Mechanism of sorafenib resistance in FLT3-ITD⁺ acute myeloid leukemia

Acute myeloid leukemia (AML) is a group of heterogeneous diseases characterized by an abnormal increase in myeloblasts in circulation and/or bone marrow. Internal tandem duplication (ITD) of the fms-like tyrosine kinase 3 (FLT3) gene occurs in about 30% of AML and is associated with an inferior prognosis. Tyrosine kinase domain (TKD) mutations occur in about 5% with uncertain prognostic significance. Intensive chemotherapy and allogeneic hematopoietic stem cell transplantation (HSCT) are the mainstays of treatment. However these approaches have reached a deadlock with a cure rate of 30-40%. Targeting FLT3 in AML with multi-tyrosine-kinase inhibitors has been evaluated in Phase II/III clinical trials. Despite an initial clearance of myeloblasts, the leukemia invariably progresses despite continuous treatment. The mechanisms of drug resistance and leukemia progression, hence the effective therapeutic strategies are currently unknown, limiting its clinical application. These issues were addressed in the present study. In the first part, 13 patients with chemo-refractory or relapsed FLT3-ITD+ AML received sorafenib 200-400 mg twice daily of whom 12 patients achieved clearance or near clearance of bone marrow blasts after a median of 27 days (range 21-84 days). There was evidence of myeloid differentiation of the leukemia blasts at remission. Leukemia progression occurred in 9 patients after a median of 72 days (range 54-287 days) and in 4 out of 6 patients it was dominated by clones carrying double FLT3-ITD and -TKD mutations. Microarray studies comparing myeloblasts before sorafenib treatment (sorafenib naïve) and at subsequent progression (sorafenib resistant) demonstrated up-regulation of 64 genes including ALDH1A1, JAK3 and TESC whose functions were unknown in AML. Transplantation of sorafenib naïve and resistant myeloblasts into NOD/SCID mice recapitulated their clinical behavior when the animals were treated with sorafenib. Both ITD and TKD mutations at D835 were identified in leukemia initiating cells (LICs) from sorafenib naïve samples. These results suggested that sorafenib have selected more aggressive sorafenib-resistant subclones carrying both FLT3-ITD and D835 mutations. In the second part, the gene encoding tescalcin (TESC), that was up-regulated at sorafenib resistance and was known to activate a sodium/hydrogen exchange (NHE1), was evaluated to examine its link with TKI resistance. TESC was highly expressed in FLT3-ITD+ AML cell lines MOLM-13 and MV4-11 and its knock-down by siRNA lowered intracellular pH and induced apoptosis. The results were recapitulated by treatment with a NHE1 inhibitor, 5-(N,N-Hexamethylene)amiloride (HMA). Induction of sorafenib resistance in MOLM-13 cell line (MOLM-13-RE) significantly increased its sensitivity to HMA. HMA treatment of MOLM-13 and MV4-11 as well as primary FLT3-ITD+ AML cells significantly reduced leukemia initiation in NOD/SCID mouse xenotransplantation. Normal CD34+ cells engraftment was not affected. HMA treatment significantly enhanced suppression of FLT3 signaling by sorafenib even in sorafenib resistant cell lines. These observations provided novel information about the pathogenetic role of TESC-NHE1-pHi in sorafenib resistance in AML. In conclusion, the information derived from the present study has provided mechanistic insights to the emergence of drug resistance during sorafenib treatment and important guide for future therapeutic strategies targeting FLT3-ITD+ AML.published_or_final_versionMedicineDoctoralDoctor of Philosoph

Suppression of SOX7 by DNA methylation and its tumor suppressor function in acute myeloid leukemia

Key PointsSOX7 expression is silenced in common myeloid malignancies. SOX7 interacts directly with β-catenin and regulates the Wnt pathway in acute myeloid leukemia.</jats:p

Functions of flt3 in zebrafish hematopoiesis and its relevance to human acute myeloid leukemia

FMS-like tyrosine kinase 3 (FLT3) is expressed in human hematopoietic stem and progenitor cells (HSPCs) but its role during embryogenesis is unclear. In acute myeloid leukemia (AML), internal tandem duplication (ITD) of FLT3 at the juxtamembrane (JMD) and tyrosine kinase (TKD) domains (FLT3-ITD(+)) occurs in 30% of patients and is associated with inferior clinical prognosis. TKD mutations (FLT3-TKD(+)) occur in 5% of cases. We made use of zebrafish to examine the role of flt3 in developmental hematopoiesis and model human FLT3-ITD(+) and FLT3-TKD(+) AML. Zebrafish flt3 JMD and TKD were remarkably similar to their mammalian orthologs. Morpholino knockdown significantly reduced the expression of l-plastin (pan-leukocyte), csf1r, and mpeg1 (macrophage) as well as that of c-myb (definitive HSPCs), lck, and rag1 (T-lymphocyte). Expressing human FLT3-ITD in zebrafish embryos resulted in expansion and clustering of myeloid cells (pu.1(+), mpo(+), and cebpα(+)) which were ameliorated by AC220 and associated with stat5, erk1/2, and akt phosphorylation. Human FLT3-TKD (D835Y) induced significant, albeit modest, myeloid expansion resistant to AC220. This study provides novel insight into the role of flt3 during hematopoiesis and establishes a zebrafish model of FLT3-ITD(+) and FLT3-TKD(+) AML that may facilitate high-throughput screening of novel and personalized agents

Homoharringtonine (omacetaxine mepesuccinate) as an adjunct for FLT3-ITD acute myeloid leukemia

An in vitro drug screening using primary AML samples identified homoharringtonine (omacetaxine mepesuccinate) as an effective adjunct for treatment of FLT3-ITD AML.</jats:p