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Hhex encodes a homeodomain transcription factor that is widely expressed in hematopoietic stem and progenitor cell populations. Its enforced expression induces T-cell leukemia and we have implicated it as an important oncogene in early T-cell precursor leukemias where it is immediately downstream of an LMO2-associated protein complex. Conventional Hhex knockouts cause embryonic lethality precluding analysis of adult hematopoiesis. Thus, we induced highly efficient conditional knockout (cKO) using vav-Cre transgenic mice. Hhex cKO mice were viable and born at normal litter sizes. At steady state, we observed a defect in B-cell development that we localized to the earliest B-cell precursor, the pro-B-cell stage. Most remarkably, bone marrow transplantation using Hhex cKO donor cells revealed a more profound defect in all hematopoietic lineages. In contrast, sublethal irradiation resulted in normal myeloid cell repopulation of the bone marrow but markedly impaired repopulation of T- and B-cell compartments. We noted that Hhex cKO stem and progenitor cell populations were skewed in their distribution and showed enhanced proliferation compared to WT cells. Our results implicate Hhex in the maintenance of LT-HSCs and in lineage allocation from multipotent progenitors especially in stress hematopoiesis. Stem Cells 2015;33:2628—2641

LIM domain Only-2 (LMO2) overexpression in T cells induces leukemia but the molecular mechanism remains to be elucidated. In hematopoietic stem and progenitor cells, Lmo2 is part of a protein complex comprised of class II basic helix loop helix proteins, Tal1and Lyl1. The latter transcription factors heterodimerize with E2A proteins like E47 and Heb to bind E boxes. LMO2 and TAL1 or LYL1 cooperate to induce T-ALL in mouse models, and are concordantly expressed in human T-ALL. Furthermore, LMO2 cooperates with the loss of E2A suggesting that LMO2 functions by creating a deficiency of E2A. In this study, we tested this hypothesis in Lmo2-induced T-ALL cell lines. We transduced these lines with an E47/estrogen receptor fusion construct that could be forced to homodimerize with 4-hydroxytamoxifen. We discovered that forced homodimerization induced growth arrest in 2 of the 4 lines tested. The lines sensitive to E47 homodimerization accumulated in G1 and had reduced S phase entry. We analyzed the transcriptome of a resistant and a sensitive line to discern the E47 targets responsible for the cellular effects. Our results suggest that E47 has diverse effects in T-ALL but that functional deficiency of E47 is not a universal feature of Lmo2-induced T-ALL.

LIM domain only 2 (Lmo2) is frequently deregulated in sporadic and gene therapy-induced acute T-cell lymphoblastic leukemia (T-ALL) where its overexpression is an important initiating mutational event. In transgenic and retroviral mouse models, Lmo2 expression can be enforced in multiple hematopoietic lineages but leukemia only arises from T cells. These data suggest that Lmo2 confers clonal growth advantage in T-cell progenitors. We analyzed proliferation, differentiation, and cell death in CD2-Lmo2 transgenic thymic progenitor cells to understand the cellular effects of enforced Lmo2 expression. Most impressively, Lmo2 transgenic T-cell progenitor cells were blocked in differentiation, quiescent, and immortalized in vitro on OP9-DL1 stromal cells. These cellular effects were concordant with a transcriptional signature in Lmo2 transgenic T-cell progenitor cells that is also present in hematopoietic stem cells (HSCs) and early T-cell precursor ALL. These results are significant in light of the crucial role of Lmo2 in the maintenance of the HSC. The cellular effects and transcriptional effects have implications for LMO2-dependent leukemogenesis and the treatment of LMO2-induced T-ALL.

Adult T-cell leukemia/lymphoma (ATLL) is an incurable disease where most patients succumb within the first year of diagnosis. Both standard chemotherapy regimens and mAbs directed against ATLL tumor markers do not alter this aggressive clinical course. Therapeutic development would be facilitated by the discovery of genes and pathways that drive or initiate ATLL, but so far amenable drug targets have not been forthcoming. Because the IL-2 signaling pathway plays a prominent role in ATLL pathogenesis, mutational analysis of pathway components should yield interesting results. In this study, we focused on JAK3, the nonreceptor tyrosine kinase that signals from the IL-2R, where activating mutations have been found in diverse neoplasms. We screened 36 ATLL patients and 24 ethnically matched controls and found 4 patients with mutations in JAK3. These somatic, missense mutations occurred in the N-terminal FERM (founding members: band 4.1, ezrin, radixin, and moesin) domain and induced gain of function in JAK3. Importantly, we show that these mutant JAK3s are inhibited with a specific kinase inhibitor already in human clinical testing. Our findings underscore the importance of this pathway in ATLL development and offer a therapeutic handle for this incurable cancer.

Proapoptotic Bax and Bak are the key B-cell lymphoma-2 family members mediating apoptosis through the intrinsic pathway. Cells doubly deficient for Bax and Bak are profoundly resistant to apoptotic stimuli originating from multiple stimuli. Here we describe mice in which Bax and Bak have been deleted specifically in T-cells using Lck-Cre. In these T cell–specific BaxBak-deficient mice, early T-cell progenitors accumulate in the thymus, with relative depletion of more mature T cells. In addition, bone marrow progenitor cells fail to progress to the double positive stage when cultured on OP9 stromal cells expressing the Notch ligand Delta-like 1, consistent with a critical role for Bax and Bak in early T-cell development. Over time, T cell–specific BaxBak-deficient mice progress to an aggressive T-cell lymphoblastic leukemia/lymphoma. Interestingly, quantitative real-time polymerase chain reaction analysis of BaxBak-deficient T-cell lymphomas does not display amplification of the Notch signal transduction pathway, commonly activated in T-cell leukemia in both mouse and man. Bax and Bak, key regulators of the intrinsic pathway of apoptosis, are thus required to prevent T-cell malignancy, and for normal T-cell differentiation, regulating early T-cell development at the stage of early T-lineage progenitor cells.

The LMO2 oncogene is deregulated in the majority of human T-cell leukemia cases and in most gene therapy-induced T-cell leukemias. We made transgenic mice with enforced expression of Lmo2 in T-cells by the CD2 promoter/enhancer. These transgenic mice developed highly penetrant T-ALL by two distinct patterns of gene expression: one in which there was concordant activation of Lyl1, Hhex, and Mycn or alternatively, with Notch1 target gene activation. Most strikingly, this gene expression clustering was conserved in human Early T-cell Precursor ALL (ETP-ALL), where LMO2, HHEX, LYL1, and MYCN were most highly expressed. We discovered that HHEX is a direct transcriptional target of LMO2 consistent with its concordant gene expression. Furthermore, conditional inactivation of Hhex in CD2-Lmo2 transgenic mice markedly attenuated T-ALL development, demonstrating that Hhex is a crucial mediator of Lmo2's oncogenic function. The CD2-Lmo2 transgenic mice offer mechanistic insight into concordant oncogene expression and provide a model for the highly treatment-resistant ETP-ALL subtype.

Cooperation of multiple mutations is thought to be required for cancer development. In previous studies, murine myeloid leukemias induced by transducing wild-type bone marrow progenitors with a SRY sex determining region Y-box 4 (Sox4)–expressing retrovirus frequently carried proviral insertions at Sfpi1, decreasing its mRNA levels, suggesting that reduced Sfpi1 expression cooperates with Sox4 in myeloid leukemia induction. In support of this hypothesis, we show here that mice receiving Sox4 virus-infected Sfpi1ko/+ bone marrow progenitors developed myeloid leukemia with increased penetrance and shortened latency. Interestingly, Sox4 expression further decreased Sfpi1 transcription. Ectopic SOX4 expression reduced endogenous PU.1 mRNA levels in HL60 promyelocytes, and decreased Sfpi1 mRNA levels were also observed in the spleens of leukemic and preleukemic mice receiving Sox4 virus-infected wild-type bone marrow cells. In addition, Sox4 protein bound to a critical upstream regulatory element of Sfpi1 in ChIP assays. Such cooperation probably occurs in de novo human acute myeloid leukemias, as an analysis of 285 acute myeloid leukemia patient samples found a significant negative correlation between SOX4 and PU.1 expression. Our results establish a novel cooperation between Sox4 and reduced Sfpi1 expression in myeloid leukemia development and suggest that SOX4 could be an important new therapeutic target in human acute myeloid leukemia.

Five X-linked severe combined immunodeficiency patients (SCID-X1) successfully treated with autologous bone marrow stem cells infected ex vivo with an IL2RG-containing retrovirus subsequently developed T-cell leukemia and four contained insertional mutations at LMO2. Genetic evidence also suggests a role for IL2RG in tumor formation, although this remains controversial. Here, we show that the genes and signaling pathways deregulated in murine leukemias with retroviral insertions at Lmo2 are similar to those deregulated in human leukemias with high LMO2 expression and are highly predictive of the leukemias induced in SCID-X1 patients. We also provide additional evidence supporting the notion that IL2RG and LMO2 cooperate in leukemia induction but are not sufficient and require additional cooperating mutations. The highly concordant nature of the genetic events giving rise to mouse and human leukemias with mutations at Lmo2 are an encouraging sign to those wanting to use mice to model human cancer and may help in designing safer methods for retroviral gene therapy., Author Summary Twenty patients with X-linked severe combined immunodeficiency (SCID-X1) have been successfully treated by gene therapy. Unfortunately, five of these patients have developed T-cell leukemia two or more years after receiving the therapeutic gene IL2RG on a retroviral vector. The leukemias developed because the vector inserted itself near cancer-causing genes and disrupted their normal regulation. Remarkably, in four patients, the vector inserted near a known T-cell oncogene, LMO2. We have found that in mice, similar retroviruses cause T-cell leukemias by inserting near Lmo2. We have found two leukemias that have retroviral insertions near Lmo2 and Il2rg in the same cell. The probability of these insertions happening by chance is exceedingly small and these results imply that these two genes are deregulated together to induce leukemia. Our data show that Lmo2 and Il2rg cooperate but may not be sufficient for leukemia development and additional mutations contribute to leukemia development. We have also found cooperating retroviral insertions in genes that are abnormally expressed in human T-cell leukemias. The mouse models provide unique insight into the pathogenesis of T-cell leukemia, and they are highly predictive of the leukemias caused by SCID-X1 gene therapy.

In this study, we present a remarkable clonal cell line, 32080, derived from a CD2-Lmo2 - transgenic T-cell leukemia with differentiation arrest at the transition from the intermediate single positive to double positive stages of T-cell development. We observed that 32080 cells had a striking variegated pattern in CD4 expression. There was cell-to-cell variability, with some cells expressing no CD4 and others expressing high CD4. The two populations were isogenic and yet differed in their rates of apoptosis and sensitivity to glucocorticoid. We sorted the 32080 line for CD4-positive or CD4-negative cells and observed them in culture. After 1 week, both sorted populations showed variegated CD4 expression, like the parental line, showing that the two populations could interconvert. We determined that cell replication was necessary to transit from CD4 + to CD4 − and CD4 − to CD4 + . Lmo2 knockdown decreased CD4 expression, while inhibition of intracellular NOTCH1 or histone deacetylase activity induced CD4 expression. Enforced expression of RUNX1 repressed CD4 expression. We analyzed the CD4 locus by Histone 3 chromatin immunoprecipitation and found silencing marks in the CD4 − cells and activating marks in the CD4 + population. The 32080 cell line is a striking model of intermediate single positive to double positive T-cell plasticity and invokes a novel mechanism for LMO2 's oncogenic functions.

Abstract 2458 LIM domain Only 2 (LMO2) is an important driver in acute T-cell lymphoblastic leukemia. Enforced expression of LMO2 in mouse models induces T-ALL with long latency suggesting that it requires cooperating oncogenic mutations for leukemia to fully manifest. To identify cooperating oncogenes, we analyzed spontaneous T-ALLs induced by retroviral insertional mutation, and T-ALLs that developed in B6.CD2-Lmo2 transgenic mice. First, we noted that the CD2-Lmo2 transgenic T-ALLs exclusively upregulated the class II basic helix-loop-helix transcription factor, Lyl1, whereas the retrovirally-induced T-ALLs showed upregulation of Tal1. We attributed this difference to the stage at which the Lmo2 activating mutations occurred. Nevertheless, we found that LYL1 protein upregulation is functionally important, as it is part of a macromolecular complex containing LMO2 that binds tandem E boxes as described for TAL1. LMO2 and LYL1 are known to be co-expressed in human T-ALL, but we found that they are part of a larger gene expression signature comprised of NMYC, HHEX, MEF2C, and BCL2 genes. The latter genes are remarkable because they are oncogenic when overexpressed; and, Mef2c and Nmyc are concordant insertions in independent tumors driven by Lmo2 or Hhex in retroviral insertional mutagenesis models. In fact, insertion site analysis suggests that Lmo2 and Hhex may actually operate in the same pathway and that Hhex may be a target of Lmo2. We tested this genetic result by chromatin immunoprecipitation and reporter assays and discovered that Lmo2 and its binding partners, Ldb1 and Lyl1, occupy an enhancer in the first intron of Hhex in T-ALL cells. Furthermore, LMO2 or LYL1 knockdown downregulated expression of HHEX in T-ALL. HHEX activation has significant functional consequences in T-ALL. We found that its upregulation is associated with T-ALL treatment resistance, clinical latency, and differential activation of Notch1 targets. These data offer important insight into the pathogenesis of Lmo2-induced T-ALL. Disclosures: No relevant conflicts of interest to declare.

Abstract 3979 Poster Board III-915 Mice that express 20% the normal levels of the Ets transcription factor Pu.1 develop AML, unlike mice that express 50% to 90% the normal levels, indicating that Pu.1 is a dosage-sensitive tumor suppressor gene. Furthermore, 3 of 13 AMLs induced by transplanting mice with cells transduced with a Sox4 oncogene-containing retrovirus were found to carry a Sox4 retroviral integration in one Pu.1 allele, suggesting that downregulation of Pu.1 may cooperate with Sox4 in AML induction. Since the other Pu.1 allele remains intact in these AMLs and a 50% decrease in Pu.1 expression is not sufficient to induce AML, we hypothesized that Sox4 might further downregulate Pu.1 expression in these AMLs. To test this hypothesis, we transfected HL60 promyelocytes with an expression vector carrying both GFP and Sox4 cDNAs or a GFP vector control. Transfected GFP+ cells were purified by flow cytometry and Pu.1 mRNA levels were analyzed by real-time RT-PCR. Pu.1 mRNA levels were consistently downregulated 4 to 10 fold in cells transfected with Sox4 cDNA compared to cells transfected with the vector control, while Beta-actin mRNA levels were maintained constant, confirming that overexpression of Sox4 downregulates Pu.1 expression in myeloid cells. The decrease of Pu.1 mRNA was observed as early as 8 hours after Sox4 transfection, further suggesting that Sox4 may directly repress the Pu.1 promoter in myeloid cells. Consistent with this, analysis of a published microarray databases comprising 285 de novo AML patient samples showed that SOX4 expression is significantly negatively correlated with Pu.1 expression (r= -0.337, p-value Disclosures: No relevant conflicts of interest to declare.

The LIM domain Only-2 (Lmo2) oncogene is deregulated in the majority of human T-cell acute lymphoblastic leukemias (T-ALL). Enforced, constitutive expression of Lmo2 in transgenic mice using the T-cell-specific Cd2 promoter/enhancer induces T-ALL with median latency of 200 days and 100% penetrance. These transgenic mice have a pre-leukemic differentiation block at the double negative stage (CD4− CD8−) of T-cell differentiation. Despite this, T-ALLs in these mice arise from various stages of T-cell differentiation. To explore the relationship between T-cell development and Lmo2 oncogene function, we established cell lines from four independent T-cell tumors representing the double negative (DN), double positive (DP), and single positive (SP) stages. Two of the lines showed variegated expression of CD4 antigen. One line, 32080, was striking in that CD4 was expressed in 50% of cells whereas CD8 was uniformly present. The cell line expressed markers consistent with the intermediate single positive stage (ISP) to double positive (DP) stage transition. We sorted the 32080 line for highly CD4 positive and negative cells and observed them in culture. After one week, both sorted populations showed variegated CD4 expression. We determined CD4 expression was regulated at the transcriptional level and not due to post-transcriptional effects and that cell replication was necessary to re-establish CD4 variegation. Importantly, variegated CD4 expression was dependent on Lmo2 because its knockdown resulted in decreased CD4 expression. HDAC inhibition by trichostatin A had the reciprocal effect of inducing more CD4 expression suggesting that chromatin modification was playing a role. The CD4 locus was analyzed by chromatin immunoprecipitation and histone H3 methylation patterns consistent with silencing were found in the CD4 negative sorted cells but not in the CD4 positive population. The 32080 cell line is a striking model of ISP to DP T-cell plasticity and invokes a novel mechanism by which Lmo2 functions.

Retroviruses induce cancer by integrating into the cellular genome and activating oncogenes or inactivating tumor suppressor genes. Human T-cell Leukemia Virus type 1 (HTLV-1), a complex retrovirus, induces Adult T-cell Leukemia/Lymphoma (ATLL) after a latency of over 30 years and in only 5% of carriers. The long latency and incomplete penetrance is similar to how slow transforming retroviruses induce cancer in mice and imply multiple oncogenic “hits” need to accumulate for clinically apparent disease. Insertional mutagenesis may be one mechanism by which ATLL develops. We used splinkerette-PCR to clone and map insertion sites from an HTLV-1 infected T-cell line, Hut-102. We identified an HTLV-1 insertion 5′ of the GLI2 gene, formerly known as Tax-Helper-Protein-1. We found GLI2 was up-regulated by promoter insertion. Interestingly, we found GLI2 protein occupied the HTLV-1 Long Terminal Repeat. The effect of GLI2 expression on viral expression was investigated by knockdown of GLI2 in Hut-102 cells. Our results show that retroviral insertional mutagenesis can be an important mechanism in HTLV-1-induced leukemias and lymphomas.