Inhibitor-induced Apoptosis Research Articles

Inhibition of ER stress-related IRE1α/CREB/NLRP1 pathway promotes the apoptosis of human chronic myelogenous leukemia cell

Endoplasmic reticulum (ER) stress is induced in chronic myelogenous leukemia (CML) cells. As an important sensor of ER stress, inositol-requiring protein-1α (IRE1α) promotes the survival of acute myeloid leukemia. NLRP1 inflammasome activation promotes metastatic melanoma growth and that IRE1α can increase NLRP1 inflammasome gene expression. This study aimed to investigate the role and molecular mechanism of IRE1α in CML cell growth. We found that overexpression of IRE1α or NLRP1 significantly promoted the proliferation and decreased the apoptosis of CML cells, whereas downregulation of these two genes showed the opposite effects. 4-phenylbutyric acid (4-PBA), an ER stress inhibitor, reduced the expression of IRE1α and NLRP1. IRE1α elevated NLRP1 expression via cAMP responsive element binding protein (CREB) phosphorylation. NLRP1 inflammasome was activated in CML cells and its activation partly reversed ER stress inhibitor-induced cell apoptosis. Furthermore, inhibition of IRE1α/NLRP1 pathway sensitized CML cells to imatinib-mediated apoptosis. Additionally, IRE1α expression was elevated and NLRP1 inflammasome was activated in primary cells from CML patients. Downregulation of IRE1α or NLRP1 suppressed the proliferation and elevated the apoptosis of primary CML cells. Collectively, this study demonstrated that the IRE1α/CREB/NLRP1 pathway contributes to the progression of CML and the development of imatinib resistance. Hence, targeting ER stress-related IRE1α expression or NLRP1 inflammasome activation may block CML development.

Abstract 895: Noncanonical c-Met activation mediates de novo and acquired resistance to polo-like kinase 1 inhibitor-induced apoptosis in non-small cell lung cancer

Abstract Introduction: Plk1 is a serine-threonine protein kinase that is overexpressed in cancer cells, and plays a major role in regulating tumor growth. Plk1 inhibitors are well tolerated, but only a few unselected patients with non-small cell lung cancer (NSCLC) respond to single-agent therapy. Our lab discovered that mesenchymal NSCLC cell lines are more sensitive to Plk1 inhibitors than epithelial cell lines in vitro and in vivo. However, mechanisms of resistance to Plk1 inhibitors have not been elucidated and this unknown is a major gap in knowledge. Experimental procedure: To study the mechanisms of Plk1 inhibitor-induced apoptosis we used 3 pairs of isogenic epithelial NSCLC cell lines induced to a mesenchymal phenotype with TGF-β. These isogenic pairs were treated with the Plk1 inhibitor volasertib for 24 h and levels of 301 proteins and phosphoproteins were simultaneously measured using reverse phase protein array (RPPA). Volasertib acquired resistance (VAR) cell lines were generated by exposing cells to increasing doses of volasertib. Results: The induction of a mesenchymal phenotype using TGF-β increased Plk1 inhibition-induced apoptosis in all 3 cell lines. To further elucidate mechanisms of resistance, we compared protein expression in these isogenic cell lines, 24 h after Plk1 inhibition. There were 33 proteins differentially regulated following Plk1 inhibition in parental vs TGF-β induced isogenic cells (p-value < 0.05). Notably, phosphorylated c-Met (Y1234/1235), FAK (Y397) and Src (Y416) were consistently inhibited following Plk1 inhibition in the mesenchymal lines. These changes were confirmed by Western blotting. Total c-Met, FAK and Src protein levels were not affected, implicating a post-translational changes. Likewise, VAR cell lines exhibited an epithelial phenotype and c-Met phosphorylation was persistent even after Plk1 inhibition. Simultaneous c-Met and Plk1 inhibition or silencing increased apoptosis in NSCLC cell lines tested compared to single agent inhibition or silencing. Combination of Plk1 and c-Met inhibitors decreased tumor volume and increased mouse survival in vivo in patient derived and cell line xenograft models. Similarly VAR cells also showed more apoptosis when treated with combination of Plk1 and c-Met inhibitors. Levels of the c-Met ligand HGF were unchanged after Plk1 inhibition and further mechanistic studies are on-going. Conclusion: NSCLC cell lines have diverse sensitivities to Plk1 inhibition, which is consistent with the results of clinical trials of Plk1 inhibitors in solid tumors. This study reveals a novel mechanism of non-canonical c-Met activation in resistant epithelial NSCLC after Plk1 inhibition. We demonstrate a profound effect of combination Plk1 and c-Met inhibition in vivo in multiple mouse models that could be a novel therapy for NSCLC patients. Citation Format: Ratnakar Singh, Pavitra Viswanath, Shaohua Peng, Vaishnavi Sambandam, Li Shen, Lerong Li, Jing Wang, Bingliang Fang, Faye M. Johnson. Noncanonical c-Met activation mediates de novo and acquired resistance to polo-like kinase 1 inhibitor-induced apoptosis in non-small cell lung cancer [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 895.

Abstract 4389: HEXIM1 is a negative regulator of H. pylori-induced BIRC3 enhancer RNA

Abstract The bacterium Helicobacter pylori inhabits the human stomach and is responsible for a number of pathologies, including being strongly associated with the development of gastric adenocarcinoma. H. pylori inhibits apoptosis in gastric cells, but the mechanism is poorly understood. We investigated how H. pylori inhibits apoptosis using cultured G27-strain H. pylori to stimulate cancer cell lines, and apoptosis was induced with Raptinal. We found that H. pylori induces the transcription of the apoptosis-inhibiting protein cIAP2, and this induction is sufficient to inhibit apoptosis in stimulated cells. We found an enhancer region upstream of the gene that codes for cIAP2 (BIRC3). H. pylori induces expression of RNA from this enhancer, and RNAi treatment targeting this eRNA inhibited induction of the BIRC3 mRNA. Chromatin immunoprecipitation revealed that positive transcription elongation factor b (P-TEFb) is recruited to the enhancer following H. pylori stimulation, and RNA polymerase II is activated. We found that the negative regulator of transcription HEXIM1 inhibits P-TEFb recruitment and eRNA production. HEXIM1 also inhibits BIRC3 mRNA transcription, cIAP2 protein induction and H. pylori's anti-apoptotic effect. Together, these findings indicate that H. pylori inhibits apoptosis induction in cultured cells, and this inhibition is mediated by the induction of BIRC3/cIAP2. H. pylori stimulates the production of RNA from the enhancer region of BIRC3, which is required for the induction of the gene, and the eRNA production is regulated by HEXIM1 and P-TEFb. Citation Format: Donald R. Sheppard. HEXIM1 is a negative regulator of H. pylori-induced BIRC3 enhancer RNA [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 4389.

Abstract 5852: FGF2 confers resistance to novel BET inhibitors in metastatic uveal melanoma

Abstract Uveal melanoma (UM) is the most common intraocular malignancy in adults and frequently metastasizes to the liver. Metastatic UM responds poorly to chemotherapy, immunotherapy and MAPK pathway inhibitors; hence, there is an urgent unmet need for new treatment strategies. UM has a low mutational burden and progression is associated with abberrant transcriptional programs. Inhibition of epigenetic modulators involved in transcriptional regulation such as the bromodomain and extraterminal domain (BET) proteins, may be feasible for the treatment of UM. Pre-clinically, JQ1, a first generation BET inhibitor, has been shown to suppress UM growth. Additionally, a multi-site clinical trial is ongoing testing a novel BET inhibitor, PLX51107 (Plexxikon Inc) in metastatic UM patients. Since growth factors in the tumor microenvironment potentially contribute to intrinsic resistance and poor responses to therapies, we determined effects of growth factors on the efficacy of BET inhibitors in metastatic UM cells. We show that FGF2 provides resistance of UM cells to the growth inhibitory effects of BET inhibitors, JQ1, PLX51107 and the third generation inhibitor, PLX72853. FGF2 rescued BET inhibitor-induced apoptosis and cell cycle arrest, which was associated with reversal of the decreased expression of cell cycle regulators (cyclin D1, phospho-RB) and increased expression of pro-apoptotic markers (cleaved PARP, Bim). Importantly, FGF receptor (FGFR) inhibitors reversed FGF2-induced BET inhibitor resistance. As UM predominantly metastasizes to the liver, we investigated FGF2 production by hepatic stellate cells. We show that hepatic stellate cells secrete FGF2 and stellate cell conditioned medium protects metastatic UM cells against the growth inhibitory effects of BET inhibitors. Interestingly, BET inhibitor treatment of UM cells elevated FGFR protein expression and this finding is also shown in UM cell xenograft tumors and in patient tumor samples following PLX51107 treatment. These results indicate that the FGF2/FGFR pathway is activated to promote resistance to BET inhibition via the secretion of FGF2 in the tumor stroma and adaptive upregulation of FGFR expression in UM. In conclusion, our studies suggest that co-targeting of FGFR signaling and BET proteins may be required to maximize the responses of metastatic UM to BET inhibitors. Citation Format: Vivian Chua, Timothy Purwin, Connie Liao, Andrew Aplin. FGF2 confers resistance to novel BET inhibitors in metastatic uveal melanoma [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 5852.

Abstract 2426: Tyrosine kinase and ERAD inhibitors promote oxidative stress-induced apoptosis through activation of ATF4 / KLF9 axis in medullary thyroid cancer

Abstract Medullary thyroid carcinoma (MTC) is a neuroendocrine malignancy, caused by activating mutations of the RET proto-oncogene, with frequent lymph node and distant metastasis at diagnosis. Tyrosine kinase inhibitors (TKIs) that target the RET have proven effective for treatment of MTC, but resistance to TKIs evolves in half of the treated patients. We have previously shown that the expression levels of Activating Transcription Factor 4 (ATF4) which is a stress-induced transcription factor, significantly decreased or lost in half of the MTC tumors. We also demonstrated that forced expression of ATF4 or TKIs-induced level of ATF4 decreases survival of MTC cells by promoting degradation of RET, blocking the activation of RET downstream signaling pathways and subsequently induces apoptosis. More importantly, ATF4 knockdown by shRNA decreased the sensitivity to tyrosine kinase inhibitor-induced apoptosis. Further thyroid gland of Atf4-knockout mice showed C-cell hyperplasia, a precancerous lesion for MTC suggesting a potential tumor suppressor role of ATF4 in MTC. Stress-induced by the accumulation of unfolded proteins in the endoplasmic reticulum (ER) is a feature of specialized secretory cells, including MTC cells that secrete calcitonin. ATF4 promotes the induction of apoptosis under persistent stress conditions, although the mechanism is not clearly understood. We hypothesize that the combination of ATF4 inducer and TKIs causes an excessive cellular oxidative stress resulting in an activation of apoptosis, all of which may prevent resistance to TKIs. Here, we show that the ER-associated protein degradation (ERAD) inhibitor, eeyarestatin sensitizes MTC cells to the TKIs including sunitinib and vandetanib. The combination of eeyarestatin and TKIs causes a synergistic induction of ATF4 expression and its target genes, an accumulation of reactive oxygen species and subsequent cell death. Chromatin immunoprecipitation followed by sequencing assay (ChIP-seq) in MTC cells treated with eeyarestatin and immunoprecipitated with ATF4 and acetylated lysine 9 of histone 3, identified transcription Kruppel-like factor 9 (KLF9) gene as a transcriptional target of ATF4 activation. KLF9 plays a functional role in oxidative stress-induced cell death. Treatment with eeyarestatin and vandetanib alone or in combination increases the occupancy of ATF4 at the promoter of KLF9 gene and stimulates the expression of KLF9. Depletion of ATF4 by shRNA leads to downregulation of KLF9 expression and prevents oxidative stress-induced cell death. These findings suggest that induction of the ATF4/KLF9 axis causes oxidative stress leading to excessive ER stress, and subsequent cell death. Thus, combining TKIs and ERAD inhibitors that promote ATF4 levels could be effective therapeutic strategies for treating MTC and preventing resistance to TKIs. Citation Format: Rozita Bagheri-Yarmand, Krishna M. Sinha, Ling Li, Yue Lu, Robert F. Gagel. Tyrosine kinase and ERAD inhibitors promote oxidative stress-induced apoptosis through activation of ATF4 / KLF9 axis in medullary thyroid cancer [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 2426.

Cytoprotective and pro-angiogenic functions of thrombomodulin are preserved in the C loop of the fifth epidermal growth factor-like domain.

We previously found that the fifth epidermal growth factor-like domain of thrombomodulin (TME5) exerts cytoprotective and pro-angiogenic functions via G-protein coupled receptor 15 (GPR15). TME5 is comprised of three S-S bonds that divide it into three loops: A (TME5A), B (TME5B), and C (TME5C). Herein we identified the minimum structure of TME5 that produces favorable effects in vascular endothelial cells (ECs). We found that TME5C, composed of 19 amino acids, but not TME5A or TME5B, stimulated the proliferation of human umbilical vein endothelial cells (HUVECs) and human hepatic sinusoidal endothelial cells (HHSECs). Matrigel plug assays showed that TME5C stimulates in vivo angiogenesis. In addition, TME5C counteracted calcineurin inhibitor-induced apoptosis and vascular permeability in HUVECs and HHSECs. Western blot analysis indicated that exposure of either HUVECs or HHSECs to TME5C increased the levels of anti-apoptotic myeloid cell leukemia-1 protein in association with the activation of signal transduction pathways, including extracellular signal-regulated kinase, AKT, and mitogen-activated protein kinase p38. Importantly, TME5C did not affect the coagulation pathway in vitro. The cytoprotective function of TME5C was mediated by cell surface-expressed GPR15, as TME5C was not able to protect vascular ECs isolated from Gpr15 knock-out (KO) mice. Strikingly, TME5C successfully ameliorated sinusoidal obstruction syndrome in a murine model by counteracting the reduction of sinusoidal EC numbers. Taken together, the cytoprotective and pro-angiogenetic functions of TM are preserved in TME5C. The use of TME5C may be a promising treatment strategy to prevent or treat lethal complications, such as sinusoidal obstruction syndrome, whose pathogenesis is based on endothelial insults.

BCL-2 selective inhibitor ABT-199 primes rhabdomyosarcoma cells to histone deacetylase inhibitor-induced apoptosis.

BH3 mimetics are emerging novel anticancer therapeutics that potently and specifically inhibit antiapoptotic BCL-2 proteins and thereby induce cell death in many cancer entities. Previously, we demonstrated that JNJ-26481585 (JNJ), a second-generation histone deacetylase inhibitor (HDACI), engages mitochondrial apoptosis via upregulation of several BH3-only proteins. In the present study, we describe synergistic interactions of JNJ with BH3 mimetics (i.e. ABT-737, ABT-199) in rhabdomyosarcoma (RMS) cells. Importantly, JNJ synergizes with ABT-199 to trigger apoptosis in primary-derived RMS cells isolated from tumor samples, underlining the translational importance of combining these compounds and their potential to improve cancer therapy. Importantly, JNJ/ABT-199 cotreatment also significantly inhibits long-term survival of RMS cells. Mechanistically, JNJ increases expression levels of the BH3-only protein BIM, while exposure to ABT-199 displaces BIM from BCL-2 and shuttles BIM to MCL-1, which also constitutively sequesters NOXA. Both BIM and NOXA contribute to JNJ/ABT-199-mediated cell death, as individual knockdown of NOXA or BIM significantly prevents cell death. Further, JNJ and ABT-199 act in concert to activate BAK and BAX, resulting in loss of the mitochondrial membrane potential (MMP) and caspase activation. These events are required for JNJ/ABT-199-mediated apoptosis, since BAK or BAX silencing or inhibition of caspases significantly protects from JNJ/ABT-199-induced cell death. Rescue experiments demonstrate that overexpression of MCL-1, but not overexpression of BCL-2, blocks JNJ/ABT-199-induced apoptosis. In conclusion, this study provides the first demonstration of ABT-199-induced priming, which sensitizes RMS cells to HDACI, such as JNJ, by engaging mitochondrial apoptosis, highlighting that BH3 mimetics show great promise for the treatment of RMS.

MiR-21 regulates the apoptosis of keloid fibroblasts by caspase-8 and the mitochondria-mediated apoptotic signaling pathway via targeting FasL.

MicroRNA-21 (miR-21) has been found to be upregulated in keloid tissue and to affect the proliferation and apoptosis of keloid fibroblasts; however, the possible mechanisms remain unclear. In this study, we aimed to evaluate the role of miR-21 in FasL-induced caspase-8 activation and the mitochondria-mediated apoptotic signaling pathway in keloid fibroblasts. Our study found that the protein level of FasL was decreased by miR-21 over-expression, while being enhanced by miR-21 inhibition in keloid fibroblasts. Subsequently, the mitochondria-mediated apoptosis of keloid fibroblasts was restrained by miR-21 over-expression, as evidenced by enhanced mitochondrial membrane potential and decreased production of mitochondrial ROS. Moreover, over-expression of miR-21 inhibited the activation of the caspase-8 and the mitochondria-mediated apoptotic signaling pathway. As expected, inhibition of miR-21 had the opposite effects. Finally, silencing of FasL suppressed miR-21 inhibition-induced apoptosis by inactivation of caspase-8 and the mitochondria-mediated apoptotic signaling pathway, which was comparable to Z-IETD-FMK, a caspase-8 inhibitor. Taken together, these results suggest that miR-21 regulates the apoptosis of keloid fibroblasts via targeting FasL, and caspase-8 and the mitochondria-mediated apoptotic signaling pathway is involved in this process. Our findings provide evidence that miR-21 may be considered to be a therapeutic target for keloids.

A variant of death-receptor 3 associated with rheumatoid arthritis interferes with apoptosis-induction of T cell

Rheumatoid arthritis (RA) is a chronic polyarthritis of unknown etiology. To unravel the molecular mechanisms in RA, we performed targeted DNA sequencing analysis of patients with RA. This analysis identified a variant of the death receptor 3 (DR3) gene, a member of the family of apoptosis-inducing Fas genes, which contains four single-nucleotide polymorphisms (SNPs) and a 14-nucleotide deletion within exon 5 and intron 5. We found that the deletion causes the binding of splicing regulatory proteins to DR3 pre-mRNA intron 5, resulting in a portion of intron 5 becoming part of the coding sequence, thereby generating a premature stop codon. We also found that this truncated DR3 protein product lacks the death domain and forms a heterotrimer complex with wildtype DR3 that dominant-negatively inhibits ligand-induced apoptosis in lymphocytes. Myelocytes from transgenic mice expressing the human DR3 variant produced soluble truncated DR3, forming a complex with TNF-like ligand 1A (TL1A), which inhibited apoptosis induction. In summary, our results reveal that a DR3 splice variant that interferes with ligand-induced T cell responses and apoptosis may contribute to RA pathogenesis.

Glycogen synthase kinase-3\u03b2 inhibition promotes lysosome-dependent degradation of c-FLIPL in hepatocellular carcinoma

Glycogen synthase kinase-3β (GSK-3β) is a ubiquitously expressed serine/threonine kinase involved in a variety of functions ranging from the control of glycogen metabolism to transcriptional regulation. We recently demonstrated that GSK-3β inhibition triggered ASK1-JNK-dependent apoptosis in human hepatocellular carcinoma (HCC) cells. However, the comprehensive picture of downstream GSK-3β-regulated pathways/functions remains elusive. In this study, we showed that GSK-3β was aberrantly activated in HCC. Pharmacological inhibition and genetic depletion of GSK-3β suppressed the growth and induced caspase-dependent apoptosis in HCC cells. In addition, GSK-3β inhibition-induced apoptosis through downregulation of c-FLIPL in HCC, which was caused by biogenesis of functional lysosomes and subsequently c-FLIPL translocated to lysosome for degradation. This induction of the lysosome-dependent c-FLIPL degradation was associated with nuclear translocation of transcription factor EB (TFEB), a master regulator of lysosomal biogenesis. Moreover, GSK-3β inhibition-induced TFEB translocation acts through activation of AMPK and subsequently suppression of mTOR activity. Thus our findings reveal a novel mechanism by which inhibition of GSK-3β promotes lysosome-dependent degradation of c-FLIPL. Our study shows that GSK-3β may become a promising therapeutic target for HCC.

Interferon regulatory factor 8 regulates caspase-1 expression to facilitate Epstein-Barr virus reactivation in response to B cell receptor stimulation and chemical induction.

Interferon regulatory factor 8 (IRF8), also known as interferon consensus sequence-binding protein (ICSBP), is a transcription factor of the IRF family. IRF8 plays a key role in normal B cell differentiation, a cellular process that is intrinsically associated with Epstein-Barr virus (EBV) reactivation. However, whether IRF8 regulates EBV lytic replication remains unknown. In this study, we utilized a CRISPR/Cas9 genomic editing approach to deplete IRF8 and found that IRF8 depletion dramatically inhibits the reactivation of EBV upon lytic induction. We demonstrated that IRF8 depletion suppresses the expression of a group of genes involved in apoptosis and thus inhibits apoptosis induction upon lytic induction by B cell receptor (BCR) stimulation or chemical induction. The protein levels of caspase-1, caspase-3 and caspase-8 all dramatically decreased in IRF8-depleted cells, which led to reduced caspase activation and the stabilization of KAP1, PAX5 and DNMT3A upon BCR stimulation. Interestingly, caspase inhibition blocked the degradation of KAP1, PAX5 and DNMT3A, suppressed EBV lytic gene expression and viral DNA replication upon lytic induction, suggesting that the reduced caspase expression in IRF8-depleted cells contributes to the suppression of EBV lytic replication. We further demonstrated that IRF8 directly regulates CASP1 (caspase-1) gene expression through targeting its gene promoter and knockdown of caspase-1 abrogates EBV reactivation upon lytic induction, partially through the stabilization of KAP1. Together our study suggested that, by modulating the activation of caspases and the subsequent cleavage of KAP1 upon lytic induction, IRF8 plays a critical role in EBV lytic reactivation.

Abstract A34: Inhibition of autophagy sensitizes gastrointestinal stromal tumor cells to TKI/Bcl-2 inhibitors-induced apoptosis

Abstract Background: Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumor of the GI tract. Most GISTs are driven by mutations in KIT or platelet-derived growth factor receptor-α (PDGFRA), which responds well to imatinib, a tyrosine kinase inhibitor (TKI) that blocks KIT and PDGFR-α signaling. Bcl-2 family plays a critical role in the regulation of cell apoptosis in GISTs. ABT-737 as an inhibitor of Bcl-2/Bcl-xL can result in a time- and dose-dependent activation of apoptosis. Autophagy is a key mechanism to promote tumor cells survival, inhibition of which can induce the cell death in GISTs. Chloroquine, an antimalarial drug, has been also identified as an autophagy inhibitor. In this study, we assessed the combinational effects of imatinib, ABT-737, and chloroquine in GIST cells. Methods: Human GIST cell lines, GIST-T1 and GIST-882, were employed in our study. Cells were treated with imatinib, ABT-737, and chloroquine either separately or in different combinations. Cell viability was tested by means of MTS and synergistic effects were analyzed by isobologram software. The levels of related proteins of apoptosis (PARP, Caspase-3) and autophagy (LC3-II, beclin-1) were measured by Western blot. Cell apoptosis and cell cycle were tested by flow cytometry. Results: Cell viability assay indicated that the cell survival percentage of double- or triple-drug combinations dramatically decreased (less than 5%) compared to that of single-drug treatment (42%, 36%, or 12%) (P<0.05). Isobologram analysis revealed a strongly synergistic drug interaction, with combination indices (CI) < 0.5 in most of double and triple combinations. Importantly, triple-drugs combination showed even more strongly synergistic effects compared to double-drugs combinations (CI =0.204 vs. 0.309 or 0.356, P<0.05). Cell apoptosis assay showed that the cell apoptosis percentage of double- (32.9% or 36.6%) or triple-drugs combinations (66.5%) also significantly increased over that of single-drug treatments (6.1%, 6.1%, or 13.1%) (P<0.05). Finally, Western blot discovered that double- or triple-drugs combinations increased the cleavage of PARP as well as expression of Caspase-3, but inhibited autophagy in comparison to single-drug treatments. Conclusions: The combination of imatinib, ABT-737, and chloroquine has collaborative effects on the treatment of GISTs in vitro. The combined strategy may enhance the clinical efficacy, which provides a rationale for the clinical evaluation of these drug combinations in GISTs treatment. Citation Format: Shuchao Zhang, Guozhi Hu, Ana cristina Paz-mejia, Luyuan Li, Jonathan C. Trent. Inhibition of autophagy sensitizes gastrointestinal stromal tumor cells to TKI/Bcl-2 inhibitors-induced apoptosis [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr A34.

Abstract A13: Genome-wide transcriptional analysis of HDAC inhibition-induced apoptosis in synovial sarcoma

Abstract Introduction: Conventional cytotoxic therapies for synovial sarcoma provide limited benefit, and no drugs specifically targeting its driving SS18-SSX fusion oncoprotein are currently available. HDAC inhibition has been shown in previous studies to disrupt the driving complex implicated in synovial sarcoma, resulting in apoptosis induction. Methods: Transcriptome analysis was undertaken in a panel of six human synovial sarcoma cell lines in order to uncover potential mechanisms of cell death following HDAC inhibition. By comparison studies with five additional publicly available related expression datasets, common class effects resulting from HDAC inhibition were investigated. Five human synovial sarcoma tumor samples were profiled by RNA-seq for comparison. A mouse model of synovial sarcoma was treated by HDAC inhibition and tumors were profiled for apoptotic markers. Results: Cell cycle arrest, differentiation, and response to oxygen-containing species and cell death were common biologic responses among the panel of post-HDAC inhibitor expression studies. Specific to synovial sarcoma, reactivation of repressed tumor suppressor CDKN2A and induction of proapoptotic transcriptional patterns results in apoptosis and decreased tumor burden in vivo. Conclusion: HDAC inhibition impedes SS18-SSX-mediated transcriptional deregulation, allowing for reactivation of normal cell cycle regulatory and apoptotic pathways. This study provides mechanistic support for a particular susceptibility of synovial sarcoma to HDAC inhibition as a means of potential clinical treatment. Citation Format: Aimee N. Laporte, Neal M. Poulin, Alireza Lorzadeh, Xiu Qing Wang, Ryan Vander werff, Jared J. Barrott, Michelle Moska, Christopher Hughes, Gregg Morin, Kevin B. Jones, Martin Hirst, T. Michael Underhill, Torsten O. Nielsen. Genome-wide transcriptional analysis of HDAC inhibition-induced apoptosis in synovial sarcoma [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr A13.

Autophagy promotes escape from phosphatidylinositol 3-kinase inhibition in estrogen receptor-positive breast cancer.

Hyperactivation of the PI3K pathway has been implicated in resistance to antiestrogen therapies in estrogen receptor α (ER)-positive breast cancer, prompting the development of therapeutic strategies to inhibit this pathway. Autophagy has tumor-promoting and -suppressing roles and has been broadly implicated in resistance to anticancer therapies, including antiestrogens. Chloroquine (CQ) is an antimalarial and amebicidal drug that inhibits autophagy in mammalian cells and human tumors. Herein, we observed that CQ inhibited proliferation and autophagy in ER+ breast cancer cells. PI3K inhibition with GDC-0941 (pictilisib) induced autophagy. Inhibition of autophagy using CQ or RNA interference potentiated PI3K inhibitor-induced apoptosis. Combined inhibition of PI3K and autophagy effectively induced mitochondrial membrane depolarization, which required the BH3-only proapoptotic proteins Bim and PUMA. Treatment with GDC-0941, CQ, or the combination, significantly suppressed the growth of ER+ breast cancer xenografts in mice. In an antiestrogen-resistant xenograft model, GDC-0941 synergized with CQ to provide partial, but durable, tumor regression. These findings warrant clinical evaluation of therapeutic strategies to target ER, PI3K, and autophagy for the treatment of ER+ breast cancer.-Yang, W., Hosford, S. R., Traphagen, N. A., Shee, K., Demidenko, E., Liu, S., Miller, T. W. Autophagy promotes escape from phosphatidylinositol 3-kinase inhibition in estrogen receptor-positive breast cancer.

Defibrotide Stimulates Angiogenesis and Protects Endothelial Cells from Calcineurin Inhibitor-Induced Apoptosis via Upregulation of AKT/Bcl-xL.

Sinusoidal obstruction syndrome is a life-threatening complication that can occur after haematopoietic stem cell transplantation. Defibrotide (DF) has been approved for the treatment of individuals with severe sinusoidal obstruction syndrome following haematopoietic stem cell transplantation in the European Union and the United States. However, the precise mechanisms by which DF protects endothelial cells remain to be elucidated. In this study, we found that DF stimulated angiogenesis in vitro and in vivo as assessed by vascular tube formation, scratch-wound repair and Matrigel plug assays. These effects were associated with an activation of pro-survival signalling pathways, including AKT (protein kinase B), ERK (extracellular signal-regulated kinases) and p38. More importantly, DF alleviated calcineurin inhibitor-induced growth inhibition and apoptosis of human umbilical vein endothelial cells and human hepatic sinusoidal endothelial cells in parallel with upregulation of anti-apoptotic protein B-cell lymphoma-extra-large (Bcl-xL), which was mediated by AKT (protein kinase B). Notably, these effects were abrogated when Bcl-xL was depleted by small interfering RNA (ribonucleic acid). In addition, DF counteracted calcineurin inhibitor-induced activation of nuclear factor-κB and Janus kinase 2 (JAK2)/Signal Transducer and Activator of Transcription 3 (STAT3) signalling and production of cytokines in vascular endothelial cell-derived EA.hy926 cells. Taken together, DF has pro-angiogenic, anti-apoptotic and anti-inflammatory effects on endothelial cells. DF is a potentially useful agent to prevent the development of, and treat individuals with, endothelial cell injury-related complications after haematopoietic stem cell transplantation.

Proteasome stress sensitizes malignant pleural mesothelioma cells to bortezomib-induced apoptosis

Based on promising results in preclinical models, clinical trials have been performed to evaluate the efficacy of the first-in-class proteasome inhibitor bortezomib towards malignant pleural mesothelioma (MPM), an aggressive cancer arising from the mesothelium of the serous cavities following exposure to asbestos. Unexpectedly, only minimal therapeutic benefits were observed, thus implicating that MPM harbors inherent resistance mechanisms. Identifying the molecular bases of this primary resistance is crucial to develop novel pharmacologic strategies aimed at increasing the vulnerability of MPM to bortezomib. Therefore, we assessed a panel of four human MPM lines with different sensitivity to bortezomib, for functional proteasome activity and levels of free and polymerized ubiquitin. We found that highly sensitive MPM lines display lower proteasome activity than more bortezomib-resistant clones, suggesting that reduced proteasomal capacity might contribute to the intrinsic susceptibility of mesothelioma cells to proteasome inhibitors-induced apoptosis. Moreover, MPM equipped with fewer active proteasomes accumulated polyubiquitinated proteins, at the expense of free ubiquitin, a condition known as proteasome stress, which lowers the cellular apoptotic threshold and sensitizes mesothelioma cells to bortezomib-induced toxicity as shown herein. Taken together, our data suggest that an unfavorable load-versus-capacity balance represents a critical determinant of primary apoptotic sensitivity to bortezomib in MPM.

Microenvironmental stromal cells abrogate NF-κB inhibitor-induced apoptosis in chronic lymphocytic leukemia.

Nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) is known to play an important role in the pathogenesis of chronic lymphocytic leukemia (CLL). Several NF-κB inhibitors were shown to successfully induce apoptosis of CLL cells in vitro. Since the microenvironment is known to be crucial for the survival of CLL cells, herein, we tested whether NF-κB inhibition may still induce apoptosis in these leukemic cells in the presence of protective stromal interaction. We used the specific NF-κB inhibitor dehydroxymethylepoxyquinomicin (DHMEQ). Microenvironmental support was mimicked by co-culturing CLL cells with bone marrow-derived stromal cell lines (HS-5 and M2-10B4). NF-κB inhibition by DHMEQ in CLL cells could be confirmed in both the monoculture and co-culture setting. In line with previous reports, NF-κB inhibition induced apoptosis in the monoculture setting by activating the intrinsic apoptotic pathway resulting in poly (ADP-ribose) polymerase (PARP)-cleavage; however, it was unable to induce apoptosis in leukemic cells co-cultured with stromal cells. Similarly, small interfering ribonucleic acid (siRNA)-mediated RELA downregulation induced apoptosis of CLL cells cultured alone, but not in the presence of supportive stromal cells. B-cell activating factor (BAFF) was identified as a microenvironmental messenger potentially protecting the leukemic cells from NF-κB inhibition-induced apoptosis. Finally, we show improved sensitivity of stroma-supported CLL cells to NF-κB inhibition when combining the NF-κB inhibitor with the SYK inhibitor R406 or the Bruton’s tyrosine kinase (BTK) inhibitor ibrutinib, agents known to inhibit the stroma-leukemia crosstalk. We conclude that NF-κB inhibitors are not promising as monotherapies in CLL, but may represent attractive therapeutic partners for ibrutinib and R406.

Non-thermal near-infrared exposure photobiomodulates cellular responses to ionizing radiation in human full thickness skin models

Ionizing and near-infrared radiation are both part of the therapeutic spectrum in cancer treatment. During cancer therapy ionizing radiation is typically used for non-invasive reduction of malignant tissue, while near-infrared photobiomodulation is utilized in palliative medical approaches, e.g. for pain reduction or impairment of wound healing. Furthermore, near-infrared is part of the solar wavelength spectrum. A combined exposure of these two irradiation qualities – either intentionally during medical treatment or unintentionally due to solar exposure – is therefore presumable for cancer patients. Several studies in different model organisms and cell cultures show a strong impact of near-infrared pretreatment on ionizing radiation-induced stress response. To investigate the risks of non-thermal near-infrared (NIR) pretreatment in patients, a human in vitro full thickness skin models (FTSM) was evaluated for radiation research. FTSM were pretreated with therapy-relevant doses of NIR followed by X-radiation, and then examined for DNA-double-strand break (DSB) repair, cell proliferation and apoptosis. Double-treated FTSM revealed a clear influence of NIR on X-radiation-induced stress responses in cells in their typical tissue environment. Furthermore, over a 24h time period, double-treated FTSM presented a significant persistence of DSBs, as compared to samples exclusively irradiated by X-rays. In addition, NIR pretreatment inhibited apoptosis induction of integrated fibroblasts, and counteracted the radiation-induced proliferation inhibition of basal keratinocytes. Our work suggests that cancer patients treated with X-rays should be prevented from uncontrolled NIR irradiation. On the other hand, controlled double-treatment could provide an alternative therapy approach, exposing the patient to less radiation.

Mitochondrial dysfunction is responsible for fatty acid synthase inhibition-induced apoptosis in breast cancer cells by PdpaMn

Targeting cellular metabolism is becoming a hallmark to overcome drug resistance in breast cancer treatment. Activation of fatty acid synthase (FASN) has been shown to promote breast cancer cell growth. However, there is no concrete report underlying the mechanism associated with mitochondrial dysfunction in relation to fatty acid synthase inhibition-induced apoptosis in breast cancer cells. The current study is aimed at exploring the effect of the novel manganese (Mn) complex, labeled as PdpaMn, on lipid metabolism and mitochondrial function in breast cancer cells. Herein, we observed that PdpaMn displayed strong cytotoxicity on breast cancer cell lines and selectively targeted the tumor without affecting the normal organs or cells in vivo. We also observed that PdpaMn could bind to TE domain of FASN and decrease the activity and the level of expression of FASN, which is an indication that FASN could serve as a target of PdpaMn. In addition, we demonstrated that PdpaMn increased intrinsic apoptosis in breast cancer cells relayed by a suppressed the level of expression of FASN, followed by the release of mitochondrial cytochrome c and the activation of caspases-9. Instigated by the above observations, we hypothesized that PdpaMn-induced apoptosis events are dependent on mitochondrial dysfunction. Indeed, we found that mitochondrial membrane potential (MMP) collapse, mitochondrial oxygen consumption reduction and adenosine triphosphate (ATP) release were deeply repressed. Furthermore, our results showed that PdpaMn significantly increased the reactive oxygen species (ROS) production, and the protection conferred by the free radical scavenger N-acetyl-cysteine (NAC) indicates that PdpaMn-induced apoptosis through an oxidative stress-associated mechanism. More so, the above results have demonstrated that mitochondrial dysfunction participated in FASN inhibition-induce apoptosis in breast cancer cells by PdpaMn. Therefore, PdpaMn may be considered as a good candidate for anti-breast cancer therapeutic option.

SGK1 inhibition induces autophagy-dependent apoptosis via the mTOR-Foxo3a pathway

Background:Although inhibition of SGK1 has been shown to delay cancer progression, the underlying mechanisms have not yet been elucidated.Methods:We investigated the cellular responses to GSK650394 treatment and SGK1 silencing (or overexpression) in human prostate cancer (PCa) cell lines and PC3 xenografts by flow cytometry, western blotting, immunofluorescence, transmission electron microscopy and immunohistochemistry.Results:In the present study, we demonstrated that SGK1 inhibition, mediated by either GSK650394 or SGK1 shRNA, induced G2/M arrest, apoptosis and autophagy. Furthermore, 3MA-mediated autophagy inhibition attenuated SGK1 inhibition-induced apoptosis, suggesting that induction of autophagy precedes apoptosis. Moreover, ectopic expression of SGK1 significantly attenuated the GSK650394-induced effects. Suppression of mTOR and Foxo3a phosphorylation is critical for blockade of SGK1-induced autophagy and apoptosis, at least partially via pFoxo3a (S253)-LC3 and pFoxo3a (S253)-p27 interactions. Dual inhibition of mTOR and SGK1 enhances autophagy activation and leads to synergistic cytocidal effects in PCa cells.Conclusions:In summary, our findings show that SGK1 inhibition exhibits significant antitumour effects against PCa in vitro and in vivo. This study uncovered a novel mechanism of SGK1 inhibition in PCa, which is mediated, at least in part, by inducing autophagy-dependent apoptosis via the mTOR-Foxo3a pathway.