Acute myeloid leukemia : apoptotic signalling and gene expression associated with treatment response

Sammanfattning: Acute myeloid leukemia (AML) is a severe, life threatening malignancy characterized by a clonal expansion of immature myeloid cells in the bone marrow, resulting in severe infections and bleedings. High dose chemotherapy is able to normalize the blood and bone marrow morphology (complete remission, CR) in a majority of treated patients, but recurrent disease, typically occurs within 1-2 years. Since further intensification of chemotherapeutic regimens is usually ineffective and accompanied by excess toxicity, novel approaches using better-targeted drugs are now being assessed. We have analysed the effects of one such new agent, gemtuzumab ozogamicin (GO) on AML cells and have also looked for biomarkers of clinical response and the role of multidrug resistance (MDR) expression utilizing biobanked cells from an AML cohort with known long-term therapuetic outcome. In paper I we analysed apoptotic signalling in response to GO, a monoclonal CD33 antibody conjugated to the DNA-double strand break-inducing toxin calicheamicin. The CD33 antigen is typically expressed on AML blast cells, but not on e.g. normal gut cells. We found that GO could induce mitochondrial depolarisation, activation of caspase-3 and decreased viability of primary cells from AML patients and AML cell lines. Moreover, we showed that GO activated the proapoptotic proteins Bak and Bax, regulators of mitochondria-mediated apoptotic signalling. Importantly, none of the above events could be observed in GO-resistant AML cells. In paper II, we looked at the role of caspase-2 in GO- or daunorubicin-induced apoptotic signalling. We noted that both drugs caused cleavage of caspase-2 into its active form. A selective caspase-2 inhibitor prevented GO-induced caspase-3 activation, yet did not influence the activation of Bak and Bax. All in all, our data indicate that both mitochondria-dependent and independent routes to caspase-3 activation are involved in GO-induced apoptotic signalling, findings that may lead to novel future therapeutic approaches for AML. Improved predictive biomarkers for treatment response are clearly needed to enable more personalized and effective therapeutic options in AML. In paper III we studied peripheral blood cells from 42 patients diagnosed with AML and subjected to induction chemotherapy, aiming to identify biomarkers of CR duration using global gene expression analysis (Affymetrix®). Prominent differences in gene expression were found with a remarkable up-regulation of the transcription factor RUNX1T1 in patients with short vs. those with long subsequent CR duration. Network analyses (Oncomine®) revealed multiple transcription factors as interactors to RUNX1T1, out of which TCF3 was also significantly up-regulated in patients with short CR duration. An in silico validation, taking advantage of previously published data from two other independent AML cohorts revealed 52 genes to be regulated in all three cohorts. Among these genes CXCL3, ZMIZ1 and PRDX2 attracted a special interest due to their reported involvement in cancer, leukemia, apoptosis and proliferation. Thus, CXCL3 and ZMIZ1, with known involvement in tumorgenesis, had increased expression in poor responders whereas PRDX2, a tumour suppressor gene, instead showed a decreased expression. In paper IV we investigated the clinical relevance of 380 genes, reported to have a role in multidrug resistance (MDR) and analyzed 11 paired sampled from AML patients, collected at diagnosis and at time of relapse. Unsupervised hierarchical clustering showed that half of the cases had a similar expression pattern at both time points, whereas in the remaining patients the MDR genes became altered, suggesting clonal evolution. Patient-by-patient analyses showed signs of unique individual patient gene signatures and in 10 out of 11 patients an increase of at least one ABC transporter was observed at relapse. These findings call for a more broad signalling analysis of diagnostic and relapse AML blasts in order to improve chemotherapy response and thereby overall survival of the individual AML patient.