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Early T cell precursor acute lymphoblastic leukemia (ETP-ALL) is an aggressive subtype of ALL distinguished by stem-cell-associated and myeloid transcriptional programs. Inactivating alterations of Polycomb repressive complex 2 components are frequent in human ETP-ALL, but their functional role is largely undefined. We have studied the involvement of Ezh2in a murine model of NRASQ61K-driven leukemia that recapitulates phenotypic and transcriptional features of ETP-ALL. Homozygous inactivation of Ezh2cooperated with oncogenic NRASQ61K to accelerate leukemia onset. Inactivation of Ezh2accentuated expression of genes highly expressed in human ETP-ALL and in normal murine early thymic progenitors. Moreover, we found that Ezh2contributes to the silencing of stem-cell- and early-progenitor-cell-associated genes. Loss of Ezh2also resulted in increased activation of STAT3 by tyrosine 705 phosphorylation. Our data mechanistically link Ezh2inactivation to stem-cell-associated transcriptional programs and increased growth/survival signaling, features that convey an adverse prognosis in patients.

ABSTRACTPrevious studies have shown that dihydrofolate reductase (DHFR) gene transfer protects marrow from methotrexate (MTX)-mediated toxicity; however, MTX treatment in vivohas not convincingly been shown to enrich DHFR-transduced progenitors or stem cells. Experiments were performed to better characterize the hematological effects of MTX, and maneuvers were tested with the aim of improving the utility of MTX as an agent for in vivoselection. Progenitors were assayed as colony forming unit cells in culture (CFU-C) and in the spleens of irradiated mice (day 11 CFU-S). A single injection of MTX at doses up to 250 mg/kg (more than three times the LD10) failed to reduce CFU-C numbers significantly in the femur or spleen assayed 1––3 days later. However, consistent declines in the number of mononuclear cells per femur reflected a significant depletion of nonclonogenic precursor cells. Preceding administration of pegylated stem cell factor (SCF), 100 μμg/kg per day, increased CFU-C killing by a single dose of 5-fluorouracil (5-FU) 15- to 65-fold in the femur, and 5- to 15-fold in the spleen, consistent with previous reports. In contrast, despite preceding SCF administration there was no significant progenitor killing by MTX. Similar results were obtained using a second folate analog, trimetrexate. These results suggest that the mechanism by which folate analogs exert their hematological toxicity is through the depletion of relatively mature, nonclonogenic precursor cells, and not by killing progenitors. This information is relevant to the use of DHFR in gene therapy protocols, and suggests that folate analogs are poorly suited agents for selection at the level of clonogenic progenitor cells in vivo.

The importance of stem cell and self-renewal programs in Acute Myeloid Leukemia (AML) is generally accepted, but the molecular details are incompletely understood. The master transcriptional regulator GATA2 is highly expressed in hematopoietic stem cells (HSCs) and has critically important roles in the hematopoietic system. Gata2is required for murine HSC development and maintenance, and heterozygous loss of Gata2compromises murine HSC- and progenitor cell-function. High levels of GATA2-expression have been correlated with adverse prognosis in human AML. GATA2is also overexpressed in human chronic myeloid leukemia. These data suggest an important role for GATA2in normal stem cells and in leukemia. However, genetic lesions resulting in compromised GATA2function can lead to MDS and in some cases AML. In a murine AML model driven by Flt3-ITDand inactivation of Tet2, Gata2is strongly downregulated. Furthermore, mouse models of leukemia suggest that high-level forced expression of Gata2can have a tumor suppressor role. To clarify the role of Gata2in AML we used homozygous genetic inactivation in established murine models of leukemia, using a a conditional allele.