AMG-176, an Mcl-1 Antagonist, Shows Preclinical Efficacy in Chronic Lymphocytic Leukemia

Abstract

Purpose: Survival of CLL cells due to the presence of Bcl-2 and Mcl-1 has been established. Direct inhibition of Bcl-2 by venetoclax and indirect targeting of Mcl-1 with transcription inhibitors have been successful approaches for CLL. AMG-176 is a selective and direct antagonist of Mcl-1, which has shown efficacy in several hematologic malignancies; however, its effect on CLL is elusive. We evaluated biological and molecular effects of AMG-176 in primary CLL cells.

Experimental design: Using samples from patients (n = 74) with CLL, we tested effects of AMG-176 on CLL and normal hematopoietic cell death and compared importance of CLL prognostic factors on this biological activity. We evaluated CLL cell apoptosis in the presence of stromal cells and identified cell death pathway including stabilization of Mcl-1 protein. Finally, we tested a couplet of AMG-176 and venetoclax in CLL lymphocytes.

Results: AMG-176 incubations resulted in time- and dose-dependent CLL cell death. At 100 and 300 nmol/L, there was 30% and 45% cell death at 24 hours. These concentrations did not result in significant cell death in normal hematopoietic cells. Presence of stroma did not affect AMG-176-induced CLL cell death. IGHV unmutated status, high β2M and Mcl-1 protein levels resulted in slightly lower cell death. Mcl-1, but not Bcl-2 protein levels, in CLL cells increased with AMG-176. Low concentrations of venetoclax (1-30 nmol/L) were additive or synergistic with AMG-176.

Conclusions: AMG-176 is active in inducing CLL cell death while sparing normal blood cells. Combination with low-dose venetoclax was additive or synergistic.

Fig 1.. Chronic lymphocytic leukemia cells are sensitive to AMG-176-mediated cell death.

CLL lymphocytes from seven CLL patients were collected and cultured in media supplemented with 10% human serum. Each sample was treated with either dimethyl sulfoxide (DMSO) (0.1%), or indicated concentration of AMG-176 for indicated times. Cell death was determined using flow cytometry after Annexin V/propidium iodide (PI) staining. A. Concentration-dependent cell death. CLL cells were incubated with 3, 10, 30, 100, 300, and 1000 nM AMG-176 for 24 hours. B. Time-dependent cell death. CLL cells were incubated with 300 or 1000 nM AMG-176 for 0, 3, 6, 9, 12, 15, 18, 21, 24 hours. We fit a two way ANOVA model using treatment and patient as fixed effects at 12 hours and obtained a p-value of 0.00007 using an F-test of no treatment effect versus treatment effect. We performed the same analysis for 24 hours and obtained a p-value of 0.00013. C. Impact of prolonged incubation on cell death. CLL cells from 10 patients were incubated with 300 nM AMG-176 for 24, 48, and 72 hours. D. Influence of microenvironment on AMG-176 mediated cell death. CLL lymphocytes from 4 CLL patients were collected and cultured either in suspension or on NK-Tert cells as indicated in the Methods section. Each sample was treated with either dimethyl sulfoxide (DMSO) (0.1%), or indicated concentration of AMG-176 for indicated times. Cell death was determined using flow cytometry after Annexin V/propidium iodide (PI) staining. We conducted two-sample paired t-tests between the two groups (without NK-Tert and with NK-Tert). The p values were 0.067, 0.12; 0.02; 0.33, 0.57, 0.59 for DMSO, 100 nM AMG-176, 300 nM AMG-176, 100 nM venetoclax, and two combinations, respectively.

Fig 2.. AMG-176 induced cell death and CLL prognostic markers.

CLL lymphocytes from 68 patients were treated in vitro with 300 nM AMG-176 for 24 h, and Annexin V/PI positivity was determined after the incubation period. The relationship between cytotoxicity and prognostic markers was plotted for immunoglobulin heavy-chain variable-region (IGHV) mutation, zeta-chain-associated protein kinase 70 (ZAP 70) status, β2M levels, WBC count in the blood, and cytogenetics status. For each prognostic factor in Figures 2 A-D, we conducted two-sample Student’s t-test assuming equal group variances. The p values are listed in each graph. For Figure 2 E, we computed p-values based on two-sample equal variance t-tests for negative (NEG) against four cytogenetic groups. The p-values are 0.72, 0.02, 0.01, and 0.11 for 11q del, trisomy 12, 13q del, and 17 p del, respectively.

Fig 3.. Biological impact of AMG-176 in normal PBMCs.

Normal PBMCs were isolated from peripheral blood sample of healthy donors. PBMCs were cultured in media supplemented with 10% human serum. Each sample was treated with either dimethyl sulfoxide (DMSO) (0.1%) or 300 nM AMG-176 for 24 hours. Cell death was determined using flow cytometry after Annexin V/propidium iodide (PI) staining and cell surface marker staining. A. PBMCs, B. CD19⁺ B-cells. C. CD4⁺ T-cells. D. CD8⁺ T-cells. E. CD56⁺ NK-cells. F. CD133⁺ plasma cells and G. CD34⁺ myeloid cells.

Fig 4.. Change in Mcl-1 protein level and caspase-dependent cell death by AMG-176.

CLL lymphocytes from 4 patients were treated in vitro with DMSO, indicated concentrations of AMG-176 or venetoclax for 24 h in presence or absence of 20 µM Q-VD-OPH. (A) Sample from one patient (#493) were stained and Annexin V/PI positivity was quantitated (% Ann/PI). Cells from same patient were used for caspase 3 activation assay (% C3 +ve) and were also analyzed for protein expression of PARP, cleaved caspase 3, Mcl-1 and Bcl-2 along with loading control. Numbers under Mcl-1 immunoblot indicate levels of Mcl-1 protein compared to DMSO (control) (B) Samples from 4 patients were treated and analyzed for cell death. For statistical analyses, we used three factor ANOVA with patient, absence or presence of caspase inhibitor and drug dosage as fixed effects. (C) Samples from 4 patients were treated and analyzed for change in Mcl-1 protein levels. For statistical analyses, we computed log2 ratios of fold changes relative to DMSO using a two factor ANOVA with patient and AMG-176 dosage as fixed effects. (D) CLL lymphocytes from 4 −8 patients were treated in vitro with DMSO, 10, 30, 100, 300 or 1000 nM AMG-176 or 100 nM venetoclax for 24 h. These were analyzed for Mcl-1 protein expression using immunoblot assay which is shown in Supplemental Figure 5. Quantitated protein levels are normalized to untreated control and plotted. Sample numbers are n=4 at 10 nM, n=8 at 30, 100, and 300 nM, and n=5 at 1000 nM of AMG-176 and n=6 at 100 nM of venetoclax. For statistical analyses, we used log2 ratios of fold change relative to DMSO using two factor ANOVA with patient and AMG-176 or venetoclax dosage as fixed effects. (E and F) Mitochondrial and cytosolic components were fractionated as described under Methods section for 10 patient samples. Immunoblots from two patients are shown to provide variability among patients. Additional two patients’ immunoblots are included in Supplementary Figure 8. Total cell, cytosolic, and mitochondrial fractions were used for western blot analyses for Mcl-1, Bax, Bak, and cytochrome C (Cyto c) along with normalizing loading control proteins. Abbreviations used are A = AMG-176; V = venetoclax.

Fig 5.. Impact of baseline endogenous levels of Bcl-2 family proteins on AMG-176-induced cell death

(A - B) CLL lymphocytes from peripheral blood of 16 patients were isolated to measure basal levels of Bcl-2 family proteins. Total protein was purified from these lymphocytes and MEC-1 cells and immunoblots were performed for the indicated Bcl-2 family proteins. (C - E) Protein levels were quantitated and normalized with GAPDH and compared with the levels in MEC-1 cells. The % Mcl-1, Bcl-2, and Bcl-xL levels were correlated with % apoptosis after a 24 hour incubation with 300 nM AMG-176. Linear regression and statistical analyses were performed using prism software, which required to put data point 0 on X-axis at 100% Y-axis value. Quantitation graphs of Bak and Bax proteins are shown in Supplemental Figure 9.

Fig 6.. Combination of venetoclax and AMG-176 in CLL cells.

CLL lymphocytes from 5 patients were either vehicle (DMSO) treated or treated with 1, 5, 10, or 30 nM venetoclax (white bars), 100 and 300 nM AMG-176 (light grey bars) or combination of both (dark grey bars) for 24 h. (A - E) Samples from patient 133, 291, 963, 897, and 689 were treated and annexin/PI positivity was measured as described in the Methods section. DMSO value was subtracted from % cell death obtained after each treatment. Abbreviations used are A = AMG-176; V = venetoclax.