Synergy of FLT3 inhibitors and a small molecule inhibitor of LIM kinase1/2 in FLT3-ITD positive acute myeloblastic leukemia (AML)

Introduction: LIM kinases 1/2 are downstream effectors of signalization pathways implicated in cytoskeleton dynamics via phosphorylation of Cofilin family proteins,matrix degradation and in activity control of Aurora kinase A. Recently, Rho kinases (ROCK) were identified to be constitutively activated by FLT3-ITD, BCR-ABL and KIT in hematologic malignancies via PI3 kinase and Rho GTPase mediated phosphorylation. Upon its activation by upstream kinases (ROCK and PAK) LIMK1/2 inactivates Cofilin by phosphorylation, leading to enhanced polymerization of Actin. Here we investigated the potential therapeutic role of LIMK1/2 inhibition in FLT3-ITD mutated AML. Materials and methods: Expression of LIMK1/2 was determined by RQ-PCR and WB. A small molecule inhibitor of LIMK1/2 (LIMKi) was tested alone or in combination with FLT3 inhibitors Midostaurin, Quizartinib and Crenolanib or the hypomethylating agent Azacitidine in FLT3-ITD driven AML cell lines MOLM-13 and MV-4-11. Cell viability and IC50 was assessed by MTT assays. In combination experiments, compounds were added simultaneously and relative cell numbers were determined at 72h with MTT assays and combination index (CI) was calculated with the Chow and Talalay model. Cell-cycle distribution was determined by cytofluorometric analysis detecting nuclear propidium iodide (PI) intercalation. Apoptosis was evaluated in cell lines and patient cells by outer Annexin V exposure and PI incorporation.Cells from healthy donors were obtained after informed consent and enriched for CD34+ cells by immunomagnetic selection and seeded in methylcellulose, FCS and cytokines with or without LIMKi. For in vivo experiments we used a bone marrow engraftment tumor model with MOLM13-LUC cells using bioluminescence imaging in NOD-SCID mice treated either with LIMKi, Midostaurin or LIMKi+Midostaurin. Results:Expression of LIMK1/2 in MOLM-13 and MV-4-11 cells could be detected by QT-PCR and at the protein level.IC50 after LIMKi exposure was 440 nM in MOLM-13 cells and 420 nM in MV-4-11 cells. Combination experiments with the LIMKi and either the FLT3 inhibitors Midostaurin, Quizartinib, Crenolanib or the hypmethylating agent Azacitidine were synergistic for treatment of MOLM-13 cells. Exposure of MOLM-13 cells to increasing doses of LIMKi induced cell cycle arrest in the G1/S transition and dose dependent apoptosis. No significant toxicity of increasing doses of LIMKi after exposure of CD34+ cells from healthy donors could be detected. In NOD-SCID mice engrafted with MOLM13-LUC cells Midostaurin and LIMKi delayed MOLM13 engraftment as detected by in vivo bioluminescence imaging and LIMKi+Midostaurin prolonged significantly survival of mice as compared to Midostaurin alone. Conclusion: LIMK1/2 inhibition seems to be promising in combination with various FLT3 inhibitors or Azacitidine in vitro as well as in vivo with Midostaurin. Citation Format: Thorsten Braun, Jeannig Berrou, Hanane Djamai, Melanie Dupont, Anna Kaci, Jan Erik Ehlert, Holger Weber, Andre Baruchel, Fabrice Paublant, Renaud Prudent, Claude Gardin, Herve Dombret. Synergy of FLT3 inhibitors and a small molecule inhibitor of LIM kinase1/2 in FLT3-ITD positive acute myeloblastic leukemia (AML) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 341.