ATM-deficient thymic lymphoma is associated with aberrant tcrd rearrangement and gene amplification

Ataxia telangiectasia mutated (ATM) deficiency predisposes humans and mice to T lineage lymphomas with recurrent chromosome 14 translocations involving the T cell receptor α/δ (Tcra/d) locus. Such translocations have been thought to result from aberrant repair of DNA double-strand breaks (DSBs) during Tcra locus V(D)J recombination, and to require the Tcra enhancer (Eα) for Tcra rearrangement or expression of the translocated oncogene. We now show that, in addition to the known chromosome 14 translocation, ATM-deficient mouse thymic lymphomas routinely contain a centromeric fragment of chromosome 14 that spans up to the 5′ boundary of the Tcra/d locus, at which position a 500-kb or larger region centromeric to Tcra/d is routinely amplified. In addition, they routinely contain a large deletion of the telomeric end of one copy of chromosome 12. In contrast to prior expectations, the recurrent translocations and amplifications involve V(D)J recombination–initiated breaks in the Tcrd locus, as opposed to the Tcra locus, and arise independently of the Eα. Overall, our studies reveal previously unexpected mechanisms that contribute to the oncogenic transformation of ATM-deficient T lineage cells

ESRRB regulates glucocorticoid gene expression in mice and patients with acute lymphoblastic leukemia

Synthetic glucocorticoids (GCs), such as dexamethasone and prednisone, remain key components of therapy for patients with lymphoid malignancies. For pediatric patients with acute lymphoblastic leukemia (ALL), response to GCs remains the most reliable prognostic indicator; failure to respond to GC correlates with poor event-free survival. To uncover GC resistance mechanisms, we performed a genome-wide, survival-based short hairpin RNA screen and identified the orphan nuclear receptor estrogen-related receptor-beta (ESRRB) as a critical transcription factor that cooperates with the GC receptor (GR) to mediate the GC gene expression signature in mouse and human ALL cells. Esrrb knockdown interfered with the expression of genes that were induced and repressed by GR and resulted in GC resistance in vitro and in vivo. Dexamethasone treatment stimulated ESRRB binding to estrogen-related receptor elements (ERREs) in canonical GC-regulated genes, and H3K27Ac Hi-chromatin immunoprecipitation revealed increased interactions between GR- and ERRE-containing regulatory regions in dexamethasone-treated human T-ALL cells. Furthermore, ESRRB agonists enhanced GC target gene expression and synergized with dexamethasone to induce leukemic cell death, indicating that ESRRB agonists may overcome GC resistance in ALL, and potentially, in other lymphoid malignancies

The TAL1 complex targets the FBXW7 tumor suppressor by activating miR-223 in human T cell acute lymphoblastic leukemia

The oncogenic transcription factor TAL1/SCL is aberrantly expressed in 60% of cases of human T cell acute lymphoblastic leukemia (T-ALL) and initiates T-ALL in mouse models. By performing global microRNA (miRNA) expression profiling after depletion of TAL1, together with genome-wide analysis of TAL1 occupancy by chromatin immunoprecipitation coupled to massively parallel DNA sequencing, we identified the miRNA genes directly controlled by TAL1 and its regulatory partners HEB, E2A, LMO1/2, GATA3, and RUNX1. The most dynamically regulated miRNA was miR-223, which is bound at its promoter and up-regulated by the TAL1 complex. miR-223 expression mirrors TAL1 levels during thymic development, with high expression in early thymocytes and marked down-regulation after the double-negative-2 stage of maturation. We demonstrate that aberrant miR-223 up-regulation by TAL1 is important for optimal growth of TAL1-positive T-ALL cells and that sustained expression of miR-223 partially rescues T-ALL cells after TAL1 knockdown. Overexpression of miR-223 also leads to marked down-regulation of FBXW7 protein expression, whereas knockdown of TAL1 leads to up-regulation of FBXW7 protein levels, with a marked reduction of its substrates MYC, MYB, NOTCH1, and CYCLIN E. We conclude that TAL1-mediated up-regulation of miR-223 promotes the malignant phenotype in T-ALL through repression of the FBXW7 tumor suppressor

Molecular rationale for the use of PI3K/AKT/mTOR pathway inhibitors in combination with crizotinib in ALK-mutated neuroblastoma

Mutations in the ALK tyrosine kinase receptor gene represent important therapeutic targets in neuroblastoma, yet their clinical translation has been challenging. The ALKF1174L mutation is sensitive to the ALK inhibitor crizotinib only at high doses and mediates acquired resistance to crizotinib in ALK-translocated cancers. We have shown that the combination of crizotinib and an inhibitor of downstream signaling induces a favorable response in transgenic mice bearing ALKF1174L/MYCN-positive neuroblastoma. Here, we investigated the molecular basis of this effect and assessed whether a similar strategy would be effective in ALK-mutated tumors lacking MYCN overexpression. We show that in ALK-mutated, MYCN-amplified neuroblastoma cells, crizotinib alone does not affect mTORC1 activity as indicated by persistent RPS6 phosphorylation. Combined treatment with crizotinib and an ATP-competitive mTOR inhibitor abrogated RPS6 phosphorylation, leading to reduced tumor growth and prolonged survival in ALKF1174L/MYCN-positive models compared to single agent treatment. By contrast, this combination, while inducing mTORC1 downregulation, caused reciprocal upregulation of PI3K activity in ALK-mutated cells expressing wild-type MYCN. Here, an inhibitor with potency against both mTOR and PI3K was more effective in promoting cytotoxicity when combined with crizotinib. Our findings should enable a more precise selection of molecularly targeted agents for patients with ALK-mutated tumors

Antileukemic Activity of Nuclear Export Inhibitors that Spare Normal Hematopoietic Cells

Drugs that target the chief mediator of nuclear export, chromosome region maintenance 1 protein (CRM1) have potential as therapeutics for leukemia, but existing CRM1 inhibitors show variable potencies and a broad range of cytotoxic effects. Here, we report the structural analysis and antileukemic activity of a new generation of small-molecule inhibitors of CRM1. Designated selective inhibitors of nuclear export (SINE), these compounds were developed using molecular modeling to screen a small virtual library of compounds against the nuclear export signal (NES) groove of CRM1. The 2.2-Å crystal structure of the CRM1-Ran-RanBP1 complex bound to KPT-251, a representative molecule of this class of inhibitors, shows that the drug occupies part of the groove in CRM1 that is usually occupied by the NES, but penetrates much deeper into the groove and blocks CRM1-directed protein export. SINE inhibitors exhibit potent antileukemic activity, inducing apoptosis at nanomolar concentrations in a panel of 14 human acute myeloid leukemia (AML) cell lines representing different molecular subtypes of the disease. When administered orally to immunodeficient mice engrafted with human AML cells, KPT-251 had potent antileukemic activity with negligible toxicity to normal hematopoietic cells. Thus, KPT-SINE CRM1 antagonists represent a novel class of drugs that warrant further testing in AML patients

T-Lymphoblastic Lymphoma Cells Express High Levels of BCL2, S1P1, and ICAM1, Leading to a Blockade of Tumor Cell Intravasation

The molecular events underlying the progression of T-lymphoblastic lymphoma (T-LBL) to acute T-lymphoblastic leukemia (T-ALL) remain elusive. In our zebrafish model, concomitant overexpression of bcl-2 with Myc accelerated T-LBL onset while inhibiting progression to T-ALL. The T-LBL cells failed to invade the vasculature and showed evidence of increased homotypic cell-cell adhesion and autophagy. Further analysis using clinical biopsy specimens revealed autophagy and increased levels of BCL2, S1P1, and ICAM1 in human T-LBL compared with T-ALL. Inhibition of S1P1 signaling in T-LBL cells led to decreased homotypic adhesion in vitro and increased tumor cell intravasation in vivo. Thus, blockade of intravasation and hematologic dissemination in T-LBL is due to elevated S1P1 signaling, increased expression of ICAM1, and augmented homotypic cell-cell adhesion.Stem Cell and Regenerative Biolog

The TAL1 complex targets the FBXW7 tumor suppressor by activating miR-223 in human T cell acute lymphoblastic leukemia

The oncogenic transcription factor TAL1/SCL is aberrantly expressed in 60% of cases of human T cell acute lymphoblastic leukemia (T-ALL) and initiates T-ALL in mouse models. By performing global microRNA (miRNA) expression profiling after depletion of TAL1, together with genome-wide analysis of TAL1 occupancy by chromatin immunoprecipitation coupled to massively parallel DNA sequencing, we identified the miRNA genes directly controlled by TAL1 and its regulatory partners HEB, E2A, LMO1/2, GATA3, and RUNX1. The most dynamically regulated miRNA was miR-223, which is bound at its promoter and up-regulated by the TAL1 complex. miR-223 expression mirrors TAL1 levels during thymic development, with high expression in early thymocytes and marked down-regulation after the double-negative-2 stage of maturation. We demonstrate that aberrant miR-223 up-regulation by TAL1 is important for optimal growth of TAL1-positive T-ALL cells and that sustained expression of miR-223 partially rescues T-ALL cells after TAL1 knockdown. Overexpression of miR-223 also leads to marked down-regulation of FBXW7 protein expression, whereas knockdown of TAL1 leads to up-regulation of FBXW7 protein levels, with a marked reduction of its substrates MYC, MYB, NOTCH1, and CYCLIN E. We conclude that TAL1-mediated up-regulation of miR-223 promotes the malignant phenotype in T-ALL through repression of the FBXW7 tumor suppressor.National Cancer Institute (U.S.) (5P01CA109901)National Cancer Institute (U.S.) (5P01CA68484)National Cancer Institute (U.S.) (1K99CA157951)National Institutes of Health (U.S.). Intramural Research ProgramCenter for Cancer Research (National Cancer Institute (U.S.)

Repression of tumor suppressor miR-451 is essential for NOTCH1-induced oncogenesis in T-ALL

miR-451 represses expression of Myc and acts as a tumor suppressor in murine and human T cell acute lymphoblastic leukemia

Targeting transcription regulation in cancer with a covalent CDK7 inhibitor

Tumor oncogenes include transcription factors that co-opt the general transcriptional machinery to sustain the oncogenic state1, but direct pharmacological inhibition of transcription factors has thus far proven difficult2. However, the transcriptional machinery contains various enzymatic co-factors that can be targeted for development of new therapeutic candidates3, including cyclin-dependent kinases (CDKs)4. Here we present the discovery and characterization of the first covalent CDK7 inhibitor, THZ1, which has the unprecedented ability to target a remote cysteine residue located outside of the canonical kinase domain, providing an unanticipated means of achieving selectivity for CDK7. Cancer cell line profiling indicates that a subset of cancer cell lines, including T-ALL, exhibit exceptional sensitivity to THZ1. Genome-wide analysis in Jurkat T-ALL shows that THZ1 disproportionally affects transcription of RUNX1 and suggests that sensitivity to THZ1 may be due to vulnerability conferred by the RUNX1 super-enhancer and this transcription factor’s key role in the core transcriptional regulatory circuitry of these tumor cells. Pharmacological modulation of CDK7 kinase activity may thus provide an approach to identify and treat tumor types exhibiting extreme dependencies on transcription for maintenance of the oncogenic state

Targeting transcription regulation in cancer with a covalent CDK7 inhibitor

Tumour oncogenes include transcription factors that co-opt the general transcriptional machinery to sustain the oncogenic state, but direct pharmacological inhibition of transcription factors has so far proven difficult. However, the transcriptional machinery contains various enzymatic cofactors that can be targeted for the development of new therapeutic candidates, including cyclin-dependent kinases (CDKs). Here we present the discovery and characterization of a covalent CDK7 inhibitor, THZ1, which has the unprecedented ability to target a remote cysteine residue located outside of the canonical kinase domain, providing an unanticipated means of achieving selectivity for CDK7. Cancer cell-line profiling indicates that a subset of cancer cell lines, including human T-cell acute lymphoblastic leukaemia (T-ALL), have exceptional sensitivity to THZ1. Genome-wide analysis in Jurkat T-ALL cells shows that THZ1 disproportionally affects transcription of RUNX1 and suggests that sensitivity to THZ1 may be due to vulnerability conferred by the RUNX1 super-enhancer and the key role of RUNX1 in the core transcriptional regulatory circuitry of these tumour cells. Pharmacological modulation of CDK7 kinase activity may thus provide an approach to identify and treat tumour types that are dependent on transcription for maintenance of the oncogenic state.National Institutes of Health (U.S.) (Grant HG002668)National Institutes of Health (U.S.) (Grant CA109901