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Patients with FLT3-ITD mutated (FLT3-ITD+) Acute Myeloid Leukemia (AML), have frequently relapsed or refractory disease and FLT3-ITD+ inhibitors have limited efficacy. Rho kinases (ROCK) are constitutively activated by FLT3-ITD+ in AML via PI3 kinase and Rho GTPase. Upon activation by ROCK, LIM kinases (LIMK) inactivate cofilin by phosphorylation which affects cytoskeleton dynamics, cell growth and apoptosis. LIMK inhibition leads to cofilin activation via dephosphorylation and activated cofilin localizes to mitochondria inducing apoptosis. Thus, we investigated the therapeutic potential of the LIMK1/2 inhibitor CEL_Amide (LIMKi) in FLT3-ITD+ AML. Expression of LIMK1/2 in FLT3-ITD+ cell lines MOLM-13 and MV-4-11 cells could be detected by RT-qPCR and at the protein level. IC50 after LIMKi monotherapy was 440 nM in MOLM-13 cells and 420 nM in MV4-11 cells. Treatment with LIMKi decreased LIMK1 protein levels and repression of inactivating phosphorylation of cofilin in FLT3-ITD+ cells. Combination experiments with LIMKi and FLT3 inhibitors including midostaurin, crenolanib and gilteritinib were synergistic for treatment of MOLM-13 cells while combinations with quizartinib were additive. Combinations of LIMKi and the hypomethylating agent azacitidine or the ROCK inhibitor fasudil were additive. In NOD-SCID mice engrafted with MOLM13-LUC cells, the FLT3 inhibitor midostaurin and LIMKi delayed MOLM13-LUC engraftment as detected by in vivo bioluminescence imaging and the LIMKi and midostaurin combination prolonged significantly survival of leukemic mice. LIMK1/2 inhibition by the small molecule CEL_Amide seems to have promising activity in combination with FLT3 inhibitors in vitro as well as in vivo and may constitute a novel treatment strategy for FLT3-ITD+ 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, Mélanie Dupont, Anna Kaci, Jan Erik Ehlert, Holger Weber, André Baruchel, Fabrice Paublant, Renaud Prudent, Claude Gardin, Hervé 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.

Introduction: LIM kinases 1 and 2 (LIMK1/2) are downstream effectors at the crossroads of different signaling pathways implicated in the dynamics of the cytoskeleton via phosphorylation of cofilin family proteins, degradation of the matrix by phosphorylating MT1-MMP and control of the activity of Aurora kinase A. Recently, the oncogenic role of Rho kinases (ROCK) was identified to be constitutively activated by BCR-ABL1, FLT3-ITD and KIT in hematologic malignancies via PI3 kinase and Rho GTPase mediated phosphorylation. Upon activation, ROCK phosphorylates LIMK1/2 leading to inactivation of cofilin by its phosphorylation and polymerization of actin and microtubules and possibly to other biological effects mediated by LIMK1/2, not yet fully understood. Here, we demonstrate synergy of a LIMK1/2 inhibitor with BCR-ABL1 tyrosine kinase inhibitors (TKI) in vitro and in vivo in different models for BCR-ABL1 driven ALL. Materials and Methods: Expression of LIMK1/2 was determined by RT-qPCR and WB in cell lines. Phosphorylation of cofilin was detected by WB. A small molecule inhibitor of LIMK1/2 was tested alone and in combination with imatinib, dasatinib, nilotinib and ponatinib in BCR-ABL1 positive ALL cell lines TOM-1 and BV-173. Cell viability and IC50 was assessed by MTS assays after exposure to LIMK1/2 inhibitor for 72h. In combination experiments, compounds were added simultaneously and relative cell numbers were determined at 72h with MTS assays and combination index (CI) values were calculated according to the Chou-Talalay model. Cell-cycle distribution was determined by cytofluorometric analysis detecting nuclear propidium iodide (PI) DNA intercalation. Induction of apoptosis was evaluated by annexin-V exposure and PI incorporation at 72h with increasing doses of LIMK1/2 inhibitor. Peripheral blood (PB) nucleated cells from apharesis products of healthy donors obtained after informed consent according to Helsinki declaration were incubated with or without LIMK1/2 inhibitor for 72h, and then enriched for CD34+ cells by immuno-magnetic selection and seeded in triplicate in methylcellulose FCS and cytokines. In vivo experiments were performed in C57Bl/6 mice injected with BCR-ABL-induced B-ALL cells. These were obtained by transduction of CDKN2A-deficient B-cell progenitors with a retrovirus coding for BCR-ABL1 (P185) and GFP, followed by transplantation in sub-lethally-irradiated recipient C57Bl/6 mice. Mice were treated either with LIMK1/2 inhibitor, nilotinib or the combination of both and compared to untreated control mice. Results: Expression of the two isoforms LIMK1 and LIMK2 in TOM-1 and BV-173 cells could be detected by RT-qPCR and at the protein level by WB. IC50 after LIMK1/2 inhibitor exposure alone was 580nM in TOM-1 cells and 1000nM in BV-173 cells. All combination experiments with the LIMK1/2 inhibitor and imatinib, dasatinib, nilotinib and ponatinib yielded synergistic CI for treatment of both TOM-1 and BV-173 cell lines. Cell cycle arrest in the G1/S transition was detected and LIMK1/2 inhibition induced dose dependent apoptosis in TOM-1 and BV-173 cells up to 40% at doses 1% GFP+ cells were detected in PB. Furthermore, nilotinib+LIMK1/2 inhibitor prolonged significantly the survival of mice compared to either nilotinib (p=0.0006) or LIMK1/2 inhibitor alone and untreated mice (p Conclusion: Combination of LIMK1/2 inhibitor with BCR-ABL targeting TKI is synergistic and has significant anti-leukemic activity in BCR-ABL1+ ALL in vitro and in vivo models. Disclosures Braun: CELLIPSE: Research Funding. Prudent:CELLIPSE: Employment. Paublant:CELLIPSE: Employment. Baruchel:Jazz Pharmaceuticals: Consultancy, Honoraria, Other: Travel, accommodations or expenses; Shire: Research Funding; Servier: Consultancy; Amgen: Consultancy; Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy; Roche: Consultancy. Dombret:CELLIPSE: Research Funding.

Acute Myeloid leukemia (AML) has a poor prognosis in part due to lack of elimination of leukemic cells and acquisition of drug resistance. In particular mutations in the activation loop of KIT and internal tandem duplication (ITD) in Flt3 receptor are associated with poor prognosis in AML. Interestingly, more than 90% patients with aggressive systemic mastocytosis (ASM) carry the KITD816V mutation which is highly resistant to conventional therapeutics and invariably associated with poor survival. Since the efforts to target the kinase activity of FLT3 and KIT have met with little success; the focus has shifted to targeting critical downstream signaling regulators. We have elucidated the role of a novel downstream regulator of FLT3-ITD and KITD816V mediated leukemic transformation - LIM kinase and evaluated the efficacy of a novel inhibitor of LIM kinase (LIMK), Liminib. LIMK, a serine/threonine kinase has 2 isoforms; LIMK1 and LIMK2. They are substrates of the Rho associated kinase (ROCK) which we have shown to play a critical role in leukemic transformation (Cancer Cell, 2011). They in turn act on cofilin and phosphorylation by LIMK inactivates cofilin and thereby modulates F-actin reorganization. Western blot analysis showed marked increase in phospho cofilin levels in cells expressing FLT3-ITD or KITD814V oncogenes as compared to their WT counterparts. Dependence of cofilin phosphorylation on activation of ROCK and LIMK was confirmed by treating the cells with an inhibitor of ROCK, H1152 and Liminib, inhibitor of LIMK. Both the inhibitors effectively reduced oncogene-induced phospho-cofilin levels. Liminib, the LIMK inhibitor also inhibited cytokine independent proliferation driven by FLT3-ITD or KITD814V (>90% at 100 nM). It also showed concentration dependent inhibition of proliferation in mutant oncogene bearing patient derived human cell lines such as MV411, Kasumi and HMC. FLT3-ITD expressing leukemic cells become resistant to Flt3 kinase inhibitor AC220 by acquiring additional point mutations. Interestingly, the cells expressing AC220 resistant FLT3-ITDD835F or FLT3-ITDF691L mutation showed similar sensitivity to Liminib as cells with FLT3-ITD. The growth inhibitory effect of Liminib was also validated in primary blasts from AML and SM patients. To further elucidate the mechanism of growth inhibition by Liminib in leukemic cells, we assayed for apoptosis induction in the treated cells. Cells expressing FLT3-ITD were significantly more susceptible (~ 73%) to induction of apoptosis as compared the WT-FLT3 cells (~33%) on treatment with Liminib. The extent of apoptosis induction by Liminib was similar in FLT3-ITD and AC220 resistant FLT3-ITDD835F or FLT3-ITDF691L mutation bearing cells. In addition to the effects of Liminib on F-actin polymerization; experimental evidence indicates involvement of mitochondrial pathway in the induction of apoptosis by Liminib. There was increase in accumulation of cofilin in the mitochondrial fraction in Liminib treated oncogene expressing cells as compared to the untreated cells. Phosphorylation of cofilin on Ser3 suppresses its mitochondrial translocation and treatment with LIMK inhibitor would inhibit cofilin phosphorylation leading to increased mitochondrial transport. Mitochondrial transport of cofilin has been observed with other inducers of apoptosis such as staurosporine and it possibly contributes to mitochondrial dysfunction during induction of apoptosis. To validate our pharmacologic findings using Liminib genetically and to assess which of the 2 isoforms of LIMK contributes to leukemogenesis, we generated LIMK1 and LIMK2 single and double knockout mice. 3 H-thymidine incorporation assay showed that deletion of either of the LIMK significantly reduced KIT D814V induced growth factor independent proliferation by approximately 50%. The extent of inhibition was more pronounced in Limk1-/- Limk2+/- or Limk1+/- Limk2-/- bone marrow cells (~80%) as compared to cells with deletion of a single isoforms of LIMK. Thus, there is some functional redundancy between the 2 isoforms and inhibition of both is likely to have better therapeutic value. Taken together we have identified a novel signaling pathway involving LIMK and cofilin that appear to be selectively activated in regulating oncogenic signaling in AML and MPN but not in normal hematopoiesis. Disclosures No relevant conflicts of interest to declare.