The BH3 mimetic ABT-737 targets selective Bcl-2 proteins and efficiently induces apoptosis via Bak/Bax if Mcl-1 is neutralized

Abstract

Since apoptosis is impaired in malignant cells overexpressing prosurvival Bcl-2 proteins, drugs mimicking their natural antagonists, BH3-only proteins, might overcome chemoresistance. Of seven putative BH3 mimetics tested, only ABT-737 triggered Bax/Bak-mediated apoptosis. Despite its high affinity for Bcl-2, Bcl-x(L), and Bcl-w, many cell types proved refractory to ABT-737. We show that this resistance reflects ABT-737's inability to target another prosurvival relative, Mcl-1. Downregulation of Mcl-1 by several strategies conferred sensitivity to ABT-737. Furthermore, enforced Mcl-1 expression in a mouse lymphoma model conferred resistance. In contrast, cells overexpressing Bcl-2 remained highly sensitive to ABT-737. Hence, ABT-737 should prove efficacious in tumors with low Mcl-1 levels, or when combined with agents that inactivate Mcl-1, even to treat those tumors that overexpress Bcl-2.

Figure 1

Many putative BH3 mimetics do not kill like BH3-only proteins A: The viability of wild-type MEFs (WT) or Bax- and Bak-deficient MEFs (DKO) 24 h after infection with the indicated retroviruses. Expression of the cDNA encoding the BH3-only protein Bim_S or tBid was linked by an IRES to that of GFP, and the viability of GFP^+ve cells determined by PI exclusion. B: Representative wells showing colony formation by wild-type (WT) or Bax/Bak-deficient (DKO) MEFs after infection with the control parental retrovirus or one expressing Bim_L. C–H: The viability (% cells excluding PI) of WT or Bax- and Bak-deficient (DKO) MEFs treated for 24 h with graded doses of the indicated putative BH3 mimetics. I: Colonies formed by wild-type (WT) or Bax/Bak-deficient (DKO) MEFs in the presence of no treatment, HA14-1 or Antimycin A. J: The relative affinities (IC₅₀ in nM) of a BimBH3 peptide (as previously reported; Chen et al., 2005) and several putative BH3 mimetic compounds for Bcl-2 and/or Bcl-w. The affinities were measured in solution competition assays (Chen et al., 2005). Data in A and C–H represent means ± SD from 3 independent experiments.

Figure 2

ABT-737 cooperates with Noxa to induce Bax/Bak-dependent killing A: The viability of wild-type MEFs (WT), Bax/Bak-deficient MEFs (DKO), and Bak- or Bax-singly deficient MEFs was determined by PI exclusion 48 h after exposure to ABT-737 (10 μM) or Etoposide (10 μM). B: ABT-737 is a Bad BH3 mimetic. Based on the relative affinities (IC₅₀ in nM) of ABT-737 for mammalian pro-survival proteins, determined in solution competition assays (Fig. S1A), ABT-737 and Bad bind to the same subset of Bcl-2 pro-survival proteins. According to our model for initiating the apoptotic program (Chen et al., 2005; Willis et al., 2005), Bad and Noxa are poor inducers of apoptosis individually because each binds only a subset of the pro-survival proteins, whereas Bim is a potent killer because it binds all of them. By this rationale, ABT-737 (like Bad) should also cooperate with Noxa to kill cells. C: Noxa triggers Mcl-1 degradation. Immunoblots of lysates prepared from the MEFs after retroviral infection with wild-type Noxa or the 3E mutant (an inactive mutant that does not bind Mcl-1) probed for Mcl-1 and HSP70 (loading control). D: Noxa sensitizes wild-type MEFs to ABT-737 killing. Wild-type MEFs expressing wild-type human Noxa or an inactive mutant (Noxa 3E) (Willis et al., 2005), were exposed to ABT-737 for 8 h and their viability determined. E: Bax/Bak-deficient MEFs (DKO) are resistant to ABT-737 even when Mcl-1 is targeted. Long-term clonogenic survival of cells exposed to ABT-737. Equal numbers of the indicated MEFs, or their counterparts stably expressing Noxa or the inactive Noxa 3E, were plated in media containing vehicle or ABT-737 (1 μM, replenished after 3 d) and the colonies formed scored after 6 d. The number of colonies obtained with ABT-737 treatment is expressed as a proportion of colonies formed with the vehicle alone. - no colonies. F: Either Bax or Bak can mediate killing by ABT-737 provided Mcl-1 is targeted. Viability of the indicated MEFs stably expressing Noxa was determined 8 h after exposure to ABT-737. Note that Bax/Bak-deficient MEFs (DKO) are resistant. G: Noxa sensitizes FDC-P1 myeloid cells to ABT-737 killing. The viabilities of FDC-P1 cells, retrovirally infected to express Noxa, mutant Noxa 3E or Bad, were compared after a 24 h treatment with graded doses of ABT-737. Data in A and D–G represent means ± SD from a representative of 3 experiments.

Figure 3

ABT-737 induces cytochrome c release and caspase-dependent apoptosis when Mcl-1 is neutralized A: Cell death triggered by ABT-737 is caspase dependent. Noxa-expressing wild-type MEFs were treated with ABT-737 (1 μM) and their viability was assessed by PI exclusion; co-incubation with the broad-spectrum caspase inhibitor zVAD. fmk (50 μM) abrogated ABT-737 killing at this time point. Data represent means ± SD from a representative of 3 experiments. B: ABT-737 induces cytochrome c release when Mcl-1 is neutralized. Noxa-expressing wild-type (WT) or Bax/Bak-deficient MEFs (DKO) were exposed to ABT-737 (10 μM for 4 h), permeabilized with digitonin to wash out any cytochrome c released to the cytosol and then fixed. Residual mitochondrial cytochrome c was detected by immunostaining and flow cytometry (Waterhouse et al., 2004). ABT-737 triggered loss of cytochrome c from the mitochondria of WT MEFs, as indicated by the peak of weaker staining (compare filled with unfilled histogram; upper), but not from the Bax/Bak-deficient DKO MEFs (lower). C: ABT-737 and Noxa cooperate in vitro to release cytochrome c. Lysates prepared from wild-type (left) or Bax/Bak-deficient MEFs (DKO; right) stably expressing Noxa or Bad were incubated with vehicle (−) or 5 μM ABT-737 (+), before fractionation into the pellet (P) and supernatant (S) fractions. Equivalent fractions were probed for cytochrome c, Bcl-2 (membrane fraction marker) and Apaf-1 (cytosolic marker).

Figure 4

Neutralizing Mcl-1 sensitizes different cell types to ABT-737 Colony formation after continuous exposure to ABT-737 (1 μM, replenished every 3 d) of HeLa (A, B) or MCF-7 cells (C, D) infected with empty vectors, or stably expressing Noxa, mutant Noxa 3E, RNAi targeting Mcl-1, or RNAi to an irrelevant target (control RNAi). Introduction of mouse Mcl-1, which is not targeted by the human specific Mcl-1 RNAi construct, restored the resistance in HeLa (B) or MCF-7 cells (D) to ABT-737. Clonogenic survival data (after 7 d) are representative means ± SD of 3 independent experiments. (A, B) The lower panels are immunoblots for Mcl-1 or HSP70 (loading control). (C, D) The lower panels are immunoblots for human Mcl-1 (top), mouse Mcl-1 (middle: * residual signal from human Mcl-1 probe) or HSP70 (lower panel). E: ABT-737 triggers Bax activation when Mcl-1 is neutralized. HeLa cells expressing mutant Noxa 3E, Noxa or Mcl-1 RNAi, were treated for 4 h with ABT-737 (10 μM), and Bax activation detected by flow cytometric analysis after staining permeabilized cells with an antibody (clone 3) that specifically recognizes activated Bax (Willis et al., 2005).

Figure 5

Pro-survival proteins differ in their ability to antagonize ABT-737 A: Noxa variants that selectively neutralize Mcl-1 or both Mcl-1 and A1. Whereas the human Noxa used in Figs. 2–4 (above) binds both Mcl-1 and A1 (Chen et al., 2005) (Fig. 2), the mouse Noxa BH3 B region (mNoxaB) only binds tightly to Mcl-1 (IC₅₀ 60 nM; IC₅₀ > 2μM for all other pro-survival proteins). The E74F mutant of mNoxaB binds tightly to both Mcl-1 and A1 (IC₅₀Mcl-1 24 nM, IC₅₀A1 12nM), but has weaker affinity (IC₅₀ > 2 μM) for all other pro-survival proteins. The affinities were measured in solution competition assays (Chen et al., 2005). B: A1 expression confers partial resistance to ABT-737. Colony formation after 6 d by parental wild-type MEFs or MEFs stably overexpressing FLAG-tagged A1 in the presence of ABT-737 (1 μM, replenished after 3 d) and the indicated BH3 domains, placed within an otherwise inert Bim_S backbone lacking its own BH3 (Chen et al., 2005) and expressed from retroviruses. C, D: Killing by ABT-737 is not inhibited by Bcl-2 and only partially by Bcl-x_L. Wild-type MEFs, or MEFs overexpressing FLAG-tagged Bcl-x_L or Bcl-2, were tested for their sensitivity to ABT-737 (1μM) in the presence human Noxa. The Bcl-2 overexpression did not rescue any colony formation, even though it inhibited apoptosis induced by 24 h exposure to Etoposide (D). - no colonies. Data in B–D represent means ± SD from a representative of 3 experiments.

Figure 6

Mcl-1 expression blunts the in vivo response of Eμ-myc/bcl-2 bi-transgenic lymphomas to ABT-737 Two independent progenitor B cell lymphomas (#9: A, C and #16: B, D) derived from Eμ-myc/bcl-2 bi-transgenic mice (Strasser et al., 1990) were infected with the control GFP expressing retrovirus, or ones co-expressing Bcl-2 or Mcl-1 and GFP. The mice were injected with 10⁶ infected tumor cells before initiating therapy 4 d later with (C, D) ABT-737 (75 mg/kg given daily for two weeks by intraperitoneal injection) or the (A, B) vehicle alone. ABT-737 improved the survival of mice transplanted with both tumors even when Bcl-2 was overexpressed. However, Mcl-1 overexpressing lymphomas were highly resistant to ABT-737 and these mice died rapidly, akin to their untreated counterparts. Kaplan-Meier survival curves were derived from an experiment with 3 mice in each cohort.

Figure 7

ABT-737 potently sensitizes cells overexpressing Bcl-2 to genotoxic agents A: Bcl-2 or Bcl-x_L overexpression renders FDC-P1 cells resistant to genotoxic agents. FDC-P1 cells or FDC-P1 cells overexpressing Bcl-2 or Bcl-x_L were treated with Etoposide (25 μM) or Cytosine Arabinoside (25 μM) for 24 h and viability determined by PI exclusion. B, C: FDC-P1 cells overexpressing Bcl-2 (B) or Bcl-x_L (C) were treated with ABT-737 (0–10 μM), and Etoposide (25μM) or Cytosine Arabinoside (Ara-C; 25 μM) or no other drug (none) for 24 h and the viability determined by PI exclusion. Filled lines - fold increase in killing efficacy; hatched lines - EC₅₀ values. D: Cytotoxic agents trigger Mcl-1 degradation. Equivalent amounts of lysates prepared from cells overexpressing Bcl-2 or Bcl-x_L that were left untreated or after 24 h exposure to Etoposide (25 μM) or Ara-C (25 μM) were probed for Mcl-1 or HSP70 (loading control). Data in A–C represent means ± SD from a representative experiment.

Figure 8

Alternative ways to target Mcl-1 and sensitize cells to ABT-737 A: IL-3 withdrawal triggers Mcl-1 degradation and Bim accumulation in FDC-P1 cells. Lysates prepared from Bcl-2-overexpressing FDC-P1 cells grown for 0–24 h in the absence of its essential growth factor IL-3 were blotted for Mcl-1, Bim or HSP70 (loading control). B: IL-3 deprivation sensitizes FDC-P1 cells overexpressing Bcl-2 (squares) or Bcl-x_L (circles) to ABT-737. Viability was determined for the cells, cultured with (filled symbols) or without (unfilled symbols) IL-3 and exposed to ABT-737 (0–10 μM) for 24 h. C: The protein synthesis inhibitor cycloheximide (CHX) and the CDK inhibitor Seliciclib both reduce Mcl-1 expression. HeLa cells were treated with 50 μg/mL cycloheximide or 30 μM Seliciclib (R-roscovitine/CYC202) for 12 h and Mcl-1 expression measured by immunoblotting (HSP-70, loading control). D: HeLa cells were left untreated, treated with 2.5 μM ABT-737, 50 μg/mL cycloheximide or 30 μM Seliciclib (R-roscovitine/CYC202), or combinations of ABT-737 with cycloheximide or Seliciclib, for 14 h. Statistical analyses were performed using two-tailed unpaired Student’s t-test. Data in B and D represent means ± SD from 3 independent experiments.