Bcl-xL Promoter Research Articles

Cross-Talk between Cytokine and NF-ĸB Signaling in the CLL Microenvironment Can Affect Sensitivity for Venetoclax

INTRODUCTION. The Bcl-2 inhibitor Venetoclax provides profound reductions in circulating chronic lymphocytic leukemia (CLL) cells in the majority of patients. The lymph node (LN) responses are less complete, which may be linked with pro-survival signals in the LN. Bcl-XL expression is prominently increased in LN compared to blood. Since Bcl-XL is a prime determinant of resistance to Venetoclax, its regulation is of clinical significance. In the present study, we investigated how candidate LN signals from T helper cytokines IL-21 or IL-4 affect CD40-signalling, specifically in relation to sensitivity/resistance to Venetoclax.RESULTS In an expression screen of changes in apoptotic regulators following CD40 stimulation, IL-4 and IL-21 had opposing effects specifically on Bcl-XL expression. This was associated with CD40-induced resistance to Venetoclax which was augmented by IL-4 and reversed by IL-21. We investigated the signalling pathway(s) involved and found that upon IL-4 or IL-21 stimulation, STAT3 and -6 were differentially phosphorylated, and pharmacological inhibition of upstream JAK kinases restored Bcl-XL expression. Molecularly, IL-21 or IL-4 signals did not significantly affect (non-)canonical NF-κB activation measured by DNA-binding of p65 or p52. STAT3 and -6 have predicted binding sites near the known p65 and p52 binding sites in the Bcl-XL promoter region. Using reporter assays with Bcl-XL promotor constructs we demonstrate synergy (STAT6) or competition (STAT3) with NF-κB. We next applied novel IKKα and NIK inhibitors to inhibit NF-kB signalling during CD40 stimulation. Especially IKKα inhibition specifically blocked Bcl-XL expression, without affecting overall viability of the CLL cells or normal T cells.Importantly, both types of NF-κB inhibitors significantly reversed CD40-induced resistance for Venetoclax. Averaged LD50 values for Venetoclax increased approximately 2500-fold from 1.8 nM to 4.6 μM after 24 hrs CD40 stimulation, but in the presence of IKKα inhibitor only to 7.4 nM (N=8). This value remains well below the average plasma Venetoclax level in CLL patients undergoing treatment.CONCLUSIONS. These data show that protective signals from the CLL microenvironment can be tipped towards apoptosis sensitivity by interfering with JAK/STAT and NF-kB signals, providing novel therapeutic clues in case of emerging resistance to targeted drugs such as Venetoclax. DisclosuresKater:Celgene: Consultancy, Research Funding; Johnson & Johnson: Research Funding; Abbvie: Research Funding. Eldering:Roche: Research Funding; Gilead: Research Funding; Celgene: Research Funding.

Regulation of Bcl-XL by non-canonical NF-\u03baB in the context of CD40-induced drug resistance in CLL

In chronic lymphocytic leukemia (CLL), the lymph node (LN) microenvironment delivers critical survival signals by inducing the expression of anti-apoptotic Bcl-2 members Bcl-XL, Bfl-1, and Mcl-1, resulting in apoptosis blockade. We determined previously that resistance against various drugs, among which is the clinically applied BH3 mimetic venetoclax, is dominated by upregulation of the anti-apoptotic regulator Bcl-XL. Direct clinical targeting of Bcl-XL by, e.g., Navitoclax is however not desirable due to induction of thrombocytopenia. Since the actual regulation of Bcl-XL in CLL in the context of the LN microenvironment is not well elucidated, we investigated various candidate LN signals to drive Bcl-XL expression. We found a dominance for NF-κB signaling upon CD40 stimulation, which results in activation of both the canonical and non-canonical NF-κB signaling pathways. We demonstrate that expression of Bcl-XL is first induced by the canonical NF-κB pathway, and subsequently boosted and continued via non-canonical NF-κB signaling through stabilization of NIK. NF-κB subunits p65 and p52 can both bind to the Bcl-XL promoter and activate transcription upon CD40 stimulation. Moreover, canonical NF-κB signaling was correlated with Bfl-1 expression, whereas Mcl-1 in contrast, was not transcriptionally regulated by NF-κB. Finally, we applied a novel compound targeting NIK to selectively inhibit the non-canonical NF-κB pathway and showed that venetoclax-resistant CLL cells were sensitized to venetoclax. In conclusion, protective signals from the CLL microenvironment can be tipped towards apoptosis sensitivity by interfering with non-canonical NF-κB signaling.

The transcription factor ETS1 promotes apoptosis resistance of senescent cholangiocytes by epigenetically up-regulating the apoptosis suppressor BCL2L1

Primary sclerosing cholangitis (PSC) is an idiopathic, progressive cholangiopathy. Cholangiocyte senescence is important in PSC pathogenesis, and we have previously reported that senescence is regulated by the transcription factor ETS proto-oncogene 1 (ETS1) and associated with overexpression of BCL2 like 1 (BCL2L1 or BCL-xL), an anti-apoptotic BCL2-family member. Here, we further explored the mechanisms regulating BCL-xL-mediated, apoptosis resistance in senescent cholangiocytes and uncovered that ETS1 and the histone acetyltransferase E1A-binding protein P300 (EP300 or p300) both promote BCL-xL transcription. Using immunofluorescence, we found that BCL-xL protein expression is increased both in cholangiocytes of livers from individuals with PSC and a mouse model of PSC. Using an in vitro model of lipopolysaccharide-induced senescence in normal human cholangiocytes (NHCs), we found increased BCL-xL mRNA and protein levels, and ChIP-PCRs indicated increased occupancy of ETS1, p300, and histone 3 Lys-27 acetylation (H3K27Ac) at the BCL-xL promoter. Using co-immunoprecipitation and proximity ligation assays, we further demonstrate that ETS1 and p300 physically interact in senescent but not control NHCs. Additionally, mutagenesis of predicted ETS1-binding sites within the BCL-xL promoter blocked luciferase reporter activity, and CRISPR/Cas9-mediated genetic deletion of ETS1 reduced senescence-associated BCL-xL expression. In senescent NHCs, TRAIL-mediated apoptosis was reduced ∼70%, and ETS1 deletion or RNAi-mediated BCL-xL suppression increased apoptosis. Overall, our results suggest that ETS1 and p300 promote senescent cholangiocyte resistance to apoptosis by modifying chromatin and inducing BCL-xL expression. These findings reveal ETS1 as a central regulator of both cholangiocyte senescence and the associated apoptosis-resistant phenotype.

Cross-Talk between Cytokine and NF-ĸb Signaling in the CLL Microenvironment Can Affect Sensitivity for Venetoclax

INTRODUCTION. The Bcl-2 inhibitor Venetoclax provides profound reductions in circulating chronic lymphocytic leukemia (CLL) cells in the majority of patients. However, lymph node (LN) responses are less robust, which may be linked to an acquired resistance imposed by pro-survival signals. Prime among these is CD40 stimulation leading to activation of NF-kB, and induction of Bcl-XL expression1. Bcl-XL is a prime determinant of resistance to Venetoclax2 and regulatory mechanisms of its expression are of clinical significance. Cytokines IL-21 and IL-4 are secreted by T helper cells and abundant in the CLL lymph node microenvironment. Importantly, IL-21 and IL-4 play an important role in CLL survival and proliferation3. In the present study, we investigated how signals from T helper cytokines IL-21 or IL-4 affect Bcl-XL expression as a model for the CLL LN microenvironment, specifically in relation to Venetoclax resistance. RESULTS. Following CD40 stimulation, IL-21 and IL-4 show opposing effects on Bcl-XL expression. Correspondingly, this was associated with CD40-induced resistance to Venetoclax which was augmented by IL-4 and reversed by IL-21. We subsequently investigated the rewiring between CD40 activation, differential cytokine signaling and Bcl-XL expression. IL-21 or IL-4 stimulation correspond with differential STAT3 or -6 phosphorylation and STAT3 and -6 have predicted binding sites near the known p65 and p52 binding sites in the Bcl-XL promoter region. Using reporter assays with Bcl-XL promotor constructs we demonstrate competition (through IL-21-induced STAT3) or synergy (through IL-4 induced-STAT6) with CD40-mediated activation of the NF-kB pathway. By applying in situ proximity ligation (isPLA) in primary CLL cells, we showed direct interaction of both (non-)canonical p65 and p52 with STAT3 and STAT6. Moreover, time-course analyses indicated that STAT3 drives NF-kB out of the nucleus, whereas STAT6 keeps NF-kB inside the nucleus, and this distinction controls Bcl-XL expression. These observations suggest that cross-talk between JAK/STAT signaling and NF-kB signaling happens by direct binding to the Bcl-XL promoter and by limiting NF-kB availability at the Bcl-XL promoter. CONCLUSIONS. These data show that protective signals from the CLL microenvironment can be tipped towards apoptosis sensitivity by interfering with JAK/STAT and NF-kB signaling, providing novel therapeutic clues in case of emerging resistance to targeted drugs such as Venetoclax. 1 J. Tromp, S. Tonino, J. Elias, A. Jaspers, D. Luijks, A. Kater, R. van Lier, M. van Oers, E. Eldering. Dichotomy in NF-kB signaling and chemoresistance in IGHV mutated versus unmutated CLL cells upon CD40/TLR9 triggering. Oncogene 2010. 2 R. Thijssen, E. Slinger, K. Weller, C. Geest, T. Beaumont, M. van Oers, A. Kater, E. Eldering. Resistance to ABT-199 induced by microenvironmental signals in chronic lymphocytic leukemia can be counteracted by CD20 antibodies or kinase inhibitors. Haematologica 2015. 3 C. Schleiss, W. Ilias, O. Tahar, Y. Güler, L. Miguet, C. Mayeur-Rousse, L. Mauvieux, L. Fornecker, E. Toussaint, R. Herbrecht, F. Bertrand. M. Maumy-Bertrand, T. Martin, S. Fournel, P. Georgel, S. Bahram, L. Vallat. BCR-associated factors driving chronic lymphocytic leukemia cells in proliferation ex vivo. Scientific Reports 2019. Disclosures Eldering: Celgene: Research Funding; Roche: Research Funding; Janssen Pharmaceutical Companies: Research Funding.

Abstract 1297: Casein kinase II (CK2) as a potential therapeutic target in acute myeloid leukemia (AML)

Abstract Casein Kinase II (CK2) is a pro-oncogenic kinase that is overexpressed in a majority of human malignancies, including leukemia. One third of AML cases without detectable high risk molecular alteration have high expression of CK2 which is strongly associated with poor outcome. Previously published data showed that CK2 directly phosphorylates several transcription factors including Ikaros (encoded by IKZF1) resulting in reduced DNA-binding affinity and loss of Ikaros function as a tumor suppressor. Here we present the evidence demonstrating role of Ikaros and CK2 in regulation of apoptosis in AML. We show that CK2 inhibitor, CX4945 has strong anti-leukemic effect in AML and one of the novel mechanisms of action involves restoration of Ikaros function as a transcriptional regulator of genes involved in apoptosis. Results: Treatment of AML cell lines and primary cells with CK2 inhibitor, CX4945 show cytotoxicity (IC50 3-5uM), increased apoptosis, cell cycle arrest and poor colony formation. Treatment of murine xenografts of AML with CX4945 showed significant anti-tumor effect. Further studies dissecting the mechanism of action of CX4945 are as follows: Genome-wide binding studies using chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-seq) demonstrated that treatment of AML cell line, U937 with CX4945 at IC50 concentration enhances binding affinity of Ikaros transcription factor at the promoter regions of several target genes. Among the significantly affected genes (more than two fold change) are anti-apoptotic genes such as BCL2A1 (B Cell Lymphoma 2 related protein A1) and Bcl-xL (B Cell Lymphoma extra-Large). Dysregulation of apoptotic pathway is hallmark of cancer cells. These findings were confirmed qChIP where CX4945 treated AML cells showed increased binding of Ikaros to the BCL2A1 and Bcl-xL promoters. We used gain-of-function and loss-of-function experiments to determine how Ikaros regulates BCL2A1 and Bcl-xL genes in AML. Ikaros overexpression using lentiviral transduction results in reduced expression of BCL2A1 and Bcl-xL both at mRNA and protein level. Similar results were seen with silencing of CK2 using CK2 SiRNA as well as CX4945. Increased expression of BCL2A1 and Bcl-xL was noted after Ikaros silencing using SiRNA which did not revert with CX4945 treatment.In conclusion, (1) Ikaros represses transcription of anti-apoptotic genes BCL2A1and Bcl-xL, (2) CK2 directly phosphorylates tumor suppressor Ikaros and inhibits its function as transcriptional regulator, (3) Inhibition of CK2 enhances Ikaros-mediated repression of BCL2A1 and Bcl-xL genes resulting in increased apoptosis, (4) CX4945 has strong anti-leukemia effect in AML xenograft models. CK2 is a promising therapeutic target in AML. Our results unveal a novel mechanism of action of CK2 inhibitor, CX4945 in AML which involves restoration of Ikaros mediated regulation of apoptosis. Citation Format: Badhauria Preeti, Soumya Iyer, Elanora Dovat, Pavan Kumar Dhanyam Raju, Jonathan Payne, Chunhua Song, Yali Ding, Claxton David, Sharma Arati, Chandrika Gowda. Casein kinase II (CK2) as a potential therapeutic target in acute myeloid leukemia (AML) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1297.

Long Noncoding RNA HEIH Promotes Colorectal Cancer Tumorigenesis via Counteracting miR-939‒Mediated Transcriptional Repression of Bcl-xL.

PurposeStudies have found that long noncoding RNA HEIH (lncRNA-HEIH) is upregulated and facilitates hepatocellular carcinoma tumor growth. However, its clinical significances, roles, and action mechanism in colorectal cancer (CRC) remains unidentified.Materials and MethodslncRNA-HEIH expression in CRC tissues and cell lines was measured by quantitative real-time polymerase chain reaction. Cell CountingKit-8, ethynyl deoxyuridine incorporation assay, terminal deoxynucleotidyl transferase dUTP nick end labeling staining, and nude mice xenografts assays were performed to investigate the roles of lncRNA-HEIH. RNA pull-down, RNA immunoprecipitation, chromatin immunoprecipitation, and luciferase reporter assays were performed to investigate the action mechanisms of lncRNA-HEIH.ResultsIn this study, we found that lncRNA-HEIH is significantly increased in CRC tissues and cell lines. lncRNA-HEIH expression is positively associated with tumor size, invasion depth, and poor prognosis of CRC patients. Enhanced expression of lncRNA-HEIH promotes CRC cell proliferation and decreases apoptosis in vitro, and promotes CRC tumor growth in vivo. Whereas knockdown of lncRNA-HEIH inhibits CRC cell proliferation and induces apoptosis in vitro, and suppresses CRC tumor growth in vivo. Mechanistically, lncRNA-HEIH physically binds to miR-939. The interaction between lncRNA-HEIH and miR-939 damages the binding between miR-939 and nuclear factor κB (NF-κB), increases the binding of NF-κB to Bcl-xL promoter, and promotes the transcription and expression of Bcl-xL. Moreover, Bcl-xL expression is positively associatedwith lncRNA-HEIH in CRC tissues. Blocking the interaction between lncRNA-HEIH and miR-939 abolishes the effects of lncRNA-HEIH on CRC tumorigenesis.ConclusionThis study demonstrated that lncRNA-HEIH promotes CRC tumorigenesis through counteracting miR-939‒mediated transcriptional repression of Bcl-xL, and suggested that lncRNA-HEIH may serve as a prognostic biomarker and therapeutic target for CRC.

Abstract 330: The Wnt signal transcription factor TCF-4 directly regulates Bcl-xL expression in human hepatocellular carcinoma cells

Abstract Background: The T-cell factor (TCF)-4 is a key transcriptional protein activated by Wnt/β-catenin signaling. Previously we identified 14 TCF-4 isoforms derived from human HCC cell lines. The TCF-4J and K pair have been characterized based on the presence (K) or absence (J) of a SxxSS motif. Furthermore, we demonstrated that TCF-4J conferred high tumorigenic potential to HCC cells in contrast to TCF-4K (PLoS ONE 2012). However, the anti-apoptotic protein Bcl-xL was much expressed in TCF-4K-overexpressing HCC cells than the level in TCF-4J-overexpressing cells, suggesting that the SxxSS was involved in Bcl-xL expression. Indeed, Wnt/β-catenin signaling needs to control cell apoptosis during embryogenesis and carcinogenesis, possible direct interaction between the TCF-4 isoforms and the bcl-xL promoter region was suggested. Thus, the AIM of this study was to assess the protein-DNA interaction and its functional consequences by using ChIP assay and luciferase-reporter assay, respectively. Methods: The human HCC cell lines HAK-1A and HAK-1B were used. HAK-1B was an aggressive sister cell line derived from HAK-1A. TCF-4K mutants (269A, 272A, and 273A) were prepared with conversion of serine (S) in the SxxSS motif to alanine (A) by site-directed mutagenesis. HAK-1A-derived stable clones overexpressing TCF-4J (J cells), K (K cells), and K-mutants (269A, 272A, and 273A cells, respectively) were established. Western blot analysis and real-time PCR were employed to evaluate protein and mRNA expression levels, respectively. ChIP assay was performed by using SimpleChIP assay kit (Cell Signaling Technology). Two primer pairs for bcl-xL promoter region (BCL2L1(-)01Kb and BCL2L1(-)02Kb) were obtained from QIAGEN. The promoter assay was done by using LightSwitch Luciferase Assay System (SWITCHGEAR GENOMICS). Results: Robust expression of Bcl-xL protein was found in HAK-1B cells, in contrast to its low expression in HAK-1A cells. Consistently, the mRNA level in HAK-1B was 2-fold of that in HAK-1A. In ChIP assay, clear binding of TCF-4 with bcl-xL promoter regions was confirmed, encouraging us to compare the binding affinity in J cells, K cells, 269 cells, and control cells. As a result, significant TCF-4-DNA interaction was found in K cells, and, of note, the interaction was abolished in 269A cells. In the promoter assay, K cells showed the highest luciferase activity. Conclusions: The findings suggest that TCF-4 directly regulates the Bcl-xL expression. We suggest that phosphorylation at serine 269 in the TCF-4K isoform is critical to fine-tune anti-apoptotic potential through increasing the Bcl-xL expression in HCC. Citation Format: Hironori Koga, Miran Kim, Anna Nakamura, Hirohisa Yano, Mitsuhiko Abe, Yu Ikezono, Toru Nakamura, Takuji Torimura, Jack R. Wands, Michio Sata. The Wnt signal transcription factor TCF-4 directly regulates Bcl-xL expression in human hepatocellular carcinoma cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 330. doi:10.1158/1538-7445.AM2014-330

Inactivation of HDAC3 and STAT3 is Critically Involved in 1-Stearoyl-sn-Glycero-3-Phosphocholine-Induced Apoptosis in Chronic Myelogenous Leukemia K562 Cells

We here investigated the anticancer mechanism of 1-stearoyl-sn-glycero-3-phosphocholine (LPC), one of the lysophosphatidylcholines, in chronic myelogenous leukemia (CML) K562 cells. LPC significantly showed cytotoxicity at 80 μM and induced apoptosis by sub-G1 accumulation, increase in Annexin V positive and caspase activation. LPC enhanced histone H3 acetylation but decreased histone deacetylase (HDAC) activity and HDAC3 expression. LPC also inhibited phosphorylation of STAT3, its DNA binding activity and nuclear co-localization of HDAC3 and STAT3. In addition, LPC effectively attenuated the expression of survival genes such as Cyclin D1, Cyclin E, Bcl-xL, Bcl-2 and survivin but did not affect COX-2 expression in K562 cells. Furthermore, LPC suppressed phosphorylation of Src and Janus activated kinase 2 while promoted the expression of tyrosine phosphatase Src homology 2 domain-containing phosphatase 1 (SHP-1). Consistently, silencing SHP-1 and pervanadate, an inhibitor of protein tyrosine phosphatase, reversed inactivation of HDAC and STAT3, cleavages of caspase 3 and poly (ADP-ribose) polymerase in LPC-induced apoptosis. Of note, chromatin immunoprecipitation assay revealed that LPC suppressed the binding of HDAC3 and STAT3 to Bcl-xL, Bcl-2 and survivin promoter. Overall, our findings indicate that inactivation of STAT3 and HDAC mediates LPC-induced apoptosis in CML K562 cells.

Targeting the IL-4/IL-13 signaling pathway sensitizes Hodgkin lymphoma cells to chemotherapeutic drugs

Hodgkin/Reed-Sternberg lymphoma (HL) is a clonal B-cell-related malignancy. Although many patients with HL can be cured by the current regimen of high-dose multi-agent chemotherapy, the treatment causes high risks of later pathologies including secondary malignancies. This fact highlights the demand to develop rational treatment for HL. Survival and growth of HL cells are largely dependent on their microenvironment. In this study, using the HL cell lines L1236 and KM-H2 as model systems, we investigated the role of IL-4/IL-13 signaling in regulation of drug sensitivity and resistance in HL. We show that specific blocking of IL-4 and IL-13-mediated STAT6 activation by either an IL-4-binding fusion protein APG598 or an IL-4R antagonist APG201 (R121D/Y124D) renders HL cells more prone to apoptotic killing by chemotherapeutic drugs such as Mitomycin C, 5-Fluorouracil, Etopside, Doxorubicin and Paclitaxel. This effect is due to inhibition of STAT6-mediated elevation of expression of the anti-apoptotic Bcl-2 family protein Bcl-xL. Employing ChIP analysis in combination with APG201 or STAT6-specific siRNA we identified a defined STAT6-binding site in the Bcl-xL promoter region from -1967 to -1957 of the transcription start site. Our data demonstrate that the IL-4/IL-13-STAT6-Bcl-xL axis may be an important target for HL treatment. This study also suggests that combination of classical chemotherapeutic drugs with the IL-4/IL-13 antagonists may enhance efficacy and reduce risks of toxicity from high dose of drugs in HL treatment.

Abstract 3115: Sumoylation enhances the stability of the proto-oncogene EVI1 however negatively regulates its DNA binding and transcriptional activity.

Abstract Aberrant expression of the proto-oncogene EVI1 (Ecotropic virus integration site1) has been implicated not only in lymphoid or myeloid malignancies but also in several other solid tumors like colon, ovarian and breast cancer. Despite its crucial role in oncogenesis, the regulatory factors and mechanisms that control the function of EVI1 are partially known. Post translational modifications play a crucial role in regulating various cellular processes by controlling the function of several proteins. Here we show that EVI1, a transcription factor is post-translationally modified by SUMO1. Indirect immunofluorescence studies revealed that EVI1 and SUMO1 colocalized in the nuclear speckles in the cells transiently transfected with EVI1 and SUMO1 as well as cells that constitutively expresses EVI1. Sumoylation is known to stabilize their target protein which was also observed in case of EVI1 through pulse chase assay with cycloheximide. Reporter gene assay revealed that sumoylation impaired EVI1 mediated transactivation of Bcl-xL and SIRT1 promoters and also reduced the DNA binding efficiency of EVI1 which was confirmed through ChIP assay. PIAS proteins are known as SUMO E3 ligase which enhances the sumoylation of the proteins. PIAS proteins are also known to regulate the function of several proteins independent of its ligase activity. Co-immunoprecipiation assay revealed that EVI1 directly interacts with SUMO E3 ligase PIASy, which was also found to enhance EVI1 sumoylation. Our study also demonstrates that PIASy negatively regulated EVI1 transcription activity which is independent of its ligase activity. Thus PIASy was found to be an interacting partner that regulates the function of EVI1 in sumoylation dependent as well as independent manner. In summary, we present a new modifier of EVI1 which controls its stability and deregulates its transcriptional activity. Further study in context with EVI1-SUMO1-PIASy may give a better idea in understanding the functions and the regulation of EVI1 in normal and diseased cells. Citation Format: Sneha Singh, Soumen Chakraborty. Sumoylation enhances the stability of the proto-oncogene EVI1 however negatively regulates its DNA binding and transcriptional activity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3115. doi:10.1158/1538-7445.AM2013-3115

Nrf2-induced antiapoptotic Bcl-xL protein enhances cell survival and drug resistance

Nuclear transcription factor Nrf2 binds with the antioxidant-response element (ARE) in the promoter regions of cytoprotective genes, leading to their increased expression and cellular protection. In this study, we investigated the role of Nrf2 in the regulation of antiapoptotic Bcl-xL protein and its effect on cellular apoptosis. Treatment of mouse Hepa-1 cells with the antioxidant tert-butylhydroquinone led to the induction of Bcl-xL gene expression. Promoter mutagenesis, transfection, and chromatin immunoprecipitation assays identified an ARE between nucleotides −608 and −600 in the forward strand of the proximal Bcl-xL promoter that bound to Nrf2 and led to increased Bcl-xL gene expression. In addition, short interfering RNA (siRNA) inhibition and overexpression of Nrf2 led to a respective decrease and increase in Bcl-xL gene expression. These results implicated Nrf2 in the regulation of expression and induction of Bcl-xL protein. Nrf2-mediated expression of Bcl-xL protein downregulated Bax and decreased caspase 3/7 activity. SiRNA inhibition of both Nrf2 and Bcl-xL increased the susceptibility of cancer cells to etoposide-mediated cell death and reduced cell survival. Moreover, dysfunctional/mutant INrf2 (inhibitor of Nrf2) in human lung cancer cells failed to degrade Nrf2, resulting in increased Bcl-xL levels and increased cell survival. These data provide the first evidence of Nrf2 in the control of Bcl-xL expression and apoptotic cell death with implications for antioxidant protection, survival of cancer cells, and drug resistance.

Targeted acetylation of NF-kappaB/RelA and histones by epigenetic drugs reduces post-ischemic brain injury in mice with an extended therapeutic window

Nuclear factor-kappaB (NF-κB) p50/RelA is a key molecule with a dual effect in the progression of ischemic stroke. In harmful ischemia, but not in preconditioning insult, neurotoxic activation of p50/RelA is characterized by RelA-specific acetylation at Lys310 (K310) and deacetylation at other Lys residues. The derangement of RelA acetylation is associated with activation of Bim promoter. ObjectiveWith the aim of producing neuroprotection by correcting altered acetylation of RelA in brain ischemia, we combined the pharmacological inhibition of histone deacetylase (HDAC) 1–3, the enzymes known to reduce global RelA acetylation, and the activation of sirtuin 1, endowed with a specific deacetylase activity on the K310 residue of RelA. To afford this aim, we tested the clinically used HDAC 1–3 inhibitor entinostat (MS-275) and the sirtuin 1 activator resveratrol. MethodsWe used the mouse model of transient middle cerebral artery occlusion (MCAO) and primary cortical neurons exposed to oxygen glucose deprivation (OGD). ResultsThe combined use of MS-275 and resveratrol, by restoring normal RelA acetylation, elicited a synergistic neuroprotection in neurons exposed to OGD. This effect correlated with MS-275 capability to increase total RelA acetylation and resveratrol capability to reduce RelA K310 acetylation through the activation of an AMP-activated protein kinase–sirtuin 1 pathway. The synergistic treatment reproduced the acetylation state of RelA peculiar of preconditioning ischemia. Neurons exposed to the combined drugs totally recovered the optimal histone H3 acetylation.Neuroprotection was reproduced in mice subjected to MCAO and treated with MS-275 (20μg/kg and 200μg/kg) or resveratrol (6800μg/kg) individually. However, the administration of lowest doses of MS-275 (2μg/kg) and resveratrol (68μg/kg) synergistically reduced infarct volume and neurological deficits. Importantly, the treatment was effective even when administered 7h after the stroke onset. Chromatin immunoprecipitation analysis of cortices harvested from treated mice showed that the RelA binding and histone acetylation increased at the Bcl-xL promoter and decreased at the Bim promoter. ConclusionOur study reveals that epigenetic therapy shaping acetylation of both RelA and histones may be a promising strategy to limit post-ischemic injury with an extended therapeutic window.

Nuclear EGFRvIII‐STAT5b complex contributes to glioblastoma cell survival by direct activation of the Bcl‐XL promoter

Aberrant EGFR signaling strongly promotes glioma malignancy and treatment resistance. The most prevalent mutation, ΔEGFR/EGFRvIII, is an in-frame deletion of the extracellular domain, which occurs in more than 25% of glioblastomas and enhances growth and survival of tumor cells. Paradoxically, the signaling of the potent oncogene ΔEGFR is of low intensity, raising the question of whether it exhibits preferential signaling to key downstream targets. We have observed levels of phosphorylation of STAT5 at position Y699 in cells expressing ΔEGFR that are similar or higher than in cells that overexpress EGFR and are acutely stimulated with EGF, prompting us to investigate the role of STAT5 activation in glioblastoma. Here, we show that in human glioblastoma samples, pSTAT5 levels correlated positively with EGFR expression and were associated with reduced survival. Interestingly, the activation of STAT5b downstream of ΔEGFR was dependent on SFKs, while the signal from acutely EGF-stimulated EGFR to STAT5b involved other kinases. Phosphorylated STAT5b and ΔEGFR associated in the nucleus, bound DNA and were found on promoters known to be regulated by STAT5 including that of the Aurora A gene. ΔEGFR cooperated with STAT5b to regulate the Bcl-XL promoter and knockdown of STAT5b suppressed anchorage independent growth, reduced the levels of Bcl-XL and sensitized glioblastoma cells to cisplatin. Together these results delineate a novel association of nuclear ΔEGFR with STAT5b, which promotes oncogenesis and treatment resistance in glioblastoma by direct regulation of anti-apoptotic gene, Bcl-XL.

Abstract 2015: Lysophosphatidylcholine induces apoptosis via suppressing STAT3 and HDAC3 activities and binding to the Bcl-xL promoter in K562 chronic myelogenous leukemia cells

Abstract Lysophosphatidylcholine (LPC) is a class of chemical compound which are derived from phosphatidylcholines usually isolated from the surface of wheat starch granules. In the present study, we investigated the molecular mechanisms responsible for LPC-induced apoptosis in chronic myeloid leukemia K562 cells. LPC exerted the inhibitory effect on the viability of K562 cells as well as other leukemic cell lines HL-60 and U937. Nonetheless, LPC significantly increased the acetylation of histone H3 at K18, 9 and 23 in K562 cells, but not in HL-60 and U937 cells. Consistently, LPC reduced histone deacetylase (HDAC) transcriptional activity and the expression of HDAC3 at protein level. LPC suppressed phosphorylation of mTOR and AKT, the upstream kinases of HDACs and induced p27 expression. We also found that LPC mediated inactivation of signal transducer and activator of transcription 3 (STAT3)-related signaling pathway in K562 cells, but not HL-60 and U937. LPC significantly inhibited phosphorylation and the DNA binding activity of STAT3, phosphorylation of Src and Janus activated kinase 2 (JAK2) as well as the expression of STAT3 target genes Cyclin D1, Cyclin E, Bcl-xL, Bcl-2, and Survivin at both protein and mRNA levels. LPC stimulated protein tyrosin phosphatases Src homology 2 domain-containing phosphatase 1 (SHP-1), but not SHP-2, SOCS-1 and PTEN. Blocking SHP-1 using sodium pervanadate or SHP-1 specific siRNA reversed LPC-mediated inactivation of STAT3 and HDAC and induction of apoptosis. Of importance, LPC blocked the nuclear translocation and interaction of HDAC3 and STAT3 as well as their binding onto the Bcl-xL promoter. Overall, our findings demonstrate that LPC has a potent anti-cancer activity through inducing apoptosis by suppressing HDAC3/STAT3 activation and binding to the Bcl-xL promoter in K562 cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2015. doi:1538-7445.AM2012-2015

Physical Interaction of the Proto-Oncogene EVI1 with Hypermethylated in Cancer 1 (HIC1) Deregulates EVI1 Mediated Apoptosis in Cancer Cells

Abstract 2449The ecotropic viral integration site I (EVII) gene has attracted considerable attention in recent years because of its reported involvement in chronic myeloid leukemia, high risk myelodysplastic syndrome and a very high percentage of acute myeloid leukemias. The most recent World Health Organization classification has designated AML with inv (3) or t(3;3) and associated RPN1/EVI1 fusion, as a distinct AML subgroup associated with an unfavorable prognosis. Studies also indicate that EVI1 may be overexpressed in a subset of human colon cancers, and that EVI1 might affect disease progression and/or sensitivity to chemotherapy. The presence of zinc finger domains that are able to bind to specific sequences of DNA suggests that EVI1 is a transcriptional regulator; however, few target genes of EVI1 have been functionally identified. In the present study, we demonstrate by co-immunoprecipitation assay that the proximal zinc finger domain of EVI1 binds to HIC1, which abolishes the ability of EVI1 to induce expression of Bcl-xL. HIC1, is an epigenetically regulated transcriptional repressor that functionally cooperates with p53 to suppress age dependent development of cancer in mice. Inactivation of HIC1 results in up regulation of SIRT1, which deacetylate and inactivates p53; allowing cells to bypass apoptosis and DNA damage survival., Our study also demonstrates via indirect immuno fluorescence analysis that EVI1 and HIC1 proteins are colocalized in speckles in the nucleus. We recently reported that EVI1 not only binds the Bcl-xL promoter but also up regulates its transcriptional activity and protein expression. We now demonstrate by EMSA and luciferase assays that physical interaction between EVI1 and HIC1 releases the binding of EVI1 from the Bcl-xL promoter, and hence promotes apoptosis. Thus by directly binding to EVI1, HIC1 displaces EVI1 from its DNA consensus site leading to down regulation of Bcl-xL. Also we observed that posttranslational modification of EVI1, mainly acetylation plays a critical role in this EVI1-HIC1 protein-protein interaction. Further elucidation of the counteracting effects of HIC1 and EVI1 on Bcl-xL expression may provide new insights in EVI1 mediated disease progression. Disclosures:No relevant conflicts of interest to declare.

Acetylation of the Proto-Oncogene EVI1 Abrogates Bcl-xL Promoter Binding and Induces Apoptosis

EVI1 (Ecotropic Viral Integration site I), which was originally identified as a myeloid transforming gene by means of retroviral insertional mutagenesis in mouse leukemia, encodes a nuclear DNA binding zinc finger protein. The presence of zinc fingers that are able to bind to specific sequences of DNA suggests that EVI1 is a transcriptional regulator; however, except a few, target genes of EVI1 are poorly functionally identified thus far. In this study we provide evidence that EVI1 directly induces the expression of Bcl-xL through the first set of zinc finger and thereby inhibits apoptosis. ChIP analysis showed that EVI1 binds to the Bcl-xL promoter in HT-29 cells, a colon carcinoma cell line, which expresses EVI1. The observation is also supported by the fact that EVI1 siRNA treated HT-29 cells, shows a down regulation of Bcl-xL expression and that over expression of EVI1 results in the induction of the Bcl-xL reporter construct. A set of EVI1 positive chronic myeloid leukemia (CML) samples also showed higher Bcl-xL expression with respect to EVI1 negative samples. Interestingly, co-expression of EVI1 with wild type, but not with dominant-negative form of PCAF, abolishes the effect of EVI1 on Bcl-xL, indicating that acetylation of EVI1 abrogates its ability not only to bind Bcl-xL promoter but also alleviate Bcl-xL activity. Finally we have shown that EVI1 expression regulates apoptosis in HT-29 cells, which is abrogated when HT-29 cells are transfected with EVI1 siRNA or PCAF. The result for the first time shows a direct pathway by which EVI1 can protect cells from apoptosis and also demonstrates that the pathway can be reversed when EVI1 is acetylated.

Dexamethasone induces transcriptional activation of Bcl-xL gene and inhibits cardiac injury by myocardial ischemia

Psychological or physical stress causes an elevation of glucocorticoids in the circulating system. Glucocorticoids regulate a variety of physiological functions, from energy metabolism and biochemical homeostasis to immune response. Synthetic steroids are among the most prescribed drugs for immune suppression and chemotherapy. While glucocorticoids are best known for inducing apoptosis in a number of cell types, we have found that corticosteroids at stress relevant levels protect cardiomyocytes from apoptosis. Current study addresses whether glucocorticoids inhibit cardiac injury in vivo. Adult male C57BL6 mice were administered with dexamethasone (20mg/kg, i.p.) or vehicle control 20h prior to left anterior descending coronary artery occlusion surgery. Myocardial infarction was measured by triphenyl tetrazoliumchloride staining in tissue slices and by levels of cardiac Troponin (cTn I) in the blood. Treatment of dexamethasone markedly reduced infarct size (19.6±4.3%, vs. 29.2±4.9%, p<0.01) and cTn I level in the blood (3.83±0.66ng/ml vs. 5.62±0.37ng/ml, p<0.01). In studying the mechanism of such protection, we found that dexamethasone induces the expression of Bcl-xL gene in the myocardium. With cardiomyocytes in culture, glucocorticoids increased transcription of Bcl-xL gene as evidenced by Bcl-xL mRNA increase and promoter activation. The glucocorticoid receptor antagonist mifepristone prevented dexamethasone from inducing cardiac protection or Bcl-xL expression. Our data suggest that activation of glucocorticoid receptor can prevent cardiac injury through transcriptional activation of Bcl-xL gene.

AZ960, a Novel Jak2 Inhibitor, Induces Growth Arrest and Apoptosis in Adult T-Cell Leukemia Cells

Adult T-cell leukemia/lymphoma (ATL) is a highly aggressive disease in which the Jak2/Stat5 pathway is constitutively activated. This study found that AZ960, a novel inhibitor of Jak2 kinase, effectively induced growth arrest and apoptosis of human T-cell lymphotropic virus type 1, HTLV-1-infected T cells (MT-1 and MT-2) in parallel with downregulation of the phosphorylated forms of Jak2 and Bcl-2 family proteins including Bcl-2 and Mcl-1. Interestingly, AZ960 increased levels of Bcl-xL in MT-1 and MT-2 cells in association with accumulation of cAMP response element-binding protein bound to the Bcl-xL promoter as measured by chromatin immunoprecipitation assay. Importantly, genetic inhibition of Bcl-xL by a small interfering RNA potentiated antiproliferative effects of AZ960 in MT-1 cells. Taken together, Jak2 is an attractive molecular target for treatment of ATL. Concomitant blockade of Jak2 and Bcl-xL may be a promising treatment strategy for this lethal disease.

Oncoproteomic Analysis Reveals Co-Upregulation of RELA and STAT5 in Carboplatin Resistant Ovarian Carcinoma

BackgroundOvarian cancer is one of the most lethal types of female malignancy. Although most patients are initially responsive to platinum-based chemotherapy, almost all develop recurrent chemoresistant tumors and succumb to their diseases. Elucidating the pathogenesis underlying drug resistance is fundamental to the development of new therapeutics, leading to improved clinical outcomes in these patients.Methods and FindingsWe compared the proteomes of paired primary and recurrent post-chemotherapy ovarian high-grade serous carcinomas from nine ovarian cancer patients using CIEF/Nano-RPLC coupled with ESI-Tandem MS. As compared to their primary tumors, more than half of the recurrent tumors expressed higher levels of several proteins including CP, FN1, SYK, CD97, AIF1, WNK1, SERPINA3, APOD, URP2, STAT5B and RELA (NF-κB p65), which were also validated by quantitative RT-PCR. Based on shRNA screening for the upregulated genes in in vitro carboplatin-resistant cells, we found that simultaneous knockdown of RELA and STAT5B was most effective in sensitizing tumor cells for carboplatin treatment. Similarly, the NF-κB inhibitor, BMS-345541, and the STAT5 inhibitor, Dasatinib, significantly enhanced cell sensitivity to carboplatin. Moreover, both RELA and STAT5 are known to bind to the promoter region of Bcl-X, regulating its promoter activity. In this regard, augmented Bcl-xL expression was detected in carboplatin-resistant cells. Combined ectopic expression of RELA and STAT5B enhanced Bcl-xL promoter activity while treatment with BMS-345541 and Dasatinib decreased it. Chromatin immunoprecipitation of the Bcl-X promoter region using a STAT5 antibody showed induction of RELA and STAT5 DNA-binding segments both in naïve cells treated with a high concentration of carboplatin as well as in carboplatin-resistant cells.ConclusionsProteomic analysis identified RELA and STAT5 as two major proteins associated with carboplatin resistance in ovarian tumors. Our results further showed that NF-κB and STAT5 inhibitor could sensitize carboplatin-resistant cells and suggest that such inhibitors can be used to benefit patients with carboplatin-resistant recurrent ovarian cancer.

Abstract 1032: Impact of HOXC9 recapitulation on cancer biology and clinical outcome in gastric cancer

Abstract The HOX genes encode transcription factors and may be implicated in the growth and the progression of many types of tumors. There are growing lines of evidence that dysregulation of HOX gene expression plays important roles in cancer. The aim of this study is to determine the level of HOXC9 expression during gastric cancer progression and the impact of its alteration on cancer biology and clinical outcome. HOXC9 expression was evaluated in normal mucosa, malignant legions, gastric cancer tissues by RT-PCR, western blot and immunohistochemistry. We established AGS gastric cancer cell in which HOXC9 was knock-down by shRNA and conditionally knock-down the HOXC9 expression by tetracycline-regulated gene expression (Tet-Off) system. The effect of HOXC9 knock-down on cell proliferation and apoptosis in AGS gastric cancer line were analyzed by tetrazolium salt-based proliferation assay (WST assay) and FACScan. Knockdown of HOXC9 by specific shRNA (67.3%) increased apoptosis, compared to scambled (7.3%) after 48 hours treatment of doxycycline, and increased active form of cellular apoptosis-related enzyme capsae-9, caspase-3 and poly (ADP-ribose) polymerase, whereas decreased expression of Bcl-xL, a mitochondria dependent anti-apoptosis factor. There was a gain or substantial increase of HOXC9 protein expression according to gastric cancer progression. Kaplan-Meier survival curves showed that HOXC9 overexpression is correlated with inferior disease-specific survival (P&amp;lt;0.001). multivariate Cox repression analysis revealed HOXC9 expression was an independent poor prognostic factor (Hazard ratio, 1.74; 95% CI, 1.01 to 2.99; P=0.047). We then demonstrated that HOXC9 directly regulated Bcl-xL transcription by binding to a HOXC9-responsive element in the Bcl-xL promoter by reporter gene assay, chromatin immunoprecipitation (ChIP), and electrophoretic mobility shift assay (EMSA). In conclusion, these results suggest that HOXC9 overexpressed in advanced gastric cancer setting and its expression should be useful as a prognostic marker in gastric cancer patients. HOXC9 dependent Bcl-xL overexpresion may be an important mechanism by which HOXC9 protects gastric cancer cells from apoptosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1032.