Abstract A24: Biomarker analysis and the role of caspase-2 in apoptotic signaling in AML cells.

Abstract

Abstract Acute myeloid leukemia (AML) is characterized by uncontrolled expansion of immature leukocytes. It is the predominant leukemia among adults and is generally treated with high dose chemotherapy. This normally results in an initial normalization of the blood- and bone marrow-cell composition (complete remission; CR). Yet, most patients relapse within 1–2 years. Conventional chemotherapy is also associated with severe and at times lethal side effects. Thus, it would be valuable to find both biomarkers capable of predicting treatment response and novel treatment strategies. Here we address both these issues. Identification of biomarkers to predict CR was analyzed by gene expression analysis of AML patient cells. To further understand the potential of gemtuzumab ozogamicin (GO) as targeted agent for AML, profiling of apoptotic signaling in AML patient derived cells and cell lines was performed. Cellular signaling was compared to the conventional drug Daunorubicin. For the biomarker study, gene expression profiling (Affymetrix U133A) was made on leukemic blast cells isolated from AML patients (n=42) with long (> 6 months).or short (< 6 months) CR time. Candidate genes were validated using real time quantitative PCR in patient cohorts and individual samples. Ingenuity pathway analysis was applied to sort out critical signaling components involved. The molecular studies of GO response were focused on caspase-2. The AML cell line HL60 was treated in vitro with GO or Daunorubicin with or without caspase-2 inhibitor z-VDVAD. Patient derived blasts were treated in vitro with GO or were untreated. Caspase-2 expression and processing was analyzed using western blotting. Caspase-3, Bak/Bax-activation assays and cell cycle profiling was analyzed using FACS. We found major discrepancies in gene expression when comparing AML patients with long or short CR time. RUNX1T1 is frequently translocated with RUNX1 to produce the characteristic fusion gene (t8;21) in AML and is blocking hematopoietic differentiation. Here we found the transcription factor RUNX1T1 (ETO) to be highly over expressed in patients with short CR duration. Examples of other up-regulated genes in patients with short CR were TKTL1, NUDT4 and CHD3. A number of genes that were lower expressed in patients with short CR were ANXA1, FLRT3 and TLR8. We have molecularly dissected the role of caspase-2 in GO-induced apoptotic signaling and compared its mechanisms of action to Daunorubicin. We found processing of caspase-2 in both GO- and Daunorubicin-induced apoptotic signaling in AML cells and after GO-treatment in patient derived leukemic blasts. A critical role of caspase-2 in GO-induced apoptosis was found as the caspase-2 inhibitor z-VDVAD-fmk reduced both caspase-3 activation and apoptotic morphology with about 50% yet having no effect on GO-induced Bak and Bax activation. These results suggest that caspase-2 is located upstream of caspase-3, in this signaling cascade. In order to further understand the clinical relevance of our findings, we are currently analyzing expression levels of caspase-2 and caspase-3 in patient cells. In conclusion, our gene expression profiling suggest RUNX1T1 to be a marker of CR duration in AML and our molecular profiling suggest caspase-2 to have a critical role in CT response of AML. Thus both these analyzes highlight possible CT resistance mechanisms in AML which warrants further studies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A24.