Enforced expression of E47 has differential effects on Lmo2-induced T-cell leukemias

Abstract

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.

Hhex is Required at Multiple Stages of Adult Hematopoietic Stem and Progenitor Cell Differentiation

“…Hhex gene expression was done in accordance with the Immgen data release policy and is available through this resource (http://www.immgen.org). Total RNA was purified by RliaPrep RNA Cell MiniPrep system (Promega, Madison, WI, http://www.promega.com) and prepared for Illumina sequencing as previously described . See Supporting Information methods for preparation of cDNA libraries for next generation sequencing (i.e., RNA‐seq).…”

“…Total RNA was purified by RliaPrep RNA Cell Mini-Prep system (Promega, Madison, WI, http://www.promega. com) and prepared for Illumina sequencing as previously described [21,32]. See Supporting Information methods for preparation of cDNA libraries for next generation sequencing (i.e., RNA-seq).…”

LMO2 Oncoprotein Stability in T-Cell Leukemia Requires Direct LDB1 Binding

“…Hence, since LMO2 mRNA was upregulated with the expression of LDB1⌬LID, we hypothesize that LDB1 normally represses LMO2 transcription in HSB-2 cells; along these lines, since the LYL1 mRNA level was decreased with the expression of LDB1⌬LID, this gene is normally activated by wild-type LDB1. We have not exhaustively analyzed the RNA-seq data, but several other intriguing mRNAs stand out for regulation by LDB1 complexes, such as ID2 and ID3, which are targets of E47 (50), and BLK, which is implicated as an oncogene (67,68). The dominant negative effects of mutant LDB1 proteins on growth and transcription suggest that the LMO2/LDB1 interaction is a rational target in T-cell leukemia and perhaps in AML as well (64,69).…”

“…RNA was ethanol precipitated, resuspended, and ethanol precipitated once more. The final resuspended RNA preparation was quantified for concentration and purity by using a NanoDrop instrument, and a portion was treated with DNase I and then processed for Illumina transcriptome sequencing (RNA-seq) library preparation and mapping as previously described (49,50). Differential gene expression was analyzed by using DEseq and EdgeR (51,52).…”

Experimental glioma with high bHLH expression harbor increased replicative stress and are sensitive toward ATR inhibition

“…E47 is a member of the bHLH family, 6 an alternative splicing product of E2A 6 , 11 and plays a crucial role in cancer, 12 neural development, and neural differentiation. 13–15 The overexpression of a dominant-negative E47 (dnE47), lacking its nuclear translocation signal leads to apoptosis, reduction of growth-capacity in vitro, and prolongation of symptom-free survival in vivo.…”