Aberrant transcriptional programs in combination with abnormal proliferative signaling drive leukemic transformation. These programs operate in normal hematopoiesis where they are involved in hematopoietic stem cell (HSC) proliferation and maintenance. Ets Related Gene (ERG) is a component of normal and leukemic stem cell signatures and high ERG expression is a risk factor for poor prognosis in acute myeloid leukemia (AML). However, mechanisms that underlie ERG expression in AML and how its expression relates to leukemic stemness are unknown. We report that ERG expression in AML is associated with activity of the ERG promoters and +85 stem cell enhancer and a heptad of transcription factors that combinatorially regulate genes in HSCs. Gene expression signatures derived from ERG promoter-stem cell enhancer and heptad activity are associated with clinical outcome when ERG expression alone fails. We also show that the heptad signature is associated with AMLs that lack somatic mutations in NPM1 and confers an adverse prognosis when associated with FLT3 mutations. Taken together, these results suggest that transcriptional regulators cooperate to establish or maintain primitive stem cell-like signatures in leukemic cells and that the underlying pattern of somatic mutations contributes to the development of these signatures and modulate their influence on clinical outcome.

Hypoxia is emerging as an important characteristic of the hematopoietic stem cell (HSC) niche, but the molecular mechanisms contributing to quiescence, self-renewal, and survival remain elusive. Vascular endothelial growth factor A (VEGFA) is a key regulator of angiogenesis and hematopoiesis. Its expression is commonly regulated by hypoxiainducible factors (HIF) that are functionally induced in low-oxygen conditions and that activate transcription by binding to hypoxia-response elements (HRE).Vegfa is indispensable for HSC survival, mediated by a cell-intrinsic, autocrine mechanism. We hypothesized that a hypoxic HSC microenvironment is required for maintenance or up-regulation of Vegfa expression in HSCs and therefore crucial for HSC survival. We have tested this hypothesis in the mouse model Vegfa ␦/␦ , where the HRE in the Vegfa promoter is mutated, preventing HIF binding. Vegfa expression was reduced in highly purified HSCs from Vegfa ␦/␦ mice, showing that HSCs reside in hypoxic areas. Loss of hypoxiaregulated Vegfa expression increases the numbers of phenotypically defined hematopoietic stem and progenitor cells. However, HSC function was clearly impaired when assessed in competitive transplantation assays. Our data provide further evidence that HSCs reside in a hypoxic microenvironment and demonstrate a novel way in which the hypoxic niche affects HSC fate, via the hypoxia-VEGFA axis. (Blood. 2011; 118(6):1534-1543) IntroductionThe process of blood formation originates with the hematopoietic stem cell (HSC) which is responsible for the life-long supply of mature blood cells through the unique ability to combine selfrenewal and differentiation. During the later stages of mammal development, HSCs are present in the fetal liver (FL) from where they seed the bone marrow (BM), which remains the main site of hematopoiesis throughout adult life. The HSC niche is a term used to describe the location and regulatory microenvironment of HSCs. In the BM, cellular HSC niche components include boneforming osteoblasts 1-2 and perivascular cells, [3][4] that through production of various secreted factors as well as through direct cell-cell interactions can affect HSC behavior. As for location it is thought that HSCs are present at a higher frequency at the endosteum close to the bone surface, because ex vivo labeled HSCs tend to localize to the endosteal areas of the BM. [5][6] However, within these areas HSCs are likely to associate with both osteoblastic and sinusoidal endothelial cells. 7 The FL HSC niche is much less defined than that in the BM, but a candidate FL niche component is a CD3 ϩ Ter119 Ϫ population that can provide HSC support. 8 Apart from HSC regulatory cues such as cell-cell contact and production of soluble factors, it is possible that variations in oxygen availability can affect HSCs, and it has been hypothesized that hypoxia is a characteristic of the HSC niche. 9 Oxygen levels are generally lower in BM compared with peripheral blood (PB). 10 Two studies have demonstrated that fractions of BM that w...

PTEN and SHP1 are tumor suppressor genes involved in the regulation of cell cycle control and apoptosis. The authors investigated the protein expression of PTEN and SHP1, by immunohistochemistry in tissue microarrays from bone marrow samples in children, diagnosed with acute lymphoblastic leukaemia and nonmalignant controls. PTEN was overexpressed in diagnostic ALL samples, while SHP1 showed a low expression. Both proteins showed a significant difference in expression compared to nonmalignant controls. The roles of PTEN and SHP1 are not well investigated in pediatric leukemia and could in the future play a role as prognostic factors.

Perturbation in the expression and signaling pathways of vascular endothelial growth factor (VEGF) has been linked to pathogenesis of hematologic malignancies. We investigated the expression and clinical importance of VEGF and two of its receptors, VEGFR-1 and VEGFR-2, in childhood precursor B-cell acute lymphoblastic leukemia (pre-B ALL) by using immunohistochemistry. These angiogenic proteins were expressed in the majority of leukemic bone marrow samples. Notably, pre-B ALL patients had significantly increased expression of VEGFR-1 compared with no expression in the nonmalignant group, indicating a link between VEGFR-1 protein expression and pre-B ALL. These novel findings suggest that VEGFR-1 may have clinical importance in childhood pre-B ALL.

3525 Leukaemic transformation is driven by aberrant transcriptional programs often in combination with abnormal proliferative signalling. These programs operate in normal hematopoiesis where they are involved in hematopoietic stem cell (HSC) proliferation and maintenance. ERG is a component of normal and leukemic stem cell signatures and high ERG expression has been proposed as a risk factor for poor prognosis in acute myeloid leukemia (AML). However, mechanisms that underlie ERG expression in AML and how its expression relates to leukemic stemness are unknown. We report that ERG expression in AML is associated with activity of the ERG+85 stem cell enhancer (SCE) and a heptad of transcription factors that combinatorially regulate genes in normal HSCs. Gene expression signatures derived from ERG+85 stem cell enhancer (Fig A) and heptad activity (Fig B) predict clinical outcome in a cytogenetically normal cohort of AML (CN-AML) patients when ERG expression alone fails. The heptad signature is an independent risk factor for poor overall and event-free survival (Fig C). There were no long-term survivors amongst patients with a heptad signature, FLT3 mutations and wild-type NPM1 (Fig D) pointing to a hitherto unappreciated link between aberrant signaling and transcriptional mediators of hematopoietic stem cell identity. In two independent cohorts, the heptad signature was as closely associated with wild-type NPM1 AML as the HOX signature was with mutant NPM1 AML (Fig E–F) suggestive of a collective role for these transcription factors in mediating the leukemic signature in the former. Taken together, these results show that key transcriptional regulators cooperate in establishing stem cell signatures in leukemic cells and that the underlying spectrum of somatic mutations contributes to the development of these signatures and modulate their influence on clinical outcome. Disclosures: No relevant conflicts of interest to declare.