Bcl-2 Phosphorylation Research Articles

Repurposing Suan-Zao-Ren-Tang as anticancer agents in apoptotic sensitization against non-small cell lung cancer cell lines through amplification of spindle assembly checkpoint activation

Treatment of non-small cell lung cancer (NSCLC) is a high unmet medical need. We repurposed Suan-Zao-Ren-Tang (SZRT), a widely used traditional Chinese medicine in alleviating insomnia, to sensitize vinorelbine-induced anti-NSCLC effect and have unveiled the underlying mechanism. B7E2, the partial purified extract from SZRT, was used through bioactivity-guided fractionation based on anti-proliferative activity in NSCLC cell lines A549 and NCI-H460. Three batches of B7E2 and their individual components were prepared for the examination of components and bioactivity and mechanistic study. B7E2 alone had no cytotoxic effect but its combination with vinorelbine displayed a synergistic activity in killing NSCLC cells. Apoptotic sensitization was verified by a remarkable increase of caspase activation and phosphorylation of Bcl-2 and Bcl-xL. Combination treatment prolonged mitotic arrest and induced a substantial activation of spindle assembly checkpoint (SAC) characterized by BUBR1 phosphorylation at Ser670 and Thr680, and cyclin B1 upregulation. Combination treatment enhanced the interaction between mitotic checkpoint complex (MCC) components, including BUBR1, MAD2, BUB3 and CDC20. Further analysis revealed that SZRT components, including senkyunolide-A, trans-neocnidilide, 3-butylidenephthalide, betulinic acid and linoleic acid, were responsible for B7E2 activities. Three B7E2 batches showed a good chemical similarity and similar activities between batches. The data suggest that B7E2 is a potential chemosensitizer in potentiating vinorelbine-induced anti-NSCLC activity through amplification and prolongation of SAC activation.

A novel HDAC6 inhibitor interferes microtubule dynamics and spindle assembly checkpoint and sensitizes cisplatin-induced apoptosis in castration-resistant prostate cancer.

Metastatic castration-resistant prostate cancer (CRPC), the most refractory prostate cancer, inevitably progresses and becomes unresponsive to hormone therapy, revealing a pressing unmet need for this disease. Novel agents targeting HDAC6 and microtubule dynamics can be a potential anti-CRPC strategy. Cell proliferation was examined in CRPC PC-3 and DU-145 cells using sulforhodamine B assay and anchorage-dependent colony formation assay. Flow cytometric analysis of propidium iodide staining was used to determine cell-cycle progression. Cell-based tubulin polymerization assay and confocal immunofluorescence microscopic examination determine microtubule assembly/disassembly status. Protein expressions were determined using Western blot analysis. A total of 82 novel derivatives targeting HDAC6 were designed and synthesized, and Compound 25202 stood out, showing the highest efficacy in blocking HDAC6 (IC50, 3.5 nM in enzyme assay; IC50, 1.0 μM in antiproliferative assay in CRPC cells), superior to tubastatin A (IC50, 5.4 μM in antiproliferative assay). The selectivity and superiority of 25202 were validated by examining the acetylation of both α-tubulin and histone H3, detecting cell apoptosis and HDACs enzyme activity assessment. Notably, 25202 but not tubastatin A significantly decreased HDAC6 protein expression. 25202 prolonged mitotic arrest through the detection of cyclin B1 upregulation, Cdk1 activation, mitotic phosphoprotein levels, and Bcl-2 phosphorylation. Compound 25202 did not mimic docetaxel in inducing tubulin polymerization but disrupted microtubule organization. Compound 25202 also increased the phosphorylation of CDC20, BUB1, and BUBR1, indicating the activation of the spindle assembly checkpoint (SAC). Moreover, 25202 profoundly sensitized cisplatin-induced cell death through impairment of cisplatin-evoked DNA damage response and DNA repair in both ATR-Chk1 and ATM-Chk2 pathways. The data suggest that 25202 is a novel selective and potent HDAC6 inhibitor. Compound 25202 blocks HDAC6 activity and interferes microtubule dynamics, leading to SAC activation and mitotic arrest prolongation that eventually cause apoptosis of CRPC cells. Furthermore, 25202 sensitizes cisplatin-induced cell apoptosis through impeding DNA damage repair pathways.

Colchicine alleviates myocardial ischemia-reperfusion injury in mice by activating AMPK

To investigate the protective effect of colchicine against myocardial ischemia-reperfusion injury (I/R) and explore the underlying mechanism. H9C2 cells exposed to hypoxia/reoxygenation (H/R) were treated with 3 nmol/L colchicine, after which the changes in cell viability were assessed using MTT assay, and AMPK phosphorylation, the expressions of NOX4, NRF2, SOD2, BAX, Bcl-2, and cleaved caspase-3 were detected with Western blotting. Male C57BL/6 mice were randomized into sham operation, I/R, I/R+colchicine, and I/R+colchicine+dorsomorphin (DSMP) groups. After the treatments, myocardial expressions of p-AMPK/AMPK, 8-OHdG, cleaved caspase-3, mitochondrial BAX (Mito-BAX), and cytoplasmic cytochrome C (Cyt-Cyto C) were examined and cardiac functions, infarct area, ATP content, and serum levels of lactic dehydrogenase (LDH) and cardiac troponin T (cTnT) levels were assessed. In H9C2 cells, H/R exposure significantly reduced AMPK phosphorylation and expressions of NRF2, SOD2, and Bcl-2, lowered cell viability, and up-regulated the expressions of NOX4, BAX, and cleaved caspase-3 (P < 0.05), and these changes were obviously alleviated by colchicine treatment (P < 0.05). In the mouse models, myocardial I/R injury significantly reduced myocardial AMPK phosphorylation level, ATP content, and expressions of NRF2, SOD2 and Bcl-2, caused cardiac function impairment, enhanced NOX4, Mito-BAX, Cyt-Cyto C, BAX, 8-OHdG, and cleaved caspase-3 expressions, and increased infarct area and serum LDH and cTnT levels (P < 0.05). Colchicine treatment significantly reversed the damaging effects of I/R (P < 0.05), but its protective effects was obviously antagonized by DSMP (P < 0.05). Colchicine alleviates myocardial I/R injury and protects cardiac function in mice by reducing myocardial oxidative stress and apoptosis via activating AMPK.

B-cell lymphoma-2 phosphorylation at Ser70 site-related autophagy mediates puerarin-inhibited the apoptosis of MC3T3-E1 cells during osteoblastogenesis.

To explore the relationship between autophagy and apoptosis regulated by puerarin during osteoblastogenesis. In this study, the effects of puerarin on the autophagic activity and apoptosis level of osteoblast precursors (MC3T3-E1 cells) was observed. Subsequently, the roles of puerarin on B-cell lymphoma-2 (Bcl-2) phosphorylation at different sites in osteoblast precursors were observed. The effect of puerarin on the interaction between Bcl-2 and autophagy regulatory molecule or pro-apoptotic molecule was also investigated using Co-immunoprecipitation assays. In addition, the effect of puerarin on mitochondrial membrane potential of osteoblast precursors was also identified by mitochondrial membrane potential fluorescence probe assays. Our results showed that puerarin can promote the autophagic activity and apoptosis level of MC3T3-E1 cells. In addition, puerarin promoted Bcl-2 phosphorylation at Ser70 site, and the dissociation of Bcl-2-Beclin1 complex. Moreover, puerarin could enhance the binding of Bcl-2-Bcl-2-Associated X (Bax) complex in MC3T3-E1 cells. Furthermore, puerarin increased the mitochondrial membrane potential of MC3T3-E1 cells. Therefore, puerarin promotes Beclin1 into autophagy flux through Bcl-2 phosphorylation at Ser70, thereby enhancing autophagy of osteoblast precursors, which mediates its anti-apoptotic role during osteoblastogenesis. Furthermore, the dissociation of Bcl-2-Beclin1 complex is conducive to the binding of Bcl-2-Bax complex, which resists the apoptosis of osteoblast precursors viathe increased mitochondrial membrane potential.

DUSP4 maintains the survival and LSD1 protein stability in esophageal squamous cell carcinoma cells by inhibiting JNK signaling-dependent autophagy.

DUSP4 is a biomarker of esophageal squamous cell carcinoma (ESCC), which is responsible for the prognosis in ESCC. However, the underlying mechanism of DUSP4-regulated ESCC carcinogenesis is unknown. As a negative regulator of JNK, DUSP4 can inhibit autophagy, which contributes to tumorigenesis. This study aimed to explore the role of autophagy in DUSP4-regulated ESCC carcinogenesis. Our results showed that DUSP4 overexpression inhibited autophagy and promoted LSD1 protein expression in ESCC cells, while DUSP4 silencing showed the opposite effects. However, DUSP4 overexpression and silencing did not affect LSD1 mRNA expression. But the regulatory ability of DUSP4 overexpression on autophagy, death level, and LSD1 protein was reversed by rapamycin. In addition, DUSP4 overexpression inhibited JNK and Bcl2 phosphorylation and the dissociation of Bcl2-Beclin1 complex, while DUSP4 silencing promoted JNK and Bcl2 phosphorylation. Moreover, the regulatory ability of DUSP4 overexpression on autophagy, death, and LSD1 protein was reversed by JNK activator anisomycin. The xenograft assays also showed that DUSP4 overexpression-promoted ESCC tumor growth in vivo and LC3II and LSD1 protein expression in tumor tissues were reversed by rapamycin or anisomycin. Overall, DUSP4 inhibits Bcl2-Beclin1-autophagy signal transduction through the negative regulation of JNK, thus suppressing autophagic death and the autophagic degradation of LSD1 in ESCC, by which DUSP4 promotes ESCC carcinogenesis.

Low-molecular-weight fucoidan inhibits the proliferation of melanoma via Bcl-2 phosphorylation and PTEN/AKT pathway.

Fucoidan, a sulfate polysaccharide obtained from brown seaweed, has various bioactive properties, including anti-inflammatory, anti-cancer, anti-viral, anti-oxidant, anti-coagulant, anti-thrombotic, anti-angiogenic, and anti-Helicobacter pylori properties. However, the effects of low-molecular-weight fucoidan (LMW-F) on melanoma cell lines and three dimensional (3D) cell culture models are not well understood. This study aimed to investigate the effects of LMW-F on A375 human melanoma cells and cryopreserved biospecimens derived from patients with advanced melanoma. Ultrasonic wave was used to fragment fucoidan derived from Fucus vesiculosus into smaller LMW-F. MTT and live/dead assays showed that LMW-F inhibited cell proliferation in both A375 cells and patient-derived melanoma explants in a 3D-printed collagen scaffold. The PTEN/AKT pathway was found to be involved in the anti-melanoma effects of fucoidan. Western blot analysis revealed that LMW-F reduced the phosphorylation of Bcl-2 at Thr 56, which was associated with the prevention of anti-apoptotic activity of cancer cells. Our findings suggested that LMW-F could enhance anti-melanoma chemotherapy and improve the outcomes of patients with melanoma resistance.

Homospisulosine induced apoptosis in cervical carcinoma cells is associated with phosphorylation of Bcl-2 and up-regulation of p27/Kip1.

Spisulosine (1-deoxysphinganine) is a sphingoid amino alcohol isolated from the sea clams that showed potent antiproliferative activity against a broad spectrum of solid tumors but failed in clinical trials due to neurotoxicity. However, its structural similarity to other bioactive sphingoids, interesting mode of action, and appreciable potency against cancer cells make it a suitable lead for future anticancer drug development. The present study was conducted to elucidate mechanisms of the antiproliferative/cytotoxic effects of newly synthesized spisulosine analog homospisulosine (KP7). The evaluation was performed on cervical carcinoma cells, representing an in vitro model of one of the most common cancer types and a significant worldwide cause of women's cancer mortality. Treatment with homospisulosine (2.0 μM) for 24, 48, and 72 h significantly inhibited the growth of HeLa cells in vitro and induced apoptosis detectable by DNA fragmentation, externalization of phosphatidylserine, dissipation of mitochondrial membrane potential, activation of caspase-3 and cleavage of PARP. In addition, treating HeLa cells with spisulosine increased p27 and Bcl-2 on protein levels and phosphorylation of Bcl-2 on Ser70 residue. These results support the potential for spisulosine analogs represented here by homospisulosine for future therapeutic development.

Phosphorylation of Bcl-2 by JNK confers gemcitabine resistance in lung cancer cells by reducing autophagy-mediated cell death.

The most common cancer-related death in the world is non-small cell lung cancer (NSCLC). Gemcitabine (GEM) is a common and effective first-line chemotherapeutic drug for the treatment of NSCLC. However, the long-term use of chemotherapeutic drugs in patients usually induces cancer cell drug resistance, leading to poor survival, and prognosis. In this study, to observe and explore the key targets and potential mechanisms of NSCLC resistance to GEM, we first cultured lung cancer CL1-0 cells in a GEM-containing medium to induce CL1-0 cells to develop GEM resistance. Next, we compared protein expression between the parental and GEM-R CL1-0 cell groups. We observed significantly lower expression of autophagy-related proteins in GEM-R CL1-0 cells than in parental CL1-0 cells, indicating that autophagy is associated with GEM resistance in CL1-0 cells. Furthermore, a series of autophagy experiments revealed that GEM-R CL1-0 cells had significantly reduced GEM-induced c-Jun N-terminal kinase phosphorylation, which further affected the phosphorylation of Bcl-2, thereby reducing the dissociation of Bcl-2 and Beclin-1 and ultimately reducing the generation of GEM-induced autophagy-dependent cell death. Our findings suggest that altering the expression of autophagy is a promising therapeutic option for drug-resistant lung cancer.

Curcumin suppresses RANKL-induced osteoclast precursor autophagy in osteoclastogenesis by inhibiting RANK signaling and downstream JNK-BCL2-Beclin1 pathway

BackgroundCurcumin ameliorates bone loss by inhibiting osteoclastogenesis. Curcumin inhibits RANKL-promoted autophagy in osteoclast precursors (OCPs), which mediates its anti-osteoclastogenic effect. But the role of RANKL signaling in curcumin-regulated OCP autophagy is unknown. This study aimed to explore the relationship between curcumin, RANKL signaling, and OCP autophagy during osteoclastogenesis. MethodsWe investigated the role of curcumin in RANKL-related molecular signaling in OCPs, and identified the significance of RANK-TRAF6 signaling in curcumin-treated osteoclastogenesis and OCP autophagy using flow sorting and lentiviral transduction. Tg-hRANKL mice were used to observe the in vivo effects of curcumin on RANKL-regulated bone loss, osteoclastogenesis, and OCP autophagy. The significance of JNK-BCL2-Beclin1 pathway in curcumin-regulated OCP autophagy with RANKL was explored via rescue assays and BCL2 phosphorylation detection. ResultsCurcumin inhibited RANKL-related molecular signaling in OCPs, and repressed osteoclast differentiation and autophagy in sorted RANK+ OCPs but did not affect those of RANK− OCPs. Curcumin-inhibited osteoclast differentiation and OCP autophagy were recovered by TRAF6 overexpression. But curcumin lost these effects under TRAF6 knockdown. Furthermore, curcumin prevented the decrease in bone mass and the increase in trabecular osteoclast formation and autophagy in RANK+ OCPs in Tg-hRANKL mice. Additionally, curcumin-inhibited OCP autophagy with RANKL was reversed by JNK activator anisomycin and TAT-Beclin1 overexpressing Beclin1. Curcumin inhibited BCL2 phosphorylation at Ser70 and enhanced protein interaction between BCL2 and Beclin1 in OCPs. ConclusionsCurcumin suppresses RANKL-promoted OCP autophagy by inhibiting signaling pathway downstream of RANKL, contributing to its anti-osteoclastogenic effect. Moreover, JNK-BCL2-Beclin1 pathway plays an important role in curcumin-regulated OCP autophagy.

Morroniside inhibits Beclin1-dependent autophagic death and Bax-dependent apoptosis in cardiomyocytes through repressing BCL2 phosphorylation.

Morroniside can prevent myocardial injury caused by ischemia and hypoxia, which can be used to treat acute myocardial infarction (AMI). Hypoxia can cause apoptosis and autophagic death of cardiomyocytes. Morroniside has the ability to inhibit apoptosis and autophagy. However, the relationship between Morroniside-protected cardiomyocytes and two forms of death is unclear. The effects of Morroniside on the proliferation, apoptosis level, and autophagic activity of rat cardiomyocyte line H9c2 under hypoxia were first observed. Next, the roles of Morroniside in the phosphorylation of JNK and BCL2, BCL2-Beclin1, and BCL2-Bax complexes as well as mitochondrial membrane potential in H9c2 cells were evaluated upon hypoxia. Finally, the significance of BCL2 or JNK in Morroniside-regulated autophagy, apoptosis, and proliferation in H9c2 cells was assessed by combining Morroniside and BCL2 competitive inhibitor (ABT-737) or JNK activator (Anisomycin). Our results showed that hypoxia promoted autophagy and apoptosis of H9c2 cells, and inhibited their proliferation. However, Morroniside could block the effect of hypoxia on H9c2 cells. In addition, Morroniside could inhibit JNK phosphorylation, BCL2 phosphorylation at the Ser70 and Ser87 sites, and the dissociation of BCL2-Beclin1 and BCL2-Bax complexes in H9c2 cells upon hypoxia. Moreover, the reduction of mitochondrial membrane potential in H9c2 cells caused by hypoxia was improved by Morroniside administration. Importantly, the inhibited autophagy, apoptosis, and promoted proliferation in H9c2 cells by Morroniside were reversed by the application of ABT-737 or Anisomycin. Overall, Morroniside inhibits Beclin1-dependent autophagic death and Bax-dependent apoptosis via JNK-mediated BCL2 phosphorylation, thereby improving the survival of cardiomyocytes under hypoxia.

Impaired Autophagy in Krabbe Disease: The Role of BCL2 and Beclin-1 Phosphorylation

Autophagic impairment was identified in many lysosomal storage diseases and adult neurodegenerative diseases. It seems that this defect could be directly related to the appearance of a neurodegenerative phenotype and could contribute to worsen metabolite accumulation and lysosomal distress. Thus, autophagy is becoming a promising target for supportive therapies. Autophagy alterations were recently identified also in Krabbe disease. Krabbe disease is characterized by extensive demyelination and dysmyelination and it is due to the genetic loss of function of the lysosomal enzyme galactocerebrosidase (GALC). This enzyme leads to the accumulation of galactosylceramide, psychosine, and secondary substrates such as lactosylceramide. In this paper, we induced autophagy through starvation and examined the cellular response occurring in fibroblasts isolated from patients. We demonstrated that the inhibitory AKT-mediated phosphorylation of beclin-1 and the BCL2-beclin-1 complex concur to reduce autophagosomes formation in response to starvation. These events were not dependent on the accumulation of psychosine, which was previously identified as a possible player in autophagic impairment in Krabbe disease. We believe that these data could better elucidate the capability of response to autophagic stimuli in Krabbe disease, in order to identify possible molecules able to stimulate the process.

Combination Therapies Targeting Apoptosis in Paediatric AML: Understanding the Molecular Mechanisms of AML Treatments Using Phosphoproteomics.

Paediatric acute myeloid leukaemia (AML) continues to present treatment challenges, as no "standard approach" exists to treat those young patients reliably and safely. Combination therapies could become a viable treatment option for treating young patients with AML, allowing multiple pathways to be targeted. Our in silico analysis of AML patients highlighted "cell death and survival" as an aberrant, potentially targetable pathway in paediatric AML patients. Therefore, we aimed to identify novel combination therapies to target apoptosis. Our apoptotic drug screening resulted in the identification of one potential "novel" drug pairing, comprising the Bcl-2 inhibitor ABT-737 combined with the CDK inhibitor Purvalanol-A, as well as one triple combination of ABT-737 + AKT inhibitor + SU9516, which showed significant synergism in a series of paediatric AML cell lines. Using a phosphoproteomic approach to understand the apoptotic mechanism involved, proteins related to apoptotic cell death and cell survival were represented, in agreement with further results showing differentially expressed apoptotic proteins and their phosphorylated forms among combination treatments compared to single-agent treated cells such upregulation of BAX and its phosphorylated form (Thr167), dephosphorylation of BAD (Ser 112), and downregulation of MCL-1 and its phosphorylated form (Ser159/Thr 163). Total levels of Bcl-2 were decreased but correlated with increased levels of phosphorylated Bcl-2, which was consistent with our phosphoproteomic analysis predictions. Bcl-2 phosphorylation was regulated by extracellular-signal-regulated kinase (ERK) but not PP2A phosphatase. Although the mechanism linking to Bcl-2 phosphorylation remains to be determined, our findings provide first-hand insights on potential novel combination treatments for AML.

DUSP4 inhibits autophagic cell death and apoptosis in colorectal cancer by regulating BCL2-Beclin1/Bax signaling.

The DUSP4 gene plays an important role in the carcinogenesis of colorectal cancer (CRC). However, the underlying mechanism of DUSP4-regulated colorectal carcinogenesis is unknown. DUSP4 is a negative regulator of the MAP kinase (MAPK) JNK, and JNK-mediated BCL2 phosphorylation is associated with apoptosis and autophagic cell death. Our study aimed to explore the significance of BCL2 phosphorylation-dependent autophagy and apoptosis in DUSP4-promoted colorectal carcinogenesis. We first investigated the roles of DUSP4 in the survival of HCT116 and SW480 CRC cell lines using gene-silencing and -overexpression techniques. Next, we explored the effects of DUSP4 on the BCL2 phosphorylation, autophagy and apoptosis of HCT116 and SW480 cells. Ultimately, with the help of pharmacological inhibitors of Beclin1 and BCL2 (spautin-1 and ABT-737), the relationship between BCL2-Beclin1/Bax signaling and DUSP4-regulated autophagy, apoptosis, survival and migration in HCT116 cells was clarified. Our results first confirmed the contribution of DUSP4 to the survival of HCT116 and SW480 cells. In addition, DUSP4 silencing resulted in BCL2 phosphorylation and the enhancement in autophagy and apoptosis in HCT116 and SW480 cells, while DUSP4 overexpression showed the opposite effect. Moreover, DUSP4 silencing inhibited the protein interaction between BCL2 and Beclin1 or Bax in HCT116 cells. Moreover, the survival and migration of HCT116 cells inhibited by DUSP4 silencing were blocked by autophagy inhibition with spautin-1. Notably, the survival and migration of HCT116 cells promoted by DUSP4 overexpression were reversed by ABT-737. It was indicated that DUSP4 can maintain the survival and function of CRC cells by inhibiting BCL2 phosphorylation-dependent autophagic cell death and apoptosis.

Inhibition of ERK1/2 Reverses Venetoclax-Induced Autophagy to Overcome Resistance in Acute Myeloid Leukemia

Background: Activation of the RAS/MAPK pathway confers inherent/acquired resistance to the Bcl-2 inhibitor, venetoclax, in AML (Zhang et al., Signal Transduct Target Ther. 2022). Pro-survival Bcl-2 inhibits apoptosis as well as autophagy. Phosphorylation of Bcl-2 by ERK1/2 leads to dissociation of the autophagic protein Beclin 1 from Bcl-2 and promotes autophagy (Pattingre et al., Cell. 2005). Thus, while Bcl-2 inhibition can induce apoptosis, its inhibition leads to induction of autophagy (Pattingre et al., Cell. 2005) that can protect tumor cells from organelle damage and nutrient deprivation. Objective: We hypothesized that terminal MAPK pathway inhibition using an ERK1/2 inhibitor would reverse venetoclax-induced autophagy and synergistically lead to cell death/apoptosis. Methods: We inhibited ERK1/2 using Compound 27 (Heightman et al., J Med Chem. 2018), a close analog of ASTX029, which is a dual-mechanism ERK1/2 inhibitor currently under clinical investigation in solid tumors (NCT03520075). We studied the potential synergy of ERK1/2 and Bcl-2 inhibition in decreasing cell proliferation and inducing apoptosis and in overcoming venetoclax resistance in AML preclinical models and primary patient samples (n=8). We performed simultaneous analysis of cell proliferation, stress response and DNA damage using single cell proteomics analysis to understand the mechanism of synergy. Autophagy was measured by lipidated LC3 quantification (flow cytometry and immunoblotting). Colony formation assay using NRAS-mutant PDX was performed to analyze the effect on progenitor cells. For insights into the mechanisms of synergy, we conducted large-scale proteomics analyses by single cell mass cytometry (CyTOF) and simultaneously measured cell proliferation, stress response, DNA damage and Bcl-2 family proteins. Results: Inhibition of ERK1/2 sensitized OCI-AML3 (NRAS-mutant) cells (Combination Index [CI]: 0.008) and venetoclax-resistant OCI-AML2 cells (CI: 0.05) to venetoclax and shifted the cytostatic effect of the single agents to a cytotoxic effect. Combination treatment induced apoptosis in leukemia initiating progenitor cells (LICs, CD34+CD38-) in primary AML samples with 54% apoptosis in combination versus 10% with venetoclax alone (CI = 0.03-0.23), while sparing normal CD34+CD38- cells. Mechanistically, basal ERK1/2 phosphorylation and autophagy (LC3 lipidation), were higher in venetoclax-resistant OCI-AML2 cells versus parental cells (p=0.0008) and both were inhibited with ERK1/2 inhibition alone and in combination with venetoclax. Simultaneous analysis of cell proliferation, stress response and DNA damage showed downregulation of proliferation (Ki-67) and an increase in ATF4 in response to ERK1/2 inhibition. The combination of Compound 27 and venetoclax inhibited autophagy (lipidated LC3) and increased apoptosis (increased cleaved PARP and cleaved caspase 3, Fig 1a). Venetoclax-induced autophagy was also confirmed in OCI-AML3 cells at 24 h post treatment (p=0.03). ERK1/2 inhibition reversed venetoclax-induced autophagy in OCI-AML3 (p=0.0003) and OCI-AML2-venetoclax-resistant cells (p=0.0007). CyTOF analysis showed decreased p-AMPK in response to ERK1/2 inhibition in OCI-AML3 cells. As AMPK/FIS1-mediated mitochondrial autophagy is required for self-renewal of AML stem cells (Pie et al., Cell Stem Cell. 2018), we confirmed decreased clonogenic potential of AML progenitor cells by reduced colony formation in response to ERK1/2 inhibition using an NRAS-mutant AML PDX model (p=0.0002). Single cell proteomics (CyTOF) showed decreased p-AMPK in response to single agent ERK1/2 inhibition or the combination with venetoclax in LIC populations (CD34+CD38-) in the NRAS-mutant AML PDX model. In addition to p-AMPK the expression of cMyc and CD44 was also reduced in the combination treatment, indicating reduced stemness (Fig 1b). Conclusion: Mechanistically, our results indicate that pharmacological inhibition of ERK1/2 impairs autophagy and sensitizes venetoclax-resistant AML cells to apoptosis, thus overcoming primary and secondary resistance to venetoclax. In addition, combination treatment induced apoptosis of LICs and reduced clonogenic potential and markers of stem cell function. These data provide a strong rationale for the combination of ERK1/2 and Bcl-2 inhibitors in treatment of AML and warrant further investigation in the clinic. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

PUMA overexpression dissociates thioredoxin from ASK1 to activate the JNK/BCL-2/BCL-XL pathway augmenting apoptosis in ovarian cancer

ASK1-JNK signaling promotes mitochondrial dysfunction-mediated apoptosis, but the bridge between JNK and apoptosis is not fully understood. PUMA induces apoptosis through BAX/BAK. Our previous study suggests a therapeutic potential of PUMA for ovarian cancer. However, whether and how PUMA activates ASK1 remains unclear. Here, we found for the first time that PUMA activated ASK1 by dissociating thioredoxin (TRX) from ASK1, however, it neither interacted with ASK1 nor TRX. Furthermore, PUMA overexpression caused ROS release from mitochondrial. H2O2 significantly impaired the interaction of ASK1 with TRX, whereas ROS scavenger NAC effectively abrogated the H2O2 effect, partly rescued PUMA-interfered interaction of ASK1 with TRX, and also abolished ASK1 phosphorylation. Interestingly, PUMA could not impair the association of ASK1 with TRX-C32S or TRX-C35S, two TRX mutants which are no longer oxidized in response to ROS. We further showed that PUMA activated ASK1-JNK axis to phosphorylate BCL-2 and BCL-XL, further augmenting apoptosis of ovarian cancer cells. In vivo, PUMA adenovirus combined with paclitaxel significantly inhibited intrinsically cisplatin-resistant ovarian cancer growth, and caused phosphorylation of BCL-2 and BCL-XL. Our results from human ovarian cancer TMA chips also revealed a positive correlation between PUMA expression and the phosphorylation of BCL-2 and BCL-XL. More importantly, all patients had no distal metastasis, implying a possibly clinical significance. Collectively, our results reveal a new pro-apoptotic signal amplification mechanism for PUMA by which PUMA overexpression first induces ROS-mediated dissociation of TRX from ASK1, and then causes JNK activation-triggering BCL-2/BCL-XL phosphorylation, ultimately augmenting apoptosis in ovarian cancer.

Inhibition of Glutamine Uptake Resensitizes Paclitaxel Resistance in SKOV3-TR Ovarian Cancer Cell via mTORC1/S6K Signaling Pathway

Ovarian cancer is a carcinoma that affects women and that has a high mortality rate. Overcoming paclitaxel resistance is important for clinical application. However, the effect of amino acid metabolism regulation on paclitaxel-resistant ovarian cancer is still unknown. In this study, the effect of an amino acid-deprived condition on paclitaxel resistance in paclitaxel-resistant SKOV3-TR cells was analyzed. We analyzed the cell viability of SKOV3-TR in culture conditions in which each of the 20 amino acids were deprived. As a result, the cell viability of the SKOV3-TR was significantly reduced in cultures deprived of arginine, glutamine, and lysine. Furthermore, we showed that the glutamine-deprived condition inhibited mTORC1/S6K signaling. The decreased cell viability and mTORC1/S6K signaling under glutamine-deprived conditions could be restored by glutamine and α-KG supplementation. Treatment with PF-4708671, a selective S6K inhibitor, and the selective glutamine transporter ASCT2 inhibitor V-9302 downregulated mTOR/S6K signaling and resensitized SKOV3-TR to paclitaxel. Immunoblotting showed the upregulation of Bcl-2 phosphorylation and a decrease in Mcl-1 expression in SKOV3-TR via the cotreatment of paclitaxel with PF-4708671 and V-9302. Collectively, this study demonstrates that the inhibition of glutamine uptake can resensitize SKOV3-TR to paclitaxel and represents a promising therapeutic target for overcoming paclitaxel resistance in ovarian cancer.

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.

Energetic metabolic reprogramming in Jurkat DFF40-deficient cancer cells.

DNA fragmentation factor 40 (DFF40), or the caspase-activated DNase (CAD), is an endonuclease specific for double-stranded DNA. Alterations in its function and expression have been linked to apoptosis resistance, a mechanism likely used by cancer cells. However, how the DFF40-related apoptosis resistance pathway occurs remains unclear. Here, we sought to determine if DFF40 expression could be linked to cell metabolism through the regulation of mitochondrial integrity and function. We demonstrated that DFF40-deficient cells are more resistant to staurosporine and tributyltin (TBT)-induced apoptosis, and express higher levels of Mcl-1 at basal state. Treatment with TBT induces higher Bcl-2 and caspase-9 mRNA transcripts in DFF40 KO Jurkat cells, as well as enhanced Bcl-2 phosphorylation. A loss of DFF40 expression induces a higher mitochondrial mass, mtDNA copy number, mitochondrial membrane potential, and glycolysis rates in resting T cells. DFF40-deficient cells exhibit the Warburg effect phenotype, where they rely significantly more on glycolysis than oxidative phosphorylation and have a higher proliferative state, demonstrated by a higher Ki-67 transcription factor expression and AKT phosphorylation. Finally, we demonstrated with cell fractioning that DFF40 can translocate to the mitochondria following apoptosis induction. Our study reveals that DFF40 may act as a regulator of mitochondria during cell death and its loss could compromise mitochondrial integrity and cause an energetic reprogramming in pathologies such as cancer.

JAK3 mutations and mitochondrial apoptosis resistance in T-cell acute lymphoblastic leukemia.

Resistance to mitochondrial apoptosis predicts inferior treatment outcomes in patients with diverse tumor types, including T-cell acute lymphoblastic leukemia (T-ALL). However, the genetic basis for variability in this mitochondrial apoptotic phenotype is poorly understood, preventing its rational therapeutic targeting. Using BH3 profiling and exon sequencing analysis of childhood T-ALL clinical specimens, we found that mitochondrial apoptosis resistance was most strongly associated with activating mutations of JAK3. Mutant JAK3 directly repressed apoptosis in leukemia cells, because its inhibition with mechanistically distinct pharmacologic inhibitors resulted in reversal of mitochondrial apoptotic blockade. Inhibition of JAK3 led to loss of MEK, ERK and BCL2 phosphorylation, and BH3 profiling revealed that JAK3-mutant primary T-ALL patient samples were characterized by a dependence on BCL2. Treatment of JAK3-mutant T-ALL cells with the JAK3 inhibitor tofacitinib in combination with a spectrum of conventional chemotherapeutics revealed synergy with glucocorticoids, in vitro and in vivo. These findings thus provide key insights into the molecular genetics of mitochondrial apoptosis resistance in childhood T-ALL, and a compelling rationale for a clinical trial of JAK3 inhibitors in combination with glucocorticoids for patients with JAK3-mutant T-ALL.

Punicalagin promotes autophagic degradation of human papillomavirus E6 and E7 proteins in cervical cancer through the ROS-JNK-BCL2 pathway

Punicalagin, which is derived from pomegranate peel, is reported to exert growth-inhibitory effects against various cancers. However, the underlying mechanisms have not been elucidated. Human papillomavirus (HPV), a major oncovirus, utilizes the host autophagic machinery to support its replication. Here, punicalagin markedly downregulated the levels of the major HPV oncoproteins E6 and E7 in cervical cancer cells through the autophagy-lysosome system. Additionally, punicalagin activated the reactive oxygen species (ROS)-JNK pathway and promoted the phosphorylation of BCL2, which led to the dissociation of BCL2 from BECN1 and the induction of autophagy. Treatment with autophagy and JNK inhibitors or ROS scavengers mitigated the punicalagin-induced degradation of E6 and E7. Moreover, the knockout of ATG5 using the clustered regularly interspaced palindrome repeat/Cas 9 system mitigated the punicalagin-induced downregulation of E6/E7. This indicated that punicalagin-induced degradation of E6 and E7 was dependent on autophagy. The results of in vivo studies demonstrated that punicalagin efficiently inhibits cervical cancer growth. In conclusion, this study elucidated a mechanism of punicalagin-induced autophagic degradation of E6 and E7. It will enable the future applications of punicalagin as a therapeutic for HPV-induced cervical cancer.