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Anti-apoptotic Mcl-1 is essential for the development and sustained growth of acute myeloid leukemia.

Acute myeloid leukemia (AML) frequently relapses after initial treatment. Drug resistance in AML has been attributed to high levels of the anti-apoptotic Bcl-2 family members Bcl-x(L) and Mcl-1. Here we report that removal of Mcl-1, but not loss or pharmacological blockade of Bcl-x(L), Bcl-2, or Bcl-w, caused the death of transformed AML and could cure disease in AML-afflicted mice. Enforced expression of selective inhibitors of prosurvival Bcl-2 family members revealed that Mcl-1 is critical for survival of human AML cells. Thus, targeting of Mcl-1 or regulators of its expression may be a useful strategy for the treatment of AML.

Acute myeloid leukemia (AML) is a heterogeneous disease, with treatment outcome after intensive chemotherapy strongly influenced by cytogenetic and molecular characteristics of the cancer. Several recurrent AML oncogenic fusion lesions have been linked to a high risk of relapse after chemotherapy, including chromosomal translocations involving the Mll (mixed-lineage leukemia) gene (MLL-ENL and MLL-AF9, particularly found in infants or individuals that had previously been treated with topoisomerase inhibitors) (Ayton et al. 2004) or Aml1 gene (AML1-ETO9, found in ;15% of human AML cases) (Yan et al. 2006;Jiao et al. 2009). Mice transplanted with bone marrow-derived hematopoietic stem/progenitor cells that had been retrovirally transduced with the AML1-ETO9a (Yan et al. 2006) oncogene succumb to myeloblastic AML highly reminiscent of the human disease induced by the same oncogenic lesion. Similar transduction/transplantation models for expression of MLL-ENL (Lavau et al. 2000), MLL-AF9 (Somervaille and Cleary 2006), Mixl1 (Glaser et al. 2006), or Hoxa9 (Thorsteinsdottir et al. 2002) oncogenes cause monocytic and myelomonocytic AML in mice, recapitulating the human subtypes of AML often associated with these oncogenes. Like human AML (Estey and Dohner 2006), these murine leukemias often respond only transiently to anti-cancer therapeutics, with relapse of drug refractory disease in a significant proportion (Zuber et al. 2009). Anticancer therapy kills many tumor cells (at least in part) through induction of apoptosis, and defects in the programmed cell death machinery are frequently associated with poor response (Kaufmann et al. 1998;Johnstone et al. 2002). Recent studies have identified somatic amplification of the genomic regions containing the antiapoptotic genes mcl-1 or bcl-x (Beroukhim et al. 2010) or post-translational stabilization of Mcl-1 protein (Schwickart et al. 2010) in diverse human cancers. We used mouse as well as human AMLs to determine which anti-apoptotic Bcl-2 family members are essential for development and sustained growth of this hematological malignancy to gain insight into which of these proteins should be targeted for therapeutic benefit.

Results and Discussion
Deletion of mcl-1 but not bcl-x kills transformed myeloid cells in culture We employed gene targeted mice expressing a tamoxifenregulated Cre recombinase estrogen receptor fusion protein (Rosa26-Cre-ERT2 mice, hereafter called Cre) (Seibler et al. 2003) to allow controlled inactivation of loxP targeted (floxed, hereafter denoted fl) bcl-x (Wagner et al. 2000) or mcl-1 (Vikstrom et al. 2010) alleles (Supplemental Fig. 1A). We used cells of six different genotypes for analysis: wild-type (wt) to reveal toxic effects of tamoxifen, cre/+ heterozygotes to detect toxicity of the activated Cre recombinase, and cre/+ cells that are either heterozygous (fl/+) or homozygous (fl/fl) for conditional bcl-x or mcl-1 alleles to examine the consequences of loss of the corresponding anti-apoptotic proteins (Bcl-x L and Mcl-1). Bone marrow-derived hematopoietic stem/progenitor cells were infected with retroviruses encoding MLL-ENL, MLL-AF9, AML1-ETO9a, Mixl1, or Hoxa9 plus GFP (from an IRES) as a marker (Supplemental Fig. 1B). Each transduced cell population readily yielded immortalized cell lines exhibiting an immature myeloid profile, with the exception of AML1-ETO9a transduced cells, which exhibited a more differentiated morphology (Supplemental Fig. 1C). Treatment with tamoxifen resulted within 24 h in near complete recombination of both conditional bcl-x fl or mcl-1 fl alleles (Supplemental Fig. 1D). Moreover, this treatment caused nearly complete loss of Bcl-x L protein in bcl-x fl/fl ;cre/+ cells and almost complete loss of Mcl-1 (and increase of Bim but not tBid or Puma) protein in mcl-1 fl/fl ;cre/+ cells (Supplemental Fig. 1E). Tamoxifen exerted no toxicity on wild-type cells, but Cre activation reduced the viability of cre/+ cells to ;70% compared with untreated cultures ( Fig. 1A; Supplemental Fig. 1F). Bcl-x fl/+ ;cre/+ and bcl-x fl/fl ;cre/+ cells showed slightly increased death compared with the cre/+ cells, displaying an initial drop in viability to 57%-74% or 45%-57%, respectively ( Fig.  1A; Supplemental Fig. 1F). Addition of tamoxifen reduced the viability of mcl-1 fl/+ ;cre/+ cells to 37%-55% and, remarkably, completely killed all mcl-1 fl/fl ;cre/+ lines (Fig. 1A). These results show that Mcl-1 but not Bcl-x L is essential for sustained survival of transformed myeloid cells in vitro.

Murine AML are more sensitive to Mcl-1 deletion than nontransformed stem cells and myeloid progenitors
We next generated AML in vivo by reconstituting lethally irradiated mice with MLL-ENL or MLL-AF9 retrovirus-infected bone marrow-derived hematopoietic stem/progenitor cells. We used the Cre/loxP system to induce loss of Bcl-x L or Mcl-1 (Supplemental Fig. 2A), or tetracyclineregulated RNAi (Zuber et al. 2011a) for knockdown of Mcl-1 (Supplemental Fig. 2B). All recipient mice developed AML within 5 wk after reconstitution (Supplemental Fig.  2C,D). Knockdown of Mcl-1 by RNAi caused apoptosis and rapid depletion of (dsRed + /Venus + ) mcl-1 shRNAexpressing AML cells that had been transformed by combined expression of the MLL-AF9 and N-ras oncogenes (Fig. 1C). In agreement with the data described above (Fig. 1B), this death could be inhibited by overexpression of Bcl-2 or Mcl-1 (Fig. 1C).
For Mcl-1 inhibition to become a feasible strategy for AML therapy, there needs to be a ''therapeutic window'' in which leukemic cells are more sensitive than normal hematopoietic stem cells (which require Mcl-1 for survival) (Opferman et al. 2005). Treatment with tamoxifen caused efficient recombination of mcl-1 fl alleles in FACSsorted nontransformed LSK (lineage marker À Sca-1 + c-Kit + ) stem and myeloid progenitors from mcl-1 fl/+ ;cre/+ or mcl-1 fl/fl ;cre/+ mice, as demonstrated by flow-cytometric detection of the hCD4 reporter (Supplemental Fig. 3A). Mcl-1 deletion lowered the viability of mcl-1 fl/+ ;cre/+ LSK cells to 80% and that of mcl-1 fl/fl ;cre/+ LSK cells to 34%, compared with untreated cells ( Fig. 2A). Myeloid progenitors were slightly more susceptible to Mcl-1 deletion, showing viability of 91% for cre/+, 66% for mcl-1 fl/+ ;cre/+, and 15% for mcl-1 fl/fl ;cre/+ cells ( Fig. 2A). When MLL-ENL and MLL-AF9 transformed AML cells were treated in vitro with tamoxifen for 48 h, no effect was observed in wildtype cells, whereas the viability of cre/+ cells dropped to ;65% (Fig. 2B). The bcl-x fl/+ ;cre/+ and bcl-x fl/fl ;cre/+ leukemic cells displayed a reduction in viability that was slightly higher compared with that of cre/+ cells (Fig. 2B). In contrast, the viability of mcl-1 fl/+ ;cre/+ cells dropped to ;35%, and most mcl-1 fl/fl ;cre/+ cells were killed within 72 h (Fig. 2B). These results demonstrate that while Mcl-1 is important in both normal stem/myeloid progenitor cells and AML cells, the AML cells were significantly more susceptible to loss of Mcl-1, even when they were cocultured (Supplemental Fig. 3B). Thus, it might be possible to establish a ''therapeutic window'' for Mcl-1 inhibitors in AML therapy.
Anti-apoptotic Bcl-x L , Bcl-2, and Bcl-w are collectively dispensable for sustained in vitro survival of AML Although loss of Bcl-x L had only a minor impact, it may have a critical function in AML cell survival that overlaps with Bcl-2 and/or Mcl-1. We therefore treated MLL-ENL transformed AML cells with ABT-737, a BH3 mimetic that potently binds and inactivates Bcl-x L , Bcl-2, and Bcl-w (but not Mcl-1 or A1) (Oltersdorf et al. 2005), but on its own this had only a minor impact on viability (Fig.  2C). Moreover, ABT-737 further decreased the viability of mcl-1 fl/+ ;cre/+ AML cells that had also been treated with tamoxifen to lose one mcl-1 allele to only a limited extent (Fig. 2C). This indicates that, collectively, Bcl-2, Bcl-x L , and Bcl-w have only a minor role in sustaining the survival of AML cells. Deletion of mcl-1 but not bcl-x kills murine AML within the whole animal The impact of loss of Mcl-1 or Bcl-x L on AML within the whole animal was assessed by administering AMLburdened mice with tamoxifen. TUNEL staining of the marrow 3 d after tamoxifen treatment revealed significant (albeit moderate) apoptosis of bcl-x fl/fl ;cre/+ AML cells, more abundant death of mcl-1 fl/+ ;cre/+ AML cells, and widespread apoptosis of mcl-1 fl/fl ;creER/+ leukemic blasts (Fig. 3A, top panels; Supplemental Fig. 5A). Accordingly, almost no leukemic blasts were evident in histological examination 5 d after tamoxifen treatment of mice that had previously developed MLL-ENL transformed mcl-1 fl/fl ;cre/+ AML (Fig. 3A, bottom panels). On the fifth day after starting treatment, we found 24 3 10 6 to 70 3 10 6 GFP + wild-type, cre/+, bcl-x fl/+ ;cre/+, or bcl-x fl/fl ;cre/+ cells and 26 3 10 6 6 21 3 10 6 GFP + mcl-1 fl/+ ;cre/+ AML cells but almost no (0.2 3 10 6 6 0.2 3 10 6 ) GFP + mcl-1 fl/fl ;cre/+ AML cells (Fig. 3B). All mice treated with tamoxifen 3 wk after transplantation with MLL-ENL transduced bone marrow cells relapsed with AML. In contrast, when treated with tamoxifen at day 10 or day 5 post-transplantation, only half or none, respectively, of the mcl-1 fl/fl ;cre/+ AMLburdened mice became sick during observation for >1 year.
In contrast, their untreated counterparts survived for only 49 6 5 d (P < 0.001), and untreated or tamoxifen-treated mcl-1 fl/+ ;cre/+ AML-burdened mice lived for 49 6 3 d or 66 6 1 d, respectively (Fig. 3C). These findings were confirmed by demonstrating that tetracycline-induced RNAi-mediated knockdown of Mcl-1 in vivo caused clearance of AML cells from the liver and spleen and substantially prolonged survival of such tumor-burdened mice (Supplemental Fig. 5B-D). All relapsed bcl-x fl/fl ;cre/+ and mcl-1 fl/+ ;cre/+ AML from tamoxifen-treated mice had efficiently deleted their floxed alleles, as reflected by the absence of Bclx L protein (Fig. 3D) or by Southern blot analysis and hCD4 reporter expression (Supplemental Fig. 6A,B). In contrast, all relapsed mcl-1 fl/fl ;cre/+ AML arose from variant leukemic cells bearing mutations that debilitate CreERT2-mediated recombination. Most (eight out of nine) of these relapsed mcl-1 fl/fl ;cre/+ AML had not recombined both mcl-1 fl alleles due to mutated CreERT2, as evidenced by the absence of hCD4 reporter (Supplemental Fig. 6B) and Cre-ERT2 protein expression (Fig. 3E). Retroviral reintroduction of CreERT2 and tamoxifen treatment allowed mcl-1 fl recombination and consequently rapidly killed such cells in vitro (Supplemental Fig. 6C). Another mcl-1 fl/fl ;cre/+ AML (#890) contained CreERT2 protein but had recombined only one mcl-1 allele due to a deletion mutation in one loxP site, as reflected by Western blotting (Fig. 3E), hCD4 expression, and DNA sequencing (Supplemental Fig. 6B,D). These results show that Mcl-1 is essential for sustained survival and expansion of AML within the whole animal-only AML cells that had escaped mcl-1 deletion could cause tumor relapse.

Mcl-1 is critical for sustained survival of human AML-derived cell lines and primary human AML
These gene deletion studies in murine AML indicated that pharmacological inhibition of Mcl-1 might be effective for treatment of this malignant disease in humans. However, small organic BH3 mimetic compounds specifically targeting Mcl-1 have yet to be developed. To test and Lin À c-Kit + Sca-1 À myeloid progenitors (right panel) were cultured in the presence or absence of tamoxifen and cell survival was determined; data are presented as in Figure 1A. (B) Leukemic cells from mice bearing MLL-ENL-induced (left panel) or MLL-AF9-induced (right panel) AML were cultured in the presence or absence of tamoxifen and cell survival was determined; data are presented as described in Figure 1A. (**) P < 0.01. (C) MLL-ENL transformed AML cells of the indicated genotypes were grown in medium containing tamoxifen, ABT-737 (1 mM), or tamoxifen plus ABT-737 (1 mM). Cell survival was determined, and data are presented as described in Figure 1A. (**) P < 0.01. (D) Leukemic cells from mice bearing ICN1-induced T-ALL of the indicated genotypes were cultured in the presence or absence of tamoxifen. Cell survival was determined, and data are presented as described in Figure 1A. (**) P < 0.01. the impact of functional inactivation of Mcl-1 in human AML, we employed lentiviral expression of Bim S -derived BH3 variant ligands: Bim S 4E, an inert Bim variant (negative control) (Chen et al. 2005); Bim S 2A to target Mcl-1 but not Bcl-2, Bcl-x L , Bcl-w, or A1 (Lee et al. 2008); Bim S Bad to target Bcl-2, Bcl-x L , and Bcl-w but not Mcl-1 or A1 (Merino et al. 2009); and Bim S wt to target all prosurvival Bcl-2 proteins (Chen et al. 2005) (Supplemental Fig. 7A). Doxycycline-induced expression of RFP or Bim S 4E for 72 h in MLL-ENL transformed mouse AML or multiple human AML-and chronic myeloid leukemia (CML)-derived cell lines did not reduce the numbers of lentivirally transduced (GFP + ) cells compared with uninduced cultures ( Fig. 4A; Supplemental Fig. 7B). In contrast, Bim S wt expression greatly reduced the viability of all tested cell lines ( Fig. 4A; Supplemental Fig. 7B). Importantly, blockade of Mcl-1 by Bim S 2A potently killed six of seven AML-derived cell lines but had only minor impact on the human erythroleukemia-derived (HEL92.1.7) and CML-derived (KU-812 and K562) cell lines (Fig.  4A). Interestingly, the few GFP + MV4,11 cells that remained viable after 3 d of doxycycline treatment exhibited low or no Bim S 2A expression (Supplemental Fig. 7C), indicating that selection against Mcl-1 blockade had occurred. In contrast, Bim S Bad expression (Fig. 4A) or ABT-737 treatment (Fig.  4B), both of which block Bcl-2, Bcl-x L , and Bcl-w (Oltersdorf et al. 2005;Merino et al. 2009), did not reduce the viability of most AML lines, except the ML-1, HL-60, and MV4,11 cells (small but significant effect), but they potently killed the human erythroleukemia (HEL92.1.7) and CML (KU-812) cell lines. Bim S 2A-mediated Mcl-1 blockade in combination with ABT-737 treatment for 40 h led to almost complete killing of all cell lines tested (Fig. 4B), consistent with the notion that cells cannot survive when a broad range of prosurvival Bcl-2 proteins are inactivated.
We next transduced primary AML cells from patients (Supplemental Fig. 7D) with the aforementioned expression constructs to block Mcl-1 (Bim s 2A) or all prosurvival Bcl-2 proteins (Bim s wt), using Bim s 4E as a negative control. Interestingly, two primary human AMLs (#1 and #3) showed substantially reduced viability after doxycyclinemediated induction of Bim S 2A or Bim S wt but not after induction of (inactive) Bim S 4E (Fig. 4C). Some primary human AML, however, did not show a significant drop in viability after doxycycline treatment. Although in some AML cells this may have been due to insufficient induction of Bim s 2A or Bim s wt, we remarkably found mutated Bak (abnormal protein size) and abnormally low Bax levels in AML #2 and very low expression levels of both Bax and Bak in AML #5 (Fig. 4C). It appears likely that the therapeutic treatments that these patients received prior to our analysis selected for outgrowth of AML with such generalized defects in the ''Bcl-2-regulated'' apoptotic pathway.
Collectively, these results demonstrate that Mcl-1 is critical for the sustained survival and expansion of mouse as well as human AML resulting from a broad range of oncogenic lesions, whereas Bcl-x L , Bcl-w, or Bcl-2, as reported (Zuber et al. 2011b), play minor prosurvival roles in these cancers. As such, these findings encourage the development of pharmacologic inhibitors that target Mcl-1 or regulators of its expression for treatment of this hematological malignancy.

Animals
Experiments with mice were conducted according to the guidelines of The Walter and Eliza Hall Institute Animal Ethics Committee. bcl-x fl/fl (Wagner et al. 2000), mcl-1 fl/fl (Supplemental Fig. 1A; Vikstrom et al. 2010) and the Rosa26-CreERT2 (Seibler et al. 2003) (Taconic Artemis) gene targeted mice (all on a C57BL/6 background) have been described. Mice received one aliquot containing 4 mg of tamoxifen (Sigma, T5648) per day for five consecutive days by oral gavage as described previously (Anastassiadis et al. 2010).   (Shen et al. 1997), and the Mixl1 retroviral construct was described previously (Glaser et al. 2006). The Bcl-2, Bcl-x L , and Mcl-1 retroviral expression constructs were provided by Dr. D.C.S. Huang. The CreER-hCD2 retroviral construct was provided by Dr. M. Bussliner (IMP, Vienna, Austria), and the retroviral construct for shRNA-mediated knockdown of Bim has been described (Bouillet et al. 2005). Lentiviral expression constructs were generated by KpnI digestion, and by cloning a synthesized 1638-base-pair (bp) fragment containing GFP-WPRE and AgeI/ClaI digestion and cloning of Bim S variants into the TRIPZ vector (Open Biosystems). Viral supernatants were produced by CaCl 2 cotransfection of 293T cells with expression constructs and packaging plasmids. Fetal liver (E13.5 [embryonic day 13.5] embryos) or bone marrow cells from 5-FU-treated mice were enriched for immature cells by immunomagnetic depletion of mature lymphoid (CD3 + , CD4 + , CD8 + , and B220 + ) myeloid (Mac1 + and Gr1 + ) and erythroid (Ter119 + ) cells. Viral transduction was performed as described (Metcalf et al. 2007). Transduced cells were cultured in Dulbecco's modified Eagle's medium supplemented with 20% FBS (HyClone), 2 mM L-glutamine, 100 ng/mL murine stem cell factor, and 10 ng/mL IL-3 (Peprotech). Infected cells were either FACS-sorted on the basis of GFP expression for in vitro studies or injected unsorted into sublethally g-irradiated (5.5 Gy) C57BL/6 mice. Cells retrovirally transduced with the MLL-ENL/neo expression vector were plated in methylcellulose medium M3234 (Stem Cell Technologies) supplemented with 100 ng/mL murine stem cell factor, 10 ng/mL IL-3, and 1 mg/mL G418 (InvivoGen). G418-resistant cells from the third plating were transduced with retroviral constructs encoding Bcl-2 family members or RNA hairpins for knockdown of Bim and were linked with IRES-GFP, and transduced cells were subsequently isolated by cell sorting. Cremediated recombination in vitro was induced by treatment with 10 À7 M 4-hydroxy tamoxifen (Sigma, H7904) (Glaser et al. 2009). For analysis of protein levels by Western blotting, mcl-1 fl/fl ;creER/+ cells were treated with tamoxifen in the presence of the pan-caspase inhibitor qVD-OPh (25 mM; MP Biomedicals) to protect them from apoptotic death.
Detailed methodology is described in the Supplemental Material.  -1), or Bim S wt-encoding (inhibits all prosurvival Bcl-2 family members) lentiviruses and were either left untreated or treated with doxycycline. Shown is a graph representing the ratio of doxycycline-treated compared with untreated GFP + AML blast cells from one representative experiment for each patient sample. Western blot analysis to detect Bax and Bak protein levels in primary AML cells. Low levels of Bax and Bak were detected in sample #5; low levels of Bax and abnormal size of Bak were detected in sample #2.