Histone Deacetylase Inhibitor Based Therapy of AML

描述（由申请人提供）：需要针对AML生物靶点的新疗法来进一步改善AML的临床结果。最近的研究表明，AML通常发生在一类基因突变或基因重排（例如，激活FLT-3， N-Ras， K-Ras和c-Kit的突变）与第二类积液癌基因(例如，AML1/ETO和TEL/AML1)作为转录的主要抑制剂，通过募集核辅阻遏物/组蛋白去乙酰化酶（HDAC）复合物来抑制造血分化和随后的造血祖细胞凋亡。因此，通过诱导组蛋白乙酰化、p21WAF1表达和促凋亡基因的HDAC抑制剂（HDI）可以解除对AML细胞分化的抑制，诱导细胞凋亡。我们实验室最近的研究结果表明，羟肟酸类似物（HAA）类有效的泛hdac抑制剂（hdi）（例如，SAHA、LAQ824和LBH589）也能诱导热休克蛋白90的乙酰化，从而破坏其伴侣蛋白与其客户蛋白（包括突变体FLT-3、c-Raf和AKT）的结合，引导它们进行多泛素化和蛋白酶体降解。因此，在AML中，haa - hdi可以破坏融合癌蛋白（募集hdac）和突变体FLT-3及其下游促生长和促生存信号之间的协同作用。此外，我们的初步数据表明，HAA hdi与FLT-3激酶抑制剂PKC412协同作用，对携带FLT-3的突变AML母细胞具有细胞毒性。基于这些发现，本研究的总体目标是阐明haa - hdi与其他新型和传统抗白血病药物在培养和原代AML细胞中的相互作用和疗效的分子机制。本研究的具体目的是：目的1：确定突变型FLT-3的表达和活性对HAA-HDI诱导的人白血病细胞凋亡的影响，以及确定HAA-HDI介导的有或没有突变型FLT-3的人AML细胞对Apo-2L/TRAIL或激动性DR4和DR5抗体诱导的外源性凋亡通路的致敏机制。目的2：确定haa - hdi诱导hsp90乙酰化的机制，并抑制hsp90与其客户蛋白（如突变体(m) FLT-3）在AML细胞中的伴侣结合。这些研究还将确定haa - hdi通过AKT、Raf/MEK/ERK和STAT5介导的下游促生长和/或存活信号的衰减，以及由此产生的含有人类AML细胞的突变体FLT-3对FLT-3抑制剂的敏化。目的3：确定haa - hdi与hsp90拮抗剂17-烯丙基氨基-去甲氧基-格尔达霉素（AAG）及其更可溶性类似物17-DMAG联合使用对人AML细胞mFLT-3及下游促生长和促存活信号的体外和体内影响。拟议的临床前研究可能有可能确定基于HAA-HDI的新组合，并为测试其对人类AML细胞的体内疗效提供依据。

DESCRIPTION (provided by applicant): Novel therapies directed against biologic targets in AML are needed to further improve the clinical outcome in AML. Recent studies have clarified that AML often results when a class of genetic mutations or gene rearrangements that confer a proliferative/and or survival advantage (e.g., activating mutations in FLT-3, N-Ras, K-Ras, and c-Kit) collaborates with a second class effusion oncogenes (e.g., AML1/ETO and TEL/AML1) that act as the dominant inhibitors of transcription through the recruitment of nuclear corepressors/histone deacetylase (HDAC) complexes known to suppress hemopoietic differentiation and subsequent apoptosis of the hemopoietic progenitor cells. Hence, HDAC inhibitors (HDI), which induce histone acetylation, p21WAF1 expression and proapoptotic genes de-repress the block in differentiation and induce apoptosis of AML cells. Recent findings from our laboratory have demonstrated that hydroxamic acid analogue (HAA) class of potent pan-HDAC inhibitors (HDIs) (e.g., SAHA, LAQ824 and LBH589) also induce acetylation of heat shock protein 90, which disrupts its chaperone binding to its client proteins, including mutant FLT-3, c-Raf and AKT, directing them to polyubiquitylation and proteasomal degradation. Consequently, HAA-HDIs can undermine the synergy between the fusion oncoproteins (recruiting HDACs) and mutant FLT-3 and its downstream pro-growth and pro-survival signaling in AML. Furthermore, our preliminary data indicate that HAA HDIs exert synergistic cytotoxicity with FLT-3 kinase inhibitor PKC412 against mutant FLT-3-harboring AML blasts. Based on these findings, the overall objectives of this proposal are to elucidate the molecular mechanisms of interaction and efficacy of HAA-HDIs combined with other novel and conventional antileukemia agents in the cultured and primary AML cells. The specific aims of this proposal are: AIM 1: To determine the effect of mutant FLT-3 expression and activity on HAA-HDI-induced apoptosis of human leukemia cells, as well as to determine the mechanism involved in HAA-HDI mediated sensitization of human AML cells with or without mutant FLT-3 to the extrinsic pathway of apoptosis induced by Apo-2L/TRAIL or the agonistic DR4 and DR5 antibodies. AIM 2: To determine the mechanism of HAA-HDI-induced hsp90 acetylation and inhibition of the chaperone association of hsp90 with its client proteins, e.g., mutant (m) FLT-3 in AML cells. These studies will also determine HAA-HDI-mediated attenuation of the downstream pro-growth and/or survival signaling through AKT, Raf/MEK/ERK and STAT5, and the resulting sensitization of mutant FLT-3 containing human AML cells to the inhibitors of FLT-3. AIM 3: To determine the in vitro and in vivo effects of combining HAA-HDIs with hsp90 antagonist 17-allylamino-demethoxy-geldanamycin (AAG) and its more soluble analogue 17-DMAG in attenuating mFLT-3 and the downstream pro-growth and pro-survival signaling in human AML cells. The proposed pre-clinical studies could potentially define HAA-HDI based novel combinations and generate the rationale to test their in vivo efficacy against human AML cells.