High Levels Of Bcl-2 Expression Research Articles

Targeted Protein Degradation for c-MYC Overcomes Therapy Resistance in T-Cell Acute Lymphoblastic Leukemias

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy characterized by diffuse infiltration of the bone marrow by immature lymphoblasts of thymic origin expressing T-cell surface markers. Early T cell precursor (ETP)-ALL is a distinct subtype of T-ALL characterized by a lack of CD1a and CD8; low expression of CD5 and the presence of myeloid or stem cell markers, and recent studies have shown resistance to conventional chemotherapies and poor outcome in ETP-ALL compared to thymic or mature T-ALL. A recent study revealed distinct gene expression profiles in adult compared to pediatric-adolescent ETP-ALL, representing higher expression levels of BCL2 and CD34 (Dai et al. Proc Natl Acad Sci 2022), however, detailed proteomic profiling in ETA-ALL has yet to be determined. Amplification of overexpression of MYC oncogene or stabilization of c-MYC protein occurs up to 70% in human cancers. Multifaceted activities of MYC include rapid proliferation of malignant cells supported by increased mitochondrial and ribosome biogenesis, dysregulated protein translation, and altered metabolism. NOTCH1-MYC signaling axis activation is one of the hallmarks that drives T-ALL leukemogenesis (Weng et al, Science 2004; Sanchez-Martin and Ferrando, Blood 2017). We have reported that targeting of BRD4 proteins induces suppression of leukemia initiating cells (LICs) in T-ALL by inhibiting the NOTCH1-MYC-CD44 axis, providing rationale to target MYC in therapy-resistant T-ALL (Piya et al. Leukemia 2022). We recently reported targeted protein degradation of c-MYC utilizing GT19715, the first-in-class cereblon modulator (CELMoD) for c-MYC exhibited promising anti-leukemia efficacy in acute myeloid leukemias (Nishida et al. ASH 2022). Here we employ GT19715 to investigate efficacy of targeting protein degradation of c-MYC in T-ALL. GT19715 induced dose-dependent apoptosis and cytoreduction in T-ALL cell lines with IC50 values below 10 nM except HPB-ALL cells. We found substantial decrease of c-MYC protein levels in sensitive but not in resistant cells (HPB-ALL). GT19715 induced apoptosis and ³ 90% cytoreduction at nanomolar concentrations in primary, therapy-resistant T-ALL. GT19715 also enhanced cell death induced by dexamethasone. In a xenograft model of CCRF-CEM cells carrying NOTCH1, PTEN, FBXW7, KRAS and TP53 mutations, GT19715 (3 mg/kg, three IP injections per week, two weeks on and one week off) reduced > 99% circulating human CD45+ leukemia cells compared to vehicle on day 16 after engraftment ( Fig. A), suggesting promising anti-leukemia efficacy in T-ALL in vivo. ETP-ALL cells from an adult patient exhibited distinct clustering compared to those from an adolescent patient as determined by single-cell mass cytometry (CyTOF) (N = 2). Adult ETP-ALL cells were characterized by increased protein levels of BCL2, CD34, CD44 and p-S6 compared to adolescent ETP-ALL cells, suggesting more stem-like properties. On the other hand, adolescent ETP-ALL cells showed higher CD7, CD33 and CD38 levels compared to the adult sample ( Fig. B). Interestingly, GT19715 treatment predominantly reduced p-S6 high clusters in adult ETP-ALL cells, potentially suggesting c-MYC degradation-induced suppression of the AKT-mTOR pathway. Conclusion: Targeted protein degradation of c-MYC induces promising anti-leukemia efficacy in T-ALL cells in vitro and in vivo. Further mechanistic and in vivo studies are ongoing.

Novel Autochthonous Mouse Models of Serve As Preclinical Tools to Investigate Pathogenesis and Treatment of MCD/C5 DLBCL

Diffuse large B cell lymphoma (DLBCL) is the most common lymphoid malignancy in adults. Both, biologically and clinically, DLBCL represents a highly heterogeneous disease. Based on gene expression profiling DLBCL is subdivided into germinal center B cell (GCB)-like and non-GCB- or activated B cell (ABC)-like DLBCL, a classifier which distinguishes DLBCL sub-entities displaying distinct biological features and clinical response to frontline anthracycline-based chemo-immune therapy. Plasmablasts have long been regarded as the cell of origin (COO) of ABC DLBCL, however, recent evidence points towards aberrant memory B cells (MB) as the true COO of these lymphomas. Several recent studies attempted to give structure to the genetic heterogeneity observed in DLBCL by clustering cases based on their mutational profiles. Clusters MCD by Schmitz et al. and C5 by Chapuy et al. are amongst the clusters enriched for ABC-DLBCL cases with particularly inferior prognosis. Both clusters are defined by mutations in MYD88 and CD79B as well as high levels of BCL2 expression and recurring mutations in transcriptional regulators of the plasmacytic differentiation process (PRDM1, SPIB). We previously reported an autochthonous mouse model of DLBCL mimicking hallmark mutations of MCD/C5 DLBCL: Combination of Myd88 p.L252P (the murine orthologue of human MYD88 L265P) and overexpression of BCL2 leads to the generation of ABC-DLBCL-like lymphomas in the Myd88p.L252P/wt;R26LSL.BCL2/wt;Cd19Cre/wt (MBC) mouse line. MBC Lymphoma cells show a plasmablastic immunophenotype with expression of CD138 and lack of expression of B220/CD19. In an attempt to refine our genetically-engineered mouse model, we incorporated other key alterations of MCD/C5 DLBCL in the MBC background: B cell specific loss of PRDM1, overexpression of SPIB and hallmark mutations in CD79B. Young Prdm1fl/fl;Myd88p.L252P/wt;R26LSL.BCL2/wt;Cd19Cre/wt (PPMBC) and Myd88p.L252P/wt;R26LSL.BCL2/LSL.Spib;Cd19Cre/wt (SMBC) compound animals display a strong reduction of the CD138+ population compared to control, accompanied by a decrease in serum immunoglobulins, in agreement with a block in plasma cell differentiation mediated by these additional genetic alterations. In contrast, cells with a marker profile of age-associated B cells (ABC, B220+/CD19+/CD21-/CD23-) as well as Memory B cells (MB, B220+/IgD-/Fas-/CD38+) accumulate markedly in all lymphoma lines, but particularly in PPMBC animals, compared to control. This is in line with recent studies hinting at ABC/ MB as the cells of origin of ABC-DLBCL. While MBC lymphomas are largely CD138+ and represent a plasmablastic/-cytic differentiation stage, PPMBC tumors express B-cell markers B220 and CD19 and lack expression of CD138. Additionally, PPMBC lymphomas frequently show a marker profile reminiscent of memory B cells, while CD21/CD23 - negativity suggests a relation to age-associated B cells, a population enlarged also in the pre-malignant setting. PPMBC tumors furthermore show an enrichment for B-cell receptor (BCR), NFkB and PI3K signaling gene signatures, when compared to MBC lesions. These signs of enhanced B cell receptor signaling translate into an increased sensitivity towards inhibition of Bruton's Tyrosine Kinase (BTK) by ibrutinib. The sensitivity of ibrutinib is further enhanced in lymphomas additionally expressing Cd79b p.Y195H, both in the Prdm1-deficient and the Prdm1-proficient model systems. We have established and characterized novel refined autochthonous mouse models of MCD/C5 DLBCL which serve as a preclinical tool to test novel therapeutic strategies in this sub-entity and pave the way to a deeper understanding of molecular mechanisms of pathogenesis, treatment response and resistance in highly aggressive DLBCL.

Abstract A21: Novel autochthonous mouse models as preclinical tools in the study of MCD/C5 DLBCL

Abstract Diffuse large B cell lymphoma (DLBCL) is a clinically and genetically very heterogeneous disease. Several [GK1] [RW2] recent studies attempted to give structure to the genetic heterogeneity by clustering cases based on their mutational profiles. Clusters MCD by Schmitz et al. and C5 by Chapuy et al. are amongst the clusters enriched for cases with particularly inferior prognosis. Both clusters are defined by mutations in MYD88 and CD79B as well as high levels of BCL2 expression and recurring mutations in transcriptional regulators of the plasmacytic differentiation process (PRDM1, SPIB). We have generated genetically-engineered mouse models that mimic key alterations of MCD/C5 DLBCL: Hallmark mutations in MYD88 and CD79B, loss of PRDM1 and overexpression of BCL2/SPIB. We could show that those mice develop clonal lesions, which morphologically resemble DLBCL. In the presence of a lesion that prevents plasmacytic differentiation (overexpression of Spib, loss of Prdm1), the forming lesions display signs of an earlier stage of B cell development, specifically histologic and transcriptomic similarities to germinal center cells. In contrast, allelic combinations without differentiation block lead to lymphomas that express CD138 and lack B220 expression, suggesting a more plasmacytic/plasmablastic differentiation status. The presence or absence of a Cd79b p.Y195H mutation has no notable effect on the immunophenotype of the lymphoma. However, its presence correlates with enrichment of BCR signaling gene set expression in lymphoma tissue and significantly increased sensitivity to inhibition of Bruton’s Tyrosine Kinase (BTK) by ibrutinib in our model systems. Taken together, we have established a set of murine models which mimic different genetic features of C5/MCD DLBCL. We have extensively characterized these models and show their usability as preclinical tools for therapeutic studies. Citation Format: Ruth Fluemann, Julia Hansen, Benedikt Pelzer, Tim Lohmann, Ilmars Kisis, Reinhard Büttner, Thorsten Persighel, Nima Abedpour, Martin Peifer, Ron Daniel Jachimowicz, Hans Christian Reinhardt, Gero Knittel. Novel autochthonous mouse models as preclinical tools in the study of MCD/C5 DLBCL [abstract]. In: Proceedings of the Third AACR International Meeting: Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2022 Jun 23-26; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2022;3(5_Suppl):Abstract nr A21.

Anti-PD-1 Checkpoint Therapy Can Promote the Function and Survival of Regulatory T Cells.

We have previously shown in a model of claudin-low breast cancer that regulatory T cells (Tregs) are increased in the tumor microenvironment (TME) and express high levels of PD-1. In mouse models and patients with triple-negative breast cancer, it is postulated that one cause for the lack of activity of anti-PD-1 therapy is the activation of PD-1-expressing Tregs in the TME. We hypothesized that the expression of PD-1 on Tregs would lead to enhanced suppressive function of Tregs and worsen antitumor immunity during PD-1 blockade. To evaluate this, we isolated Tregs from claudin-low tumors and functionally evaluated them ex vivo. We compared transcriptional profiles of Tregs isolated from tumor-bearing mice with or without anti-PD-1 therapy using RNA sequencing. We found several genes associated with survival and proliferation pathways; for example, Jun, Fos, and Bcl2 were significantly upregulated in Tregs exposed to anti-PD-1 treatment. Based on these data, we hypothesized that anti-PD-1 treatment on Tregs results in a prosurvival phenotype. Indeed, Tregs exposed to PD-1 blockade had significantly higher levels of Bcl-2 expression, and this led to increased protection from glucocorticoid-induced apoptosis. In addition, we found in vitro and in vivo that Tregs in the presence of anti-PD-1 proliferated more than control Tregs PD-1 blockade significantly increased the suppressive activity of Tregs at biologically relevant Treg/Tnaive cell ratios. Altogether, we show that this immunotherapy blockade increases proliferation, protection from apoptosis, and suppressive capabilities of Tregs, thus leading to enhanced immunosuppression in the TME.

Визначення експресії імуногістохімічних маркерів апоптозу та проліферації у тканинах фолікулярних неоплазій щитоподібної залози

Мета. Визначення експресії імуногістохімічних маркерів апоптозу р53 та bcl-2 і маркера проліферації Ki-67 у фолікулярних неоплазіях щитоподібної залози (ЩЗ). Матеріали та методи. Як критерії диференціальної діагностики фолікулярних неоплазій ЩЗ пропонуються численні імуногістохімічні маркери, серед яких найбільш корисними є маркери проліферації Ki-67 та апоптозу р53, bcl-2. Проведено імуногістохімічне дослідження операційного матеріалу 50 пацієнтів, оперованих у клініці ДУ «Інститут проблем ендокринної патології імені В.Я. Данилевського НАМН України» з приводу доброякісних (25 аденом) та злоякісних (25 карцином) фолікулярних новоутворень. Результати. Отримані дані показують, що фолікулярні аденоми солідної і фетально-ембріональної будови мають високий рівень спорідненості з фолікулярним раком ЩЗ за показниками проліферативної активності та порушень механізмів апоптозу, що, ймовірно, передбачає найбільший потенціал до малігнізації і подальшого метастазування. Висновки. Високий рівень експресії р53 та стабільно високий рівень експресії bcl-2 в клітинах фолікулярного раку ЩЗ і фолікулярної аденоми солідної та фетально-ембріональної будови вказує на глибокі порушення апоптотичних процесів при розвитку фолікулярних неоплазій ЩЗ.

P–125 Evaluation of N-acetylcysteine (NAC) effect on in vitro culture of immature mouse testis following vitrification

Abstract Study question Can the Culture of cryopreserved immature mouse testicular tissue in the presence of NAC improves the developmental process and prevent apoptosis induction during the culture? Summary answer An appropriate dosage of NAC in the culture medium of immature mouse testicular tissue was associated with increased cell survival and spermatogonia stem cell regeneration. What is known already Spermatogonial stem cells (SSCs) are the most advanced type of stem cells in the testes of prepubertal boys which are the main targets of oncological treatments. Therefore, the only possible alternative to maintain fertility in prepubertal boys is to preserve SSCs before their depletion by cryopreserving the testicular tissue. Despite the possibility of obtaining viable spermatozoa using cryopreserved testicular tissue cultivated in vitro,cryopreservation methods and damages caused by the culture procedure would be obstacles for maintaining the testicular tissueand it seems that optimizing the culture condition is vital. Study design, size, duration Testis tissues were harvested from 6-days-old immature NMRI male mice (n = 100) after cervical dislocation and vitrified. After 3 days testicular biopsies were warmed and distributed into control, culture Ӏ (not supplemented with NAC) and culture ӀӀ (supplemented with NAC) groups. To determine the appropriate NAC concentration 8 different dosages of NAC were evaluated in terms of cell survival and the best dose, a culture medium containing 125mmol/L NAC was selected to continue the study. Participants/materials, setting, methods Vitrified-warmed fragments (2mm3) obtaining from immature NMRI mice were cultured in vitro for 7 days on agar gel. The effects of culture conditions were assessed by Morphological evaluation of seminiferous tubules (using Hematoxylin-eosin staining). Cell viability, protein expression (caspase–3), and gene expression (Bax, Bcl2, Caspase–3, plzf) were evaluated by flow cytometry, immunofluorescence staining, and real time polymerase chain reaction respectively. Additionally, Malondialdehyde (MDA) concentration in the culture medium was measured by MAD Assay Kit. Main results and the role of chance Significant (p < 0.01) increase in cell viability was observed in the culture ӀӀ group after 7 days of culture compared to the culture Ӏ. Bax/Bcl2 ratio was significantly (p < 0.01) lower in the culture ӀӀ group compared to the control and culture Ӏ group. The expression of caspase–3 showed a significant (p < 0.001) increase in the culture ӀӀ group while immunofluorescence analysis showed low expression of it in all groups. These results were consistent with the high level of Bcl2expression that inhibited Caspase–3 expression and consequently the inhibition of apoptosis, and on the other hand, the presence of NAC showed that plzf expressions significantly (p < 0.001) increased in culture ӀӀ group compared to the control and culture Ӏ group. Although the presence of NAC did not inhibit all the deleterious effects of culture medium on tissue morphology, NAC was able to maintain better integrity of tissue and seminiferous tubules within central regions compared to the group without NAC. The decrease in MDA level in the presence of NAC (culture ӀӀ) was also a good indicator to confirm the desired results obtained from the presence of NAC in the culture medium. Limitations, reasons for caution Although the findings of the study were satisfactory in mice tissue after 1 week of culture, it is essential to replicate the experiments using human tissue and evaluate the quality and reproductive potential of surviving spermatogonia after long-term storage to become clinically applicable. Wider implications of the findings: This study highlights the necessity for further experiments to improve the testicular tissue culture conditions for better spermatogonial survival and differentiation to sperm, as the prepubertal fertility restoration methods are promising to be implemented in the clinic in the near future. Trial registration number Not applicable

BCL2 Expression Identifies a Population with Unmet Medical Need in Previously Untreated (1L) Patients with DLBCL

▪Introduction.High BCL2 protein expression has been associated with inferior outcome in multiple studies in diffuse large B-cell lymphoma (DLBCL) (Gascoyne et al. 1997; Salles et al. 2011; Horn et al. 2013; Tsuyama et al. 2017). Patients with high levels of BCL2 expression may benefit from treatment with venetoclax, a first-in-class, highly selective BCL2 inhibitor with demonstrated activity in cell lines and xenograft models (Souers et al. 2013, Punnoose et al. 2016). The evaluation of BCL2 as a prognostic marker has been complicated by the variability in recognized immunohistochemistry (IHC) thresholds (de Jong et al. 2007; Tsuyama et al. 2017) and the impact of cell-of-origin (COO) (Iqbal et al. 2011). Using a BCL2 IHC scoring algorithm that was developed to identify cell lines that are sensitive to BCL2 inhibition, and that could potentially identify patients likely to benefit from venetoclax, we evaluated the prognostic capability of BCL2 IHC in three independent cohorts of 1L DLBCL patients. Cohorts included two Phase 3 studies (MAIN [NCT00486759] and GOYA [NCT01287741]), and a representative cohort from the British Columbia Cancer Agency (BCCA) registry-based population, in which subjects received chemoimmunotherapy (either rituximab or obinutuzumab, combined with cyclophosphamide, doxorubicin, vincristine and prednisone [CHOP]).Patients and Methods. Pretreatment tumor samples were tested for BCL2 IHC using an algorithm that assesses both the percent of tumor cells stained and the intensity of staining, similar to the BCL2 algorithm proposed by Tsuyama et al. (Table 1).Whole-tissue slides from184 patients from MAIN were tested at Genentech , whole tissue slides from 366 patients from GOYA were tested at Hematogenex (Ventana assay) and tissue microarray slides from 310 patients from BCCA were tested at Ventana (Ventana assay). The NanoString Lymphoma Subtyping gene expression assay was used for classification of COO into germinal center B-cell (GCB), activated B-cell (ABC) or unclassified subtypes in MAIN (n=159), GOYA (n=353) and BCCA (n=302). Multivariate Cox regression analysis of progression-free survival (PFS) was performed in each study with the International Prognostic Index (IPI; The International NHL Prognostic Factors Project 1993) and/or COO as covariates. Given the similar treatment effect, patient data from both treatment arms in each study were merged for this analysis.Results. Baseline characteristics were similar for the BCL2 IHC evaluable and intent-to-treat (ITT) populations of these three cohorts. Prevalence of BCL2 positive patients was 48% in MAIN, 49% in GOYA and 58% in the BCCA cohort. The prevalence of BCL2 positive samples within the ABC subtype was 64%, 75% and 60% in the MAIN, GOYA and BCCA cohorts, respectively, and 40%, 38% and 56% within the GCB subtype, respectively. In univariate analysis, BCL2 positivity was associated with shorter PFS in all three cohorts (Figure 1, A-C). In the MAIN study, the prognostic effect of BCL2 appeared to be in the GCB subgroup, not ABC, as previously reported (Punnoose et al. ASH 2015). In the two largest studies of GOYA and BCCA cohorts, the prognostic effect of BCL2 was similar in ABC and GCB (not shown), and was independent of IPI (Table 2).Conclusions. Using a newly developed BCL2 IHC scoring algorithm, we demonstrate that DLBCL patients positive for BCL2 have a consistently inferior prognosis compared with patients negative for BCL2. The prognostic value of BCL2 IHC is independent of IPI and COO in GOYA and BCCA. The Ventana assay is currently being used in a Phase 2 study of venetoclax in combination with R-CHOP to evaluate the correlation (if any) of BCL2 expression with venetoclax + R-CHOP treatment (CAVALLI - NCT02055820). [Display omitted] DisclosuresSzafer-Glusman:Genentech: Employment. Peale:Genentech / Roche: Employment, Equity Ownership. Lei:Roche: Employment. Ray:Genentech/Roche: Employment. Farazi:Genentech: Employment. Kornacker:Roche: Equity Ownership; F. Hoffmann-La Roche: Employment. Oestergaard:F. Hoffmann-La Roche Ltd: Employment. Liu:F. Hoffmann-La Roche: Employment. Punnoose:Genentech: Employment.

Bexarotene enhances heart structure and function in diabetic rats by inhibiting ventricular remodeling and cardiomyocyte apoptosis

Purpose: To investigate the influence of bexarotene (Bex) on cardiac structure and function in streptozotocin (STZ)-induced diabetes mellitus (DM) rats, and the mechanism of action involved.
 Methods: Four groups of Sprague Dawley rats (n = 40) were used: normal control, DM, DM+ Bex (10 mg/kg/day), and DM+ Bex (20 mg/kg/day) (n = 10). The DM rat model was established by intraperitoneal injection of STZ. Cardiac structure and function of rats were determined and compared. Whole heart and left ventricle were weighed. The protein expressions of Bcl2 and Bax in rat myocardial tissue were determined using Western blotting.
 Results: Compared to control group, there was significant reduction in the levels of IVSd (inlet ventricular septal defect) and LVPWd (left ventricle posterior wall in diastole) in DM group, but significant increase in these parameters in DM +Bex (20 mg/kg/day) group, relative to DM-treated rats (p < 0.05). Moreover, there were higher expression levels of Bcl2 and Bax in DM group, when compared with normal control, but Bcl2/Bax ratio was significantly lower (p < 0.05). Furthermore, Bcl2 and Bax levels in DM + Bex (20 mg/kg/day) group were significantly lower than those in DM group, while Bcl2/Bax ratio increased significantly (p < 0.05).
 Conclusion: Bexarotene improves the cardiac structure of DM rats by lowering blood glucose, and by inhibiting ventricular remodeling and cardiomyocyte apoptosis. These findings may be beneficial in the development of new anti-DM drugs.

Biomarkers of Overall Survival and Response to Glasdegib and Intensive or Non-Intensive Chemotherapy in Patients with Acute Myeloid Leukemia

Background: In a previously reported Phase 2 randomized study of patients with acute myeloid leukemia (AML), addition of the investigational agent glasdegib (PF-04449913) to low-dose cytarabine (LDAC) improved overall survival (OS) when compared with LDAC alone. In a non-randomized study arm, glasdegib together with 7+3 chemotherapy was well tolerated and associated with clinical activity. We used a comprehensive biomarker analysis, evaluating gene expression, circulating cytokine levels, and gene mutations, to identify molecular drivers that predict overall response (OR) and OS.Methods: In this Phase 2 multicenter study (NCT01546038), patients with AML who were suitable for non-intensive therapy were randomized (2:1) to LDAC + glasdegib 100 mg QD or LDAC alone, and patients suitable for intensive therapy were assigned 7+3 plus glasdegib 100 mg QD. Whole blood, serum, and bone marrow aspirate samples were collected at baseline, and used to assess 19 genes for expression analysis, 38 analytes for circulating cytokine levels, and 109 genes for mutation analysis. Gene expression was analyzed using TaqMan Low Density Array Cards (TLDCs), cytokine levels were analyzed using quantitative, multiplexed immunoassays (Myriad RBM), and mutation analysis was performed using the Illumina® MiSeq instrument (San Diego, CA). All correlations were performed either for OS or for OR. For gene expression and cytokine analysis, a cut-off value above or below the median expression level for each treatment arm was used to separate samples into two subgroups (< or ≥ the median value) to explore the relationship of expression levels with OS data. Criteria for significance in the non-intensive cohort required one subgroup to have a p-value of <0.05 in the between-treatment arms comparison and the HR difference between the two subgroups to be ≥2 fold. Responses were defined as patients with a complete remission (CR), CR with incomplete blood count recovery (CRi), morphologic leukemia-free state, partial remission (PR), or PRi. For response correlations, genes or cytokines were considered to be differentially expressed if they had a p-value <0.05 and were differentially expressed by ≥2-fold.Results: Within the non-intensive arm (LDAC + glasdegib, n=68; LDAC alone, n=30), expression levels of several genes correlated with improved OS with glasdegib plus LDAC. Lower levels of expression of FOXM1 and MSI2, and higher expression levels of BCL2 and CCND2 correlated with improved OS with the combination. Additionally, lower levels of the cytokines 6CKINE (CCL21), ICAM-1, MIP-1α, and MMP-3 correlated with improved OS. An analysis of correlations of gene expression and cytokine levels with OR could not be completed due to the low number of responders in the LDAC only group (n=2). In the intensive treatment arm (glasdegib and 7+3, n=59), higher PTCH1 expression correlated with improved OS (p=0.0219, median OS 10.8 versus 39.5 months). In this cohort, lower levels of IL-8 (p=0.0225) and MIP-3β (p=0.0403) correlated with lower OS. Expression levels of no genes or cytokines significantly correlated with OR in this arm. We also examined correlations between gene mutation status and OS in both study arms. In the non-intensive arm (LDAC + glasdegib, n=58; LDAC alone, n=25), no genes mutated in at least 5 patients correlated with OS. In the intensive treatment arm (n=47), mutations in FLT3, TP53, CEP170, NPM1, and ANKRD26 correlated with OS (all p<0.05). Patients in this arm with FLT3 mutations responded better than patients with wild type FLT3 (p=0.0336, median OS of 13.1 months versus unreached for FLT3 mutant).Conclusions: In this biomarker analysis, we found that expression levels of a select number of genes and circulating cytokines implicated in AML correlated with OS in the non-intensive and the intensive arms. The improved response for patients with FLT3 mutations and high PTCH1 expression levels in the intensive arm deserves further investigation. These findings need to be verified in larger controlled studies, which are ongoing. DisclosuresCortes:Novartis: Consultancy, Research Funding; Daiichi Sankyo: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Astellas Pharma: Consultancy, Research Funding; Arog: Research Funding. Pollyea:Argenx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Consultancy; Celyad: Consultancy, Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Research Funding; Curis: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Heuser:Astellas: Research Funding; Daiichi Sankyo: Research Funding; Sunesis: Research Funding; Tetralogic: Research Funding; Bayer Pharma AG: Consultancy, Research Funding; StemLine Therapeutics: Consultancy; Janssen: Consultancy; Pfizer: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; BergenBio: Research Funding; Karyopharm: Research Funding. Chan:Pfizer: Employment, Equity Ownership. Wang:Pfizer: Employment, Equity Ownership. Ching:Pfizer Inc: Employment, Equity Ownership. Johnson:Pfizer Inc: Employment, Equity Ownership. O'Brien:Pfizer Inc: Employment, Equity Ownership.

Abstract 307: Targeting BCL-2 and BCL-xL with a novel dual inhibitor APG-1252 triggers cell death and inhibits tumor growth in small cell lung cancer models

Abstract Small cell lung cancer (SCLC) is one of the most deadly diseases with a dismal five year survival rate less than 7%. Even though most SCLC patients respond to the initial platinum-based cytotoxic or radiation therapies, they inevitably relapse and succumb to the disease. Anti-apoptotic proteins BCL-2 and BCL-xL, which are highly expressed in 40-60% of SCLCs and play a critical role in tumorigenesis and drug resistance, have been emerging as a promising target for therapeutic intervention. We have recently developed a novel dual BCL-2/BCL-xL inhibitor APG-1252 for cancer therapy. In this study, the effect of APG-1252 was evaluated in a panel of SCLC cell lines for discovery of indications and predictive biomarkers. The results show that the sensitivity of SCLC cell lines with sub-µM or nM IC50 values are correlated with the higher expression levels of BCL-2/BCL-xL, BIM and/or PUMA but lower levels of MCL-1. Conversely, the resistant cell lines either lack of BCL-2/BCL-xL protein, or exhibit higher level of MCL-1 protein. In xenograft tumor models, consistent with in vitro results, APG-1252 exhibits antitumor activities in the models derived from the sensitive cells but not in those from the resistant cells. Interestingly, while ABT-263 failed to inhibit H146 xenograft tumor growth, despite of its similar in vitro killing ability as APG-1252, APG-1252 showed potent antitumor activity in the xenograft model. To overcome the drug resistance conferred by MCL-1 in the resistant cells, we explore the combination therapy with other targeted agents. We found that our novel MDM2 inhibitor APG-115 was able to overcome the intrinsic resistance and sensitize those cells to APG-1252 in vitro, suggesting that reducing the apoptotic threshold by inhibiting other anti-death proteins like MCL-1 or increasing apoptotic function through p53 can enhance SCLC sensitivity to APG-1252. Collectively, APG-1252 represents a novel opportunity that can neutralize the protection from BCL-2/BCL-xL and trigger cell death and inhibit tumor growth in SCLC models. With the significance of these preclinical data, APG-1252 has been granted for phase 1 clinical trials in USA (NCT03080311) and China. Citation Format: GuangFeng Wang, Ping Min, MiaoYi Wu, Shuo Dang, ChuanYan Tang, Fei Zhang, Ming Guo, Shaomeng Wang, Jing Deng, Douglas D. Fang, DaJun Yang, YiFan Zhai. Targeting BCL-2 and BCL-xL with a novel dual inhibitor APG-1252 triggers cell death and inhibits tumor growth in small cell lung cancer models [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 307.

Atorvastatin inhibits cholesterol-induced caspase-3 cleavage through down-regulation of p38 and up-regulation of Bcl-2 in the rat carotid artery.

SummaryAim:Atherosclerotic lesions in the carotid arteries lead to a broad range of cerebrovascular disorders such as vascular dementia and ischaemic stroke. Recent studies have verified the beneficial role of atorvastatin (AV) in atherosclerosis. Despite a large body of studies, the mechanisms underlying this effect have not been completely explained. In this study, several experiments were performed on atherosclerotic rat models to investigate the anti-inflammatory and anti-apoptotic effect of AV in the carotid artery.Methods:In this experimental study, 40 male Wistar rats (250 ± 25 g) were randomly divided into four groups: rats on a normal diet (ND; n = 10); a high-cholesterol diet (HD; n = 10); a high-cholesterol diet plus AV (HD + AV; n = 10); and the AV control group (AV; n = 10). Cleavage of caspase-3 protein, expression of B-cell lymphoma 2 (Bcl-2) as well as phosphorylation of p38 mitogen-activated protein kinase (MAPK) were determined by immunoblotting assay in the carotid artery homogenate. Plasma atherogenic indices, including total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) were measured by colorimetric assay at the end of the experiment. Plasma levels of oxidised LDL (oxLDL) were measured by sandwich enzyme-linked immunosorbent assay (ELISA).Results:After eight weeks of feeding with a high-cholesterol diet, an elevated level of oxLDL was observed in the plasma in the HD group compared with the ND group [214.42 ± 17.46 vs 69.13 ± 9.92 mg/dl (5.55 ± 0.45 vs 1.78 ± 0.26 mmol/l); p < 0.01]. AV administration significantly reduced oxLDL levels in the HD + AV compared to the HD group [126.52 ± 9.46 vs 214.42 ± 17.46 mg/dl (3.28 ± 0.25 vs 5.55 ± 0.45 mmol/l); p < 0.01]. Results also showed that compared with the HC group, the HC + AV group had lower levels of p38 phosphorylation (p < 0.05) and higher levels of Bcl-2 expression (p < 0.05). Lower levels of cleaved caspase-3 were observed in the HC + AV group in comparison with the HC group (p < 0.05).Conclusions:The resultant data suggest that the anti-apoptotic effect of AV could be partially mediated by the pro-inflammatory protein p38 MAPK and the anti-apoptotic protein Bcl-2 in the rat carotid artery. Atorvastatin can therefore be considered a target drug in the prevention or development of atherosclerotic events.

Effects of Fu Shen ke li on myocardial cell apoptosis and endoplasmic reticulum stress-related protein expressions in rats with adriamycin induced heart failure

Objective To explore the contribution of endoplasmic reticulum stress to cardiac myocyte apoptosis in mouse congestive heart failure induced by Doxorubicin. Methods Doxorubicin (DOX) was administered intraperitoneally to rats, 2 mg/kg doses over 8 weeks. Echocardiographic and hemodynamics measurements were obtained at 8 weeks after treatment. Then left ventricular (LV) samples were collected. The successfully established rats were divided into 3 groups (Fu Shen Ke Li 40 mg/kg, 60 mg/kg, and 80 mg/kg). After 5 weeks post the operation, an echocardiographic was performed on all the rats under narcotism, Western blot to test the B-cell lymphoma-2 (Bcl-2), myeloid cell leukemia-1 (Mcl-1), Bcl-2-Associated X (Bax), Caspase-3, C/EBP homologous protein (CHOP), and 78-kDa glucose-regulated protein (GRP78), and reverse transcription-polymerase chain reaction (RT-PCR) to test the mRNA expression levels of Mcl-1, and NOXA. Results The left ventricular end-diastolic diameter (LVEDD), left ventricular end systolic diameter (LVESD), left ventricular ejection fraction (LVEF) prompted modeling success The left ventricular end-diastolic diameter (LVEDD), left ventricular ejection fraction (LVEF), and fractional shortening (FS) have been improved in high and middle groups of Fu Shen Ke Li. Compared to the model group, higher expression levels of Bcl-2, and Mcl-1 were, lower expressions of Bax, NOXA, CHOP, and GRP78 were (P<0.05) in 3 groups of Fu Shen Ke Li. Compared to the model group, there were higher mRNA expression level of Mcl-1 was , lower mRNA expression of NOXA was (P<0.05) in 3 groups of Fu Shen Ke Li. Conclusions Fu Shen Ke Li depresses endoplasmic reticulum stress, which can reduce the apoptosis of myocardial cells, and ultimately achieve the purpose of treatment of heart failure. Key words: SALVIA MILTIORRHIZA△/PD; GINSENG△/PD; SCROPHULARIA NINGPOENSIS/PD; Epirubicin/AE; Heart failure; Apoptosis; Endoplasmic reticulum; Stress

Inhibition of c-Jun N-terminal kinase signaling suppresses skin flap apoptosis in a rat ischemia and/or reperfusion model

BackgroundThe goals of this study were to validate the role of c-Jun N-terminal kinase (JNK) activation in skin flap apoptosis in a rat model of abdomen skin ischemia and/or reperfusion (IR) and to compare the protective effect of SP600125 and hydrogen-rich saline in skin IR injury. MethodsMale Sprague–Dawley rats were divided into five groups: one sham surgery group and four surgery groups. Before undergoing 3 h of IR management, the surgery groups were treated with normal saline (IR), dimethyl sulfoxide, SP600125 (SP), and hydrogen-rich saline (H). On the third postoperative day, blood perfusion of the flap was measured using Laser Doppler flowmeters. Hematoxylin and eosin staining was used to observe morphologic changes. Early apoptosis was observed using TdT-mediated dUTP-X nick end-labeling staining. pASK-1, pJNK, Bcl-2, and Bax were examined by immunodetection. Caspase-3 activity was also measured 24 h after reperfusion. ResultsCompared to the IR group and the dimethyl sulfoxide group, the SP group and the H group had larger skin flap survival area, more blood perfusion and lower levels of caspase-3 activity. The SP and the H groups had high expression levels of Bcl-2 and low expression levels of pASK-1 and pJNK. Bax was significantly decreased in the SP group. In addition, cell apoptosis was decreased in both the sham surgery and the H groups. ConclusionsIR-induced JNK phosphorylation was reduced by SP600125, indicating that JNK mediates the apoptosis pathways in rat skin. In the SP and the H groups, the apoptotic factors measured showed similar expression levels, indicating that JNK inhibition during IR may be associated with H-mediated protection against skin IR apoptosis.

Abstract P1-07-16: Triple-negative breast cancer: A single-centre retrospective cohort study of 408 TNBC cases with a focus on elderly patients

Abstract Background: TNBC represents a heterogeneous group of breast cancers that do not express ER-α, PgR and Her-2 receptors. Generally, these tumors are aggressive and more common in younger women. The aim of our study was to create a representative set of patients with TNBC, which could be analyzed and the data gathered to build basic epidemiological, molecular and clinical characteristics of Czech patients with TNBC. In particular, we focused on older patients (≥70 and ≥75 y.o.). Methods: We retrospectively studied a consecutive cohort of 408 patients diagnosed and/or treated for TNBC at the Masaryk Memorial Cancer Institute between 2004 and 2010. Some clinical-pathologic/molecular correlations were performed to identify prognostically different groups of patients. Results: The median age of patients was 56 years (25–88). A total of 9.3% of TNBC cases were diagnosed in patients under the age of 34, another 15,2% and 15,0% of cases were in the age group of 35 to 44 years and ≥70 years, respectively. In the group of patients aged ≥70 years (61), 59 % (36) were ≥75 y.o. Incidence of CK5/6+ and BRCA1 mutated tumors decreased with increasing age of patients, while the number of AR+ tumors increased (Chi-square test for trends: p=0,0245, p=0,0049 and p=0,0047, respectively). We confirmed the aggressive nature of this disease: in the follow-up period (median 77,2 months) we observed a relapse in 27,2 % (111) of patients: 71 % of deaths due to disease progression occured within 2 years after diagnosis of the disease. Patients ≥70 and especially ≥75 years of age had, together with patients ≤30 y.o., the highest risk of death due to tumor progression. DFS and OS of patients ≥75 y.o. was significantly worse in comparison with other patients (OS: p=0,035, HR 0,515; DFS: p=0,0077, HR 0,475). Simultaneously, adjuvant chemotherapy and anthracyclines were much less frequently administered in this age group (p&amp;lt;0,0001), despite the fact that the distribution of clinical stages did not differ among the age groups. In the whole cohort, the most important negative prognostic factors in relation to disease specific OS were: higher clinical stage and pT (both p&amp;lt;0.0001), pN–positive status (p&amp;lt;0.0001), absence or early withholding of chemotherapy (p&amp;lt;0.0001) and minimal disease response to neoadjuvant treatment (TRG4-TRG5) (p=0.005). High levels of BCL2 expression predicted poor OS in basal-like TNBC patients treated with adjuvant anthracycline-based regimens (p=0.033, HR 3.04). Contrariwise, longer OS was associated with the presence of tumor infiltrating lymphocytes (p=0,0004, HR 2,40). Conclusion: TNBC is an aggressive form of breast cancer, which may occur in patients of all ages, but more frequently in younger patients. Early detection and intensive treatment od these tumors gives a high chance of cure. Patients ≥70 and especially ≥75 years of age, together with patients ≤30 y.o., are at the highest risk of death due to tumor progression. While in younger patients it is likely due to the aggressiveness of disease, in older patients, in particular, because of an absence of adjuvant systemic therapy. The introduction of targeted therapies could potentially improve prognosis in both groups of patients (eg. PARP inhibitors, antiandrogens). Supported by IGA MZ CR: NT14599-3. Citation Format: Svoboda M, Navrátil J, Palácová M, Fabian P, Bareková L, Kabut T, Coufal O, Fait V, Jurácek J, Selingerová I. Triple-negative breast cancer: A single-centre retrospective cohort study of 408 TNBC cases with a focus on elderly patients. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P1-07-16.

Therapeutic effects of dendrosomal solanine on a metastatic breast tumor

AimsOur previous studies showed that alpha-solanine can inhibit tumor growth in cell culture and animal models of breast cancer. However, solanine is insoluble in common solvents; therefore, we developed a special nanoparticle with high-capacity solubility. The present study is aimed to deliberate the therapeutic effects of dendrosomal solanine (DNS) on a metastatic breast tumor in vitro and in vivo. Main methodsAfter DNS preparation and dosing procedures, forty-five mice were equally divided into five groups to investigate the anti-metastatic effects of DNS on mammary tumor-bearing mice. Key findingsCompared to solanine, DNS significantly suppressed the proliferation of 4 T1 cells in a dose- and time-dependent manner. DNS showed a remarkable safety rate of up to 10mg/kg. A significant decrease in white blood-cell count was seen at 20mg/kg DNS in comparison with control animals. Mice treated with DNS had smaller tumor volume (mm3) in comparison with control and solanine groups. Moreover, the incidence of the breast tumor metastases was about 67% in the control animals, where as solanine and DNS 1mg/kg were about 22% and 0%, respectively. Furthermore, the number of metastases per mouse varied from one to three. The tissues of tumor, brain, liver, spleen, and lung showed higher expression levels of Bcl-2 but lower expression levels of Bax, MMP-2, MMP-9, mTOR, and Akt in DNS-treated mice than control and solanine groups. SignificanceThe findings suggest that DNS has a more impactful therapeutic effect than solanine on 4 T1-induced breast tumorigenesis via influencing the tissue microenvironment.

Synergistic Co-Targeting of BTK and BCL2 in Mantle Cell Lymphoma

Mantle cell lymphoma (MCL), a B-cell non-Hodgkin lymphoma, remains incurable with current treatment modalities. Bruton tyrosine kinase (BTK), a key component of the early BCR signaling pathway, has emerged as a promising therapeutic target. Ibrutinib, a specific inhibitor that binds covalently to the active site of BTK at cysteine 481, has been approved for the treatment of MCL. In a recent Phase II study, treatment for relapsed or refractory MCL with Ibrutinib alone achieved a response rate of 68%. Although this response rate is striking, approximately one third of patients show primary resistance, and acquired resistance with a C481S mutation in BTK can also develop. To overcome primary and acquired resistance to BTK inhibition, a combinatory strategy that targets multiple pathways is needed.Here, we focused on the BCL2 anti-apoptotic pathway. Our previous study established FBXO10 as the E3 ubiquitin ligase that targets BCL2 for proteasomal degradation and revealed reduced levels of FBXO10 expression in MCL cell lines. To examine BCL2 and FBXO10 expression in patient samples, we performed immunohistochemical (IHC) analysis in a tissue microarray (TMA) that contained 62 MCL cases. Our data showed high levels of BCL2 expression but no or low FBXO10 expression in MCL cases. Based on this initial expression analysis, we hypothesized that a defect in FBXO10-mediated proteasomal degradation contributes to high BCL2 expression in MCL. To test this hypothesis, we genetically manipulated FBXO10 expression in MCL cell lines. Flow cytometric analysis revealed that overexpression of FBXO10 resulted in reduced BCL2 expression. Inversely, we silenced endogenous FBXO10 by shRNA. Indeed, after blocking protein synthesis by cycloheximide, the amount of BCL2 protein was sustained in these FBXO10 shRNA-expressing cells. These results indicate that BCL2 is the predominantly expressed anti-apoptotic protein in MCL, due to a specific defect in its proteasomal degradation. Inhibition of BCL2 by the specific inhibitor ABT-199 killed cancer cells in culture and also inhibited xenografted tumor growth.BCL2 is a target gene of the BTK/canonical NF-κB signaling pathway. We next analyzed BTK expression in the above TMA and found that BTK was highly expressed and positively correlated with BCL2 expression in these cases. BTK inhibition reduced BCL2 expression as shown in the BCR-dependent cell lines Jeko and Mino. RNA-seq analysis confirmed that a set of anti-apoptotic genes (e.g. BCL2, BCL-XL and DAD1) was downregulated by BTK shRNA. The downregulated genes also included those that are critical for B cell growth and proliferation, such as BCL6, MYC, PIK3CA and BAFF-R. Elevated BCL2, however, can also result from other mechanisms, such as amplification. Indeed, this genomic alteration is present in some MCL cell lines, including Granta-519 and Z138 used for this study. Since these cell lines are insensitive or less sensitive to Ibrutinib, we hypothesized that BCL2 upregulation is a mechanism underlying primary resistance to Ibrutinib, and thus targeting BCL2 by ABT-199 increases or restores sensitivity of these cells to Ibrutinib. The results supported this hypothesis and demonstrated a synergistic effect of ABT-199 and Ibrutinib on growth inhibition of Granta-519 and Z138 cells both in vitro and in vivo.In summary, this study elucidated mechanisms of BCL2 overexpression and association with the BCR/BTK signaling pathway in MCL. Our data provided a mechanistic rationale for co-targeting of these two oncogenic pathways by ABT-199 and Ibrutinib as a new therapeutic strategy in a pre-clinical setting. Most importantly, the findings indicate that this combination has a synergistic effect on the killing of both BCR-dependent and independent MCL cells. Since these two signaling pathways are deregulated in DLBCL and follicular lymphoma, co-targeting by Ibrutinib and ABT-199 may provide an attractive therapeutic strategy for these additional lymphoma patients as well. DisclosuresKahl:Roche/Genentech: Consultancy; Seattle Genetics: Consultancy; Millennium: Consultancy; Cell Therapeutics: Consultancy; Celgene: Consultancy; Infinity: Consultancy; Pharmacyclics: Consultancy; Juno: Consultancy.

BCL2 is an independent predictor of outcome in basal-like triple-negative breast cancers treated with adjuvant anthracycline-based chemotherapy.

Neither targeted therapies nor predictors for chemotherapy sensitivity are available for triple-negative breast cancer (TNBC). Our study included 187 patients with TNBC, 164 of whom were treated with anthracycline-based adjuvant chemotherapy. Eleven molecular biomarkers were analyzed. BCL2, epidermal growth factor receptor (EGFR), MYC, TOP2A, and Ki-67 protein expression was evaluated by immunohistochemistry. The status of the EGFR, MYC, and TOP2A genes and chromosomes 7, 8, and 17 was assessed using fluorescence in situ hybridization. High BCL2 expression predicted poor relapse-free survival (RFS) in patients treated with anthracycline-based adjuvant chemotherapy (p = 0.035), poor breast cancer-specific survival (BCSS) (p = 0.048), and a trend to poor overall survival (OS) (p = 0.085). High levels of BCL2 expression predicted poor OS in basal-like (BL) TNBC patients treated with adjuvant anthracycline-based regimens (log-rank p = 0.033, hazard ratio (HR) 3.04, 95 % confidence interval (CI) 1.04-8.91) and a trend to poor RFS (log-rank p = 0.079) and poor BCSS (log-rank p = 0.056). Multivariate analysis showed that BCL2 status, tumor size, and nodal status all had independent predictive significance for RFS (p = 0.005, p = 0.091, p = 0.003, respectively; likelihood ratio test for the whole model, p = 0.003), BCSS (p = 0.012, p = 0.077, p = 0.01, respectively; likelihood ratio test for the whole model, p = 0.016), and OS (p = 0.008, p = 0.004, p = 0.004, respectively; likelihood ratio test for the whole model, p = 0.0006). Similarly, multivariate analysis for BL TNBC showed BCL2, tumor size, and nodal status all had independent predictive significance for RFS (likelihood ratio test for the whole model, p = 0.00125), BCSS (p = 0.00035), and OS (p = 0.00063). High EGFR expression was associated with poor BCSS (p = 0.039) in patients treated with anthracycline-based adjuvant chemotherapy. Patients who underwent anthracycline-based adjuvant chemotherapy and exhibited CMYC amplification had a trend to worse BCSS (p = 0.066). In conclusion, high BCL2 expression is a significant independent predictor of poor outcome in TNBC patients treated with anthracycline-based adjuvant chemotherapy, and this is the first study showing the BCL2 prediction in BL TNBC. BCL2 expression analysis could facilitate decision making on adjuvant treatment in TNBC patients.

Neuroprotective effects of Bcl-2 overexpression on nerve cells of rats with acute cerebral infarction

We aimed to investigate the influence of lentiviral-mediated Bcl-2 overexpression in cerebral tissues of rats with acute cerebral infarction. Forty-five rats were randomly divided into sham, model, and treatment groups. The sham and model groups were administered a control lentiviral vector via the intracranial arteries 10 days before surgery, while the treatment group received lentivirus encoding a Bcl-2 overexpression vector. We induced cerebral artery infarction using a suture-occlusion method and analyzed the cerebral expression levels of apoptosis-related genes (caspase-3, Bax), total cerebral apoptosis, range of cerebral tissue infarction, and changes in nerve cell function after 72 h. The Bcl-2-encoding lentivirus was well expressed in rat cerebral tissues. The treatment group had significantly higher expression levels of Bcl-2 than the other two groups. After cerebral infarction, the model group had significantly increased expression levels of caspase-3 and Bax protein in cerebral tissues than the sham (P < 0.05). Expression of these apoptosis-related proteins in the treatment group was obviously lower than that in the model group (P < 0.05), but significantly higher than in the sham group (P < 0.05). Compared to sham, neuronal apoptosis levels and infarction range of cerebral tissues was increased in the model and treatment groups; however, these values in the treatment group were significantly lower than that in the model group (P < 0.05). Importantly, the treatment group had significantly decreased neurological impairment scores (P < 0.05). In conclusion, Bcl-2 over-expression can decrease neuronal apoptosis in rat cerebral tissue, and thus is neuroprotective after cerebral ischemia.

Combination Therapy with Gossypol Reveals Synergism against Gemcitabine Resistance in Cancer Cells with High BCL-2 Expression

Although gemcitabine is highly active in several cancer types, intrinsic and acquired drug resistance remains a major challenge. Overexpression of Bcl-2 has been associated with gemcitabine resistance. The aim of this study is to determine whether gossypol can overcome gemcitabine resistance in cell lines with high level of Bcl-2 expression in combination drug therapy. Our study demonstrated that in 10 cell lines derived from different cancers, high Bcl-2 baseline expression was observed in cell lines that were resistant to gemcitabine (GEM-R). Furthermore, synergistic effect of combination therapy was observed in gemcitabine-resistant (GEM-R) cell lines with high Bcl-2 expression, but not in a gemcitabine-sensitive (GEM-S) cell lines regardless of Bcl-2 expression. Gossypol treatment resulted in the decrease of anti-apoptotic genes such as Bcl-2 and Bcl-xl and an upregulation of the pro-apoptotic gene, Noxa. Furthermore, the addition of gossypol to gemcitabine resulted in lower expressions of anti-apoptotic genes compared to gemcitabine alone. Gene expression profiling in GEM-R and GEM-S cell lines suggest that anti-apoptotic genes such as pAkt and PI3KR2 may play important role in gemcitabine resistance, while pro-apoptotic Bcl-2 related genes (Bad, Caspase-6 and Calpain-1) may regulate synergistic interaction in combination therapy.

Protein Kinase C-β Dependent Activation of NF-κB in Stromal Cells Is Indispensable for the Survival of Chronic Lymphocytic Leukemia B-Cells in Vivo

AbstractAbstract 314Defects in the apoptosis program are a hallmark of chronic lymphocytic leukemia (CLL), characterized by high expression levels of bcl2 and Mcl1. Notably, this de-regulation of anti-apoptotic proteins is not sufficient to maintain long-term survival of CLL cells, which remain highly dependent on pro-survival factors provided by the leukemia-microenvironment. Bone marrow stromal cells (BMSCs) play an important role for microenvironment mediated survival of CLL cells, based on the provision of soluble and membrane-bound factors. The stroma-CLL interactions not only protect CLL cells from spontaneous, but also from drug-induced apoptosis, clinically recognized as minimal-residual disease. Therefore, understanding the molecular mechanisms of CLL-stroma interactions may offer new therapeutic options and help in eradicating CLL cells from the bone-marrow niche.Here we describe that monoclonal B-cells from CLL patients impose morphological and genetic changes in stromal cells, which become reminiscent of cancer-associated fibroblast (CAF). Comparative gene expression profiles indicate that contact with primary CLL cells induce the expression of pro-inflammatory genes in stromal cells. Further characterization of the underlying signaling pathways activated in stromal cells revealed that CLL cells induce the expression of protein-kinase C-β in BMSCs. Blocking the up-regulation of PKC-β by siRNA abrogated the pro-survival effects of stromal cells on CLL cells. Furthermore, following induction of PKC-β, BMSCs activate NF-kappaB through a Bcl10-independent, but NEMO/IKKgamma-dependent pathway. Gene expression profiling of NEMO-proficient and deficient BMSCs indicated that NF-kappaB regulates the expression of pro-inflammatory cytokines and adhesion molecules by stromal cells, required to promote survival of CLL. Interference with the NF-kappaB activation in BMSCs abrogated the pro-survival effects of stromal cells on CLL, similar to PKC-β deficient stromal cells.To demonstrate that this pathway is also important in vivo, Tcl1-CLL was transplanted into syngeneic PKC-β knock-out and wild-type mice. Notably, all PKC-β wild-type mice died of a CLL-like disease, whereas PKC-β kock-out animals were entirely resistant to CLL transplants. Immunofluorescence staining of PKC-β in bone marrow trephine biopsies indicated that this pathway is also activated in mesenchymal stromal cells of CLL patients. Importantly, our data provide further evidence that the PKC-β – NF-kappaB pathway is also activated in stomal cells by monoclonal B-cells from ALL and mantle-cell lymphoma (MCL) patients.Conclusively, we describe a novel survival signaling pathway activated by monoclonal B-cells in BMSCs. Interference with the PKC-β-NF-kappaB pathway activated in the leukemia/lymphoma microenvironment may offer new therapeutic options to fully eradicate malignant B-cells from bone-marrow niches. Disclosures:No relevant conflicts of interest to declare.