Synergistic antileukemic activity of JAK inhibition combined with cytarabine in acute megakaryoblastic leukemia

Background: Acute megakaryoblastic leukemia (AMKL) remains a therapeutic challenge, with limited responsiveness to standard cytarabine-based regimens and frequent microenvironment-mediated resistance. Aberrant activation of the JAK-STAT signaling pathway has been implicated in AMKL pathogenesis but has not been fully exploited therapeutically in combination with cytarabine. Methods: We evaluated the antileukemic efficacy of combining a JAK inhibitor with cytarabine across multiple preclinical models: (i) AMKL cell lines (including CMK, CMY, MKPL1, UT7, MOLM16, Mo7e) assessing viability, apoptosis, and combination index; (ii) three-dimensional (3D) co-culture with mesenchymal cells (MSCs) modelling bone-marrow niche-mediated protection; and (iii) murine xenograft models of AMKL assessing leukemic burden and survival. Results: In cell-line experiments, the JAK inhibitor plus cytarabine demonstrated marked synergy (combination index < 0.7) with significantly increased apoptosis (Annexin V+) and reduced proliferation compared to either agent alone. In the 3D MSC co-culture model, the microenvironment protected AMKL cells from cytarabine-induced death, but this protective effect was abrogated by JAK inhibition, restoring cytarabine sensitivity especially in MOLM16 and MKPL1 cells. In vivo, combination therapy significantly reduced leukemic burden (p < 0.05) and significantly prolonged median survival compared to cytarabine alone. Mechanistically, ruxolitinib suppressed phosphorylation of STAT3 and STAT5 and exerted a synergistic effect in combination with cytarabine; however, this inhibitory effect on phosphorylation was attenuated under the 3D co-culture condition. Conclusions: Our results provide robust preclinical evidence that combining JAK inhibition with cytarabine yields synergistic antileukemic effects in AMKL, including in a niche-protected context, and significantly improves in vivo outcomes. This data strongly supports the rationale for clinical evaluation of this combination in AMKL patients, especially those with JAK-STAT pathway activation.