RUXOLITINIB TARGETS STAT1-STAT3 COOPERATIVELY IN LARGE GRANULAR LYMPHOCYTIC LEUKEMIA

Introduction: Large granular lymphocytic (LGL) leukemia is a clonal T- or NK-cell disorder frequently associated with cytopenias. Standard treatments rely on immunosuppressive therapies with limited efficacy and toxicity concerns. Given that up to 40% of LGL cases harbor activating STAT3 mutations, JAK/STAT oncogenic dependence has emerged as a potential therapeutic target.

Methods: We recently completed a multicenter investigator-initiated phase II clinical trial that evaluated ruxolitinib (20 mg PO twice daily) in LGL patients, with treatment continuing until progression (Moskowitz et al., Blood 2021 and ASH 2023). Peripheral blood samples collected before and during treatment were analyzed using single-cell Combined Indexing of Transcriptome and Epitopes (CITE-seq) and plasma proteomic profiling to elucidate Ruxolitinib mechanism of action. Functional experiments, including confocal microscopy, Cut&Run, and western blot analyses, were conducted in STAT3-wild type (WT) and STAT3-mutant Jurkat cells to validate key findings (Figure 1A).

Results: Among 22 evaluable patients, ruxolitinib achieved a 68% clinical benefit rate and a 45% overall response rate. Single-cell analysis revealed that Ruxolitinib efficacy stems not only from direct targeting of LGL cells but also from reducing JAK/STAT-driven myeloid inflammation. Specifically, ruxolitinib suppressed IL6/JAK/STAT3 target gene expression in WT but not in STAT3-mutant LGL cells, consistent with these mutations conferring kinase-independent activity. Further analysis indicated that non-malignant circulating myeloid cells, which showed high JAK/STAT target gene enrichment at baseline, exhibit significant downregulation of JAK/STAT activity on-treatment in responding patients. SCENIC analysis was performed to investigate the heightened inflammatory signaling in STAT3-mutant cells, revealing increased STAT1 and IRF8 expression before ruxolitinib exposure. Functional assays confirmed increased nuclear translocation of STAT1 and stronger binding to IFNg-responsive genes in STAT3 mutant Jurkat cells (Figure 1B,C). This suggested that STAT3 gain-of-function mutations stabilize STAT3 homodimers, enhancing STAT1 signaling and interferon-gamma (IFNg) production (Figure 1D). Among IFNg-stimulated genes, we identified macrophage migration inhibitory factor (MIF) as an LGL-derived factor linked to treatment response. Further functional studies demonstrated that MIF enhances monocyte-induced inflammation by specific activation of JAK/STAT in these myeloid cells.

Conclusion: These findings establish a previously unrecognized STAT3-STAT1 interplay in LGL, where STAT3 mutations enhance STAT1 signaling, promoting IFNg-mediated MIF secretion. Finally, STAT3 and STAT1 cooperatively induce myeloid-driven inflammation and cytopenia in patients with STAT3-mutant LGL, this loop being a key therapeutic target of ruxolitinib.

Research funding declaration: S.A.V. was supported by a Steven A. Greenberg Lymphoma Research Grant and a generous donation from Joshua and Lisa Bernstein.

Keywords: other lymphoid cancers; other basic and translational science; molecular targeted therapies

No potential sources of conflict of interest.