ALBERT

Description: Abstract 3867 The generation of B cells from multipotent hematopoietic stem cells involves activation of B cell specific and repression of alternative lineage programs. This process to a large extent is under the control of transcription factors such as EBF1, E2A, IKAROS and PAX5. Inactivation of PAX5 and EBF1, by point mutations or deletions can be found in 30% of the acute lymphocytic leukemia (ALL) cases, probably due to a disturbance of differentiation in early B cell development. Conditional deletion of PAX5 and EBF1 leads to a complete block in B cell production and subsequent accumulation of early immature B cell progenitors. Although the roles of these transcription factors have been established in B cell development, the presence of a feedback loop between PAX5 and EBF1 as well as lack of well defined early B cell developmental events and cell populations, lead to some discrepancies in the current literature regarding the role of these factors in restricting non-B cell lineages and the establishment of B cell fate. By using a combination of RAG1/EBF1 reporter mice and newly identified surface markers i.e Ly6D, we have shown the exact point of myeloid (M), natural killer (NK) and T cells restriction within conventional common lymphoid progenitors (CLP) population. The Lin-IL7r+flt3+kitloSca1lo(CLP)Ly6D-λ5- population retains the residue of myeloid potential together with dendritic (D), Nk, T and B potentials. Upon expression of Ly6D, these cells lose the residual myeloid, NK and D potentials, and eventually the expression of λ5, associated with loss of T cell potential, marks the B-cell committed cells. Based on this new model of lymphoid development we have revisited the functional roles of EBF1 and PAX5 in B-cell commitment. This work suggests that EBF1 restricts the myeloid, NK and D potentials in the transition from CLPLy6D- to CLPLy6D+ stage while the expression of PAX5 in the transition from CLPLy6D+λ5- to CLPLy6D+λ5+ restricts the T cell potential by possibly by counteracting Notch1 signaling in the bone marrow. Considering the high prevalence of PAX5 and EBF1 mutations in ALL and diffuse large B cell lymphoma (DLBL) cases and the fact that inactivation of PAX5 and EBF1 leads to accumulation of B cell progenitors in mice models, understanding the temporal and spatial expression of EBF1 and PAX5 in regard to restriction of non-B cell fates in early stages of lymphoid development will allow us to identify the leukemia-initiating cells and the mechanisms of leukemogenesis in these diseases. Disclosures: No relevant conflicts of interest to declare.

Description: The NOD.SCID xenotransplantation assay is a key model system for interrogating the biology of leukemic stem cells (LSCs) in human acute myeloid leukemia (AML). Approximately 50% of AMLs can generate human grafts in immunodeficient mice that recapitulate the features of the parent sample. However, some AML samples generate non-leukemic grafts upon xenotransplantation. We recently reported that multilineage (ML) grafts, comprised of both B-lymphoid and myeloid cells, are generated by preleukemic hematopoietic stem cells (preL-HSCs; Shlush et al. Nature 2014). PreL-HSCs contain a subset of the mutations present in leukemic blasts and have a competitive growth advantage over wildtype HSCs leading to clonal expansion in vivo, yet retain multilineage differentiation capacity. To investigate the relationship between patient outcomes and the biological properties of LSCs and preL-HSCs as reflected by different engraftment patterns, we transplanted 272 diagnostic patient samples, representing a broad cross-section of adult AML, into sublethally irradiated NOD.SCID mice by intrafemoral injection. Human chimerism was assessed 8-10 weeks post-transplant by flow cytometry. 41% of samples generated AML xenografts, defined as a human graft containing 〉90% myeloid (CD33+CD19-CD45+) cells. Three patterns of engraftment were seen with the remaining samples: no human graft, defined as 10% CD19+CD33-CD45+ B-cells (22%). Among patients whose samples generated ML grafts compared to AML grafts or TC/no graft, secondary AML was less common (10% vs. 27% vs. 26%, respectively; P=0.03). Associations between engraftment pattern and other baseline clinical characteristics, including age, white blood cell (WBC) count and cytogenetics, did not reach statistical significance in this cohort. However, there was a strong correlation between AML engraftment capacity and response to standard induction chemotherapy. AML engrafters had lower complete remission (CR) rates compared to all other patients as a group (51% vs. 81%; P

Description: Abstract 2370 Aplastic anemia (AA) encompasses a spectrum of marrow failure disorders that include paroxysmal nocturnal hematuria (PNH), myelodysplastic syndrom (MDS) and acute myeloid leukemia (AML). Despite advances in therapy, approximately a tenth of patients will evolve to severe MDS or AML. A major unresolved question is the nature of the initiating cell that eventually expands during the aplastic phase and gives rise to secondary disease. A critical first step to approach this problem is to characterize the primitive stem and progenitor compartment in AA, taking advantage of the recent advances in phenotypic profiling of primitive human hematopoiesis (Doulatov et al Cell Stem Cell 2012). To this end, we established a 12-parameter flow-sorting scheme for deep phenotypic characterization of the CD34 compartment in AA patients that we used to quantify the gains and losses of all major cellular entities during the aplastic phase. These studies represent the first comprehensive analysis of AA. In 7 out of 10 patients, the proportion of mature myeloid, B cells and NK cells was reduced by greater than 5 fold, whereas the percentage of T cells remained indistinguishable against controls. Within the CD34+CD38-primitive progenitor compartment, the relative number of CD38-CD90+CD45RA- hematopoietic stem cells (HSC) and multipotent progenitors (MPP) reduced. The CD34+CD38+ progenitor populations including common myeloid progenitors (CMP) and megakaryocyte erythroid progenitors (MEP) were either dramatically reduced (〉5 fold) or virtually undetectable. Although we hypothesized that absence of phenotypic HSC would result in the absence of its immediate downstream progeny, this was not the case in most of patients. Within the CD34+CD38+ progenitor compartment, MEPs were the most affected population compare to CMPs or GMPs in more than 80% of cases. Interestingly we noticed in 8 of 12 patients, the proportion of granulocyte macrophage progenitors (GMP), defined by FLT3 and CD45RA expression, was unperturbed. To validate whether GMPs from AA patients were functional, we measured the in vitro colony forming capacity of GMP sorted populations. Clonal analyses of these cells in methylcellulose culture showed that these cells have similar potentials and cloning efficiency as normal donor cells. Cloning frequency, size of the clones and number of clones generated in methylcellulose and morphology was also comparable between GMPs from patients and normal donors. We then asked whether these GMPs are the result of a clonal expansion using mitochondrial DNA (MtDNA) analysis. This assay assesses the mutation rate of the D loop region of MtDNA from clones derived from single GMP cells from a patient or healthy donor to gain insight into the diversity within the clones. Our preliminary data suggest that the aplastic anemia GMPs share a closer ancestry compare to GMPs from healthy donor. The in depth quantification of the CD34+ compartment in AA patients that our study provides has directly established that phenotypically defined HSC are profoundly reduced, and this loss has a subsequent impact on downstream components of the hematopoietic hierarchy. The impact of HSC depletion is not a universal loss of progenitors, some progenitors can become overrepresented. Despite the small scale of this study, the overrepresentation of GMP versus other myeloid progenitors in patients with limited clonal heterogeneity suggests that GMP may be a potential candidate for the initiating cell that eventually evolves to MDS or AML. Disclosures: No relevant conflicts of interest to declare.

Description: Therapy resistance and relapse in acute myeloid leukemia (AML) are driven by leukemia stem cells (LSCs). Recent evidence highlighting functional and genetic heterogeneity among LSC subclones underscores the importance of capturing the entire LSC compartment in studies of LSC biology. Although LSCs are often enriched in the CD34+CD38- cell fraction, they are frequently detected in other phenotypic fractions, and in some cases are restricted to the CD34- population. In order to discover novel LSC markers, we examined genes differentially expressed between functionally-validated LSC+ and LSC- cell fractions obtained from primary AML samples, and identified CD200 as a candidate cell surface marker for LSCs. CD200 expression in 57 primary AML samples was analyzed by flow cytometry using anti-human CD200 clone 1B9(kindly provided by Trillium Therapeutics Inc.). CD200 was present on a greater proportion of CD45dim blasts compared to more differentiated CD45high non-blast populations (54.4% versus 21.7%, p

Description: Commitment of hematopoietic progenitor cells to B-lymphoid cell fate has been suggested to coincide with the development of PAX5-expressing B220+CD19+ pro–B cells. We have used a transgenic reporter mouse, expressing human CD25 under the control of the B-lineage–restricted Igll1 (λ5) promoter to investigate the lineage potential of early progenitor cells in the bone marrow. This strategy allowed us to identify a reporter expressing LIN−B220−CD19−CD127+FLT3+SCA1lowKITlow population that displays a lack of myeloid and a 90% reduction in in vitro T-cell potential compared with its reporter-negative counterpart. Gene expression analysis demonstrated that these lineage-restricted cells express B-lineage–associated genes to levels comparable with that observed in pro–B cells. These data suggest that B-lineage commitment can occur before the expression of B220 and CD19.

Description: Key Points Diverse patient groups with GATA2 mutation develop mononuclear cytopenia and elevated Flt3 ligand. Progressive cytopenias, rising Flt3 ligand, and terminal differentiation of lymphoid cells accompany clinical progression.

Description: Development of B-cell lineage from hematopoietic Stem cells in bone marrow is a stepwise process associated with a gradual loss of myeloid and T cell potential. This process involves a complex interaction of transcription factors like EBF1 and E2A, and extrinsic signals including IL7. It has been suggested that IL7 plays an inductive role in B-cell commitment through EBF activation in early B-Cell development. Mice deficient in Il-7 signaling show a dramatic reduction in the number of B-cell progenitors and reduced expression of EBF1 in the common lymphoid progenitor (CLP) compartment and ectopic expression of EBF can partially rescue the B-cell phenotype. However, the rather limited ability of EBF1 to rescue the phenotype as well as the powerful function of Il-7 in the expansion of committed cells creates a complex situation with an inherent difficulty to separate instructive and permissive actions of Il-7. Using transgenic mice carrying a reporter gene under the control of the EBF1 dependent Igll1 promoter, we were able to identify a B220−CD19− committed B-cell progenitor likely to represent the earliest committed population in the mouse bone marrow. This has opened the possibility to investigate lineage commitment in cells not expressing classical surface markers creating increased possibilities to study lineage choices. In order to investigate the inductive role of Il-7 we crossed the Igll1 reporter mice to Il-7 deficient mice. Analysis of reporter gene expression, gene expression by multiplex single cell PCR as well as functional analysis by in vitro differentiation assays, all supported that the committed lineage negative population was dramatically decreased in the absence of Il-7. These data all support the idea that Il-7 is critical not only for expansion of B-lineage progenitors but also for commitment per se. Investigation of the expression of EBF-1 by Real time and single cell PCR suggested that the expression level of EBF was on an average 50% lower in Il-7 deficient progenitors as compared to wild type cells. This expression level was recapitulated in mice heterozygote for a mutation in the EBF1 gene but since the formation of the early committed cells was not as dramatically effected in these mice, we found a need to look for a further function of Il-7 in its instructive role in B-cell development. This prompted us to investigate a potential function of Il-7 in the modulation of Notch signals known to counteract B-cell commitment and EBF function. This revealed that the addition of Il-7 largely inhibits the Notch response in pro-B cells in vitro. Therefore we suggest that Il-7 is directly involved in B-cell commitment and that this function is achieved by modulation of EBF1 both at the transcriptional and functional level.

Description: Deficiencies in the IL-7 signaling pathway result in severe disruptions of lymphoid development in adult mice. To understand more about how IL-7 deficiency impacts early lymphoid development, we have investigated lineage restriction events within the common lymphoid progenitor (CLP) compartment in IL-7 knockout mice. This revealed that although IL-7 deficiency had a minor impact on the development of LY6D− multipotent CLPs, the formation of the lineage restricted LY6D+ CLP population was dramatically reduced. This was reflected in a low-level transcription of B-lineage genes as well as in a loss of functional B-cell commitment. The few Ly6D+ CLPs developed in the absence of IL-7 displayed increased lineage plasticity and low expression of Ebf-1. Absence of Ebf-1 could be linked to increased plasticity because even though Ly6D+ cells develop in Ebf-1–deficient mice, these cells retain both natural killer and dendritic cell potential. This reveals that IL-7 is essential for normal development of Ly6D+ CLPs and that Ebf-1 is crucial for lineage restriction in early lymphoid progenitors.

Description: There is a growing body of evidence that the molecular properties of leukemia stem cells (LSCs) are associated with clinical outcomes in acute myeloid leukemia (AML), and LSCs have been linked to therapy failure and relapse. Thus, a better understanding of the molecular mechanisms that contribute to the persistence and regenerative potential of LSCs is expected to result in the development of more effective therapies. We therefore interrogated functionally validated data sets of LSC-specific genes together with their known protein interactors and selected 64 candidates for a competitive in vivo gain-of-function screen to identify genes that enhanced stemness in human cord blood hematopoietic stem and progenitor cells. A consistent effect observed for the top hits was the ability to restrain early repopulation kinetics while preserving regenerative potential. Overexpression (OE) of the most promising candidate, the orphan gene C3orf54/INKA1, in a patient-derived AML model (8227) promoted the retention of LSCs in a primitive state manifested by relative expansion of CD34+ cells, accumulation of cells in G0, and reduced output of differentiated progeny. Despite delayed early repopulation, at later times, INKA1-OE resulted in the expansion of self-renewing LSCs. In contrast, INKA1 silencing in primary AML reduced regenerative potential. Mechanistically, our multidimensional confocal analysis found that INKA1 regulates G0 exit by interfering with nuclear localization of its target PAK4, with concomitant reduction of global H4K16ac levels. These data identify INKA1 as a novel regulator of LSC latency and reveal a link between the regulation of stem cell kinetics and pool size during regeneration.