PO-299 In vivo shRNA screening to identify quiescence-related genes required for AML growth

Introduction AML is hierarchically organised with at the apex Leukaemia Stem Cells (LSCs), a rare cell population able to initiate and sustain the tumour growth. LSCs share many functional properties with normal Hematopoietic Stem Cells (HSCs) including self-renewal capacity and quiescence. Quiescent LSCs can survive to radiation and chemotherapy acting as a reservoir for leukaemia relapse, the major cause of death for AML patients. Therefore, LSCs quiescence is critical for leukaemia maintenance and few evidences suggest that quiescence regulation in pre-leukemic phase plays a pivotal role for leukemogenic process as well. Material and methods We analysed the transcriptional deregulations induced by the expression of different leukemic oncogenes in HSCs and we examined the contribution of representative quiescence related genes in AML growth by in vivo RNA interference screening. Results and discussions The transcriptional profile of oncogene-expressing HSCs is enriched in a quiescent stem cell gene signature, compared to normal HSCs. Therefore, we hypothesised that enhancement of the quiescent phenotype in HSCs could be a shared mechanism for leukaemia development and maintenance. The in vivo shRNA screening allowed the identification of genes whose silencing in AML blasts was sufficient to significantly decrease in vitro self-renewal and delay leukaemia growth in vivo . Conclusion We identified quiescence-related genes, commonly deregulated by leukemic oncogenes at pre-leukemic level, which may offer new therapeutic targets in a wide group of AML patients

PO-272 Leukemia-associated NPM mutations promote quiescence of hematopoietic stem cells and prevent their functional exhaustion upon oncogene-induced hyper-proliferation

Introduction Acute Myeloid Leukaemia (AML) is a heterogeneous and multi-step disease. The serial acquisition of mutations and the environmental pressure allow one or more clones to expand and contribute to the disease. In particular, 6% of AMLs are characterised by an initial mutation in the DNMT3a gene, followed by mutations in NPM (NPMc) and FLT3 loci (FLT3-ITD). We previously shown that NPMc can drive AML development in mouse model and highly cooperates with FLT3-ITD. Moreover, it has been reported that normal Hematopoietic Stem Cells (HSCs) of elderly people may bear some somatic early AML mutations and this correlate with an increased risk of hematologic diseases suggesting that mutations can shape pre-leukemic HSCs to be more prone to the acquisition of further mutations giving rise to Leukaemia Initiating Cells (LIC). While the ability of FLT3-ITD to drive HSC compartment exhaustion has been already shown, the impact of NPMc on HSCs remains unclear. Material and methods Taking advantage of the extended pre-leukemic phase of our inducible NPMc mouse model, we elucidate the role of NPMc in HSCs by functional and transcriptional analysis. Moreover, to investigate the basis of NPMc and FLT3-ITD cooperation we generate mice carrying both the conditional NPMc transgene and the FLT3-ITD constitutive mutation and, before AML onset, we analyse double mutant HSCs behaviour. Results and discussions We have found that NPMc expression lead to the expansion of the HSC compartment through the enforcement of a stem-cell transcriptional program that increases self-renewal by promoting quiescence. We then investigated how the NPMc dependent quiescence program is linked to its oncogenic function. The expression of NPMc +in the FLT3-ITD background prevents the HSCs exhaustion imposed by FLT3-ITD and restores their repopulating capacity. Accordingly, gene expression analysis revealed a strong dominance of NPMc +with the restoration of the same transcriptional program observed in the NPMc HSCs. These data strongly suggest that NPMc imposes a HSC-specific program that, in combination with the oncogenic signal provided by FLT3-ITD, allows the selection of the LIC and the occurrence of AML. Conclusion In conclusion, enforcement of quiescence might be a critical function for the maintenance of the transformed clone during both the pre-leukemic and the leukemic phase. As consequence, interfering with quiescence key determinants may eradicate the reservoir of quiescent cells responsible for disease recurrence