Overexpression Of Bcl-2 Research Articles

Bcl-3 promotes multi-modal tumour cell migration via NF-κB1 mediated regulation of Cdc42.

A key challenge in the implementation of anti-metastatics as cancer therapies is the multi-modal nature of cell migration, which allows tumour cells to evade the targeted inhibition of specific cell motility pathways. The nuclear factor-kappaB (NF-κB) co-factor B-cell lymphoma 3 (Bcl-3) has been implicated in breast cancer cell migration and metastasis, yet it remains to be determined exactly which cell motility pathways are controlled by Bcl-3 and whether migrating tumour cells are able to evade Bcl-3 intervention. Addressing these questions and the mechanism underpinning Bcl-3's role in this process would help determine its potential as a therapeutic target. Here we identify Bcl-3 as an upstream regulator of the two principal forms of breast cancer cell motility, involving collective and single-cell migration. This was found to be mediated by the master regulator Cdc42 through binding of the NF-κB transcription factor p50 to the Cdc42 promoter. Notably, Bcl-3 depletion inhibited both stable and transitory motility phenotypes in breast cancer cells with no evidence of migratory adaptation. Overexpression of Bcl-3 enhanced migration and increased metastatic tumour burden of breast cancer cells in vivo, whereas overexpression of a mutant Bcl-3 protein, which is unable to bind p50, suppressed cell migration and metastatic tumour burden suggesting that disruption of Bcl-3/NF-κB complexes is sufficient to inhibit metastasis. These findings identify a novel role for Bcl-3 in intrinsic and adaptive multi-modal cell migration mediated by its direct regulation of the Rho GTPase Cdc42 and identify the upstream Bcl-3:p50 transcription complex as a potential therapeutic target for metastatic disease.

Bcl-2 expression in colorectal carcinoma and its correlation with clinicopathological parameters

Background: Bcl-2 gene is a 26-kD protein that blocks apoptosis and inhibits programmed cell death. Its mutation is frequently detected genetic alteration, in human cancers. Its expression can easily be detected by immunohistochemistry (IHC). Objective: The objective was to estimate the frequency of Bcl-2 expression in colorectal carcinoma and its correlation with clinicopathologic parameters. Materials and Methods: The overexpression of Bcl-2 was studied in 112 paraffin-preserved colorectal carcinoma samples using E-17 clone antibody; Biogenex. The number of cells stained was scored semiquantitatively as none (no immunoreactive cells detectable), weak (1+, 50%). All tumors showing Bcl-2 immunoreactivity (at least +) were considered positive. The correlation between Bcl-2 protein expression and clinicopathological parameters was evaluated using Chi-square analysis. Results: Bcl-2 staining was positive in 45 of 112 cases. Of these, 15 cases were weak positive (score 1), 17 cases were moderately positive (score 2), and 13 cases were intensely positive (score 3) for Bcl-2 protein expression. Sixty-seven cases were negative for Bcl-2 staining (score 0). There was statistically significant correlation between Bcl-2 staining and sex (49% of females expressed Bcl-2 in tumors; P = −0.04), pathological type (mucinous vs. nonmucinous; P = −0.02), and tumor stage (P = −0.04). There was no significant correlation between Bcl-2 staining and age (P = −0.38), site of tumor (left colon vs. right colon; P = −0.13), pathological differentiation (P = −0.73), and between Bcl-2 scoring and tumor differentiation (P = −0.91). Conclusion: Bcl-2 protein seems to play an important role in the carcinogenesis of colorectal cancer. Considering the acceptable reliability and feasibility of IHC method for the detection of Bcl-2 immunoreactivity, this technique may be expected to serve as an important diagnostic and prognostic marker in colorectal carcinoma.

BCL-XL expression is essential for human erythropoiesis and engraftment of hematopoietic stem cells

The anti-apoptotic BCL-2 proteins (BCL-2, BCL-XL, MCL-1, A1, BCL-W) counteract apoptotic signals emerging during development and under stress conditions, and are thus essential for the survival of every cell. While the “BCL-2 addiction” of different cell types is well described in mouse models, there is only limited information available on the role of different anti-apoptotic BCL-2 proteins in a given human cell type. Here we characterize the role of BCL-XL for survival and function of human hematopoietic cells, with the aim to predict hematological side effects of novel BCL-XL-inhibiting BH3-mimetics and to identify hematological malignancies potentially responsive to such inhibitors. Earlier clinical studies have shown that the combined BCL-2/BCL-XL/BCL-W inhibitor, Navitoclax (ABT-263) induces severe thrombocytopenia caused by direct platelet demise and counteracted by increased megakaryopoiesis. In contrast, murine studies have reported important contribution of BCL-XL to survival of late erythroid cells and megakaryocytes. Using lentiviral knockdown, we show that the roles of BCL-XL for human hematopoietic cells are much more pronounced than expected from murine data and clinical trials. Efficient genetic or chemical BCL-XL inhibition resulted in significant loss of human erythroid cells beginning from very early stages of erythropoiesis, and in a reduction of megakaryocytes. Most importantly, BCL-XL deficient human hematopoietic stem cells and multipotent progenitors were reduced in numbers, and they showed a severely impaired capacity to engraft in mice during xenotransplantation. BCL-XL deficiency was fully compensated by BCL-2 overexpression, however, loss of its antagonist BIM did not result in any rescue of human erythroid or stem and progenitor cells. We thus conclude that novel and specific BCL-XL inhibitors might be efficient to treat malignancies of erythroid or megakaryocytic origin, such as polycythemia vera, acute erythroid leukemia, essential thrombocytosis or acute megakaryocytic leukemia. At the same time, it can be expected that they will have more severe hematological side effects than Navitoclax.

BCL-6 Overexpression as a Predictor for Endometriosis in Patients Undergoing In Vitro Fertilization.

Objective:To evaluate the positive predictive value (PPV) of endometrial BCL-6 overexpression as a noninvasive screening test endometriosis in patients undergoing in vitro fertilization (IVF).Methods:Retrospective cohort study at a university-affiliated private practice. Inclusion criteria were reproductive age females currently undergoing IVF with a diagnosis of unexplained infertility or unexplained recurrent pregnancy loss. Those with endometrial BCL-6 overexpression underwent laparoscopic surgery with an indication for treatment of suspected endometriosis. The primary outcome was the PPV of endometrial BCL-6 testing to surgically diagnose endometriosis. Statistical analysis was performed using SPSS v.25.0.Results:Seventy-five patients met inclusion criteria for our study. The PPV of BCL-6 testing for endometriosis was 96%. Of those patients without endometriosis, 100% had other inflammatory pelvic pathologies, which were diagnosed and treated at the time of laparoscopy.Conclusions:Endometrial BCL-6 overexpression has a high PPV for diagnosing endometriosis and can help identify a patient population that may require surgical treatment before embryo transfer.

Association of Monoamine Oxidase A with Tumor Burden and Castration Resistance in Prostate Cancer

BackgroundMetastatic burden and aggressive behavior determine severity stratification and guide treatment decisions in prostate cancer (PCa). Monoamine oxidase A (MAOA) may promote tumor burden and drug/castration resistance in PCa. A positive association will pave the way for MAOA inhibitors such as moclobemide for PCa therapy. ObjectiveTo analyze MAOA in peripheral blood mononuclear cells qualitatively and p38, c-Jun N-terminal kinases, nuclear factor kappa B, and their phosphorylated forms, vascular endothelial growth factor (angiogenesis), transforming growth factor beta, interleukin 6, and tumor necrosis factor-α (cytokines), Bcl-2 associated X, B-cell lymphoma 2, and P53 (apoptosis), prostate-specific membrane antigen, and epithelial cell adhesion molecules (surface markers) in plasma of patients with PCa. MethodsThis was a 1-year pilot study in which patients with PCa were recruited and stratified into 2 groups and subgroups: treatment-naive with (M1) (n = 23) or without (M0) (n = 23) bone metastasis; hormone-sensitive prostate cancer (n = 26) or hormone/castration-resistant prostate cancer (n = 26). MAOA was detected using ELISA and other proteins were detected using immunoblotting technique. ResultsMAOA was detected in 8.6% of M0 compared with 30.4% of M1 patients, and in 7.7% of hormone-sensitive compared with 27% of hormone/castration resistant PCa patients, associating it with bone metastasis and castration resistance. Multivariable regression analysis showed a correlation of MAOA with serum prostate-specific antigen, a marker for progression in PCa (Pearson correlation coefficient r = 0.30; P < 0.01). In patients with positive MAOA, there was overexpression of p38, phosphorylated-p38, c-Jun N-terminal kinases, phosphorylated c-Jun N-terminal kinases, nuclear factor kappa B, phosphorylated nuclear factor kappa B, transforming growth factor beta, vascular endothelial growth factor, interleukin 6, tumor necrosis factor α, Bcl-2 associated X, B-cell lymphoma 2, prostate-specific membrane antigen, and epithelial cell adhesion molecule in M1 compared with M0 group patients, associating these proteins with tumor burden. Overexpression of Bcl-2 associated X, tumor protein 53, c-Jun N-terminal kinases, nuclear factor kappa B, transforming growth factor beta, vascular endothelial growth factor, and prostate-specific membrane antigen and underexpression of B-cell lymphoma 2 and phosphorylated nuclear factor kappa B were observed in hormone-sensitive prostate cancer compared with hormone/castration-resistant prostate cancer, associating these proteins with castration resistance. ConclusionsAssociation of key molecules of oncogenesis and metastasis with MAOA suggests that MAOA inhibitors such as moclobemide might be effective in the management of PCa.

MicroRNA-214 Prevents Traits of Cutaneous Squamous Cell Carcinoma via VEGFA and Bcl-2.

Background:Dysregulation of microRNA-214 (miR-214) has been indicated in different tumors. The function of miR-214 in cutaneous squamous cell carcinoma (CSCC) is yet to be deciphered. The current study aimed to investigate the specific mechanism underpinning CSCC development with the involvement of miR-214 and its putative targets.Methods:Microarray analysis of CSCC and adjacent tissues was carried out to filter the most significant downregulated miRNA. Survival analysis of patients was subsequently implemented, followed by miRNA expression determination in CSCC cells. Gain-of-function assays were performed to evaluate its function on cellular level. The targets of the determined miRNA were predicted and their expression in CSCC and adjacent tissues was evaluated. The targeting relationship was analyzed by dual-luciferase assays. Finally, rescue experiments were conducted.Results:miR-214 was reduced in CSCC tissues and cells, and the survival of patients harboring overexpression of miR-214 was higher. miR-214 restoration increased CSCC cell apoptosis, while decreased proliferative, invasive and migratory activities. miR-214 interacted with vascular endothelial growth factor A (VEGFA) and B-cell CLL/lymphoma 2 (Bcl-2). VEGFA and Bcl-2, overexpressed in CSCC tissues and cells, were negatively correlated with miR-214. Moreover, VEGFA and Bcl-2 overexpression reversed the anti-tumor phenotypes of miR-214 on CSCC cells. miR-214 disrupted the Wnt/β-catenin pathway through VEGFA and Bcl-2 in the CSCC cells.Conclusion:Our data demonstrates that miR-214 exerts a suppressing role in CSCC. The discovery of novel targets such as miR-214 and VEGFA/Bcl-2 may facilitate the development of therapeutic options.

MiR-636 represses cell survival by targeting CDK6/Bcl-2 in cervical cancer.

Cervical cancer is widely known as one of the most common types of cancer diagnosed in women, and microRNAs (miRNAs) has been characterized as an important regulator in tumor progression, such as cervical cancer. MiR-636 was found to play a tumor suppressor role in hepatocellular carcinoma tumorigenesis. However, the tumorigenic mechanism of miR-636 on cervical cancer has not yet been found. In the present study, we first found that miR-636 was significantly downregulated in cervical cancer tissues and cell lines. in vitro gain- and loss-of-function assays revealed that overexpression of miR-636 inhibited cell proliferation and induced cell apoptosis, while knockdown of miR-636 reversed the effect on cervical tumorigenesis. Furthermore, cyclin-dependent kinase 6 (CDK6) and B-cell lymphoma 2 (Bcl-2) were characterized as targets of miR-636. Notably, overexpression of CDK6 or Bcl-2 could reverse the inhibitory effect of miR-636 on cervical cancer progression. Mechanistically, miR-636 repressed cell survival by targeting CDK6/Bcl-2 in cervical cancer, which may be the underlying mechanism of miR-636-inhibited cervical progression. In conclusion, our findings clarified the biologic significance of miR-636/CDK6/Bcl-2 axis in cervical cancer progression and suggested the potential therapeutic target ability of miR-636 in treatment of cervical cancer.

Superoxide induced inhibition of death receptor signaling is mediated via induced expression of apoptosis inhibitory protein cFLIP

The death inhibitory proteins, cFLIP and Bcl-2, canonically act at different steps to regulate receptor-mediated apoptosis in cancer cells. Here we report that pharmacological or genetic means to effect an increase in intracellular superoxide result in cFLIP upregulation. Interestingly, Bcl-2 overexpression is associated with a concomitant increase in cFLIP, and reducing superoxide sensitizes Bcl-2 overexpressing cancer cells to receptor-mediated apoptosis via downregulation of cFLIP. Moreover, inhibiting glycolytic flux overcomes apoptosis resistance by superoxide-dependent downregulation of cFLIP. Superoxide-induced upregulation of cFLIP is a function of enhanced transcription, as evidenced by increases in cFLIP promoter activity and mRNA abundance. The positive effect of superoxide on cFLIP is mediated through its reaction with nitric oxide to generate peroxynitrite. Corroborating these findings in cell lines, subjecting primary cells derived from lymphoma patients to glucose deprivation ex vivo, as a means to decrease superoxide, not only reduced cFLIP expression but also significantly enhanced death receptor sensitivity. Based on this novel mechanistic insight into the redox regulation of cancer cell fate, modulation of intracellular superoxide could have potential therapeutic implications in cancers in which these two death inhibitory proteins present a therapeutic challenge.

The transcription factor ETS1 promotes apoptosis resistance of senescent cholangiocytes by epigenetically up-regulating the apoptosis suppressor BCL2L1

Primary sclerosing cholangitis (PSC) is an idiopathic, progressive cholangiopathy. Cholangiocyte senescence is important in PSC pathogenesis, and we have previously reported that senescence is regulated by the transcription factor ETS proto-oncogene 1 (ETS1) and associated with overexpression of BCL2 like 1 (BCL2L1 or BCL-xL), an anti-apoptotic BCL2-family member. Here, we further explored the mechanisms regulating BCL-xL-mediated, apoptosis resistance in senescent cholangiocytes and uncovered that ETS1 and the histone acetyltransferase E1A-binding protein P300 (EP300 or p300) both promote BCL-xL transcription. Using immunofluorescence, we found that BCL-xL protein expression is increased both in cholangiocytes of livers from individuals with PSC and a mouse model of PSC. Using an in vitro model of lipopolysaccharide-induced senescence in normal human cholangiocytes (NHCs), we found increased BCL-xL mRNA and protein levels, and ChIP-PCRs indicated increased occupancy of ETS1, p300, and histone 3 Lys-27 acetylation (H3K27Ac) at the BCL-xL promoter. Using co-immunoprecipitation and proximity ligation assays, we further demonstrate that ETS1 and p300 physically interact in senescent but not control NHCs. Additionally, mutagenesis of predicted ETS1-binding sites within the BCL-xL promoter blocked luciferase reporter activity, and CRISPR/Cas9-mediated genetic deletion of ETS1 reduced senescence-associated BCL-xL expression. In senescent NHCs, TRAIL-mediated apoptosis was reduced ∼70%, and ETS1 deletion or RNAi-mediated BCL-xL suppression increased apoptosis. Overall, our results suggest that ETS1 and p300 promote senescent cholangiocyte resistance to apoptosis by modifying chromatin and inducing BCL-xL expression. These findings reveal ETS1 as a central regulator of both cholangiocyte senescence and the associated apoptosis-resistant phenotype.

Analysis of apoptosis related genes in nurses exposed to anti-neoplastic drugs

BackgroundAnti-neoplastic agents are widely used in the treatment of cancer and some non-neoplastic diseases. These drugs have been proved to be carcinogens, teratogens, and mutagens. Concern exists regarding the possible dangers of the staff handling anti-cancer drugs. The long-term exposure of nurses to anti-neoplastic drugs is still a controversial issue. The purpose of this study was to monitor cellular toxicity parameters and gene expression in nurses who work in chemotherapy wards and compare them to nurses who work in other wards.MethodsTo analyze the apoptosis-related genes overexpression and cytotoxicity effects, peripheral blood lymphocytes obtained from oncology nurses and the control group.The resultsSignificant alterations in four analyzed apoptosis-related genes were observed in oncology nurses. In most individual samples being excavated, Bcl-2 overexpression is superior to that of Bax. Prominent P53 and Hif-1α up-regulation were observed in oncology nurses. Moreover, all cytotoxicity parameters (cell viability, ROS formation, MMP collapse, Lysosomal membrane damage, Lipid peroxidation, Caspase 3 activity and Apoptosis phenotype) in exposed oncology nurses were significantly (p < 0.001) higher than those of unexposed control nurses. Up-regulation of three analyzed apoptosis-related genes were observed in nurses occupationally exposed to anti-cancer drugs.ConclusionOur data show that oxidative stress and mitochondrial toxicity induced by anti-neoplastic drugs lead to overexpression of apoptosis-related genes in oncology nurses.

P53, HER2/neu, Bcl-2 and PCNA overexpression in ductal carcinoma in situ lesions of the breast and their role in progression to invasive carcinoma

Amac: Proliferatif aktivitenin, onkogenlerde ve tumor supresor genlerdeki degisikliklerin anlasilmasi, duktal karsinoma in situnun invaziv meme kanserine gelisme ihtimalinin yuksek oldugu olgulari belirlemede onemli olabilir. Bu calismada p53, HER2/neu, bcl-2 ve PCNA overekspresyonunun duktal karsinoma in situnun progresyonu ile iliskisi degerlendirildi. Gerec ve Yontem: Biz 20 duktal karsinoma in situ (grup 1) ve 20 invaziv duktal karsinoma birlikteliginde duktal karsinoma in situ (grup 2) olgusunu degerlendirdik. Olgulara immunhistokimyasal yontemler kullanilarak p53, HER2/neu, bcl-2 ve PCNA calisildi. Bulgular: Grup 2 olgularinda p53, HER2/neu ve PCNA overekspresyonu daha yuksekti. Ancak farklar istatistiksel olarak anlamli degildi (p>0,05). Grup 1 olgularinin %36,8’inde, grup 2 olgularinin %70’inde bcl-2 overekspresyonu izlendi. Iki grup arasindaki bu fark istatistiksel olarak anlamliydi (p<0,05). Calismamizda onemli bulgulardan biri de, p53, HER2/neu, bcl-2 ve PCNA overekspresyonunun ayni anda grup 2 olgularinin 3’unde izlenirken grup 1 olgularinin hicbirinde gozlenmemesiydi. Sonuc: Bulgular bize, bcl-2 overekspresyonunun duktal karsinoma in situnun invaziv duktal karsinomaya gelisim ihtimalinin yuksek oldugu olgulari belirlemede daha degerli olabilecegini, invaziv duktal karsinoma gelisiminde farkli progresyon yollarinin varoldugunu, preinvaziv evrede bu degisikliklerin birden fazlasinin ayni olguda tespit edilmesinin invaziv duktal karsinomaya progresyon riskini belirlemede tek baslarina oldugundan daha degerli olabilecegini gosterdi. Bu sonuclar farkli biyolojik davranisa sahip DKIS alt tiplerinin tanimlanmasina katki saglayacaktir.

The synthetic flavanone 6-methoxy-2-(naphthalen-1-yl)chroman-4-one induces apoptosis and activation of the MAPK pathway in human U-937 leukaemia cells.

Synthetic flavonoids containing a naphthalene ring have attracted attention as potential cytotoxic compounds. Here, we synthesized ten chalcones and their corresponding flavanones and evaluated their antiproliferative activity against the human tumour cell line U-937. This series of chalcone derivatives was characterized by the presence of a naphthalene ring which was kept unaltered- and attached to the β carbon of the 1-phenyl-2-propen-1-one framework. The structure-activity relationship of these chalcone derivatives and their corresponding cyclic compounds was investigated by the introduction of different substituents (methyl, methoxy, benzyloxy, chlorine) or by varying the position of the methoxy or benzyloxy groups on the A ring. The results revealed that both the chalcone containing the methoxy group at 5' position of the A ring as well as its corresponding flavanone [6-methoxy-2-(naphthalen-1-yl)chroman-4-one] were the most cytotoxic compounds, with IC50 values of 2.8±0.2 and 1.3±0.2μM, respectively, against U-937 cells. This synthetic flavanone was as cytotoxic as the antitumor etoposide in U-937 cells and displayed strong cytotoxicity against additional human leukaemia cell lines, including HL-60, MOLT-3 and NALM-6. Human peripheral blood mononuclear cells were more resistant than leukaemia cells to the cytotoxic effects of the flavanone. Treatment of U-937 cells with this compound induced G2-M cell cycle arrest, an increase in sub-G1 ratio and annexin-V positive cells, mitochondrial cytochrome c release, caspase activation and poly(ADP-ribose)polymerase processing. Apoptosis induction triggered by this flavonoid was blocked by overexpression of the anti-apoptotic protein Bcl-2. This flavanone induces phosphorylation of p38 mitogen-activated protein kinases, extracellular-signal regulated kinases and c-jun N-terminal kinases/stress-activated protein kinases (JNK/SAPK) following different kinetics. Moreover, cell death was attenuated by the inhibition of mitogen-activated extracellular kinases and JNK/SAPK and was independent of reactive oxygen species generation.

Molecular and clinical progress in follicular lymphoma lacking the t(14;18) translocation (Review).

Although the majority of patients with follicular lymphoma (FL) harbor the t(14;18)(q32;q21) IGH/BCL2 gene rearrangement that leads to the overexpression of BCL2 protein, approximately 20% of FL cases lack t(14;18)(q32;q21). It is considered that BCL2 overexpression underscores the development of the majority of cases of FL and their transformation to more aggressive lymphoma [known as transformed FL (tFL)]. However, FL cases lacking the t(14;18)(q32;q21) translocation exhibit symptoms analogous to their t(14;18)‑positive counterparts. An important goal of recent research on FL has been to clarify the distinctions between the two different forms of FL. Numerous studies have shed light onto the genetic and molecular features of t(14;18)‑negative FL and the related clinical manifestations. In this review, we summarize the current knowledge of t(14;18)‑negative FL occurring in the lymph nodes with an emphasis on the underlying molecular and clinical features. In addition, novel treatment directions are discussed.

Inhibiting Mitochondria Function By Bcl-2 Inhibitor Venetoclax and Complex I Inhibitor Iacs-010759 Eliminate Leukemia Cells in Pre-Clinical AML Models

Previous studies have demonstrated that AML is BCL-2 dependent malignancy, and leukemia stem cell (LSC) rely on BCL-2 for survival (Pan, Cancer Discovery 2014;Lagadinou, Cell Stem Cell, 2013). Selective Bcl-2 inhibitor venetoclax (ABT-199) combined with azacitidine was reported to inhibit complex II of the mitochondrial transport chain in AML (Pollyea, Nat Med 2018). IACS-010759, a novel complex I inhibitor, demonstrated effective inhibition of cell respiration and potent anti-leukemia effect in AML pre-clinical models (Molina, Nat Med 2018). We designed the experiments to study the combined efficacy and mechanisms of action of venetoclax and IACS-010759 in AML. In vitro, priming of MOLM-13 cells with 20nM venetoclax for 24hrs followed by 10nM IACS-010759 for 1hr triggered 60% reduction in oxygen consumption rate (OCR), while only partial inhibition (&lt;15% OCR reduction) was seen in cells treated with these agents separately. This translated in accelerated loss of the mitochondrial membrane potential by JC-1 flow cytometry, cleavage of caspase 3 and potent (&gt;70%) reduction of viable cell numbers (OCI-AML2, MV-4-11, and MOLM-13). We have further shown by co-immunoprecipitation studies that venetoclax disrupts interaction of BCL-2 with the mitochondrial protein VDAC known to regulate ADP/ATP exchange during electron transport across mitochondria membrane. In addition, an MS-based metabolomics analysis indicated that ATP and CTP intracellular levels dropped to undetectable levels following treatment with ABT-199 (with or without IACS). ADP, GDP and UDP levels were unchanged with ABT-199; however, GDP levels dropped to undetectable levels following the combined treatment. Moreover, ABT-199 significantly increased intracellular levels of AMP, UMP, CMP and GMP and this accumulation of mono-nucleotides was enhanced by the combination of ABT-199 and IACS-010759. In primary AML samples (n=3) and PDX cells (n=4) cultured ex vivo, combined venetoclax and IACS-010759 at low nanomolar doses reduced viable cell numbers in an additive or synergistic fashion. To better understand the role of BCL-2 in cellular respiration, we examined the oxygen consumption rates (OCR) in control or Bcl-2-overexpressing HL-60 cells (a kind gift of Dr. K. Bhalla, MDACC). The HL-60/BCL-2 cells had higher basal and maximal OCR than the control cells by Seahorse analysis, and higher mitochondrial ROS production by H2DCFDA and MitoSOX Red flow cytometry. BCL-2 inhibition with 100nM venetoclax for 2 hrs induced ROS production in control HL-60 cells but not in cells with BCL-2 overexpression. Further, cells with BCL-2 overexpression were less sensitive to IACS-010759. These data suggest that BCL-2 facilitates cellular respiration and reduces efficacy of the mitochondrial inhibitors. Given recent accelerated FDA approval of venetoclax and azacitidine combination for elderly unfit AML, we next tested the efficacy of the "triple" combination of venetoclax, azacitidine and IACS-010759 in the in vivoAML PDX model. We injected AML PDX cells 3747422 harboring IDH1, NMP1, NRAS, CEBPA, FLT3-ITD mutations into NRG mice and upon engraftment, randomized mice into 4 groups to receive vehicle, venetoclax (50mg/kg, 5 days on/2 days off, day 1-21) with azacitidine (1.25mg/kg daily , day 1-7), IACS-010759 (1mg/kg, 5 days on/2 days off, day 1-14), or the triple combination. Therapy was well tolerated, without any apparent weight loss or toxicities. All therapies reduced circulating leukemia burden with the best efficacy seen in the triple-therapy cohort, with average circulating tumor burden of 31.2%, 6.9%, 5.1% and 0.4% in vehicle, IACS-010759, venetoclax/azacitidine and triple-therapy cohorts, respectively. Survival analysis and additional PDX models are ongoing and will be reported. In summary, these findings indicate that BCL-2 modulates mitochondrial respiration in addition to its established anti-apoptotic role. Venetoclax disrupts the BCL-2/VDAC interactions and reduces mitochondrial respiration, which is facilitated by the combined therapy with mitochondrial complex I inhibitor IACS-010759. Our preliminary findings indicate potent anti-AML activity of the dual and triple (with hypomethylating agent) combinations in vitroand in vivo. Disclosures Zhang: The University of Texas M.D.Anderson Cancer Center: Employment. Kuruvilla:The University of Texas M.D.Anderson Cancer Center: Employment. Konopleva:Stemline Therapeutics: Consultancy, Honoraria, Research Funding; Forty-Seven: Consultancy, Honoraria; Eli Lilly: Research Funding; Calithera: Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Cellectis: Research Funding; Amgen: Consultancy, Honoraria; F. Hoffman La-Roche: Consultancy, Honoraria, Research Funding; Genentech: Honoraria, Research Funding; Ascentage: Research Funding; Kisoji: Consultancy, Honoraria; Reata Pharmaceuticals: Equity Ownership, Patents &amp; Royalties; Ablynx: Research Funding; Astra Zeneca: Research Funding; Agios: Research Funding.

Response to Venetoclax in Combination with Low Intensity Therapy (LDAC or HMA) in Untreated Patients with Acute Myeloid Leukemia Patients with IDH, FLT3 and Other Mutations and Correlations with BCL2 Family Expression

Background Acute myeloid leukemia (AML) is a highly heterogeneous disease with multiple prognostic indicators of outcomes to conventional treatments; including age, cytogenetic risk, and presence of genomic abnormalities (NPM1, TP53, FLT3 and IDH1/2). Additionally, overexpression of BCL-2 is a predictor for poor response to chemotherapy and can lead to therapeutic resistance in AML (Campos et. al. Blood, 1993). Venetoclax (Ven), an oral selective BCL-2 inhibitor, in combination with hypomethylating agents (HMA) or low dose cytarabine (LDAC), was recently approved for treatment naive patients (pts) with AML who were not fit for standard induction therapy on the basis of two phase 1b/2 studies (DiNardo et.al, Lancet Onclogy, 2018; Wei et.al., J Clin. Oncol., 2019). Here, we present clinical outcome results from the phase 1b/2 study populations in subgroups defined by molecular markers and correlations with BCL-2 family expression. Methods: The data cut-off dates were 31-Aug-2018 (VEN+HMA study M14-358, NCT02203773) and 22-Aug-2018 (VEN+LDAC study M14-387 NCT02287233). Pt responses were assessed according to the IWG criteria for AML (Cheson et.al., J Clin. Oncol.,2003). The presence of AML associated mutations (NPM1, IDH1, IDH2, TP53 and FLT3) were determined centrally in bone marrow aspirates (BMA) at baseline. BCL-2 (BCL2) gene expression was defined centrally by quantitative polymerase chain reaction (qPCR) using the deltaCt (ΔCt) method. BCL2 expression normalized to a reference gene (2-ΔCt) was evaluated for pts with &amp;gt; 50% AML blasts in baseline BMA (median 75%; range 50-99%). The composite complete remission (CR) and CR with incomplete marrow recovery (CRi) rate, overall survival (OS), time to first response (TTR), and duration of response (DOR), and BCL-2 gene expression were correlated with presence of baseline molecular markers. Results: Data from 209 pts who received Ven 400 mg or 600 mg in combination with either HMA or LDAC are reported here. The median age was 74 years (range: 61-90 years) and 61% (n=127) were male (Table 1). Of the 167 pts with centrally assessed molecular markers, IDH1 or IDH2 mutations were detected in 25.7%, NPM1 mutations in 15.6%, TP53 mutations in 22.2%, and FLT3 mutations in 18.0% pts. Pts may have more than one of these mutations (i.e co-mutations of NPM1 and IDH1or IDH2, NPM1 with FLT3, or NPM1 with TP53) identified in baseline BMA. The CR/CRi rates were 83.7% for pts with IDH1/IDH2 mutations, 84.6% for pts with NPM1 mutations, 59.5% for pts with TP53 mutations, and 53.3% for pts with FLT3 mutations (Table 2). The median OS was not reached (NR) for pts with IDH1, IDH2 or NPM1 mutations, while the median OS for pts with detectable TP53 mutations or FLT3 mutations was 8.9 mos and 12.4 mos respectively. The remissions (CR or CRi) were rapid (median TTR between 1.1 mos to 1.8 mos) for each group (Table 2) and durable responses were observed for pts with IDH1, IDH2, NPM1, or FLT3 mutations (median DOR was either NR for IDH and NPM1 and 19.9 mos for FLT3). Pts with TP53 mutations are known to have poor prognosis and the DOR in this cohort was 5.6 mos. The median time on study was 11.6 mos (range: 0.3-44 mos). The range of BCL2 mRNA expression (2-ΔCt) in bone marrow blasts was 0.03 to 2.93, with a median of 0.86. The Cox hazard ratio for OS was 1.06, p=0.8565 based on BCL2 above the median (&amp;gt; 0.86 2-ΔCt). The multivariate analysis included age, cytogenetic risk, AML type (primary vs secondary) and trial (M14-387 vs M14-358). Albeit not statistically significant and small sample sets, pts with either IDH1 or IDH2 mutations (N = 21) tended to have higher BCL2 expression (median 2-ΔCt 1.1 ; range 0.19 -2.93) while pts with TP53 mutations (N = 7) had lower levels of BCL2 expression (median 2-ΔCt 0.55; range 0.03 - 1.27). Determination of baseline expression of other family members (BCL2L1, MCL1, BCL2A1, BCLw, NOXA, etc) is ongoing and additional univariate and multivariate analyses will be presented at the meeting. Conclusions: These analyses demonstrate that VEN + HMA or LDAC has efficacy across multiple molecular markers in AML. This activity is rapid and durable, and is observed across different levels of BCL2 expression in AML blasts. Disclosures Chyla: Abbvie, Inc: Employment, Other: Stock or options. Harb:AbbVie Inc: Employment, Other: Stock/stock options. Mantis:AbbVie Inc: Employment, Other: Stock/stock options. Riehm:AbbVie Inc.: Employment, Other: Stock/stock options. Ross:AbbVie: Employment, Other: Stock/stock options. Sun:AbbVie: Employment, Other: Stock/stock options. Huang:AbbVie Inc: Employment, Other: Stock/stock options. Jiang:AbbVie Inc.: Employment, Other: Stock/stock options. Dail:Genentech: Employment, Equity Ownership. Peale:Genentech Inc.: Employment, Other: Stock/stock options. Potluri:AbbVie, Inc.: Employment, Other: Stock/stock options. Hayslip:AbbVie Inc: Employment, Other: Stock/stock options. OffLabel Disclosure: Venetoclax is a BCL-2 inhibitor that is FDA-approved in some indications. This presentation will focus on venetoclax for treatment in acute myeloid leukemia, which is not an approved indication.

A Phase I Trial of Venetoclax in Combination with BEAM Conditioning Chemotherapy (V-BEAM) with Autologous Stem Cell Transplantation in Non-Hodgkin Lymphoma

Background: High-dose chemotherapy (HDCT) followed by autologous stem cell transplantation (ASCT) is a standard treatment both as consolidation after induction chemotherapy and as second-line therapy depending on the histologic subtype of NHL. BEAM (BCNU, etoposide, cytarabine, and melphalan) is a commonly used conditioning regimen for NHL but often fails to produce durable remission, likely due to inherent chemoresistance. Many B-cell NHL subtypes including diffuse large B-cell lymphoma and double-hit lymphoma are resistant to chemotherapy-induced apoptosis by overexpression of Bcl-2. Venetoclax is an orally administered selective Bcl-2 inhibitor with significant single agent activity in CLL and mantle cell lymphoma. In addition, the efficacy of venetoclax is potentiated by combination with rituximab [Seymour 2018, Kater 2018] and multiple lines of preclinical data show synergistic efficacy with a range of both novel and conventional antineoplastic agents [Johnson-Farley 2015, Li 2015]. Study Design and Methods: This is a single arm, open-label, dose-escalation phase I trial to evaluate the safety of venetoclax in combination with BEAM (V-BEAM) conditioning chemotherapy. Key inclusion criteria include patients with pathologically confirmed NHL, regardless of specific histology, who have received one prior systemic therapy and are eligible for and proceeding with HDCT followed by ASCT. The trial employs a standard 3+3 cohort-based dose escalation design of venetoclax (800 mg daily days -7 through -6 on dose level 1, 800 mg daily days -7 through -5 on dose level 2, 800 mg daily days -7 through -4 on dose level 3, 800 mg daily days -7 through -3 on dose level 4, and 800 mg daily days -7 through -2 on dose level 5) with standard doses of BEAM followed by ASCT. The primary objectives are to assess safety, describe dose-limiting toxicities, engraftment of stem cells and to identify the recommended phase II dose. Secondary objectives include evaluation of progression-free survival (PFS) and overall survival (OS) compared to historical controls treated with BEAM as part of ASCT. Neutrophil and platelet engraftment will be estimated with cumulative incidence and compared to controls with Gray test. OS and PFS will be estimated with Kaplan-Meier and compared to controls with the log-rank test. As of 8/1/2019, the first dosing cohort of 3 patients have been enrolled and successfully completed study treatment. There are 3 patients in screening to be enrolled in dose level 2. Additional accrual will be presented at the time of the meeting. Clinical trial information: NCT03713580. Disclosures Anwer: Seattle Genetics: Membership on an entity's Board of Directors or advisory committees; In-Cyte: Speakers Bureau. Gerds:Celgene Corporation: Consultancy, Research Funding; Sierra Oncology: Research Funding; Pfizer: Consultancy; Incyte: Consultancy, Research Funding; CTI Biopharma: Consultancy, Research Funding; Roche: Research Funding; Imago Biosciences: Research Funding. Majhail:Nkarta: Consultancy; Atara Bio: Consultancy; Mallinckrodt: Honoraria; Incyte: Consultancy; Anthem, Inc.: Consultancy. Hill:TG therapeutics: Research Funding; Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Consultancy, Research Funding; Kite: Consultancy, Honoraria; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AstraZeneca: Consultancy, Honoraria; Celegene: Consultancy, Honoraria, Research Funding; Seattle Genetics: Consultancy, Honoraria; Takeda: Research Funding; Amgen: Research Funding. OffLabel Disclosure: Our trial in progress is investigating the use of venetoclax in combination with BEAM conditioning chemotherapy prior to autologous stem cell transplantation. Venetoclax is not currently approved in this setting.

COO, MYC / BCL2 Status and R-IPI Define a Group of Poor Prognosis DLBCL Patients in a Real-World Clinical Setting

BACKGROUND In DLBCL, and mainly in advanced-stage, the presence of an activated B-cell phenotype or a non-GCB phenotype, and the coexpression of Myc and Bcl2 protein by IHC, are usually considered to be associated with inferior outcomes. Some recent studies however found that non-GCB COO did not have prognostic impact in limited stage DLBCL patients. Further, in real life patient management, the clinical relevance of the assessment of the COO, and of Myc and Bcl2 protein overexpression (DE), is still not fully clarified. The aim of the research was to evaluate in a real-life context the prognostic impact of the COO and of DE status together with R-IPI. With this purpose we have therefore evaluated 142 DLBCL patients in stage I/IV by PET-CT. METHODS This is a retrospective multicenter study which recruited 142 DLBC patients with PET-CT-defined stage I/IV treated with R-CHOP or R-CHOP like between 2010 and 2016 in Modena, Rome and Pisa (Italy) and Haifa (Israel). Eligible patients had a morphologic diagnosis of DLBCL. All clinical data were recorded at diagnosis and during follow up including response assessment and survival outcome. COO was determined on FFPE diagnostic specimens either by IHC applying Hans algorithm, or at RNA level with Lymph2Cx assay by Nanostring technology. Expression of Myc and Bcl2 to define DE status was evaluated by IHC considering Myc and Bcl2 positive ≥40% and ≥50%, respectively. PET-CT imaging was interpreted locally utilizing Deauville criteria. The end points of the study were to determine the impact of COO, Myc/Bcl2 status and R-IPI on PFS and OS. PFS and OS were calculated using the Kaplan-Meyer method with 95 confidence interval (95C%CI), comparison between groups were made with logrank test. The effect of covariates was estimate by means of Cox PH regression, as hazard ratio (HR) with 95%CI. Statistical tests with P&lt;0.10 were considered clinically interesting. RESULTS We identified 142 patients with morphological diagnosis of DLBCL and with PET-CT-defined Stage I/IV treated with R-CHOP or CHOP-like regimens between 2011 and 2018. Patients with evidence of histologic transformation from indolent lymphoma, and double-hit high grade lymphoma were excluded. Median age was 63 yrs, 55% were more than 65 yrs old, 64% had stage III/IV, and 38% had R-IPI 3-5. Bcl2 and Myc were positive in 65% and 20%, respectively. Double expression of Bcl2 and Myc was found in 20% of cases. By Lympho2Cx, COO subtypes were ABC, CGB and Unclassified in 37 %, 46 % and 16 %, respectively. We observed a good concordance between COO determined by Lympho2Cx and Hans algorithm (0.718). Analysis of association between COO subtype by Lympho2Cx and baseline characteristics showed significant association of ABC subtype with B symptoms, R-IPI 3-5, BCL2+, but not with Myc+ , end of treatment PET+, and Response. After a median follow up of 49 months, 3 yrs and 5 yrs PFS and OS were 65% and 59%, respectively in PFS and 80% and 73 % in OS. By univariable analysis we observed significant differences between R-IPI 3-5 vs 0-2, stage III-IV vs I-II, and Bcl2 + vs -. GCB and Unclassified subtypes, Myc - and DE - showed better PFS and OS in comparison with ABC subtype, Myc + and DE +, but the differences were not statistically significant. Forty-seven of the 142 patients underwent SNC prophylaxis with MTX IT or IV. While in patients who received consolidation treatment no difference in PFS or OS between GCB and ABC were observed, patients with GCB subtype showed better PFS and OS in comparison with ABC patients (P=0.05). Based on the results of the univariable analysis, we have assembled a Score from 0 to 3 with R-IPI 3-5 (weight 1), Bcl2 + (weight 1) and ABC subtype (weight 1). The differences in 5 yrs PFS and OS between Score 0, 1, 2-3 were statistically significant (Fig 1). Discussion Although COO is considered a relevant prognostic factor, several studies have produced conflicting results. Our retrospective study conducted on a relatively small group of cases shows that patients with GCB and Unclassified subtypes and DE have a moderately improvement of survival outcome. However, a score based on ABC subtype, Bcl2 overexpression and a high R-IPI score is able to identify a group of poor prognosis patients. As determination of COO by Lympho2Cx is rapid to perform and potentially applicable to clinical practice as well as the determination of Bcl2 and Myc protein expression by IHC, we believe that the use of the Score is useful in patient management in the real life context. Figure 1 Disclosures No relevant conflicts of interest to declare.

Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN) Commonly Presents in the Setting of Prior or Concomitant Hematologic Malignancies (PCHM): Patient Characteristics and Outcomes in the Rapidly Evolving Modern Targeted Therapy Era

Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN) Commonly Presents in the Setting of Prior or Concomitant Hematologic Malignancies (PCHM): Patient Characteristics and Outcomes in the Rapidly Evolving Modern Targeted Therapy Era

Smart Start: Rituximab, Lenalidomide, and Ibrutinib Alone and in Combination with Standard Chemotherapy for Patients with Newly Diagnosed Diffuse Large B-Cell Lymphoma: Final Phase II Results

Smart Start: Rituximab, Lenalidomide, and Ibrutinib Alone and in Combination with Standard Chemotherapy for Patients with Newly Diagnosed Diffuse Large B-Cell Lymphoma: Final Phase II Results

Palmitoylation of GNA13 Is Essential for Its Tumor Suppressor Function and Its Inhibition Confers Hypersensitivity of B-Cell Lymphoma to BCL2 Inhibitors

GNA13, encoding G alpha subunit 13 protein, is a major tumor suppressor gene frequently mutated in germinal center B-cell-like diffuse large B-cell lymphoma (GCB-DLBCL) and Burkitt's lymphoma (BL). GNA13 is one of the key mediators of sphingosine-1-phosphate (S1P) signaling to confine B cells in germinal center. Inactivation of GNA13 coupled signaling could release tumor B cells from germinal center of lymphoid organs to peripheral blood and promote lymphomagenesis. Due to the "undruggable" nature of GNA13 as a tumor suppressor, targeted therapy remains an unsolved clinical issue. Protein palmitoylation is a unique post-translational lipid modification in regulating protein trafficking, stability and functionality. Emerging evidence reveals that palmitoylation plays vital roles in control of G-protein-coupled receptor (GPCR) signaling transduction. In this study, we discovered that GNA13 protein relies on amino-terminal palmitoylation for its membrane association and signal transduction, which is essential for its tumor suppressor activities. By using a Isobaric iodoTMT switch labeling based mass spectrometry method, we firstly identified that palmitoylation mainly occurs on two cysteine residues, Cys14 and Cys18, one of which site was also found mutated in DLBCL patients. To validate this result, we genetically mutated the cysteine to serine residues on these two potential palmitoylation sites, respectively. We found that both single and double mutants could largely reduce the palmitoylation level of GNA13. We also found that the loss of palmitoylation dissociates GNA13 protein from cell plasma membrane. Similar results were obtained using a pan-palmitoylation inhibitor, 2-bromopalmitate (2-BP). In order to examine the role of GNA13 palmitoylation in human DLBCL cells, we reintroduced either wildtype (wt) GNA13 or palmitoylation mutant GNA13 into Su-DHL-4 lymphoma cells after knocking down the endogenous wt GNA13 expression with short hairpin RNA interference. Our data show that the loss of palmitoylation promotes GCB-DLBCL cell proliferation and tumor growth both in vitro and in vivo, indicating that palmitoylation of GNA13 is essential to its tumor suppressor function. Mechanically, inactivation of GNA13, either by knocking down GNA13 expression or mutating the palmitoylation sites of GNA13, leads to phosphoinositide 3-kinase (PI3K)/AKT activation and BCL2 overexpression. Consistent to the increased BCL2 expression, we found that BCL2 inhibitors are among the most effective drugs to kill GNA13-deficient cells in a high-throughput chemical screen. Furthermore, we show that inhibition of palmitoylation by 2-BP enhances the drug sensitivity of GNA13 wildtype GCB-DLBCL cells to BCL2 inhibitors. These results indicate that the palmitoylation of GNA13 could serve as a target for treating B-cell lymphoma in combination with a BCL2 inhibitor. Disclosures No relevant conflicts of interest to declare.