Dephosphorylation Of Bcl-2 Research Articles

Marine Polyether Phycotoxin Palytoxin Induces Apoptotic Cell Death via Mcl-1 and Bcl-2 Downregulation

Palytoxin is considered one of the most potent biotoxins. As palytoxin-induced cancer cell death mechanisms remain to be elucidated, we investigated this effect on various leukemia and solid tumor cell lines at low picomolar concentrations. As palytoxin did not affect the viability of peripheral blood mononuclear cells (PBMC) from healthy donors and did not create systemic toxicity in zebrafish, we confirmed excellent differential toxicity. Cell death was characterized by a multi-parametric approach involving the detection of nuclear condensation and caspase activation assays. zVAD-sensitive apoptotic cell death was concomitant with a dose-dependent downregulation of antiapoptotic Bcl-2 family proteins Mcl-1 and Bcl-xL. Proteasome inhibitor MG-132 prevented the proteolysis of Mcl-1, whereas the three major proteasomal enzymatic activities were upregulated by palytoxin. Palytoxin-induced dephosphorylation of Bcl-2 further exacerbated the proapoptotic effect of Mcl-1 and Bcl-xL degradation in a range of leukemia cell lines. As okadaic acid rescued cell death triggered by palytoxin, protein phosphatase (PP)2A was involved in Bcl-2 dephosphorylation and induction of apoptosis by palytoxin. At a translational level, palytoxin abrogated the colony formation capacity of leukemia cell types. Moreover, palytoxin abrogated tumor formation in a zebrafish xenograft assay at concentrations between 10 and 30 pM. Altogether, we provide evidence of the role of palytoxin as a very potent and promising anti-leukemic agent, acting at low picomolar concentrations in cellulo and in vivo.

Effective tumor cell abrogation via Venetoclax-mediated BCL-2 inhibition in KMT2A-rearranged acute B-lymphoblastic leukemia

Dysregulation of the intrinsic BCL-2 pathway-mediated apoptosis cascade is a common feature of hematological malignancies including acute B-lymphoblastic leukemia (B-ALL). The KMT2A-rearranged high-risk cytogenetic subtype is characterized by high expression of antiapoptotic protein BCL-2, likely due to the direct activating binding of KMT2A fusion proteins to the BCL2 gene. The BCL-2 inhibitor venetoclax (VEN) has proven great clinical value in other blood cancers, however, data on B-ALL is sparse and past studies have not so far described the effects of VEN on gene and protein expression profiles. Using cell lines and patient-derived in vivo xenograft models, we show BCL-2 pathway-mediated apoptosis induction and decelerated tumor cell counts in KMT2A-rearranged B-ALL but not in other cytogenetic subtypes. VEN treatment of cell line- and patient-derived xenografts reduced blast frequencies in blood, bone marrow, and spleen, and tumor cell doubling times were increased. Growth rates are further correlated with VEN concentrations in blood. In vitro incubation with VEN resulted in BCL-2 dephosphorylation and targeted panel RNA sequencing revealed reduced gene expression of antiapoptotic pathway members BCL2, MCL1, and BCL2L1 (BCL-XL). Reinforced translocation of BAX proteins towards mitochondria induced caspase activation and cell death commitment. Prolonged VEN application led to upregulation of antiapoptotic proteins BCL-2, MCL-1, and BCL-XL. Interestingly, the extrinsic apoptosis pathway was strongly modulated in SEM cells in response to VEN. Gene expression of members of the tumor necrosis factor signaling cascade was increased, resulting in canonical NF-kB signaling. This possibly suggests a previously undescribed mechanism of BCL-2-independent and NF-kB-mediated upregulation of MCL-1 and BCL-XL. In summary, we herein prove that VEN is a potent option to suppress tumor cells in KMT2A-rearranged B-ALL in vitro and in vivo. Possible evasion mechanisms, however, must be considered in subsequent studies.

Abstract 3959: Targeting BCL2 family protein phosphorylation in venetoclax resistant lymphoid malignancies

Abstract Introduction: BCL2 family protein dysfunction is an important mediator of chemoresistance in lymphoid malignancies. Venetoclax (VEN), a BH3 mimetic that selectively targets the anti-apoptotic protein BCL2, is FDA approved for treatment of chronic lymphocytic leukemia (CLL), with clinical trials underway for other lymphoid malignancies. Despite the clinical efficacy of VEN monotherapy, complete remission and progression free rates are still relatively low, thereby indicating the existence of inherent or acquired mechanisms of resistance. Preclinically, increases in anti-apoptotic BCL2 and MCL1 phosphorylation (BCL2-P/MCL1-P) and MCL1 protein level have been implicated in chemoresistance. We therefore question if phosphorylation of these proteins may drive VEN resistance and if removing this modification may re-sensitize resistant cells to VEN induced cell death. Methodology: BH3 profiling (a functional assay that assesses mitochondrial apoptotic priming and anti-apoptotic protein dependence), western blot and cell death assays were performed. Result: We report that VEN acquired resistant DLBCL cell line (OCI-Ly1R) has increased BCL2-P, MCL1-P and MCL1 protein levels as compared to sensitive parental cell line (OCI-Ly1S), while inherently resistant DHL cell line (Su-DHL4) possesses higher MCL1-P and MCL1 protein levels as compared to OCI-Ly1S. We further show that this increase in phosphorylation status contributes to the induction of VEN resistance. Using BH3 profiling, we demonstrate that anti-apoptotic protein phosphorylation-driven VEN resistance involves a change in sensitivity to pro-apoptotic proteins, with an increase in sensitivity to MCL1 inhibition and reciprocal decrease in sensitivity to BCL2 inhibition. We further show that BCL2 and MCL1 dephosphorylation by PP2A activating drugs (PADs) re-sensitizes resistant cells to VEN treatment. Mechanistically, PADs such as the fingolimod (FTY720) not only dephosphorylate BCL2-P and MCL1-P, but also dissociate BAX from BCL2 and destabilize MCL1 protein. These changes lead to the rewiring of cellular sensitivity to BCL2 inhibition, thereby re-sensitizing both VEN acquired and inherent resistant cells to VEN induced cell death. Importantly, the PP2A activating effects of FTY720 were recapitulated in primary cells from the peripheral blood of 16 CLL patients, where FTY720 treatment also reduced BCL2-P, MCL1-P and MCL1 protein levels and dissociated BAX from BCL2. This led to an increased sensitivity to BCL2 inhibition and enhanced cell death upon combined treatment with FTY720 and VEN. Conclusion: Our work defines a new targetable mechanism of VEN resistance in lymphoid malignancies due to the phosphorylation of anti-apoptotic BCL2 family proteins. PADs re-sensitize resistant malignant cells to VEN treatment via dephosphorylation of anti-apoptotic proteins, suggesting a promising combination approach to explore further. Citation Format: Stephen J. Chong, Fen Zhu, Jolin X. Lai, Liam Hackett, Mary C. Collins, Shazib Pervaiz, Jean-Philippe Coppe, Matthew S. Davids. Targeting BCL2 family protein phosphorylation in venetoclax resistant lymphoid malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3959.

Relationship between osteoporosis and benign paroxysmal positional vertigo based on evidence-based medicine and bioinformatics.

We analyzed the correlation between osteoporosis and BPPV and the possible mechanism by performing evidence-based medicine meta-analysis and bioinformatics analysis. Initially, English articles related to osteoporosis and BPPV were obtained through PubMed and EMBASE databases. Stata12.0 software was used for meta-analysis to calculate the odd ratio (OR) and 95% confidence interval (CI) of outcome indicators, and the heterogeneity was evaluated by subgroup analysis, publication bias evaluation, and sensitivity analysis. In addition, microarray datasets related to BPPV and osteoporosis were obtained from gene expression omnibus (GEO) database to screen differentially expressed genes. At last, a mouse model of osteoporosis was established by bilateral oophorectomy for validation. RT-qPCR and Western blot analysis were performed to determine expression of related factors in mouse tissues. Osteoporosis was suggested as an important risk factor for BPPV through meta-analysis of these 12 articles. It was found that PPP2CA was upregulated in BPPV and low bone mineral density (BMD) samples. Moreover, PPP2CA induced dephosphorylation of BCL2, which may be involved in BPPV through regulation of BMD. Through this mechanism, silencing of PPP2CA could elevate the incidence of BPPV by promoting bone remodeling and reducing the density of otoconia around the macula. PPP2CA reduces BMD expression by inducing dephosphorylation of BCL2, which may be one of the mechanisms responsible for the onset of BPPV in osteoporosis.

Targeting the Allosteric Pathway That Interconnects the Core-Functional Scaffold and the Distal Phosphorylation Sites for Specific Dephosphorylation of Bcl-2.

Protein phosphorylation is the most significant post-translational modification for regulating cellular activities, but site-specific modulation of phosphorylation is still challenging. Using three-dimensional NMR spectra, molecular dynamics simulations, and alanine mutations, we identified that the interaction network between pT69/pS70 and R106/R109 residues prevents the phosphorylation sites from exposure to phosphatase and subsequent dephosphorylation. A Bcl-2-dephosphorylation probe, S1-6e, was designed by installing a carboxylic acid group to a Bcl-2 inhibitor. The carboxyl group competitively disrupts the interaction network between R106/R109 and pT69/pS70 and subsequently facilitates Bcl-2 dephosphorylation in living cells. As a result, S1-6e manifests a more effective apoptosis induction in pBcl-2-dependent cancer cells than other inhibitors exhibiting a similar binding affinity for Bcl-2. We believe that targeting the allosteric pathways interconnecting the core-functional domain and the phosphorylation site can be a general strategy for a rational design of site-specific dephosphorylating probes, since the allosteric pathway has been discovered in a variety of proteins.

BDA-366, a putative Bcl-2 BH4 domain antagonist, induces apoptosis independently of Bcl-2 in a variety of cancer cell models

Several cancer cell types, including chronic lymphocytic leukemia (CLL) and diffuse large B-cell lymphoma (DLBCL) upregulate antiapoptotic Bcl-2 to cope with oncogenic stress. BH3 mimetics targeting Bcl-2’s hydrophobic cleft have been developed, including venetoclax as a promising anticancer precision medicine for treating CLL patients. Recently, BDA-366 was identified as a small molecule BH4-domain antagonist that could kill lung cancer and multiple myeloma cells. BDA-366 was proposed to switch Bcl-2 from an antiapoptotic into a proapoptotic protein, thereby activating Bax and inducing apoptosis. Here, we scrutinized the therapeutic potential and mechanism of action of BDA-366 in CLL and DLBCL. Although BDA-366 displayed selective toxicity against both cell types, the BDA-366-induced cell death did not correlate with Bcl-2-protein levels and also occurred in the absence of Bcl-2. Moreover, although BDA-366 provoked Bax activation, it did neither directly activate Bax nor switch Bcl-2 into a Bax-activating protein in in vitro Bax/liposome assays. Instead, in primary CLL cells and DLBCL cell lines, BDA-366 inhibited the activity of the PI3K/AKT pathway, resulted in Bcl-2 dephosphorylation and reduced Mcl-1-protein levels without affecting the levels of Bcl-2 or Bcl-xL. Hence, our work challenges the current view that BDA-366 is a BH4-domain antagonist of Bcl-2 that turns Bcl-2 into a pro-apoptotic protein. Rather, our results indicate that other mechanisms beyond switching Bcl-2 conformation underlie BDA-366’s cell-death properties that may implicate Mcl-1 downregulation and/or Bcl-2 dephosphorylation.

Targeting BCL2 with venetoclax is a promising therapeutic strategy for "double-proteinexpression" lymphoma with MYC and BCL2 rearrangements.

The so-called “double-hit” and “double-protein-expression” lymphoma with MYC and BCL2 rearrangements is a rare, mature B-cell neoplasm characterized by a germinal center B-cell phenotype, abundant protein expression of MYC and BCL2, rapid disease progression, and a poor prognosis. In this study, we showed the potential benefit of the BCL2 inhibitor venetoclax in the treatment of this disease. Immunohistochemical studies of the lymphoma tissues confirmed that overexpression of MYC and BCL2 was observed more frequently in this subtype than in other germinal center B-cell-like diffuse large B-cell lymphomas. In contrast, another pro-survival protein MCL1 was less expressed in this subtype, even when compared with its expression in the non-“double-hit” and “double-protein-expression” type. Furthermore, in vitro studies using two “double-hit” and “double-protein-expression” lymphoma-derived cell lines, Karpas231 and OCI-Ly8, clearly showed that a low concentration of venetoclax, but not the MCL1 inhibitor S63845, was sufficient to induce apoptosis in the two lines, compared with in other germinal center B-cell-derived cell lines, BJAB and SU-DHL10. These results indicate that the survival of this type of lymphoma depends predominantly on BCL2 rather than on MCL1. Unexpectedly, we found that venetoclax not only disrupts the interaction between BCL2 and the pro-apoptotic protein BIM, but also leads to dephosphorylation of BCL2 and further downregulates MCL1 protein expression, probably through modulation of the protein phosphatase 2A B56α activity in Karpas231 and OCI-Ly8. Indeed, a low concentration of venetoclax induced substantial apoptosis in the primary lymphoma cells, regardless of high protein expression of MCL1 associated with venetoclax resistance. Venetoclax clearly triggers the signal transduction related to BCL2 and MCL1 in “double-hit” and “double-protein-expression” lymphoma cells.

151 - Rac1-induced ROS is implicated in the sustained activation of anti-apoptotic protein Bcl-2 in cancer cells

The anti-apoptotic protein Bcl-2 is strongly associated with the development of therapy resistance in a host of hematopoietic and other cancers. To that end, the post-translational modification of Bcl-2, in particular its mono- or multi-site phosphorylation, have been reported to impact its apoptosis inhibitory activity. While multi-site phosphorylation compromises its death inhibitory activity, our recent work has highlighted the critical involvement of serine 70 phosphorylation (S70pBcl-2) in stabilizing its anti-apoptotic activity. S70pBcl-2 is a function of either an amplification of the activities of various kinases involved in this post-translational modification or a loss of function of the phosphatase regulating this modification. The pleiotropic phosphatase, PP2A, is specifically involved in the dephosphorylation of Bcl-2 at S70. In our search to identify redox dependent pathways impacting the sustained activation of Bcl-2 via S70P, we provided evidence that redox modification of the B56δ sub-unit of PP2A resulted in the sustained S70P of Bcl-2, thereby promoting its death inhibitory activity in cancer cells. Interestingly, here we present substantial evidence for the existence of a similar redox-dependent regulation of Bcl-2 phosphorylation in cells expressing GTP-bound active Rac1. We show that active Rac1 promotes sustained S70pBcl2 levels in cancer cells via physical interaction with Bcl-2, thereby permitting Rac1-induced production of superoxide (O2–) in close proximity to inactivate PP2A holoenzyme assembly and specific activity. Corroborating this, pharmacological inhibition of the interaction with BH3 mimetics such as HA14-1 or ABT199 as well as scavengers of O2– such as Tiron reduced S70pBcl2. Importantly, the interaction between Rac1 and Bcl-2 to promote S70pBcl2 as well as the oxidative inhibition of PP2A are re-capitulated in primary cells derived from lymphoma patients. Collectively, our findings demonstrate a novel redox mechanism of active Rac1 in sustaining S70pBcl2 in cancer cells.

Abstract 5442: Dose-dependent toxicity of FTY720, a sphingosine-1-phosphate receptor agonist, on double hit lymphoma cells via PP2A-mediated dephosphorylation of c-myc and bcl-2

Abstract High grade B cell lymphoma with translocations involving MYC and rearrangements involving BCL2 and/or BCL6 define an aggressive subtype of diffuse large B cell lymphoma (DLBCL) with poor prognosis and high rates of relapse. The standard treatment for DLBCL is not considered effective and targeted molecular therapy has yet to be identified for this disease. The mechanisms that make this type of lymphoma particularly aggressive and refractory to treatment include cell growth signals from the c-myc transcriptional regulator working together with the anti-apoptotic activity of the bcl-2 protein. Protein Phosphatase 2A (PP2A) is a ubiquitous cellular enzyme that acts as a tumor suppressor through dephosphorylation of specific cellular phosphoproteins. In fact, PP2A has been shown to dephosphorylate bcl-2 at ser70 leading to inhibition of its function. Further, PP2A-mediated dephosphorylation of c-myc at ser62 promotes its degradation. In many cancers, PP2A activity is inhibited due to overexpression of binding proteins such as SET and CIP2A. Small molecules can act as PP2A activators by releasing PP2A from these inhibitory binding proteins. In the present study we investigated the efficacy of FTY720, a high-affinity agonist of the sphingosine 1-phosphate receptor-1 and PP2A activator, on the double hit lymphoma derived cell line, WSU DLCL2. WSU DLCL2 cells were treated with various concentrations of FTY720, a derivative of myriocin, a fungal metabolite of the Chinese herb Iscaria sinclarii known to promote PP2A activation. Cell viability and PP2A activity were measured 24 hours after FTY720 treatment. In addition, cell lysates were assayed by Western blot using antibodies to detect PP2A subunits, as well as total and phosphorylated c-myc and bcl-2 proteins. A dose-dependent decrease in cell viability was observed over the range of FTY720 concentrations from 1 to 10 micromolar with concomitant increase in in vitro PP2A activity. Western blots showed decreases in c-myc protein levels with FTY720 treatment as well as decreases in ser70 phosphorylation levels of bcl-2 and ser62 phosphorylation levels of c-myc. The results indicate that PP2A activation via FTY720, a small molecule activator of PP2A, leads to a dose-dependent induction of cell death in double hit lymphoma. The data are consistent with an underlying cell death mechanism that involves the targeted dephosphorylation of c-myc and bcl2. Taken together, these data suggest that FTY720 may act as a targeted treatment with therapeutic potential as a single agent or as a combination with other therapies in the treatment of this subtype of lymphoma. There is also the potential of a therapeutic role in other lymphoma subtypes with poor prognosis including transformed follicular lymphoma and relapsed burkitt's lymphoma that rely individually on bcl-2 and c-myc, respectively, for their growth. Citation Format: Geoffrey P. Shouse, Rosalia de Necochea-Campion, Saied Mirshahidi, Chien-Shing Chen, Kimberly J. Payne. Dose-dependent toxicity of FTY720, a sphingosine-1-phosphate receptor agonist, on double hit lymphoma cells via PP2A-mediated dephosphorylation of c-myc and bcl-2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5442.

133 - Tumor Pro-oxidant Enviroment Stabilizes Onco-protein c-Myc via Sustained Phosphorylation at Serine 62 that Promotes its Oncogenic Activity

c-Myc is arguably one of the most notorious oncoproteins known. c-Myc activity and oncogenic potential, are in turn, intricately regulated by its stabilizing phosphorylation at the Serine 62 (S62) residue. Phosphorylation at the S62 site is mediated by RAS activated RAF/MEK/ERK signaling cascade, while its dephosphorylation is under the governance of the protein phosphatase 2A (PP2A). Our recent work highlighted the critical involvement of cellular redox status, in particular superoxide anion, in inhibiting PP2A-mediated dephosphorylation of Bcl-2 at serine 70, thus stabilizing its anti-apoptotic activity1. Here we report that the stabilization and activity of the onco-protein c-myc is significantly amplified by the 'pro-oxidant' tumor microenvironment, which profoundly impacted PP2A phosphatase activity via nitrative inhibition of the B56α regulatory subunit. The B56α regulatory arm is responsible for delivering PP2A substrate phosphoprotein-c-Myc to the catalytic C subunit within the AC core dimer. Tyrosine nitration of the B56α isoform renders it incapable of binding the core dimer causing c-Myc to remain hyperphosphorylated at the serine 62 site. Sustained serine 62 phosphorylated c-Myc inhibits its ubiquitination and degradation by the 26S proteasome. Hitherto driven c-Myc stability is further complemented with a moderate increase in its activity and gain of chemoresistance in osteosarcoma cells. This paradigm shift in our understanding of c-Myc stability as a function of tumor redox microenvironment could give us valuable cues in providing a pathway for therapeutic intervention targeting post-translational regulation of c-Myc in human cancer.

Bisdemethoxycurcumin enhances X-ray-induced apoptosis possibly through p53/Bcl-2 pathway

Bisdemethoxycurcumin (BDMC), which is isolated from the rhizomes of Curcuma longa, has anti-inflammatory and anti-carcinogenic activities. Here we found that BDMC enhanced X-ray-induced apoptosis in human T-cell leukemia MOLT-4 cells. Knockdown of p53 significantly attenuated the radiosensitizing effect of BDMC. However, BDMC did not enhance X-ray-mediated activation of the p53 signaling pathway via p53’s transactivation or mitochondrial translocation. On the other hand, BDMC promoted the X-ray-induced dephosphorylation at Ser 70 in Bcl-2’s flexible loop regulatory domain and Bcl-2 binding to p53. Overexpressing Bcl-2 completely blocked the BDMC’s radiosensitization effect. Our results indicate that BDMC stimulates the dephosphorylation and p53-binding activity of Bcl-2 and suggest that BDMC may induce a neutralization of Bcl-2’s anti-apoptotic function, thereby enhancing X-ray-induced apoptosis.

Reactive oxygen species mediate heat stress-induced apoptosis via ERK dephosphorylation and Bcl-2 ubiquitination in human umbilical vein endothelial cells

Heat stress can induce the mitochondrial apoptotic pathway in HUVEC cells, indicating that apoptosis may be a prominent pathological feature of heat stroke, however, little is known about the precise mechani sms involved in it. In this study, we describe the apoptotic effect of intense heat stress on HUVEC cells and our investigation of its underlying mechanisms. Treatment of cells with intense heat stress induced production of reactive oxygen species (ROS) and a concomitant increase in activation of the mitochondrial apoptotic pathway. Furthermore, by over-expression of MnSOD and GPx in cells, we show that ROS, and especially superoxide, is the primary oxidative species induced by intense heat stress and responsible for cell death. In addition, we explored the mechanism by which superoxide regulates the apoptotic effect of intense heat stress, and found that it involved Bcl-2 down-regulation through ubiquitin - proteasomal degradation. Superoxide production also led to Bcl-2 dephosphorylation through inactivation of MAP kinase ERK1/2, which promoted Bcl-2 ubiquitination. Taken together, these findings describe a novel pathway downstream of heat stress-induced apoptosis in HUVEC cells, and provide new insight into the process of redox-mediated down-regulation of Bcl-2 and apoptosis induction. These results could be important in the understanding of pathogenesis of heat stroke and for the development of preventive and treatment measures, both of which are currently lacking.

The plant-derived triterpenoid tingenin B is a potent anticancer agent due to its cytotoxic activity on cancer stem cells of breast cancer in vitro

Despite the rapid advances in chemotherapy regimens, the outcome of patients with breast cancer is not satisfactory. One of the reasons of this dissatisfaction is that subsets of cells in tumors which referred as cancer stem cells (CSCs) show and/or gain resistance to therapies. Thus, compounds that target CSCs are urgently needed. Since some are already used in the clinic, natural products have great potential for further development as anti cancer drugs. The aim of this study is to investigate the cytotoxic activity of tingenin b (or 22β-hydroxytingenone) which is a quinone-methide triterpenoid structurally related to tingenone, against breast CSCs (stem-cell enriched population from MCF-7 cell line, MCF-7s). It has been found that tingenin b was cytotoxic against MCF-7s (IC50 value for 48 h was found to be 2.38 μM) by inducing apoptosis. It was evident by Annexin V staining positivity, decreased mitochondrial membrane potential and Bcl-2 dephosphorylation with a concomitant increase in Bax protein expression. In addition, endoplasmic reticulum stress was also found to be involved in tingenin b-induced cell death. In conclusion, the results warrant further studies aimed at elucidating and corroborating its possible use in the treatment of breast cancer.

Ser70 phosphorylation of Bcl-2 by selective tyrosine nitration of PP2A-B56δ stabilizes its antiapoptotic activity

AbstractBcl-2 is frequently overexpressed in hematopoietic malignancies, and selective phosphorylation at ser70 enhances its antiapoptotic activity. Phospho-ser70 is dephosphorylated by specific heterotrimers of protein phosphatase 2A (PP2A). We report here that a mild pro-oxidant intracellular milieu induced by either pharmacological inhibition or genetic knockdown of superoxide dismutase 1 (SOD1) inhibits the functional holoenzyme assembly of PP2A and prevents Bcl-2 ser70 dephosphorylation. This redox-dependent regulation of Bcl-2 phosphorylation is due to nitrosative modification of B56δ, which we identify as the regulatory subunit mediating PP2A-dependent Bcl-2 dephosphorylation. Redox inhibition of PP2A results from peroxynitrite-mediated nitration of a conserved tyrosine residue within B56δ (B56δY289). Although nitrated B56δY289 binds efficiently to ser70-phosphorylated Bcl-2, this specific modification inhibits the recruitment of the PP2A catalytic core (A and C subunits). Furthermore, inhibition of B56δY289 nitration restores PP2A holoenzyme assembly, thereby permitting S70 dephosphorylation of Bcl-2 and inhibiting its antiapoptotic activity. More important, in primary cells derived from clinical lymphomas, Bcl-2 phosphorylation at S70 directly correlates with B56δ nitration and repression of SOD1, but inversely correlates with B56δ interaction with the PP2A-C catalytic subunit. These data underscore the role of a pro-oxidant milieu in chemoresistance of hematopoietic and other cancers via selective targeting of tumor suppressors such as PP2A.

Gossypol induces apoptosis in multiple myeloma cells by inhibition of interleukin-6 signaling and Bcl-2/Mcl-1 pathway.

Multiple myeloma (MM) is a clonal plasma cell disorder affecting the immune system with various systemic symptoms. MM remains incurable even with high dose chemotherapy using conventional drugs, thus necessitating development of novel therapeutic strategies. Gossypol (Gos) is a natural polyphenolic compound extracted from cotton plants, and has been shown to possess anti-neoplastic activity against various tumors. Recent studies have shown that Gos is an inhibitor for Bcl-2 or Bcl-XL acting as BH3 mimetics that interfere interaction between pro-apoptotic BH3-only proteins and Bcl-2/Bcl-XL. Since most of the patients with MM overexpress Bcl-2 protein, we considered Gos might be a promising therapeutic agent for MM. We herein show that Gos efficiently induced apoptosis and inhibited proliferation of the OPM2 MM cell line, in a dose- and time-dependent manner. Gos induced activation of caspase-3 and cytochrome c release from mitochondria, showing mitochondrial dysfunction pathway is operational during apoptosis. Further investigation revealed that phosphorylation of Bcl-2 at serine-70 was attenuated by Gos treatment, while protein levels were not affected. In addition, Mcl-1 was downregulated by Gos. Interestingly, phosphorylation of JAK2, STAT3, ERK1/2 and p38MAPK was inhibited by Gos-treatment, indicating that Gos globally suppressed interleukin-6 (IL-6) signals. Moreover, JAK2 inhibition mimicked the effect of Gos in OPM2 cells including Bcl-2 dephosphorylation and Mcl-1 downregulation. These results demonstrated that Gos induces apoptosis in MM cells not only through displacing BH3-only proteins from Bcl-2, but also through inhibiting IL-6 signaling, which leads to Bcl-2 dephosphorylation and Mcl-1 downregulation.

Chemosensitization of rhabdomyosarcoma cells by the histone deacetylase inhibitor SAHA

Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma in childhood with a dismal prognosis, highlighting the need for novel treatment strategies. Here, we identify a novel synthetic lethal interaction between the histone deacetylase inhibitor (HDACI) SAHA and anticancer drugs in RMS cells. Importantly, SAHA significantly increases chemotherapeutic drug-induced apoptosis in both embryonal and alveolar RMS cell lines, including several anticancer agents that are used in the clinic for the treatment of RMS such as Doxorubicin, Etoposide, Vincristine and Cyclophosphamide. Calculation of combination index (CI) reveals that the interaction of SAHA and Doxorubicin or Etoposide is synergistic. Mechanistically, SAHA causes acetylation of histone H3 protein in RMS cells, indicating that SAHA alters the chromatin context. Also, cotreatment with SAHA and Doxorubicin changes the ratio of pro- and antiapoptotic Bcl-2 proteins with downregulation of Mcl-1 and Bcl-xL, dephosphorylation of Bcl-2 and upregulation of BimEL, thus shifting the balance towards apoptosis. Consistently, SAHA and Doxorubicin cooperate to stimulate activation of Bax and Bak, caspase activation and caspase-dependent apoptosis. Overexpression of Bcl-2 significantly rescues SAHA/Doxorubicin-mediated apoptosis, underscoring the requirement of the mitochondrial apoptotic pathway for the synergistic induction of apoptosis by SAHA and Doxorubicin. Caspase-dependent apoptotic cell death is confirmed by the use of the broad-range caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD.fmk), which significantly decreases SAHA/Doxorubicin-triggered apoptosis. In conclusion, these findings demonstrate that the HDACI SAHA represents a promising strategy to prime RMS cells for chemotherapy-induced apoptosis and warrants further investigation in combination regimens.

PKR negatively regulates leukemia progression in association with PP2A activation, Bcl-2 inhibition and increased apoptosis

Reduced expression and activity of the proapoptotic, double-stranded RNA-dependent protein kinase, PKR (protein kinase R) is observed in breast, lung and various leukemias, suggesting that loss of PKR potentiates transformation. Now we report that decreased PKR activity inhibits chemotherapy-induced apoptosis of leukemia cells both in vitro and in vivo. Inhibition of PKR expression or activity reduces protein phosphatase 2A (PP2A) activity, a B-cell lymphoma 2 (Bcl-2) phosphatase, resulting in enhanced Bcl-2 phosphorylation. Thus, inhibition of PKR activity leads to hyperphosphorylation of Bcl-2, stabilization of Bcl-2/Bax interaction and decreased Bax insertion into the outer mitochondrial membrane. Treatment with the PP2A activator, FTY720, restores Bcl-2 dephosphorylation and apoptosis in cells with reduced PKR expression following stress. Significantly, xenografts of REH leukemic cells with reduced PKR display significantly increased tumor volume, increased resistance to doxorubicin treatment and shorter survival. Importantly, FTY720 treatment restores sensitivity to chemotherapy and prolongs overall survival of these mice. Collectively, these findings suggest that PP2A activation is a downstream target of PKR and the PKR/PP2A signaling axis is required for rapid and potent stress-induced apoptosis. Importantly, loss of PKR promotes leukemia progression and may serve as a biomarker for predicting chemosensitivity.

Abstract 5673: Novel inhibitors of the interferon-inducible, dsRNA-activated kinase PKR for myelodysplastic syndrome.

Abstract In hematopoietic stem/progenitor cells, the interferon-inducible, dsRNA-activated protein kinase, PKR, is a central mediator for the anti-proliferative effects of a wide variety of hematopoietic, inflammatory or cytotoxic stresses such as deprivation of IL-3, IL-5 or GM-CSF from hematopoietic factor-dependent cells. Once activated, PKR inhibits protein synthesis by phosphorylation of eIF2alpha, inhibits mitochondrial function by activating PP2A-dependent Bcl2 dephosphorylation, and activates signaling pathways such as NF-kappaB, p53 and STAT1 to regulate proliferation and apoptosis in response to cellular stress. Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematologic malignancies characterized by peripheral cytopenias, a hypercellular marrow with ineffective hematopoiesis and risk of transformation to acute myeloid leukemia (AML). Significantly, PKR is highly activated in myeloid progenitor cells from MDS patients and inhibition of PKR activity or siRNA-knockdown partially reverses the suppressive effects of IFN-gamma and TNF-alpha on hematopoietic colony formation using normal or MDS-derived CD34+ cells. Thus, increased PKR activity in hematopoietic stem cells may cooperate with driver/founder mutations to promote bone marrow failure and apoptosis characteristic of MDS, and inhibitors of PKR may be therapeutically useful to delay the onset and/or reduce the severity of MDS and its evolution to AML. Since there is only one commercially available pharmacologic PKR inhibitor that targets ATP binding and has been reported to have off target effects, we performed studies to identify novel small molecule compounds that specifically inhibit PKR-substrate association. Using the crystal structure of the PKR kinase domain-eIF2alpha complex, we employed a molecular docking strategy to screen in silico the NIH Developmental Therapeutic Program's repository of over 300,000 small molecules for compounds that interact with and inhibit PKR. The top 195 scoring small molecule compounds identified were obtained and validated in vitro using a LanthaScreen PKR kinase assay and 32P PKR assay. In addition, we screened the NIH/DTP Diversity and Challenge Compound Sets for inhibitors of PKR (2047 compounds total). Using these methods, we have identified 27 novel PKR inhibitors with an IC50 < 100 μM. Significantly, five are competitive inhibitors for eIF2alpha binding to PKR while the remaining inhibit ATP binding. These results identify a novel class of PKR inhibitors that specifically disrupt association of PKR with its substrate, eIF2alpha, and lay the groundwork for future studies that test whether these PKR inhibitors may be therapeutically useful to delay the onset of cytopenias and bone marrow failure associated with MDS and MDS evolution to AML. Citation Format: Richard L. Bennett, David A. Ostrov, W. Stratford May. Novel inhibitors of the interferon-inducible, dsRNA-activated kinase PKR for myelodysplastic syndrome. [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 5673. doi:10.1158/1538-7445.AM2013-5673

Abstract 4606: Loss of PKR promotes leukemia progression by activating Bcl-2 to inhibit apoptosis.

Abstract The interferon-inducible, dsRNA-dependent protein kinase, PKR, is activated by diverse cellular stresses. Loss of PKR expression or activity has been found in tumors including various types of leukemia, suggesting that loss of PKR may contribute to malignancy. However, no convincing conclusion has been reached regarding the relationship between PKR and leukemia progression. We analyzed 511 AML primary bone marrow samples by reverse phase protein array (RPPA) and discovered that PKR protein expression is > 50% reduced in CD34+ blasts from AML patients compared to CD34+ cells from healthy donors. This finding prompted us to further study how PKR affected progression of leukemia. PKR expression was either knocked down (by 82%) by specific SiRNA or directly inhibited by specific PKR inhibitor in leukemia cell lines. Significantly, inhibition of PKR decreased the rate of apoptosis following treatment with hydrogen peroxide (H2O2) or doxorubicin for 48 hours. In addition, bone marrow mononuclear cells form wild type, PKR null (-/-) or PKR transgenic mice were harvested and subjected to growth factor withdrawal for up to 120 hours. Cells isolated from PKR null mice had decreased apoptosis while cells from PKR transgenic mice had increased apoptosis compared to cells from wild type littermate mice. Further investigation showed that PKR inhibition led to reduction of PP2A activity, and a 2.4 fold increase in Bcl-2 phosphorylation. In addition, okadaic acid (an inhibitor of PP2A) had a similar effect to PKR inhibition on both Bcl-2 phosphorylation (1.74 fold increase) and stress-induced apoptosis. By contrast, treatment of cells with a PP2A activator, FTY 720, resulted in decreased Bcl-2 phosphorylation (by 2.24 fold) and enhanced apoptosis in response to H2O2. Furthermore, our study showed that following PKR inhibition, phosphorylation of Bcl-2 stabilized Bcl-2/Bax complex following H2O2 treatment, preventing Bax from being released, anchoring and perforating the mitochondrial outer membrane, which would initiate apoptotic process. To confirm our in vitro findings, a leukemia xenograft model was established in NSG mice using REH cells expressing luciferase. Mice receiving REH SiPKR cells displayed significantly faster tumor progression compared to mice receiving Sicontrol cells. Furthermore, tumors from REH SiPKR cells were more resistant to doxorubicin treatment compared to tumors from REH sicontrol cells. Taken together our results demonstrate that loss of PKR function inhibits PP2A-dependent Bcl-2 dephosphorylation resulting in increased leukemia progression and survival. In addition, since our studies indicate that PKR activation enhances the effectiveness of chemotherapy, PKR level/function status may serve as a clinical predictor of prognosis, disease relapse and resistance to chemotherapy. Citation Format: Xiaodong Cheng, Richard L. Bennett, Xiangfei Liu, Stratford May. Loss of PKR promotes leukemia progression by activating Bcl-2 to inhibit apoptosis. [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 4606. doi:10.1158/1538-7445.AM2013-4606

Abstract 2009: PKR induces apoptosis via PP2A activation and Bcl-2 dephosphorylation in IL-3 dependent murine bone marrow stem/progenitor cells

Abstract The Interferon-induced double-stranded RNA-activated protein kinase (PKR) was originally found to be activated by viral infection leading to inhibition of new protein synthesis and apoptosis. Recently multiple studies have reported decreased expression and function of PKR in leukemia cells, although the exact role of PKR in the process of oncogenesis remains unclear. Since leukemias are clonal disorders originating in a primitive pluripotent leukemic stem cell (LSC) which is regulated by multi-potential hematopoietic growth factors including interleukin-3 (IL-3). Thus, studying how PKR regulates apoptosis in IL-3 dependent (CD123+) pluripotent stem/progenitor cells is expected to reveal PKR's role in leukemia. PKR expression was either knocked down (by 78%) using a specific ShRNA or directly inhibited (57%) by treating cells with a specific PKR inhibitor. Results revealed that inhibition of PKR was associated with a reduction of PP2A activity, a 2.3 fold increase in Bcl-2 phosphorylation and inhibition of apoptosis following treatment with 100uM hydrogen peroxide for 48 hours. In addition, okadaic acid (an inhibitor of PP2A) had a similar effect to PKR inhibition on both Bcl-2 phosphorylation (1.74 fold increase) and stress-induced apoptosis. By contrast, treatment of cells with the PP2A activator, FTY 720, resulted in decreased Bcl-2 phosphorylation (by 2.24 fold) and enhanced apoptosis in response to treatment of cells with chemotherapy (etoposide or doxorubicin) or hydrogen peroxide. To test the role of PKR in growth factor- withdrawal mediated apoptosis in bone marrow stem/progenitor cells, CD123+(IL-3R[[Unsupported Character - Symbol Font ]]+) bone marrow cells from wild type, PKR transgenic (over-expressed) and PKR null(-/-) mice were exposed to different stresses including IL-3 deprivation or hydrogen peroxide. CD123+ cells isolated from PKR null mice had a significantly lower percentage of apoptosis while CD123+ cells from PKR transgenic mice had increased apoptosis compared to cells from wild type littermate mice. These results demonstrate that PKR negatively affects hematopoietic cell survival by enhancing apoptosis following stress in a mechanism that involves PP2A activation and Bcl2 dephosphorylation. Taken together these results may indicate that inhibition of PKR's pro-apoptotic function in bone marrow pluripotent stem/progenitor cells may contribute to tumorigenesis in hematopoietic malignancies. 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 2009. doi:1538-7445.AM2012-2009