Harakiri Research Articles

ER stress-induced LINC00173 promotes the apoptosis of ovarian granulosa cells by regulating the HRK/PI3K/AKT pathway in polycystic ovary syndrome

Polycystic ovary syndrome (PCOS) is a prevalent endocrine disorder and metabolic abnormality disease that mainly affects women of reproductive age. LINC00173, a novel long noncoding RNA (lncRNA), has emerged as an important factor in the development of PCOS. However, the role of LINC00173 in PCOS development and its specific upstream and downstream mechanisms remain to be further clarified. Here, we found that LINC00173 was significantly upregulated in granulosa cells (GCs) of PCOS patients, and played a crucial role in promoting apoptosis of GCs. Mechanistically, we observed the activation of endoplasmic reticulum (ER) stress in the GCs of PCOS patients, and the ER stress sensor ATF4 could directly induce LINC00173 expression by binding to its promoter. LINC00173 further upregulated the expression of Harakiri (HRK) and subsequently inhibited downstream PI3K/AKT pathway. In conclusions, our study uncovered that ER stress-induced upregulation of LINC00173 leads to increased HRK expression and inhibition of the PI3K/AKT pathway, thereby promoting the progression of PCOS. These findings provide a new therapeutic strategy for the treatment of PCOS.

Abstract 3552: YAP-TEAD2 binding mediates therapy resistance in RAS-driven neuroblastoma

Abstract Background/Objectives: Despite intensive multimodal therapy, greater than 50% of children with high-risk neuroblastoma (HR NB) relapse with incurable disease. Next generation sequencing of primary HR NB tumors identified an increase in activating mutations in the RAS/RAF/MAPK pathway. Moreover, gene set enrichment analyses showed a significant decrease in expression of genes suppressed by the Yes-Associated Protein (YAP) at relapse, suggesting increased YAP transcriptional repression. YAP binds with TEAD family transcription factors to regulate gene expression. We have shown that YAP promotes chemotherapy and MEK inhibitor resistance in RAS-mutated NB tumors in vivo by suppressing the expression of Harakiri (HRK), a BH3-only pro-apoptotic protein activated in response to tumor environmental stress such as serum starvation. Our overall objective is to elucidate how YAP represses HRK and tumor suppressor genes globally, and to enhance MEK inhibitor potency by combining MEK inhibition with agents that inhibit YAP or induce HRK to restore the tumor environmental stress response and apoptosis in RAS-mutated NB. Design/Methods: We used publicly available databases to identify TEAD binding sites on the HRK gene locus in NB. To assess the global state of methylation, we treated NB cells, SK-N-AS (NRASQ61K mutation, MYCN non-amplified) and NLF (NF1 deletion, MYCN amplified), with demethylating agent azacitidine and evaluated HRK expression. To identify the specific TEAD (1-4) binding partner to YAP, we performed siRNA and co-immunoprecipitation studies. We further tested novel YAP-TEAD small molecule inhibitors with varying TEAD1-4 inhibition specificity in SK-N-AS and NLF cells in vitro. Results: We observed that TEAD binds near cis-regulatory regions on the HRK gene locus in NB. We found that HRK expression is restored when SK-N-AS and NLF cells are treated with azacitidine despite YAP expression increasing. We also identified TEAD2 as the specific binding partner to YAP in NB and found that TEAD2 is necessary for HRK regulation. Novel YAP-TEAD small molecule inhibitors affect NB cell viability under serum-deprived conditions in vitro, especially the inhibitor with highest specificity against TEAD2, and affect YAP-TEAD downstream targets. Conclusions: YAP-TEAD2 binding is essential for HRK regulation in RAS-mutated NB and thus is a logical therapeutic target to restore therapy response. Further studies are ongoing to test YAP-TEAD small molecule inhibitors in combination with MEK inhibitors and in vivo. Citation Format: Jenny Shim, Andrew Ho, Hunter C. Jonus, Adeiye A. Pilgrim, Benjamin G. Barwick, Tracy T. Tang, Lawrence H. Boise, Kelly C. Goldsmith. YAP-TEAD2 binding mediates therapy resistance in RAS-driven neuroblastoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3552.

Rational design of Harakiri (HRK)-derived constrained peptides as BCL-xL inhibitors.

Using the HRK BH3 domain, sequence hybridization and in silico methods we show dibromomaleimide staple scanning can be used to inform the design of BCL-xL selective peptidomimetic ligands. These HRK-inspired reagents may serve as starting points for the discovery of therapeutics to target BCL-xL-overexpressed cancers.

The gallium complex KP46 sensitizes resistant leukemia cells and overcomes Bcl-2-induced multidrug resistance in lymphoma cells via upregulation of Harakiri and downregulation of XIAP in vitro

Tris-(8-quinolinolato)gallium(III) (KP46, AP-002) is an orally administered investigational anticancer and bone-protective drug currently being evaluated in patients with advanced solid tumors with bone involvement. Despite the clinical efficacy of other gallium compounds in non-Hodgkin’s lymphoma, effects of KP46 in hematological tumor settings have not been studied systematically before. We report here intriguing activities in various human cell lines, including such with multidrug resistance (MDR): In Nalm-6 lymphoblastic leukemia cell sublines, KP46 was capable of overcoming P-gp-related as well as P-gp-unrelated MDR. Apoptosis induction by KP46 was unaffected by bcl2-mediated vincristine-induced MDR in a BJAB lymphoma cell subline and even enhanced in a K562 leukemia subline with daunorubicin-induced MDR, which could be re-sensitized to daunorubicin by KP46. As the latter resistance is associated with lowered Harakiri (HRK) protein levels, a modulating effect of KP46 on HRK expression is suggested. This is consistent with the significant high upregulation of HRK on RNA and protein levels observed in KP46-treated parental BJAB cells according to qPCR and Western blot analysis, respectively. Furthermore, KP46 significantly reduces the protein level of X-linked inhibitor of apoptosis (XIAP) in BJAB cells, the most potent known inhibitor of apoptosis. Overall, these results indicate both a higher potential of HRK and XIAP as cellular targets for cancer therapy and a broader therapeutic potential of KP46 than hitherto envisaged.

P332: MIR-625-5P IS NOVEL CANDIDATE ONCOMIR IN T-CELL ACUTE LYMPHOBLASTIC LEUKEMIA IMPLICATED IN REGULATION OF APOPTOSIS VIA REPRESSION OF HARAKIRI

Background: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive subtype of ALL, arising from T-cell precursors. miRNAs are non-coding RNAs, contributing to leukemogenesis by involvement in key cellular functions such as proliferation, apoptosis, and signaling. We previously found in miRNA transcriptome analysis that miR-625-5p is overexpressed in T-ALL patients, thus is a potential oncomiR in T-ALL. Here, we confirm the implication of miR-625-5p in the regulation of apoptosis in T-ALL cells in vitro. We show that it acts via repression of HARAKIRI (HRK). HRK was originally identified as a proapoptotic gene induced by reduced levels of cytokine in hematopoietic cells and repressed by the expression of death-repressor proteins. Although its proapoptotic function is well described, little is known about the post-transcriptional mechanisms that may participate in HRK inactivation. Aims: The aim of this study was to investigate the mechanism of miR-625-5p oncogenic action via unraveling its targetome and functional effects in T-ALL in vitro. Methods: T-ALL JURKAT cells (with high endogenous miR-625-5p level) were transduced for stable inhibition of this miRNA. To evaluate the influence of miR inhibition on growth of T-ALL cells, we used flow cytometry GFP competition assay and CCK8 proliferation assay. To identify the genes mediating the effect of miR-625-5p, we subjected JURKAT cells to Ago2-RIP-seq. We used magnetic beads coated with anti-AGO2 antibody to immunoprecipitate RNA bound to RISC complexes. Ago2-IP and total RNA fractions were sequenced (polyA RNA-seq, 150 bp reads, 60M PE reads/sample, Illumina NovaSeq 6000). Transcripts depleted in Ago2-IP fraction upon miR inhibition were screened for the presence of miR-625-5p binding sites. Luciferase assay was performed to confirm direct interaction between hsa-miR-625-5p and 3’UTR of HRK gene. The changes of HRK protein level upon miR-625-5p inhibition were evaluated via Western Blot. To estimate the apoptosis rate in T-ALL cells we used two flow cytometry assays based on Annexin V and Caspase 3/7 staining. Results: Inhibition of miR-625-5p decreased the growth of JURKAT cells. In RIP-seq analysis we identified 384 transcripts depleted in RISC by at least 20% upon miR-625-5p inhibition and having at least one putative 3’UTR binding site for this miRNA. Overrepresentation analysis performed for these genes indicated their involvement in the apoptotic process. Among transcripts depleted in RIP-seq with predicted binding sites for miR-625-5p, we found HRK gene, previously reported to have proapoptotic activity. We postulate that repression of this gene by overexpressed miR-625-5p contributes to decreased apoptosis and to growth advantage of T-ALL cells. To further confirm that miR-625-5p affects growth of T-ALL cells via negative regulation of apoptosis, we performed apoptotic assays in JURKAT cells and showed that inhibition of this miRNA increased apoptosis rate, in line with our hypothesis on the oncogenic potential of miR-625-5p. We confirmed the direct interaction between miR-625-5p and HRK 3’UTR by luciferase assay. Additionally, in Western blot a subtle (approaching statistical significance) increase of HRK protein upon miR-625-5p inhibition was observed, supporting our notion that this miRNA is indeed a negative regulator of HRK. Image:Summary/Conclusion: miR-625-5p is a novel candidate oncogenic miRNA in T-ALL, implicated in the negative regulation of apoptosis. Post-transcriptional repression of the proapoptotic HRK gene is a putative mechanism contributing to oncogenic properties of this miRNA when overexpressed in T-ALL cells.

A novel endoplasmic stress mediator, Kelch domain containing 7B (KLHDC7B), increased Harakiri (HRK) in the SubAB-induced apoptosis signaling pathway

Locus for Enterocyte Effacement (LEE)-positive Shiga-toxigenic Escherichia coli (STEC) contributes to many global foodborne diseases, with infection characterized by severe gastrointestinal symptoms, including bloody diarrhea. The incidence of LEE-negative STEC-mediated disease is also increasing globally. Subtilase cytotoxin (SubAB) is released by some LEE-negative STEC strains. It cleaves BiP, which is a chaperone protein located in the endoplasmic reticulum (ER), thereby causing apoptosis induced by ER stress. To date, the apoptotic signaling pathway mediated by SubAB has not been identified. In the current study, RNA-seq analysis showed that SubAB significantly induced the expression of Kelch domain containing 7B (KLHDC7B). We explored the role of KLHDC7B in the SubAB-induced apoptotic pathway. SubAB-induced KLHDC7B mRNA expression was increased after 12 h of incubation of toxin with HeLa cells. KLHDC7B expression was downregulated by knockdown of PKR-like endoplasmic reticulum kinase (PERK), CEBP homologous protein (CHOP), activating transcription factor 4 (ATF4), and CEBP β (CEBPB). KLHDC7B knockdown suppressed SubAB-stimulated CHOP expression, poly(ADP-ribose) polymerase (PARP) cleavage, and cytotoxicity. The over-expressed KLHDC7B was localized to the nucleus and cytosolic fractions. Next, we used RNA-seq to analyze the effect of KLHDC7B knockdown on apoptosis induced by SubAB, and found that the gene encoding for the pro-apoptotic Bcl-2 family protein, Harakiri (HRK), was upregulated in SubAB-treated control cells. However, this effect was not observed in SubAB-treated KLHDC7B-knockdown cells. Therefore, we identified the pathway through which SubAB-induced KLHDC7B regulates HRK expression, which is essential for apoptosis in toxin-mediated ER stress.

Abstract PO-132: Rosmarinic acid-induced apoptosis and cell cycle arrest in triple-negative breast cancer cells

Abstract Rosmarinic acid (RA) is a polyphenolic compound with various pharmacological properties, including anti-inflammatory, immunomodulatory, anti-mutagenic, antioxidant, and anticancer activities. This study evaluated the effects of RA in two racially different triple-negative breast cancer (TNBC) cell lines. One cell line was derived from Caucasian origin (MDA-MB-231), and another one was derived from an African American origin (MDA-MB-468). The apoptotic effect of RA was performed using An Annexin V-FITC Apoptosis Detection Kit, while the Propidium Iodide Flow Cytometry Kit was used to detect the cell cycle distribution. Gene expression profiling was established using Human Apoptosis PCR array. The results obtained show that RA significantly caused cytotoxic and antiproliferative effects in both cell lines in a dose- and time-dependent manner. It was noted that RA induced cell cycle arrest-related apoptosis and altered the expression of many apoptosis-involved genes differently. In MDA-MB-231 cells, RA arrested the cells in the G0/G1 phase. In contrast, RA causes S-phase arrest in MDA-MB-468 cells, leading to a 2-fold increase in the apoptotic effect compared to MDA-MB-231 cells. Furthermore, in MDA-MB- 231 cells, RA significantly upregulated the mRNA expression of three genes: Harakiri (HRK), tumor necrosis factor receptor superfamily 25 (TNFRSF25), and BCL-2 interacting protein 3 (BNIP3). In contrast, in the MDA-MB-468 cell line, RA induced a significant transcription activation in three genes, including TNF, growth arrest, and DNA damage-inducible 45 alpha (GADD45A), and BNIP3. Furthermore, RA repressed the expression of TNF receptor superfamily 11B (TNFRSF11B) in MDA-MB-231 cells in comparison to the ligand TNF superfamily member 10 (TNFSF10) and baculoviral IAP repeat-containing 5 (BIRC5) in MDA-MB-468 cells. In conclusion, the data suggest that the polyphenol RA may have a potential role in TNBC therapies, particularly in African American cells. Citation Format: Karam F.A. Soliman, Samia S. Messeha, Najla O. Zarmouh, Abrar Asiri. Rosmarinic acid-induced apoptosis and cell cycle arrest in triple-negative breast cancer cells [abstract]. In: Proceedings of the AACR Virtual Conference: Thirteenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2020 Oct 2-4. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(12 Suppl):Abstract nr PO-132.

YAP-Mediated Repression of HRK Regulates Tumor Growth, Therapy Response, and Survival Under Tumor Environmental Stress in Neuroblastoma.

Following chemotherapy and relapse, high-risk neuroblastoma tumors harbor more genomic alterations than at diagnosis, including increased transcriptional activity of the Yes-associated protein (YAP), a key downstream component of the Hippo signaling network. Although YAP has been implicated in many cancer types, its functional role in the aggressive pediatric cancer neuroblastoma is not well-characterized. In this study, we performed genetic manipulation of YAP in human-derived neuroblastoma cell lines to investigate YAP function in key aspects of the malignant phenotype, including mesenchymal properties, tumor growth, chemotherapy response, and MEK inhibitor response. Standard cytotoxic therapy induced YAP expression and transcriptional activity in patient-derived xenografts treated in vivo, which may contribute to neuroblastoma recurrence. Moreover, YAP promoted a mesenchymal phenotype in high-risk neuroblastoma that modulated tumor growth and therapy resistance in vivo. Finally, the BH3-only protein, Harakiri (HRK), was identified as a novel target inhibited by YAP, which, when suppressed, prevented apoptosis in response to nutrient deprivation in vitro and promoted tumor aggression, chemotherapy resistance, and MEK inhibitor resistance in vivo. Collectively, these findings suggest that YAP inhibition may improve chemotherapy response in patients with neuroblastoma via its regulation of HRK, thus providing a critical strategic complement to MEK inhibitor therapy. SIGNIFICANCE: This study identifies HRK as a novel tumor suppressor in neuroblastoma and suggests dual MEK and YAP inhibition as a potential therapeutic strategy in RAS-hyperactivated neuroblastomas.

Rosmarinic acid-induced apoptosis and cell cycle arrest in triple-negative breast cancer cells

Rosmarinic acid (RA) is a polyphenolic compound with various pharmacological properties, including, anti-inflammatory, immunomodulatory, and neuroprotective, as well as having antioxidant and anticancer activities. This study evaluated the effects and mechanisms of RA in two racially different triple-negative breast cancer (TNBC) cell lines. Results obtained show that RA significantly caused cytotoxic and antiproliferative effects in both cell lines in a dose- and time-dependent manner. Remarkably, RA induced cell cycle arrest-related apoptosis and altered the expression of many apoptosis-involved genes differently. In MDA-MB-231 cells, RA arrested the cells in the G0/G1 phase. In contrast, the data suggest that RA causes S-phase arrest in MDA-MB-468 cells, leading to a 2-fold increase in the apoptotic effect compared to MDA-MB-231 cells. Further, in MDA-MB-231 cells, RA significantly upregulated the mRNA expression of three genes: harakiri (HRK), tumor necrosis factor receptor superfamily 25 (TNFRSF25), and BCL-2 interacting protein 3 (BNIP3). In contrast, in the MDA-MB-468 cell line, the compound induced a significant transcription activation in three genes, including TNF, growth arrest and DNA damage-inducible 45 alpha (GADD45A), and BNIP3. Furthermore, RA repressed the expression of TNF receptor superfamily 11B (TNFRSF11B) in MDA-MB-231 cells in comparison to the ligand TNF superfamily member 10 (TNFSF10) and baculoviral IAP repeat-containing 5 (BIRC5) in MDA-MB-468 cells. In conclusion, the data suggest that the polyphenol RA may have a potential role in TNBC therapies, particularly in MDA-MB-468 cells.

A Mission for MARS: The Success of Climate Change Skeptic Rhetoric in the US

Radio and television broadcasters accuse climate scientists of “promoting a global warming hoax”, recommending that they be “named and fi red, drawn and quartered” (Rush Limbaugh); commit “hara kiri” (Glenn Beck); and be “publicly flogged” (Mark Morano). Conservative media are crucial in promoting climate skepticism. Likewise, climate skepticism resonates well with white middle-class men. But why does the middle class continue to support “radical” positions? This article focuses on Anti-Intellectualism to explain why climate skeptic rhetoric resonates with “Middle American Radicals” (MARS).

Rosmarinic Acid‐Induced Apoptosis and Cell Cycle Arrest in Triple‐Negative Breast Cancer Cells

Rosmarinic acid (RA) is one of the potent polyphenolic compounds in rosemary (Rosmarinus officinalis) extract. The compound has shown various bioactive properties, including, anti‐inflammatory, immunomodulatory, anti‐mutagenic, hepatoprotective, and neuroprotective, as well as having antioxidant and anti‐cancer activity. This study evaluated the effects and mechanisms of RA in two triple‐negative breast cancer (TNBC) cell lines; MDA‐MB‐231(MM‐231) and MDA‐MB‐468 (MM‐468); which were derived from a Caucasian and an African American woman, respectively. In this study, RA significantly and similarly inhibited cell viability and proliferation in both cell lines in a dose‐ and time‐dependent manner. The results obtained show that RA differentially affected the two cell lines in cell cycle arrest‐related apoptosis and in the alteration of the expression of many apoptosis‐involved genes. In MM‐231 cells, RA arrested the cells in the G0/G1 phase. In contrast, the data suggest that RA causes S‐phase arrest in MM‐468 cells and leads to a 2‐fold greater apoptotic effect than in MM‐231 cells. In MM‐231 cells, RA significantly upregulated the mRNA expression of three genes; harakiri (HRK), tumor necrosis factor receptor superfamily 25 (TNFRSF25), and BCL2 interacting protein 3 (BNIP3). In contrast, in MM‐468 cell line, the compound induced a significant transcription activation in five genes, including (TNF), baculoviral IAP repeat‐containing 3 (BIRC3), receptor‐interacting serine/threonine‐protein kinase 2 (RIPK2), BNIP3, and growth arrest and DNA damage‐inducible 45 alpha (GADD45A). Furthermore, RA repressed the expression of TNF receptor superfamily 11B (TNFRSF11B) in MM‐231 cells in comparison to the ligand TNF superfamily member 10 (TNFSF10) in MM‐468 cells. In conclusion, the data suggest that the polyphenol RA may have therapeutic potential for TNBC. However, RA should be further evaluated for in‐vivo studies and in non‐cancer cells.Support or Funding InformationU54 RCMI

Mitochondrial dysfunction and role of harakiri in the pathogenesis of myositis.

The etiology of myositis is unknown. Although attempts to identify viruses in myositis skeletal muscle have failed, several studies have identified the presence of a viral signature in myositis patients. Here we postulate that in individuals with susceptible genetic backgrounds, viral infection alters the epigenome to activate the pathological pathways leading to disease onset. To identify epigenetic changes, methylation profiling of Coxsackie B infected human myotubes and muscle biopsies from polymyositis (PM) and dermatomyositis (DM) patients were compared to changes in global transcript expression induced by in vitro Coxsackie B infection. Gene and protein expression analysis and live cell imaging were performed to examine the mechanisms. Analysis of methylation and gene expression changes identified that a mitochondria-localized activator of apoptosis-harakiri (HRK) - is upregulated in myositis skeletal muscle cells. Muscle cells with higher HRK expression have reduced mitochondrial potential and poor ability to repair from injury as compared to controls. In cells from myositis patient toll-like receptor 7 (TLR7) activates and sustains high HRK expression. Forced over expression of HRK in healthy muscle cells is sufficient to compromise their membrane repair ability. Endurance exercise that is associated with improved muscle and mitochondrial function in PM and DM patients decreased TLR7 and HRK expression identifying these as therapeutic targets. Increased HRK and TLR7 expression causes mitochondrial damage leading to poor myofiber repair, myofiber death and muscle weakness in myositis patients and exercise induced reduction of HRK and TLR7 expression in patients is associated with disease amelioration. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

The pro-apoptotic Bcl-2 family member Harakiri (HRK) induces cell death in glioblastoma multiforme

Harakiri (HRK) is a BH3-only protein of the Bcl-2 family and regulates apoptosis by interfering with anti-apoptotic Bcl-2 and Bcl-xL proteins. While its function is mainly characterized in the nervous system, its role in tumors is ill-defined with few studies demonstrating HRK silencing in tumors. In this study, we investigated the role of HRK in the most aggressive primary brain tumor, glioblastoma multiforme (GBM). We showed that HRK is differentially expressed among established GBM cell lines and that HRK overexpression can induce apoptosis in GBM cells at different levels. This phenotype can be blocked by forced expression of Bcl-2 and Bcl-xL, suggesting the functional interaction of Bcl-2/Bcl-xL and HRK in tumor cells. Moreover, HRK overexpression cooperates with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a known tumor-specific pro-apoptotic agent. Besides, secondary agents that augment TRAIL response, such as the histone deacetylase inhibitor MS-275, significantly increases HRK expression. In addition, GBM cell response to TRAIL and MS-275 can be partly abolished by HRK silencing. Finally, we showed that HRK induction suppresses tumor growth in orthotopic GBM models in vivo, leading to increased survival. Taken together, our results suggest that HRK expression is associated with GBM cell apoptosis and increasing HRK activity in GBM tumors might offer new therapeutic approaches.

Targeted inhibition of PI3Kα/δ is synergistic with BCL-2 blockade in genetically defined subtypes of DLBCL

AbstractInhibition of the B-cell receptor (BCR) signaling pathway is a promising treatment strategy in multiple B-cell malignancies. However, the role of BCR blockade in diffuse large B-cell lymphoma (DLBCL) remains undefined. We recently characterized primary DLBCL subsets with distinct genetic bases for perturbed BCR/phosphoinositide 3-kinase (PI3K) signaling and dysregulated B-cell lymphoma 2 (BCL-2) expression. Herein, we explore the activity of PI3K inhibitors and BCL-2 blockade in a panel of functionally and genetically characterized DLBCL cell line models. A PI3K inhibitor with predominant α/δ activity, copanlisib, exhibited the highest cytotoxicity in all BCR-dependent DLBCLs. The proapoptotic effect of copanlisib was associated with DLBCL subtype-specific dysregulated expression of BCL-2 family members including harakiri (HRK) and its antiapoptotic partner BCL extra large (BCL-xL), BCL2 related protein A1, myeloid cell leukemia 1 (MCL-1), and BCL2 interacting mediator of cell death. Using functional BH3 profiling, we found that the cytotoxic activity of copanlisib was primarily mediated through BCL-xL and MCL-1–dependent mechanisms that might complement BCL-2 blockade. For these reasons, we evaluated single-agent activity of venetoclax in the DLBCLs and identified a subset with limited sensitivity to BCL-2 blockade despite having genetic bases of BCL-2 dysregulation. As these were largely BCR-dependent DLBCLs, we hypothesized that combined inhibition of PI3Kα/δ and BCL-2 would perturb BCR-dependent and BCL-2–mediated survival pathways. Indeed, we observed synergistic activity of copanlisib/venetoclax in BCR-dependent DLBCLs with genetic bases for BCL-2 dysregulation in vitro and confirmed these findings in a xenograft model. These results provide preclinical evidence for the rational combination of PI3Kα/δ and BCL-2 blockade in genetically defined DLBCLs.

KDM2B, an H3K36-specific demethylase, regulates apoptotic response of GBM cells to TRAIL

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can selectively kill tumor cells. TRAIL resistance in cancers is associated with aberrant expression of the key components of the apoptotic program. However, how these components are regulated at the epigenetic level is not understood. In this study, we investigated novel epigenetic mechanisms regulating TRAIL response in glioblastoma multiforme (GBM) cells by a short-hairpin RNA loss-of-function screen. We interrogated 48 genes in DNA and histone modification pathways and identified KDM2B, an H3K36-specific demethylase, as a novel regulator of TRAIL response. Accordingly, silencing of KDM2B significantly enhanced TRAIL sensitivity, the activation of caspase-8, -3 and -7 and PARP cleavage. KDM2B knockdown also accelerated the apoptosis, as revealed by live-cell imaging experiments. To decipher the downstream molecular pathways regulated by KDM2B, levels of apoptosis-related genes were examined by RNA-sequencing upon KDM2B loss, which revealed derepression of proapoptotic genes Harakiri (HRK), caspase-7 and death receptor 4 (DR4) and repression of antiapoptotic genes. The apoptosis phenotype was partly dependent on HRK upregulation, as HRK knockdown significantly abrogated the sensitization. KDM2B-silenced tumors exhibited slower growth in vivo. Taken together, our findings suggest a novel mechanism, where the key apoptosis components are under epigenetic control of KDM2B in GBM cells.

SUZ12 Promotes Human Epithelial Ovarian Cancer by Suppressing Apoptosis via Silencing HRK

Epithelial ovarian cancer (EOC) ranks first as the cause of death for gynecological cancers in the United States. SUZ12 is a component of the polycomb repressive complex 2 (PRC2) and is essential for PRC2-mediated gene silencing by generating trimethylation on lysine 27 residue of histone H3 (H3K27Me3). The role of SUZ12 in EOC has never been investigated. Here, we show that SUZ12 is expressed at significantly higher levels in human EOC (n = 117) compared with either normal human ovarian surface epithelium (n = 35, P < 0.001) or fallopian tube epithelium (n = 15, P < 0.001). There is a positive correlation between expression of SUZ12 and EZH2 in human EOC (P < 0.001). In addition, expression of SUZ12 positively correlates with Ki67, a marker of cell proliferation (P < 0.001), and predicts shorter overall survival (P = 0.0078). Notably, knockdown of SUZ12 suppresses the growth of human EOC cells in vitro and in vivo in both orthotopic and subcutaneous xenograft EOC models. In addition, SUZ12 knockdown decreases the levels of H3K27Me3 and triggers apoptosis of human EOC cells. Mechanistically, we identified Harakiri (HRK), a proapoptotic gene, as a novel SUZ12 target gene, and showed that HRK upregulation mediates apoptosis induced by SUZ12 knockdown in human EOC cells. In summary, we show that SUZ12 promotes the proliferation of human EOC cells by inhibiting apoptosis and HRK is a novel SUZ12 target gene whose upregulation contributes to apoptosis induced by SUZ12 knockdown.

Synergistic Silencing by Promoter Methylation and Reduced AP-2α Transactivation of the Proapoptotic HRK Gene Confers Apoptosis Resistance and Enhanced Tumor Growth

The Harakiri (HRK) gene encodes an important proapoptotic mitochondrial protein of the Bcl-2 family. HRK is expressed in normal tissues but is decreased in many cancers such as melanoma, the mechanisms of which have not been fully elucidated. Here, we demonstrate that HRK is silenced by hypermethylation of a major proximal CpG island in the HRK promoter. Furthermore, we show that HRK is a novel target gene regulated by the transcription factor AP-2α, which interacts with an AP-2α binding site in the HRK promoter. Hypermethylation of the major proximal CpG island (which contains the AP-2α binding site within the most densely methylated -218- to -194-bp region) inhibited AP-2α binding and transcriptional activity. Artificial overexpression of AP-2α in melanoma cells up-regulated HRK transcription, which was further restored by treatment with DNA methyltransferase inhibitor 5-azacytidine. Artificial overexpression of HRK by recombinant adenovirus induced caspase-dependent apoptosis, inhibited melanoma cell growth invitro, and markedly reduced invivo melanoma growth in a nude mouse xenograft model. RNA interference by siHRK or siAP-2α reversed the above effects. We conclude that the synergistic effects of HRK promoter hypermethylation and loss of AP-2α transactivation lead to HRK gene silencing and confer resistance to apoptosis and enhanced tumor growth. These novel molecular lesions may provide the basis for new therapeutic approaches to treating AP-2α- and HRK-deficient cancers.

ATF4 orchestrates a program of BH3-only protein expression in severe hypoxia

Intratumoral hypoxia is associated with poor prognosis, regardless of the mode of therapy. Cancer cells survive this condition through activating several adaptive signaling pathways, including the integrated stress response (ISR) and autophagy. Activating transcription factor 4 (ATF4) is the major transcriptional mediator of the ISR, which we have shown to be involved in autophagy regulation to protect cells from severe hypoxia. Here we demonstrate that ATF4 orchestrates a program of BH3-only protein expression in severe hypoxia. We find that the BH3-only proteins HRK, PUMA, and NOXA are transcriptionally induced in severe hypoxia and that their expression is abrogated by RNA interference against ATF4. In particular, we show that the BH3-only protein harakiri (HRK) is transactivated by ATF4 in severe hypoxia through direct binding of ATF4 to the promoter region. Furthermore, we demonstrate through siRNA knockdown that HRK induces autophagy and promotes cancer cell survival in severe hypoxia.

E2F‐1‐ and E2Ftr‐mediated apoptosis: the role of DREAM and HRK

E2F-1-deleted mutant, ‘truncated E2F’ (E2Ftr, E2F-1[1–375]), lacking the carboxy-terminal transactivation domain, was shown to be more potent at inducing cancer cell apoptosis than wild-type E2F-1 (wtE2F-1; full-length E2F-1). Mechanisms by which wtE2F-1 and E2Ftr induce apoptosis, however, are not fully elucidated. Our study demonstrates molecular effects of pro-apoptotic BH3-only Bcl-2 family member Harakiri (Hrk) in wtE2F-1- and E2Ftr-induced melanoma cell apoptosis. We found that Hrk mRNA and Harakiri (HRK) protein expression was highly up-regulated in melanoma cells in response to wtE2F-1 and E2Ftr overexpression. HRK up-regulation did not require the E2F-1 transactivation domain. In addition, Hrk gene up-regulation and HRK protein expression did not require p53 in cancer cells. Hrk knockdown by Hrk siRNA was associated with significantly reduced wtE2F-1- and E2Ftr-induced apoptosis. We also found that an upstream factor, ‘downstream regulatory element antagonist modulator’ (DREAM), may be involved in HRK-mediated apoptosis in response to wtE2F-1 and E2Ftr overexpression. DREAM expression levels increased following wtE2F-1 and E2Ftr overexpression. Western blotting detected increased DREAM primarily in dimeric form. The homodimerization of DREAM resulting from wtE2F-1 and E2Ftr overexpression may contribute to the decreased binding activity of DREAM to the 3′-untranslated region of the Hrk gene as shown by electromobility shift assay. Results showed wtE2F-1- and E2Ftr-induced apoptosis is partially mediated by HRK. HRK function is regulated in response to DREAM. Our findings contribute to understanding the mechanisms that regulate wtE2F-1- and E2Ftr-induced apoptosis and provide insights into the further evaluation of how E2Ftr-induced apoptosis may be used for therapeutic gain.

Impact of Lenalidomide on Gene Expression Profiles of Malignant and Immune Cells in Patients with Chronic Lymphocytic Leukemia

Abstract 976Lenalidomide has recently been demonstrated to have significant activity in chronic lymphocytic leukemia (CLL). Its mechanism of action in this disease is not well understood, but it is thought to act primarily by enhancing anti-tumor immunity and reducing production of pro-tumoral factors in the CLL microenvironment. We have previously demonstrated alterations in the expression of cytoskeletal genes in T-cells from patients with CLL and have subsequently shown that these changes translate into a deficit in T-cell function, due to impaired actin polymerization resulting in defective immunological synapse formation. Treatment of both autologous T-cells and CLL cells with lenalidomide was necessary to repair this defect, suggesting that this may be a key component of this agent's activity in CLL. Therefore we examined the effect of lenalidomide on the global gene expression profiles of isolated B-cells and T-cell subsets from CLL patients and healthy donors. Peripheral blood mononuclear cells from patients with untreated CLL or healthy donors were cultured in the presence of 1 μM lenalidomide or vehicle control for 48 hours. The lymphocyte subsets were isolated, followed by RNA extraction and gene expression profiling using the Affymetrix HGU133Plus2.0 platform. Lenalidomide treatment had similar effects on gene expression in T-cells from both patients with CLL and healthy donors. The most prominent changes in expression were of genes involved in cytoskeletal signaling including a 20-fold increase in WASF1 (Wiskott Aldrich Syndrome protein family, member 1), and greater than 2-fold increases in the expression of Rac-family member RHOC, (Ras homolog gene family, member C), actin binding proteins CORO1B (Coronin 1B), PARVA (Parvin alpha), and the Rho guanine nucleotide exchange factors (GEFs), ARHGEF5 and ARHGEF7. We also observed changes in genes regulating integrin signaling including PXN (Paxilin) and FAK (Focal adhesion kinase), and a shift towards Th1 differentiation with upregulation of TNF, IL-12R, and IL-18R. In addition, we noted increased expression of the transcription factors IKZF1, IKZF4 and IRF4, genes involved in the Ikaros pathways that are essential for hematopoiesis and control of lymphoid proliferation. These changes in gene expression provide further evidence that an important mechanism of action of lenalidomide is the upregulation of the actin cytoskeletal network including Rho-GTPases and integrin activation signaling, and are consistent with our previous observations concerning the functional repair of T-cells in CLL.Initial analysis of the effect of lenalidomide on the gene expression profiles of the CLL B-cells showed similar changes to those previously described in vivo from CLL patients receiving single agent lenalidomide in a clinical trial (Chen et al. JCO 2010). In our system, lenalidomide treatment resulted in a greater than 2-fold upregulation of 189 genes, and a greater than 2-fold downregulation of 85 genes in CLL B-cells. We observed increased expression of several genes belonging to the TNF superfamily including TNF-α, OX40L, and APRIL, and the receptors DR5, DCR2, and OX40. Many of these are known to mediate apoptosis signaling, and we also observed increased expression of pro-apoptotic genes such as FAS, BID (BH3 interacting domain death agonist), HRK (Harakiri), and CFLAR (CASP8 and FADD-like apoptosis regulator), and cell cycle regulators CDKN1A and CDKN1C (Cyclin-dependent kinase inhibitors 1A and 1C). Lenalidomide also upregulated expression of several genes of known importance in the CLL microenvironment, including the chemokines CCL3 and CCL4, CD40, CD274 (PD-L1), CD279 (PD-1), and adhesion molecules LFA3 and ICAM1. The effect of lenalidomide on the gene expression profiles of normal B-cells was less marked, with greater than 2-fold upregulation of 51 genes and downregulation of 12 genes. However, we did observe that lenalidomide treatment induced upregulation of genes involved in cytoskeletal pathways such as RND1 (Rho family GTPase 1), RHOQ (Ras homolog gene family, member Q), and MYO1B (myosin 1B).In conclusion, investigation of the effect of lenalidomide on gene expression profiling in CLL suggests that the drug acts both to enhance T-cell function, and to render the CLL cells more susceptible to immune cell mediated killing. Disclosures:Gribben:Roche: Honoraria; Celgene: Honoraria; GSK: Honoraria; Mundipharma: Honoraria; Gilead: Honoraria; Pharmacyclics: Honoraria.