Expression of the BAD pathway is a marker of triple-negative status and poor outcome

Triple-negative breast cancer (TNBC) has few therapeutic targets, making nonspecific chemotherapy the main treatment. Therapies enhancing cancer cell sensitivity to cytotoxic agents could significantly improve patient outcomes. A BCL2-associated agonist of cell death (BAD) pathway gene expression signature (BPGES) was derived using principal component analysis (PCA) and evaluated for associations with the TNBC phenotype and clinical outcomes. Immunohistochemistry was used to determine the relative expression levels of phospho-BAD isoforms in tumour samples. Cell survival assays evaluated the effects of BAD pathway inhibition on chemo-sensitivity. BPGES score was associated with TNBC status and overall survival (OS) in breast cancer samples of the Moffitt Total Cancer Care dataset and The Cancer Genome Atlas (TCGA). TNBC tumours were enriched for the expression of phospho-BAD isoforms. Further, the BPGES was associated with TNBC status in breast cancer cell lines of the Cancer Cell Line Encyclopedia (CCLE). Targeted inhibition of kinases known to phosphorylate BAD protein resulted in increased sensitivity to platinum agents in TNBC cell lines compared to non-TNBC cell lines. The BAD pathway is associated with triple-negative status and OS. TNBC tumours were enriched for the expression of phosphorylated BAD protein compared to non-TNBC tumours. These findings suggest that the BAD pathway it is an important determinant of TNBC clinical outcomes.

Ibrutinib Therapy Increases BCL-2 Dependence and Enhances Sensitivity to Venetoclax in CLL

Recent studies have demonstrated promising preclinical efficacy for the combination of ibrutinib with venetoclax (formerly ABT-199/GDC-0199) in CLL; however, the biology underlying how ibrutinib enhances the sensitivity of CLL cells to BCL-2 antagonism by venetoclax has heretofore remained obscure. Our group previously developed BH3 profiling, a functional assay that determines the proximity of a cell to the threshold of apoptosis, a property that we call "mitochondrial priming". A new variant of this technique known as dynamic BH3 profiling (DBP) measures early changes in net pro-apoptotic signaling at the mitochondrion that are induced in cancer cells treated with drugs. This technique can be used to determine "delta priming", which reflects the degree of short term change in mitochondrial priming in response to various drug treatments. We hypothesized that DBP would allow us to determine how ibrutinib and venetoclax influence mitochondrial priming and anti-apoptotic dependence in primary CLL cells. Mononuclear cells were isolated from CLL patients and treated in vitro with ibrutinib and/or venetoclax. Co-culture was performed with the StromaNKtert cell line seeded at a 1:10 ratio relative to CLL cells. BH3 profiling was performed as previously described by measuring the release of cytochrome C on a BD FACS Fortessa. Cell viability was determined by AnnexinV/PI by FACS. Western blot analyses were performed by standard protocols with signals captured by an LAS 4000 imager. The student's paired t-test was used to compare drug treatments with a one-tailed p ≤ 0.05 considered significant. We performed DBP on samples from 6 previously untreated CLL patients and found that BIM BH3 peptide (a measure of general priming) induced delta priming of 10% or less in all 6 samples treated in vitro with ibrutinib, whereas all 6 samples treated with venetoclax had delta priming of 20% or greater (Figure 1A). The results observed with BAD BH3 peptide (a measure of BCL-2 dependence) were exactly the opposite (Figure 1B), suggesting that while venetoclax increases the overall level of mitochondrial priming of CLL cells, ibrutinib selectively increases the dependence of CLL cells on BCL-2. Consistent with this finding, samples treated in vitro with ibrutinib also had increased BAD protein expression by Western blot compared to samples treated with venetoclax. Importantly, we confirmed these findings ex vivo in primary CLL cells isolated from 3 patients treated with ibrutinib on the recently reported phase 3 PCYC-1112 (RESONATE) trial. In all 3 patients, delta priming with BAD BH3 peptide was increased by day 15 of treatment (Figure 2), with 2 patients showing increases as early as 6 hours post first dose. By Western blot, increased BAD protein was observed after ibrutinib treatment in 2 of these 3 patients. Cells from patients on ibrutinib were also found to be more sensitive to venetoclax by Annexin/PI compared to their pre-treatment samples. These data suggest that ibrutinib makes mitochondria more BCL-2 dependent, providing further justification for moving forward with clinical trials of the ibrutinib plus venetoclax combination in CLL. Disclosures Letai: AstraZeneca: Consultancy, Research Funding; AbbVie: Consultancy, Research Funding; Tetralogic: Consultancy, Research Funding. Davids:Janssen: Consultancy; Pharmacyclics: Consultancy; Genentech: Other: ad board.

The Quality of Porcine Mesenchymal Stem Cells and Their Osteo- and Adipogenic Cell Derivatives – The Level of Proapoptotic Bad Protein Expression / Jakość Mezenchymalnych Komórek Macierzystych Świni Oraz Ich Pochodnych Zróżnicowanych W Kierunku Komórek Szeregu Osteo- I Adipogennego – Poziom Ekspresji Proapoptotycznego Białka Bad

The aim of the research was to evaluate the quality of porcine mesenchymal stem cells (MSCs) and MSC-derived osteoblasts/osteocytes (bone cells) and adipocytes (fat cells). This evaluation was performed on the basis of molecular analysis for proapoptotic BAD protein expression. MSCs isolated from the pig bone marrow were cultured in vitro for five weeks in three types of culture media: differentiating towards the osteoblasts/osteocytes (O) and adipocytes (A) and non-differentiating, control medium (C). In all groups of cells, the relative extent of BAD protein expression was estimated by western blotting. Significant differences in the posttranslational abundance of BAD proteins were noted between MSCs differentiated into the osteogenic and the adipogenic cell lineages (P<0.05). Summarizing the results, we conclude that posttranslational level of BAD protein expression can be used as a reliable marker for assessing the quality of both MSCs and their cell derivatives. Interestingly, the semi-quantitative profile of BAD protein expression in differentiated cells turned out to be lower than that observed in undifferentiated cells, demonstrating that the culture conditions used for pro-osteogenic or pro-adipogenic cellular transformation did not affect negatively the quality of MSCs.