PMC

(–)BI97D6 effectively kills primary AML myeloblasts from refractory patients. (A) (–)BI97D6 IC₅₀ values in primary AML samples. Fresh mononuclear cells from AML patients were isolated and incubated with (–)BI97D6 for 48 hours. Calcusyn software was used to calculate the IC₅₀ values based on live cell numbers (Annexin V⁻/PI⁻). Patient information is provided in . (B) Protein expression measured by quantitative immunoblot was plotted against the corresponding (–)BI97D6 IC₅₀ values of patient samples (n = 12). No significant correlations were observed between IC₅₀ values and the expression levels of Bcl-2, Bcl-xL, or Mcl-1 (Spearman’s rank correlation). (C) The IC₅₀ values of (–)BI97D6 against refractory or nonrefractory primary AML samples. There is no significant difference in the median IC₅₀ values (Mann-Whitney test).

Pan–Bcl-2 inhibition by (–)BI97D6 potently kills leukemia stem/progenitor cells at doses sparing normal hematopoietic stem/progenitor cells. (A-B) Representative FACS plots of (A) AML blast cells or (B) normal MNCs following treatment with 100 nM of (–)BI97D6 for 48 hours. (C-D) Change in live cell numbers (AnnV⁻/PI⁻) as a function of (–)BI97D6 concentration. Fresh AML or normal MNCs were isolated and treated with indicated doses of (–)BI97D6 for 48 hours. Live cell numbers were enumerated and normalized to untreated controls. (E) Diagram showing the experimental design. AML patient cells (1.0 × 10⁶) were intravenously injected into NSG mice following 24-hour incubation with 100 nM (–)BI97D6 or DMSO (vehicle). (F) Representative FACS plots showing engraftment of human AML cells in mouse peripheral blood 6 weeks after injection. (–)BI97D6 pretreatment significantly reduced engraftment/progression of AML patient cells. (G) Kaplan-Meier survival curves for the mouse experiment described in E (5 mice per arm).

BM MSC coculture increases Mcl-1 protein level in OCI-AML3 cells and Mcl-1 inhibition by the pan–Bcl-2 inhibitor (–)BI97D6 abrogates BM MSC-mediated protection. (A) Representative FACS plots showing the gating strategy used to determine apoptosis and viability of OCI-AML3 cells cocultured with BM MSCs. (B) Viability of OCI-AML3 cells following treatment with increasing concentrations of (–)BI97D6 for 72 hours, with or without coculture with protective MSCs. The live cell number (AnnV⁻/PI⁻) was enumerated by FACS analysis using CountBright counting beads and normalized to the counts of untreated controls. (C) Apoptosis (AnnV⁺) as assessed by FACS in OCI-AML3 cells after incubation with indicated compounds for 72 hours; cells were cultured alone or cocultured with protective MSCs. (D) Live cell numbers after incubation with indicated compounds for 72 hours. The methodology is as described in B. Both Sabutoclax and (–)BI97D6 effectively killed OCI-AML3 cells cocultured with BM MSCs. (E) Coculture with BM MSCs for 24 hours significantly increased Mcl-1 protein level in OCI-AML3 cells. (F-H) (–)BI97D6 (100 nM) had minimal effects on BM MSC (F) morphology, (G) proliferation, and (H) viability. NS, not significant. All data in bar graphs represent the means of triplicate experiments, with error bars indicating the standard deviations.

(–)BI97D6 induces multiple hallmarks of mitochondrial apoptosis. (A-B) Phosphatidylserine externalization as determined by (A) Annexin V (AnnV) staining and (B) loss of mitochondrial membrane potential as measured by CMXRos assay in OCI-AML3 cells after 48-hour treatment with 50 nM (–)BI97D6. (C) Cleavage of caspase-3, caspase-9, and PARP-1 in OCI-AML3 cells after (–)BI97D6 treatment of 36 or 48 hours. (D) Apoptosis induction (AnnV⁺) by 50 nM (–)BI97D6 in OCI-AML3 cells pretreated with a caspase-3 or -9 inhibitor for 2 hours. ***P < .001. (E) Bax conformational change as measured by immunoprecipitation (IP) with an anti-Bax 6A7 monoclonal antibody. OCI-AML3 cells were treated with 100 nM (–)BI97D6 for 24 hours. WCL, whole cell lysate. (F) Disruption of Bcl-2/Bax association by (–)BI97D6 as shown by Bax/Bcl-2 co-IP. Bax was immunoprecipitated from OCI-AML3 cells following (–)BI97D6 treatment of 24 hours. (G) Disruption of Mcl-1/Bim association by (–)BI97D6. Bim was immunoprecipitated from OCI-AML3 cells treated with 100 nM (–)BI97D6 or vehicle for 24 hours. (H-I) Bim deficiency reduced apoptosis induction by (–)BI97D6. (H) Representative flow cytometry plots of MEF control and Bim^−/− cells following treatment with 300 nM (–)BI97D6 for 48 hours. (I) Apoptosis induction as measured by AnnV/PI flow cytometry after 48-hour treatment with 300 nM (–)BI97D6.

(–)BI97D6 effectively induces apoptosis in AML cells regardless of high expression of Mcl-1. (A) Representative FACS plots showing gating strategy and apoptosis induction by (–)BI97D6 in OCI-AML3 cells. (B-C) Time- and dose-dependent (B) induction of apoptosis and (C) reduction of live cell numbers of OCI-AML3 cells after (–)BI97D6 treatment. (D) Mcl-1 expression in 3 AML cell lines as determined by immunoblot densitometry using the Odyssey Infrared Imaging System. α-Tubulin served as loading control. The ratio of Mcl-1/α-tubulin was normalized to that of untreated OCI-AML3 cells. (E-F) The effects of (E) ABT-737 and (F) (–)BI97D6 on 3 AML cell lines with low, intermediate, or high expression of Mcl-1 protein. Effect = 1 – live cell number^treated/live cell number^control. (G) Immunoblot showing stable Mcl-1 knockdown by lentiviral shRNA in OCI-AML3 cells. (H-J) Comparison of the sensitivities of OCI-AML3 shGFP and shMCL1 cells to ABT-737, (–)BI97D6, and Sabutoclax. The cells were treated with indicated concentrations for 72 hours. (K) Immunoblot showing stable Mcl-1 overexpression in HL-60 cells. (L-N) Comparison of the sensitivities of HL60-GFP and HL60-Mcl-1 overexpressing cells to ABT-737, (–)BI97D6, and Sabutoclax. The cells were treated for 48 hours. The live cell numbers were enumerated by FACS analysis using CountBright counting beads and normalized to those of untreated controls. Calcusyn software was used to calculate the IC₅₀ values based on live cell numbers. All data in line/bar graphs represent the means of triplicate experiments, with error bars indicating the standard deviations.