Overexpression Of Bcl-2 Protein Research Articles

Bclw Overexpression Predicts Aggressive Disease in B-Cell Lymphomas

B-cell lymphomas encompass a phenotypically and genetically heterogenous subgroup within hematologic malignancies. Despite this heterogeneity, the ability to evade apoptosis is a unifying feature across B-cell lymphomas, and alterations within the Bcl-2 family of apoptosis regulatory proteins is a key mechanism for this evasion. While overexpression of the anti-apoptotic Bcl-2 family member BCL2 has been widely described in multiple B-cell lymphomas, altered expression of other anti-apoptotic proteins within this family, including BCLX, MCL1, A1 and, in particular, BCLW has been under-investigated. This is highlighted by clinical trials of the BCL2 inhibitor, venetoclax, which found poor single agent activity in B-cell lymphomas that were thought to be highly BCL2-dependent. Therefore, we investigated the contributions of anti-apoptotic Bcl-2 proteins in B-cell lymphomas. Analysis of gene expression profiling datasets of patient samples of major B-cell lymphoma subtypes as compared to normal B-cells demonstrated differential overexpression of anti-apoptotic Bcl-2 proteins, with BCL2, BCLX and BCLW being significantly overexpressed in multiple B-cell lymphoma subtypes. Focusing on aggressive B-cell lymphoma subtypes, BCLW was consistently overexpressed across all major B-cell lymphoma subtypes. Notably, BCLW overexpression was maintained in high grade follicular lymphoma compared to low grade, whereas BCL2 levels decreased as grade increased. These findings indicate that BCLW overexpression is selected for in more aggressive B-cell lymphoma subtypes. BCLW overexpression in aggressive B-cell lymphoma cell lines conferred resistance to the non-specific BH3 mimetic navitoclax as well as to venetoclax. Further, analysis of diffuse large B-cell lymphoma (DLBCL) cell lines demonstrated that the combination of high BCLW/low BCL2 in GCB DLBCL and high BCLW/moderate BCL2 in non-GCB DLBCL is also associated with decreased navitoclax sensitivity, suggesting that high BCLW may confer treatment resistance. In accordance with this, analysis of patient outcomes in DLBCL demonstrates that patients exhibiting high BCLW levels with low BCL2 levels have poorer survival than those with low BCLW. Moreover, Cox regression analysis showed BCLW was an independent determinant of overall survival in DLBCL. Together, our results indicate that BCLW predicts for more aggressive disease in B-cell lymphomas and increased resistance to therapy. Disclosures Eischen: AbbVie Inc.:Research Funding.

Abstract PO-18: Comprehensive correlation between genetic alterations in DLBCL and deregulated activation of the PI3K-AKT pathway isolates unique players in lymphoma dissemination and inferior outcome

Abstract Diffuse large B-cell lymphoma (DLBCL) is characterized by deregulation of numerous signaling pathways that direct cell proliferation and survival during B-cell maturation in the germinal center reaction. The subtype of germinal center B-cell DLBCL (GCB-DLBCL) demonstrates addiction to tonic B-cell receptor (BCR) signaling in part through the PI3K-AKT pathway. However, a comprehensive catalogue of the somatic genetic alterations leading to this deregulation is currently lacking and represents a limitation in advancing precision medicine options. In this study, we present a comprehensive analysis in 347 DLBCL biopsies uniformly treated with R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone), describing recurrent mutations, copy number alterations, and clinical parameters that are associated with AKT activation (pAKT) as determined by immunohistochemistry. We observed a significantly enhanced pAKT population including players in the Gα13 signaling (GNA13, P2RY8, ARHGEF1, RHOA, and S1PR2) and PI3K activation pathways (NFATC1, USP7, PTEN, INPP4B). Following this correlation, we observed a co-occurrence of BCL2 protein overexpression and mutation in Gα13 signaling associated with advanced clinical stage (P = .008) in 183 GCB-DLBCL cases, as well as activation of cell migration and cell-cell adhesion pathways, indicating their contribution to lymphoma dissemination. This co-occurrence was shown to be associated with shorter time to progression in GCB-DLBCL (P = .005). We furthermore uncovered recurrent deletions of INPP4B, a lipid phosphatase involved in AKT deactivation, to be associated with increased pAKT in GCB-DLBCL. We observed that INPP4B loss is similarly associated with shorter time to progression (P = .036) in GCB-DLBCL, and demonstrated altered AKT phosphorylation in a GCB-DLBCL-derived cell line with a monoallelic INPP4B deletion. We present here a useful comprehensive tool, through a positive correlation to enhanced pAKT staining, to uncover novel prognostic indicators related to genetic alteration status in DLBCL. Our findings provide novel insight into somatic genomic alterations underlying prosurvival signaling of GC-derived lymphomas, and present new avenues for therapeutic targeting. Citation Format: Shannon Healy, Daisuke Ennishi, Elena Viganò, Aixiang Jiang, Anja Mottok, Stacy Hung, Olga Kutovaya, Gerben Duns, Bruce Woolcock, David Scott, Christian Steidl. Comprehensive correlation between genetic alterations in DLBCL and deregulated activation of the PI3K-AKT pathway isolates unique players in lymphoma dissemination and inferior outcome [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr PO-18.

Phase I study of the Bcl-2 inhibitor venetoclax with DA-EPOCH-R as initial therapy for aggressive B-cell lymphomas.

8003 Background: Dose-adjusted (DA) EPOCH-R is a frontline treatment for aggressive B-cell lymphomas. Bcl-2 is associated with chemoresistance due to BCL2 gene rearrangement or protein overexpression in lymphomas and is antagonized by BH3 mimetic venetoclax (ven). We conducted a phase I study combining ven with DA-EPOCH-R in aggressive B-cell lymphomas. Methods: This phase 1 study used Bayesian optimal interval design with dose expansion. Eligible patients (pts) were 18 years with newly diagnosed diffuse large B-cell (DLBCL), primary mediastinal, and high grade B-cell lymphoma (HGBCL) with double hit (DHL) or not otherwise specified (NOS). Ven was dosed at 400 mg (DL1), 600 mg (DL2), and 800 mg (DL3) daily for 10 days with 6 cycles of DA-EPOCH-R. A subsequent cohort received ven 600 mg daily for 5 days (DL2B). Toxicities were graded by CTCAE v4.0 and response assessed by Lugano criteria. Dose limiting toxicity (DLT) period was cycle 1 and primarily included grade (gr) ≥3 neutropenia on cycle 2 day 1, gr ≥4 febrile neutropenia/thrombocytopenia, and gr ≥3 nausea, vomiting, diarrhea despite supportive care. Results: 30 pts enrolled with median age 64 (24-79), and 50% female. Ann Arbor stage was III-IV in 23. IPI was high risk in > 50%. Diagnosis was DHL (15), DLBCL NOS (13), and HGBCL NOS (2). 18 had MYC and 14 had BCL2 rearrangements. Bcl-2 was expressed ≥50% by IHC in 21/26 with data. There were no DLTs in DL1 (3 pts) or DL2 (9 pts). 1/6 had DLT in DL3 (gr 4 thrombocytopenia). Ven dose reductions occurred in subsequent cycles in 4 (2 in DL2; 2 in DL3). Of 18 in DL1-3, EPOCH was escalated above level 1 in 1 and de-escalated below level 1 in 7. Because of delays and ven dose reductions in DL2-3 due in part to cytopenias, infections and GI toxicities, we accrued DL2B. In DL2B, 0/12 pts had DLTs or ven dose reductions. EPOCH was escalated above level 1 in 4 and de-escalated below level 1 in 3. 1 died of sepsis during cycle 3. Most common gr 3-4 toxicities across all dose levels were cytopenias; febrile neutropenia occurred in 57%. Most common non-hematologic toxicities of all grades were hypocalcemia, nausea, diarrhea, hypokalemia and fatigue. ORR (N = 30) ITT was 97% with 27 (90%) complete and 2 (7%) partial responses; 1 was not evaluable. Follow up is ongoing. Of 15 DHL, ORR and CRR were 93% and 80%. Conclusions: We identified ven 600 mg for 5 days per cycle as RP2D with DA-EPOCH-R. DL2B was well tolerated and required no ven dose reductions. Further efficacy and safety is being evaluated in Alliance 51701, DA-EPOCH-R/R-CHOP in DH/double expressor lymphomas, using the dosing regimen defined by this study. Clinical trial information: NCT03036904 .

A novel recombinant expression and purification approach for the full-length anti-apoptotic membrane protein Bcl-2

Programmed cell death (apoptosis) is an essential mechanism in life that tightly regulates embryogenesis and removal of harmful cells. Besides an extrinsic pathway, an intrinsic (mitochondrial) apoptotic pathway exists where mitochondria are actively involved in cellular clearance in response to internal stress signals. Pro-apoptotic (death) and anti-apoptotic (survival) members of the B cell CLL/lymphoma-2 (Bcl-2) protein family meet at the mitochondrion's surface where they accurately regulate apoptosis. Overexpression of the anti-apoptotic Bcl-2 protein is a hallmark for many types of cancers and in particular for many treatment resistant tumors. Bcl-2 is a membrane protein residing in the mitochondrial outer membrane. Due to its typical membrane protein features including very limited solubility, it is difficult to express and to purify. Therefore, most biophysical and structural studies have used truncated, soluble versions. However, to understand its membrane-coupled function and structure, access to sufficient amount of full-length human Bcl-2 protein is a necessity. Here, we present a novel, E. coli based approach for expression and purification of preparative amounts of the full-length human isoform 2 of Bcl-2 (Bcl-2(2)), solubilized in detergent micelles, which allows for easy exchange of the detergent.

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.

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<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.

T(11;14) and High BCL2 Expression Are Predictive Biomarkers of Response to Venetoclax in Combination with Bortezomib and Dexamethasone in Patients with Relapsed/Refractory Multiple Myeloma: Biomarker Analyses from the Phase 3 Bellini Study

T(11;14) and High BCL2 Expression Are Predictive Biomarkers of Response to Venetoclax in Combination with Bortezomib and Dexamethasone in Patients with Relapsed/Refractory Multiple Myeloma: Biomarker Analyses from the Phase 3 Bellini Study

Addiction of B Cell Lymphomas to microRNA-17-92 Is Mediated By a Single Target Gene

Mature microRNAs (miRNA) are short non-coding RNAs that regulate gene expression by binding to messenger RNAs (mRNA) in a sequence-specific manner, causing translational repression and/or mRNA decay. The mammalian genome harbors thousands of miRNA genes, many of which are organized into transcriptionally co-regulated clusters such as miR-17-92. Knockout of the miR-17-92 cluster gene in mice blocked B lymphopoiesis, and ectopic miR-17-92 expression sufficed to initiate B cell lymphomas and autoimmunity. In humans, the miR-17-92 gene is commonly amplified or overexpressed via MYC-driven transcription in diffuse large B cell and Burkitt's lymphomas. A computationally predicted shared target of the miR-17-92 miRNAs is the pro-apoptotic BCL-2 family protein BIM, central to life-death decisions in mammalian cells. To clarify the contribution of miR-17-92:Bim interactions to the miR-17-92 knockout and overexpression phenotypes, we engineered a unique in vivo system of conditional mutagenesis of the nine Bim 3'UTR miR-17-92 binding sites. Instead of causing the predicted B cell developmental block, interruption of miR-17-92:Bim interactions produced a selective inability of B cells to resist cellular stress; and prevented lymphocyte hyperplasia caused by Bim haploinsufficiency. Surprisingly, partial genetic disruption of miR-17-92:Bim interactions was sufficient to fully prevent B cell lymphoma formation in two out of three mice using two independent pre-clinical MYC-driven cancer models. This protective effect could be attributed to an increased activity of the mitochondrial apoptosis pathway in pre-malignant B cells, as apoptosis was abolished by concomitant overexpression of an anti-apoptotic BCL-2 protein. MYC-driven B lymphoma cells are addicted to miR-17-92 function. Our data build on these results and strongly suggest that miR-17-92:Bim interactions are vital in this context as acute ablation of miR-17-92:Bim interactions effectively promoted lymphoma cell apoptosis, both in vitro and in vivo. In conclusion, among hundreds of putative miR-17-92 target mRNAs a single direct binding partner is vital for lymphoma development and maintenance, a discovery whose therapeutic exploitation is of major relevance. Disclosures No relevant conflicts of interest to declare.

Immunohistochemical overexpression of BCL-2 protein predicts an inferior survival in patients with primary central nervous system diffuse large B-cell lymphoma

This study was designed to analyze the clinical characteristics and prognostic value of c-MYC and BCL-2 proteins expression in patients with primary central nervous system diffuse large B-cell lymphoma (PCNS-DLBCL).82 patients newly diagnosed with PCNS-DLBCL, from January 2008 to November 2018, were enrolled in this study. Clinical characteristics, immunohistochemical features, laboratory examinations, and treatment outcome were analyzed among these patients.Among these 82 cases, 45 were males (54.9%) and 37 were females (45.1%). Age ranged from 16 to 78 years old, and 29 patients (35.4%) were elder than 60 years old, with median age at 57 years old. According to Hans classification, 25 were accounted for origin of germinal center B-cell (GCB) subtype (30.5%) and 49 were accounted for non-GCB subtype (59.8%), respectively. Eight patients were unclassified due to lack of detailed pathological results. The median survival of these 82 patients was 30 months, and 1-year, 3-year, and 5-year overall survival (OS) rate was 59.7%, 44.6%, and 34.1%, respectively. Patients treated with sequential HD-MTX based chemotherapies showed a superior prognosis than those without. In combination with rituximab, the outcome was further improved. The median OS was 55 months in HD-MTX + R group, 27 months in HD-MTX group, and 9 months in other groups, respectively. Univariate analysis identified age ≥60, ECOG score ≥ 2 points, and overexpression of BCL-2 protein (≥85%) were adverse prognostic factors for OS. Co-expression of c-MYC (≥40%) and BCL-2 (≥50%) proteins was associated with poor ECOG score, high Ki-67 expression, and trended towards an inferior outcome. Gender, lesion location, number of lesions, lactic dehydrogenase (LDH), cell of origin, BCL-6 protein expression, expression of c-MYC protein alone and Ki-67 ≥85% had no significant impact on OS.In patients with PCNS-DLBCL, age ≥60 years old, ECOG score ≥2 points, and overexpression of BCL-2 protein (≥85%) were associated with a poor survival. HD-MTX based chemotherapies in combination with rituximab could improve the prognosis.

Abstract 2497: FCN-338, a novel and selective Bcl-2 inhibitor, exhibits potent anti-tumor activity in B-cell lymphoma

Abstract B-cell lymphoma-2 (Bcl-2) family proteins are key regulators of apoptosis, which is vital for proper tissue development and cellular homeostasis. Apoptosis occurs via activation of two different pathways: the extrinsic pathway, triggered by a death ligand binding to a death receptor, such as TNF-α to TNFR1; and the intrinsic pathway, regulated by Bcl-2 family proteins and their complex protein-protein interactions. Bcl-2 family proteins are functionally classified as either anti-apoptotic, such as Bcl-2, Bcl-xL and Mcl-1, or pro-apoptotic, including Bid, Bim, Bad, Bak and Bax. Anti-apoptotic Bcl-2 family members are often found to be up-regulated in many types of human cancers, such as different subtypes of B-cell lymphoma and diverse solid tumors, and are frequently been correlated with decreased susceptibility to chemotherapeutics and to increased radio-resistance. Overexpression of Bcl-2 proteins is an independent indicator of poor prognosis in tumors including chronic lymphocytic leukemia (CLL), prostate cancer, and small cell lung cancer (SCLC). Therefore, targeting Bcl-2 and its pro-survival relatives that form the core anti-apoptotic machinery, has emerged as a promising therapeutic approach in cancer. To that end, we have developed a novel and selective Bcl-2 inhibitor, FCN-338, with nanomolar affinity to Bcl-2 and physicochemical properties suitable for oral administration. FCN-338 exhibits much greater affinity to Bcl-2 than Bcl-xL, suggesting its good potential to avoid thrombocytopenia caused by the inhibition of Bcl-xL. FCN-338 has shown remarkable anti-proliferation potency against a panel of Bcl-2-addicted human B-cell lymphoma cell lines, including DOHH2 follicular lymphoma (FL) cell line (IC50 2 nM), Mino diffuse large B-cell lymphoma (DLBCL) cell line (IC50 23 nM), and RS4;11 acute lymphoblastic leukemia (ALL) cell line (IC50 35 nM), but not in Bcl-xL-dependent H146 SCLC cell line (IC50 >4,500 nM). Correspondingly, in vivo efficacy of FCN-338 was demonstrated in a variety of xenograft models derived from FL, DLBCL and ALL tumors. FCN-338 dramatically caused tumor regression in a dose-dependent manner. In the non-clinical studies, FCN-338 exhibits much improved pharmacokinetic properties with higher T1/2, dose-normalized AUC and bioavailability in dogs compared with the FDA-approved Bcl-2 inhibitor ABT-199. Preferable safety profiles of FCN-338 were shown with no potential hERG inhibitory effect and less drug-drug interaction potential, as evidenced by no inhibitory effect (IC50 >50 μM) on CYP2C9 enzyme compared with ABT-199 (IC50 1.05 μM). Together, our findings highlight the therapeutic potential of FCN-338 to be used as effective therapy across a broad range of Bcl-2-addicted B-cell malignancies. Citation Format: Shu Lin, Xingdong Zhao, Hongbin Liu, Huajie Zhang, Zhifang Chen, Lihua Jiang, Yanxin Liu, Min Lin, Yue Rong, Rui Tan, Zuwen Zhou, Zongyao Zou, Yuwei Gao, Weibo Wang. FCN-338, a novel and selective Bcl-2 inhibitor, exhibits potent anti-tumor activity in B-cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2497.

PF239 COMBINED TARGETING OF BCL-2 AND SET-PP2A INTERACTION INDUCES POTENT AND SYNERGISTIC ANTI-LEUKEMIC EFFECTS ON ACUTE MYELOID LEUKEMIA

Background: Acute myeloid leukemia (AML) is an aggressive malignancy that arises from the transformation of hematopoietic stem cells. Most AML patients are older than 60 years, and in this group only 5–15% of cases are cured (Döhner et al., 2015). Therefore, it is necessary to explore new therapeutic options. Overexpression of the anti-apoptotic BCL-2 protein is a common finding in AML. ABT-199 (Venetoclax), a potent and selective inhibitor of BCL-2, has been recently developed. However, clinical activity of ABT-199 monotherapy is modest in AML (Konopleva et al., 2016), which could be explained by the genetic and epigenetic heterogeneity of AML. Our group has shown that inactivation of PP2A is present in 78% of AML patients, and that FTY720 treatment restores PP2A activity by disrupting the SET-PP2A interaction, thereby inhibiting proliferation of AML cells (Pippa et al., 2014). Since PP2A promotes apoptosis by affecting the phosphorylation state of a variety of pro- and anti-apoptotic factors, we hypothesized that BCL-2 targeting could synergize with PP2A re-activation and induce synergistic anti-leukemic effects. Aims: To investigate whether the combination of ABT-199 with FTY720 could induce synergistic anti-leukemic effects in vitro and in vivo. Methods: A panel of six human AML cell lines was used to perform cell culture experiments. To generate zebrafish AML xenograft models, AML cell lines were treated for 24 h with ABT-199, FTY720 or their combination. Subsequently, cells were stained with a fluorescent cell tracker and injected into the yolk sac of 48 hours-post-fecundation wild type zebrafish embryos. After injection, cell proliferation and colonization index were measured. Colony-Forming-Unit (CFU) assays in semi-solid medium were performed on patient-derived AML cells isolated from three patients with AML at diagnosis. Results: GI50 values for ABT-199 and FTY720 showed that HL-60, MV4–11 and MOLM-13 were the most sensitive cell lines to ABT-199, while OCIAML-3 and HEL cells were resistant to BCL-2 specific inhibition (GI50 value greater than 10 μM). Regarding FTY720 treatment, we observed that OCIAML-3 and MV4–11 were the most sensitive cell lines, while HEL cells were FTY720-resistant. By western-blot analyses, we ascertained that sensitivity to ABT-199 was dependent on BCL-2, MCL-1 and BCL-XL expression levels, and sensitivity to FTY720 was dependent on PP2A and SET expression. Next, survival of AML cells was analyzed after exposure to ABT-199, FTY720, or their combination at different concentrations (accordingly on the above GI50 calculated values). Analyses of the combination index values showed that combination of ABT-199 and FTY720 had a potent and synergistic anti-leukemic effect, even in OCI-AML3 cells, which were ABT-199-resistant cells. Mechanistically, combination of ABT-199 and FTY720 induced a synergistic pro-apoptotic activity by increasing significantly the number of Annexin/IP positive cells, the activity of Caspases 3/7, and PARP cleavage. Compared to single treatments, treatment with ABT-199 plus FTY720 also reduced significantly cell proliferation and/or cell invasive potential of HL-60, MOLM-13, and MV4–11 cells in zebrafish xenograft models. Furthermore, we demonstrated in primary-AML cells that the combined treatment significantly decreased the number as well as the size of CFUs. Summary/Conclusion: Combined targeting of BCL-2 and the SET-PP2A interaction has potent and synergistic anti-leukemic effects in vitro and in vivo. Our findings provide a rational basis for further testing of novel combined therapies with ABT-199 and FTY720 in AML patients.

5-Chlorobenzofuran-2-carboxamides: From allosteric CB1 modulators to potential apoptotic antitumor agents

Cannabinoids as THC and the CB1 allosteric modulator CBD were reported to have antiproliferative activities with no reports for other CB1 allosteric modulators as the 5-chloroindole-2-carboxamide derivatives and their furan congeners. Based on the antiproliferative activity of two 5-chlorobenzofuran-2-carboxamide allosteric CB1 modulators, a series of novel derivatives was designed and synthesized. The synthesized compounds were tested in a cell viability assay using human mammary gland epithelial cell line (MCF-10A) where all the compounds exhibited no cytotoxic effects and more than 85% cell viability at a concentration of 50 μM. Some derivatives showed good antiproliferative activities against tumor cells as compounds 8, 15, 21 and 22. The most active compound 15 showed equipotent activity to doxorubicin. Compounds 7, 9, 15, 16, 21 and 22 increased the level of active caspase 3 by 4–8 folds, compared to the control cells in MCF-7 cell line and doxorubicin as a reference drug. Compounds 15 and 21, the most activecaspase-3 inducers, increase the levels of caspase 8 and 9 indicating activation of both intrinsic and extrinsic pathways and showed potent induction of Bax, down-regulation of Bcl-2 protein levels and over-expression of Cytochrome C levels in MCF-7 cell lines. Compound 15 exhibited cell cycle arrest at the Pre-G1 and G2/M phases in the cell cycle analysis of MCF-7 cell line. The drug Likeness profile of the synthesized compounds showed that all the compounds were predicted to have high oral absorption complying with different pharmacokinetics filters.

Synthesis and biological evaluation of novel xanthine derivatives as potential apoptotic antitumor agents

A series of novel xanthine/NO donor hybrids containing 1,3,8-trisubstituted or 1,8-disubstituted xanthine derivatives were designed and synthesized. The synthesized compounds were tested in a cell viability assay using human mammary gland epithelial cell line (MCF-10A) where all the compounds exhibited no cytotoxic effects and more than 90% cell viability at a concentration of 50 μM. The oxime containing compounds 7a-b and 17-24 were more active as antiproliferative agents than their non-oxime congeners 6a-b and 9-16. Hydroxyimino-phenethyl scaffold compounds 17-24 were more active than the hydroxyimino-ethyl phenyl acetamide 7a-b derivatives. Compounds 18–20 and 22-24 exhibited inhibition of EGFR with IC50 ranging from 0.32 to 2.88 μM. Compounds 18-20 and 22-24 increased the level of active caspase 3 by 4–8 folds, compared to the control cells in Panc-1 cell lines compared to doxorubicin as a reference drug. Compounds 18, 22 and 23 were the most caspase-3 inducers. Compounds 22 and 23 increased the levels of caspase-8 and 9 indicating activation of both intrinsic and extrinsic pathways and showed potent induction of Bax, down-regulation of Bcl-2 protein levels and over-expression of cytochrome c levels in Panc-1 human pancreas cancer cells. Compound 23 exhibited mainly cell cycle arrest at the Pre-G1 and G2/M phases in the cell cycle analysis of Panc-1 cell line. The drug likeness profiles of compounds 18-20 and 22-24 were predicted to have good to excellent drug likeness profiles specially compounds 18-20 and 23. Finally molecular docking study was performed at the EGFR active site to suggest thier possible binding mode. The hydroxyimino-phenethyl scaffold compounds 17-24 represent an interesting starting point to optimize their pharmacokinetics and pharmacodynamics profiles.

Correlation Between C-MYC, BCL-2, and BCL-6 Protein Expression and Gene Translocation as Biomarkers in Diagnosis and Prognosis of Diffuse Large B-cell Lymphoma.

This study investigates the protein expression of C-MYC, BCL-2, and BCL-6 in diffuse large B-cell lymphoma (DLBCL) and their relationship with genetic abnormalities. A retrospective study of 42 cases on paraffin-embedded tissue specimens diagnosed with DLBCL was performed using immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). The expression of C-MYC, BCL-2, BCL-6 protein, and gene abnormalities in these tissue samples was analyzed. The relationship in genetic abnormalities and Ki-67, Hans classification, gender, and age was also evaluated. It was found that the positive rate of C-MYC expression was 47.6% (20/42), the rate of C-MYC gene abnormality was 26.2% (11/42), in which gene translocation accounted for 23.8% (10/42) and gene amplification 2.4% (1/42); C-MYC protein expression was positively correlated with C-MYC gene translocation (χ2 = 11.813; P = 0.001); C-MYC gene translocation was mainly found in germinal center B cell type (χ2 = 4.029; P = 0.045). The positive rate of BCL-2 protein expression was 85.71% (36/42), the positive rate of translocation was 42.86% (18/42) and the amplification rate was 26.19% (11/42); the overexpression of BCL-2 protein was correlated with the BCL-2 translocation (χ2 = 3.407; P = 0.029). The positive rate of BCL-6 protein expression was 45.24% (19/42), the positive rate of BCL-6 translocation was 14.29% (6/42) and the positive rate of BCL-6 amplification was 7.14% (3/42); the overexpression of BCL-6 protein was significantly correlated with BCL-6 translocation (χ2 = 6.091; P = 0.014). The Ki-67 index was significantly higher in C-MYC translocation cases than in non-C-MYC translocation cases (χ2 = 4.492; P = 0.034). Taken together, our results suggest that the protein expression of C-MYC, BCL-2, and BCL-6 are positively correlated with their gene translocation. Overexpression of C-MYC, BCL-2, BCL-6 protein suggests the possibility of translocation. Therefore, immunohistochemical detection of C-MYC, BCL-2, and BCL-6 are useful in diagnosis and prognosis of DLBCL.

Immunohistochemical expression of Bcl-2 in oral squamous cell carcinoma: a clinicopathological correlation

Background Oral malignancy includes a group of neoplasms affecting any district of the oral cavity, salivary glands, and pharyngeal regions. Notwithstanding, this term has a tendency to be utilized conversely with oral squamous cell carcinoma (OSCC), which represents the most incessant of every oral neoplasm. It is evaluated that a greater than of 90% of oral neoplasm are OSCC malignancies. The Bcl-2 protein is the encoding result of Bcl-2 proto-oncogene. The overexpression of Bcl-2 protein assumes an imperative part in apoptosis protection in many tumors, including head and neck squamous cell carcinoma. Aim/objectives This study was conducted to scope and find out the possible role of Bcl-2 in the biological behavior of OSCC. Methods A total of 20 OSCC cases were immunohistochemically examined for expression of Bcl-2. Results and conclusions There was significant difference among grades of OSCC regarding Bcl-2 expression. A significant correlation between Bcl-2 expression and clinical staging, nodal infiltration, as well as distant metastasis was observed. We concluded that Bcl-2 may be used as a prognostic factor for OSCC and may aid in decisions on cancer diagnosis and therapy.

Prognostication of Diffuse Large B-Cell Lymphoma By Lympho2Cx Assay and BCL2 and MYC Expression: Application in a Real Life Context of 154 Patients

BACKGROUND: About one third of patients with diffuse large B-cell lymphoma (DLBCL) eventually die from their disease. Thus, it is extremely important to identify powerful and reliable predictive and prognostic markers. In real life patient management, the clinical relevance of the assessment of the Cell of Origin (COO), of MYC and BCL2 protein overexpression, is still not fully clarified. The aim of the study is to evaluate the predictive and prognostic value of COO with Lympho2Cx assay by Nanostring technology and BCL2 and MYC overexpression by immunohistochemistry (IHC), in a real-life context.METHODS: This is a retrospective multicenter study which recruited 154 DLBCL patients treated with R-containing regimen between June 2010 and December 2016 in Modena, Rome (Italy) and Haifa (Israel). All clinical data were recorded at diagnosis and during follow up, including response assessment and survival outcome. COO was determined on formalin-fixed paraffin-embedded diagnostic specimens either by IHC using a panel of antibodies which included CD10, BCL6, MUM1 (i.e. Hans algorithm), or at RNA level with Lymph2Cx assay by Nanostring technology. Expression of MYC and BCL2 was evaluated by IHC. Event free survival (EFS) is defined as the time from diagnosis to the time of last follow-up, or to one of the following events: any response other than complete remission (CR) at the end of therapy or death from any cause. It is assessed by Kaplan-Meier estimates and groups of risk are compared using the log rank test.RESULT: We have currently evaluated the data of 60% (95 out of the 154) of the patients enrolled in the study (Tab 1). After a median follow up of 49 months, EFS is 63% (95CI 51-72%).In univariate analysis, the variables with the greatest impact on the response and on EFS are absolute granulocyte count and BCL2 expression (MYC ongoing). Patients with low IPI showed better survival in comparison with patients with high IPI, but the difference is not statistically significant. By IHC non-GCB subtype was more common than GCB (56% vs. 44%); by Nanostring ABC, GCB and unclassified subtypes were 33%, 50% and 17%, respectively, and K statistics was 0,647, showing a substantial agreement between the results obtained by IHC and by Lympho2Cx assay. No statistically significant differences were observed in EFS among ABC, GCB and unclassified subtypes. However, BCL2 protein overexpression in ABC subtype is associated with shorter EFS.CONCLUSION: Early retrospective studies showed a survival advantage for GCB-type disease. Clinical trial data evaluating the impact of COO determined by GEP on prognosis have shown inconsistent results with 2 studies observing inferior survival of ABC subtype and 2 German studies showing no significant differences in PFS or OS among COO subtypes. Determination of COO by Lympho2Cx is attractive as it is relatively easy and rapid to perform and potentially applicable to clinical practice. However, these preliminary results do not support, at this point, its use as prognostic factor in clinical practice. The analysis of the remaining 59 patients will help to clarify the role Lympho2Cx assay in the context of real life.Table 1. Patients baseline characteristics. DisclosuresTadmor:ABBVIE: Consultancy; ROCHE: Research Funding; JNJ: Consultancy; NOVARTIS: Consultancy; PFIEZER: Consultancy.

ABT-199-mediated inhibition of Bcl-2 as a potential therapeutic strategy for nasopharyngeal carcinoma

BackgroundAberrant overexpression of Bcl-2 protein has been detected in 80% of nasopharyngeal carcinoma (NPC), and Bcl-2 family proteins are implicated in both NPC oncogenesis and chemotherapy resistance. Previous studies have shown that while treatment of NPC cells with Bcl-2 family inhibitors alone is rarely effective, concomitant treatment with a cytotoxic reagent such as cisplatin can increase efficacy through a synergistic effect. The aim of the current work was to determine how we might increase the efficacy of Bcl-2 family inhibitors in the absence of cytotoxic reagents, which are associated with negative side effect profiles. MethodsWe assessed cell proliferation in Bcl-2 high-expressing NPC cells by CCK-8 assay after treatment with the Bcl-2 inhibitor ABT-199 and/or the Mcl-1 inhibitor S63845. Apoptotic induction by ABT-199 was evaluated by Annexin V-FITC and PI double staining. We also evaluated Bcl-2 family protein expression (Bim, Mcl-1, Bcl-xL, Noxa) after treatment with ABT-199 by western blotting. Finally, xenografted Balb/c nude mice were used to test ABT-199 efficacy in vivo, H&E and immunohistochemistry assay were used to analyze tumor samples. ResultsABT-199 effectively induced NPC cell apoptosis in vitro and in the xenograft model. Following ABT-199 treatment in NPC cells, upregulation of Mcl-1 and Bcl-xL can lead to drug resistance, while concomitant Noxa overexpression partially neutralized the Mcl-1-caused resistance. Given that ABT-199 induces apoptosis in NPC cells through the Bcl-2/Noxa/Mcl-1 axis, treatment avenues further targeting this pathway should be promising. Indeed, the newly developed Mcl-1 inhibitor S63845 in combination with ABT-199 had a synergistic effect on NPC cell apoptosis. ConclusionBcl-2 inhibition in NPC cells with ABT-199 triggers apoptosis through the Bcl-2/Noxa/Mcl-1 axis, and dual inhibition of the anti-apoptotic Bcl-2 family proteins Bcl-2 and Mcl-1 provided a strong synergistic effect without the need for adjunctive cytotoxic agent treatment with cisplatin.

Targeted and Sustained Corelease of Chemotherapeutics and Gene by Injectable Supramolecular Hydrogel for Drug-Resistant Cancer Therapy.

Coadministration of chemotherapeutics as well as therapeutic gene could play a synergistic effect on cancer treatment. It is noteworthy that targeted and sustained codelivery of chemotherapeutic and therapeutic gene was rarely achieved in previous reports, while it might serve as an important platform for treating solid tumor with possible surrounding lesions. Herein, an injectable supramolecular hydrogel formed by α-cyclodextrin (α-CD) and cationic amphiphilic copolymer made of methoxy-poly(ethylene glycol)-b-poly(ε-caprolactone)-b-poly(ethylene imine) with folic acid targeted group (MPEG-PCL-PEI-FA), is rationally designed to achieve sustained codelivery of chemotherapeutic paclitaxel (PTX) and B-cell lymphoma-2 (Bcl-2) conversion gene Nur77 in the form of nanocomplex up to 7 days, to effectively inhibit the growth of folate receptor overexpressing H460/Bcl-2 therapeutic-resistant tumors (induced by overexpression of anti-apoptotic Bcl-2 protein), with peritumoral injection rather than direct intratumoral injection of hydrogel. To the best of our knowledge, this is a pioneer report on injectable MPEG-PCL-PEI-FA/α-CD supramolecular hydrogel with the ability to codeliver and sustainedly release PTX and Nur77gene to combat Bcl-2 overexpressed therapeutic-resistant tumors in a targeted manner, which might be beneficial for further design in personalized medicine.

Abstract 951: Direct small-molecule BAX activation in acute myeloid leukemia

Abstract Resistance to apoptosis is a hallmark of human cancer. The BCL-2 protein family includes both pro- and anti-apoptotic proteins and has a central role in regulating mitochondrial apoptosis. Cancer cells most frequently ensure their survival and resistance to treatments by overexpression of anti-apoptotic BCL-2 proteins to maintain BAX and other pro-apoptotic members suppressed. Pro-apoptotic BAX is the cardinal executioner BCL-2-family member that, upon conformational activation and oligomerization at the mitochondrial outer membrane (MOM), causes permeabilization of the MOM and release of mitochondrial factors, e.g., cytochrome c, that activate the caspase cascade of apoptosis. Interestingly, the vast majority of cancer cells contain functional BAX in an inactive conformation or suppressed by anti-apoptotic proteins; mutations or alterations in BAX that may cause its inactivation to occur at a low frequency. Therefore, we hypothesized that induction of apoptosis by direct activation of BAX offers the possibility of a new anticancer strategy. Using a structure-based drug design approach we developed BTSA1, a pharmacologically optimized BAX activator that binds with high affinity and specificity to the N-terminal activation site (trigger site) and induces conformational changes to BAX, resulting in BAX mitochondrial translocation, oligomerization and mitochondrial dysfunction (Reyna et al., Cancer Cell 2017). BTSA1 induces activation of cytosolic BAX, leading to prompt and robust mitochondrial apoptosis in acute myeloid leukemia (AML) cell lines. The efficacy of BTSA1 is regulated by the availability of anti-apoptotic BCL-2 proteins to inhibit activated BAX and therefore, higher levels of cytosolic BAX monomer correlated with higher efficacy of BAX-mediated mitochondrial dysfunction. BTSA1-induced BAX activation promotes effectively apoptosis in primary AML samples and has significant antitumor activity in human AML xenograft models, increasing host survival. BTSA1 is orally bioavailable with excellent pharmacokinetics at therapeutically effective doses; it does not show any detectable toxicity in the hematopoietic system or other tissues. Importantly, BTSA1 is also effective in inducing apoptosis in leukemic stem cell-enriched fractions (CD34+CD38−) while sparing healthy counterparts. Consistent with their sensitivity to direct BAX activation, AML blasts and highly purified stem and progenitor cell populations from AML patients when compared to healthy counterparts displayed higher expression of BAX. Overall, our data provide proof of concept for BAX as a druggable target and demonstrate the therapeutic potential of direct BAX activation by BTSA1 as an effective treatment strategy in AML. Citation Format: Denis Reyna, Thomas Garner, Andrea Lopez, Felix Kopp, Gaurav Choudhary, Ashwin Sridharan, Swathi-Rao Narayanagari, Kelly Mitchell, Baoxia Dong, Boris Bartholdy, Loren Walensky, Amit Verma, Ulrich Steidl, Evripidis Gavathiotis. Direct small-molecule BAX activation in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 951.

Prognostic significances of overexpression MYC and/or BCL2 in R-CHOP-treated diffuse large B-cell lymphoma: A Systematic review and meta-analysis

Numerous studies have investigated the prognostic values of MYC and/or BCL2 protein overexpression in diffuse large B-cell lymphoma (DLBCL). However, the results still demonstrate discrepancies among different studies. We aimed to do a systematic review and meta-analysis on the relationships between overexpression MYC and/or BCL2 and DLBCLs treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). This study followed the guidelines of PRISMA and Cochrane handbook. The hazard ratios (HRs) for overall survival (OS) were pooled to estimate the main effect size. Twenty studies recruited a total of 5576 patients were available for this meta-analysis. The results showed that MYC (HR = 1.96, 95%CI (confidence interval) = 1.69–2.27)without heterogeneity(I2 = 17.2%, P = 0.280), BCL2 (HR = 1.65, 95%CI = 1.43–1.89, I2 = 20.7%, P = 0.234) protein overexpression, and co-overexpression (HR = 2.58, 95%CI = 2.19–3.04, I2 = 17.2%, P = 0.275) had a poor prognosis in R-CHOP treated DLBCL patients, respectively. The current analysis indicated that MYC and/or BCL2 protein overexpression, and particularly co-overexpression was related to short overall survival in R-CHOP treated DLBCL patients, showing that application of the two new biomarkers can help to better stratify DLBCL patients and guide targeted treatment.