Overexpression Of Bcl-2 Research Articles

CD3xCD19 Dart Treatment Is Efficient in Venetoclax Resistant CLL and Reverses T Cell Dysfunction

Intro - Agents targeting the apoptosis pathway, like the Bcl-2 inhibitor venetoclax, are highly effective in chronic lymphocytic leukemia (CLL). However, not all patients experience deep responses and acquired resistance has already been described. T cell mediated lysis is another tool currently exploited in hematologic malignancies. In contrast to acute lymphoblastic leukemia (ALL) however, efficacy of autologous based T cell therapy, such as CAR T cells, in CLL has been low. This is linked to a CLL mediated acquired T cell dysfunction. Bispecific T cell engagers targeting CD19 are successfully applied in ALL, but whether it overcomes the acquired T cell dysfunction in CLL is unknown. We therefore tested efficacy of a CD3xCD19 Dual Affinity Re-Targeting molecule (DART) in CLL. Since it has been observed that bispecific antibodies can overcome deficient synapse formation in CLL (Robinson et al, 2018) and based on our assumption that T cell mediated lysis differs from venetoclax-mediated killing, we hypothesized that usage of a CD3xCD19 DART in CLL overcomes T cell dysfunction and will be effective against venetoclax resistant CLL. Methods - Co-culture of CLL derived or aged-matched healthy donor (HD) CD4+ and/or CD8+ T cells with (CD40 activated) primary CLL or CD19+ cell lines JeKo-1 or Ramos in presence of CD3xCD19 (JNJ-64052781), CD3xFITC, anti-CD3/28 antibodies was performed. R esults - JeKo-1 cells were highly sensitive to CD3xCD19 mediated HD T cell killing with close to 70% of lysis in a concentration of 10ng/mL using an E:T ratio of 4:1. In the same conditions, primary CLL cells proved sensitive for CD3xCD19 mediated HD T cell killing with 50% of lysis. Killing was observed irrespective of IGHV mutation or chemorefractory status. We next compared HD with CLL-derived T cells by measuring activation levels between direct TCR (anti-CD3/CD28) and CD3xCD19 stimulation. As described, TCR stimulation resulted in impaired CLL CD4+ and CD8+ T cell activation and proliferation when compared to HD. In contrast, treatment of CLL derived PBMCs with CD3xCD19 did not resulted in dysfunctional CLL-derived T cell responses (Fig 1A-C). Consistently, co-culture of CLL derived CD4+, CD8+ or a combination with either JeKo-1 or allogeneic CLL cells in the presence of CD3xCD19 resulted in significant cytotoxicity (Fig. 1D). In the allogeneic setting, cytotoxic capacity of CD4+ T cells was similar to their CD8+ counterparts. When targeting autologous CLL, a benefit was observed when both CD4+ and CD8+ T cells were present (Fig. 1D). We then studied whether venetoclax resistant CLL cells could be targeted by CD3xCD19 mediated T cell killing. Bcl-2 overexpressing Ramos were equally lysed in presence of the CD3xCD19 DART as their wildtype counterpart, indicating that Bcl-2 expression does not inhibit CD3xCD19 mediated cell death. Following CLL cell stimulation by CD40 ligation, anti-apoptotic Bcl-XL, Bfl-1 and Mcl-1 are highly induced (Thijssen et al., 2015) resulting in venetoclax resistance (Fig 1E). Nevertheless, CD40L stimulated CLL cells were as efficiently lysed upon CD3xCD19 treatment as unstimulated CLL. (Fig 1F). This indicates that an augmented apoptotic threshold does not impact efficacy of CD3xCD19. Further examination of the mechanism of CD3xCD19 mediated killing showed that lysis depended on granzymes, as blocking granule exocytosis prevented cell death. Independence of the mitochondrial apoptotic pathway was shown by equal sensitivity to CD3xCD19 mediated T cell lysis comparing BAX/BAK knockout Jeko-1 cells to the parental cell line. Also, caspase blockage did not inhibit cell death, pointing to apoptosis independent killing. In concordance, PARP cleavage could only be detected when caspase activity was not blocked. Conclusion - This is the first report describing reversal of CLL mediated T cell dysfunction by applying a CD3xCD19 DART. Furthermore, it shows that venetoclax resistant CLL can still be efficiently targeted by T cells, in a non-apoptotic fashion. These results imply that T cell mediated therapy could be used alongside venetoclax. Figure 1 Disclosures Eldering: Celgene: Research Funding; Roche: Research Funding; Janssen Pharmaceutical Companies: Research Funding. van der Windt:Genmab: Employment. Kater:Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Acerta: Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche/Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Research Funding.

Recapitulation of Prognostic Mutational Subtyping Utilizing F1H in GOYA and CAVALLI and the Potential to Highlight Benefit of Targeted Therapy in De Novo DLBCL

Introduction: Diffuse large B-cell lymphoma (DLBCL), is a genetically and clinically heterogeneous disease. Building on existing clinical prognostic scores in the era of genomic risk modeling is critical to improve patient (pt) outcomes and inform future trials. With advances in next-generation sequencing, the interrogation of genetic drivers to refine prognostic subgroups over and above histological cell of origin (COO) subtypes has been of keen interest. The application of an unsupervised prognostic clustering approach, non-negative matrix factorization (NMF) algorithm, to whole exome sequence (WES) data has led to identification of six distinct molecular clusters with potential clinical relevance (Chapuy et al. Nature Med, 2018). Here, we applied the NMF clustering approach to targeted exome-sequencing data utilizing the FoundationOne HemeTM panel and analyzed RNA gene expression data from the randomized Phase 3 GOYA and Phase 1b/2 CAVALLI studies in de novo DLBCL. Methods: Biopsy samples, clinical outcomes (overall survival [OS], progression-free survival [PFS]), and targeted exome-sequencing data were available for 499 pts from the GOYA trial (NCT01287741; ITT=1418; obinutuzumab [G]-CHOP vs rituximab [R]-CHOP) and 106 pts from the CAVALLI trial (NCT02055820; ITT=267, venetoclax+[G/R]-CHOP). The FoundationOne HemeTM panel of 465 genes (F1H, Foundation Medicine Inc. [FMI]) was used to define single nucleotide variants, copy number amplifications, and rearrangements. Clustering methodology was implemented using the R package NMF (Gaujoux and Seoighe, BMC Bioinformatics 2010). All models were adjusted for treatment (where applicable), age, International Prognostic Index (IPI), sex, COO (NanoString), and BCL2 by IHC. Results: Applying the NMF algorithm, de novo pts in GOYA clustered similarly into four of the six groups recently reported by Chapuy et al.: MYD88/CD79B, BCL2/EZH2, NOTCH2/TNFAIP3, and no mutations (Fig 1A). The mutation profiles and COO subset distribution were also similar to those previously described. Furthermore, the MYD88/CD79B and BCL2/EZH2 groups remained associated with inferior clinical outcomes (Fig 1B), with observed poor prognosis over the NOTCH2/TNFAIP3 and no mutations groups (OS: hazard ratio [HR], 2.0; 95% CI: 0.97, 3.90; PFS: HR, 1.60; 95% CI: 0.93, 2.80), even when adjusting for known prognostic factors. Application of NMF to pts treated with the BCL2-inhibitor venetoclax in combination with R-CHOP (CAVALLI) yielded only MYD88/CD79B- and BCL2/EZH2-like groups due to enrollment. However, in contrast to the BCL2/EZH2 subgroup reported in both Chapuy et al. and GOYA, these pts demonstrated a trend towards improved outcomes over MYD88/CD79B (OS: HR, 0.12; 95% CI: 0.01, 1.60; PFS: HR, 0.11; 95% CI: 0.01, 1.10; Fig 1D). The difference between studies is potentially explained by the ability of venetoclax treatment to overcome the adverse prognostic impact of BCL2 over-expression. BCL2 IHC positive (BCL2+) pts were enriched in high-risk groups in GOYA (MYD88/CD79B and BCL2/EZH2) (odds ratio [OR], 5.0; 95% CI: 3.13, 8.33) and CAVALLI (MYD88/CD79B) (OR, 2.0; 95% CI: 0.63, 6.67). In GOYA, a prognostic trend for BCL2+ status is observed within low-risk pts (OS BCL2+ HR, 1.9; 95% CI: 0.55, 6.3; PFS BCL2+ HR, 2.7; 95% CI: 1.0, 6.8), but not within high-risk groups (OS BCL2+ HR, 0.55; 95% CI: 0.29, 1.00; PFS BCL2+ HR, 0.76; 95% CI: 0.45, 1.30; Fig 1C). Additional genetic risk models will be presented, e.g. as per Reddy et al. (Cell, 2017), as well as differential expression analysis. Conclusions: Important findings from the present work include: 1) utility of targeted mutational data from F1H in recapitulating molecular clusters, previously identified using WES, that further subclassify established prognostic groups such as COO and IPI; and 2) a trend towards improved survival outcomes for patients in the BCL2/EZH2 cluster upon treatment with the BCL2 inhibitor venetoclax. This highlights the potential for utilizing novel genetic signatures as a means of identifying patients suitable for targeted therapies in the era of personalized health care. Disclosures Kim: Genentech, Inc.: Employment. Fan:Genentech, Inc.: Employment. Bolen:F. Hoffmann-La Roche: Equity Ownership; Genentech, Inc.: Employment. Bazeos:Roche Products Ltd: Employment, Equity Ownership. Jiang:Genentech: Employment, Equity Ownership; F. Hoffman-La Roche: Equity Ownership. Balasubramanian:Roche-Genentech: Employment; F. Hoffman-La Roche: Equity Ownership. Balakrishnan:Genentech, Inc.: Employment. Knapp:F. Hoffmann-La Roche Ltd: Employment. Humphrey:F. Hoffmann-La Roche Ltd: Employment. Nielsen:F. Hoffmann-La Roche Ltd: Employment, Equity Ownership. Venstrom:F. Hoffmann-La Roche Ltd: Employment. Hernandez:Genentech, Inc.: Employment, Equity Ownership. Paulson:F. Hoffman-La Roche: Equity Ownership; Genentech: Employment.

Efficacy of the Pan-Bcl-2 Inhibitor (Obatoclax) As a Single Agent to Treat Myeloproliferative Neoplasm in JAK2V617F Murine Transplantation Model

Background: The Jak2V617F mutation represents the most common molecular abnormality present in Philadelphia chromosome-negative Classical Myeloproliferative Neoplasms (Ph-neg MPNs). The differential expression of genes/proteins between the hematopoietic stem cells (HSC) and the more committed bone marrow progenitors may reveal, at least in part, the contribution of each cell type to the pathogenesis of the disease. Recently, it has been shown that the deregulation of molecules of the BCL2 family in MPN increases the survival of the transformed hematopoietic stem and progenitor cells (HSPCs), which may contribute the progression of the disease (Figueiredo-Pontes et al, Blood #122:2852 2013). In this context, it is possible that the inhibition of Bcl-2 proteins may increase the sensibility of transformed HSPC to apoptosis leading to the control of myeloid proliferation and potential reduction of the Jak2 mutation allele burden. To date, the treatment of Ph-neg MPNs remains non-curative despite the development of Jak2 inhibitors, which improve the clinical features but fail to eliminate leukemic initiating cells. Aim: Therefore, we aimed to study if the modulation of Bcl-2 proteins could have an impact in the control of MPN. Methods: Wild type CD45.1 C57BL/6 mice (age: 10-12 weeks) were transplanted with 5x106 total bone marrow from CD45.2 C57BL/6 mice (age: 10-12 weeks old) harboring the Jak2V617F mutation after lethal irradiation. All transplanted mice presented at least 80% of chimerism and developed MPN features (increased red blood cells (RBC), white blood cells (WBC) and platelets counts, elevated hematocrit and hemoglobin) 6 weeks post-transplantation. Treatment with vehicle (5% dimethylacetamide 0.5% methylcellulose, n=5) or Bcl-2 inhibitor (GX15-070 - the BH3 mimetic obatoclax 3 mg/Kg/day, n=6) once a day by oral gavage was started 7 weeks after transplant for a time period of 4 weeks. Spleen and bone marrow were collected from euthanized mice to evaluate both chimerism and frequency of HSC by using fluorescence-conjugated antibodies against CD45.1, CD45.2, Ter119, CD19, CD4, CD8 CD3, CD71, cKit, Sca-1, CD48, CD150, CD16/32 and CD34. Data are represented as means and standard error of mean (mean ± SEM). The statistical significance was defined as p≤0.05 and analyzed by the Mann Whitney test. Results: Mice treated with obatoclax exhibited a reduction of 32% in spleen weight compared to the vehicle group, but did not reach statistical significance (Control=0.19 ±0.01 g vs Obatoclax=0.13 ±0.03 g; p=0.3). No differences were observed in blood counts (RBC, WBC or platelets) between vehicle and obatoclax-treated mice. In addition, treatment with the pan-Bcl-2 inhibitor did not affect the number of Lineage negative, Sca-1+, c-Kit+ (LSK) cells (Control=5.8x104±2.8x104vs Obatoclax=4.7x104±1.2x104, p=0.9), MPP (Control=1.9x104±2.8x104vs Obatoclax=1.9x104±8.5x103, p=0.9), ST-HSC (Control=2.8x104±1.9x104vs Obatoclax=4.4x104±4x103, p=0.2) and LT-HSC (Control=9.8x103±4.7x103vs Obatocalx=1x104±2.4x103, p=0.9). Similarly, no differences were observed in the number of myeloid progenitors between the groups: (Control=6x105±1.7x105vs Obatoclax=1x106±1.9x105, p=0.1), MEP (Control=2.7x105±7.7x104vs Obatoclax=2.9x105±6.3x104,p=0.9), CMP (Control=9.8x104±2.1x104vs Obatoclax=2.3x105±4.4x104, p=0.06) and GMP (Control=1.5x105±6.5x104vs Obatoclax=3.6x105±8.8x104, p=0.08). The frequency of erythroid progenitors in the BM (Early progenitors, Control=18.0% ±3% vs Obatoclax=26.0%±1.4%, p=0.052, and late erythroid cells, Control=38.4%±9.5% vs Obatoclax=49.0%±2.7%, p=0.4) was also not altered by Bcl-2 inhibition. Conclusion: Our findings are in accordance with a phase II clinical trial demonstrating that obatoclax has no clinical activity in patients with Myelofibrosis. (Parikh et al, CLML 2010). Thus, we concluded that obatoclax did not reduce MPN burden in our model, when used as a single agent administrated orally. Yet, we cannot exclude the possibility that that the modulation of Bcl-2 overexpression at MPN initiating cells level could render these cells sensitive to the JAK2 inhibition. Hence, further studies will be necessary to assess whether the combination of obatoclax with other drugs such as JAK2 inhibitors could result in improved therapeutic response. Disclosures Mullally: Janssen: Research Funding. Kobayashi:Taiho Pharmaceutical: Research Funding; MiNA therapeutics: Research Funding; Pfizer: Consultancy; Ono Pharmaceutica: Consultancy; Chugai Pharmaceutical: Honoraria; Boehringer Ingelheim: Honoraria; Roche Diagnostic: Honoraria. Figueiredo-Pontes:Novartis: Honoraria.

Rituximab and Obinutuzumab Induce Direct B-Cell Death Via B-Cell Receptor (BCR) Signaling, but Rituximab Elicits Stronger BCR-Derived Pro-Survival Signals Diminishing Apoptosis

The anti-CD20 monoclonal antibody (mAb) rituximab in combination with chemotherapy has improved outcomes for patients with CD20+ B-cell lymphoma. Obinutuzumab was developed as an anti-CD20 mAb with enhanced induction of direct B-cell death and antibody-dependent cellular cytotoxicity. In direct comparison to rituximab, improved outcomes with obinutuzumab have been observed for chronic lymphocytic leukemia (CLL) and follicular lymphoma (FL), but not for diffuse large B-cell lymphoma (DLBCL). The molecular basis behind these discrepancies remained unknown. Our aim was to define intracellular signaling events in the induction of direct B-cell death upon rituximab and obinutuzumab treatment. We performed LC-MS/MS phosphoproteomics on SU-DHL4 lymphoma cells treated with rituximab or obinutuzumab (0, 1 and 24 h time-points). Kinase activities were inferred by kinase-substrate enrichment analysis linking kinases to their phosphosite substrates. Immunoblotting was done where necessary to understand the nature of specific signaling events. The activity of 41 and 40 protein kinases was altered upon rituximab or obinutuzumab treatment, respectively, 32 of which were affected by both mAbs. Pathway enrichment analyses revealed up-regulated B-cell receptor (BCR) signaling and down-regulated cell cycle progression with both treatments. To delineate differences between the two mAbs we investigated signaling events in the BCR cascade in more detail. The proximal BCR kinase SYK was strongly phosphorylated on Tyr352 by both mAbs, whereas SYK Tyr525/526 phosphorylation, essential for normal kinase function, was detectable at a low level only with rituximab. Phospho-SYK Tyr352 in the absence of phospho-SYK Tyr525/526 has been associated with autoimmune checkpoint activation and B-cell apoptosis. Both mAbs phosphorylated the key BCR kinase BTK on Tyr551 but not on Tyr223. The latter site was essential for full kinase activity and, in line with incomplete BTK activation, downstream PCLγ2 phosphorylation was delayed. No evidence was found for PLCγ2 dependent de-phosphorylation of NFAT2/NFAT3 and PKCβ activation. Instead, we observed NFAT1 de-phosphorylation and PKCδ activation, both of which have been associated with an anergic B-cell reaction. The MAPK pathway and MYK were strongly activated by both mAbs serving as potent inducers of cell death in the absence of pro-survival signals. To determine if pro-survival signals were generated, we assessed PI3K and NF-κB activation. While we found no evidence for NF-κB activation, differences in the phosphorylation status of PI3K binding sites on CD19 and BCAP suggest stronger PI3K activation by rituximab than obinutuzumab. In keeping with this, the PI3K effector AKT was much more strongly activated by rituximab. AKT activity was likely also increased by Ca2+-flux following rituximab but not obinutuzumab treatment and modified by differential SHIP1 phosphorylation, a phosphatase that hydrolyzes PI3K-generated PIP3. A central AKT target in apoptosis regulation is BAD. We found increased BAD Ser99 and Ser118 phosphorylation only after rituximab treatment. Rituximab thereby sequestered BAD in the cytosol and impaired its inhibitory effects on anti-apoptotic BCL-2 and BCL-xL. Moreover, signaling events protecting against caspase 2 activation were exclusively found for rituximab. In summary, here we show that rituximab and obinutuzumab both induce BCR signaling capable of promoting B-cell apoptosis. However, rituximab generates signals that increase anti-apoptotic BCL-2 effects and diminish B-cell death, whereas obinutuzumab more readily overcomes resistance through BCL-2 overexpression characteristic for FL and CLL. The differences found between rituximab and obinutuzumab are likely less pronounced in cases with strong tonic BCR signaling, which is more prevalent in DLBCL. Thus, our findings provide a mechanistic rationale for the observations made in clinical trials comparing rituximab and obinutuzumab head-to-head. We believe that our results will help to identify new patient subgroups benefitting from obinutuzumab and to better assess the role of anti-CD20 treatment in combination therapies. Disclosures Vilventhraraja: Janssen Pharmaceutical Companies: Employment. Döhner:AbbVie, Agios, Amgen, Astellas, Astex, Celator, Janssen, Jazz, Seattle Genetics: Consultancy, Honoraria; AROG, Bristol Myers Squibb, Pfizer: Research Funding; Celgene, Novartis, Sunesis: Honoraria, Research Funding. Gribben:Acerta/Astra Zeneca: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding.

IRF4 Activity Is Required in Established Plasma Cells to Regulate Gene Transcription and Mitochondrial Homeostasis

The transcription factor interferon regulatory factor 4 (IRF4) is critical for the development, maintenance, and function of plasma cells. The mechanism by which IRF4 exerts its action in mature plasma cells has been elusive due to the death of all such cells upon IRF4 loss. While we identify apoptosis as a critical pathway for the death of plasma cells caused by IRF4 loss, we also determine that IRF4 did not regulate the intrinsic apoptotic pathway directly. By using an inducible IRF4 deletion system in the presence of the overexpression of anti-apoptotic BCL2, we identify genes whose expression is coordinated by IRF4 and that in turn specify plasma cell identity and mitochondrial homeostasis.

Pooled Genomic Screens Identify Anti-apoptotic Genes as Targetable Mediators of Chemotherapy Resistance in Ovarian Cancer.

High-grade serous ovarian cancer (HGSOC) is often sensitive to initial treatment with platinum and taxane combination chemotherapy, but most patients relapse with chemotherapy-resistant disease. To systematically identify genes modulating chemotherapy response, we performed pooled functional genomic screens in HGSOC cell lines treated with cisplatin, paclitaxel, or cisplatin plus paclitaxel. Genes in the intrinsic pathway of apoptosis were among the top candidate resistance genes in both gain-of-function and loss-of-function screens. In an open reading frame overexpression screen, followed by a mini-pool secondary screen, anti-apoptotic genes including BCL2L1 (BCL-XL) and BCL2L2 (BCL-W) were associated with chemotherapy resistance. In a CRISPR-Cas9 knockout screen, loss of BCL2L1 decreased cell survival whereas loss of proapoptotic genes promoted resistance. To dissect the role of individual anti-apoptotic proteins in HGSOC chemotherapy response, we evaluated overexpression or inhibition of BCL-2, BCL-XL, BCL-W, and MCL1 in HGSOC cell lines. Overexpression of anti-apoptotic proteins decreased apoptosis and modestly increased cell viability upon cisplatin or paclitaxel treatment. Conversely, specific inhibitors of BCL-XL, MCL1, or BCL-XL/BCL-2, but not BCL-2 alone, enhanced cell death when combined with cisplatin or paclitaxel. Anti-apoptotic protein inhibitors also sensitized HGSOC cells to the poly (ADP-ribose) polymerase inhibitor olaparib. These unbiased screens highlight anti-apoptotic proteins as mediators of chemotherapy resistance in HGSOC, and support inhibition of BCL-XL and MCL1, alone or combined with chemotherapy or targeted agents, in treatment of primary and recurrent HGSOC. IMPLICATIONS: Anti-apoptotic proteins modulate drug resistance in ovarian cancer, and inhibitors of BCL-XL or MCL1 promote cell death in combination with chemotherapy.

Microvesicle-containing miRNA-153-3p induces the apoptosis of proximal tubular epithelial cells and participates in renal interstitial fibrosis.

To uncover the role of microvesicle-containing (MV-containing) miRNA-153-3p in inducing the apoptosis of proximal tubular epithelial cells and RIF (renal interstitial fibrosis), and its potential mechanism. Mice were subjected to unilateral ureteral obstruction (UUO) to establish the in vivo RIF model. MVs were extracted from the obstructed kidney tissues of mice, to further isolate the RNAs. MiRNA-153-3p levels in RIF mice and MVs were determined. In vitro RIF model was constructed by TGF-β1 induction in NRK-52E and NRK-49F cells. The regulatory effect of miRNA-153-3p on the apoptosis of tubular epithelial cells was examined. Subsequently, potential target gene of miRNA-153-3p was predicted and identified. Rescue experiments were finally carried out to uncover the role of miRNA-153-3p/Bcl-2 in influencing RIF. MiRNA-153-3p was upregulated in mice undergoing UUO, MVs extracted from obstructed kidney tissues of mice and TGF-β1-induced NRK-52E and NRK-49F cells. The overexpression of miRNA-153-3p remarkably induced apoptosis in tubular epithelial cells. Bcl-2 was verified to be the target gene of miRNA-153-3p, and the Bcl-2 level was negatively regulated by miRNA-153-3p. Overexpression of Bcl-2 reversed the effect of miRNA-153-3p on inducing cell apoptosis. MV-containing miRNA-153-3p released by tubulointerstitial fibroblasts transmits to the proximal tubular epithelial cells via the damaged tubule basement membrane. It induces the apoptosis of proximal tubular epithelial cells by inhibiting Bcl-2 level and further aggravates RIF.

The Deubiquitinating Enzyme Inhibitor PR-619 Enhances the Cytotoxicity of Cisplatin via the Suppression of Anti-Apoptotic Bcl-2 Protein: In Vitro and In Vivo Study.

After chemotherapy for the treatment of metastatic bladder urothelial carcinoma (UC), most patients inevitably encounter drug resistance and resultant treatment failure. Deubiquitinating enzymes (DUBs) remove ubiquitin from target proteins and play a critical role in maintaining protein homeostasis. This study investigated the antitumor effect of PR-619, a DUBs inhibitor, in combination with cisplatin, for bladder UC treatment. Our results showed that PR-619 effectively induced dose- and time-dependent cytotoxicity, apoptosis, and ER-stress related apoptosis in human UC (T24 and BFTC-905) cells. Additionally, co-treatment of PR-619 with cisplatin potentiated cisplatin-induced cytotoxicity in UC cells and was accompanied by the concurrent suppression of Bcl-2. We also proved that Bcl-2 overexpression is related to the chemo-resistant status in patients with metastatic UC by immunohistochemistry (IHC) staining. In a xenograft mice model, we confirmed that PR-619 enhanced the antitumor effect of cisplatin on cisplatin-naïve and cisplatin-resistant UCs. Our results demonstrated that PR-619 effectively enhanced the cisplatin-induced antitumor effect via concurrent suppression of the Bcl-2 level. These findings provide promising insight for developing a therapeutic strategy for UC treatment.

Overexpression of BCL-2 in the Intestinal Epithelium Prevents Sepsis-Induced Gut Barrier Dysfunction via Altering Tight Junction Protein Expression.

Sepsis induces both intestinal hyperpermeability and epithelial apoptosis. While each has been implicated in mediating sepsis mortality, the relationship between these two processes is unclear. We hypothesized that preventing intestinal apoptosis would prevent gut barrier dysfunction. To test this hypothesis, transgenic mice that overexpress the anti-apoptotic protein Bcl-2 in the gut epithelium (Fabpl-Bcl-2 mice) and wild-type (WT) mice were subjected to sham laparotomy or cecal ligation and puncture and orally gavaged with fluorescein isothiocyanate conjugated-dextran (FD-4) 5 h before sacrifice. Serum FD-4 concentration was assayed to measure intestinal permeability, and jejunal tight junctions were assayed for mRNA and protein expression. Baseline FD-4 concentration was similar between WT and Fabpl-Bcl-2 mice. Intestinal permeability increased 6, 12, 24, and 48 h following sepsis in WT mice; however, FD-4 concentration was significantly lower at each timepoint in Fabpl-Bcl-2 mice. In addition, there were no statistically significant changes in permeability between septic and sham transgenic mice. Intestinal mRNA expression of claudin 3, claudin 5, and occludin was lower in septic Fabpl-Bcl-2 mice, while claudin 4 mRNA levels were higher in Fabpl-Bcl-2 mice. In contrast, no differences were detected in claudins 2, 7, 15, JAM-A, or ZO-1. Protein levels followed the same trend for all tight junction mediators different between WT and Fabpl-Bcl-2 mice except occludin was significantly higher in transgenic mice. Together these results demonstrate that decreasing intestinal epithelial apoptosis prevents hyperpermeability following sepsis via tight junction alterations which may be at least partially responsible for improved survival conferred by Bcl-2 overexpression.

MicroRNA dysregulation and multi-targeted therapy for cancer treatment.

We established that loss of miR-15a/16-1 genes on chromosome 13q14 is the most common alteration in Chronic Lymphocytic Leukemia (CLL) and that miR-15/16 are crucial negative regulator of BCL-2, an antiapoptotic gene overexpressed in most CLLs and in many other malignancies. We have also shown that miR-15/16 target ROR1, a cell surface receptor for Wnt5a which can enhance growth/survival of CLL cells. Interestingly, ROR1 is expressed by many cancers, but not by normal adult tissues. Moreover, Venetoclax, the anti-Bcl-2 drug, and Cirmtuzumab, the monoclonal antibody against ROR1, are synergistic in killing CLL cells. Since an additional miR-15/16 locus exists on chromosome 3q25 (miR-15b/16-2), we generated a knocked out mouse model to study its the role in cancer. We observed that the KO mice developed predominantly CLL. Thus, we generated a double knock out mouse model where both miR-15/16 loci were deleted. Surprisingly we observed that 77% of double KO mice developed Acute Myeloid Leukemia (AML). Based on these evidences, we anticipate that also AMLs with low miR-15/16 expression, overexpression of BCL2 and expression of ROR1, would show an excellent response to a combination therapy with Venetoclax and Cirmtuzumab, since both drugs target the same malignant cells that have lost miR-15/16.

Bovine ephemeral fever virus triggers autophagy enhancing virus replication via upregulation of the Src/JNK/AP1 and PI3K/Akt/NF-\u03baB pathways and suppression of the PI3K/Akt/mTOR pathway

Autophagy plays an important role in cellular response to pathogens. However, the impact of the autophagy machinery on bovine ephemeral fever virus (BEFV) infection is not yet determined. A recent study in our laboratory demonstrated that BEFV triggers simultaneously the PI3K/Akt/NF-κB and Src/JNK-AP1 pathways in the stage of virus binding to enhance virus entry. In this work, we report that BEFV induces autophagy via upregulation of the PI3K/Akt/NF-κB and Src/JNK/AP1 pathways in the early to middle stages of infection and suppresses the PI3K/Akt/mTOR pathway at the late stage of infection. To activate NF-κB, BEFV promotes degradation of IκBα and activates Akt to stimulate NF-κB translocation into the nucleus. Immunoprecipitation assays revealed that BEFV disrupts Beclin 1 and Bcl-2 interaction by JNK-mediated Bcl-2 phosphorylation, thereby activating autophagy. Overexpression of Bcl-2 reversed the BEFV-induced increase in the LC3 II levels. Suppression of autophagy either by knockdown of autophagy-related genes with shRNAs or treatment with a pharmacological inhibitor 3-MA reduced BEFV replication, suggesting that BEFV-induced autophagy benefits virus replication. Our results revealed that the BEFV M protein is one of the viral proteins involved in inducing autophagy via suppression of the PI3K/Akt/mTORC1 pathway. Furthermore, degradation of p62 was observed by immunoblotting, suggesting that BEFV infection triggers a complete autophagic response. Disruption of autophagosome-lysosome fusion by depleting LAMP2 resulted in reduction of virus yield, suggesting that formation of autolysosome benefits virus production.

2030P - Doxorubicin resistance: early and advanced tumors can use two different strategies based on initial and profound abnormalities in microRNA expression signature

BackgroundDoxorubicin is an anthracycline antibiotic that acts as a DNA intercalating agent, inhibiting the topoisomerase II and inducing the apoptotic cell death, mainly due to the accumulation of double-strand DNA breaks (DSB). This research aims to identify in what way the abnormalities in microRNA (miRNA) expression profile in cancer cells can confer them the resistance to doxorubicin. MethodsMiRNA targets within gene transcripts were predicted in silico using the TargetScan software. ResultsBinding sites for miRNAs miR-21, miR-96, miR-183 and miR-365, which are usually upregulated in cancer cells, were revealed in transcript of TOP2A gene encoding topoisomerase II-alpha. Transcripts of proapoptotic genes BID, BCL2L11 (BIM), BMF, BAX, BAK1, PMAIP1 (NOXA), and BBC3 (PUMA) as well as transcripts of tumor suppressor genes TP53 and PTEN carry targets for at least one of hyperexpressed miRNAs miR-19, miR-21, miR-23, miR-27, miR-29, miR-155, miR-181, miR-221/222 and miR-375. Downregulation of anti-onco-miRNAs let-7, miR-22, miR-34, miR-101, miR-125, miR-140, miR-143, miR-199, miR-200, miR-203, miR-204 and miR-205 allows overexpression of antiapoptotic genes BCL2, BCL2L1 (Bcl-XL), MCL1 and AKT1. In the same way, downregulation of the anti-onco-miRNAs can lead to overexpression of ABCA1/4/12, ABCB1 (MDR1), ABCB6, ABCC1 (MRP1) and ABCC3/5/8/11 genes encoding the ATP binding cassette (ABC) transporters. Moreover, multiple targets for the both up- and downregulated miRNAs were found in transcripts of XRCC5/6, PRKDC, LIG4, DCLRE1C, NHEJ1 (XLF), RAD50/51/51B/51D/52/54B/54L, MRE11A, NBN (NBS1), GEN1, ATM and ATR genes encoding the key elements of non-homologous end joining and homologous recombination pathways. ConclusionsObviously, in case of doxorubicin administration, strategy of early tumors consists in strengthening of miR-21 expression with the purpose to silence TOP2A gene and escape from immediate apoptotic death. Advanced tumors with more profound abnormalities in miRNA signature overexpress antiapoptotic genes as well as genes responsible for DSB repair and drug efflux and, therefore, can do without the TOP2A silencing. Legal entity responsible for the studyPalladin Institute of Biochemistry of the National Academy of Sciences of Ukraine. FundingHas not received any funding. DisclosureAll authors have declared no conflicts of interest.

Xanthones from the Bark of Garcinia xanthochymus and the Mechanism of Induced Apoptosis in Human Hepatocellular Carcinoma HepG2 Cells via the Mitochondrial Pathway.

Xanthones are important chemical constituents of Garcinia xanthochymus and varied bioactivities including cytotoxicity. However, their anti-tumor mechanism has remained unknown. Here, we isolated and identified a new xanthone named garciniaxanthone I (1) and five known compounds from the bark of G. xanthochymus. Their structures were elucidated by NMR analysis and HRESIMS. The anti-proliferation activities of all isolated compounds were evaluated on four human tumor cell lines (HepG2, A549, SGC7901, MCF-7). The results demonstrated that the anti-proliferation activity of xanthone was related to the number and location of prenyl groups. We further found that garciniaxanthone I (GXI) could induce HepG2 apoptosis and enhance the expression of cleaved caspase-8, caspase-9, and caspase-3. GXI could also increase Bax level and concurrently reduce the overexpression of Bcl-2, Bcl-XL, Mcl-1, and surviving in HepG2 cells. Moreover, GXI could inhibit cell migration of HepG2 cells by inhibiting the expressions of MMP-7 and MMP-9. In summary, our study suggests that GXI could induce HepG2 apoptosis via the mitochondrial pathway and might become a lead compound for liver cancer treatment.

CRISPR/Cas9 Knockout of Bak Mediates Bax Translocation to Mitochondria in response to TNFα/CHX-induced Apoptosis.

TNFα/CHX-induced apoptosis is dependent on caspase-8 activation and regulated by Bcl-2. However, the specific participants and precise mechanisms underlying this apoptotic pathway are poorly understood. The proapoptotic proteins Bak and Bax—members of the Bcl-2 family—are essential for the functioning of the mitochondrial apoptotic pathway. In this study, we used the CRISPR/Cas9 system to knockout Bak in the human SH-SY5Y cell line and determined the effects of this knockout on TNFα/CHX-induced apoptosis. Our data showed that overexpression of Bcl-2 dramatically prevented TNFα/CHX-induced apoptosis, and then pro-apoptotic protein Bak was downregulated and became more resistant to TNFα/CHX-induced apoptosis, because both TNFα/CHX-induced PARP cleavage and caspase activation were blocked in BAK−/− cells or using specific siRNA, whereas Bax was dispensable in TNFα/CHX-induced apoptosis, as evidenced using specific siRNA. Bax translocated from the cytosol into the mitochondria in response to TNFα/CHX, and CRISPR/Cas9 knockout of Bak significantly decreased this translocation. These results indicate that TNFα/CHX-induced apoptosis does not occur in Bak−/− cells, suggesting that TNFα/CHX-induced apoptosis is Bak-dependent but Bax-independent.

Bcl-2 Overexpression Induces Neurite Outgrowth via the Bmp4/Tbx3/NeuroD1 Cascade in H19-7 Cells.

Bcl-2 is overexpressed in the nervous system during neural development and plays an important role in modulating cell survival. In addition to its anti-apoptotic function, it has been suggested previously that Bcl-2 might act as a mediator of neuronal differentiation. However, the mechanism by which Bcl-2 might influence neurogenesis is not sufficiently understood. In this study, we aimed to determine the non-apoptotic functions of Bcl-2 during neuronal differentiation. First, we used microarrays to analyze the whole-genome expression patterns of rat neural stem cells overexpressing Bcl-2 and found that Bcl-2 overexpression induced the expression of various neurogenic genes. Moreover, Bcl-2 overexpression increased the neurite length as well as expression of Bmp4, Tbx3, and proneural basic helix-loop-helix genes, such as NeuroD1, NeuroD2, and Mash1, in H19-7 rat hippocampal precursor cells. To determine the hierarchy of these molecules, we selectively depleted Bmp4, Tbx3, and NeuroD1 in Bcl-2-overexpressing cells. Bmp4 depletion suppressed the upregulation of Tbx3 and NeuroD1 as well as neurite outgrowth, which was induced by Bcl-2 overexpression. Although Tbx3 knockdown repressed Bcl-2-mediated neurite elaboration and downregulated NeuroD1 expression, it did not affect Bcl-2-induced Bmp4 expression. While the depletion of NeuroD1 had no effect on the expression of Bcl-2, Bmp4, or Tbx3, Bcl-2-mediated neurite outgrowth was suppressed. Taken together, these results demonstrate that Bcl-2 regulates neurite outgrowth through the Bmp4/Tbx3/NeuroD1 cascade in H19-7 cells, indicating that Bcl-2 may have a direct role in neuronal development in addition to its well-known anti-apoptotic function in response to environmental insults.

Identification of Aloperine as an anti-apoptotic Bcl2 protein inhibitor in glioma cells.

ObjectiveAloperine (ALO), an alkaloid isolated from the leaves of Sophora alopecuroides, has been suggested to exhibit anti-inflammatory and anti-tumor properties and is traditionally used to treat various human diseases, including cancer. However, limited information is available about the mechanisms that determine the anti-tumor activities of ALO.MethodsHerein, through comprehensive bioinformatics methods and in vitro functional analyses, we evaluated the detailed anti-tumor mechanisms of ALO.ResultsUsing the databases Bioinformatics analysis tool for molecular mechanism of traditional Chinese medicine and PubChem Project, we identified the potential targets of ALO. A protein–protein interaction network was constructed to determine the relationship among these probable targets. Functional enrichment analysis revealed that ALO is potentially involved in the induction of apoptosis. In addition, molecular docking demonstrated that ALO expectedly docks into the active pocket of the Bcl2 protein, suggesting Bcl2 as a direct target of ALO. Moreover, western blot and qPCR analysis showed that ALO downregulated Bcl2 expression in human glioma cell lines, SK-N-AS and U118. Using flow cytometry methods, we further confirmed that ALO significantly promotes apoptosis in SK-N-AS and U118 cell lines, similar to the effect induced by ABT-737, a well-known Bcl2 inhibitor. In addition, Bcl-2 overexpression could rescue ALO-induced Bcl-2 inhibition and suppress pro-apoptotic effects in glioma cells.ConclusionTaken together, these findings suggest that the natural agent ALO effectively enhances apoptosis by acting as a potential Bcl2 inhibitor in human glioma cells.

Concomitant targeting of Hedgehog signaling and MCL-1 synergistically induces cell death in Hedgehog-driven cancer cells.

In the present study, we show that concomitant inhibition of Hedgehog (HH) signaling by the glioma-associated oncogene homolog1 (GLI1)-targeting agent GANT61 and the antiapoptotic BCL-2 protein family member MCL-1 by A-1210477 synergistically induces cell death in HH-driven cancers, i.e. rhabdomyosarcoma (RMS) and medulloblastoma (MB) cells. Combined genetic and pharmacological inhibition emphasized that co-treatment of GANT61 and A-1210477 indeed relies on inhibition of GLI1 (by GANT61) and MCL-1 (by A-1210477). Mechanistic studies revealed that A-1210477 triggers the release of BIM from MCL-1 and its shuttling to BCL-xL and BCL-2. Indeed, BIM proved to be required for GANT61/A-1210477-induced cell death, as genetic silencing of BIM using siRNA significantly rescues cell death upon GANT61/A-1210477 co-treatment. Similarly, genetic silencing of NOXA results in a significant reduction of GANT61/A-1210477-mediated cell death. Also, overexpression of MCL-1 or BCL-2 significantly protects RMS cells from GANT61/A-1210477-triggered cell death. Addition of the pan-caspase inhibitor zVAD.fmk significantly decreases GANT61/A-1210477-stimulated cell demise, indicating apoptotic cell death. In conclusion, GANT61 and A-1210477 synergize to engage mitochondrial apoptosis. These findings provide the rationale for further evaluation of dual inhibition of HH signaling and MCL-1 in HH-driven cancers.

Zika Virus Infection Induces Acute Kidney Injury Through Activating NLRP3 Inflammasome Via Suppressing Bcl-2.

Zika virus (ZIKV) is a newly emerging flavivirus that broadly exhibits in various bodily tissues and fluids, especially in the brain, and ZIKV infection often causes microcephaly. Previous studies have been reported that ZIKV can infect renal cells and can be detected in the urine samples of infected individuals. However, whether ZIKV infection causes renal diseases and its pathogenic mechanisms remains unknown. Here, we identified that ZIKV infection resulted in acute kidney injury (AKI) in both newborn and adult mouse models by increasing the levels of AKI-related biomarkers [e.g., serum creatinine (Scr), kidney injury molecular−1 (Kim-1), and neutrophil gelatinase-associated lipocalin (NGAL)]. ZIKV infection triggered the inflammatory response and renal cell injury by activating Nod-like receptor 3 (NLRP3) inflammasome and secreting interleukin-1β (IL-1β). IL-1β inhibited aquaporins expression and led to water re-absorption disorder. Furthermore, ZIKV infection induced a decreased expression of B-cell lymphoma-2 (Bcl-2) in the kidney. Overexpression of Bcl-2 attenuated ZIKV-induced NLRP3 inflammasome activation in renal cells and down-regulated PARP/caspase-3-mediated renal apoptosis. Overall, our findings demonstrated that ZIKV infection induced AKI by activating NLRP3 inflammasome and apoptosis through suppressing Bcl-2 expression, which provided potential therapeutic targets for ZIKV-associated renal diseases.

RESISTANCE TO VENETOCLAX IN CHRONIC LYMPHOCYTIC LEUKAEMIA (CLL)

Venetoclax, the BCL2 inhibitor, is highly effective for the treatment of chronic lymphocytic leukaemia (CLL), a disease marked by BCL2 overexpression. The highly encouraging results from clinical trials has led to its approval in many jurisdictions, including recent listing in Australia on the Pharmaceutical Benefits Scheme (PBS). In spite of its efficacy, many treated patients will eventually experience disease progression after several years. At the current time, we do not fully understand why resistance emerges. Our research group is well placed to address this question as we have access to one of the largest cohorts of venetoclax-treated patients, and access to serially collected samples such as prior to venetoclax therapy and at progression. Our preliminary data indicates that amongst patients who have progressive CLL while on venetoclax, multiple mechanisms drive resistance even within a patient. While the recently discovered BCL2 G101V mutation accounted significantly for the resistance, we found evidence for co-existing adverse genetic changes (e.g. BCLxL upregulation). Moreover, we observed that CLL cells from the lymph nodes at disease progression are less sensitive to venetoclax compared to ones present in peripheral blood. Strikingly, we could recapitulate this phenotype by maintaining CLL cells in culture under conditions that mimic the lymphoid tissue microenvironment. Interestingly, such culture conditions promote the growth of more activated CLL cells which express an altered repertoire of BCL2 family proteins that probably accounts for the reduced sensitivity to venetoclax. Taken together, our studies will help us understand why resistance emerges and have significant implications for how venetoclax is best used, for preventing the development of therapy resistance or to develop strategies to tackle resistance once it emerges.

Bcl6 Suppresses Cardiac Fibroblast Activation and Function via Directly Binding to Smad4.

Bcl6, a critical pro-oncogene of human B-cell lymphomas, can promote tumor progress. Previous studies have found that Bcl6 participates in hypoxia injury in cardiomyocytes. However, the effect of Bcl6 on cardiac fibroblasts is still unclear. The aim of this study was to elucidate the functional role of Bcl6 in cardiac fibroblast activation and function. The neonatal rat cardiac fibroblasts were isolated and cultured. First, transforming growth factor ß1 (TGFß1) was used to stimulate fibroblast activation. A decreased expression level of Bcl6 was observed in fibroblasts after stimulation with TGFß1. Then, cells were transfected with adenovirus Bcl6 to overexpress Bcl6. The results showed that Bcl6 overexpression induced decreased proliferation and reduced activation of fibroblasts which were stimulated with TGFß1. It was found that activated smad2 and smad3 were not changed by overexpressing Bcl6, but smad4 was decreased. Furthermore, co-immunoprecipitation results showed that Bcl6 directly bound to smad4, and induced down-regulation of smad4. At last, smad4 activator could counteract the anti-fibroblast effects of Bcl6. In conclusion, Bcl6 may negatively regulate cardiac fibroblast activation and function by directly binding to smad4.