HSP90 Inhibition Has Potent Activity Against T-Cell Acute Lymphoblastic Leukemia (T-ALL) Through Degradation Of TYK2 Kinase

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2528-2528
Author(s):  
Koshi Akahane ◽  
Takaomi Sanda ◽  
Marc R. Mansour ◽  
Julia Etchin ◽  
Ng Cherry ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Cell Viability ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Hsp90 Inhibitors ◽  
Hsp90 Inhibition ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Bcl2 Expression

Abstract The intensification of therapy for patients with T-cell acute lymphoblastic leukemia (T-ALL) has improved clinical outcomes substantially. However, first-line therapy still continues to fail in approximately 25% of children and in more than 50% of adults, clearly indicating that further therapeutic improvement is urgently needed. Recently, we identified a novel oncogenic pathway that involves aberrant activation of the TYK2 tyrosine kinase and its downstream substrate, STAT1, which ultimately promotes T-ALL cell survival through the upregulation of BCL2 expression (Sanda et al. Cancer Discovery 2013). This finding indicates that in many T-ALL cases, the leukemic cells are dependent upon the TYK2-STAT1-BCL2 pathway for continued survival, suggesting that drugs able to potently inhibit or degrade the TYK2 kinase are likely to provide a therapeutic advantage in patients with T-ALL. However, there are no kinase inhibitors with potent activity to TYK2 under current clinical trials. Heat shock protein 90 (HSP90) is an ATP-dependent molecular chaperone that is exploited by cancer cells to support activated oncoproteins including many cancer-associated kinases, and recent reports on the early clinical efficacy of HSP90 inhibitors are encouraging in some tumors. Based on the finding that TYK2 is related to JAK2 and the reports implicating JAK2 as an HSP90 client protein (Weigert et al. JEM 2012 and Marubayashi et al. J Clin Invest 2010), we investigated the therapeutic efficacy of HSP90 inhibition in T-ALL. First, we tested the effect of two HSP90 inhibitors (AUY922 and HSP990) on the cell viability of T-ALL cell lines. The growth of 10 T-ALL lines, which harbor different genetic aberrations, was dramatically reduced in response to treatment with both compounds, with IC50 values of 5-280 nM (AUY922) and 18-221 nM (HSP990) after 72 hours of exposure. Treatment with AUY922 induced significant apoptosis in KOPT-K1, HPB-ALL and Jurkat “TYK2-dependent” T-ALL cell lines, which are sensitive to shRNA knockdown of the TYK2 gene, as indicated by increased levels of Annexin V staining, which was not observed in Loucy and TALL-1 “TYK2-independent” cells. AUY922 also induced strong cell-cycle arrest at the G2/M phase in some of T-ALL cell lines. Western blotting analysis using T-ALL cell lines after exposure to AUY922 demonstrated rapid reduction of the TYK2 kinase and dephosphorylation of STAT1 in concentrations less than 30 nM of AUY922. The decrease of BCL2 expression after AUY922 treatment was seen only in “TYK2-dependent” T-ALL cell lines, not in “TYK2-independent” lines. These results indicate that pharmacological HSP90 inhibition resulted in TYK2 degradation and subsequent downregulation of its downstream pathway, which includes phosphorylation of STAT1 and activation of BCL2, and this effect is critical especially for the survival of “TYK2-dependent” T-ALL cells. To assess the significance of BCL2 on the cell viability of T-ALL cells after HSP90 inhibition, we tested AUY922 on specific Jurkat cell lines overexpressing each of the pro-survival BCL2 family proteins BCL2, BCLXL, or MCL1. The growth curve after 72 hours of treatment demonstrated that BCL2 overexpression could partially rescue the AUY922-induced decrease of cell viability, but overexpression of BCLXL or MCL1 had no effect. Immunoprecipitation assay using Jurkat cells overexpressing BCL2 indicated that treatment with AUY922 resulted in increased interaction between overexpressed BCL2 and the pro-apoptotic BH3-only protein, Bim. These results suggest that decreasd BCL2 expression is at least partially responsible for the ability of HSP90 inhibitors to induce apoptosis in T-ALL cells. In conclusion, these results provide preclinical evidence of the therapeutic potential of HSP90 inhibitors against T-ALL, providing a rationale for the evaluation of these inhibitors in clinical trials of patients with T-ALL. Disclosures: No relevant conflicts of interest to declare.

TYK2-STAT1 Pathway Positively Regulates BCL2 Gene Expression in T-Cell Acute Lymphoblastic Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1470-1470
Author(s):  
Takaomi Sanda ◽  
Jeffrey W Tyner ◽  
Alejandro Gutierrez ◽  
Vu N Ngo ◽  
Jason M Glover ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Protein Expression ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Wild Type ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Bcl2 Protein ◽  
Bcl2 Expression

Abstract Abstract 1470 To discover oncogenic pathways that are characteristically deregulated in T-cell acute lymphoblastic leukemia (T-ALL), we performed RNA interference screens both in T-ALL cell lines and primary specimens. We found that the JAK tyrosine kinase family member, TYK2, and its downstream effector, STAT1, are each required for the survival of T-ALL cells. To identify the effector molecules downstream of the TYK2-STAT1 pathway in T-ALL, we analyzed global gene expression profiles in TYK2-dependent T-ALL cell lines after silencing of TYK2 or STAT1. As expected, gene set enrichment analysis revealed that genes downregulated by TYK2 knockdown were generally also downregulated by knockdown of STAT1. Importantly, we found that expression of the anti-apoptotic gene BCL2 was significantly downregulated after silencing of both TYK2 and STAT1. Analysis by quantitative PCR of additional T-ALL cell lines revealed that silencing of TYK2 resulted in significant reductions of BCL2 mRNA expression in multiple TYK2-dependent cell lines. Expression of the wild-type but not the kinase-dead TYK2 protein was sufficient to rescue BCL2 protein expression and to prevent apoptosis after knockdown of endogenous TYK2, indicating that the tyrosine kinase activity of TYK2 is required for BCL2 upregulation. Similarly, expression of the shRNA-resistant wild-type STAT1A protein partially rescued BCL2 protein expression and prevented apoptosis, while a variant of STAT1A (Y701F) that is incapable of becoming phosphorylated on a requisite tyrosine residue did not rescue BCL2 levels. Taken together, our findings indicate that aberrant activation of a TYK2-STAT1 pathway upregulates BCL2 expression in T-ALL cells, and that the T-ALL cells develop pathway dependence, in that they require these sustained high levels BCL2 expression for survival. 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<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.

Notch Signaling Represses Mir-223 in T-Cell Acute Lymphoblastic Leukemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4630-4630
Author(s):  
Samuel D Gusscott ◽  
Florian Kuchenbauer ◽  
Andrew P Weng
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Cell Growth ◽  
Notch Signaling ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Gamma Secretase ◽  
Protein Levels ◽  
Cell Acute Lymphoblastic Leukemia ◽  
T Cell Leukemogenesis

Abstract Abstract 4630 T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive cancer of immature T cells that often shows aberrant activation of the Notch1 signaling pathway. Several studies have utilized mRNA expression profiling to identify downstream mediators of oncogenic Notch signaling in this context. Since microRNAs (miRNAs) have in recent years been shown to play important roles in hematological maliganancy, we performed a microarray-based screen for Notch-dependent miRNA expression in T-ALL. Jurkat and P12-Ichikawa cell lines were treated with gamma-secretase inhibitor to block Notch signaling vs. DMSO control for 4 days and profiled using Exigon miRCURY LNA miRNA microarrays. Surprisingly few miRNAs were found to be regulated by this approach; however, one of the hits, miR-223, showed consistent upregulation after gamma-secretase treatment in Jurkat cells and 5 additional human T-ALL cell lines assessed by miRNA qPCR. This observation was unique to human T-ALL as murine models of T-ALL showed no evidence for Notch-dependent miR-223 expression. Given that canonical Notch signaling results in transcriptional activation, our observation that Notch signaling is associated with reduced miR-223 expression suggests an intermediary repressor may be involved. miR-223 has been reported to play an important role in normal granulopoiesis, to be expressed relatively highly in T-ALL with myeloid-like gene features, and most recently to accelerate Notch-mediated T-cell leukemogenesis. To explore potential functional consequences for Notch-dependent miR-223 repression in T-ALL, candidate miR-223 targets identified by TargetScan software were analyzed with Ingenuity Pathway Analysis software, which indicated IGF-1, insulin receptor, PTEN, and ERK5 signaling pathways as the top hits. We recently reported IGF1R signaling to be important for growth and viability of bulk T-ALL cells as well as for leukemia-initiating cell activity. Additionally, we reported that Notch signaling directly upregulates IGF1R transcription by binding to an intronic enhancer which is present between exons 21/22 in the human, but not mouse IGF1R locus. As miR-223 has previously been reported to target IGF1R mRNA and reduce its translation, we hypothesized that Notch signaling may also upregulate net IGF1R protein expression by repressing miR-223. To test this hypothesis, we transduced several human T-ALL cell lines with miR-223 retrovirus and observed a modest decrease in total IGF1R protein levels by western blot; however, no significant change was observed in surface IGF1R levels as assessed by flow cytometry. Addtionally, knockdown of miR-223 by lentiviral expression miR-223 target sequences (miR-223 “sponge”) resulted in modestly increased total IGF1R protein levels, but again showed no demonstrable effect on surface IGF1R levels. Of note, we also observed no apparent effect of either overexpression or knockdown of miR-223 on bulk cell growth or viability. We interpret these findings to suggest that Notch signaling does not have major effects on the miR transcriptome, and that up- or down-modulation of miR-223 in established T-ALL cells does not have significant effects on overall cell growth/viability. Further studies will be required to determine if miR-223 may act in concert with other Notch target genes to modulate cell physiology. Disclosures: No relevant conflicts of interest to declare.

Down-Regulation of Mir-26b and Its Inhibition on the Growth of T Cell Acute Lymphoblastic Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2440-2440
Author(s):  
Tian Yuan ◽  
Yaling Yang ◽  
Jeffrey You ◽  
Daniel Lin ◽  
Kefeng Lin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Cell Growth ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Expression Level ◽  
Rt Pcr ◽  
Altered Expression ◽  
Cell Acute Lymphoblastic Leukemia

Abstract Introduction: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy accounting for 15% of pediatric and 25% of adult acute lymphoblastic leukemia (ALL) cases. With current chemotherapies and transplantation therapy, there are still 25-50% T-ALL patients that suffer from relapse and have a poor outcome. MicroRNAs (miRNAs or miRs) are endogenous small non-coding RNAs (containing about 22 nucleotides in length). miRs function at posttranscriptional level as negative regulators of gene expression and exert their regulatory function through binding to target mRNAs and silencing gene expression. To better understand the pathogenesis and develop the new therapeutic targets of T-ALL, we have developed a Pten tumor suppressor knockout T-ALL mouse model and profiled miRs from the mouse Pten deficient T-ALL. miR-26b was one of the miRs that were found down-regulated in the mouse Pten deficient T-ALL. Recent studies showed that the aberrant expression of miR-26b is implicated in several types of cancer. The expression level of miR-26b and its role of in T-ALL, however, are unknown. We investigated if the expression level of miR-26b is aberrant in T-ALL and the effect of potentially altered expression on the growth of human T-ALL cells. Methods: We conducted miR array profiling to identify differentially expressed miRs in the mouse Pten deficient T-ALLs compared with preneoplastic thymocyte controls. We validated expression levels of several miRs, including miR-26b, that are differentially expressed in mouse and human T-ALL cells using quantitative RT-PCR. We also overexpressed miR-26b using a lentivirus based vector in human T-ALL cell lines to assess its effect on cell growth and apoptosis. Results: Employing miR array profiling, we identified a subset of miRs that exhibited marked altered expression in the mouse Pten deficient T-ALL cells. Quantitative RT-PCR validated that the expression level of miR-26b in the mouse Pten deficient T-ALL cells was markedly lower in comparison to that of preneoplastic thymocytes. To determine if miR-26b expression level is also altered in human T-ALL, we performed quantitative RT-PCR on a panel of human T-ALL cell lines. Indeed, the expression level of miR-26b is significantly lower in the human T-ALL cell lines when compared with that of normal thymocytes. To functionally assess if miR-26b plays a role in the cell growth of human T-ALL cells, we expressed exogenous miR-26b in a panel of human T-ALL cell lines. We demonstrated that the expression of exogenous miR-26b significantly reduced the proliferation and promoted apoptosis of several human T-ALL cell lines. Conclusions: Our results demonstrated that miR-26b is down-regulated in T-ALL and the expression of exogenous miR-26b elicits deceased cell proliferation and increased apoptosis of human T-ALL. These results suggest that miR-26b may function as a tumor suppressor in the development of T-ALL and further characterization of the target and regulation of miR-26b may have therapeutic implications. Disclosures No relevant conflicts of interest to declare.

scholarly journals Glucocorticoids Paradoxically Induce Intrinsic Steroid Resistance through a STAT5-Mediated Survival Mechanism in T-Cell Acute Lymphoblastic Leukemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 913-913
Author(s):  
Lauren K. Meyer ◽  
Benjamin Huang ◽  
Ritu Roy ◽  
Aaron Hechmer ◽  
Anica Wandler ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Lymphoblastic Leukemia ◽  
Gene Set Enrichment Analysis ◽  
Steroid Resistance ◽  
Luciferase Reporter ◽  
Index Analysis ◽  
Stat Signaling ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Bcl2 Expression

Abstract Upfront resistance to glucocorticoids (GCs) confers a poor prognosis for children with T-cell acute lymphoblastic leukemia (T-ALL). Using primary diagnostic samples from the Children's Oncology Group trial AALL1231, we previously demonstrated that one-third of patient T-ALL samples are intrinsically resistant to GCs when cultured in the presence of interleukin-7 (IL7), a cytokine that is abundant in the T-ALL microenvironment. Furthermore, we demonstrated that inhibiting JAK/STAT signaling downstream of the IL7 receptor (IL7R) with the JAK1/2 inhibitor ruxolitinib (RUX) overcomes GC resistance in these samples. The objective of the present study was to determine the mechanism of IL7-induced GC resistance in T-ALL and to identify novel therapeutic targets to enhance GC sensitivity. We utilized CCRF-CEM cells, a human T-ALL cell line, as a model system in conjunction with primary patient samples. Exposing CCRF-CEM cells to IL7 induced phosphorylation of STAT5, the predominant downstream effector of IL7R signaling. When cultured in the presence of IL7 and the GC dexamethasone (DEX), CCRF-CEM cells recapitulated the IL7-induced GC resistance phenotype observed in patient samples. This resistance could be overcome with RUX, and Bliss index analysis demonstrated a synergistic relationship between DEX and RUX in the presence of IL7. Furthermore, CRISPR/Cas9 mediated knockout of STAT5 (STAT5 KO) was sufficient to overcome resistance, implicating STAT5 as the critical mediator of IL7-induced GC resistance. DEX exposure potently induced upregulation of IL7R expression in CCRF-CEM cells. Using a luciferase reporter construct containing a series of STAT5 response elements, we demonstrated that in the presence of IL7, DEX-induced upregulation of IL7R expression is associated with increased downstream signal transduction, leading to a significant increase in STAT5 transcriptional output. We then performed RNA-seq to further assess the functional consequences of this enhanced STAT5-mediated transcription. Gene set enrichment analysis (GSEA) revealed that STAT5 target genes were significantly upregulated in cells exposed to DEX and IL7 relative to IL7 alone (normalized enrichment score -2.27; p < 0.001; FDR < 0.001), suggesting that DEX exposure augments activation of the STAT5 transcriptional program. One critical component of this program that was induced by the combination of DEX and IL7 was the anti-apoptotic family member BCL2, which was not induced by DEX alone. Further analysis of its protein expression in CCRF-CEM cells confirmed this paradoxical upregulation of BCL2 specifically by the combination of DEX and IL7. Furthermore, BCL2 was not upregulated by DEX and IL7 in STAT5 KO cells, consistent with this being a STAT5-mediated effect. IL7-induced GC resistance could be overcome with shRNA-mediated knockdown of BCL2 and with pharmacologic inhibition of BCL2 by venetoclax. Similar to the effect observed with RUX, Bliss index analysis demonstrated synergy between DEX and venetoclax in the presence of IL7. Consistent with our observations in CCRF-CEM cells, an analysis of primary diagnostic T-ALL samples revealed DEX-induced upregulation of IL7R expression in samples with IL7-induced GC resistance, which was associated with increased BCL2 expression in the presence of DEX and IL7. Finally, we performed a similar analysis in healthy murine thymocytes, and found that CD4/CD8 double negative (DN) and CD4 or CD8 single positive (SP) thymocytes, but not double positive (DP) thymocytes, exhibited profound IL7-induced GC resistance that was associated with DEX-induced upregulation of IL7R expression and increased BCL2 expression in the presence of DEX and IL7. These data are consistent with the known role of IL7 specifically at the DN and SP stages of development, and suggests that IL7-induced GC resistance is a physiologic mechanism of GC resistance in normal thymocyte development that is retained during leukemogenesis in a subset of T-ALL samples. Taken together, these data demonstrate that GCs paradoxically induce their own resistance in a subset of T-ALLs and in normal developing T-cells by augmenting a STAT5-mediated pro-survival program that results in upregulation of BCL2. Furthermore, we demonstrate that inhibition of JAK/STAT signaling or of BCL2 may have considerable therapeutic benefit to enhance GC sensitivity in T-ALL patients with IL7-induced GC resistance. Disclosures Teachey: La Roche: Consultancy; Amgen: Consultancy.

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.

Sign in / Sign up

Export Citation Format

Share Document