Simultaneous functional and phenotypic flow cytometry (SFPC) to elucidate the genetics of drug resistance in acute
myeloid leukemia (AML) stem cells
Chemotherapy regimens induce complete remission of acute myeloid leukemia (AML) in 60-70%. However, about 80% of these AML patients will ultimately relapse due to persistent leukemic cells (PLC) that remain in the patient. The phenotype of PLC is currently unknown. In the previous funding period, we have therefore characterized stem and progenitor compartments from bone marrow of AML patients at diagnosis, in remission and relapse employing simultaneous functional and descriptive flow cytometry analysis (SFDC). We demonstrate that high activity of functional stem cells parameters such as high aldehyde dehydrogenase and drug transporter function are associated with refractory disease or high relapse rates in our AML patient cohort. In order to track PLC in stem and progenitor cell compartments from AML patients we established a genomic single cell PCR analysis in order to detect nucleophosmin 1 (NPM1) mutations in different cell compartments. Preliminary data indicate, that more NPM1 mutated cells can be detected in progenitor cell compartments compared to the primitive stem cell compartment. Thus, the PLC immunophenotype may not resemble the phenotype of normal hematopoietic stems cells. In the second funding period we will now ask, which progenitor cell compartment from AML remission patients does have the highest NPM1 mutation rate, so that these cells can be selected by SFDC for further genetic analyses to reveal gene signaling cascades that are relevant for survival of PLC in AML patients in vivo.
In order to analyze leukemic progenitor cells in vivo, we have established a murine model of human AML. To this end, we transplant s tem and progenitor cell compartments of primary human AML patients and found engraftment within immunodeficient Rag2-/-γ-/- mice from only 2500 human sorted AML patient cells. After application of leukemia cells (KG1a) Rag2-/-γ-/- mice developed a human AML within 5-6 weeks. These mice were then treated by chemotherapy. Whole genome microarray analysis from human PLC that remained in the mice after in vivo chemotherapy revealed some genes that are known to be involved in cell survival in addition to previously unknown interesting new resistance candidate genes. These genes shall be further validated and studied in the second funding period. Our AML- Rag2-/-γ-/- model is a good platform for human leukemia cells that are lentivirally transduced. AML cells are now being transduced with a lentiviral shRNA library and can be applied to our Rag2-/-γ-/- mice in order to select for resistance-associated genes via bar code screen by in vivo chemotherapy.
Both strategies employing either a murine AML in vivo model or primary patient bone marrow resemble real life scenarios in order to characterize PLC. Uncovering survival signaling cascades in PLC is a prerequisite for the development of novel targeted therapeutic approaches to overcome PLC resistance mechanisms in AML. Final eradication of these persistent leukemic clones would lead to long-lasting cure for many AML patients.
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