Pathway Dependence on the Tyrosine Kinase TYK2 and Its Mediator STAT1 In T-Cell Acute Lymphoblastic Leukemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3155-3155
Author(s):  
Takaomi Sanda ◽  
Jeffrey W Tyner ◽  
Alejandro Gutierrez ◽  
Vu N Ngo ◽  
Richard Moriggl ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Tyrosine Kinase ◽  
Cell Lines ◽  
Small Molecule ◽  
Lymphoblastic Leukemia ◽  
B Cell Lymphoma ◽  
Knock Down ◽  
A Genome ◽  
Cell Acute Lymphoblastic Leukemia

Abstract Abstract 3155 The often aggressive and unpredictable behavior of T-cell lymphoblastic malignancies continues to pose both major clinical challenges in children and adults. To discover oncogenic pathways downstream of critical genetic abnormalities that are characteristically deregulated in T-cell acute lymphoblastic leukemia (T-ALL), and to identify novel molecular targets for anti-leukemic agents with T-cell specificity, we carried out a genome-wide functional screen in T-ALL cell lines using a retroviral library of inducible short-hairpin RNAs (shRNAs). Among the genes that are required for the growth of T-ALL cells, we found that loss of TYK2, a JAK family tyrosine kinase, was specifically lethal in each of three T-ALL cell lines that we tested in this screen. By contrast, TYK2 knock-down did not affect the growth of cell lines from diffuse large B-cell lymphoma or multiple myeloma, indicating that TYK2 is specifically required for the growth of T-ALL cells. We confirmed by knock-down with multiple independent shRNAs that the loss of TYK2 induces apoptosis in T-ALL, whereas knock-down of other JAK proteins (JAK1, JAK2 or JAK3) had no effect. We found that the TYK2 protein is constitutively phosphorylated in many T-ALL cell lines, and that these cells are sensitive to small molecule JAK/TYK2 inhibitors, including JAK inhibitor-I, AG-490 and CEP-701. To identify upstream receptors involved in TYK2 activation, we knocked down IFNAR, IFNGR, IL10R and IL12RB, and found that loss of IFNAR1 and IFNAR2 specifically inhibit the growth of T-ALL cells, as strongly as TYK2 knock-down. In addition, targeted knock-down analysis of downstream STAT proteins revealed that loss of STAT1 also inhibits the growth of T-ALL cells, indicating that this transcription factor is involved in the TYK2 pathway and required for cell survival. In fact, STAT1 protein was constitutively phosphorylated in many T-ALL cell lines and this phosphorylation was inhibited by both TYK2 knock-down and treatment with JAK/TYK2 inhibitors. Although interferon-mediated pathways are cytostatic in many cell types, our results indicate a requirement for the IFNAR-TYK2-STAT1 pathway in promoting the growth and survival of T-ALL cells. Dependence on this pathway confers unique sensitivity of T-ALL cells to TYK2 inhibition by small molecule inhibitors, thus providing a novel therapeutic target for clinical testing in patients with this disease. Disclosures: Druker: Molecular MD: Equity Ownership.

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.

scholarly journals Protein Tyrosine Phosphatase 4A3 (PTP4A3) modulates Src signaling in T-cell Acute Lymphoblastic Leukemia to promote leukemia migration and progression

2019 ◽  
Author(s):  
M Wei ◽  
MG Haney ◽  
JS Blackburn
Keyword(s):  
Cell Migration ◽  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Small Molecule ◽  
Protein Tyrosine Phosphatase ◽  
Lymphoblastic Leukemia ◽  
Tyrosine Phosphatase ◽  
Knock Down ◽  
Src Signaling ◽  
Cell Acute Lymphoblastic Leukemia

AbstractT-cell Acute Lymphoblastic Leukemia (T-ALL) is an aggressive blood cancer, and currently, there are no immunotherapies or molecularly targeted therapeutics available for treatment of this malignancy. The identification and characterization of genes and pathways that drive T-ALL progression is critical for development of new therapies for T-ALL. Here, we determined that Protein Tyrosine Phosphatase 4A3 (PTP4A3) plays a critical role in disease initiation and progression by promoting cell migration in T-ALL. PTP4A3 expression was upregulated in patient T-ALL samples at both the mRNA and protein levels compared to normal lymphocytes. Inhibition of PTP4A3 function with a small molecule inhibitor and knock-down of PTP4A3 expression using short-hairpin RNA (shRNA) in human T-ALL cells significantly impeded T-ALL cell migration capacityin vitroand reduced their ability to engraft and proliferatein vivoin xenograft mouse models. Additionally, PTP4A3 overexpression in aMyc-induced zebrafish T-ALL model significantly accelerated disease onset and shortened the time needed for cells to enter blood circulation. Reverse phase protein array (RPPA) revealed that manipulation of PTP4A3 expression levels in T-ALL cells directly affected the SRC signaling pathway, which plays a well-characterized role in migratory behavior of several cell types. Taken together, our study revealed that PTP4A3 is a key regulator of T-ALL migration via SRC signaling, and suggests that PTP4A3 plays an important role as an oncogenic driver in T-ALL.HighlightsA subset of T-cell Acute Lymphoblastic Leukemia (T-ALL) highly express the phosphatase PTP4A3PTP4A3 expression promotes leukemia development in zebrafish T-ALL modelsLoss of PTP4A3 prevents T-ALL engraftment in mouse xenograft modelsKnock-down or small molecule inhibition of PTP4A3 prevents T-ALL migration in part via modulation of SRC signaling.

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.

Anti-Leukemic Activity of the TYK2 Selective Inhibitor Ndi-031301 in T-Cell Acute Lymphoblastic Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1596-1596
Author(s):  
Koshi Akahane ◽  
Zhaodong Li ◽  
Julia Etchin ◽  
Alla Berezovskaya ◽  
Evisa Gjini ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Tyrosine Kinase ◽  
P38 Mapk ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Parp Cleavage ◽  
Immunodeficient Mice ◽  
Tyrosine Kinase 2 ◽  
Cell Acute Lymphoblastic Leukemia

Abstract T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy resulting from the transformation of T-cell progenitors. Although the prognosis of this disease has substantially improved due to the introduction of intensified chemotherapy, the clinical outcome of T-ALL patients with primary resistant or relapsed disease remains poor, indicating that further therapeutic improvement is urgently needed. We have previously demonstrated that activation of tyrosine kinase 2 (TYK2) contributes to aberrant survival of human T-ALL cells. TYK2 is a member of the Janus-activated kinase (JAK) tyrosine kinase family and our report was the first to implicate TYK2 in T-ALL pathogenesis. Indeed, our gene knockdown experiments showed TYK2 dependency in 14 of 16 (88%) T-ALL cell lines and 5 of 8 (63%) patient-derived T-ALL cells tested, suggesting that inhibition of TYK2 would be beneficial in most patients with T-ALL. Based on these findings, we investigated the therapeutic potential of a novel small-molecule TYK2 kinase inhibitor NDI-031301 in T-ALL. We found that NDI-031301 shows potent and selective inhibitory activity against TYK2 in a cellular context, because this compound strongly inhibited the growth of TYK2-transfomed Ba/F3 cells when compared to the JAK inhibitors tofacitinib and baricitinib, whereas Ba/F3 cells transformed by other tyrosine kinases showed decreased sensitivity to NDI-031301. NDI-031301 induced robust growth inhibition in each of 4 human T-ALL cell lines representing different molecular subtypes of the disease (DU.528, KOPT-K1, HPB-ALL and SKW-3), with IC50 values of 0.8186 - 2.380 μM after 72 hours of exposure. NDI-031301 treatment of human T-ALL cell lines resulted in induction of apoptosis that was not observed with tofacitinib and baricitinib. To elucidate the mechanism of apoptosis induced by NDI-031301 in T-ALL cells, we next investigated cellular signaling pathways that are associated with cell survival and specifically affected by TYK2 inhibition with NDI-031301. Western blotting analysis demonstrated that treatment with 3 μM of NDI-031301 resulted in reduction of STAT1 Tyr-701 phosphorylation and BCL2 levels in KOPT-K1 cells, consistent with our previous finding that TYK2 phosphorylates STAT1 and upregulates BCL2 expression in most T-ALL cells. Surprisingly, the treatment also uniquely led to activation of three mitogen-activated protein kinases (MAPKs), resulting in phosphorylation of ERK, SAPK/JNK and p38 MAPK coincident with PARP cleavage, which was not observed with tofacitinib and baricitinib. NDI-031301-mediated activation of SAPK/JNK and p38 MAPK pathways are likely mediated through inhibition of TYK2, because increased phosphorylation levels of SAPK/JNK and p38 MAPK were observed in the cells transfected with TYK2-targeting shRNAs, while the levels of ERK1/2 phosphorylation were not upregulated. Further investigation revealed that activation of p38 MAPK occurred within 1 hour of NDI-031301 treatment and was responsible for NDI-031301-induced T-ALL cell death, as pharmacologic inhibition of p38 MAPK by SB203580 partially rescued apoptosis induced by TYK2 inhibitor, while inhibition of ERK or SAPK/JNK showed no rescue effects. Finally, we found that daily oral administration of NDI-031301 at 100mg/kg BID to immunodeficient mice engrafted with KOPT-K1 T-ALL cells was well tolerated, and led to decreased tumor burden and a significant survival benefit. After 29 days of treatment, the mice receiving NDI-031301 had marked reductions in infiltration of leukemia cells into spleen and bone marrow by comparison with controls. Thus, our findings clearly support TYK2 inhibition with NDI-031301 or a related compound as a potential therapeutic strategy for patients with T-ALL, and also raise the possibility that enhancing p38 MAPK activation in T-ALL cells may be an approach to accentuate its anti-leukemic activity. Disclosures Masse: Nimbus Therapeutics: Employment. Miao:Nimbus Therapeutics: Employment. Rocnik:Nimbus Therapeutics: Employment. Kapeller:Nimbus Therapeutics: Employment.

Abstract 2809: Pathway dependence on the tyrosine kinase TYK2 in T-cell acute lymphoblastic leukemia

2011 ◽  
Author(s):  
Takaomi Sanda ◽  
Jeffrey W. Tyner ◽  
Alejandro Gutierrez ◽  
Vu N. Ngo ◽  
Richard Moriggl ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Tyrosine Kinase ◽  
Lymphoblastic Leukemia ◽  
Cell Acute Lymphoblastic Leukemia

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.

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