Targeting FL3 and SRC kinases for AML therapy

摘要 大约三分之一的急性髓性白血病（AML）患者体内存在一种 FLT 3基因的串联重复（FLT 3-ITD），临床上与FLT 3基因突变率增加相关。 标准治疗后复发。因此，靶向小分子如quizartinib， 诱导AML细胞分化的2型FLT 3抑制剂，作为一种通过 可有效治疗复发的FLT 3-ITD+ AML。不幸的是， 这些患者的quizartinib被发现是12.1周，由于获得继发性突变 在FLT 3基因的D835位点。开发了FLT 3-ITD的第二种1型抑制剂米多替林， 诺华，作为单一药物未能诱导完全缓解（CR），当骨髓 分析后，米多鲁肽治疗组和安慰剂组之间没有显著差异 组然而，联合化疗，CR率为59%，CR率为54% 是由化疗引起的米多替林，与柔红霉素和阿糖胞苷联合， 与安慰剂相比，将AML相关死亡风险降低22%，并获得FDA批准 用于治疗AML。为了解释米多替林作为单一药物缺乏疗效， 提出AML细胞通常过表达SRC家族激酶，它们的高表达可能 提供生存优势。这一理论得到了研究的进一步支持，研究表明， 米多替尼和达沙替尼（一种SRC抑制剂）联合使用在杀死FLT 3-ITD+ 细胞系（体外）比单一药剂疗法。这些观察表明，有必要制定 其他FLT 3抑制剂可诱导携带突变型AML患者完全缓解 FLT 3.为了解决这一医疗需求，我们开发了一种抑制FLT 3和SRC家族的化合物， 激酶（150030）。150030是一种I型抑制剂，可抑制FLT 3-ITD以及D835突变形式。 细胞活力测定显示，150030诱导FLT 3突变AML细胞凋亡，而保留了FLT 3突变AML细胞的细胞活力。 AML伴WT-FLT 3。150030具有口服生物利用度，表现出优异的药理学特征， 在裸鼠异种移植试验中诱导肿瘤完全消退。拟议的研究旨在 进一步评价150030的作用机制和治疗潜力， 这种化合物进行临床试验目的：（1）比较米多塞林和150030对大鼠心肌细胞凋亡的影响。 共表达FLT 3-ITD和SRC小鼠骨髓细胞系32 Dcl 3的生长和分化 家族激酶，并确定这两种化合物对同基因白血病进展的影响。 （2）比较150030和米多替林在临床相关PDX模型中的疗效 制定单一药物和联合治疗方案;（3）确定 与150030相关的耐药机制。

Abstract Approximately one third of patients suffering from acute myeloid leukemia (AML) harbor an internal tandem duplication in the FLT3 gene (FLT3-ITD), which is clinically associated with an increased rate of relapse in response to standard therapies. As a result, targeted small molecules such as quizartinib, a type 2 FLT3 inhibitor that induces differentiation of AML cells, have garnered attention as a means by which relapsed FLT3-ITD+ AML can be effectively treated. Unfortunately, the median duration response of these patients to quizartinib was found to be 12.1 weeks due to the acquisition of secondary mutations at the D835 locus in the FLT3 gene. A second type 1 inhibitor of FLT3-ITD, midostaurin, was developed by Novartis, which, as a single agent failed to induce complete remission (CR) and when bone marrow was analyzed, there was no significant difference seen between the midostaurin-treated and placebo groups. However, in combination with chemotherapy, the CR rate was 59% compared to 54% CR induced by chemotherapy alone. Midostaurin, in combination with daunorubicin and cytarabine, decreased the risk of AML-related death by 22% compared with placebo and was approved by the FDA for the treatment of AML. To explain the lack of efficacy of midostaurin as a single agent, it has been proposed that AML cells often overexpress SRC family kinases and their elevated expression may provide a survival advantage. This theory was further supported by studies which showed that combination of midostaurin and dasatinib (a SRC inhibitor) were more effective in killing FLT3-ITD+ cell lines (in vitro) than single agent therapy. These observations suggest that there is a need to develop additional FLT3 inhibitors which can induce complete remission in AML patients harboring mutant FLT3. To address this medical need, we developed a compound that inhibits both FLT3 and SRC family kinases (150030). 150030 is a Type I inhibitor that inhibits the FLT3-ITD as well as the D835 mutant forms. Cell viability assays showed that 150030 induced apoptosis of FLT3-mutant AML cells while sparing AMLs with WT-FLT3. 150030 is orally bioavailable, exhibited excellent pharmacological profile and induced complete regression of tumors in nude mouse xenograft assays. The studies proposed are aimed at further evaluating the mechanism of action and therapeutic potential of 150030 in an effort to advance this compound to clinical trials. The aims are: (1) To compare the effects of midostaurin and 150030 on the growth and differentiation of murine myeloid cell line, 32Dcl3, co-expressing FLT3-ITD and SRC family kinases and determine the effects of these two compounds on leukemia progression in syngeneic animal models; (2) To compare the efficacy of 150030 and midostaurin in clinically relevant PDX models of AML to develop protocols for a single agent and combination therapy; and (3) To determine the resistance mechanisms associated with 150030.