C-Myc, but Not Akt, Can Substitute for Notch in Lymphomagenesis.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 7-7
Author(s):  
Mark Y Chiang ◽  
M. Eden Childs ◽  
Candice Romany ◽  
Olga Shestova ◽  
Jon Aster ◽  
...  
Keyword(s):  
T Cell ◽  
Mouse Model ◽  
Selective Pressure ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Gamma Secretase ◽  
Growth And Survival ◽  
Strong Selective Pressure ◽  
Human T Cell ◽  
Notch1 Mutations

Abstract Abstract 7 Notch signaling is activated in ∼70% of human T-cell acute lymphoblastic leukemia/lymphoma (T-ALL) samples and many human and mouse T-ALL cell lines require Notch signals for growth and survival. To gain insight into the role of Notch during induction of T-ALL, we used a fully penetrant, conditional, transgenic KrasG12D mouse model in which ∼80% of T-ALLs acquire activating Notch1 mutations in the endogenous locus. We crossed mice bearing this transgene with Rosa26-DNMAMLf/f mice, which conditionally express the pan-Notch inhibitor DNMAML. T-ALL developed in these mice despite the expression of DNMAML throughout T-cell development. ∼75% of T-ALL tumors acquired activating Notch1 mutations and suppressed expression of DNMAML, which is consistent with frequent “escape” of Notch from inhibition for efficient T-ALL development. We next compared T-ALL cells that lacked DNMAML expression with T-ALL cells that continued to express DNMAML. T-ALL cells lacking DNMAML expressed the direct Notch target c-Myc at higher levels, proliferated at a higher rate, and contained ∼10-fold higher levels of leukemia-initiating cells. Moreover, DNMAML-positive T-ALLs lost DNMAML after transfer into secondary recipients. These data underscore the strong selective pressure for Notch signals during generation and maintenance of T-ALL. We next sought a mechanistic answer for the strong selective pressure for Notch activation. c-Myc and Akt have both been posited to be critical targets of oncogenic Notch signals. To compare the relative contributions of c-Myc and Akt to lymphomagenesis, we overexpressed c-Myc and activated AKT in the KrasG12D-driven mouse model. T-ALLs induced by KrasG12D and Akt acquired activating Notch1 mutations in ∼70% of tumors, which were sensitive to Notch inhibitors (gamma-secretase inhibitors [GSI]). In contrast, T-ALLs induced by KrasG12D and c-Myc did not acquire Notch1 mutations and were resistant to GSI. We conclude that upregulation of c-Myc is sufficient to substitute for Notch in lymphomagenesis, whereas activation of Akt signaling is not. These data identify c-Myc not AKT as the driving force behind Notch-induced lymphomagenesis. These data emphasize the Notch/c-Myc axis as an attractive, rational, therapeutic target in T-ALL. Disclosures: No relevant conflicts of interest to declare.

scholarly journals A New Mechanism for T-ALL Leukemogenesis: Early Expression of Alpha-Beta TCR Occurs in a Natural Subset of CD4-CD8- Double Negative (DN) Thymocytes Where MHC Engagement May Drive NOTCH Mutation and Tumor Initiation

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1463-1463
Author(s):  
Kimberly G Laffey ◽  
Robert J Stiles ◽  
Melissa Ludescher ◽  
Tessa Davis ◽  
Shariq S Khwaja ◽  
...  
Keyword(s):  
T Cell ◽  
Mouse Model ◽  
Antigen Presentation ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Dependent Manner ◽  
Double Negative ◽  
Tcr Signaling ◽  
Deficient Mice ◽  
Notch1 Mutations

T cell lymphoblastic leukemia (T-ALL) is an aggressive cancer arising from transformed thymocytes. Most human T-ALL involves hyperactive NOTCH signaling that is often caused by activating NOTCH mutations. However, the identification of specific molecular signals that might induce or select for mutation and transformation are incompletely understood. We report that an understudied low-frequency, natural thymocyte subset expresses αβ T cell antigen receptor (TCR) earlier than most cells in mice and humans; engagement of the early αβTCR by major histocompatibility complexes (MHC) can cause outgrowth of NOTCH1 mutant clones and T-ALL leukemogenesis in a mouse model of T-ALL. Assessment of 5 recent human T-ALL cases found one to present this unique CD4-CD8- double- negative (DN) stage as the earliest identifiable developmental stage. These studies present a model of T-ALL leukemogenesis that identifies (i) a natural cell stage of origin susceptible to transformation, (ii) a matching mouse model showing that a signaling receptor (αβTCR) and its ligand (MHC) drive leukemogeneis and outgrowth of tumors bearing activating NOTCH1 mutations, and (iii) a human case that presents with a tumor consistent with this model and mechanism. In past work, the pre-TCR has been shown to impact T-ALL development in mice (Campese et al, Blood 2006), but an oncogenic role for the mature αβTCR is less well characterized and somewhat surprising. This is because, although T-ALL tumor cells may express variable levels of surface αβTCR/CD3, the earliest cell stages that are thought to transform are also thought to precede stages with αβTCR expression. Most conventional αβ thymocytes rearrange TCRβ and TCRα loci in separate, ordered developmental stages. However, some thymocytes in the conventional pathway rearrange both at DN stage thus exhibiting 'precocious' αβTCR (PAT) expression. Importantly, these PAT cells are indeed part of the conventional αβ lineage, being a 'subset' only due to early αβTCR expression but without known distinction in ultimate immune function (Aifantis et al, JEM 2006). We found that ~0.01% of mouse and human thymocytes are such PAT cells at steady state. To interrogate the PAT thymocyte surface phenotype, we performed multi-parametric flow cytometry with Spanning-tree Progression Analysis of Density-normalized Events (SPADE). This revealed that PAT thymocytes constitute a DN subset that is not associated with other well-described unconventional DN thymocytes known to express αβTCR, consistent with as the expectation that PATs are part of the conventional developmental pathway. We observed that the OT1 TCR transgene is expressed in mice with parallel timing and level to the natural PAT subset, allowing use of this model to study antigen-dependent signaling and oncogenesis. In a cohort study, no T-ALL was observed in wild-type C57BL/6 or OT-1.β2M-/- mice (deficient in endogenous antigen presentation), but MHC-sufficient OT-1 mice developed PAT-stage-specific T-ALL with activating NOTCH1 mutations. Transplant experiments corroborated a requirement for antigen presentation and TCR signaling for tumor maintenance as transplanted tumors grew in MHC+ but not MHC-deficient mice. This predicted that PAT thymocytes might have an unusual ability to signal through αβTCR even without coreceptor expression. When cultured in the presence of either exogenously added β2M or antigen presenting cells, both untransformed and neoplastic PAT cells upregulated CD69 in response to the OT-1 antigenic peptide, OVA. Furthermore, ex vivo analysis of PAT cells from polyclonal C57BL/6 versus MHC-deficient mice showed intrinsic upregulation of TCR-signaling-dependent Nur77 in an MHC-dependent manner. These data revealed a unique ability of PAT cells to engage in co-receptor independent but antigen-dependent signaling. Microarray analysis showed that the gene expression profile of neoplastic PAT cells from OT-1 T-ALL most closely resembled that of conventional post β-selection DN thymocytes, in agreement with the natural PAT stage during normal T cell development. These data support a model in which transformation occurred in the naturally occurring αβ PAT thymocyte subset as cell-of-origin. Collectively, our data suggest that precocious αβTCR expression and coreceptor-independent antigen engagement can cause activating NOTCH mutation and T-ALL development. Disclosures No relevant conflicts of interest to declare.

Kruppel-Like Factor, Klf9, Promotes Proliferation of Notch-Independent Precursor T-Cell Lymphoblastic Lymphomas

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3496-3496
Author(s):  
Peggy P. C. Wong ◽  
Daniele Merico ◽  
Irina Matei ◽  
Vicki Ling ◽  
Shaheena Bashir ◽  
...  
Keyword(s):  
T Cell ◽  
Conformational Changes ◽  
Conflicts Of Interest ◽  
Expression Profiles ◽  
Functional Dependence ◽  
Lymphoblastic Leukemia ◽  
Gene Expression Profiles ◽  
Notch1 Signaling ◽  
The Difference ◽  
Notch1 Mutations

Abstract Abstract 3496 Notch1 signaling is required at multiple stages of normal T-lymphocyte development. Notch1 is a transmembrane receptor that is physiologically activated when Notch ligands induce conformational changes that allow Notch1 cleavage by the intramembranous γ-secretase complex, releasing active intracellular Notch1 (ICN1) fragment from the plasma membrane. Activating NOTCH1 mutations are very frequent in human and mouse T-cell lymphoblastic leukemia/lymphoma (T-LL). Typically, these mutations promote ligand-independent Notch1 cleavage by γ-secretase or increase ICN1 stability by truncating the C-terminal PEST domain. Understandably, much effort has focused on elucidating mechanisms of normal and oncogenic Notch1 signaling. However, some studies suggest that the absence of NOTCH1 mutations portends a worse prognosis for human T-LL. Therefore, we set out to define signals that promote proliferation and survival of T-LL cells lacking activated Notch1. We used Western blotting to detect γ-secretase cleaved ICN1 protein in a cohort of 35 primary T-LLs that developed spontaneously in mice lacking the Ataxia telangiectasia mutated (Atm) tumor suppressor. We identified 3 ICN1 subgroups: 63% expressed PEST-truncated ICN1 (T-ICN1); 17% expressed non-truncated ICN1 (NT-ICN1); and 20% had undetectable ICN1 (UD-ICN1), most lacked Notch1 mRNA. We confirmed the difference in Notch transcriptional activity and functional dependence between the UD-ICN1 and T-ICN1 subgroups and then compared their gene expression profiles to define pathways unique to the UD-ICN1 group. Gene set enrichment analyses revealed that UD-ICN1 T-LLs expressed higher levels of Klf9 and other transcription factors associated with a highly proliferative stage of normal T-cell development. siRNA knock-down studies demonstrated that Klf9 promoted proliferation of UD-ICN1 but not T-ICN1 T-LL cells. Collectively, these data demonstrate that Klf9 can regulate proliferation of Notch-independent T-LLs and suggest that Klf9 may provide a novel therapeutic target for human T-LLs lacking activating NOTCH1 mutations. Disclosures: No relevant conflicts of interest to declare.

A CXCL10/CXCR3 Driven Thymic Epithelium-Leukemia Cell Crosstalk Augments T Cell Acute Lymphoblastic Leukemia Notch1 Signalling

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2537-2537
Author(s):  
Ashwini M Patil ◽  
Stefanie Kesper ◽  
Vishal Khairnar ◽  
Marco Luciani ◽  
Michael Möllmann ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Mouse Model ◽  
Cell Line ◽  
Cell Lines ◽  
Research Funding ◽  
Lymphoblastic Leukemia ◽  
Thymic Epithelial Cells ◽  
Hematopoietic Organ ◽  
Notch1 Mutations

Introduction: The thymus is a specialized hematopoietic organ, which is responsible for the generation of T cells. The central thymic cell type controlling T cell development are thymic epithelial cells (TECs). Based on their specific function and anatomic location TECs are separated into cortical and medullary subsets (cTECs and mTECs). cTECs express pivotal NOTCH-ligands such as DLL4 controlling T cell lineage commitment while mTECs play a central role in negative selection of developing T cells. Acquisition of NOTCH1 gain-of-function mutations play a central role in acute T cell lymphoblastic leukemia (T-ALL) development. During T-ALL leukemogenesis aberrant expression of transcription factors such as SCL/TAL1 and LMO1 block T cell differentiation and increase self-renewal while NOTCH1 mutations promote survival and proliferation. Since most acquired NOTCH1 mutations still require ligand binding to exert augmented signaling we propose DLL4-expressing TECs playing a critical role during T-ALL leukemogenesis. Methods: In the present study, we used a Scl/Lmo1 T-ALL transgenic mouse model, murine ANV and TE71 TEC cell lines and human T-ALL cell lines (Jurkat, ALL-SIL, DND-41, and HPB-ALL) to investigate TEC dynamics and function in the T-ALL context. Results: First, we demonstrated T-ALL supporting potential of TEC cell lines in vitro, which was comparable to the mesenchymal cell line OP9. Next, we showed in the Scl/Lmo1 T-ALL mouse model which had a mean survival rate of 90 days that preleukemic thymocytes displayed a striking upregulation of Notch1 target genes. Interestingly, fluorescence microscopy revealed a relative expansion of cortical and a relative reduction of the medullary thymic areas in Scl/Lmo1 thymi (Fig. 1A). Correspondingly, absolute numbers of cTECs expanded while mTEC numbers declined (Fig. 1B). Gene expression profiling of sorted preleukemic Scl/Lmo1 cTECs revealed upregulation of the chemokine CXCL10 (Fig. 1C). Moreover, increased CXCL10 chemokine concentrations were detected in Scl/Lmo1 thymic interstitial fluid (Fig.1D). Strikingly, we demonstrated T-ALL dependence of TEC Cxcl10 upregulation. We showed that Cxcl10 upregulation in TEC cell lines was only induced by direct cellular contact with Scl/Lmo1 thymocytes while wild-type control thymocytes did not alter TEC cell line Cxcl10 expression (Fig. 1E). Next, a high proportion of the CXCL10 receptor CXCR3 expressing cells was revealed in Scl/Lmo1 thymi (Fig. 1F) and by human T-ALL cell lines. Finally, we demonstrated a CXCL10 dependent pro-survival effect within cultured SCL/LMO1 thymocytes (Fig. 1G), which was associated with the activation of NOTCH1 signaling (Fig. 1H). Conclusions: In summary, the data support a novel T-ALL-promoting regulatory circuit in which emerging T-ALL lymphoblasts induce CXCL10 in expanding TECs which positively feeds back to T-ALL cells via the CXCL10 receptor CXCR3. Disclosures Dührsen: Celgene: Research Funding; Takeda: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Research Funding; Teva: Honoraria; Novartis: Consultancy, Honoraria; Alexion: Honoraria; Roche: Honoraria, Research Funding; CPT: Consultancy, Honoraria; Janssen: Honoraria. Göthert:Proteros Biostructures: Consultancy; Novartis: Consultancy, Honoraria, Other: Travel support; Pfizer: Consultancy, Honoraria; Incyte: Consultancy, Honoraria, Other: Travel support; Bristol-Myers Squibb: Consultancy, Honoraria, Other: Travel support; AOP Orphan Pharmaceuticals: Honoraria, Other: Travel support.

KLF4 Acts As a Tumor Suppressor in T-Acute Lymphoblastic Leukemia and Negatively Regulates Human T Cell Development and Homeostasis

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2405-2405
Author(s):  
Bing Xu ◽  
Peng Li
Keyword(s):  
T Cells ◽  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Tumor Suppressor ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
T Cell Development ◽  
Cell Development ◽  
Dependent Manner ◽  
Human T Cell

Abstract The transcription factor Kruppel-like factor 4 (KLF4) may induce tumorigenesis or suppress tumor growth in a tissue-dependent manner. We found that overexpression of KLF4 induced not only human acute T-acute lymphoblastic leukemia (T-ALL) cell lines but also primary samples from T-ALL patients to undergo apoptosis through the BCL2/BCLXL pathway in vitro. T cell-associated genes including BCL11B, GATA3, and TCF7 were negatively regulated by KLF4 overexpression. Especially, KLF4 induced SUMOylation and degradation of BCL11B. However, the KLF4-induced apoptosis in T-ALL was rescued by the in vivo microenvironment. Furthermore, the invasion capacity of T-ALL to hosts was compromised when KLF4 was overexpressed. In normal human T cells, the overexpression of KLF4 severely impaired T cell development at early stages, but the blockage of T cell development was resumed by restoration of GATA3 or ICN1. In summary, our data demonstrate that KLF4 acts as a tumor suppressor in malignant T cells and that downregulation of KLF4 may be a prerequisite for early human T cell development and homeostasis. Disclosures No relevant conflicts of interest to declare.

scholarly journals Clinical Significance of Co-Existence of PHF6 and NOTCH1 mutations in Adult T-Cell Acute Lymphoblastic Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1300-1300
Author(s):  
Zheng Ge ◽  
Min Li ◽  
Lichan Xiao ◽  
Run Zhang ◽  
Jianyong Li
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Synergistic Effect ◽  
Clinical Significance ◽  
Conflicts Of Interest ◽  
Survival Curve ◽  
Lymphoblastic Leukemia ◽  
Mrna Level ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Notch1 Mutations

Abstract Objective: T-cell acute lymphoblastic leukemia (T-ALL) is caused by collaboration of multiple genetic abnormalities in the transformation of T-cell progenitors. PHF6 is founded as a new key tumor suppressor and mutated in T-ALL. The clinical significance of PHF6 mutations has not been fully determined in adult T-ALL. This study aimed to screen the PHF6 mutations in adult T-ALL and explore the associations of PHF6 mutations with other genetic lesions, as well as their clinical relevance in adult T-ALL patients. Methods: We amplified the exons of PHF6, NOTCH1, FBXW7, PTEN and JAK1 following by DNA sequencing to identify the genomic mutations and examined the PHF6 mRNA level by qPCR in adult T-ALL patients. We also analyzed the correlations of PHF6 and NOTCH1 mutations with clinical features using a χ2 test and survival curve using the Kaplan-Meier method. Results: The 27.1% (16/59) PHF6 mutations including 10 novel mutations were detected in Chinese adult T-ALL. Six of 16 (37.5%) were frame-shift mutations, which could result in the deletion of the protein. We also observed PHF6 expression was significantly lower in T-ALL patients with PHF6 mutations compared with wide type cases (0.00423 vs. 0.06464, P=0.035) , indicating PHF6 mutations could be loss of function. Moreover, PHF6 mutation was significantly associated with NOTCH1 mutation(P=0.035). We further analyzed the domains involving co-existence mutations of NOTCH1 with PHF6. The most commonly mutated domains in NOTCH1 co-existed with PHF6 were HD-N only 6/12 (50.0%), followed by HD-C only 2/12(16.7%), PEST only 2/12(16.7%), HD-C+PEST 1/12(8.3%) and HD-N+HD-C 1/12(8.3%), indicating that HD domain (especially HD-N) of NOTCH1 may contribute to the synergistic effect on oncogenesis of the two genes. Furthermore, the patients with co-existence of PHF6 and NOTCH1 mutations had lower hemoglobin and higher incidence of splenomegaly or lymphadenopathy compared to that without co-existence of the mutations (95.0 vs 122.0, P=0.007; 81.8% vs 38.3%, P=0.009; 90.9% vs 44.7%, P=0.006). Importantly, the patients with co-existence of mutations in PHF6 and NOTCH1 (PHF6mutNOTCH1mut) had significant shorter event-free survival (EFS) compared with that without co-existence (non-PHF6mutNOTCH1mut)(2.0 months vs. 12.0 months, P=0.027). Conclusion: PHF6 is inactivated in T-ALL due to its low expression and mutations. PHF6 mutation is co-existed with NOTCH1 mutations, and the patients with PHF6mutNOTCH1mut had a poor prognosis. Our results indicated synergistic effect of PHF6 and NOTCH1 mutations on leukemogenesis and PHF6mutNOTCH1mut may be potential prognostic marker in adult T-ALL. Disclosures No relevant conflicts of interest to declare.

scholarly journals High selective pressure for Notch1 mutations that induce Myc in T-cell acute lymphoblastic leukemia

Blood ◽  
2016 ◽  
Vol 128 (18) ◽  
pp. 2229-2240 ◽  
Author(s):  
Mark Y. Chiang ◽  
Qing Wang ◽  
Anna C. Gormley ◽  
Sarah J. Stein ◽  
Lanwei Xu ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Selective Pressure ◽  
Lymphoblastic Leukemia ◽  
Key Points ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Notch Inhibition ◽  
Notch1 Mutations

Key Points Notch1 mutations are selected in the murine T-ALL model despite genetic pan-Notch inhibition; other pathways do not easily substitute it. Myc is the key Notch target responsible for Notch-selective pressure in T-ALL as it can substitute for Notch; by contrast, Akt cannot.

Gain-of-Function NOTCH1 Mutations Occur Frequently in Human T Cell Acute Lymphoblastic Leukemia.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4-4
Author(s):  
Andrew P. Weng ◽  
Adolfo A. Ferrando ◽  
Woojoong Lee ◽  
John P. Morris ◽  
Lewis B. Silverman ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Cell Lines ◽  
Reporter Gene ◽  
Lymphoblastic Leukemia ◽  
Reporter Gene Activity ◽  
Human T Cell ◽  
Cell Acute Lymphoblastic Leukemia ◽  
In Cis ◽  
Notch1 Mutations

Abstract NOTCH1 was discovered originally through its involvement in a rare (7;9) translocation found in human T cell acute lymphoblastic leukemia (T-ALL). Here, we report that >50% of human T-ALLs have activating NOTCH1 mutations, occurring as amino acid substitutions in an extracellular heterodimerization (HD) domain and/or as frameshift and stop codon mutations that result in the deletion of a C-terminal PEST destruction box. Normal pro-NOTCH1 is processed into a heterodimer consisting of an extracellular subunit and a transmembrane subunit, which associate non-covalently through the HD domain. NOTCH1 activation is triggered by binding of Serrate or Delta-like ligands to the extracellular subunit, which induces successive proteolytic cleavages in the transmembrane subunit that are dependent on i) metalloproteases and ii) gamma-secretase. The γ-secretase cleavage releases intracellular NOTCH1 (ICN1), which translocates to the nucleus and forms a transcriptional activation complex with the transcription factor CSL and co-activators of the Mastermind family. Normal turnover of ICN1 is regulated by the C-terminal PEST sequence. Data pointing to the existence of frequent abnormalities of NOTCH1 in T-ALL stemmed from a functional screen of 30 T-ALL cell lines. This identified five T-ALL cell lines that underwent growth arrest in response to i) treatment with an inhibitor γ-secretase, and ii) retroviral transduction of dominant negative Mastermind-like-1. Sequencing of of cDNAs from 4 of these 5 cell lines demonstrated both a missense mutation in the HD domain and a frameshift mutation in the PEST domain lying in cis in the same NOTCH1 allele. Subsequent sequencing of genomic DNA obtained from bone marrow lymphoblasts of 96 children and adolescents with T-ALL demonstrated identical or similar mutations in NOTCH1 in 53 samples (55.2%). Mutations in the HD domain alone were observed in 26 cases (27.1%), in the PEST domain alone in 11 cases (11.4%), and in both the HD and PEST domains in 16 cases (16.7%). Mutations were observed in tumors associated with expression of HOX11 (2/3), HOX11L2 (10/13; 77%), TAL1 (12/31; 39%), LYL1 (9/14; 64%), MLL-ENL (1/3) or CALM-AF10 (1/2), which span the major molecular T-ALL subtypes. In contrast, NOTCH1 mutations were not observed in genomic DNAs samples obtained from B-ALL lymphoblasts (N=89), or from T-ALL patients with NOTCH1-associated disease at the time of clinical remission (N=4). Reporter gene assays conducted with plasmids expressing normal and mutated forms of NOTCH1 showed that a PEST deletion or various HD mutations alone caused ~1.5-fold and 3–9-fold stimulations of reporter gene activity, respectively, whereas normal NOTCH1 lacked intrinsic signaling activity. More strikingly, the combination of various HD mutations and a PEST deletion in cis caused synergistic 20–40-fold stimulations of reporter gene activity that were completely abrogated by a γ-secretase inhibitor, indicating that signaling depends on proteolysis. These results suggest a model in which HD domain mutations promote ICN1 production, and PEST domain mutations enhance ICN1 stability. Our findings greatly expand the role of NOTCH1 in the pathogenesis of human T-ALL, and provide a rationale for targeted therapies that interfere with NOTCH signaling.

Acquired Notch1 Mutations in Murine Precursor-T Leukemia/Lymphoma Models.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2612-2612
Author(s):  
Yingwei Lin ◽  
Rebecca A. Nichols ◽  
John J. Letterio ◽  
Peter D. Aplan
Keyword(s):  
T Cell ◽  
Transgenic Mice ◽  
Cell Lines ◽  
Nude Mice ◽  
Lymphoblastic Leukemia ◽  
Gamma Secretase ◽  
Deletion Mutations ◽  
Wide Range ◽  
Notch1 Mutations ◽  
Thymic Tumors

Abstract NOTCH1 has been implicated in hematopoiesis, T-cell differentiation, and the pathogenesis of precursor T-cell lymphoblastic leukemia/lymphoma (pre-T LBL). Although rare patients with pre-T LBL have chromosomal translocations that activate NOTCH1, it has recently been shown that over 50% of human pre-T LBL samples which did not have chromosomal aberrations involving NOTCH1 had activating mutations in the NOTCH1 heterodimerization (HD) and/or the PEST domain. We examined murine pre-T LBL cell lines as well as primary thymic tumors that arose in SCL/LMO1, OLIG2, OLIG2/LMO1, LMO1, NUP98/HOXD13 transgenic mice, and [p27(− /−) /SMAD3(−/+)] mice for evidence of Notch1 mutations. We also investigated the timing of Notch1 mutation in SCL/LMO1 transgenic mice. We found that 13/19 cell lines and 29/49 primary thymic tumors had Notch1 mutations in either the HD or PEST domain, but not both. Of the thirteen cell lines with Notch1 mutations, twelve had mutations in the PEST domain. The cell lines with Notch1 mutations were sensitive to gamma-secretase inhibitor treatment, indicating that the mutations were important for the survival of these cells. Of twenty-nine tumors with Notch1 mutations, 23 were in the PEST domain and 6 in the HD. All HD mutations were single base substitutions, whereas all PEST domain mutations were insertion/deletion mutations. Intriguingly, half of the PEST domain mutations mapped to one of two mutational hot spots, suggesting that these regions may be prone to insertion/deletion mutations. Thymocytes from clinically healthy SCL/LMO1 mice aged 5 weeks showed evidence of oligoclonal T-lymphocyte expansion, but did not have Notch1 mutations and did not form tumors when injected into nude mice (pre-malignant thymocytes), whereas thymocytes from SCL/LMO1 mice aged 8–12 weeks had Notch1 mutations and formed tumors upon transplantation into nude mice. Thus, Notch1 mutations are very frequent secondary events that can cooperate with a wide range of primary events as cells progress from a pre-malignant to a fully transformed state.

Genome-Wide Analysis Reveals Conserved and Divergent Features of Notch1/RBPJ Binding in Human and Murine T Lymphoblastic Leukemia Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 5236-5236
Author(s):  
Jon C Aster ◽  
Hongfang Wang ◽  
James Zou ◽  
Yumi Yashiro-Ohtani ◽  
Bo Zhao ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cell ◽  
Cell Lines ◽  
Binding Sites ◽  
Target Genes ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Human T Cell ◽  
Two Factors

Abstract Abstract 5236 Activated Notch1 regulates gene expression by associating with the DNA-binding factor RBPJ and is an important oncoprotein in murine and human T cell acute lymphoblastic leukemia/lymphoma (T-ALL), yet the interplay between Notch1 and other factors that regulate the transcriptional output of T-ALL cells is poorly understood. Using ChIP-Seq and starting with Notch1-dependent human and murine T-ALL cell lines, we find that Notch1 binds preferentially to promoters, to RBPJ binding sites, and near sites for ZNF143, as well as Ets and Runx factors. By ChIP-Seq, ZNF143 binds to ∼40% of Notch1 sites, whereas Ets1 binding is observed within 100 basepairs of ∼70% of genomic Notch1 binding sites. Notch1/ZNF143 “co-sites” have high Notch1 and ZNF143 signals, frequent co-binding of RBPJ to sites embedded within ZNF143 motifs, strong promoter bias, and low mean levels of “activated” chromatin marks. RBPJ and ZNF143 binding to DNA is mutually exclusive in vitro, suggesting RBPJ/Notch1 and ZNF143 complexes exchange on these sites in T-ALL cell lines. In contrast, Ets1 binding sites flank RBPJ/Notch1 binding sites and are associated with high levels of activated chromatin marks, whereas Runx sites are predominantly intergenic. Although Notch1 predominantly binds promoters, ∼75% of direct Notch1 target genes lack promoter binding and appear to be regulated by enhancers, which were identified near MYC, DTX1, IGF1R, IL7R and the GIMAP gene cluster. Both Ets1 and Notch1 binding to an intronic enhancer located in DTX1 were required for expression of this well characterized Notch1 target gene, suggesting that these two factors coordinately regulate DTX1 expression. Although the association of Notch1 binding with ZNF143, Ets, and Runx sites was highly conserved, binding near certain important genes showed substantial divergence. For example, in human T-ALL lines Notch1/RBPJ bind a 3' enhancer near the IL7R gene, whereas in murine T-ALL lines no binding was observed near Il7r. Similarly, in human T-ALL lines Notch1/RBPJ bound an enhancer located ∼565 kb 5' of MYC, whereas in murine T-ALL cells Notch1/RBPJ bound an enhancer located ∼1 Mb 3' of Myc. Human and murine T-ALL genomes also have many sites that bind only RBPJ. Murine RBPJ “only” sites are highly enriched for imputed sites for the corepressor REST, whereas human RPBJ “only” sites lack REST motifs and are more highly enriched for imputed CREB binding sites. Thus, there is a conserved network of cis-regulatory factors that interacts with Notch1 to regulate gene expression in T-ALL cell lines, as well as novel classes of divergent RBPJ “only” sites that also likely regulate transcription. To extend these findings to normal and pathophysiologic tissues, ChIP-Seq was used to identify RBPJ/Notch1 binding sites in primary murine thymocytes and primary murine T-ALL associated with Notch1 gain-of-function mutations. Early findings appear to indicate that primary T-ALLs closely resemble normal DN3a thymocytes in terms of the distribution of Notch1 binding sites and associated chromatin marks. These data suggest that Notch1-driven T-ALLs epigenetically resemble the DN3a stage of T cell development, during which Notch1 signaling is high and cells are rapidly proliferating. Disclosures: No relevant conflicts of interest to declare.

scholarly journals LIM Domain Protein 1 (Ldb1) Is Required for Lmo2 Oncogene-Induced Thymocyte Self-Renewal and T-Cell Leukemia in a Mouse Model of Human T-ALL

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2538-2538
Author(s):  
Liqi Li ◽  
Apratim Mitra ◽  
Bin Zhao ◽  
Seeyoung Choi ◽  
Jan Lee ◽  
...  
Keyword(s):  
T Cell ◽  
Mouse Model ◽  
Transgenic Mice ◽  
Progenitor Cells ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Conditional Knockout ◽  
Lim Domain ◽  
Self Renewal ◽  
Hematopoietic Stem

LIM domain Only-2 (LMO2) is one of the most frequently deregulated oncogenes in T-cell acute lymphoblastic leukemia (T-ALL) and is generally expressed in the clinically aggressive Early Thymocyte Precursor ALL. LMO2 encodes a small protein with 2 LIM domains that is part of a large multiprotein complex in hematopoietic stem and progenitor cells (HSPC), where it is required for HSC specification and maintenance. Many of LMO2's protein partners in HSPCs are expressed in T-ALL implying that protein complexes like those scaffolded by LMO2 in HSPCs also play a role in leukemia. LDB1 is concordantly expressed with LMO2 in human T-ALL although its expression is more widespread than LMO2. In this study, we analyzed a critical component of the Lmo2 associated complex, LIM domain binding 1 (Ldb1), in the CD2-Lmo2 transgenic mouse model of human T-ALL. To further define Ldb1's role in leukemia, we induced its conditional knockout in CD2-Lmo2 transgenic mice with the use of Lck-Cre, Rag1-Cre, and Il7r-Cre transgenic mice. CD2-Lmo2 transgenic mice develop T-ALL with high penetrance and closely model the human disease. We discovered that the penetrance and latency of Lmo2-induced T-ALL were markedly attenuated in the Lck-Cre model and T-ALL onset was completely abrogated in the Rag1-Cre and Il7r-Cre models. The latter two models induced more efficient deletions of Ldb1, earlier in the T-cell differentiation program compared to Lck-Cre. Interestingly, Lck-Cre deletion was efficient in thymocytes without the Lmo2 transgene. In striking contrast, Ldb1 deletion could not be induced in CD2-Lmo2 transgenic T-cell progenitors. Consistent with this finding, T-ALLs that developed in CD2-Lmo2/floxed-Ldb1/Lck-Cre mice had incomplete deletion of Ldb1. These results imply that Ldb1 is a required factor for Lmo2 to induce T-ALL. To further probe the pathogenesis of Lmo2-induced T-ALL, we analyzed preleukemic phenotypes in the Rag1-Cre (or Il7r-Cre) conditional knockout models. Our results showed that Ldb1 is required for the induction of thymocyte self-renewal and radioresistance. Ldb1 was also required for the acquisition of the pre-leukemic ETP gene expression signature observed in immature CD2-Lmo2 transgenic thymocytes. Detailed biochemical experiments show that LMO2 protein is directly stabilized by LDB1 in leukemia cells perhaps on chromatin. In conclusion, these results support a model where Lmo2-induced T-ALL is caused by a failure to downregulate Ldb1/Lmo2 nucleated transcription complexes that normally function to enforce self-renewal in bone marrow hematopoietic progenitor cells. Disclosures No relevant conflicts of interest to declare.

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