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.

Transcriptional Regulation of Oncogenic MEF2C In T-Cell Acute Lymphoblastic Leukemia (T-ALL).

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3636-3636
Author(s):  
Stefan Nagel ◽  
Letizia Venturini ◽  
Corinna Meyer ◽  
Maren Kaufmann ◽  
Michaela Scherr ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Ectopic Expression ◽  
Regulatory Elements ◽  
Hematopoietic Stem ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Bhlh Proteins

Abstract Abstract 3636 Myocyte enhancer factor 2C (MEF2C) is a transcription factor of the MADS-box family which is physiologically expressed in hematopoietic stem cells and during development of B-cells. Recently, we identified ectopic expression of MEF2C in T-cell acute lymphoblastic leukemia (T-ALL) cell lines activated either via chromosomally mediated ectopic expression of homeodomain protein NKX2-5 or via deletion of non-coding exon and promoter regions at 5q14, suggesting loss of negative regulatory elements. Our aim was to identify additional transcriptional regulators of MEF2C in T-ALL. Therefore, we analyzed the sequence of the MEF2C 5′-region, thus identifying potential regulatory binding sites for GFI1B, basic helix-loop-helix (bHLH) proteins, STAT5 and HOXA9/HOXA10. Overexpression studies demonstrated MEF2C activation by GFI1B (strong), LYL1 and TAL1 leukemic bHLH proteins (weak), and inhibition by STAT5 (strong) and HOXA9/HOXA10 (weak). Chromatin-Immuno-Precipitation analysis demonstrated direct binding of GFI1B, LYL1 and STAT5 but not of HOXA10 to the MEF2C 5′-region in T-ALL cells. However, HOXA9/HOXA10 activated expression of NMYC which in turn mediated MEF2C repression, indicating an indirect mode of MEF2C regulation. Chromosomal deletion of the 5′-MEF2C STAT5 binding site in LOUCY cells by del(5)(q14), reduced expression levels of STAT5 protein in some MEF2C-positve T-ALL cell lines, and the presence of inhibitory IL7-JAK-STAT5-signaling highlighted the repressive impact of this factor in MEF2C regulation. Taken together, our results indicate that ectopic expression of MEF2C in T-ALL cells is mainly regulated via activating leukemic transcription factors GFI1B or NKX2-5 and by escaping inhibitory STAT5-signaling. Disclosures: No relevant conflicts of interest to declare.

Activation of Paired-Homeobox Gene PITX1 by Del(5)(q31) In T-Cell Acute Lymphoblastic Leukemia (T-ALL)

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4644-4644
Author(s):  
Stefan Nagel ◽  
Letizia Venturini ◽  
Grzegorz K. Przybylski ◽  
Piotr Grabarczyk ◽  
Björn Schneider ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Regulatory Region ◽  
Homeobox Genes ◽  
Homeobox Gene ◽  
Activation Mechanism ◽  
Cell Acute Lymphoblastic Leukemia

Abstract Abstract 4644 Aberrant activation of various oncogenes, including homeobox genes encoding fundamental transcription factors, during T-cell development contributes to T-cell acute lymphoblastic leukemia (T-ALL). Neoplastic chromosome rearrangements are known to deregulate Antennapedia-class homeobox genes of the NKL-family (TLX1, TLX3, NKX2-5) and HOXA-cluster genes of the Extended-Hox-family. Following analysis of T-ALL cell lines and primary cells, we describe leukemogenic involvement of a third homeobox gene group, the Paired (PRD)-class. Ascertainment was performed in an early stage-arrested T-ALL cell line (LOUCY) which revealed chromosomal deletion at 5q31, removing the downstream regulatory region of the PRD-homeobox gene PITX1. Comparative expression analysis confirmed ectopic PITX1 expression, consistent with aberrant activation by del(5)(q31) which removes a STAT1 binding site. STAT1 mediates repressive IL2-STAT1 signaling, implicating IL2-pathway avoidance as a possible activation mechanism. Furthermore, we detected expression of the physiologically similar PITX2 in 11/24 (46%) T-ALL cell lines, 5 with genomic PITX2 gains. Among primary T-ALL samples, 2/22 (9%) – both pediatric pre-T-ALL - ectopically expressed PITX1 but not PITX2. Forced expression of PITX1 by lentiviral transduction of JURKAT cells and subsequent analysis by expression profiling, prompted upregulation of RUNX2 and JUN and inhibition of RUNX1 and NKX3-1, indicating impaired T-cell differentiation. Taken together, our data show leukemic activation of PITX1, a novice PRD-class homeobox gene in the T-ALL repertoire, which may promote leukemogenesis by inhibiting differentiation. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Phloridzin Docosahexaenoate (PZ-DHA) : A Novel Naturally Derived Drug Active in T-Cell Acute Lymphoblastic Leukemia (T-ALL)

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5197-5197
Author(s):  
Niroshaathevi Arumuggam ◽  
Nicole Melong ◽  
Catherine K.L. Too ◽  
Jason N. Berman ◽  
H.P. Vasantha Rupasinghe
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Xenograft Model ◽  
Jurkat Cells ◽  
Interferon Α ◽  
Omega 3 ◽  
Cell Acute Lymphoblastic Leukemia

Abstract T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignant disease that accounts for about 15% of pediatric and 25% of adult ALL. Although risk stratification has provided more tailored therapy and improved the overall survival of T-ALL patients, clinical challenges such as suboptimal drug responses, morbidity from drug toxicities, and drug resistance still exist. Plant polyphenols have therapeutic efficacy as pharmacological adjuvants to help overcome these challenges. They can be acylated with fatty acids to overcome issues concerning bioavailability, such as poor intestinal absorption and low metabolic stability. Phloridzin (PZ), a flavonoid found in apple peels, was acylated with an omega-3 fatty acid, docosahexaenoic acid (DHA), to generate a novel ester called phloridzin docosahexaenoate (PZ-DHA). The cytotoxic effect of PZ-DHA was studied in the human Jurkat T-ALL cell line. PZ-DHA significantly reduced the viability and cellular ATP levels of treated cells. PZ-DHA was found to selectively induce apoptosis in Jurkat cells, while sparing normal murine T-cells. Apoptosis was further confirmed by demonstrating the ability of PZ-DHA to induce morphological alterations, DNA fragmentation, caspase activation, and the release of intracellular lactate dehydrogenase. PZ-DHA also significantly inhibited cell division in Jurkat cells. Furthermore, interferon-α-induced phosphorylation of the transcription factor, STAT3, was downregulated following PZ-DHA treatment. The in vitro efficacy of PZ-DHA was recapitulated in vivo in an established zebrafish xenograft model, where the proliferation of transplanted Jurkat cells was inhibited when PZ-DHA was added to the embryo water. Overall, these findings provide evidence for PZ-DHA as a novel therapeutic agent with activity in T-ALL. Studies examining the effect of PZ-DHA on patient-derived ALL cells engrafted in zebrafish are currently underway. Disclosures No relevant conflicts of interest to declare.

scholarly journals The Role of Protein Tyrosine Phosphatase PTP4A3 in T-Cell Acute Lymphoblastic Leukemia Progression

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2539-2539
Author(s):  
Min Wei ◽  
Jessica Blackburn
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
T Cell Activation ◽  
Protein Tyrosine Phosphatase ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Tyrosine Phosphatase ◽  
Causative Role ◽  
Protein Tyrosine ◽  
Cell Acute Lymphoblastic Leukemia

The tyrosine protein tyrosine phosphatase PTP4A3 has been extensively reported to play a causative role in numerous cancers, including several types of acute leukemia. We found PTP4A3 to be highly expressed in T-cell Acute Lymphoblastic Leukemia samples, and show that PTP4A3 accelerates T-ALL onset and increases the invasive ability of T-ALL cells in a zebrafish model, and is required for T-ALL engraftment and progression in mouse xenograft. Our in vitro studies showed that PTP43A3 enhances T-ALL migration, in part via modulation of SRC signaling. However, whether SRC is a direct substrate of PTP4A3, and whether the phosphatase activity of PTP4A3 actually plays a role in T-ALL or other types of leukemia progression is unknown and remains a major question in the field. We used a BioID-based proximity labeling approach combined with PTP4A3 substrate trapping mutant pull down assay to capture the PTP4A3 substrates candidates. BioID, a biotin ligase, was fused to PTP4A3 to generate a Biotin-PTP4A3 (BP) fusion protein. The overexpression of BP in T-ALL cell lines led to biotin modification of 288 PTP4A3 proximal proteins, including the potential direct PTP4A3 substrates. PANTHER pathway analysis showed that PTP4A3 interacting proteins are largely clustered in the T-cell activation, PDGF signaling, and angiogenesis. We are in process of validating potential substrates using immunoprecipitation and phosphoenrichement assays. Finally, we are using a novel zebrafish Myc+PTP4A3 induced T-ALL model to assess the function of PTP4A3 in leukemia progression. We have created several PTP4A3 protein mutants, including a phosphatase-dead mutant, a mutant unable to bind magnesium transporter, and a prenylation deficient mutant, and are in process of assessing the effects of these mutants in T-ALL onset and progression in our in vivo model. In total, these studies will allow us to better understand function of PTP4A3 in T-ALL progression, and may provide a strong rationale for the development of PTP4A3 inhibitors for use in leukemia. Disclosures No relevant conflicts of interest to declare.

Frequency of NUP214-ABL1 Oncogene in Patients with T-Cell Acute Lymphoblastic Leukemia (T-ALL) and Analysis of the Activity of Imatinib and Nilotinib in NUP214-ABL1-Expressing T-ALL Cell Lines.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 710-710
Author(s):  
Alfonso Quintas-Cardama ◽  
Weigang Tong ◽  
Taghi Manshouri ◽  
Jan Cools ◽  
D. Gary Gilliland ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Cell Lines ◽  
Inhibitory Activity ◽  
Lymphoblastic Leukemia ◽  
Fusion Transcript ◽  
Lymphoblastic Lymphoma ◽  
Abl Kinase ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Kinase Inhibitory Activity

Abstract The fusion of ABL1 with BCR results in the hybrid BCR-ABL1 oncogene that encodes the constitutively active Bcr-Abl tyrosine kinase encountered in the majority of patients with chronic myeloid leukemia (CML) and in approximately 30% of pts with B-cell acute lymphoblastic leukemia (B-ALL). Recently, the episomal amplification of ABL1 has been described in 6% of pts with T-ALL (Nat Genet2004;36:1084–9). Molecular analysis demonstrated the oncogenic fusion of ABL1 with the nuclear pore complex protein NUP214 (NUP214-ABL1). We screened 29 pts with T-cell lymphoblastic lymphoma (T-LBL) and T-ALL for the presence of the NUP214-ABL1 fusion transcript by RT-PCR using specific primers for the 5 different transcripts thus far described. Three (10%) pts were found to express this fusion transcript, including 2 with T lymphoblastic lymphoma (NUP214 exon 31) and 1 with T-ALL (NUP214 exon 29). This was confirmed by direct sequencing in all cases. All pts received therapy with hyperCVAD and achieved a complete remission (CR). However, 2 of them died 6 and 9 months into therapy, respectively. One other pt remains in CR (19+ months) by morphologic and flow cytometry criteria. However, NUP214-ABL1 is still detectable in peripheral blood by nested PCR, thus suggesting minimal residual disease (MRD). We then studied the activity of the tyrosine kinase inhibitors imatinib and nilotinib in the NUP214-ABL1-expressing cell lines PEER and BE-13. Although PEER and BE-13 cell viability was reduced with both agents, the IC50 was almost 10-fold higher for imatinib (643 nM) than for nilotinib (68 nM) (F test, p&lt;0.001), which parallels the 10− to 30− fold higher Abl kinase inhibitory activity of nilotinib compared to imatinib in BCR-ABL-expressing cells. Nilotinib also potently inhibited the cell proliferation of BE-13 cells (IC50 131 nM). In contrast, Jurkat cells, a T-ALL cell line which does not carry NUP214-ABL1, were remarkably resistant to both imatinib and nilotinib with an IC50 values greater than 5 μM indicating that the cytotoxicity mediated by both TKIs is not related to a general toxic effect on T-ALL cell lines. The inhibition of cellular proliferation by imatinib and nilotinib was associated with a dose- and time-dependent induction of apoptosis in both PEER and BE-13 cells. In Western blotting, higher inhibition of phospho-Abl and phospho-CRKL (a surrogate of Bcr-Abl kinase status) was observed in PEER cells upon exposure to nilotinib as compared with imatinib at their respective IC50 concentrations for cell growth inhibition. We conclude that NUP214-ABL1 can be detected in 10% of pts with T-cell malignancies and its detection can be used as a sensitive marker of MRD. Imatinib and nilotinib potently inhibits the growth of NUP214-ABL1-expressing cells. Given the higher Abl kinase inhibitory activity of nilotinib with respect to imatinib, this agent must be further investigated in clinical studies targeting patients with T-ALL and T-LBL expressing the NUP214-ABL1 fusion kinase.

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.

Essential Role for Mir-181a1/b1 In T-Cell Acute Lymphoblastic Leukemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 470-470
Author(s):  
Ana Rita Fragoso ◽  
Tin Mao ◽  
Song Wang ◽  
Steven Schaffert ◽  
Hyeyoung Min ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Essential Role ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Cancer Development ◽  
Loss Of Function ◽  
Control Of Gene Expression ◽  
Mirna Genes ◽  
Cell Acute Lymphoblastic Leukemia

Abstract Abstract 470 MiRNA-mediated gene regulation represents a fundamental layer of post-transcriptional control of gene expression with diverse functional roles in normal development and tumorigenesis. Whereas some studies have shown that over-expression of miRNA genes may contribute to cancer development and progression, it is yet to be rigorously tested by the loss-of-function genetic approaches whether miRNA genes are required for cancer development and maintenance in mice. Here we show that mir-181a1/b1 coordinates Notch and pre-TCR signals during normal thymocyte differentiation and plays an essential role in development and onset of T-cell acute lymphoblastic leukemia (T-ALL) induced by some Notch mutations. Using gain-of-function and loss-of-function approaches, we demonstrated that mir-181a1/b1 controls Notch and pre-TCR receptor signals during the early stages of T cell development in the thymus by repressing multiple negative regulators of both pathways, including Nrarp, PTPN-22, SHP2, DUSP5, and DUSP6. These results illustrate that a single miRNA can coordinate multiple signaling pathways by modulating the timing and strength of signaling at different stages. Intriguingly, synergistic signaling between Notch and pre-TCR pathways is necessary for the development of T-ALL, and miR-181 family miRNAs are aberrantly expressed in T-ALL patients. These observations raise the possibility that mir-181a1/b1 might contribute to the onset or maintenance of T-ALL by targeting similar pathways in tumor cells as it does in normal thymic progenitor cells. In support of this notion, we found that loss of mir-181a1/b1 significantly delayed the onset and development of T-ALL induced by intracellular domain of Notch1 (ICN1) and caused a 32% increase in the median survival time from 41 days to 54 days in T-ALL mice. Importantly, we noted that loss of mir-181a1/b1 more efficiently repressed the leukemogeneic potential of cells with lower levels of ICN1 expression, suggesting that mir-181a1/b1 may be more effective in inhibiting T-ALL development induced by a Notch mutant with weaker signal strength. Indeed, we demonstrated that loss of mir-181a1b1 essentially blocked T-ALL development induced by the weaker Notch mutant and dramatically decreased mortality from 60% to 10% in these T-ALL mice. Since human Notch mutations identified in T-ALL patients generally have weaker signaling strength and lower oncogenic potential than that of ICN1, our findings indicate that mir-181a1/b1 may play an essential role in development of normal thymic progenitors and Notch-induced T-ALL and may be targeted to treat T-ALL patients harboring Notch mutations. Disclosures: No relevant conflicts of interest to declare.

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