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MicroRNAs (miRs) are involved in many aspects of normal and malignant hematopoiesis, including hematopoietic stem cell (HSC) self-renewal, proliferation, and terminal differentiation. However, a role for miRs in the generation of the earliest stages of lineage committed progenitors from HSCs has not been identified. Using Dicer inactivation, we show that the miR complex is not only essential for HSC maintenance but is specifically required for their erythroid programming and subsequent generation of committed erythroid progenitors. In bipotent pre-MegEs, loss of Dicer up-regulated transcription factors preferentially expressed in megakaryocyte progenitors (Gata2 and Zfpm1) and decreased expression of the erythroid-specific Klf1 transcription factor. These results show a specific requirement for Dicer in acquisition of erythroid lineage programming and potential in HSCs and their subsequent erythroid lineage differentiation, and in particular indicate a role for the miR complex in achieving proper balance of lineage-specific transcriptional regulators necessary for HSC multilineage potential to be maintained.

Abstract 1585 Recent studies utilising surrogate leukaemic stem cell (LSC) assays have suggested that LSCs in acute lymphoblastic leukaemias (ALLs) might be neither rare, nor phenotypically or functionally distinct. However, studies of candidate LSCs in surrogate assays might not recapitulate the full leukaemic potential of candidate LSCs in patients, and in particular their responsiveness and resistance to therapeutic targeting. Therefore, we have investigated the identity, molecular and functional properties, and persistence of different subsets of candidate LSCs in childhood ALL, at diagnosis and during the course of clinical and molecular remissions in response to chemotherapy, and their relationship to subsequent relapses. First, we investigated 6 patients diagnosed with “good prognosis” TEL-AML1+ ALL, and at diagnosis we found TEL-AML1+ leukaemic cells within the immature B cell progenitor compartment (proB: 34+38+19+), mature B-cells (34-19+), as well as in a population expressing an aberrant combination of stem cell (34+38-/lo) and B-cell (19+) cell surface markers. These stem/B (34+38-/lo19+) cells were all TEL-AML1+ and not present in age-matched normal bone marrow controls. In contrast, haematopoietic stem cells (HSC: 34+38-19-) were not part of the TEL-AML1+ leukaemic clone in any of the patients. 15 days into chemotherapy, all TEL-AML1+ mature B-cells were eliminated in all patients, and this was followed by a clearance of leukaemic proB cells by day 28 of treatment. In striking contrast, leukaemic stem/B cells were still detectable at day 28, but in all TEL-AML1 patients, at later stages all leukaemic cells including the stem/B cells were undetectable, and at the same time these patients went into complete remission with less than 1 leukaemic cell in 10e4 cells detectable. A similar pattern was observed in a case of “high risk” BCR-ABL+ ALL: BCR-ABL+ proB and B-cells were efficiently eliminated by day 90 of the course of chemotherapy, and up to 180 days into the treatment only 34+38-/lo19+ stem/B cells remained part of the BCR-ABL+ clone. In agreement with the persistence of BCR-ABL+ 34+38-/lo19+ stem/B cells, this patient relapsed 17 months after the initiation of chemotherapy. In order to understand the underlying mechanisms of the observed functional and therapeutic heterogeneity seen in leukaemic subpopulations, we performed comparative gene-expression analysis of diagnostic leukaemic stem/B and proB cells of TEL-AML1+ patients. This analysis revealed a differential gene expression pattern between leukaemic stem/B and proB cells, with positive regulators of cell cycle being the most distinctly up regulated genes in leukaemic proB cells. In agreement with this, cell cycle analysis of 3 diagnostic TEL-AML1+ cases also showed proB cells to be more actively cycling compared to the more quiescent state of the leukaemic stem/B compartment (proB: G0 42%; G1 40%; S,G2,M 18% vs. stem/B: G0 81%; G1 18%; S,G2,M 1%), providing a potential mechanistic basis for the relative therapy resistance of ALL stem/B cells. Taken together the present studies suggest that quiescent 34+38-/lo19+ stem/B cells are selectively resistant to chemotherapy, and most likely the origin of relapses when these occur in childhood ALL. Disclosures: No relevant conflicts of interest to declare.

Background: T-cell activity is regulated by immune checkpoints to maintain the sensitive balance of co-stimulatory and inhibitory immune signals. Therapeutic blockade of checkpoint molecules on tumor or T cells such as CTLA-4 or PD-L1 has shown clinical success in several tumor types including Hodgkin´s disease. Furthermore, Blinatumomab, a bispecific T-cell engager (BiTE antibody construct) directing cytotoxic T cells to CD19 positive leukemic cells has been approved for treatment of acute lymphoblastic leukemia. The CD33 specific BiTE antibody construct AMG 330 has been developed for the therapy of acute myeloid leukemia (AML) and will be evaluated in phase I studies shortly. In our current study, we investigated the therapeutic utility of blockade of the novel checkpoint proteins PVR (poliovirus receptor) and PVRL2 (poliovirus receptor-related 2) alone and in combination with AMG 330 in AML. Methods and results: Samples from 140 treatment naive patients with newly diagnosed AML (AMLSG 07-04, NCT00151242) were analyzed by RT-qPCR for expression of the immune checkpoint molecules PVR, PVRL2 and Galectin-9 (Gal-9). Expression was correlated with patient demographics (age, karyotype, FLT3 mutation status) and clinical survival data by multivariate cox regression. The majority of patients showed mRNA expression of PVR (94%), PVRL2 (95%) and Gal-9 (92%). In a multivariate stepwise cox regression for overall survival, an unfavorable karyotype, high PVR and high Gal-9 expression were identified as independent prognostic markers (p In a second, independent patient cohort containing microarray-based gene expression and clinical data of 291 AML patients (Verhaak et.al., Haematologica 2009;94) a high PVR and PVRL2 expression in contrast to expression of CD80, CD86 or PD-L1 was associated with poor overall survival (log-rank test p=0.003 and p=0.032, respectively). In in vitro killing assays the therapeutic effect of PVR and PVRL2 blockade was studied by FACS using 7-AAD staining. AML cell lines MV4-11, Kasumi-1 and Molm-13 were preincubated with blocking antibodies against PVR, PVRL2 or both and co-cultured for 24h with peripheral blood mononuclear cells (PBMCs) of healthy donors in the presence or absence of AMG 330. In the absence of AMG 330, the cell kill of MV4-11 increased from 12.6±4.7% (control) to 33.0±8.8% (PVR), to 40.4±10.4% (PVRL2) and to 56.0±12.0% (both PVR + PVRL2). In the presence of suboptimal concentration of AMG 330 (0.1 ng/ml) MV4-11 cell lysis was 29.4±9.0% (AMG 330 alone), 49.7±12.6% (AMG 330 + PVR), 57.9±11.3% (AMG 330 + PVRL2) and 70.0±9.8% (AMG 330 + PVR + PVRL2; n=4, p Conclusion: The expression of immune checkpoint ligands PVR and PVRL2 confers a negative prognosis to AML patients possibly due to immune evasion. We could further show that the killing of AML cells by PBMCs could be augmented by blockade of these novel checkpoint inhibitors. Furthermore, addition of PVR and/or PVRL2 blocking antibodies to AMG 330 could enhance cytotoxicity. Therefore, blockade of PVR and PVRL2 represents a promising target for the treatment of AML. Disclosures Kischel: Amgen Research (Munich) GmbH: Employment. Stienen:Amgen Research (Munich) GmbH: Employment. Friedrich:Amgen Research (Munich) GmbH: Employment. Lutteropp:Amgen Research (Munich) GmbH: Employment. Nagorsen:Amgen: Employment, Equity Ownership, Patents & Royalties: Inventor on blinatumomab-related patent.

Abstract 1380 Although the growth factor receptor (GFR) FLT3 has a crucial role in normal early B- and T-lymphoid development, constitutively activating internal tandem duplications (ITDs) of FLT3 are almost entirely restricted to patients with adverse-risk acute myeloid leukemia. We used a murine knock-in model of FLT3-ITD myeloproliferative disease (MPD) to gain a better understanding of the cellular and molecular basis for the myeloid-bias of FLT3-ITD-induced hematological malignancies. As Flt3 cell surface expression is lacking in homozygous Flt3-itd mice, we used CD48 and CD150 expression to investigate the distribution of multipotent progenitors (MPPs) and hematopoietic stem cells (HSCs) within the primitive Lin-Sca1+Kit+ (LSK) compartment. Notably, phenotypic (LSKCD150+CD48-) and functional HSCs were markedly reduced in adult Flt3-itd mice. Competitive transplantation experiments using fetal liver confirmed that HSC numbers were reduced (20-fold reduction) by Flt3-ITDs, in a cell-extrinsic manner. Rather, LSKCD48+150- cells (MPPs) were expanded 2.7-fold in Flt3-itd mice comprising >90% of LSK cells. Similarly to Flt3high wild type (WT) lymphoid-primed multipotent progenitors (LMPPs), nanofluidic gene-expression analysis demonstrated that WT MPPs and Flt3-itd MPPs were myeloid-primed (Csf1r, Csf2r, Cebpa, Mpo) with loss of megakaryocyte and erythroid (MkE) priming (Eklf, Epor, Vwf, Gata1). In contrast, the lymphoid (Il7r, Rag1, sIgH) transcriptional priming of WT MPPs was downregulated in Flt3-itd MPPs. In agreement with this, Flt3-itd MPPs sustained extensive GM potential in vitro, with no MkE potential and, unlike WT MPPs, considerably reduced lymphoid potential. Furthermore, microarray analysis demonstrated global upregulation of the myeloid program in Flt3-itd MPPs. These findings demonstrate that primitive lympho-myeloid MPPs, are expanded and biased towards myeloid development by Flt3-ITDs. In agreement with reduced lymphoid-priming of Flt3-itd MPPs, analysis of early thymic development demonstrated a 10-fold reduction of early thymic progenitors (DN1 Kit+) in Flt3-itd mice. Subsequent stages of thymic development were also reduced, as was overall thymic cellularity. Interestingly, expression of the chemokine receptor CCR9 was 5.5-fold reduced in Flt3-itd MPPs suggesting that thymic seeding progenitors in the bone marrow are suppressed by FLT3-ITDs. Previous studies have suggested that the earliest stage of B-cell development, pre-pro-B cells, retain both B-cell and myeloid potential. Lin-CD19-CD24-AA4.1+CD43+B220+ pre-pro-B cells were expanded 13.7-fold in Flt3-itd mice, whereas subsequent stages of CD19+ B-lymphopoiesis were all reduced. The expanded pre-pro-B cells in Flt3-itd mice were myeloid biased at the transcriptional level with markedly reduced expression of lymphoid genes. Pu1 is a master-regulator of myeloid commitment in early hematopoiesis and a STAT3 target gene. As FLT3-ITDs are known to activate STAT3, unlike WT FLT3, we therefore investigated Pu1 expression in Flt3-itd mice using a Pu1-YFP reporter. Expression of Pu1 was significantly increased in LSK cells (1.4 fold) and in pre-pro-B cells (2.6 fold) in Flt3-itd mice. Furthermore, other STAT3 target genes (Cish, Id1, Pim1, Socs1, Junb) were also upregulated in these cell populations in Flt3-itd mice. Moreover, gene-set enrichment analysis in MPPs demonstrated upregulation of Pu1 target genes in Flt3-itd mice, thus providing a link between aberrant ITD signaling and the observed myeloid bias. In order to determine the functional relevance of this myeloid-bias of Flt3-itd MPPs for disease transformation, we targeted a conditional Aml1-ETO fusion-gene to the earliest B-cell progenitors in Flt3-itd mice using Mb1-Cre. Expression of AML1-ETO in WT mice did not induce any phenotype. However, Mb1-Cre induced AML1-ETO expression in Flt3-itd mice led to a high-penetrance, short latency acute leukaemia. All leukaemias expressed myeloid markers (Mac1 and Gr1) but lacked CD19 and B220 expression. These data demonstrate that Flt3-ITDs expand primitive MPPs with a myeloid lineage bias at the molecular and cellular level, at the expense of HSCs and early lymphoid development. This provides insight into the mechanisms by which mutations resulting in activation of a GFR introduce a lineage bias of resulting hematological malignancies. Disclosures: No relevant conflicts of interest to declare.