Noxa Expression Research Articles

Dendrosomal Curcumin Showed Cytotoxic Effects on Breast Cancer Cell Line by Inducing Mitochondrial Apoptosis Pathway and Cell Division Arrest

Background: Mutations in the p53 gene have been linked to the initiation and progression of breast cancer, as well as resistance to chemotherapy. Therefore, the development of novel treatment approaches is essential to combat this disease. Objectives: This study aimed to evaluate the effects of dendrosomal curcumin (DNC) on the breast cancer cell line MDA-MB231. Methods: MDA-MB231 cells were treated with 20 μM DNC, and the apoptosis rate and cell proliferation cycles were assessed using flow cytometry. Additionally, after RNA extraction and cDNA synthesis, the expression levels of Lnc-DANCR, EZH2, Noxa, bcl-2, bax, PUMA, p21, and p53 genes were analyzed using RT-PCR. Protein expression levels of P53, P21, Bcl-2, and Bax were evaluated through western blotting. Results: Dendrosomal curcumin induced apoptosis in MDA-MB231 cells and caused cell cycle arrest at the SubG1 phase. Dendrosomal curcumin treatment downregulated Lnc-DANCR, EZH2, bcl-2, and p53 gene expression, while upregulating bax, Noxa, PUMA, and p21 gene expression in a time-dependent manner. Bax and P21 protein levels were significantly upregulated following DNC treatment, whereas Bcl-2 and P53 protein levels were downregulated in DNC-treated breast cancer cells. Conclusions: In summary, dendrosomal nanocurcumin demonstrated potent anti-tumor effects against breast cancer cells, suggesting its potential as a therapeutic agent in breast cancer treatment.

Combination of bortezomib and venetoclax targets the pro-survival function of LMP-1 and EBNA-3C of Epstein-Barr virus in spontaneous lymphoblastoid cell lines.

Epstein-Barr virus (EBV) manipulates the ubiquitin-proteasome system and regulators of Bcl-2 family to enable the persistence of the virus and survival of the host cells through the expression of viral proteins in distinct latency patterns. We postulate that the combination of bortezomib (proteasome inhibitor) and venetoclax (Bcl-2 inhibitor) [bort/venetoclax] will cause synergistic killing of post-transplant lymphoproliferative disorder (PTLD) through targeting the pro-survival function of latent viral proteins such as latent membrane protein-1 (LMP-1) and EBV nuclear antigen-3C (EBNA-3C). Bort/venetoclax could synergistically kill spontaneous lymphoblastoid cell lines (sLCLs) derived from patients with PTLD and EBV-associated hemophagocytic lymphohistiocytosis by inducing DNA damage response, apoptosis and G1-S cell cycle arrest in a ROS-dependent manner. Bortezomib potently induced the expression of Noxa, a pro-apoptotic initiator and when combined with venetoclax, inhibited Mcl-1 and Bcl-2 simultaneously. Bortezomib prevented LMP-1 induced proteasomal degradation of IκBα leading to the suppression of the NF-κB signaling pathway. Bortezomib also rescued Bcl-6 from EBNA-3C mediated proteasomal degradation thus maintaining the repression of cyclin D1 and Bcl-2 causing G1-S arrest and apoptosis. Concurrently, venetoclax inhibited Bcl-2 upregulated by either LMP-1 or EBNA-3C. Bort/venetoclax decreased the expression of phosphorylated p65 and Bcl-2 at serine 70 thereby suppressing the NF-κB signaling pathway and promoting apoptosis, respectively. These data corroborated the marked suppression of the growth of xenograft of sLCL in SCID mice (p<0.001). Taken together, the combination of bortezomib and venetoclax targets the pro-survival function of LMP-1 and EBNA-3C of Epstein-Barr virus in spontaneous lymphoblastoid cell lines.

CRISPR screen of venetoclax response-associated genes identifies transcription factor ZNF740 as a key functional regulator

BCL-2 inhibitors such as venetoclax offer therapeutic promise in acute myeloid leukemia (AML) and other cancers, but drug resistance poses a significant challenge. It is crucial to understand the mechanisms that regulate venetoclax response. While correlative studies have identified numerous genes linked to venetoclax sensitivity, their direct impact on the drug response remains unclear. In this study, we targeted around 1400 genes upregulated in venetoclax-sensitive primary AML samples and carried out a CRISPR knockout screen to evaluate their direct effects on venetoclax response. Our screen identified the transcription factor ZNF740 as a critical regulator, with its expression consistently predicting venetoclax sensitivity across subtypes of the FAB classification. ZNF740 depletion leads to increased resistance to ventoclax, while its overexpression enhances sensitivity to the drug. Mechanistically, our integrative transcriptomic and genomic analysis identifies NOXA as a direct target of ZNF740, which negatively regulates MCL-1 protein stability. Loss of ZNF740 downregulates NOXA and increases the steady state protein levels of MCL-1 in AML cells. Restoring NOXA expression in ZNF740-depleted cells re-sensitizes AML cells to venetoclax treatment. Furthermore, we demonstrated that dual targeting of MCL-1 and BCL-2 effectively treats ZNF740-deficient AML in vivo. Together, our work systematically elucidates the causal relationship between venetoclax response signature genes and establishes ZNF740 as a novel transcription factor regulating venetoclax sensitivity.

ONC212 enhances YM155 cytotoxicity by triggering SLC35F2 expression and NOXA-dependent MCL1 degradation in acute myeloid leukemia cells

Although the anticancer activity of ONC212 has been reported, the precise mechanism underlying its apoptotic effects remains unclear. In this study, we investigated the apoptotic mechanism of ONC212 in acute myeloid leukemia (AML) cells. ONC212 induces apoptosis, MCL1 downregulation, and mitochondrial depolarization in AML U937 cells. Ectopic MCL1 expression alleviates mitochondria-mediated apoptosis in ONC212-treated U937 cells. ONC212 triggers AKT phosphorylation, inducing NOX4-dependent ROS production and promoting HuR transcription. HuR-mediated ATF4 mRNA stabilization stimulates NOXA and SLC35F2 expression; ONC212-induced upregulation of NOXA leads to MCL1 degradation. The synergistic effect of ONC212 on YM155 cytotoxicity was dependent on increased SLC35F2 expression. In addition, YM155 feedback facilitated the activation of the ONC212-induced signaling pathway. A similar mechanism explains ONC212- and ONC212/YM155-induced AML HL-60 cell death. The continuous treatment of U937 cells with the benzene metabolite hydroquinone (HQ) generated U937/HQ cells, exhibiting enhanced responsiveness to the cytotoxic effects of ONC212. In U937/HQ cells, ONC212 triggered apoptosis through NOXA-mediated MCL1 downregulation, enhancing YM155 cytotoxicity. Collectively, our data suggested that ONC212 upregulated SLC35F2 expression and triggered NOXA-mediated MCL1 degradation in U937, U937/HQ, and HL-60 cells by activating the AKT/NOX4/HuR/ATF4 pathway. The ONC212-induced signaling pathway showed anti-AML activity and enhanced YM155 cytotoxicity.

Dual drug nanoparticle synergistically induced apoptosis, suppressed inflammation, and protected autophagic response in rheumatoid arthritis fibroblast-like synoviocytes

Rheumatoid arthritis (RA) is a chronic immune-mediated joint inflammatory disorder associated with aberrant activation of fibroblast-like synoviocytes (FLS). Recently, FLS gained importance due to its crucial role in RA pathogenesis, and thus, targeting FLS is suggested as an attractive treatment strategy for RA. FLS-targeted approaches may be combined with disease-modifying antirheumatic drugs (DMARDs) and natural phytochemicals to improve efficacy in RA control and negate immunosuppression. In this study, we assessed the therapeutic effectiveness of DD NP HG in primary RA-FLS cells isolated from the synovial tissue of FCA-induced RA rats. We observed that DD NP HG had good biosafety for healthy FLS cells and, at higher concentrations, a mild inhibitory effect on RA-FLS. The combination therapy (DD NP HG) of MTX NP and PEITC NE in RA-FLS showed a higher rate of apoptosis with significantly reduced LPS-induced expression of pro-inflammatory cytokines (TNF-α, IL-17A, and IL-6) in arthritic FLS. Further, the gene expression studies showed that DD NP HG significantly down-regulated the mRNA expression of IL-1β, RANKL, NFATc1, DKK1, Bcl-xl, Mcl-1, Atg12, and ULK1, and up-regulated the mRNA expression of OPG, PUMA, NOXA and SQSTM1 in LPS-stimulated RA-FLS cells. Collectively, our results demonstrated that DD NP HG significantly inhibited the RA-FLS proliferation via inducing apoptosis, down-regulating pro-inflammatory cytokines, and further enhancing the expression of genes associated with bone destruction in RA pathogenesis. A nanotechnology approach is a promising strategy for the co-delivery of dual drugs to regulate the RA-FLS function and achieve synergistic treatment of RA.

Abstract 4579: DNMT1 determines the sensitivity of Class I HDAC inhibitor Psammaplin A to induce apoptosis and DNA damage in hematological malignant cells

Abstract Histone deacetylases (HDACs) and DNA methyltransferases (DNMTs) are overexpressed in cancer cells associated with dysregulated gene expression and tumor progression. DNMT1 partnered with Class I HDAC as complex to inhibit gene transcription. Several pan-HDAC inhibitors have been approved for T-cell lymphoma with evident side effects. More selective HDAC inhibitors need to be developed. Psammaplin A (PsA), a symmetrical bromotyrosine-derived disulfide dimer isolated from the Psammaplysilla sponge, is a selective Class I HDAC inhibitor. We tested its cell growth inhibition and apoptosis induction in 12 hematological malignant cell lines with 8 lines sensitive (more than 50% growth inhibition at 1 μM) and 4 lines insensitive (less than 20% growth inhibition at 1 μM). PsA induces acetylation of H3, but not α-tubulin, supporting the Class I HDAC inhibition selection. In the sensitive cell lines, PsA induces apoptosis with down-regulation of c-Flip and upregulation of NOXA as well as DNA damage detected by γ-H2A.X. RNA sequence analysis results confirm the regulation of PsA on NOXA, c-Flip and p21 gene expression. The four insensitive cell lines express high levels of DNMT1 with resistance to apoptosis induction. Silencing DNMT1 with siRNA sensitizes PsA-induced apoptosis with induction of NOXA and γ-H2A.X. Azacytidine, the DNMT1 degrader, in combination with PsA induce synergistic cell growth inhibition and apoptosis in vitro. PsA (20 mg/kg) plus Azacytidine (2.5 mg/kg) synergistically inhibit tumor growth in insensitive mantle cell lymphoma Jeko-1 xenografts, without detected toxic signs in organ pathological analysis. We find a new mechanism of DNMT1 controlling the sensitivity of Class I HDAC inhibitor PsA and provide a rationale of PsA with azacytidine for combination therapy. Citation Format: Yu Bao, Dan Liu, Zi Wu, Yumei He, Ping Gong, Samuel Waxman, Yongkui Jing. DNMT1 determines the sensitivity of Class I HDAC inhibitor Psammaplin A to induce apoptosis and DNA damage in hematological malignant cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4579.

Abstract 6484: Overcoming resistance to EGFR-TKI in NSCLC by simultaneously inhibition of anti-apoptotic proteins

Abstract Lung cancer is the second most common cancer worldwide with 1.8 million deaths in 2020 being the leading cause of cancer related death. With 86% of cases, non-small cell lung cancer (NSCLC) is the most prominent subtype. The survival rate improved over the past 20 years by application of targeted therapies. Driver mutations in protein kinases such as the epidermal growth factor receptor (EGFR) are frequent and have evolved as therapeutic targets in anti-cancer therapy. Inhibitors of receptor tyrosine kinase (RTKIs), prominently EGFR-TKIs, are effective in anti-cancer therapy but tumor evolution inevitably generates resistance. Thus, strategies for the treatment of patients with acquired resistance to RTKIs remains an urgent need. Resistance in solid tumors is overcome by a combination of bortezomib with the BH3-mimetic ABT-199 (Muenchow et al. 2020). Also, overcoming ALK-TKI resistance in NSCLC was described by Tanimoto et al. (2021) by proteasome inhibiton that mediates induction of NOXA. Furthermore, the novel described BCL-2 independent activity of ABT-199 to transactivate NOXA expression (Weller et al. 2022) represents a generally active principle by combining drugs with independent mechanism of action. We hypothesize, that combined treatment of EGFR-TKI Osimertinib (OSI) with BH3-mimetics enhances sensitivity to apoptosis induction. To explore this hypothesis, we investigated cell death induction by combined administration of OSI and BH3-mimetics or PIs in NSCLC differing in their mutational status. In addition, we analyzed the expression of BCL-2 family proteins and their relevance and regulation during RTKI-induced cell death by RNA sequencing and Western Blot. These analyses will be performed in cellular model systems of NSCLC with specific resistance mediating mutations of EGFR or activated parallel survival pathways. We found that BCL-2 inhibitors enhance efficacy of OSI in NSCLC, resulting in synergistic cell death induction. Additionally, the sensitivity to BCL-2 inhibitors tends to increase in OSI-resistant NSCLC. Western Blot analysis and RNA sequencing data of OSI-resistant NSCLC identified BCL-2 overexpression and an EMT signature as resistance-mediating mechanisms, which can be overcome by combination of OSI with BCL-2 inhibitors. Indeed, our data suggest that combination of OSI with BCL-2 inhibitors disrupts resistance to OSI in NSCLC. We propose that BCL-2 inhibitors sensitize OSI-resistant NSCLC to effectively induce cell death. Thus, studying the molecular mechanism of synergistic efficacy of RTKI/BCL-2 inhibitor combination therapies, should prompt future efforts in the clinical evaluation of combinatorial regimens. We hypothesize that a general therapeutic concept which relies on simultaneous blocking of driver mutations and survival-promoting BCL-2 proteins represents a generally active principle of a tumor-agnostic mechanism of action. Citation Format: Sandra Weller, Tobias Beigl, Frank Essmann, Annika Harsch, Hans-Georg Kopp, Tom Ekstrom. Overcoming resistance to EGFR-TKI in NSCLC by simultaneously inhibition of anti-apoptotic proteins [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6484.

Steroid-free combination of 5-azacytidine and venetoclax for the treatment of multiple myeloma.

Multiple myeloma (MM) is an incurable plasma cell malignancy that, despite an unprecedented increase in overall survival, lacks truly risk-adapted or targeted treatments. A proportion of patients with MM depend on BCL-2 for survival, and, recently, the BCL-2 antagonist venetoclax has shown clinical efficacy and safety in t(11;14) and BCL-2 overexpressing MM. However, only a small proportion of MM patients rely on BCL-2 (approx. 20%), and there is a need to broaden the patient population outside of t(11;14) that can be treated with venetoclax. Therefore, we took an unbiased screening approach and screened epigenetic modifiers to enhance venetoclax sensitivity in 2 non-BCL-2 dependent MM cell lines. The demethylase inhibitor 5-azacytidine was one of the lead hits from the screen, and the enhanced cell killing of the combination was confirmed in additional MM cell lines. Using dynamic BH3 profiling and immunoprecipitations, we identified the potential mechanism of synergy is due to increased NOXA expression, through the integrated stress response. Knockdown of PMAIP1 or PKR partially rescues cell death of the venetoclax and 5-azacytidine combination treatment. The addition of a steroid to the combination treatment did not enhance the cell death, and, interestingly, we found enhanced death of the immune cells with steroid addition, suggesting that a steroid-sparing regimen may be more beneficial in MM. Lastly, we show for the first time in primary MM patient samples that 5-azacytidine enhances the response to venetoclax ex vivo across diverse anti-apoptotic dependencies (BCL-2 or MCL-1) and diverse cytogenetic backgrounds. Overall, our data identify 5-azacytidine and venetoclax as an effective treatment combination, which could be a tolerable steroid-sparing regimen, particularly for elderly MM patients.

Noxa inhibits oncogenesis through ZNF519 in gastric cancer and is suppressed by hsa-miR-200b-3p

While Phorbol-12-myristate-13-acetate-induced protein 1 (Noxa/PMAIP1) assumes a pivotal role in numerous tumors, its clinical implications and underlying mechanisms of gastric cancer (GC) are yet enigmatic. In this investigation, our primary objective was to scrutinize the clinical relevance and potential mechanisms of Noxa in gastric cancer. Immunohistochemical analysis was conducted on tissue microarrays comprising samples from a meticulously characterized cohort of 84 gastric cancer patients, accompanied by follow-up data, to assess the expression of Noxa. Additionally, Noxa expression levels in gastric cancer clinical samples and cell lines were measured through quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analysis. The effect of Noxa expression on the prognosis of patients with gastric cancer was evaluated using Kaplan–Meier survival. Further insight into the role of Noxa in driving gastric cancer progression was gained through an array of experimental techniques, including cell viability assays (CCK8), plate cloning assays, transwell assays, scratch assays, and real-time cell analysis (RTCA). Potential upstream microRNAs (miRNAs) that might modulate Noxa were identified through rigorous bioinformatics analysis, substantiated by luciferase reporter assays and Western blot experiments. Additionally, we utilized RNA sequencing, qRT-PCR, and Western blot to identify proteins binding to Noxa and potential downstream target. Finally, we utilized BALB/c nude mice to explore the role of Noxa in vivo. Our investigation unveiled a marked downregulation of Noxa expression in gastric cancer and underscored its significance as a pivotal prognostic factor influencing overall survival (OS). Noxa overexpression exerted a substantial inhibitory effect on the proliferation, migration and invasion of GC cells. Bioinformatic analysis and dual luciferase reporter assays unveiled the capacity of hsa-miR-200b-3p to interact with the 3′-UTR of Noxa mRNA, thereby orchestrating a downregulation of Noxa expression in vitro, consequently promoting tumor progression in GC. Our transcriptome analysis, coupled with mechanistic validation, elucidated a role for Noxa in modulating the expression of ZNF519 in the Mitophagy-animal pathway. The depletion of ZNF519 effectively reversed the oncogenic attributes induced by Noxa. Upregulation of Noxa expression suppressed the tumorigenesis of GC in vivo. The current investigation sheds light on the pivotal role of the hsa-miR-200b-3p/Noxa/ZNF519 axis in elucidating the pathogenesis of gastric cancer, offering a promising avenue for targeted therapeutic interventions in the management of this challenging malignancy.

Clarithromycin overcomes stromal cell-mediated drug resistance against proteasome inhibitors in myeloma cells via autophagy flux blockage leading to high NOXA expression.

We previously reported that macrolide antibiotics, such as clarithromycin (CAM), blocked autophagy flux, and simultaneous proteasome and autophagy inhibition by bortezomib (BTZ) plus CAM resulted in enhanced apoptosis induction in multiple myeloma (MM) cells via increased endoplasmic reticulum (ER) stress loading. However, in actual therapeutic settings, cell adhesion-mediated drug resistance between bone marrow stromal cells (BMSC) and MM cells has been known to be a barrier to treatment. To investigate whether CAM could enhance BTZ-induced cytotoxicity in MM cells under direct cell adhesion with BMSC, we established a co-culture system of EGFP-labeled MM cells with BMSC. The cytotoxic effect of BTZ on MM cells was diminished by its interaction with BMSC; however, the attenuated cytotoxicity was recovered by the co-administration of CAM, which upregulates ER stress loading and NOXA expression. Knockout of NOXA in MM cells canceled the enhanced cell death by CAM, indicating that NOXA is a key molecule for cell death induction by the co-administration of CAM. Since NOXA is degraded by autophagy as well as proteasomes, blocking autophagy with CAM resulted in the sustained upregulation of NOXA in MM cells co-cultured with BMSC in the presence of BTZ. Our data suggest that BMSC-associated BTZ resistance is mediated by the attenuation of ER stress loading. However, the addition of CAM overcomes BMSC-associated resistance via upregulation of NOXA by concomitantly blocking autophagy-mediated NOXA degradation and transcriptional activation of NOXA by ER stress loading.

Extracellular and intracellular destruction of Pseudomonas aeruginosa by Dictyostelium discoideum phagocytes mobilize different antibacterial mechanisms.

Ingestion and killing of bacteria by phagocytic cells are critical processes to protect the human body from bacterial infections. In addition, some immune cells (neutrophils, NK cells) can release microbicidal molecules in the extracellular medium to eliminate non-ingested microorganism. Molecular mechanisms involved in the resulting intracellular and extracellular killing are still poorly understood. In this study, we used the amoeba Dictyostelium discoideum as a model phagocyte to investigate the mechanisms allowing intracellular and extracellular killing of Pseudomonas aeruginosa. When a D. discoideum cell establishes a close contact with a P. aeruginosa bacterium, it can either ingest it and kill it in phagosomes, or kill it extracellularly, allowing a direct side-by-side comparison of these two killing modalities. Efficient intracellular destruction of P. aeruginosa requires the presence of the Kil2 pump in the phagosomal membrane. On the contrary, extracellular lysis is independent on Kil2 but requires the expression of the superoxide-producing protein NoxA, and the extracellular release of the AplA bacteriolytic protein. These results shed new light on the molecular mechanisms allowing elimination of P. aeruginosa bacteria by phagocytic cells.

Transcriptional Changes in Patients with CLL Starting Venetoclax Therapy Identifies Inflammatory and Adaptive Stress Responses

Venetoclax is a selective BCL2 inhibitor that was recently approved for CLL treatment. Venetoclax inhibits the pro-survival protein BCL2 and induces deep and durable responses in a significant proportion of CLL patients. Herein, we aimed to study gene expression changes in leukemic cells from CLL patients during the ramp-up period of venetoclax treatment and to assess the transcriptional effect of escalating doses of BCL2 inhibition on tumor biology. We performed RNA sequencing on CD19+ selected tumor cells from 29 patients, ramped-up according to the standard Venetoclax schedule, with weekly increments from 20 mg to 400mg. Four samples were collected per patient, at baseline, and after completing one week on 50mg,100mg, and 400mg. Twelve (41%) patients switched from BTK inhibitor therapy to venetoclax due to disease progression on the BTKi. Those patients discontinued BTKi the day before they started Venetoclax ramp-up. 19 (59%) patients were either treatment-naïve or relapsed with at least months of treatment free-intervals before starting venetoclax. Circulating CD19+ cell counts, typically composed of &amp;gt;95% CLL cells, were enumerated using flow-cytometry. during the ramp-up period, revealed gradual reduction in CLL burden with escalating doses. In the off-BTKi group, median CLL cell counts started to decline on the 100mg dose and were reduced to 98% of baseline at the end of the ramp-up. In the other patients, reductions in median counts were already apparent after one week on 20mg and reached 99% at the end of the ramp-up. Principal component analysis (PCA) of transcriptome data grouped samples by patient, as we have commonly observed in such unsupervised analysis. After batch correcting the patient effect, PCA clearly separated on treatment timepoints from baseline (Figure). Interestingly, patients who recently stopped BTKi had distinct distribution on PCA plots in comparison to the other patients (Others). Indicating distinct transcriptomic changes in patients based on their recent treatment histories. To identify the impact of venetoclax on the CLL transcriptome, we carried out Gene set enrichment analysis using the Hallmark and C3 gene sets from the molecular signature database. Gene sets were identified using a false discovery rate (FDR) of &amp;lt; 0.05, and a normalized enrichment score (NES) of ≥ |1.8|. Gene sets were ranked by NES. In the off-BTKi patient group, 27 gene sets were identified as statistically significantly enriched at 100mg and 400 mg doses. In the “other” patients group, 15 gene sets were found significantly enriched at the same doses. Eight gene sets relating to inflammatory, interferon, and reactive oxygen species responses, cytokine signaling, and fatty acid metabolism. In the off-BTKi we prominently found enriched gene sets regulated by TP53 and related to DNA repair, cell cycle checkpoints, hypoxia and apoptosis. Consistent with an oxidative stress response we identified upregulation of NRF2, NFE2, TCF11, and BACH motif gene sets. The apparent more pronounced transcriptional response to venetoclax in the off-BTKi group prompted us to look more closely at changes in gene. At baseline, expression of BCL2L2 and NOXA was significantly lower in the off-BTKi patients in comparison to the “others” patient group. Surprisingly, on the 50mg dose expression of both the pro-apoptotic and anti-apoptotic BCL2 family members was significantly increased in the off-BTKi group in comparison to the others group; at the 100mg dose only pro-apoptotic genes remained significantly overexpressed in the off-BTKi group. In conclusion, venetoclax induced a distinct and prominent transcriptional response in CLL patients indicating broad impact on tumor biology, even at doses below 400mg. Intriguingly, CLL cells from patients who had progressed on BTKi therapy and switched to venetoclax showed a more extensive stress response that included activation of DNA damage and anti-oxidant pathways. It will be interesting to correlate the identified transcriptional changes with the depth and durability of the venetoclax response.

The UBE2J2/UBE2K-MARCH5 Ubiquitination Machinery Regulates Apoptosis in Response to Venetoclax in Acute Myeloid Leukemia

Evasion of apoptosis is crucial for the growth, survival and chemoresistance of many cancer types, including acute myeloid leukemia (AML); thus, the reactivation of apoptosis can be exploited as a therapeutic approach. Apoptosis induction is mainly controlled by the balance between anti-apoptotic and pro-apoptotic BCL2 family proteins on the mitochondrial membrane. Venetoclax, a selective inhibitor antagonizing the anti-apoptotic protein BCL2, has emerged as a promising therapy in AML. Despite high response rates in combination with hypomethylating agents, however, some patients display upfront resistance, and most patients will ultimately relapse. Therefore, identification of synergistic targets for combination therapies with venetoclax is important for improving the clinical application of this drug. To systematically identify key genes that can modulate the venetoclax effect, we performed a genome-scale CRISPR-Cas9 screen in the AML cell line MV4-11. Consistent with previous reports, loss of the apoptosis effector BAX or the pro-apoptotic gene NOXA ( PMAIP1) caused venetoclax resistance, while sgRNAs targeting the anti-apoptotic genes BCLXL ( BCL2L1), BCL2L2 and BCL2A1 were significantly more depleted in venetoclax-treated cells. Furthermore, depletion of the E2 ubiquitin-conjugating enzymes, UBE2J2 and UBE2K, ranked highly among the venetoclax sensitizers. We validated that deletion of either UBE2J2 or UBE2K increased induction of apoptosis in multiple AML cell lines and patient-derived xenograft (PDX) cells upon venetoclax treatment and was deleterious as single gene perturbation in some models. Exploiting the Broad Institute Cancer DepMap dataset, which includes genome-scale CRISPR-Cas9 screens in over 1000 cancer cell lines, revealed that dependency on UBE2J2 or UBE2K significantly correlated with a dependency on the E3 ligase MARCH5. We previously identified the repression of MARCH5 as a strong inducer of apoptosis in AML. Since E2s coordinate with ubiquitin E3 ligases to execute ubiquitination, this DepMap result suggested that UBE2J2 and UBE2K serve as MARCH5 E2 partners. To test this hypothesis, we utilized NanoBiT technology, a structural complementation reporter system, to detect protein interactions between MARCH5 and the E2 candidates. LgBIT and SmBIT, two split subunits of luciferase, were fused with MARCH5 and the E2 proteins, respectively. The luminescent signal was activated upon the co-expression of LgBIT-MARCH5 with either of the SmBIT-tagged E2 proteins but not an empty vector, suggesting that MARCH5 and UBE2J2/UBE2K constitute ubiquitination machinery that regulates apoptosis in AML. We next tested the possible redundancy between the two E2s and showed that the double knockout of UBE2J2/ UBE2K further enhanced the venetoclax sensitivity. MARCH5 depletion results in increased NOXA expression, an important node in dictating venetoclax response. Several reports indicated that NOXA is also a critical downstream mediator of MARCH5. In contrast, we previously showed that MARCH5 can regulate apoptosis independently of NOXA in AML. To reassess the role of NOXA and other pro-apoptotic proteins in MARCH5-mediated apoptosis, we conducted an unbiased CRISPR rescue screen in a MARCH5-dTAG degradation system derived from PDX17-14, a complex karyotype, MLL-AF10 PDX model. BAX was the top rescuing target, emphasizing that apoptosis induction is the main mechanism accounting for the growth inhibition of MARCH5-depleted cells. However, depletion of other pro-apoptotic BCL2 members, including NOXA and BIM, did not rescue MARCH5 depletion in this screen consistent with our previously published data. Here, we confirmed that NOXA KO does not rescue MARCH5 depletion in additional AML models. Similarly, KO of UBE2J2 or UBE2K can repress AML cell growth and increase venetoclax sensitivity even in the context of NOXA KO. Our study highlights that UBE2J2 and UBE2K are two important functional partners of MARCH5 in regulating apoptosis in AML cells and can serve as additional targets for enhancing venetoclax efficacy. Unbiased screening and low-throughput target validation further emphasize that the MARCH5 ubiquitination machinery regulates apoptosis in AML cells largely in a NOXA-independent manner. Additional studies are needed to dissect the MARCH5/UBE2J2/UBE2K complex-mediated apoptosis regulation in AML.

Mutant N- and K- Ras Show Distinct Effects on Growth, Signaling and Drug Response in CRISPR-Cas Edited Alleles of a Myeloma Cell Line

Myeloma is well known to have significant genetic heterogeneity among patients' tumors, affecting both progression as well as therapeutic responses. Moreover, subclonal diversity can lead to selective outgrowths that are influenced by new mutations, as well as the bone marrow microenvironment and therapeutic exposures. The ras family of genes (N -and K-) are the most common mutations (40-50% ) in myeloma. Notably, ras mutations occur among a variety of other co-existing chromosomal abnormalities. Ras mutations were found to be more commonly associated with cyclin D1 high expression (e.g., resulting from 11;14 translocations). We and others have also shown that rasmutations may not be disease-initiating but may arise during disease progression and relapse; or are subclonal events that impart selective growth advantage and emerging drug resistance. There is evidence that activating mutations (codons 12,13, 61) in K- ras may have different clinical impact than activating mutations in N- ras yet there are conflicting clinical outcomes that may be due to therapies analyzed or differences in other co-existing genetic mutations. Previous studies using transfection of genes with targeted mutations show random integration and often in multiple copies, thus, limiting interpretations. We chose to create N ras61 and K ras12 mutations by CRISPR-cas directed edits in a single endogenous ras allele in a myeloma cell line (FLAM76) that initially had two wild type alleles (and an 11;14 translocation). We then compared the impact of these edits on growth, signaling, gene expression, and drug responses. We find both the mutN ras and mutK ras edited cells have increased proliferation compared to wild type. However, we find that the N ras mutation increases MAPK signaling and downstream gene activation to a greater extent than the K rasmutation. Based on RNAseq analysis, a MAPK gene activation score was higher in mutN ras edited cells than mutK ras edited cells. Further, we see distinct gene expression patterns between mutN ras versus mutK ras edited cells (See heat map Fig) impacting distinct pathways. Notably, mutNras cells have much higher NOXA expression, increasing sensitivity to apoptosis-inducing drugs (e.g., proteasome inhibitors); and Kras induces anti-apoptotic Bcl-xL, reducing sensitivity. We have conducted a high throughput drug screen with 84 drugs, and while some drugs are active against both mutN ras and mutK ras edits (Proteasome inhibitors), we have identified drugs with differential response to K versus N ras mutations. For example, mutK ras FLAM76 cells are more resistant to proteasome inhibitors; and particularly more resistant to HSP inhibitors and HDAC inhibitors compared to wild type or mutN ras cells. In contrast, mutK ras cells are more sensitive to PikFyve and IL-12 inhibitors than mutNras or wild type cells. Gene expression analysis shows that mutKras cells result in more downregulation of apoptotic genes and higher upregulation of cell survival genes, as well as ER stress pathway genes. Our results suggest that treating myelomas containing K versus N ras activating mutations may benefit from understanding distinct pathway activations and targeting selective drug therapies. We are currently testing new pan-Ras inhibitors to determine if there are distinct responses between our N- and K- ras edited cells, and we are expanding testing to a wider panel of myeloma cell lines containing ras mutations in the context of other genetic abnormalities. Details of gene expression distinctions, pathways, and drug responses that distinguish mutN ras and mutK ras in myeloma will be presented.

The chromatin remodeling protein BRG1 contributes to liver ischemia-reperfusion injury by regulating NOXA expression

AimsHepatic ischemia-reperfusion injury (IRI) is a common complication secondary to liver transplantation. Extensive death of hepatocytes, typically in the form of apoptosis, is observed in and contributes to IRI. In the present study we investigated the role of BRG1 (encoded by Smarca4), a chromatin remodeling protein, in the pathogenesis of liver IRI focusing on the transcriptional mechanism and translational potential. MethodsSmarca4f/f mice were crossed to Alb-Cre mice to generate hepatocytes-specific BRG1 knockout mice (CKO). Alterations in cellular transcriptome were evaluated by RNA-seq. ResultsBRG1 expression was up-regulated in liver tissues of mice subjected to I/R and in hepatocytes exposed to hypoxia-reoxygenation (H/R). Compared to wild type (WT) littermates, the BRG1 CKO mice displayed significant amelioration of liver injury following ischemia-reperfusion as evidenced by decreased ALT/AST levels and cell apoptosis. Primary hepatocytes isolated from the CKO mice were protected from H/R-induced apoptosis compared to those from the WT mice. RNA-seq analysis revealed phorbol-12-myristate-13-acetate-induced protein 1 (PMAIP1, also known as NOXA) as a novel target for BRG1. Consistently, NOXA knockdown attenuated liver IRI in mice. More importantly, administration of a small-molecule BRG1 inhibitor (PFI-3) protected the mice from liver IRI. ConclusionsOur data uncover a pivotal role for BRG1 in liver IRI and suggest that targeting BRG1 with small-molecule inhibitors can be considered as a reasonable therapeutic strategy.

GPER deletion triggers inhibitory effects in triple negative breast cancer (TNBC) cells through the JNK/c-Jun/p53/Noxa transduction pathway

The G protein-coupled estrogen receptor (GPER) mediates estrogen action in different pathophysiological conditions, including cancer. GPER expression and signaling have been found to join in the progression of triple-negative breast cancer (TNBC), even though controversial data have been reported. In present study, we aimed at providing new mechanistic and biological discoveries knocking out (KO) GPER expression by CRISPR/Cas9 technology in MDA-MB-231 TNBC cells. GPER KO whole transcriptome respect to wild type (WT) MDA-MB-231 cells was determined through total RNA sequencing (RNA-Seq) and gene ontology (GO) enrichment analysis. We ascertained that anti-proliferative and pro-apoptotic gene signatures characterize GPER KO MDA-MB-231 cells. Thereafter, we determined that these cells exhibit a reduced proliferative, clonogenic and self-renewal potential along with an increased mitochondria-dependent apoptosis phenotype. In addition, we recognized that decreased cAMP levels trigger the JNK/c-Jun/p53/Noxa axis, which in turn orchestrates the pro-apoptotic effects observed in GPER KO cells. In accordance with these data, survival analyses in TNBC patients of the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) dataset indicated that high Noxa expression correlates with improved outcomes in TNBC patients. Furthermore, we demonstrated that GPER KO in TNBC cells impairs the expression and secretion of the well-acknowledged GPER target gene named CTGF, thus resulting in the inhibition of migratory effects in cancer-associated fibroblasts (CAFs). Overall, the present study provides novel mechanistic and biological insights on GPER KO in TNBC cells suggesting that GPER may be considered as a valuable target in comprehensive therapeutic approaches halting TNBC progression.

NOXA expression is downregulated in human breast cancer undergoing incomplete pathological response and senescence after neoadjuvant chemotherapy

Neoadjuvant chemotherapy (NAC) is a frequently utilized approach to treat locally advanced breast cancer, but, unfortunately, a subset of tumors fails to undergo complete pathological response. Apoptosis and therapy-induced senescence (TIS) are both cell stress mechanisms but their exact role in mediating the pathological response to NAC is not fully elucidated. We investigated the change in expression of PAMIP1, the gene encoding for the pro-apoptotic protein, NOXA, following NAC in two breast cancer gene datasets, and the change in NOXA protein expression in response to NAC in 55 matched patient samples (pre- and post-NAC). PAMIP1 expression significantly declined in post-NAC in the two sets, and in our cohort, 75% of the samples exhibited a downregulation in NOXA post-NAC. Matched samples that showed a decline in NOXA post-NAC were examined for TIS based on a signature of downregulated expression of Lamin-B1 and Ki-67 and increased p16INK4a, and the majority exhibited a decrease in Lamin B1 (66%) and Ki-67 (80%), and increased p16INK4a (49%). Since our cohort consisted of patients that did not develop complete pathological response, such findings have clinical implications on the role of TIS and NOXA downregulation in mediating suboptimal responses to the currently established NAC.

MDA5 Enhances Invasive Candida albicans Infection by Regulating Macrophage Apoptosis and Phagocytosis/Killing Functions.

Candida albicans is a common opportunistic pathogenic fungus. The innate immune system provides the first-line host defense against fungal infection. Innate immune receptors and downstream molecules have been shown to play various roles during fungal infection. The innate immune receptor MDA5, encoded by the gene Ifih1, enhances host resistance against viral and Aspergillus fumigatus infection by inducing the production of interferons (IFNs). However, the role of MDA5 in C. albicans infection is still unclear. Here, we found that the gene expression levels of IFIH1 were significantly increased in innate immune cells after C. albicans stimulation through human bioinformatics analysis or mouse experiments. Through in vivo study, MDA5 was shown to enhance host susceptibility to C. albicans infection independent of IFN production. Instead, MDA5 exerted its influence on macrophages and kidneys by modulating the expression of Noxa, Bcl2, and Bax, thereby promoting apoptosis. Additionally, MDA5 compromised killing capabilities of macrophage by inhibition iNOS expression. The introduction of the apoptosis inducer PAC1 further impaired macrophage functions, mimicking the enhancing effect of MDA5 on C. albicans infection. Furthermore, the administration of macrophage scavengers increased the susceptibility of Ifih1-/- mice to C. albicans. The founding suggests that MDA5 promote host susceptibility to invasive C. albicans by enhancing cell apoptosis and compromising macrophage functions, making MDA5 a target to treat candidiasis.

NOXA Accentuates Apoptosis Induction by a Novel Histone Deacetylase Inhibitor.

Epigenetic modifiers of the histone deacetylase (HDAC) family are often dysregulated in cancer cells. Experiments with small molecule HDAC inhibitors (HDACi) have proven that HDACs are a vulnerability of transformed cells. We evaluated a novel hydroxamic acid-based HDACi (KH16; termed yanostat) in human pancreatic ductal adenocarcinoma (PDAC) cells, short- and long-term cultured colorectal cancer (CRC) cells, and retinal pigment epithelial cells. We show that KH16 induces cell cycle arrest and apoptosis, both time and dose dependently in PDAC and CRC cells. This is associated with altered expression of BCL2 family members controlling intrinsic apoptosis. Recent data illustrate that PDAC cells frequently have an altered expression of the pro-apoptotic BH3-only protein NOXA and that HDACi induce an accumulation of NOXA. Using PDAC cells with a deletion of NOXA by CRISPR-Cas9, we found that a lack of NOXA delayed apoptosis induction by KH16. These results suggest that KH16 is a new chemotype of hydroxamic acid HDACi with superior activity against solid tumor-derived cells. Thus, KH16 is a scaffold for future research on compounds with nanomolar activity against HDACs.

The Effect of Simvastatin on the Dynamics of NF-κB-Regulated Neurodegenerative and Neuroprotective Processes in the Acute Phase of Ischemic Stroke

Statins are lipid-lowering drugs that act by inhibiting 3-hydroxy-3-methylglutaryl coenzyme A reductase, a rate-limiting enzyme in cholesterol biosynthesis. Animal studies have shown neuroprotective effects of statins in cerebral stroke. However, the underlying mechanisms are not fully understood. The nuclear factor-kappa B (NF-κB) transcription factor is involved in the regulation of apoptosis in stroke. Different dimers of NF-κB regulate the gene expression of proteins involved in both neurodegeneration and neuroprotection. We aimed to determine whether simvastatin improves stroke outcome via inhibition of the RelA/p65-containing subunit and downregulation of stroke-induced pro-apoptotic genes or via activation of NF-κB dimers containing the c-Rel subunit and upregulation of anti-apoptotic genes during the acute stroke phase. Eighteen-month-old Wistar rats, subjected to permanent MCAO or sham surgery, were administered simvastatin (20 mg/kg b.w.) or saline for 5 days before the procedure. Stroke outcome was determined by measuring cerebral infarct and assessing motor functions. The expression of NF-κB subunits in various cell populations was investigated using immunofluorescence/confocal microscopy. RelA and c-Rel were detected by WB. The NF-κB-DNA binding activity was investigated using EMSA, and expression of Noxa, Puma, Bcl-2, and Bcl-x genes was analyzed by qRT-PCR. Results showed a 50% infarct size reduction and significant motor function improvement in the simvastatin-treated animals which correlated with a decrease in RelA and a transient increase in the c-Rel level in the nucleus, normalization of the NF-κB-DNA binding activity, and downregulation of the NF-κB-regulated genes. Our results provide new insights into the statin-mediated neuroprotective action against stroke based on NF-κB pathway inhibition.