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

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.

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Epigenetic silencing of SOCS 5 potentiates JAK ‐ STAT signaling and progression of T‐cell acute lymphoblastic leukemia

Cancer Science ◽

10.1111/cas.14021 ◽

2019 ◽

Vol 110 (6) ◽

pp. 1931-1946

Author(s):

Nitesh D. Sharma ◽

Christian K. Nickl ◽

Huining Kang ◽

Wojciech Ornatowski ◽

Roger Brown ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

T Cell ◽

Lymphoblastic Leukemia ◽

Epigenetic Silencing ◽

Stat Signaling ◽

Cell Acute Lymphoblastic Leukemia

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Effect of Tissue Transglutaminase on Steroid Resistance in T-Cell Acute Lymphoblastic Leukemia

Anticancer Research ◽

10.21873/anticanres.13824 ◽

2019 ◽

Vol 39 (11) ◽

pp. 6165-6173

Author(s):

HYUN JOO JUNG ◽

EUN HEE HAN ◽

IN KEUN JANG ◽

SEUNG-HYUN YOON ◽

JUN EUN PARK

Keyword(s):

Acute Lymphoblastic Leukemia ◽

T Cell ◽

Tissue Transglutaminase ◽

Lymphoblastic Leukemia ◽

Steroid Resistance ◽

Cell Acute Lymphoblastic Leukemia

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Distribution and Clinical Features of NOTCH1 Signaling Activating Alterations in Pediatric T-Cell Acute Lymphoblastic Leukemia (T-ALL)

Blood ◽

10.1182/blood-2018-99-111092 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4089-4089

Author(s):

Shunsuke Kimura ◽

Masafumi Seki ◽

Kenichi Yoshida ◽

Hiroo Ueno ◽

Yasuhito Nannya ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

T Cell ◽

Childhood Leukemia ◽

Transmembrane Domain ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Luciferase Reporter ◽

Internal Deletion ◽

Notch1 Signaling ◽

Cell Acute Lymphoblastic Leukemia

Abstract Introduction NOTCH1 and FBXW7 alterations leading to aberrant activation of NOTCH1 signaling, classified into two patterns; ligand-independent activation (LIA) and impaired degradation (ID) of NOTCH1. In general, activation of NOTCH1 axis is a hallmark of T-cell acute lymphoblastic leukemia (T-ALL), though comprehensive studies regarding subclonal mutations inducing NOTCH1 activation are still elusive. In the present study, we explored the clinicopathological relevance of NOTCH1/FBXW7 aberrations considering subclonal alterations. Methods A total of 176 cases with pediatric T-ALL were enrolled in this study. We reanalyzed our previous data of targeted-capture sequencing (n=176) for 158 ALL-related genes/regions and combined with previous expression profiling data based on whole transcriptome sequencing (WTS; n=121). We defined as a subclonal mutation when variant allele frequency was below 0.15 and/or multiple alterations were found within the same pattern of NOTCH1 activation (LIA or ID). All patients were received Berlin-Frankfurt-Münster based chemotherapies with non-minimal residual disease (MRD) based risk stratification, which were mainly offered from the Tokyo Children's Cancer Study Group (TCCSG) and the Japan Association of Childhood Leukemia Study (JACLS). Results In total, we detected aberrations activating NOTCH1 signaling in 81.3% (143/176) of cases including subclonal mutations. Subclonal alterations were observed in 26.7% (n=47). Single nucleotide variations in the heterodimerization domain (HD-SNV) were the most frequent (43.2%; n=76), followed by PEST domain mutations (33.0%; n=58), FBXW7 mutations (26.1%; n=46), non-frameshift indels of NOTCH1 (19.9%; n=35), and in-frame internal duplication known as juxta-membrane expansion (6.3%; n=11). Amplification of NOTCH1 region and 5' NOTCH1 deletion were not detected in our cohort. Both LIA and ID patterns were detected in 43.2% (n=76). Most mutations were mutually exclusive within each LIA and ID pattern. Intriguingly, we detected four (2.3%) internal deletion of NOTCH1 gene (DEL; missing exon 3-27 (DEL3) or 21-27 (DEL21)), three cases (1.7%) of SNV at 3' untranslated region, and two (1.1%) SEC16A-NOTCH1 fusions. These alterations were previously reported to activate NOTCH1 signaling in breast cancer or chronic lymphoblastic leukemia, except for DEL21. We confirmed that DEL21 strongly activates NOTCH1 signaling by luciferase reporter assay (over 100 times compared to wild type NOTCH1). As previously reported in DEL3 and CUTLL cell line, transcripts might initiate at methionine 1737 located within the NOTCH1 transmembrane domain and seem to be sensitive to γ-secretase inhibitors. Analysis of frequency of detected NOTCH1 activating alterations in each previously reported WTS-based cluster (ETP, SPI1, TLX, TAL1-RA, and TAL1-RB) revealed that alterations were frequently detected in TLX (100%; 24/24) and TAL1-RB (95.1%; 39/41), whereas less frequent in TAL1-RA (61.1%; 11/18). In TAL1-RA, all SEC16A-NOTCH1 fusions were observed despite significantly low rate of HD-SNV (11.1%; 2/18). In SPI1 cluster, PEST domain alterations were frequently detected (71.4%; 5/7). Importantly, cases harboring subclonal NOTCH1/FBXW7 alterations showed significantly worse outcome (log-rank P = 0.01), although there was no prognostic difference between cases with and without NOTCH1/FBXW7 mutations. Conclusions We observed NOTCH1 activating alterations in 81.3% of pediatric T-ALL cases and detected rare internal deletion of NOTCH1 gene and NOTCH1 fusions recurrently in T-ALL. Furthermore, the presence of subclonal NOTCH1/FBXW7 mutations might be relevant to unfavorable outcome. Despite several limitations such as non-MRD based treatment, our results might be useful for developing a new anti-NOTCH1 therapeutic strategy for pediatric T-ALL patients. Disclosures No relevant conflicts of interest to declare.

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The NOTCH1 Driven Long Non-Coding RNA Repertoire in T-Cell Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v124.21.900.900 ◽

2014 ◽

Vol 124 (21) ◽

pp. 900-900

Author(s):

Annelynn Wallaert ◽

Kaat Durinck ◽

Pieter Rondou ◽

Inge van de Walle ◽

Wouter Vanloocke ◽

...

Keyword(s):

T Cells ◽

Acute Lymphoblastic Leukemia ◽

T Cell ◽

Lymphoblastic Leukemia ◽

Gene Set Enrichment Analysis ◽

Protein Coding ◽

Gene Set ◽

Notch1 Signaling ◽

Cell Acute Lymphoblastic Leukemia ◽

The Individual

Abstract Introduction: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive cancer resulting from the malignant transformation of T-cell precursors. Genetic studies in T-cell acute lymphoblastic leukemia (T-ALL) have uncovered a remarkable heterogeneity of genetic defects. Amongst these, NOTCH1activating mutations are the most frequently occurring events (>50%) in T-ALL. Since long non-coding RNAs (lncRNAs) are emerging as important players in oncogenesis, we decided to decode the NOTCH1 driven lncRNA transcriptional landscape in T-ALL and normal T-cell development. Methods and Results: RNA-sequencing was performed following pharmacological inhibition (GSI) of the NOTCH1 mutant and gamma secretase inhibitor (GSI) sensitive T-ALL cell line CUTLL1 in a time series experiment as well as for human CD34+ thymic progenitor T-cells cultured on an OP9 feeder layer with or without DL1-triggered NOTCH1 stimulation. First, we validated both model systems by confirming robust regulation of multiple canonical known protein coding NOTCH1 target genes including DTX1, NOTCH3 and NRARP. Next, we identified distinct subsets of NOTCH1 regulated lncRNAs in both experiments with an overlap of 27 commonly regulated NOTCH1 driven and previously annotated lncRNAs. An even larger number of novel, unannotated T-ALL/T-cell specific lncRNAs was found to be NOTCH1 regulated. Next, we took advantage of publically available ChIP-sequencing data for ICN1 and enhancer specific chromatin marks in CUTLL1 (Wang et al., PNAS, 2013), allowing the selection for direct regulated lncRNAs with enhancer properties. Amongst these lncRNAs, the recently described LUNAR1 lncRNA (Trimarchi et al., Cell, 2014) was present as top candidate in our dataset, thus validating this approach for further selecting bona fide NOTCH1 regulated lncRNAs. In a first step towards functional annotation of this subset of selected lncRNAs, we performed so-called guilt-by-association analysis through correlating expression levels of the individual lncRNAs with transcriptome data for all protein coding genes followed by gene set enrichment analysis in a large cohort of primary T-ALL patients. Subsequent enrichment mapping of significant gene sets yielded markedly different gene set clustering patterns for each of the individual analyzed lncRNAs, as well as distinct annotated related functionalities such as cytokine signaling, TCA-cycle, DNA replication and repair and translation. Prioritarization of lncRNAs for further functional validation was performed by measuring their expression in an extended panel of GSI-treated T-ALL cell lines (HPB-ALL, DND-41, T-ALL1 and ALL-SIL), sorted subsets of CD34+ and CD4+/CD8+ double positive thymocytes and an independent T-ALL patient cohort. Conclusion: We present the landscaping of an integrated lncRNA network acting downstream of NOTCH1 signaling in T-ALL and normal T-cells. These data pave the way towards the development of novel therapeutic strategies impacting on hyperactive NOTCH1 signaling. Disclosures No relevant conflicts of interest to declare.

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

Blood ◽

10.1182/blood.v120.21.1470.1470 ◽

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.

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Targeting steroid resistance in T-cell acute lymphoblastic leukemia

Blood Reviews ◽

10.1016/j.blre.2019.100591 ◽

2019 ◽

Vol 38 ◽

pp. 100591 ◽

Cited By ~ 7

Author(s):

Renate De Smedt ◽

Julie Morscio ◽

Steven Goossens ◽

Pieter Van Vlierberghe

Keyword(s):

Acute Lymphoblastic Leukemia ◽

T Cell ◽

Lymphoblastic Leukemia ◽

Steroid Resistance ◽

Cell Acute Lymphoblastic Leukemia

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JDP2: An oncogenic bZIP transcription factor in T cell acute lymphoblastic leukemia

Journal of Experimental Medicine ◽

10.1084/jem.20170484 ◽

2018 ◽

Vol 215 (7) ◽

pp. 1929-1945 ◽

Cited By ~ 6

Author(s):

Marc R. Mansour ◽

Shuning He ◽

Zhaodong Li ◽

Riadh Lobbardi ◽

Brian J. Abraham ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

T Cell ◽

Insertional Mutagenesis ◽

Lymphoblastic Leukemia ◽

Steroid Resistance ◽

Bzip Transcription Factor ◽

Transgenic Zebrafish ◽

Cell Acute Lymphoblastic Leukemia ◽

Resistance To Steroids

A substantial subset of patients with T cell acute lymphoblastic leukemia (T-ALL) develops resistance to steroids and succumbs to their disease. JDP2 encodes a bZIP protein that has been implicated as a T-ALL oncogene from insertional mutagenesis studies in mice, but its role in human T-ALL pathogenesis has remained obscure. Here we show that JDP2 is aberrantly expressed in a subset of T-ALL patients and is associated with poor survival. JDP2 is required for T-ALL cell survival, as its depletion by short hairpin RNA knockdown leads to apoptosis. Mechanistically, JDP2 regulates prosurvival signaling through direct transcriptional regulation of MCL1. Furthermore, JDP2 is one of few oncogenes capable of initiating T-ALL in transgenic zebrafish. Notably, thymocytes from rag2:jdp2 transgenic zebrafish express high levels of mcl1 and demonstrate resistance to steroids in vivo. These studies establish JDP2 as a novel oncogene in high-risk T-ALL and implicate overexpression of MCL1 as a mechanism of steroid resistance in JDP2-overexpressing cells.

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

Blood ◽

10.1182/blood.v122.21.2528.2528 ◽

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.

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