Histone Deacetylase Inhibitor Based Therapy of AML

描述(申请人提供)：需要针对急性髓细胞白血病生物靶点的新疗法来进一步改善急性髓细胞白血病的临床结果。最近的研究阐明，AML通常是当一类具有增殖和/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的突变)与第二类渗出性癌基因(如AML1/ETO和TEL/AML1)协同作用时产生的，后者通过募集已知的抑制造血祖细胞造血分化和随后的凋亡的核转录抑制因子/组蛋白脱乙酰酶(HDAC)复合体而发挥主要的转录抑制作用。因此，诱导组蛋白乙酰化、p21WAF1表达和促凋亡基因的HDAC抑制物(HDI)解除了AML细胞分化的阻断，诱导了AML细胞的凋亡。我们实验室最近的研究结果表明，异羟肟酸类似物(HAA)类有效的PAN-HDAC抑制剂(如SAHA、LAQ824和LBH589)也可以诱导热休克蛋白90的乙酰化，破坏其与其客户蛋白(包括突变的Flt-3、c-Raf和AKT)的伴侣结合，导致它们发生多泛素化和蛋白酶体降解。因此，HAA-HDI可以破坏AML中融合癌蛋白(招募HDAC)和突变的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对含有突变Flt-3的AML细胞具有协同细胞毒作用。基于这些发现，这项建议的总体目标是阐明HAA-HDIs与其他新型和传统的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：研究突变型Flt-3的表达和活性对HAA-HDI诱导人白血病细胞凋亡的影响，以及HAA-HDI增敏有无突变型Flt-3的AML细胞对Apo-2L/TRAIL或激动型DR4和DR5抗体诱导的外源性细胞凋亡的作用机制。目的：探讨HAA-HDI诱导急性髓系白血病细胞HSP90乙酰化及抑制HSP90与其客户蛋白(M)flt-3伴侣结合的机制。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/或生存信号的减弱，以及由此导致的含有突变的Flt-3的人AML细胞对Flt-3抑制剂的敏感性。目的：研究HAA-HDIs与HSP90拮抗剂17-烯丙氨基-去甲氧基格尔达那霉素(AAG)及其可溶性类似物17-DMAG联用抑制mFlt-3及其下游促生长和促生存信号通路的体内外效应。拟议的临床前研究可能会潜在地定义基于HAA-HDI的新组合，并产生测试其对人类AML细胞体内疗效的理论基础。

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.