Hierarchy for targeting prosurvival BCL2 family proteins in multiple myeloma: pivotal role of MCL1

Multiple myeloma is a malignancy of antibody-secreting plasma cells.¹  Despite recent advances in our understanding of the genetic basis of this disease and the introduction of novel therapies, the outcome for many patients remains poor.^2,3  For example, 20% of newly diagnosed patients with stage 3 myeloma have disease marked by high lactate dehydrogenase levels or unfavorable cytogenetic abnormalities (eg, 17p deletion, t[4;14]). Half these patients die within 2 years of diagnosis, in spite of advanced therapies. Hemizygous loss of the tumor suppressor TP53, typically because of the deletion of chromosome 17p, with or without concomitant loss of protein expression⁴  or mutation of the remaining allele,⁵  is associated with a poor prognosis.^6,7  In such patients, responses to targeted therapies such as immune modulator agents remain suboptimal, and allogeneic stem cell transplantation, with its associated risks, remains the only curative treatment. Hence, there is great interest in discovering and developing novel therapeutic approaches for this disease.^2,3 

Our focus is on targeting the BCL2-regulated cell survival pathway, also known as the mitochondrial or intrinsic cell survival pathway.⁸  Results from early-phase clinical trials with ABT-263 (navitoclax),⁹  and more recently ABT-199 (venetoclax),¹⁰  reveal that the prosurvival protein BCL2 is a highly promising target for treating some types of B-cell malignancies. About 80% of patients with refractory or relapsed chronic lymphocytic leukemia respond to ABT-199. In some, their responses have been dramatic and durable. Interestingly, studies using myeloma cell lines have suggested that the targeting BCL2 holds promise for patients with multiple myeloma.^11,12  However, in a clinical trial of ABT-199 in patients with heavily pretreated myeloma, objective responses were observed in only 2, both t(11;14), of 32 evaluable patients, suggesting only a minor subset of myeloma is BCL2-dependent.^13,14 

We set out to determine which prosurvival BCL2 proteins are responsible for the survival of a panel of myeloma cell lines, in anticipation that our findings are likely to be highly relevant to patients with multiple myeloma. Normally, BCL2 and its prosurvival relatives such as BCLXL and MCL1 maintain cellular viability by restraining the activity of cell death mediators BAX and BAK.¹⁵  When a cell is no longer required, stressed, or damaged, these prosurvival proteins are inhibited by the BH3-only proteins (eg, BIM), thus allowing BAX and BAK to drive mitochondrial outer membrane permeabilization.¹⁶  ABT-199 selectively inhibits BCL2, but not its other prosurvival relatives, thereby triggering apoptosis in cells that rely on BCL2 for their survival.¹⁷  It mimics the action of the killer BH3-only proteins; hence, this class of small molecule inhibitors is known as the BH3 mimetics.¹⁸ 

By pharmacologically inhibiting the prosurvival BCL2 proteins, we found that a fraction of the myeloma cell lines were rapidly killed when BCL2 (25%) or BCLXL (25%) were targeted with small-molecule inhibitors. In contrast, the majority (∼70%) of the cell lines tested are heavily reliant on MCL1. Moreover, targeting MCL1 halted the otherwise inevitable progression of this disease when tumor cells are inoculated into mice. Our studies lend strong support to the notion that the MCL1 is a high-value therapeutic target for advancing the treatment of multiple myeloma, particularly for those patients with characteristics that currently carry a poor prognosis.

All myeloma cell lines used are described in supplemental Table 1, available on the Blood Web site. The low-passage patient-derived myeloma cell lines ALF-1/TK-2 and ALF-2/TK-1 have been described.¹⁹  Using similar approaches, additional low-passage lines (TK-3 to TK-8) were generated and used in this study. BAX^−/−BAK^−/− cells were generated using CRISPR/Cas9.

ABT-737, ABT-199, and A-1155463 were provided by AbbVie; A-1210477 (Active Biochem), obatoclax (Selleck), doxycycline (Sigma), and Q-VD-OPH (MP Biomedicals) were purchased. A-1331852 was synthesized as previously described.²⁰ 

The pFTRE3G_pGK3G_GFP (expressing the BIM variants) and pFTRE3G_pGK3G_GFP_Luc2 (expressing luciferase) lentiviral constructs have been described.²¹  Constitutive Cas9 and inducible guide RNA vectors have been described.²²  The guide RNA sequences used are listed in supplemental Table 2.

Myeloma cells were serially infected with lentiviruses expressing Cas9 mCherry and guide RNAs (sgRNA) GFP. To induce expression of the sgRNA, doxycycline was added to tissue culture medium at a final concentration of 1 μg/mL. After 72 hours, cell lysates were prepared for immunoblotting. In addition, genomic DNA was sequenced to confirm the mutation of targeted DNA by using the Illumina MiSeq platform.²²  For sequencing, PCR primers with overhang sequences for each sgRNA are described (supplemental Table 3).

GraphPad Software was used for statistical analysis. All data are expressed as means ± SD and analyzed with an unpaired Student t test for statistical significance. P values < .05 were considered to be significant.

All animal experiments were approved by the Walter and Eliza Hall Institute Animal Ethics Committee and conducted according to its guidelines. Written informed consents were obtained from patients for the collection of bone marrow samples. The studies were performed with the approval of the Alfred Hospital’s Human Research Ethics Committee.

Methods for lentivirus production and infection, drug sensitivity of primary myeloma cells, cell viability assays, immunoblotting, in vivo imaging, and histology are provided as supplemental data.

To determine whether human myeloma cell lines rely on 1 or more of the prosurvival BCL2 proteins for their survival, we treated a panel of 25 well-established myeloma cell lines with validated inhibitors of these proteins (Figure 1A; experimental strategy is outlined in supplemental Figure 1). ABT-737 targets BCL2, BCLXL, and BCLW,^23,24  whereas ABT-199 has significantly lower affinity for BCLXL and BCLW.¹⁷  In contrast, 2 recently described compounds, A-1155463 and A-1331852 (hereafter, referred to as A5463 and A1852), act to target BCLXL selectively^20,25 ; they have no appreciable affinity for the other prosurvival BCL2 proteins.

Of the 25 lines tested, 5 (20%) were highly sensitive (defined as IC₅₀ < 0.5 μM) to ABT-737 (Figure 1B; supplemental Table 4). Interestingly, these same lines were also very sensitive to killing induced by the BCL2 inhibitor ABT-199. This strongly suggested that the selective targeting of BCL2 alone was sufficient to kill these 5 cell lines. Of note, 2 of these 5 lines (KMS-12-BM, KMS-12-PE) harbored the t(11;14) chromosomal translocation.

Our finding that 20% of the myeloma cell lines rely on BCL2 is in good general agreement with previously reported studies.^11,12  Our data also suggest that previously unrecognized cases of myeloma, such as those harboring the t(4;14) chromosomal translocation (eg, KMS-28-BM, KMS-18), are also likely to be promising candidates for therapy with a BCL2 inhibitor. Having confirmed the role for BCL2 in myeloma, we next asked whether BCLXL might also have a role.

Although ABT-737 can inhibit BCLXL, our previous functional studies had indicated that it is a relatively ineffective BCLXL inhibitor in lymphoid cells.²⁶  Given this caveat, and to fully define the role of BCLXL in myeloma, we used a novel and highly potent inhibitor of BCLXL.^20,25,27  Importantly, A5463 is structurally distinct from ABT-737 and ABT-199.

We found that, of the 25 cell lines, 5 (20%) of them were readily killed by A5463. Of these, only 1 (WL-2) was also readily killed by ABT-737 (and ABT-199; Figure 1B). In contrast, 4 other cell lines highly sensitive to A5463 (KMS-34, RPMI-8226, MM1R, MM1S) were relatively insensitive to ABT-737 (10-80-fold weaker) and completely resistant to ABT-199. In summary, our initial screens identified subsets of human myeloma cell lines that rely on BCL2, as previously described,^11,12  or interestingly, on BCLXL^2,7  with very little overlap between these subsets (Figure 1C). Although all sensitive cells prominently expressed the target (BCL2 or BCLXL), high expression level was not predictive of sensitivity (supplemental Figure 2).

Our data suggest that BCLXL on its own should be considered a potential therapeutic target in multiple myeloma, in addition to BCL2. It is also noteworthy that suboptimal small molecule inhibitors may not fully reveal the full effect of targeting specific prosurvival proteins, as is the case with using ABT-737 to functionally inhibit BCLXL (Figure 1B). Our data strongly suggest that studies to test for sensitivity to BCLXL inhibition are best conducted using a selective BCLXL inhibitor, such as A5463, not just with ABT-737, as the latter does not appear to be as potent at inhibiting BCLXL as A5463 (supplemental Figure 3).²⁶ 

Having established the potency of A5463, we also formally tested whether it acts specifically to induce apoptosis (Figure 2; supplemental Figures 4-5). In mechanistic studies using KMS-12-PE or MM1S cells, cell lines sensitive to BCL2 or BCLXL inhibition, respectively, we found that the absence of the downstream mediators of apoptosis, BAX and BAK (Figure 2A-B; supplemental Figure 4), completely abolished killing by these BH3 mimetics, thereby precluding any off-target or nonspecific cytotoxic effects these compounds might harbor. We also established that A5463 killed MM1S cells by a caspase-dependent mechanism (supplemental Figure 5C), whereas this compound had negligible effect on the viability of KMS-12-PE cells, a line sensitive to BCL2 inhibition.

As only a fraction of the myeloma cell line panel tested was killed by inhibition of BCL2 or BCLXL, we next asked whether this conclusion is borne out in samples freshly isolated from patients with multiple myeloma (Figure 2C). Consistent with our cell line data, we found that a similarly minor fraction of these primary patient-derived samples were killed by ABT-199 treatment. Our data indicate that the majority (75%, 9/12 patient samples) were refractory to the selective inhibition of BCL2 or BCLXL, or when both of these prosurvival proteins are targeted.

On the basis of these studies, we thus conclude that only some patients with myeloma are likely to respond to ABT-199, and that inhibition of BCLXL on its own may have a role for treating multiple myeloma.

We then asked which other prosurvival BCL2 protein is responsible for keeping the other myeloma cell lines alive. A prime candidate is MCL1, as normal plasma cells rely on it^28,29  and previous studies had implicated MCL1 in myeloma.^30,31  We tested 2 recently described MCL1 inhibitors, obatoclax³²  and A-1210477,^33,34  for their cytotoxic activity in 2 myeloma cell lines that depend on MCL1; namely, OPM-2³⁰  and H929.³⁵ 

We readily discounted obatoclax,³²  as it killed both the parental wild-type (WT) cells as well as ones engineered to be devoid of BAX and BAK (Figure 2D-E). This is consistent with the notion that the cytotoxic action of obatoclax cannot be solely accounted for by inducing apoptosis.^36,37  In contrast, A-1210477^33,34  appeared much more promising, as it acted selectively (Figure 2D-E). However, its lack of potency (IC₅₀ > 5 μM), unlike that of ABT-199 or A5463 (Figure 2A-B), made it a less than optimal tool compound for our myeloma studies. Thus, neither obatoclax nor A-1210477 is optimal for our attempts to clearly define the role of MCL1 in myeloma.

As there are no validated small molecules suitable to specifically and efficiently inhibit MCL1, we next investigated the feasibility of alternative approaches to address the question of what maintains their survival. Initially, we turned to targeting the expression of the pro-survival BCL2 proteins, using CRISPR/Cas9 genome editing technology.^38,39  Using OPM-2 cells as a MCL1-dependent model cell line,³⁰  we evaluated the effect of inactivating BCL2, BCLXL, BCLW, MCL1, or BCL2A1 in this cell line when expression of sgRNAs targeting these genes was induced (Figure 3A).²² 

Interestingly, inducing the expression of sgRNAs to MCL1 in OPM-2 cells led to the rapid loss of cell viability, whereas targeting other prosurvival BCL2 genes had no effect, consistent with their MCL1 dependence. Moreover, we confirmed that the loss of cell viability in OPM-2 cells is mediated by apoptosis, as co-incubating the cells with a broad-spectrum caspase inhibitor (Q-VD-OPh) ameliorated cell death (Figure 3B). We obtained similar results with another MCL1-dependent cell line, H929 (data not shown).

Our studies with small molecule inhibitors, as well as observations by others,^11,12,27  had identified the KMS-12-PE cell line as one highly sensitive to BCL2 inhibition. Consistent with these observations, this cell line rapidly died when BCL2 was genetically targeted, using CRISPR/Cas9 (Figure 3C). Interestingly, this cell line was also killed when MCL1 was targeted.

Unlike OPM-2, H929, and KMS-12-PE, the viability of U266B1 cells was unaffected when BCL2, BCLXL, BCLW, MCL1, or BCL2A1 were genetically targeted on their own (Figure 3D). Interestingly, although targeting MCL1 alone had no effect in this cell line, cotreating MCL1-ablated cells with the well-validated small molecules allowed us to ascertain whether combinations of them keep U266B1 cells viable. Our data strongly suggest these cells rely on MCL1 and BCLXL, as targeting the former genetically using CRISPR/Cas9 and the latter by A5463 cotreatment rapidly killed U266B1 cells (Figure 3E). However, the combined targeting of MCL1 by CRISPR/Cas9 and BCL2 with ABT-199 had no effect.

As there was no adverse effect on the viability of the U266B1 cell line when the prosurvival BCL2 genes were singly targeted, we could readily confirm the highly efficient targeting of these genes in U266B1 cells by immunoblotting for the proteins (Figure 3F) or by DNA sequencing (Figure 3G; supplemental Figure 6-14). Moreover, the efficiency and specificity of guides targeting BCL2 or BCLXL were further confirmed in other cell lines (supplemental Figure 15) or in a recent study.²² 

These studies (Figure 3) to assess the effect of targeting MCL1 using CRISPR/Cas9 suggested this technique has broad applicability to screen a larger panel of cell lines. First, cells that have been previously reported to rely on MCL1 (OPM-2 and H929) had significantly reduced viability when expression of MCL1 in these cells was ablated by CRSIPR/Cas9 targeting (Figure 3A-B and data not shown). Second, a cell line (KMS-12-PE) sensitive to a small molecule BCL2 inhibitor (Figure 1B) was killed when BCL2 was targeted genetically (Figure 3C). In addition to these functional studies, our sequencing analysis revealed that a significant fraction (∼50% up to 100%) of the cells in the pools harbored mutations in the target genes (Figure 3G; supplemental Figures 6-14), suggesting we can readily screen pools of cells that conditionally express a guide RNA to MCL1 to establish whether or not they rely on this prosurvival protein. Using the sgRNA for MCL1 (guide 1) introduced into the myeloma cell lines, we found that many lines, in addition to the ones previously recognized, die when MCL1 was targeted genetically (Figure 4; supplemental Table 5): the viability of 14/19 cell lines screened was reduced by greater than 50% when MCL1 was targeted using CRISPR/Cas9.

However, this screen had a number of potential limitations. First, we only analyzed the effect on cell viability at a few defined times (72 and 96 hours; data for the later time are not shown). Second, although the efficiency of MCL1 mutations in the U266B1 pool that express sgMCL1 #1 was high (∼90%; supplemental Figure 12), we had not determined the mutation frequency in all the cell lines screened. Moreover, the consequences of gene deletion may not be the same as using a small molecule to disrupt protein function.

Given these caveats, we adopted an alternative approach to validate our findings obtained using CRISPR/Cas9 genome editing (Figure 4), with the goal of definitively establishing whether or not MCL1 has a central role in maintaining the survival of these myeloma cell lines.

To target the prosurvival BCL2 proteins with an orthogonal approach that closely recapitulates the pharmacologic action of the BH3 mimetics, we exploited a recently developed panel of BIM variants that has been fully characterized (Figure 5A).^21,40,41  By comparing the viability of cells inducibly expressing WT BIM or 1 of its variants, we could deduce whether the cells depend on 1 or more of the prosurvival BCL2 proteins for their survival. Another BIM variant, BIM4E, which does not bind to any of the prosurvival proteins, served as the negative control in these studies.

As anticipated, all the cell lines were killed by BIM expression, suggesting they rely on 1 or more of the prosurvival BCL2 proteins for their viability (Figure 5B). We next focused on the subset (6/25 cell lines) that was sensitive to BIMBAD (arbitrarily defined as percentage viability ≤ 25%) and compared them with the activity of ABT-199, which targets BCL2, or A5463, which targets BCLXL. About half the cells lines sensitive to BIMBAD were sensitive to ABT-199, whereas the other half was killed by A5463 (supplemental Figure 16). This is anticipated, as BIMBAD binds both BCL2 and BCLXL.⁴⁰  Given these encouraging results, we exploited the MCL1-selective activity of BIM2A in the follow-up studies.

Very interestingly, we found that expression of the MCL1 selective ligand BIM2A rapidly killed a sizeable fraction (17/25 cell lines) of the myeloma cell line panel (Figure 5B; supplemental Table 6). Moreover, the activity observed with the peptidyl antagonist BIM2A correlated with the genetic targeting of MCL1 by CRISPR/Cas9 (Figure 5C), whereas the killing by BIMBAD, which does not target MCL1, did not (supplemental Figure 17).

From these approaches, using small molecule inhibitors (Figure 1), gene editing (Figures 3-4), or a peptide-based approach (Figure 5), we discerned which prosurvival protein is critical for the myeloma cell lines. Although there is some overlap (Figure 5D), it appears that most myeloma cell lines are readily killed when MCL1 is targeted (Figure 5B), whereas BCL2 or BCLXL play lesser roles (Figures 1C and 5B). Using BIM2A, strong killing (defined as viability of ≤25% of the control) was observed in 17/25 (∼68%) cell lines, and moderate killing (viability ≤50%) was observed in another 7/25 (∼28%) cell lines. We conclude that a highly significant fraction of the myeloma cell lines will be susceptible to apoptosis when MCL1 is targeted. Importantly, even cell lines that harbor the poor prognostic t(4;14) chromosomal translocation were readily killed by BIM2A (Figure 5B), and the cell lines were killed regardless of their TP53 status (Figure 5E).

Given the key role of MCL1 in myeloma, according to our results (Figures 3-5), and as in vitro studies cannot accurately reflect the in vivo scenario in which microenvironmental factors can modulate therapeutic responses,^42,43  we next asked whether targeting this prosurvival protein might ameliorate the disease in vivo. To undertake these studies, we focused on 2 myeloma cell lines which we, and others, have identified to be reliant on MCL1: AMO1 (Figures 4-5) and H929 (Figures 2-5, and data not shown).^33,35  We exploited our panel of BIM variants (Figure 5A), as the available small molecule inhibitors are not suitable (Figure 2) for in vivo studies.

Strikingly, expression of BIM2A to inhibit MCL1 in AMO1 (Figure 6; supplemental Figure 18) significantly delayed disease progression compared with mice expressing the inert BIM variant, or when BIM2A was not expressed. In the mice inoculated with the AMO1 cell line, we found significantly reduced overall disease burden (Figure 6A-B), CD38⁺ plasma cells in the peripheral circulation, bone marrow involvement, or serum paraprotein levels (Figure 6C-D). Equally impressive results were observed in mice inoculated with another cell line, H929 (supplemental Figure 19).

Our data from studies, both in vitro (Figures 4-5) and in vivo (Figure 6; supplemental Figures 18-19), using the panel of cell lines, strongly suggests MCL1 is an important therapeutic target to consider for myeloma. To extend our studies beyond these immortalized cell lines that are well adapted to growth under tissue culture conditions, we evaluated the effect of BCL2, BCLXL, or MCL1 inhibition on a panel of low-passage cell lines, some with complex poor-risk karyotypes.¹⁹  Three of 7 and 2/7 lines were readily killed by inhibition of BCL2 or of BCLXL, respectively (Figure 7A; supplemental Table 7). Strikingly, we found that 5/7 of these cell lines tested were readily killed (viability, ≤25%) by expressing the MCL1-selective ligand BIM2A (Figure 7B; supplemental Table 7), further defining MCL1 as a therapeutic target of high potential in multiple myeloma.

The main focus of our study was to definitively establish which of the prosurvival BCL2 proteins is required to maintain the viability of multiple myeloma cells and to establish a hierarchy of targets for clinical evaluation. Using pharmacologic (Figure 1) as well as genetic (Figure 4) and peptide-based (Figure 5) approaches, we showed that the majority of the myeloma cell lines tested, including low-passage ones, rely on the prosurvival protein MCL1, with a smaller fraction dependent on BCL2 or on BCLXL (Figures 5D and 7). Taken together with recent findings,⁴⁴  our results provide strong impetus for testing potent and selective MCL1 inhibitors for the treatment of this disease. Although some BH3 mimetics such as ABT-199 (venetoclax) and ABT-263 (navitoclax) are undergoing clinical trials, ones to target MCL1 are in a much earlier developmental stage.^33,45,46 

The ever-expanding repertoire of small molecule inhibitors of 1 or more of the prosurvival BCL2 proteins is proving invaluable to preclinical studies to establish which of BCL2 proteins a particular tumor relies on.^{11,17,27,44,47,48}  Importantly, the potency, selectivity, and specificity of these compounds must be determined in mechanism-of-action studies; our finding that a significant subset of the myeloma cell lines is susceptible to BCLXL inhibition (Figure 1) is not widely appreciated. Our conclusion relies on using a potent inhibitor of BCLXL, A-1155463, which is significantly more active than ABT-737 (supplemental Figure 3; supplemental Table 4). Precisely why this is the case is unclear and is the subject of our ongoing studies.

Although similar studies using small molecule inhibitors to selectively target MCL1 were not feasible, we were able to exploit the power of CRISPR/Cas9 genome editing to rapidly screen the panel of myeloma cell lines (Figure 4). This technique proved rapid and reliable, as we were able to confirm our findings using a peptide ligand BIM2A that is highly selective for MCL1 (Figure 5). Thus, our studies, using 2 distinct approaches (CRISPR/Cas9 genome editing to target MCL1 or the MCL1-selective ligand BIM2A), lend support to previous studies that suggested the potential importance of MCL1 for multiple myeloma.^30,31,35  We anticipate that our studies pinpointing the central role of MCL1 in multiple myeloma, including in vivo studies (Figure 6), are very likely to closely mirror the clinical setting.

Our results also draw attention to ongoing efforts attempting to identify optimal targets for developing novel cancer therapeutics. Although the notion that targeting the molecular lesion driving a cancer is compelling and validated, such as the case with targeting the BCR-ABL fusion oncoprotein in chronic myelogenous leukemia or mutant B-RAF V600E in melanoma, it has been argued that myeloma cells may be more susceptible to some classes of targeted therapeutics, such as the proteasome inhibitors or to immune modulator agents, because they retain fundamental properties of their normal cellular counterparts, the plasma cells, from which they are derived.⁴⁹  In this regard, it is noteworthy that previous biological studies, mainly in the mouse, have identified MCL1 as the critical survival factor required to sustain plasma cells.^28,29  As such, one could hypothesize that the targeting of critical prosurvival proteins such as MCL1 will prove to be agnostic to the recognized adverse biological features of myeloma⁵⁰  that continue to define the limitations of presently available immune modulator agents and proteasome inhibitor-based therapeutic approaches.

In summary, our results highlight the pressing urgency for the development of high-quality, potent inhibitors of MCL1 for efficacy and safety studies, particularly in the context of treating multiple myeloma. MCL1 should be added to the list of highly promising targets for the development of novel therapies in this disease, and we anticipate that it would prove clinically effective, provided safety concerns can be adequately addressed.

The authors thank J. M. Adams, S. Cory, A. Strasser, M. F. van Delft, and J. G. Zhang for discussions and suggestions; R. Anderson and W. Welch for gifts of reagents; D. Cooper, C. Hay, S. Oliver, and G. Siciliano for animal husbandry; and AbbVie for providing ABT-737, ABT-199, and A-1155463.

This work is supported by scholarships, fellowships, and grants from the Australian National Health and Medical Research Council (research fellowships to A.W.R. and D.C.S.H.; project grant 1057742 [D.C.S.H.]; program grants 1016647 and 1016701; and Independent Research Institutes Infrastructure Support Scheme [grant 9000220]), the Cancer Council Victoria (grant-in-aid to A.W.R. and D.C.S.H.), the Leukemia and Lymphoma Society (Specialized Centers of Research grants 7001-13), the Australian Cancer Research Foundation, a Victorian State Government Operational Infrastructure Support grant, and the China Scholarship Council (award to Y.Y.). A.W.R. holds the Metcalf Chair of Leukaemia Research at the University of Melbourne.

Contribution: J.-N.G., A.W.R., and D.C.S.H. devised the study; J.-N.G., T.K., D.S., Y.Y., and C.D.R. performed the experiments; J.-N.G., T.K., D.S., S.L.K., and M.J.H. developed methodology; T.K. and A.S. provided the low-passage myeloma cell lines; J.-M.G. and G.L. provided A-1331852; J.-N.G., T.K., D.S., Y.Y., C.D.R., A.S., A.W.R., and D.C.S.H. analyzed the data; J.-N.G. wrote the first draft of the manuscript, which was edited and revised by A.W.R. and D.C.S.H.; all authors contributed to review and analysis of data in the manuscript. The study was supervised by A.W.R. and D.C.S.H.

Conflict-of-interest disclosure: J.-N.G., D.S., C.D.R., S.L.K., G.L., M.J.H., A.W.R., and D.C.S.H. are employees of the Walter and Eliza Hall Institute of Medical Research, which receives research funding and milestone payments in relation to venetoclax (ABT-199). The laboratories of A.W.R. and D.C.S.H. receive research funding from Servier. The rest of the authors declare no competing financial interests.

The current affiliation for J.-M.G. is SYNthesis Med Chem, Melbourne, Australia.

Correspondence: Andrew W. Roberts, The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, VIC 3052, Australia; e-mail: roberts@wehi.edu.au; and David C. S. Huang, The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, VIC 3052, Australia; e-mail: huang_d@wehi.edu.au.