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.

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.

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 and Early T-cell Precursor ALL. These results are significant in light of the crucial role of Lmo2 in the maintenance of the hematopoietic stem cell. The cellular effects and transcriptional effects have implications for LMO2-dependent leukemogenesis and the treatment of LMO2-induced T-ALL.

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.

These results underscore the utility of profiling occurrences of resistance to standard regimens and support JAK enzymes as rational therapeutic targets for T-cell leukemias and lymphomas.

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 (ISP) to double positive (DP) stages of T-cell development. 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 negative cells and observed them in culture. After one 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 HDAC activity, induced CD4 expression. Enforced expression of Runx1 repressed CD4 expression. We analyzed the CD4 locus by H3 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 ISP to DP T-cell plasticity and invokes a novel mechanism for Lmo2's oncogenic functions.

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.

Background: T-cell leukemias and lymphomas (TL&Ls) are rare when compared with other hematologic malignancies, and pose a challenge for clinical management. TL&Ls are characteristically resistant to chemotherapeutic regimens used to treat other hematologic malignancies (PMID 17369126, 22649104, 25042790). Recently, activating JAK1 and JAK3 mutations have been described in a subset of mature TL&Ls including adult T-cell leukemias/lymphoma (ATLL), NK/T-cell lymphoma, and T-cell prolymphocytic leukemia (T-PLL). We used a comprehensive hybrid capture-based sequencing assay to identify genomic alterations that might suggest benefit from targeted therapy. Methods and Results. Using FoundationOne® and FoundationOne® Heme assays, we performed genomic profiling on 28 consecutive TL&Ls (leukemias, n=10; lymphomas, n=18) for all alterations in 370 or 405 cancer-related genes and select introns of 19 or 31 genes commonly rearranged in cancer, or both DNA and RNA were extracted and captured using custom baits for 405 genes and 265 frequently rearranged genes, respectively. The samples were sequenced to high uniform coverage, averaging >500X for DNA and >8M total pairs for RNA (PMID 24142049). JAK3 alterations were the most common at 25% (7/28) with an enrichment in T-cell derived leukemias (2 T-PLL, 3 T-ALL, and 1 cutaneous T-cell lymphoma (CTCL)). Other altered genes in the 28 cases included CDKN2A/B (21%), TP53 (18%), and TET2 , DNMT3A , and STAT3 (11% each). A complex rearrangement involving FCHO1 and JAK3 resulting from a ~70kb genomic segment deletion was also identified in a CTCL. The most frequently co-altered genes identified in the JAK3 mutated cases were relevant to the chromatin remodeling pathway. Focal STAT3, STAT5A, and STAT5B amplifications were identified in 2 cases (CTCL and ALCL) as well as one STAT3 D661Y in a T-cell large granular lymphocytic leukemia (T-LGL). All 3 cases with STAT gene alterations were mutually exclusive with JAK3 alterations. Additionally, 2 patients with T-PLL and T-ALL respectively were both found to harbor multiple co-existing JAK1 and JAK3mutations (2/28; 7%). One heavily pre-treated T-PLL patient harbored a major JAK1 V658F clone (40%) and a minor JAK3 M5111 clone (5%), and loss of heterozygosity for mutant ATM and TP53. Based on assay results, the patient was treated with ruxolitinib, a potent and specific inhibitor of JAK1 and JAK2. The patient had an impressive clinical response of duration of 4 months after which her T-PLL cell count rose markedly to pre-treatment levels and she developed worsening thrombocytopenia, indicating acquired resistance to therapy. The resistant T-PLL was assayed and was found to harbor an increased frequency of JAK3 M5111 (28%), decrease of JAK1 V658F (18%) and a new EZH2mutation. The patient’s disease progressed through another line of cytotoxic chemotherapy and she succumbed to her disease. Signal transduction analysis was conducted which showed in T-PLL, the JAK1 and JAK3 mutations were functionally significant as they induced constitutive phosphorylation of downstream STAT proteins. Importantly, these phosphorylations could be inhibited by JAK inhibitors. Collection of clinical outcome on additional cases is ongoing. Conclusion. In this study, we discovered a high frequency of JAK-STAT alterations in 28 T-cell neoplasms with 25% of cases harboring JAK3 missense mutations and two cases with concordant JAK1 and JAK3 mutations. In one case, treatment with the JAK1/2 inhibitor, ruxolitinib, resulted in an impressive clinical response. Our data argue for oncogene dependence upon the JAK-STAT pathway in diverse T-cell neoplasms and underscore the importance of genomic profiling by FoundationOne Heme® of rare and difficult to treat cases by revealing alterations that may be targeted by specific inhibitors, such as ruxolitinib, and may also be predictive of treatment resistance. Disclosures Off Label Use: Ruxolitinib is FDA approved for myeloproliferative disorders.. Palma:Foundation Medicine, Inc.: Employment, Equity Ownership. Wang:Foundation Medicine Inc: Employment. Ali:Foundation Medicine, Inc.: Employment, Equity Ownership. Stephens:Foundation Medicine: Employment, Equity Ownership. Miller:Foundation Medicine, Inc.: Employment, Equity Ownership.