Histone Deacetylase Inhibitor Based Therapy of AML

描述（由申请人提供）：需要针对AML生物靶点的新型疗法来进一步改善AML的临床结局。 最近的研究已经阐明，AML通常是在一类赋予增殖和/或生存优势的基因突变或基因重排（例如，激活FLT-3、N-Ras、K-Ras和c-Kit中的突变）与第二类渗出癌基因（例如，AML 1/ETO和TEL/AML 1），其通过募集已知抑制造血祖细胞的造血分化和随后的凋亡的核辅阻遏物/组蛋白脱乙酰酶（HDAC）复合物而作为主要的转录抑制剂。 因此，诱导组蛋白乙酰化、p21 WAF 1表达和促凋亡基因的HDAC抑制剂（HDI）解除对AML细胞分化的阻滞并诱导AML细胞凋亡。 我们实验室的最新研究结果表明，异羟肟酸类似物（HAA）类有效的泛HDAC抑制剂（HDIs）（例如，SAHA、LAQ 824和LBH 589）还诱导热休克蛋白90的乙酰化，这破坏了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，将它们引导至多泛素化和蛋白酶体降解。 因此，HAA-HDI可以破坏融合癌蛋白（招募HDAC）和突变FLT-3之间的协同作用及其在AML中的下游促生长和促存活信号传导。 此外，我们的初步数据表明，HAA HDIs与FLT-3激酶抑制剂PKC 412对携带突变型FLT-3的AML母细胞发挥协同细胞毒性。 基于这些发现，本提案的总体目标是阐明HAA-HDIs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。 目的1：确定突变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介导的人AML细胞（有或无突变FLT-3）对Apo-2L/TRAIL或激动性DR 4和DR 5抗体诱导的细胞凋亡外源性途径的致敏机制。 目标2：为了确定HAA-HDI诱导的hsp 90乙酰化和抑制hsp 90与其客户蛋白的伴侣缔合的机制，例如，AML细胞中的突变体（m）FLT-3。 这些研究还将确定HAA-HDI介导的通过AKT、Raf/MEK/ERK和STAT 5的下游促生长和/或存活信号传导的减弱，以及含有突变体FLT-3的人AML细胞对FLT-3抑制剂的致敏性。 目标3：确定HAA-HDI与hsp 90拮抗剂17-烯丙基氨基-去甲氧基-格尔德霉素（AAG）及其更可溶的类似物17-DMAG组合在减弱人AML细胞中的mFLT-3和下游促生长和促存活信号传导中的体外和体内作用。 拟议的临床前研究可能会定义基于HAA-HDI的新型组合，并产生测试其对人AML细胞的体内疗效的基本原理。

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.