Analysis Of Leukemic Stem Cell Population Comparing NPM1wt and NPM1mut AML Patients and Potential Therapeutic Targets

Abstract

Treatment of acute myeloid leukemia (AML) patients became more effective, yet the relapse rate is still high and cure rate still low. Different therapeutic approaches, based on molecular targeting are offering new treatment strategies using markers like FLT3, NPM1, cKIT and DNMT3A. Many others are currently under investigation. Leukemic stem cells (LSC) might be the source for leukemic disease self-renewal and account for disease relapse after treatment. Therefore LSC represent a critical target for further therapeutic options. AML with mutated nucleophosmin (NPM1) gained attention because of its distinctive molecular, clinical, and prognostic features. NPM1 mutation is also an interesting target for specific immunotherapy (Greiner et al., Blood 2012), however LSC of this new entity have not been completely characterized. Differentially expressed genes might influence molecular and immunological relevant pathways which could have an impact on the better overall survival of the mutated patients.In this study, we were interested in expression differences of the LSC enriched cell fraction descendent of NPM1wt and NPM1mut primary AML patients. We suggest that expression differences between the patients groups could be a factor for the better overall survival of NPM1mut patients. In addition we aimed to study new targets on NPM1mut LSC for new therapeutical purposes.We enriched the CD34+CD38- fraction of primary AML peripheral blood mononuclear cells (PBMC), by Fluorescence-activated cell sorting (FACS) and Magnetic-activated cell sorting (MACS), comparing both methods in efficiency and feasibility. We chose FACS for further enrichment assays, as it resulted in a mean purity of 92% vs 88% using MACS, and a higher cell number after sorting (9% more) in the first pilot trials. We sorted 21 AML patient samples; 12 NPM1wt and 9 NPM1mut. We also enriched healthy donor samples for HSC purification using FACS, in order to compare expression levels. We showed that enriched CD34+CD38- cells in NPM1mut AML samples harbor cytoplasmic NPM1 via immunocytochemical staining, indicating that these cells belong to the leukemic clone. The cell number and RNA quality was sufficient for further Microarray studies in which we analysed the CD34+CD38- enriched compartments descendent from NPM1mut and NPM1wt patients. Those showed significant differences in gene expression patterns which noticeably are immunologically coined, for example: immunoglobulin superfamily, member 10 (p = 0.0003405; fold change: 6.22) and the interleukin 12 receptor, beta 1 (p = 0.000834, fold change: 1.87). This impression was confirmed by pathway analysis indicating deregulation of pathways like the NO2-dependent IL 12 Pathway in NK cells and the Th1/Th2 Differentiation and the Platelet Amyloid Precursor Protein Pathway.Functional assays, confirming the biological importance of these factors have been performed for example an assay to test the effect of IL12 on AML cellines.Furthermore we screened our data for new potential therapeutic target structures, specifically on enriched CD34+CD38- cells of NPM1mut patients, comparing the expression level of target genes to NPM1wt CD34+CD38- cells, and the expression level on HSC. Amongst others, our most promising candidates are SERPINA1 (p = 0.0062344, fold change: 14.32), OSCAR (p = 6.11E-05, fold change: 9.03) and several further interesting genes. These genes could be used in order to target CD34+CD38- cells of NPM1mut patients in a therapeutic manner.Taken together, we successfully enriched the CD34+CD38- fraction of NPM1mut and NPM1wt AML patients and performed Microarray analysis, unraveling gene expression differences between the two patients groups, which seem to be of an immunological nature. This could be a factor, amongst others, for the better overall survival of the NPM1mut patient group. Furthermore we described new potential targets structures on CD34+CD38- cells for NPM1mut patient's treatment. Disclosures:No relevant conflicts of interest to declare.