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. Author manuscript; available in PMC: 2016 Nov 21.

Published in final edited form as: Clin Cancer Res. 2016 Mar 29;22(18):4712–4726. doi: 10.1158/1078-0432.CCR-15-2522

(A) Evaluation of carfilzomib apoptotic response in CLL. PBMCs of 30 patients with CLL were subjected to a concentration-dependent response of carfilzomib or were treated with vehicle (DMSO) or were left untreated for 16 h. Cell death was evaluated by annexin V/PI double positivity. U, untreated; D, DMSO; CFZ, carfilzomib. (B) Relative cytotoxic effect of carfilzomib at 50 nM and 100 nM. The percentage of cell death recorded in DMSO-treated cells was subtracted from the percentage of cell death recorded at 50 nM and 100 nM of carfilzomib, respectively. Results are shown with histogram graphs. (C) Correlation between IgVH unmutated or mutated status and cytotoxic response to carfilzomib in CLL patient samples. CLL patient samples with umutnated IgVH status (IgVH Unmut) (n=7; red dots) were less sensitive than CLL patient samples with mutated IgVH status (IgVH Mut) (n=12; blue dots) to the cytotoxic effect of carfilzomib at 50 nM (p=0.0157) and 100 nM (p=0.0070), respectively. Cytotoxic response was evaluated by annexin V/PI double positivity. Graph prism software was used to evaluate the mean (horizontal lines) and p values. (D and E) Intracellular molecular changes in CLL patient samples in response to carfilzomib treatment. PBMCs from 7 CLL patient samples, representing a wide range of cytotoxic profiles (median cytotoxicity at 100 nM: 63% (range 33%–86%), were isolated and either left untreated or treated with the indicated concentration of carfilzomib or with vehicle for 16 h. Cell lysates for each condition were processed for immunoblot analysis with the indicated antibodies. Cell death evaluated by annexin V/PI double positivity is indicated beneath the immunoblot panels. For each sample, patients’ characteristics are shown below the western blot. Arrow and rounded arrow indicate cleaved and full-length of the corresponding protein, respectively. U, untreated; D, DMSO; CFZ, carfilzomib; LE, long exposure; SE, short exposure; Pr Rx, prior treatments; WBC, white blood cell count (K/mL of blood); IgVH, immunoglobulin variable region heavy chain; ZAP-70, zeta-chain-associated protein kinase-70; IHC, immunohistochemistry; B2M, β-2-microglobulin level (mg/mL); ATM, Ataxia telangiectasia mutated; *, Percentage of positive cell with cytogenetic abnormality for the corresponding locus.

(A) Evaluation of carfilzomib apoptotic response in CLL. PBMCs of 30 patients with CLL were subjected to a concentration-dependent response of carfilzomib or were treated with vehicle (DMSO) or were left untreated for 16 h. Cell death was evaluated by annexin V/PI double positivity. U, untreated; D, DMSO; CFZ, carfilzomib. (B) Relative cytotoxic effect of carfilzomib at 50 nM and 100 nM. The percentage of cell death recorded in DMSO-treated cells was subtracted from the percentage of cell death recorded at 50 nM and 100 nM of carfilzomib, respectively. Results are shown with histogram graphs. (C) Correlation between IgVH unmutated or mutated status and cytotoxic response to carfilzomib in CLL patient samples. CLL patient samples with umutnated IgVH status (IgVH Unmut) (n=7; red dots) were less sensitive than CLL patient samples with mutated IgVH status (IgVH Mut) (n=12; blue dots) to the cytotoxic effect of carfilzomib at 50 nM (p=0.0157) and 100 nM (p=0.0070), respectively. Cytotoxic response was evaluated by annexin V/PI double positivity. Graph prism software was used to evaluate the mean (horizontal lines) and p values. (D and E) Intracellular molecular changes in CLL patient samples in response to carfilzomib treatment. PBMCs from 7 CLL patient samples, representing a wide range of cytotoxic profiles (median cytotoxicity at 100 nM: 63% (range 33%–86%), were isolated and either left untreated or treated with the indicated concentration of carfilzomib or with vehicle for 16 h. Cell lysates for each condition were processed for immunoblot analysis with the indicated antibodies. Cell death evaluated by annexin V/PI double positivity is indicated beneath the immunoblot panels. For each sample, patients’ characteristics are shown below the western blot. Arrow and rounded arrow indicate cleaved and full-length of the corresponding protein, respectively. U, untreated; D, DMSO; CFZ, carfilzomib; LE, long exposure; SE, short exposure; Pr Rx, prior treatments; WBC, white blood cell count (K/mL of blood); IgVH, immunoglobulin variable region heavy chain; ZAP-70, zeta-chain-associated protein kinase-70; IHC, immunohistochemistry; B2M, β-2-microglobulin level (mg/mL); ATM, Ataxia telangiectasia mutated; *, Percentage of positive cell with cytogenetic abnormality for the corresponding locus.