The main therapeutic challenge in the treatment of acute myelogenous leukemia (AML) is the refractory behavior of residual leukemic cells. The factors regulating the survival and subsequent clonal expansion of residual leukemic cells causing relapse remain to be elucidated and may be different from those determined in leukemia samples at diagnosis. This project is designed to investigate the dysregulated proliferation and apoptosis of leukemic progenitor cells which leads to resistance and subsequent expansion of the leukemic clone. We will determine the relationship between cell proliferation and cell death in normal and in AML progenitor cells at diagnosis, remission and relapse. To this end, we have developed new methods to determine gene expression (quantitative multiparametric FACS analysis of cellular proteins, and FACS/RT-PCR) and clonality (FACS/FISH) in very rare progenitor and minimal residual disease (MRD) cells. MRD was detectable in all patients studied in the first 4 years of this program project, but the mechanism of survival of residual leukemic cells in the bone marrow is still unknown and sensitive detection methods are necessary to evaluate them. We will characterize the proliferation of immunophenotypically characterized residual leukemic cells so that any differences found between normal diploid and genetically distinct neoplastic cells may later be exploited in the design of new therapeutic approaches. We will study the molecular determinants affecting survival and proliferation within progenitor cell compartments following induction and consolidation chemotherapy. By monitoring residual disease in AML patients, we will quantitate the level of MRD which requires therapeutic intervention. We will then test the effects of ATRA, G-CSF and Flt-3 ligand (already shown to regulate antiapoptotic genes) on chemotherapy- induced apoptosis in vitro and in vivo better. By understanding the differences which exist between leukemic and normal progenitors in their responses to stimuli of proliferation and apoptosis, using concomitant FISH with BUdR incorporation and Annexin V labeling of apoptotic cells, we will find ways to selectively sensitize leukemic cells to apoptosis by chemotherapeutic agents. Relapsed leukemia shows increased resistance to conventional chemotherapy because of the development of multidrug resistance and because residual leukemic cells have perhaps been selected for intrinsic resistance to the factors which normally regulate hematopoiesis. We will therefore determine the expression of the multidrug resistance gene (MDR-1) and the expression of molecules which regulate proliferation and apoptosis (Bcl-2 family proteins) in residual leukemic progenitors and at relapse. The respective contribution of the different mechanisms of resistance (proliferation, apoptosis, MDR) will be tested in appropriate mathematical models with the aim of identifying the key resistance factors and devising methods to sensitize and eradicate residual AML cells.
Showing the most recent 10 out of 422 publications
