scholarly journals Krüppel-like Factor 4 (KLF4) Suppresses T-Cell Acute Lymphoblastic Leukemia By Inhibiting Expression of MAP2K7 and Expansion of Leukemia Initiating Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3569-3569
Author(s):  
Ye Shen ◽  
Chun Shik Park ◽  
Koramit Suppipat ◽  
Takeshi Yamada ◽  
Toni-Ann Mistretta ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Tumor Suppressor ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cells ◽  
Brdu Incorporation ◽  
Jnk Pathway

Abstract Acute lymphoblastic leukemia (ALL) is the most common hematological malignancy in children. Although risk-adaptive therapy, CNS-directed chemotherapy and supportive care have improved the survival of ALL patients, disease relapse is still the leading cause of cancer-related death in children. Therefore, new drugs or novel multi-drug combinations are needed as frontline treatments for high-risk patients and as salvage agents for relapsed disease. T-cell ALL (T-ALL) is a subset of ALL that exhibits activating mutations of NOTCH1 in more than 50% of the patients. However, the use of gamma-secretase inhibitors to reduce NOTCH1 activity has not been successful in patients due to limited response and toxicity. Therefore, identification of genetic factors that cooperate with T-ALL leukemogenesis is needed for the development of alternative therapies. KLF4 is a transcription factor that functions as a tumor suppressor or an oncogene depending on cellular context. Our data showed significant reduction of KLF4 transcripts in lymphoblasts from T-ALL patients compared to blood and bone marrow cells from healthy individuals. In consistent with reduced KLF4 levels, these patients exhibit hyper-methylation of CpG islands located between nt -811 and +1190 relative to KLF4 transcription start site. From these findings we hypothesized that KLF4 has tumor suppressor function in T-ALL leukemogenesis. To test our hypothesis, we transduced 5-FU treated bone marrow (BM) cells from control (Klf4fl/fl), Klf4 null (Klf4fl/fl; Vav-iCre) and Klf4 heterozygous (Klf4fl/+; Vav-iCre) mice with retrovirus carrying a NOTCH1 activating mutant (L1601P-ΔP) and then transplanted these BM cells into irradiated recipient mice. In contrast to controls, mice transplanted with transduced Klf4-null BM cells developed T-ALL with significantly higher penetrance (Klf4 null 76.5% v.s. control 21.3%) and shorter latency (Klf4 null 93 days v.s. control 130 days). Interestingly, Klf4 heterozygous group shows similar survival kinetics as Klf4 null group, suggesting that Klf4 haploinsufficiency is enough to accelerate onset of leukemia. To investigate the effect of Klf4 deletion in established leukemia cells, we transplanted NOTCH1 L1601P-ΔP transduced BM cells from Klf4fl/fl; CreER+ mice to induce leukemia. Post-transplantation deletion of the Klf4 gene by tamoxifen administration was able to accelerate T-ALL development compared to mice injected with vehicle. On the cellular level, loss of KLF4 led to increased proliferation of leukemia cells as assessed by in vivo BrdU incorporation, which correlated with decreased levels of p21 protein. Limited dilution transplantation of primary leukemia cells into secondary recipients showed a 9-fold increase of leukemia initiating cells (LIC) frequency in Klf4null leukemia cells compared to controls, suggesting that KLF4 controls expansion of LIC in T-ALL. To elucidate molecular mechanism underlying KLF4 regulation in T-ALL cells, we performed microarray and ChIP-Seq in control and Klf4 null CD4+CD8+ leukemia cells. Combined analyses revealed 202 genes as KLF4 direct targets, of which 11 genes are also deregulated in human T-ALL cells by comparing with published microarray datasets. One of the top upregulated genes is Map2k7, which encodes a kinase upstream of the JNK pathway. Immunoblots in leukemia cells confirmed increased expression of MAP2K7 protein and enhanced phosphorylation of its downstream targets JNK and ATF2. To further investigate the role of JNK pathway in T-ALL, we tested JNK inhibitor SP600125 in human T-ALL cell lines (KOPTK1, DND41, CCRF-CEM, MOLT3). Interestingly, SP600125 showed dose-dependent cytotoxicity in all human T-ALL cell lines tested regardless of their NOTCH1 status. Overall our results showed for the first time that KLF4 functions as a tumor suppressor in T-ALL by regulating proliferation of leukemia cells and frequency of LIC. Additional study elucidated that KLF4 suppresses the JNK pathway via direct transcriptional regulation of MAP2K7. Moreover, the vulnerability of human T-ALL cell lines to JNK inhibition provides a novel target for future therapy in T-ALL patients. Disclosures No relevant conflicts of interest to declare.

Activation of the JNK Pathway Mediates Chemoresistance in T-Cell Acute Lymphoblastic Leukemia by Phosphorylation and Proteosomal Degradation of BimEL.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2372-2372
Author(s):  
Kam Tong Leung ◽  
Karen Kwai Har Li ◽  
Samuel Sai Ming Sun ◽  
Paul Kay Sheung Chan ◽  
Yum Shing Wong ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Cytochrome C ◽  
Cell Lines ◽  
Caspase 3 ◽  
Lymphoblastic Leukemia ◽  
Chemotherapeutic Agents ◽  
Jnk Pathway ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Induced Apoptosis

Abstract Despite progress in the development of effective treatments against T-cell acute lymphoblastic leukemia (T-ALL), about 20% of patients still exhibit poor response to the current chemotherapeutic regimens and the cause of treatment failure in these patients remains largely unknown. In this study, we aimed at finding mechanisms that drive T-ALL cells resistant to chemotherapeutic agents. By screening etoposide sensitivity of a panel of T-ALL cell lines using DNA content and PARP cleavage as apoptosis markers, we identified an apoptosis-resistant cell line, Sup-T1. Western blot analysis and caspase activity assay showed that Sup-T1 cells were deficient in etoposide-induced activation of caspase-3 and caspase-9. In addition, mitochondrial cytochrome c release was not evident in etoposide-treated Sup-T1 cells. However, addition of exogenous cytochrome c in cell-free apoptosis reactions induced prominent caspase-3 activation, indicating that the chemoresistance observed in Sup-T1 cells was due to its insusceptibility to the drug-induced mitochondrial alterations. Analysis of the basal expression of the Bcl-2 family proteins revealed that the levels of Bcl-2 was higher in Sup-T1 cells, while Bax and BimEL levels were lower, when compared to etoposide-sensitive T-ALL cell lines. Gene silencing using antisense oligonucleotide to Bcl-2 and overexpression of Bax did not resensitize cells to etoposide-induced apoptosis. On the contrary, transient transfection of BimEL into Sup-T1 cells significantly restored etoposide sensitivity. Further experiments revealed that the lack of BimEL expression in Sup-T1 cells was due to the rapid degradation of newly-synthesized BimEL by the proteosomal pathway, as treatment of Sup-T1 cells with a proteosome inhibitor significantly restored the protein level of BimEL. Moreover, treatment with proteosome inhibitor resulted in mobility shift of BimEL, which was sensitive to phosphatase digestion. Furthermore, treatment of Sup-T1 cells with JNK inhibitor resulted in accumulation of BimEL, and pretreatment with JNK inhibitor restored sensitivity of Sup-T1 cells to etoposide-induced apoptosis, indicating that constitutive activation of the JNK pathway in Sup-T1 cells was responsible for promoting BimEL phosphorylation, and this may serve as a signal targeting BimEL to the proteosome for degradation. Altogether, our findings provide the first evidence that JNK activation correlates inversely with BimEL level by promoting its phosphorylation and degradation. This, in turn, reduces the sensitivity of T-ALL cells to chemotherapeutic agents.

Transient Responses to Notch and Tlx1/Hox11 Inhibition In T-Cell Acute Lymphoblastic Leukemia/Lymphoma.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1026-1026
Author(s):  
Erica A. Lehotzky ◽  
Mark Y. Chiang
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Cell Line ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Ectopic Expression ◽  
Leukemia Cells ◽  
Inhibitory Effects ◽  
Notch Inhibition

Abstract Abstract 1026 Despite numerous advances in the past few decades, treatment of acute lymphoblastic leukemia/lymphoma (ALL) remains a common and considerable challenge. Further efforts to define the molecular lesions that drive ALL are needed to improve clinical management. The Hox subfamily of T-cell ALL (T-ALL) represents 30–40% of pediatric and adult cases. TLX1/HOX11 is the prototypical member of the Hox group. To generate a resource for developing targeted therapies for Hox T-ALLs, we developed a doxycycline-regulated mouse model of Tlx1-initiated T-ALL. Dysregulated thymic expression of Tlx1 induces T-ALL after ∼5-7 months with penetrance of 15–60%. The lymphoblasts are arrested at the early CD4+/CD8+/CD24hi stage of T-cell development, similar to human T-ALLs of the TLX1 subtype. Spontaneous activation of the Notch1 oncogene occurred in the tumors. In about two-thirds of samples, Notch was activated through acquired mutations in the heterodimerization and PEST domains that resemble the Notch1 mutations found in human patients. Inhibition of Notch signaling with g-secretase inhibitors completely abrogated cell line growth and induced apoptosis. Notch inhibition also transiently delayed leukemia progression by ∼17 days in vivo. In contrast, suppression of Tlx1 expression had more moderate inhibitory effects on cell line growth in vitro. However, suppression of Tlx1 expression in transgenic mice transiently delayed leukemia progression by ∼11 days. Tlx1 suppression had the strongest inhibitory effects on expression of CCR7 and lymph node size. These effects were fully reversed with ectopic expression of Tlx1. These data show that Tlx1 can convert normal thymocytes into leukemia cells, but the leukemia cells are not fully dependent on continued Tlx1 expression. The leukemia cells recruit secondary factors and pathways such as Notch and c-Myc to sustain growth and survival. Our study highlights a strong resiliency of T-ALL cells to both Tlx1 and Notch inhibition. Our study has important implications for targeting these pathways for the treatment of T-ALL. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Identification of a New Targeted Therapy for T-Cell Acute Lymphoblastic Leukemia and Studies on Its Mechanism of Action

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2752-2752
Author(s):  
Kinjal Shah ◽  
Julhash U. Kazi
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Predictive Biomarkers ◽  
Protein Kinase Inhibitors ◽  
Maturation Stage ◽  
Mrna Levels ◽  
Cell Acute Lymphoblastic Leukemia

Background: Acute lymphoblastic leukemia (ALL) is the most frequent pediatric malignancy, of which T- cell acute lymphoblastic leukemia (T-ALL) constitutes an aggressive subset. Due to the advent of new therapies, T-ALL now has a 5-year event-free survival (EFS) rate exceeding 85%. However, some patients still relapse and display resistance to therapy. Moreover, adverse side-effects of intensive chemotherapy worsen the duration of treatment. Therefore, we still need to improve our current treatment beyond that of the chemotherapeutic approaches. It has been shown that the maturation stage of T-ALL decides its dependency on Bcl-2/Bcl-xL. The immature early T cell progenitor ALL (ETP-ALL) rely on Bcl-2 for their survival while all the other stages of T-ALL and primary patient samples depend on Bcl-xL. Bcl-2 inhibitors have thus shown to display promising antitumor activity against ETP-ALL, a subgroup with a high risk of relapse, but with a variable response across these patients. Therefore, there is a need for predictive biomarkers and further investigation towards finding a combination of drugs for the treatment of these patients. Methodology & Aim: We screened 10 different T-ALL cell lines with a combination of Bcl-2 inhibitor and a panel of 378 protein kinase inhibitors and identified polo-like kinase inhibitor as a promising candidate. We thus aimed to study the combined effect of Bcl-2 and PLK1 inhibition in a panel of T-ALL cell lines and in a PDX model of chemo-resistant childhood T-ALL. We also investigated the underlying mechanism of drug synergy by various biochemical assays. Results: Cell viability of 14 T-ALL cell lines was determined after being subjected to Bcl-2 inhibitor (ABT-199) and PLK1 inhibitor (BI-6727). All cell lines responded well to BI6727 with an EC50 of less than 70nM. However, they showed differential response to ABT199 with only 3 cell lines being sensitive with an EC50 of less than 40nM. The mRNA levels of Bcl-2, Bcl-xL and PLK 1, 2, 3 and 4 were determined by qRT-PCR. PLK1 was found to be highly expressed in all the cell lines as compared to the rest of the 3 PLK family proteins. ABT-199-sensitive cell lines showed lower Bcl-xL mRNA levels irrespective of their Bcl-2 expression, and displayed synergy with BI-6727. A higher degree of apoptosis was also observed in the combination treatment as compared to a single drug. Immunoblot analysis revealed cleavage of PARP1 and lower levels of c-Myc and MCL1 expression in the presence of both ABT-199 and BI-6727. Conclusions: Upregulation of the anti-apoptotic BCL2 family members is one of the canonical ways for cancer cells to escape apoptosis. In the past years, several highly selective and potent BCL2 inhibitors have been developed and showed promising efficacy in various cancers. We found that the sensitivity of T-ALL cell lines to ABT-199 is largely determined by the lower levels of Bcl-xL expression. Furthermore, ABT-199 displays synergy with the PLK inhibitor. T-ALL cell lines predominantly express PLK1 and thus the combinatorial effect of ABT-199 and BI-6727 is mediated through the pharmacological inhibition of both BCL2 and PLK1. Currently, we are generating iRFP-expressing T-ALL cell lines which will be used to check drug efficacy in vivo. Furthermore, we have collected chemo-resistant PDX cell lines which will be used to verify the cell line data. Besides its role in cell cycle control, we still have very limited knowledge about the function of PLK1 in leukemia. Thus, studying its role in T-ALL cell lines by knocking down PLK1 with CRISPR/Cas9 technology will provide an important insight. Disclosures No relevant conflicts of interest to declare.

New LMO2 Translocations in T-Cell Acute Lymphoblastic Leukemia, Involving STAG2 at Xq25 and MBNL1 at 3q25: A Positive Change?.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2641-2641
Author(s):  
Suning Chen ◽  
Stefan Nagel ◽  
Bjoern Schneider ◽  
Maren Kaufmann ◽  
Ursula R. Kees ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
B Cell Lymphoma ◽  
Small Sample ◽  
Upstream Region ◽  
Cell Acute Lymphoblastic Leukemia

Abstract Abstract 2641 Poster Board II-617 Background: In T-cell acute lymphoblastic leukemia (T-ALL) the LMO2 transcription factor locus is juxtaposed with T-cell receptor (TCR) genes by a recurrent chromosome translocation, t(11;14)(p13;q11). Recent molecular cytogenetic data indicate that unlike classical TCR rearrangements, t(11;14) operates synonymously with submicroscopic del(11)(p13p13) by removing a negative upstream LMO2 regulator (Dik et al., Blood 2007;110:388). The combined incidence of both LMO2 rearrangements is ∼10-15% (Van Vlierberghe and Huret, Atlas Genet Cytogenet Oncol Haematol, November 2007). However, aberrant LMO2 expression occurs in nearly half of all T-ALL cases, a discrepancy which may indicate a significant contribution by cryptic chromosome alterations. We attempted the extended characterization of the LMO2 genomic region in T-ALL cell lines to look for such rearrangements. Cells and Methods: We investigated a panel of 26 well characterized and authenticated T-ALL cell lines using parallel fluorescence in situ hybridization (FISH) with a tilepath BAC/fosmid contig and both conventional and quantitative reverse transcriptase (Rq)-PCR. Global gene expression was additionally measured in some cell lines by Affymetrix array profiling. Results: LMO2 rearrangements were detected in 5/26 (19.2%) cell lines including both established rearrangements, t(11;14) and del(11)(p13p13) in one cell line apiece (3.8%). Interestingly, we found two novel LMO2 translocations: t(X;11)(q25;p13) in 2/26 (7.7%), and t(3;11)(q25;p13) in 1/26 (3.8%) cell lines, respectively. Comparing transcription levels in cell lines with and without genomic rearrangements showed that LMO2 expression was significantly higher in T-ALL cell lines carrying LMO2 rearrangements (P<0.001). Rq-PCR revealed that 5 of the top 10 (50%) LMO2 expressing cell lines carry cytogenetic rearrangements at this locus, compared to 0/16 remaining examples. Loss of a recently defined LMO2 negative regulatory element was identified in the del(11)(p13p13) cell line but no other deletions were detected. Two genes STAG2 at Xq25 and MBNL1 at 3q25 were identified as novice LMO2 partners in t(X;11) and t(3;11), respectively. In both genes breakpoints lay at intron 1 close to deeply conserved noncoding regulatory regions. Both t(X;11) cell lines displayed conspicuous silencing of the ubiquitously expressed STAG2 gene highlighting the transcriptional significance of the region displaced. Unlike t(11;14)/del(11)(p13p13) both new rearrangements carry LMO2 breakpoints in the far upstream region (at minus 80–150 Kbp), and appear to result in upregulation of LMO2 by juxtaposition rather than via covert deletion. STAG2 is a component of the chromosomal cohesin complex which acts as a transcriptional coactivator, and which has been recently identified as a potential driver of oncogene transcription in acute myeloid leukemia (Walter et al., Proc Natl Acad Sci U S A. 2009;106:1295). MBNL1 controls RNA splicing and is a rare BCL6 partner gene in B-cell lymphoma, but this is the first report of its involvement in T-ALL. Conclusion: Given their frequency and variety in a small sample, we propose that cryptic chromosome rearrangements targeting LMO2 upregulation may be significantly more frequent than hitherto appreciated in T-ALL. Unlike canonical LMO2 rearrangements, both t(X;11) and t(3;11) would appear to function positively by upregulation of LMO2 via juxtaposition with noncoding driver elements within these novel partner genes. Perspectives: Future work will address the regulatory potential of candidate enhancer sequences embedded within conserved noncoding intronic sequences of MBNL1 and STAG2. Cytogenetically inconspicuous cell lines displaying LMO2 upregulation will be subjected to more detailed scrutiny using high density genomic SNP arrays. Disclosures: No relevant conflicts of interest to declare.

Identification of JAK1 Mutation In 40 Cases of Adult Acute T Lymphoblastic Leukemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4652-4652
Author(s):  
Aining Sun ◽  
Wenjuan Wang ◽  
Guanghua Chen ◽  
Wu Depei ◽  
Suning Chen ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Amino Acid Sequences ◽  
Insertion Mutation ◽  
Newly Diagnosed ◽  
Cell Acute Lymphoblastic Leukemia

Abstract Abstract 4652 Objective This study was purposed to analyze the mutations of JAK1 in bone marrow cells from adult patients with newly-diagnosed T cell acute lymphoblastic leukemia (T-ALL). Methods The entire coding sequence of JAK1 in bone marrow mononuclear cells (MNCs) from 40 adult T cell ALL patients were screened by polymerase chain reaction (PCR) and direct sequencing, then the clinical features of JAK1 mutation positive patients were analyzed. Results JAK1 mutations were identified in 5%(2/40) patients, one is point mutation, the other is insertion mutation, resulting in changes in amino acid sequences, and the two types of mutations are different from those mutations found before. Conclusion The JAK1 mutations were found in about 5% newly-diagnosed T cell acute lymphoblastic leukemia, which was lower than those report abroad. Disclosures: No relevant conflicts of interest to declare.

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.

T Cell Lymphoblastic Lymphoma Resulting From Expression of Self-Reactive TCRs During Early Thymopoiesis

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2057-2057
Author(s):  
Yongzhi Cui ◽  
Haven Garber ◽  
Masahiro Onozawa ◽  
Haiying Qin ◽  
Terry J Fry ◽  
...  
Keyword(s):  
T Cells ◽  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Insertional Mutagenesis ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Brdu Incorporation ◽  
Immune Reactivity ◽  
Antitumor Effects ◽  
Cell Acute Lymphoblastic Leukemia

Abstract Abstract 2057 Survivin has been considered a potential tumor antigen due to high expression in most cancers and limited expression in normal tissues. To explore the potential for survivin reactive TCRs to mediate antitumor effects in mice, we generated several founders of TCR transgenic (Tg) mice with specificity for the H-2b restricted immunodominant epitope of survivin. In survivin TCR Tg mice, survivin reactive T cells were predominantly CD8+ and mediated specific immune reactivity toward survivin peptide pulsed targets. Some antitumor reactivity was observed, but it was not potent, and the survivin reactive transgenic T cells were unable to mediate objective tumor regression of survivin bearing tumors in vivo. Surprisingly, spontaneous T cell acute lymphoblastic leukemia (T-ALL) was observed beginning at 4–6 months of age in both survivin TCR+Rag+/− and survivin TCR+Rag−/− mice. By one year of age, all mice had succumbed to T-cell ALL. The leukemic cells were CD3+, survivin TCR+, and CD8+ or CD4−/CD8−. Analysis of alpha gene rearrangements in tumor tissues revealed oligoclonality but the cells were malignant since they grew continuously in vitro without growth factors and induced tumors in C57BL/6 immunocompetent recipients. The occurrence of T-ALL in 3 founders suggests that the transgene itself, rather than insertional mutagenesis, is causative. We postulate that the survivin reactive TCR serves as an oncogene via recognition of survivin peptides within the thymus, leading to expansion of early thymic progenitors. In support of this, survivin itself is expressed in thymic tissue and premalignant survivin TCR Tg+ thymi show expanded frequencies and absolute numbers of CD4−CD8−CD44−CD25− thymocytes and increased BrdU incorporation within this subset compared to controls. Subsequent to the premalignant phase characterized by expansion of early thymic progenitors, surviving TCR Tg+ cells acquired NOTCH mutations and upregulated CD25, consistent with NOTCH signaling as a 2nd hit in this oncogenic process. At least one NOTCH1 mutation was found in all leukemias, with mutations in the PEST domain being most common (8/8), but 5' deletions (19/25) and mutations in the heterodimerization domain were also observed. Interestingly, T cell acute lymphoblastic leukemia with NOTCH mutations were also observed, albeit at reduced frequencies, in TCR Tg mice with specificity to WT1 and gp100. We propose a 2-hit model of oncogenesis for self-reactive TCR expression in the thymus. Early thymic expression of TCRs recognizing self antigens expressed in the thymus induces proliferation of early thymocytes, followed by acquisition of NOTCH mutations and ultimately lymphoblastic leukemia. We conclude that genetic engineering aimed at endowing hematopoietic or T lymphoid progenitors with the capacity to recognize tumor antigens expressed in the thymus could pose a risk for neoplasia, independent of insertional mutagenesis. Disclosures: No relevant conflicts of interest to declare.

Collaboration Between RUNX and NOTCH Pathways in T-Cell Acute Lymphoblastic Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1279-1279 ◽  
Author(s):  
Christopher R Jenkins ◽  
Hongfang Wang ◽  
Olena O Shevchuk ◽  
Sonya H Lam ◽  
Vincenzo Giambra ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Notch Signaling ◽  
Cell Lines ◽  
Target Genes ◽  
Lymphoblastic Leukemia ◽  
Brdu Incorporation ◽  
Knock Down ◽  
Primary Mouse ◽  
Cell Acute Lymphoblastic Leukemia

Abstract Abstract 1279 T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy characterized by the clonal outgrowth of developmentally arrested T-lymphoid blasts. Notch signaling is activated by mutation of NOTCH1 and/or FBW7 in over half of cases, and ultimately results in increased expression of target genes via the NOTCH/CSL transcriptional complex. Enforced expression of activated NOTCH1 in mouse hematopoietic progenitors leads to the development of clonal T-cell leukemias, suggesting that collaborating mutations are required for establishment and/or propagation of malignant clones. To identify candidate collaborating loci, Beverly and Capobianco performed a retroviral insertional mutagenesis screen in mice expressing a relatively weak activated Notch1 transgene and found recurrent insertions into Ikaros (Ikzf1). These insertions resulted in expression of dominant negative isoforms of Ikaros and thus potentiated Notch signaling since Ikaros and Notch/CSL compete for occupancy at target gene regulatory elements. In an attempt to identify collaborating mutations outside of the Notch pathway, we performed a similar screen, but employed instead a very potent activated NOTCH1 allele (ΔE) in hopes of saturating the Notch signaling pathway. We thus cloned out the insertion sites from 88 primary mouse leukemias generated by transduction of bone marrow with ΔE retrovirus. While recurrent insertions into Ikzf1 were again identified, we also observed frequent insertions into other regions including the Runx3 locus. The Runx3 integrations were tightly clustered in a region 40–60kb upstream of the transcriptional start site, suggesting the retroviral LTR might be inducing an increase in Runx3 expression. A single integration upstream of Runx1 was also identified in a region frequently mutated in similar screens. Of note, analysis of publically available gene expression profile data revealed that RUNX1 and RUNX3 are ubiquitously expressed in patient T-ALL samples. In order to functionally characterize the roles of RUNX1 and RUNX3 in T-ALL, we utilized lentiviral shRNAs to knock down RUNX1 and/or RUNX3 across a broad panel of 26 human T-ALL cell lines. Despite recent studies suggesting RUNX1 may act as a tumor suppressor in T-ALL, we observed the overwhelming majority of cell lines to show substantial growth defects after knock-down of RUNX1/3 as measured by competitive growth assay. These results were confirmed in a subset of cell lines and also in xenograft-expanded primary T-ALL samples by BrdU incorporation/DNA content assays which showed reduced proliferation/G1 cell cycle arrest following RUNX1/3 knock-down. Conversely, overexpression of RUNX3 induced T-ALL cells to proliferate more rapidly and to resist ABT-263-induced apoptosis. To explore potential target genes responsible for these pro-growth/survival effects, we mined available ChIP-Seq data and found NOTCH1/CSL and RUNX1 binding sites to co-localize within IGF1R and IL7R loci at intronic enhancer-like regions with associated H3K4me1>H3K4me3 marks and reduced H3K27me3 marks. Importantly, we show that NOTCH1 and RUNX factors co-regulate surface protein expression of IGF1R and IL7R in a synergistic/additive manner. As we and others have previously demonstrated important roles for both IGF1R and IL7R in T-ALL cell growth and leukemia-initiating activity, these studies reveal a novel collaborative mechanism between NOTCH1 and RUNX proteins in supporting propagation of established T-ALL disease. Disclosures: No relevant conflicts of interest to declare.

scholarly journals MiR-7 Functions as a Tumor Suppressor by Targeting the Oncogenes TAL1 in T-Cell Acute Lymphoblastic Leukemia

2020 ◽  
Vol 19 ◽  
pp. 153303382093413
Author(s):  
Hongbo Sun ◽  
Zhifu Zhang ◽  
Wei Luo ◽  
Junmin Liu ◽  
Ye Lou ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Tumor Suppressor ◽  
Lymphoblastic Leukemia ◽  
Critical Role ◽  
Ectopic Expression ◽  
Lymphocytic Leukemia ◽  
Leukemia Cells ◽  
Cell Acute Lymphoblastic Leukemia ◽  
And Migration

Background: T-cell acute lymphoblastic leukemia is a hematologic malignancy characterized by T-cell proliferation, and in many cases, the ectopic expression of the oncogenic transcription factor T-cell acute lymphocytic leukemia protein 1 (TAL1). MicroRNA-7 has been shown to play a critical role in proliferation, migration, and treatment sensitivity in a diverse array of cancers. In this study, we sought to establish a novel link between microRNA-7 and T-cell acute lymphoblastic leukemia oncogenesis. Material and Method: To do so, we characterized gene expression of microRNA-7 as well as TAL1 in both T-cell acute lymphoblastic leukemia patient-derived tissue and cell lines, as well as performing functional luciferase assays to assess microRNA-7 binding to the TAL1 3′-untranslated region. We also performed growth, apoptosis, and migration experiments using 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide, Annexin V, and transwell assays in the context of microRNA-7 overexpression. Results: We found that microRNA-7 expression is attenuated and inversely correlated with TAL1 expression in TAL1 + T-cell acute lymphoblastic leukemia cells. Additionally, microRNA-7 directly targets and suppresses TAL1 levels. Finally, microRNA-7 overexpression reduces growth, motility, and migration while inducing apoptosis in T-cell acute lymphoblastic leukemia cells, phenotypes that can be rescued by concomitant overexpression of TAL1. Conclusions: These results indicate that microRNA-7 functions as a potent tumor suppressor by inhibiting the oncogene TAL1 and suggest microRNA-7 could function as a prognostic biomarker and possible therapeutic in the clinical management of T-cell acute lymphoblastic leukemia.

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