Matthew P. McCormack

The Lmo2 Oncogene Initiates Leukemia in Mice by Inducing Thymocyte Self-Renewal

Its All About Self-Renewal The Lmo2 oncogene was identified as a contributing factor in human T cell acute lymphoblastic leukemia (T-ALL) nearly two decades ago, but the gene rose to prominence in 2003 when its inadvertent activation by a retroviral vector was shown to cause leukemia in two patients in a gene therapy trial. The cellular mechanism by which the gene product of Lmo2, a transcriptional regulator, induces T-ALL is poorly understood. Studying transgenic mice, McCormack et al. (p. 879, published online 21 January) now show that Lmo2 confers self-renewal activity to committed T cells in the thymus without affecting their capacity for T cell differentiation. These self-renewing cells, which were detectable 8 months prior to the onset of overt leukemia in the mice, expressed genes in common with hematopoietic stem cells (HSCs), suggesting that Lmo2 might reactivate an HSC-specific transcriptional program. Expression of an oncogene confers self-renewal activity to committed T cells in the thymus long before disease onset. The LMO2 oncogene causes a subset of human T cell acute lymphoblastic leukemias (T-ALL), including four cases that arose as adverse events in gene therapy trials. To investigate the cellular origin of LMO2-induced leukemia, we used cell fate mapping to study mice in which the Lmo2 gene was constitutively expressed in the thymus. Lmo2 induced self-renewal of committed T cells in the mice more than 8 months before the development of overt T-ALL. These self-renewing cells retained the capacity for T cell differentiation but expressed several genes typical of hematopoietic stem cells (HSCs), suggesting that Lmo2 might reactivate an HSC-specific transcriptional program. Forced expression of one such gene, Hhex, was sufficient to initiate self-renewal of thymocytes in vivo. Thus, Lmo2 promotes the self-renewal of preleukemic thymocytes, providing a mechanism by which committed T cells can then accumulate additional genetic mutations required for leukemic transformation.

The LMO2 T-cell oncogene is activated via chromosomal translocations or retroviral insertion during gene therapy but has no mandatory role in normal T-cell development.

ABSTRACT The LMO2 gene encodes a LIM-only protein and is a target of chromosomal translocations in human T-cell leukemia. Recently, two X-SCID patients treated by gene therapy to rescue T-cell lymphopoiesis developed T-cell leukemias with retroviral insertion into the LMO2 gene causing clonal T-cell proliferation. In view of the specificity of LMO2 in T-cell tumorigenesis, we investigated a possible role for Lmo2 in T-lymphopoiesis, using conditional knockout of mouse Lmo2 with loxP-flanked Lmo2 and Cre recombinase alleles driven by the promoters of the lymphoid-specific genes Rag1, CD19, and Lck. While efficient deletion of Lmo2 was observed, even in the earliest detectable lymphoid cell progenitors of the bone marrow, there was no disturbance of lymphopoiesis in either T- or B-cell lineages, and in contrast to Lmo2 transgenic mice, there were normal distributions of CD4− CD8− thymocytes. We conclude that there is no mandatory role for LMO2 in lymphoid development, implying that its specific role in T-cell tumorigenesis results from a reprogramming of gene expression after enforced expression in T-cell precursors.

Functional but Abnormal Adult Erythropoiesis in the Absence of the Stem Cell Leukemia Gene

ABSTRACT Previous studies have indicated that the stem cell leukemia gene (SCL) is essential for both embryonic and adult erythropoiesis. We have examined erythropoiesis in conditional SCL knockout mice for at least 6 months after loss of SCL function and report that SCL was important but not essential for the generation of mature red blood cells. Although SCL-deleted mice were mildly anemic with increased splenic erythropoiesis, they responded appropriately to endogenous erythropoietin and hemolytic stress, a measure of late erythroid progenitors. However, SCL was more important for the proliferation of early erythroid progenitors because the predominant defects in SCL-deleted erythropoiesis were loss of in vitro growth of the burst-forming erythroid unit and an in vivo growth defect revealed by transplant assays. With respect to erythroid maturation, SCL-deleted proerythroblasts could generate more mature erythroblasts and circulating red blood cells. However, SCL was required for normal expression of TER119, one of the few proposed target genes of SCL. The unexpected finding that SCL-independent erythropoiesis can proceed in the adult suggests that alternate factors can replace the essential functions of SCL and raises the possibility that similar mechanisms also explain the relatively minor defects previously observed in SCL-null hematopoietic stem cells.

The Hedgehog receptor Patched1 regulates myeloid and lymphoid progenitors by distinct cell-extrinsic mechanisms.

Hedgehog (Hh) ligands bind to the Patched1 (Ptch1) receptor, relieving repression of Smoothened, which leads to activation of the Hh signaling pathway. Using conditional Ptch1 knockout mice, the aim of this study was to determine the effects of activating the Hh signaling pathway in hematopoiesis. Surprisingly, hematopoietic-specific deletion of Ptch1 did not lead to activation of the Hh signaling pathway and, consequently, had no phenotypic effect. In contrast, deletion of Ptch1 in nonhematopoietic cells produced 2 distinct hematopoietic phenotypes. First, activation of Hh signaling in epithelial cells led to apoptosis of lymphoid progenitors associated with markedly elevated levels of circulating thymic stromal lymphopoietin. Second, activation of Hh signaling in the bone marrow cell niche led to increased numbers of lineage-negative c-kit(+) Sca-1(+) bone marrow cells and mobilization of myeloid progenitors associated with a marked loss of osteoblasts. Thus, deletion of Ptch1 leads to hematopoietic effects by distinct cell-extrinsic mechanisms rather than by direct activation of the Hh signaling pathway in hematopoietic cells. These findings have important implications for therapeutics designed to activate the Hh signaling pathway in hematopoietic cells including hematopoietic stem cells.

The Molecular Basis of Lmo2-Induced T-Cell Acute Lymphoblastic Leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is commonly caused by the overexpression of oncogenic transcription factors in developing T cells. In a mouse model of one such oncogene, LMO2, the cellular effect is to induce self-renewal of committed T cells in the thymus, which persist long-term while acquiring additional mutations and eventually giving rise to leukemia. These precancerous stem cells (pre-CSC) are intrinsically resistant to radiotherapy, implying that they may be refractory to conventional cancer therapies. However, they depend on an aberrantly expressed stem cell–like self-renewal program for their maintenance, in addition to a specialized thymic microenvironmental niche. Here, we discuss potential approaches for targeting pre-CSCs in T-ALL by using therapies directed at oncogenic transcription factors themselves, downstream self-renewal pathways, and the supportive cell niche. Clin Cancer Res; 16(23); 5618–23. ©2010 AACR.

ZEB2 drives immature T-cell lymphoblastic leukaemia development via enhanced tumour-initiating potential and IL-7 receptor signalling

Early T-cell precursor leukaemia (ETP-ALL) is a high-risk subtype of human leukaemia that is poorly understood at the molecular level. Here we report translocations targeting the zinc finger E-box-binding transcription factor ZEB2 as a recurrent genetic lesion in immature/ETP-ALL. Using a conditional gain-of-function mouse model, we demonstrate that sustained Zeb2 expression initiates T-cell leukaemia. Moreover, Zeb2-driven mouse leukaemia exhibit some features of the human immature/ETP-ALL gene expression signature, as well as an enhanced leukaemia-initiation potential and activated Janus kinase (JAK)/signal transducers and activators of transcription (STAT) signalling through transcriptional activation of IL7R. This study reveals ZEB2 as an oncogene in the biology of immature/ETP-ALL and paves the way towards pre-clinical studies of novel compounds for the treatment of this aggressive subtype of human T-ALL using our Zeb2-driven mouse model.

Requirement for Lyl1 in a model of Lmo2-driven early T-cell precursor ALL.

Lmo2 is an oncogenic transcription factor that is frequently overexpressed in T-cell acute lymphoblastic leukemia (T-ALL), including early T-cell precursor ALL (ETP-ALL) cases with poor prognosis. Lmo2 must be recruited to DNA by binding to the hematopoietic basic helix-loop-helix factors Scl/Tal1 or Lyl1. However, it is unknown which of these factors can mediate the leukemic activity of Lmo2. To address this, we have generated Lmo2-transgenic mice lacking either Scl or Lyl1 in the thymus. We show that although Scl is dispensable for Lmo2-driven leukemia, Lyl1 is critical for all oncogenic functions of Lmo2, including upregulation of a stem cell-like gene signature, aberrant self-renewal of thymocytes, and subsequent generation of T-cell leukemia. Lyl1 expression is restricted to preleukemic and leukemic stem cell populations in this model, providing a molecular explanation for the stage-specific expression of the Lmo2-induced gene expression program. Moreover, LMO2 and LYL1 are coexpressed in ETP-ALL patient samples, and LYL1 is required for growth of ETP-ALL cell lines. Thus, the LMO2-LYL1 interaction is a promising therapeutic target for inhibiting self-renewing cancer stem cells in T-ALL, including poor-prognosis ETP-ALL cases.

Acute myeloid leukemia requires Hhex to enable PRC2-mediated epigenetic repression of Cdkn2a

Unlike clustered HOX genes, the role of nonclustered homeobox gene family members in hematopoiesis and leukemogenesis has not been extensively studied. Here we found that the hematopoietically expressed homeobox gene Hhex is overexpressed in acute myeloid leukemia (AML) and is essential for the initiation and propagation of MLL-ENL-induced AML but dispensable for normal myelopoiesis, indicating a specific requirement for Hhex for leukemic growth. Loss of Hhex leads to expression of the Cdkn2a-encoded tumor suppressors p16(INK4a) and p19(ARF), which are required for growth arrest and myeloid differentiation following Hhex deletion. Mechanistically, we show that Hhex binds to the Cdkn2a locus and directly interacts with the Polycomb-repressive complex 2 (PRC2) to enable H3K27me3-mediated epigenetic repression. Thus, Hhex is a potential therapeutic target that is specifically required for AML stem cells to repress tumor suppressor pathways and enable continued self-renewal.

Loss-of-function mutations of Dynamin 2 promote T-ALL by enhancing IL-7 signalling.

Mutations in the DYNAMIN2 (DNM2) gene are frequently detected in human acute T-cell lymphoblastic leukemia (T-ALL), although the mechanisms linking these mutations to disease pathogenesis remain unknown. Using an ENU-based forward genetic screen for mice with erythroid phenotypes, we identified a heterozygous mouse line carrying a mutation in the GTPase domain of Dnm2 (Dnm2V265G) that induced a microcytic anemia. In vitro assays using the V265G mutant demonstrated loss of GTPase activity and impaired endocytosis that was comparable to other DNM2 mutants identified in human T-ALL. To determine the effects of DNM2 mutations in T-ALL, we bred the Dnm2V265G mice with the Lmo2 transgenic mouse model of T-ALL. Heterozygous Dnm2 mutants lacking the Lmo2 transgene displayed normal T-cell development, and did not develop T-ALL. In contrast, compound heterozygotes displayed an accelerated onset of T-ALL compared with mice carrying the Lmo2 oncogene alone. The leukemias from these mice exhibited a more immature immunophenotype and an expansion in leukemic stem cell numbers. Mechanistically, the Dnm2 mutation impaired clathrin-mediated endocytosis of the interleukin (IL)-7 receptor resulting in increased receptor density on the surface of leukemic stem cells. These findings suggest that DNM2 mutations cooperate with T-cell oncogenes by enhancing IL-7 signalling.

A crucial role for the homeodomain transcription factor Hhex in lymphopoiesis

The hematopoietically expressed homeobox gene, Hhex, is a transcription factor that is important for development of definitive hematopoietic stem cells (HSCs) and B cells, and that causes T-cell leukemia when overexpressed. Here, we have used an Hhex inducible knockout mouse model to study the role of Hhex in adult hematopoiesis. We found that loss of Hhex was tolerated in HSCs and myeloid lineages, but resulted in a progressive loss of B lymphocytes in the circulation. This was accompanied by a complete loss of B-cell progenitors in the bone marrow and of transitional B-cell subsets in the spleen. In addition, transplantation and in vitro culture experiments demonstrated an almost complete failure of Hhex-null HSCs to contribute to lymphoid lineages beyond the common lymphoid precursor stage, including T cells, B cells, NK cells, and dendritic cells. Gene expression analysis of Hhex-deleted progenitors demonstrated deregulated expression of a number of cell cycle regulators. Overexpression of one of these, cyclin D1, could rescue the B-cell developmental potential of Hhex-null lymphoid precursors. Thus, Hhex is a key regulator of early lymphoid development, functioning, at least in part, via regulation of the cell cycle.