FF-10101

Purpose:FLT3 mutations occurred in approximately one third of patients with acute myeloid leukemia (AML). FLT3-ITD mutations caused the constitutive activation of the RAS/MAPK signaling pathway. Ribosomal S6 Kinases (RSKs) were serine/threonine kinases that function downstream of the Ras/Raf/MEK/ERK signaling pathway. However, roles and mechanisms of RSKs inhibitor LJH-685, and combinational effects of LJH-685 and FLT3 inhibitor FF-10101 on AML cells were till unclear.Methods:Cell viability assay, CFSE assay, RT-qPCR, Colony formation assay, PI stain, Annexin-V/7-AAD double stain, Western blot, and Xenogeneic transplantation methods were used to used to investigate roles and mechanisms of LJH-685 in the leukemogenesis of AML.Results:LJH-685 inhibited the proliferation and clone formation of AML cells, caused cell cycle arrest and induced the apoptosis of AML cells via inhibiting the RSK-YB-1 signaling pathway. MV4-11 and MOLM-13 cells carrying FLT3-ITD mutations were more sensitive to LJH-685 than that of other AML cell lines. Further studies suggested that LJH-685 combined with Daunorubicin or FF- 10101 synergistically inhibited the cell viability, promoted the apoptosis and caused cycle arrest of AML cells carrying FLT3-ITD mutations. Moreover, in vivo experiments also indicated that LJH-685 combined with FF-10101 or Daunorubicin prolonged the survival time of NSG mice and reduced the leukemogenesis of AML.Conclusion:Thus, these observations demonstrated combination of RSK inhibitor LJH-685 and FLT3 inhibitor FF-10101 showed synergism anti-leukemia effects in AML cell lines with FLT3-ITD mutations via inhibiting MAPK-RSKs-YB-1 pathway and provided new targets for therapeutic intervention especially for AML with FLT3-ITD mutations and Daunorubicin-resistant AML.

Small-molecule FLT3 inhibitors have recently improved clinical outcomes for patients with FLT3-mutant acute myeloid leukemia (AML) after many years of development, but resistance remains an important clinical problem. FF-10101 is the first irreversible, covalent inhibitor of FLT3 which has previously shown activity against FLT3 tyrosine kinase inhibitor resistance-causing FLT3 F691L and D835 mutations. We report that FF-10101 is also active against an expanded panel of clinically identified FLT3 mutations associated with resistance to other FLT3 inhibitors. We also demonstrate that FF-10101 can potentially address resistance mechanisms associated with growth factors present in the bone marrow microenvironment but is vulnerable to mutation at C695, the amino acid required for covalent FLT3 binding. These data suggest that FF-10101 possesses a favorable resistance profile that may contribute to improved single-agent efficacy when used in patients with FLT3-mutant AML.

FLT3-ITD and FLT3-TKD mutations were observed in approximately 20 and 10% of acute myeloid leukemia (AML) cases, respectively. FLT3 inhibitors such as midostaurin, gilteritinib and quizartinib show excellent response rates in patients with FLT3-mutated AML, but its duration of response may not be sufficient yet. The majority of cases gain secondary resistance either by on-target and off-target abnormalities. On-target mutations (i.e., FLT3-TKD) such as D835Y keep the TK domain in its active form, abrogating pharmacodynamics of type II FLT3 inhibitors (e.g., midostaurin and quizartinib). Second generation type I inhibitors such as gilteritinib are consistently active against FLT3-TKD as well as FLT3-ITD. However, a "gatekeeper" mutation F691L shows universal resistance to all currently available FLT3 inhibitors. Off-target abnormalities are consisted with a variety of somatic mutations such asNRAS,AXLandPIM1that bypass or reinforce FLT3 signaling. Off-target mutations can occur just in the primary FLT3-mutated clone or be gained by the evolution of other clones. A small number of cases show primary resistance by an FL-dependent, FGF2-dependent, and stromal CYP3A4-mediated manner. To overcome these mechanisms, the development of novel agents such as covalently-coupling FLT3 inhibitor FF-10101 and the investigation of combination therapy with different class agents are now ongoing. Along with novel agents, gene sequencing may improve clinical approaches by detecting additional targetable mutations and determining individual patterns of clonal evolution.