Djamai Hanane, Berrou Jeannig, Dupont Mélanie, Kaci Anna, Ehlert Jan Erik, Weber Holger, Baruchel André, Paublant Fabrice, Prudent Renaud, Gardin Claude, Dombret Hervé, Braun Thorsten
Laboratoire de Transfert des Leucémies, EA3518, Institut de Recherche Saint Louis, University of Paris, Paris, France.
Reaction Biology GmbH, Freiburg, Germany.
Leuk Res. 2021 Jan;100:106490. doi: 10.1016/j.leukres.2020.106490. Epub 2020 Dec 13.
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.
伴有FLT3内部串联重复突变（FLT3-ITD+）的急性髓系白血病（AML）患者常出现疾病复发或难治，且FLT3-ITD+抑制剂疗效有限。在AML中，Rho激酶（ROCK）通过PI3激酶和Rho GTP酶被FLT3-ITD+持续激活。ROCK激活后，LIM激酶（LIMK）通过磷酸化使丝切蛋白失活，这会影响细胞骨架动力学、细胞生长和凋亡。LIMK抑制通过去磷酸化导致丝切蛋白激活，而激活的丝切蛋白定位于线粒体诱导凋亡。因此，我们研究了LIMK1/2抑制剂CEL_Amide（LIMKi）在FLT3-ITD+ AML中的治疗潜力。通过RT-qPCR和蛋白水平检测，可在FLT3-ITD+细胞系MOLM-13和MV-4-11细胞中检测到LIMK1/2的表达。LIMKi单药治疗后，在MOLM-13细胞中的IC50为440 nM，在MV4-11细胞中为420 nM。LIMKi处理可降低FLT3-ITD+细胞中LIMK1蛋白水平并抑制丝切蛋白的失活磷酸化。LIMKi与包括米哚妥林、克伦诺尼和吉列替尼在内的FLT3抑制剂联合实验对MOLM-13细胞的治疗具有协同作用，而与quizartinib联合则具有相加作用。LIMKi与低甲基化剂阿扎胞苷或ROCK抑制剂法舒地尔联合具有相加作用。在植入MOLM13-LUC细胞的NOD-SCID小鼠中，通过体内生物发光成像检测发现，FLT3抑制剂米哚妥林和LIMKi可延迟MOLM13-LUC细胞的植入，且LIMKi与米哚妥林联合可显著延长白血病小鼠的生存期。小分子CEL_Amide抑制LIMK1/2似乎在体外和体内与FLT3抑制剂联合时具有有前景的活性，可能构成FLT3-ITD+ AML的一种新治疗策略。
