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

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.

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

Blood ◽

10.1182/blood.v114.22.7.7 ◽

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.

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KDM2B in polycomb repressive complex 1.1 functions as a tumor suppressor in the initiation of T-cell leukemogenesis

Blood Advances ◽

10.1182/bloodadvances.2018028522 ◽

2019 ◽

Vol 3 (17) ◽

pp. 2537-2549 ◽

Cited By ~ 6

Author(s):

Yusuke Isshiki ◽

Yaeko Nakajima-Takagi ◽

Motohiko Oshima ◽

Kazumasa Aoyama ◽

Mohamed Rizk ◽

...

Keyword(s):

T Cell ◽

Tumor Suppressor ◽

Zinc Finger ◽

Target Genes ◽

Lymphoblastic Leukemia ◽

Dependent Manner ◽

Polycomb Repressive Complex ◽

Human T Cell ◽

T Cell Leukemogenesis ◽

Deficient Mice

Abstract KDM2B together with RING1B, PCGF1, and BCOR or BCORL1 comprise polycomb repressive complex 1.1 (PRC1.1), a noncanonical PRC1 that catalyzes H2AK119ub1. It binds to nonmethylated CpG islands through its zinc finger-CxxC DNA binding domain and recruits the complex to target gene loci. Recent studies identified the loss of function mutations in the PRC1.1 gene, BCOR and BCORL1 in human T-cell acute lymphoblastic leukemia (T-ALL). We previously reported that Bcor insufficiency induces T-ALL in mice, supporting a tumor suppressor role for BCOR. However, the function of BCOR responsible for tumor suppression, either its corepressor function for BCL6 or that as a component of PRC1.1, remains unclear. We herein examined mice specifically lacking the zinc finger-CxxC domain of KDM2B in hematopoietic cells. Similar to Bcor-deficient mice, Kdm2b-deficient mice developed lethal T-ALL mostly in a NOTCH1-dependent manner. A chromatin immunoprecipitation sequence analysis of thymocytes revealed the binding of KDM2B at promoter regions, at which BCOR and EZH2 colocalized. KDM2B target genes markedly overlapped with those of NOTCH1 in human T-ALL cells, suggesting that noncanonical PRC1.1 antagonizes NOTCH1-mediated gene activation. KDM2B target genes were expressed at higher levels than the others and were marked with high levels of H2AK119ub1 and H3K4me3, but low levels of H3K27me3, suggesting that KDM2B target genes are transcriptionally active or primed for activation. These results indicate that PRC1.1 plays a key role in restricting excessive transcriptional activation by active NOTCH1, thereby acting as a tumor suppressor in the initiation of T-cell leukemogenesis.

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

Blood ◽

10.1182/blood.v120.21.3496.3496 ◽

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.

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Dnmt3a Haploisufficiency Cooperates with Oncogenic Kras to Promote an Early-Onset T-Cell Leukemia

Blood ◽

10.1182/blood.v128.22.2729.2729 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2729-2729

Author(s):

Yuan-I Chang ◽

Guangyao Kong ◽

Jing Zhang ◽

Erik A. Ranheim

Keyword(s):

T Cell ◽

Dna Methyltransferase ◽

Bone Marrow Cells ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Myeloproliferative Neoplasm ◽

Dominant Negative ◽

Dependent Manner ◽

Myeloid Malignancies ◽

Dose Dependent

Abstract Recent whole genome/exome sequencing efforts in myeloid malignancies identified that mutations in DNA methyltransferase 3A (DNMT3A) are prevalent in acute myeloid leukemia (AML). In addition, DNMT3A mutations are also identified in various T cell malignancies. Of note, DNMT3A mutations are typically heterozygous and some WT DNMT3A functions thus remain in this state. However, the predominant DNMT3A R882 mutations, which locate in the catalytic domain, seem to inhibit the methyltransferase activity of the remaining WT DNMT3A due to its dominant-negative function (Yang L, Rau R, Goodell MA, Nat. Rev. Cancer 15: 152-165, 2015). COSMIC database analysis reveals different prevalence of DNMT3A R882 mutations in various hematopoietic malignancies. Approximately 60% of DNMT3A mutations in AML are R882 mutations, while the frequency of R882 mutations drops to ~40% in myelodysplastic syndrome (MDS) and myeloproliferative neoplasm (MPN). In contrast, the frequency of R882mutations is less than 25% in T-cell acute lymphoblastic leukemia (T-ALL). The significantly different frequencies of DNMT3A R882 mutations in AML versus T-ALL inspired us to investigate whether downregulation of DNMT3A regulates malignancies of different lineages in a dose-dependent manner. We previously showed that Dnmt3a-/- promotes MPN progression in KrasG12D/+ mice and ~1/3 compound mice develop AML-like disease (Chang et al. Leukemia 29: 1847-1856, 2015). Here, we generated KrasG12D/+; Dnmt3afl/+; Mx1-Cre mice to determine how Dnmt3a haploisufficiency affects KrasG12D/+-induced leukemogenesis. After pI-pC injections to induce Mx1-Cre expression, primary KrasG12D/+; Dnmt3a+/- mice died quickly as primary KrasG12D/+ mice; the survival rates of these two groups of animals were not significantly different. However, in a competitive transplant setting, recipients transplanted with KrasG12D/+; Dnmt3a+/- bone marrow cells displayed a significantly shortened survival than recipients with KrasG12D/+ cells. Moreover, all of the recipients with KrasG12D/+; Dnmt3a+/- cells developed a lethal T-ALL without significant MPN phenotypes, while ~20% of recipients with KrasG12D/+ cells developed MPN with or without T-ALL. This is in sharp contrast to the recipients with KrasG12D/+; Dnmt3a-/- cells, in which ~60% developed a lethal myeloid malignancy (MPN or AML). Our data suggest that in the context of oncogenic Kras, loss of Dnmt3a promotes myeloid malignancies, while Dnmt3a haploisufficiency induces T-ALL. This dose-dependent phenotype is highly consistent with the prevalence of DNMT3A R882 mutations in AML versus T-ALL in human. We are currently investigating the underlying mechanisms. Disclosures No relevant conflicts of interest to declare.

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

Blood ◽

10.1182/blood-2019-125331 ◽

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.

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

Blood ◽

10.1182/blood.v124.21.2405.2405 ◽

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.

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

Blood ◽

10.1182/blood.v126.23.1300.1300 ◽

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.

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Endogenous Oncogenic Nras Mutation Initiates T-Cell Leukemia/Lymphoma In a Dose-Dependent Manner

Blood ◽

10.1182/blood.v116.21.4183.4183 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4183-4183

Author(s):

Jinyong Wang ◽

Zeyang Li ◽

Zhongde Wang ◽

Yangang Liu ◽

Myung-Jeom Ryu ◽

...

Keyword(s):

T Cell ◽

Tumor Cells ◽

Lymphoblastic Leukemia ◽

Dependent Manner ◽

T Cell Leukemia ◽

Cell Leukemia ◽

Nras Mutation ◽

Low Incidence ◽

Dose Dependent ◽

Notch1 Mutations

Abstract Abstract 4183 The oncogenic NRAS mutations are frequently identified in myeloid diseases but rare in lymphoid diseases. They occur in 4% of acute T-cell lymphoblastic leukemia/lymphoma (T-ALL) patients and 22% of human T-ALL cell lines. Its differential roles in myeloid versus lymphoid disease development remain unclear. Here we examine the tumorigenic potential of oncogenic Nras in T-cells using two conditional Nras G12D murine knock-in models that either hypomorphically (NrasG12D Hypo) or normally (NrasG12D Norm) expresses oncogenic Nras G12D from its endogenous locus. Mice expressing monoallelic or biallelic NrasG12D Hypo develop normally and are tumor free. However, NrasG12D Norm leads to acute T-cell leukemia/lymphoma (TAL/L) in a bone marrow transplantation model, with a low incidence (∼8%) when expressing one allele (TAL/L-het) and a complete penetrance when expressing two alleles (TAL/L-homo). TAL/L-het tumors are associated with spontaneous up-regulation of oncogenic Nras in ∼67% of animals, and tumor cells are TdT positive, suggesting that they are transformed at an immature stage. In contrast, TAL/L-homo tumors express comparable levels of Nras to control thymocytes, and tumor cells are TdT negative, suggesting that they are transformed at a more mature stage. Both TAL/L-het and TAL/L-homo tumors are oligoclonal or polyclonal. Above 70% of these tumors contain clonal Notch1 mutations and are sensitive to gamma-secretase inhibitor. These data indicate that Notch1 mutations are acquired at an early stage and play an important role in the development of TAL/L-het and TAL/L-homo tumors. Together, our results show that engdogenous oncogenic Nras mutation leads to TAL/L in a dose-dependent manner, and thus explain the low incidence of oncogenic NRAS mutations in human T-cell diseases. Disclosures: No relevant conflicts of interest to declare.

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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 ◽

10.1182/blood-2019-127534 ◽

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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KLF4 induces apoptosis in T-ALL through the BCL2/BCLXL pathway

Blood ◽

10.1182/blood.v122.21.4902.4902 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4902-4902

Author(s):

Bing Xu ◽

Xiangmeng Wang ◽

Peng Li ◽

Wei Li ◽

Huijuan Dong ◽

...

Keyword(s):

Transcription Factor ◽

T Cell ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Ectopic Expression ◽

B Cell Lymphoma ◽

Dependent Manner ◽

Over Expression ◽

Yamanaka Factors ◽

Induced Apoptosis

Abstract KLF4, also known as GKLF (gut KLF), is a member of the KLF zinc finger-containing transcription factor family. Klf4 together with Oct4, Sox2, and c-Mycare widely referred to as ‘Yamanaka factors’ because mouse somatic cells can be reprogrammed into pluripotent stem cells following their ectopic expression. The transcription factor Kruppel-like factor 4 (KLF4) may induce tumorigenesis or suppress tumor growth in a tissue-dependent manner. In T cell leukemia and pre-B cell lymphoma cells, KLF4 acts as a tumor suppressor. We found that over expression of KLF4 induced human acute T cell lymphoblastic leukemia (T-ALL) cell lines to undergo apoptosis through the BCL2/BCLXL pathway, and we confirmed KLF4-induced apoptosis in primary samples from T-ALL patients. We further characterized KLF4 function in human early and mature T cells. Our analysis uncovered that KLF4 suppressed the transcription of other T cell-associated genes in T-ALL. Disclosures: No relevant conflicts of interest to declare.

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