Journal: bioRxiv

Article Title: MCL1 dependence across MDS subtypes and dual inhibition of MCL1 and BCL2 in MISTRG6 mice

doi: 10.1101/2020.06.05.133090

Figure Lengend Snippet: Dual inhibition of MCL1 and BCL2 is synergistic in MDS cells resulting in increased apoptosis and loss of clonogenicity. a Cell viability of primary MDS samples was measured by CellTiter-Glo at 48 hr after treatment with three-fold dilutions of S63845, venetoclax (VEN) or a combination of both. Contour plots of synergy scores generated from the cell viability dose matrix of S63845 and VEN using the zero interaction potency (ZIP) model. The synergy scores were represented by pseudocoloring 2-D contour plots over the dose matrix, giving rise to the overall synergy landscape. Red color indicates synergy, while green color indicates antagonism for the various concentrations of the S63845/VEN combination. (Note different pseudocoloring scale for ZIP synergy scores between the samples) b-c Apoptosis in CD34 + cells was measured by annexin V/PI staining using flow cytometry after 24 hr of treatment in all samples combined ( b ) or broken down into MDS subtypes ( c ). The ratio of live cells relative to DMSO control were calculated. Individual patient samples in b-c are represented by symbols with means ± SEM, and statistical comparisons between S63845 or VEN monotherapy and the combination are shown (two-tailed t-test; n.s. not significant, *p < 0.05, **p <0 .01, ***p<0 .001, ****p < 0.0001). d-e Colony forming assays of frozen ( d ) or freshly-obtained ( e ) MDS cells. The number of colonies per 1 × 10 4 cells plated were calculated for each of the treated samples and for a DMSO control. The percent reduction of CFU-GM colonies in the combination treatment group compared to the DMSO control group is listed above each combination bar. Data is represented as an average of two replicates ± SEM. MDS-RS-SLD is MDS with ring sideroblast with single lineage dysplasia; MLD is multi-lineage dysplasia; EB1 is excess blasts 5-9%; EB2 is 10-19% blasts; CFU-GEMM is colony-forming unit – granulocyte, erythroid, macrophage, megakaryocyte; CFU-GM is colony-forming unit – granulocyte, macrophage; BFU-E is burst-forming unit – erythroid.

Article Snippet: Cells were plated into a 384-well dish with IMDM + 10% FBS + 1% Pennicillin/Streptomycin + cytokines and supplements (10ng/ml hIL3, 10ng/ml hFLT3 ligand, 10ng/ml hTPO, 10ng/ml hSCF, 5ng/ml hIL6, 4μg/ml hLDL, 10μM 2-Mercaptoethanol, and 1x L-GlutaMAX) and treated for 48 hours with an MCL1 inhibitor (S63845; Chemietek, Indianapolis, IN), BCL2 inhibitor (venetoclax; Chemietek), or BCL-xL inhibitor (A-1155463; Chemietek) at concentrations from 3nM to 10µM.

Techniques: Inhibition, Generated, Staining, Flow Cytometry, Two Tailed Test

Journal: bioRxiv

Article Title: MCL1 dependence across MDS subtypes and dual inhibition of MCL1 and BCL2 in MISTRG6 mice

doi: 10.1101/2020.06.05.133090

Figure Lengend Snippet: Evidence of reduced MDS engraftment in MISTRG6 mice after co-inhibition of BCL2 and MCL1. a-c Confirmed engraftment of 3 primary MDS patient cells in MISTRG6 mice. a Flow cytometric analysis of mouse vs human CD45 (mCD45 vs. huCD45; left panels) and huCD34 vs huCD33 (right panels). b Percent of huCD45 + cells in the bone marrow (BM) for each patient sample. c Relative distribution of myeloid CD33 + (red), B-lymphoid CD19 + (blue), and T-lymphoid CD3 + (gray) cells as percent of human CD45 + cells for each patient sample. d-g Patient sample MDS019-EB1 was allowed to engraft in MISTRG6 mice for 12 weeks before beginning treating with vehicle, VEN (15 mg/kg; 5 days on, 2 days off), or a combination of VEN (15 mg/kg; 5 days on, 2 days off) and S63845 (12.5 mg/kg; 2 days on, 5 days off) for a total of 4 weeks (n = 4 per treatment group). The percent ( d ) or total number ( e ) of huCD45 + cells in the BM were compared between pre and post-treatment for each treatment group. f After completion of the treatment, the percent of huCD45 + /lin - /huCD38 - /huCD34 + in the BM was determined. g Representative histologic images of BM from mice in d-f that were treated with vehicle, VEN, or S63845/VEN and underwent immunohistochemical staining for huCD34 (top panel, original magnification 10x, scale bars: 100μm; lower panel, original magnification 60x, scale bars:10μm). The data in e-f are represented by means ± SEM, and statistical comparisons between vehicle and VEN or combination treatments are shown (two-tailed t-test; n.s. not significant, *p < 0.05, **p <0 .01, ***p<0 .001).

Article Snippet: Cells were plated into a 384-well dish with IMDM + 10% FBS + 1% Pennicillin/Streptomycin + cytokines and supplements (10ng/ml hIL3, 10ng/ml hFLT3 ligand, 10ng/ml hTPO, 10ng/ml hSCF, 5ng/ml hIL6, 4μg/ml hLDL, 10μM 2-Mercaptoethanol, and 1x L-GlutaMAX) and treated for 48 hours with an MCL1 inhibitor (S63845; Chemietek, Indianapolis, IN), BCL2 inhibitor (venetoclax; Chemietek), or BCL-xL inhibitor (A-1155463; Chemietek) at concentrations from 3nM to 10µM.

Techniques: Inhibition, Immunohistochemical staining, Staining, Two Tailed Test

Journal: Cell Death & Disease

Article Title: GNA13 regulates BCL2 expression and the sensitivity of GCB-DLBCL cells to BCL2 inhibitors in a palmitoylation-dependent manner

doi: 10.1038/s41419-020-03311-1

Figure Lengend Snippet: A , B Relationship between survival of GCB-DLBCL and expression levels of GNA13 and BCL-2 was analyzed based on public datasets (PPISURV). ( A ) High-level expression of GNA13 is significantly associated with positive outcome of GCB-DLBCL. ( B ) Overexpression of BCL-2 is significantly associated with negative outcome of GCB-DLBCL. C Plot of GNA13 expression vs. BCL2 expression in 102 GCB-DLBCL cases (R2: Genomics Analysis and Visualization Platform, https://hgserver1.amc.nl/cgi-bin/r2/main.cgi ). The transcript levels of GNA13 and BCL2 are inversely related ( r = −0.729, P = 7.155 × 10 −6 ). D Western blot analysis of GNA13 and BCL2 expressions in SU-DHL4 cells, which harbor the WT GNA13, OCI-LY1, and SU-DHL6 cells, both of which harbor mutant GNA13. α-Tubulin was used as a loading control. E Western blot analysis of GNA13 and BCL2 expressions in SU-DHL4 cells expressing scrambled shRNA (Scr) or GNA13 knockdown shRNA (UTR) (left two lanes), and the SU-DHL4-shGNA13 UTR cells ectopically overexpressing (OE) HA-tagged WT, C14S, C18S, or C14/18S mutant GNA13 (right four lanes). GAPDH was used as a loading control. F Western blot analysis of GNA13 and BCL2 expressions in OCI-LY1 cells expressing scrambled shRNA (Scr) or GNA13 knockdown shRNA (UTR) (left two lanes) and the OCI-LY1 cells ectopically expressing HA-tagged WT, C14S, C18S, or C14/18S mutant GNA13 (right four lanes). GAPDH was used as a loading control.

Article Snippet: The BCL2 inhibitor venetoclax (ABT-199), the pan-caspase inhibitor Emricasan and Q-VD-Oph, the group II caspases inhibitor Ac-DEVD-CHO, the pan-PI3K inhibitor GDC-0941, and the PI3Kα/δ inhibitor Copanlisib were purchased from CSNpharm (Arlington Heights, IL, USA).

Techniques: Expressing, Over Expression, Western Blot, Mutagenesis, Control, shRNA, Knockdown

Journal: Cell Death & Disease

Article Title: GNA13 regulates BCL2 expression and the sensitivity of GCB-DLBCL cells to BCL2 inhibitors in a palmitoylation-dependent manner

doi: 10.1038/s41419-020-03311-1

Figure Lengend Snippet: A Volcano plot of FDA-approved drugs and bioactive compounds with known targets on SU-DHL4-shGNA13 Scr and SU-DHL4-shGNA13 UTR cells. Drug effect size ratio between SU-DHL4-shGNA13 Scr and SU-DHL4-shGNA13 UTR vs. statistical significance ( P value) were plotted. Red and blue points indicate drug identified as differentially inhibited between the two types of cells. B Dose–response curves for SU-DHL4-shGNA13 Scr or SU-DHL4-shGNA13 UTR treated with AKT inhibitor MK-2206. C IC 50 of BCL2 inhibitors ABT-199 and ABT-263 in DLBCL cell lines with either wild-type (blue dots) or mutant (red dots) GNA13 . D Dose–response curves for SU-DHL4 and OCI-LY1 cells treated with ABT-199. E SU-DHL4 or OCI-LY1 cells were transfected with constructs containing shGNA13-600, shGNA13-UTR, or a scrambled shRNA (Scr). Levels of total (T)-AKT and phosphorylated (P)-AKT S473 in these cells were examined by western blot analysis. GAPDH was used as loading controls. F Western blot analysis of BCL2, total (T)-AKT, and phosphorylated (P)-AKT S473 level in SU-DHL4 cells treated with PI3K inhibitors Copanlisib or GDC-0941 at indicated concentrations for 6 h, respectively. G Western blot analysis of BCL2 level in SU-DHL4-shGNA13 Scr or SU-DHL4-shGNA13 UTR cells treated with PI3K inhibitors Copanlisib or GDC-0941 at indicated concentrations for 6 h, respectively. α-Tubulin was used as a loading control.

Article Snippet: The BCL2 inhibitor venetoclax (ABT-199), the pan-caspase inhibitor Emricasan and Q-VD-Oph, the group II caspases inhibitor Ac-DEVD-CHO, the pan-PI3K inhibitor GDC-0941, and the PI3Kα/δ inhibitor Copanlisib were purchased from CSNpharm (Arlington Heights, IL, USA).

Techniques: Mutagenesis, Transfection, Construct, shRNA, Western Blot, Control

Journal: Cell Death & Disease

Article Title: GNA13 regulates BCL2 expression and the sensitivity of GCB-DLBCL cells to BCL2 inhibitors in a palmitoylation-dependent manner

doi: 10.1038/s41419-020-03311-1

Figure Lengend Snippet: A Dose–response curves for SU-DHL4-shGNA13 Scr and SU-DHL4-shGNA13 UTR cells treated with ABT-199. B Tumor growth curves of SU-DHL4-shGNA13 UTR cells either expressing the WT GNA13 (SU-DHL4-shGNA13 UTR -OE WT ) or the C14/C18S palmitoylation mutant of GNA13 (SU-DHL4-shGNA13 UTR -OE C14/18S ) in mice treated with either BCL2 inhibitor ABT-199 or vehicle. n , number.

Article Snippet: The BCL2 inhibitor venetoclax (ABT-199), the pan-caspase inhibitor Emricasan and Q-VD-Oph, the group II caspases inhibitor Ac-DEVD-CHO, the pan-PI3K inhibitor GDC-0941, and the PI3Kα/δ inhibitor Copanlisib were purchased from CSNpharm (Arlington Heights, IL, USA).

Techniques: Expressing, Mutagenesis