Disease relapse is a common cause of treatment failure in FMS-like tyrosine kinase 3 (FLT3) mutated acute myeloid leukemia (AML). In this study, to identify therapeutic targets responsible for the survival and proliferation of leukemic cells (blasts) with FLT3 mutations after gilteritinib (GILT, a 2nd generation tyrosine kinase inhibitor (TKI)) treatment, we performed proteomic screening of cytokine release and in vitro/ex vivo studies to investigate their associated signaling pathways and transcriptional regulation. Here, we report that macrophage migration inhibition factor (MIF) was significantly increased in the supernatant of GILT-treated blasts when compared to untreated controls. Additionally, the GILT-treated blasts that survived were found to exhibit higher expressions of the CXCR2 gene and protein, a common receptor for MIF and pro-inflammatory cytokines. The supplementation of exogenous MIF to GILT-treated blasts revealed a group of CD44High+ cells that might be responsible for the relapse. Furthermore, we identified the highly activated non-classical NFKB2 pathway after GILT-treatment. The siRNA transient knockdown of NFKB2 significantly reduced the gene expressions of MIF, CXCR2, and CXCL5. Finally, treatments of AML patient samples ex vivo demonstrated that the combination of a pharmaceutical inhibitor of the NFKB family and GILT can effectively suppress primary blasts’ secretion of tumor-promoting cytokines, such as CXCL1/5/8. In summary, we provide the first evidence that targeting treatment-activated compensatory pathways, such as the NFKB2-MIF/CXCLs-CXCR2 axis could be a novel therapeutic strategy to overcome TKI-resistance and effectively treat AML patients with FLT3 mutations.

Despite significant advancements in our understanding of acute myeloid leukemia (AML), relapsed and refractory disease remains a major cause of treatment failure. Approximately 50% of patients with AML will develop relapsed disease following induction chemotherapy, which results in a dismal 5-year overall survival rate of 29%. The development of FMS-like tyrosine kinase 3 (FLT3) inhibitors have led to improved outcomes among patients with FLT3-mutated AML. However many patients eventually relapse and succumb to chemo-resistant disease, highlighting the need to characterize the molecular pathways which confer early TKI resistance. To explore the possible mechanisms responsible for the survival and proliferation of tyrosine kinase inhibitor (TKI) resistant blasts, we compared in vitro and ex vivo cytokine expression of gilteritinib (GILT)-treated and untreated blasts. Here we report that the pro-inflammatory cytokine macrophage migration inhibitory factor (MIF) is significantly expressed by GILT-treated blasts when compared to untreated controls. We further demonstrate that MIF expression promotes blast proliferation through the upregulation of its receptor CXCR2. Most remarkably we found that combination of CXCR2-inhibitor plus GILT works synergistically to reduce the percentage of viable blasts. Together these findings support that targeting the TKI-activated MIF/CXCR pathway could be a novel therapeutic strategy for both newly diagnosed and relapsed/refractory AML. This study is supported by Loma Linda University GRASP and Loma Linda University Research Innovation Grant