gene expression were assessed from peripheral blood, spleen or bone marrow (BM). Inflammatory cytokines implicated in AA including IFN-g, TNF-a, IL-5, IL-1a, CCL2, FasL, CCL5, and IL-10 in the plasma were measured by Luminex. Colony-forming unit (CFU) assays were performed on BM mononuclear cells mixed in semisolid methylcellulose media containing interleukin (IL)-3, IL-6, stem cell factor and erythropoietin with colonies counted at day 10; results were compared with both untreated BMF and TBI controls.

Results: All RUX-treated BMF mice (days +2, +4, and +6) achieved long term survival and all untreated BMF mice died (Figure 1A). RUX rescued blood counts in BMF mice, even with delayed treatment, that were sustained with drug discontinuation. Improvement in bodyweight (BW) was seen in Rux-fed mice. Infiltration and activation of BM T-cells were suppressed with a reduction in CD4+ and CD8+ T cells, decrease in CD25+ T-cells, and an increase in splenic FOXP3+ Tregs. Gene expression in RUX treated mice showed downregulation of immune pathways including cytokine signaling in the immune system, the JAK/STAT signaling pathway, and the IFN-g pathway, indicating inhibition of T cell function. RUX therapy preserved HSPCs phenotypically and functionally: hematopoietic progenitors were significantly greater among BM cells from BMF+RUX mice, at 2 weeks and at 8 weeks following BM failure induction, compared to both untreated BMF mice and TBI controls (Figure 1B). Inflammatory cytokines IFN-g, TNF-a, IL-5, CCL-2, CCL-5, FasL, and IL-10 were suppressed on RUX at two weeks but gradually increased when drug was discontinued (except IFN-g which remained at low levels long term).

Conclusion: RUX resulted in prolonged overall survival achieved by rescue of BMF as demonstrated by a sustained improvement in peripheral blood counts; this occurred even with delayed administration up to 6 days after BMF induction. RUX alleviated T-cell mediated immune destruction by suppression of T cell infiltration and activation, expansion of Tregs, as well as functional preservation of HSPCs. RUX, an orally administered medication with a favorable toxicity profile, is a potential new therapy for aplastic anemia and related syndromes. A clinical trial using RUX in patients with immune BMF is currently under development.

Figures:1A: Survival with delayed RUX therapy.

1B: CFU assays in RUX treated mice, TBI controls, and BMF controls

No relevant conflicts of interest to declare.