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.
