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

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.

Download Full-text

Distinct Roles for the NF-κB Pathway In Myeloid and Lymphoid Transformation and Leukemogenesis by BCR-ABL.

Blood ◽

10.1182/blood.v116.21.1225.1225 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1225-1225

Author(s):

Mo-Ying Hsieh ◽

Richard A. Van Etten

Keyword(s):

Stem Cells ◽

Bone Marrow Cells ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Myeloproliferative Neoplasm ◽

Dominant Negative ◽

Lymphoid Cells ◽

Leukemic Cells ◽

Mutant Form ◽

B Lymphoid

Abstract Abstract 1225 The BCR-ABL tyrosine kinase, product of the t(9;22) Ph chromosome, activates multiple signaling pathways in leukemic cells from patients with chronic myeloid leukemia (CML) and Ph+ B-cell acute lymphoblastic leukemia (B-ALL). Previous studies have shown that NF-κB is activated in BCR-ABL-expressing cell lines and contributes to transformation of primary B-lymphoid cells by BCR-ABL (Reuther et al., Genes Dev. 1998;12:968), but the mechanism of activation has not been defined (Kirchner et al., Exp. Hematol. 2003;31:504), and importance of NF-kB to myeloid and lymphoid leukemogenesis by BCR-ABL is unknown. To interrogate the role of NF-κB in BCR-ABL-mediated transformation, we utilized a super-repressor mutant form of IκBα (IκBαSR), which has been used to block NF-κB nuclear localization and transactivation by constitutively sequestering NF-κB in the cytoplasm. Using retrovirus co-expressing BCR-ABL and IκBαSR, we found that IκBαSR blocked nuclear p65/RelA expression and inhibited the IL-3 independent growth of Ba/F3 cells and primary B-lymphoid cells transformed by BCR-ABL. The effect of NF-κB inhibition was primarily on proliferation rather than on cell survival, as there was no increase in apoptosis in cells expressing IκBαSR. When primary bone marrow cells were transduced and transplanted under conditions favoring induction of B-ALL or CML-like myeloproliferative neoplasm in recipient mice, co-expression of IκBαSR significantly attenuated disease development and prolonged survival of diseased mice. Molecular analysis of these leukemias demonstrated that NF-κB inhibition decreased the frequency of leukemia-initiating (“stem”) cells in the CML model, but not in the B-ALL model, and was associated with decreased expression of c-Myc, an NF-κB target. To clarify the mechanism of activation of NF-κB in BCR-ABL-expressing cells, we targeted two upstream kinases that negatively regulate IκBα, IKKα/IKK1 or IKKβ/IKK2. To accomplish this, we engineered retroviruses co-expressing BCR-ABL and kinase-inactive, dominant-negative mutants of IKK1 (IKK1KM) or IKK2 (IKK2KM). Co-expression of either IKK mutant inhibited both B-lymphoid transformation and leukemogenesis by BCR-ABL, as well as induction of CML-like MPN, with IKK1 inhibition more effective than IKK2. Together, these results demonstrate that NF-κB is activated in part through the canonical IKK pathway in BCR-ABL-expressing leukemia cells, and that NF-κB signaling plays distinct roles in the pathogenesis of myeloid and lymphoid leukemias induced by BCR-ABL. In CML, NF-κB may play a role for in generation and/or maintenance of leukemic stem cells. These results validate IKKs as targets for therapy in Ph+ leukemias, and motivate the evaluation of small molecule IKK inhibitors in these diseases. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Altered Expression of Hoxa1 Perturbs Haematopoiesis in a Dose-Dependent Manner and Induces Myelodysplasia in Mice

Blood ◽

10.1182/blood.v128.22.1132.1132 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1132-1132

Author(s):

Shuhying Tan ◽

Alistair Chalk ◽

Ana C Maluenda ◽

Lenny Straszkowski ◽

Jack O'Halloran ◽

...

Keyword(s):

Bone Marrow ◽

Platelet Count ◽

Bone Marrow Cells ◽

Conflicts Of Interest ◽

Significant Proportion ◽

Dominant Negative ◽

Proliferative Potential ◽

Dependent Manner ◽

Altered Expression ◽

Dose Dependent

Abstract Homeobox A1 (Hoxa1) is a highly conserved transcription factor. Through pre-mRNA splicing within exon 1, wildtype Hoxa1 (WT-Hoxa1) expresses two transcript variants: a full-length Hoxa1 (Hoxa1-FL) containing a homeobox-binding domain, and a truncated homeoboxless Hoxa1 (Hoxa1-T). We have demonstrated that the two spliceforms have opposing proliferative potential, suggesting a dominant negative role exerted by Hoxa1-T. Using in silico analysis, we found that 50% of patients with myelodysplastic syndromes (MDS) have upregulated expression of HOXA1 in CD34+ cells. Delineating this further by quantitative real-time PCR, we observed that a significant proportion of patients with high-risk MDS also lacked expression of HOXA1-T. To explore how Hoxa1 alters haematopoiesis and whether the magnitude of Hoxa1-FL expressionand/or loss of Hoxa1-T results in a more severe MDS phenotype, we generated conditional knock-in mice with altered Hoxa1 expression. Two strains of transgenic mice were created: WT-Hoxa1 (which express both Hoxa1-FL and Hoxa1-T) and mutated Hoxa1 (MUT-Hoxa1, which can only express Hoxa1-FL due to oligomutagenesis at the acceptor splice site). They were crossed to tamoxifen-inducible haematopoietic-specific Cre (hSclCreERT) mice to express WT-Hoxa1 or MUT-Hoxa1 in a heterozygous (WT-Hoxa1ki/+, MUT-Hoxa1ki/+)or homozygous (WT-Hoxa1ki/ki, MUT-Hoxa1ki/ki)manner at the Rosa26 locus. Four weeks after induction of the knock-in, WT-Hoxa1ki/+ and MUT-Hoxa1ki/+ mice displayed significant thrombocytopenia (mean platelet count (x106/ml): controls=848; WT-Hoxa1ki/+=646; MUT-Hoxa1ki/+=566, p<0.01 and p<0.0001 for WT-Hoxa1ki/+ and MUT-Hoxa1ki/+ respectively vs controls). By 4 months, heterozygous knock-in mice showed relative pancytopenia compared to controls, with associated myeloid bias. There was no difference in bone marrow cellularity, and mature and immature progenitors were present in similar proportions. However, knock-in mice showed significant reductions in Lin-cKit+Sca1+ (LKS+) cells (WT-Hoxa1ki/+: p<0.001, MUT-Hoxa1ki/+: p<0.0001, vs controls) with a trend to reduction in the proportions of multipotent progenitors (MPPs), accompanied by increased apoptosis. Homozygous knock-in mice showed more dramatic haematopoietic changes. Four weeks after induction of the knock-in, WT-Hoxa1ki/ki and MUT-Hoxa1ki/ki mice were profoundly thrombocytopenic (mean platelet count (x106/ml): controls=804; WT-Hoxa1ki/ki=238; MUT-Hoxa1ki/ki=50, p<0.0001 for WT-Hoxa1ki/ki and MUT-Hoxa1ki/ki vs controls; p<0.05 for WT-Hoxa1ki/ki vs MUT-Hoxa1ki/ki). This was accompanied by myeloid bias at 4 months (mean granulocytes (x106/ml): controls=0.91; WT-Hoxa1ki/ki=1.88; MUT-Hoxa1ki/ki=2.21, p<0.05 and p<0.001 for WT-Hoxa1ki/ki and MUT-Hoxa1ki/ki respectively vs controls). The mice also demonstrated reduced LKS+ cells (mean LKS+ (%): controls=4.04; WT-Hoxa1ki/ki=0.43; MUT-Hoxa1ki/ki=0.11, p<0.0001, vs controls) with significant reductions in MPPs (WT-Hoxa1ki/ki: p<0.0001, MUT-Hoxa1ki/ki: p<0.05, vs controls). Strikingly, there was skewed lineage commitment towards granulopoiesis at the expense of B-lymphopoiesis in the bone marrow. These phenotypes were transplantable, with the majority of recipients of bone marrow from WT-Hoxa1ki/+ and MUT-Hoxa1ki/+ mice developing MDS at 12 to 15 months (WT-Hoxa1ki/+: 60%; MUT-Hoxa1ki/+: 71%). They displayed normocytic or macrocytic anaemia with or without thrombocytopenia. Transplant recipients of knock-in cells showed near complete exhaustion of LKS+ cells (mean LKS+ (%): controls=2.2; WT-Hoxa1ki/+=0.2; MUT-Hoxa1ki/+=0.7, p<0.01, vs controls). Morphologic evidence of trilineage dysplasia was present without an increase in the blast count. Transplantation studies of homozygous knock-in bone marrow cells have been performed, and the mice are being monitored for MDS and leukaemia. In summary, knock-in mouse models of WT-Hoxa1 and MUT-Hoxa1 recapitulated features of human MDS, with findings reminiscent of MDS with multilineage dysplasia. Bone marrow function was more severely compromised in homozygous mice than in heterozygous mice in keeping with a dose-dependent phenotype. Collectively, these models are clinically tractable, making them valuable pre-clinical platforms for understanding MDS and developing novel therapies. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Endogenous Nras G12D/+ and G12D/G12D Distinctly Regulate Self-Renewal and Differentiation of Haematopoietic Stem Cells

Blood ◽

10.1182/blood.v120.21.4086.4086 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4086-4086

Author(s):

Guangyao Kong ◽

Jinyong Wang ◽

Yangang Liu ◽

Juan Du ◽

Alisa Damnernsawad ◽

...

Keyword(s):

Stem Cells ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Myeloproliferative Neoplasm ◽

Erk Signaling ◽

Dependent Manner ◽

Self Renewal ◽

Haematopoietic Stem Cells ◽

Leukemia Development ◽

Dose Dependent

Abstract Abstract 4086 How oncogenes regulate adult stem cells to promote tumorigenesis is poorly understood. We and others previously reported that recipient mice transplanted with Nras G12D/+ or Nras G12D/G12D bone marrow cells develop distinct haemtopoietic malignancies. Mice with G12D/+ cells predominantly develop a myeloproliferative neoplasm (MPN) closely resembling chronic myelomonocytic leukemia (CMML), while animals with G12D/G12D cells develop acute T- or B-cell lymphoblastic leukemia (TALL or BALL) and/or MPN, with varying penetrance, which appear to be regulated by the activity of G12D/G12D haematopoietic stem cells (HSCs). Consistent with this notion, we found that G12D/+ HSCs are required to initiate and maintain CMML-like phenotypes in recipient mice and serve as MPN initiating cells. Therefore, we further investigated how endogenous oncogenic Nras signaling regulates the self-renewal and differentiation of HSCs to promote leukemia development in different lineages of cells. Here we show that G12D/+ signaling results in moderate hyperproliferation and increased self-renewal of HSCs, promoting expansion of myeloid progenitors and consequently myeloid malignancies. In contrast, G12D/G12D leads to excessive hyperproliferation, decreased self-renewal, and depletion of HSCs, which promote expansion of myeloid and lymphoid progenitors and subsequently malignancies in both compartments. Because leukemia development in Nras G12D/+ and G12D/G12D models is tightly associated with ERK1/2 hyperactivation in haematopoietic stem/progenitor cells (HSPCs), we studied the MEK/ERK signaling in HSCs and their downstream multipotent progenitors (MPPs) using a “HSC phosphor-flow” method we developed. Our data demonstrate that ERK1/2 is hyperactiavated in G12D/+ and G12D/G12D HSCs in a dose-dependent manner, while AKT is not affected in G12D/+ and G12D/G12D HSCs. In contrast, both ERK1/2 and AKT are not changed in G12D/+ and G12D/G12D MPPs. As expected, inhibition of MEK/ERK signaling by AZD6244 (a MEK1 inhibitor) rescues the HSC phenotypes and attenuates myeloproliferative neoplasm phenotypes in G12D/+ and G12D/G12D mice. Mechanistic analysis identifies that a cohort of MAPK pathaway genes regulating cell cycle and signaling are significantly differentially expressed in G12D/+ HSCs compared to control or G12D/G12D HSCs. Unlike the prevailing theory based on Ras overexpression studies, depletion of G12D/G12D HSCs is not associated with overexpression of cell senescence genes. Rather, the Wnt and Notch pathways are significantly downregulated in G12D/G12D but not G12D/+ HSCs. Therefore, we propose that endogenous Nras G12D signaling differentially regulates HSCs self-renewal and differentiation through a dose-dependent hyperactivation of ERK1/2. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

CITED2 Facilitates Apoptosis Induced By Histone Deacetylase Inhibitor SAHA In Human Pediatric Pre-B Acute Lymphoblastic Leukemia Cells

Blood ◽

10.1182/blood.v122.21.2913.2913 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2913-2913

Author(s):

Jinwei Du ◽

Shigemi Matsuyama ◽

Yu-Chung Yang

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Side Effects ◽

Mononuclear Cells ◽

Bone Marrow Cells ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Dependent Manner ◽

Mouse Development ◽

Childhood All ◽

Normal Peripheral Blood

Abstract Acute lymphoblastic leukemia (ALL) is the most common malignancy in childhood, representing 31% of all tumors, and about 85% of children with ALL have B-cell ALL. Although the survival rate is approaching 90%, ALL remains the main cause of death from disease in children and young adults. The activity of histone deacetylases (HDAC) in childhood ALL is increased compared with that in normal peripheral blood mononuclear cells or bone marrow cells. Treatment of mice engrafted with T or B-ALL cells with HDAC inhibitor (HDACi) increases the acetylation of Histone 3 and Histone 4 and prolongs survival of these mice. <>Vorinostat (Suberoylamilide Hydroxamic Acid, SAHA) was the first HDACi approved by the FDA for the treatment of refractory cutaneous T-cell lymphoma. Currently, several clinical trials are being conducted to evaluate its effects on other cancers, including ALL. However, some patients are resistant to HDACi therapy, and concerns regarding toxic side effects of HDACi exist due to the roles of HDACs in multiple pathways. Therefore, identification of new therapeutic targets is required which could improve the efficacy of HDACi by reducing the dose of HDACi administered without compromising the treatment benefits but alleviating the side effects of HDACi. <>CBP/p300-interacting transactivator with glutamic acid (E) and aspartic acid (D)–rich tail 2 (CITED2) is a cytokine-inducible gene that plays various roles during mouse development and, in particular, is essential for normal hematopoiesis. While the role of CITED2 in the pathogenesis of leukemia is currently unclear, dysregulation of CITED2 has been implicated in various types of leukemia, including ALL, in which downregulation of CITED2 is frequently observed. In this study, we tested the hypothesis that CITED2 may enhance the sensitivity of human pediatric pre-B ALL cells to HDACi SAHA. <>SAHA treatment of NALM-6 and 697 cells (human pediatric pre-B ALL cell lines) significantly induced apoptosis and cell cycle arrest in a dose dependent manner. The protein level of CITED2 was not affected by SAHA treatment. Although overexpression of CITED2 alone only slightly increased apoptosis, it significantly enhanced apoptosis resulted from SAHA treatment in both NALM-6 (15.1% versus 39.2%) and 697 cells (9.4% versus 14.6%) as assessed by annexin V/Propidium Iodide double staining and flow cytometry analysis (Figure 1). Accordingly, compared with control (i.e. NALM-6 cells transduced with GFP), overexpression of CITED2 also greatly reduced mitochondrial membrane potential of NALM-6 cells caused by SAHA treatment. To explore the potential mechanisms underlying enhanced apoptosis by overexpression of CITED2 in NALM-6 cells treated with SAHA, we determined the levels of pro- and anti- apoptotic proteins by Western blot and real-time quantitative PCR. We found that SAHA treatment increased the levels of pro-apoptotic molecules Bak, Puma, and Noxa, and decreased the levels of anti-apoptotic molecule Bcl-xL and apoptosis inhibitors XIAP and survivin. Importantly, overexpression of CITED2 markedly increased the protein levels of pro-apoptotic molecules Bak and Bim. Furthermore, knockdown of Bim by shRNA significantly attenuated apoptosis in Cited2 overexpressing NALM-6 cells treated with SAHA. Taken together, these results suggest that modulation of the CITED2 activity may confer its cooperative effect with SAHA in pre-B ALL cells and warrant future evaluation of such a combination in inducing apoptosis of primary ALL cells. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

NKL Homeobox Gene MSX1 Reactivates An Oncogenic Network In Lymphoid and Myeloid Malignancies

Blood ◽

10.1182/blood.v122.21.3765.3765 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3765-3765

Author(s):

Stefan Nagel ◽

Stefan Ehrentraut ◽

Corinna Meyer ◽

Maren Kaufmann ◽

Hans G. Drexler ◽

...

Keyword(s):

T Cell ◽

Cell Lines ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Notch Pathway ◽

Molecular Cytogenetics ◽

Homeobox Gene ◽

In Silico Analysis ◽

Reciprocal Regulation ◽

Myeloid Malignancies

Abstract Homeobox genes encode conserved transcription factors (TFs) which regulate fundamental cellular processes during development. Many members of the NKL homeobox gene subfamily are aberrantly expressed in T-cell leukemia and compromise cell differentiation. NKL homeobox gene MSX1 is expressed during embryonic hematopoiesis and its deregulation in Hodgkin lymphoma suggests an oncogenic role of this gene in hematopoietic malignancies. After screening 114 leukemia/lymphoma cell lines by microarray profiling, we detected MSX1 overexpression in three examples each from T-cell acute lymphoblastic leukemia (T-ALL) and mantle cell lymphoma (MCL), and one from acute myeloid leukemia (AML). In silico analysis by R-based statistical tools identified conspicuous expression of MSX1 in 11% of pediatric T-ALL patients, and in 3% each of MCL and AML patients. Thus, we found aberrant MSX1 expression in subsets of both lymphoid and myeloid malignancies. Focusing on MCL and AML we excluded chromosomal rearrangements by classical and molecular cytogenetics at the MSX1 locus underlying overexpression in affected cell lines. However, comparative expression profiling data indicted aberrant histone acetylation involving PHF16 and RTN1, together with TFs FOXC1, HLXB9 and TAL1, as activators of MSX1 transcription. Their involvement was confirmed by siRNA-mediated knockdown and overexpression studies. Reciprocal regulation of MSX1 involved CCND1 and NOTCH signalling. Reporter gene analyses demonstrated that CCND1 and CDKN2D are direct transcriptional targets of MSX1 and its repressive cofactor histone H1C. Fluorescence in situ hybridization showed that t(11;14)(q13;q32) in MCL results in detachment of CCND1 from its corresponding repressive MSX1 binding site. In conclusion, we uncovered a regulatory network around MSX1 in leukemia/lymphoma cells, involving factors and pathways implicated in embryonic hematopoiesis. The reciprocal regulation of MSX1 and the NOTCH pathway in B-cells parallels that of MSX2 in T-cells. These data support the view of a recurrent genetic network involved in hematopoietic development which is reactivated in malignant transformation. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

The Synthetic Hest Shock Protein (Hsp) 90 Inhibitor EC141 Induces Degradation of the Bcr-Abl p190 Protein and Apoptosis of Philadelphia Chromosome (Ph)-Positive Acute Lymphoblastic Leukemia (ALL) Cells.

Blood ◽

10.1182/blood.v110.11.2784.2784 ◽

2007 ◽

Vol 110 (11) ◽

pp. 2784-2784

Author(s):

Alessandra Ferrajoli ◽

Yongtao Wang ◽

Susan M. O’Brien ◽

Stefan H. Faderl ◽

David M. Harris ◽

...

Keyword(s):

Kinase Inhibitors ◽

Bone Marrow Cells ◽

Apoptotic Cell ◽

Lymphoblastic Leukemia ◽

Philadelphia Chromosome ◽

Protein Kinase Inhibitors ◽

High Dose ◽

Dependent Manner ◽

Protein Levels ◽

Dose Dependent

Abstract High dose chemotherapy of Ph+ ALL is rarely curative and clinical responses to protein kinase inhibitors have been transient. Although new regimens combining chemotherapy with Bcr-Abl kinase inhibitors improve survival, the long-term prognosis of patients with Ph+ ALL remains guarded. Thus, novel therapeutic strategies are needed. Hsp90 is a ubiquitous molecular chaperone protein required for the folding, activation and assembly of mediators of signal transduction, cell cycle control, and transcription regulation. The Hsp90 inhibitor EC141 (Biogen Idec, Inc.) blocks the chaperone activity of Hsp90 and induces proteasomal degradation of it’s client proteins. Because Hsp90 is a chaperone of Bcr-Abl we investigated the activity of EC141 against the Ph+ ALL B-cell lines Z-119, Z-181 and Z-33 (Estrov et al. J Cell Physiol166: 618, 1996; Leukemia10:1534, 1996). First we studied the effect of EC141 on Hsp levels in Ph+ ALL cells. EC141 (50 nM) down-regulated the protein levels of Hsp90 and upregulated those of Hsp70. Then, the effect of EC141 on the proliferation of Ph+ ALL cells was evaluated using the MTT assay. EC141 inhibited the growth and metabolic activity of Z-119, Z-181 and Z-33 Ph+ ALL cells in a dose-dependent manner at concentrations ranging from 1 to 100 nM. Similar results were obtained with primary bone marrow cells from patients with Ph+ ALL. Using the ALL blast colony culture assay we found that EC141 inhibited the proliferation of marrow-derived ALL colony-forming cells in a dose-dependent fashion. To explore the mechanism of action Z-181 were incubated cells with increasing concentrations of EC141; immunoprecipitation and Western immunoblotting were used to detect changes in cellular protein levels. EC141 degraded the Bcr-Abl p190 protein and inhibited the phosphorylation of CrkL in a dose-dependent manner. Furthermore, exposure of Z-181 cells to EC141 resulted in a time- and dose-dependent activation of procaspase 3, cleavage of poly (adenosine diphosphate-ribose) polymerase and apoptotic cell death as assessed by Annexin V. Taken together, our data suggest that EC141 degrades the Bcr-Abl p190 protein, inhibits proliferation, and induces apoptosis of Ph+ ALL cells. Additional studies aimed at investigating the in vivo activity of EC141 in Ph+ ALL are warranted.

Download Full-text

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.

Download Full-text

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.

Download Full-text

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.

Download Full-text

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 ◽

10.1182/blood-2019-131897 ◽

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.

Download Full-text