Members of the B-cell lymphoma 2 (BCL-2) gene family are attractive targets for cancer therapy as they play a key role in promoting cell survival, a long-since established hallmark of cancer. Clinical utility for selective inhibition of specific anti-apoptotic Bcl-2 family proteins has recently been realized with the Food and Drug Administration (FDA) approval of venetoclax (formerly ABT-199/GDC-0199) in relapsed chronic lymphocytic leukemia (CLL) with 17p deletion. Despite the impressive monotherapy activity in CLL, such responses have rarely been observed in other B-cell malignancies, and preclinical data suggest that combination therapies will be needed in other indications. Additional selective antagonists of Bcl-2 family members, including Bcl-XL and Mcl-1, are in various stages of preclinical and clinical development and hold the promise of extending clinical utility beyond CLL and overcoming resistance to venetoclax. In addition to direct targeting of Bcl-2 family proteins with BH3 mimetics, combination therapies that aim at down-regulating expression of anti-apoptotic BCL-2 family members or restoring expression of pro-apoptotic BH3 family proteins may provide a means to deepen responses to venetoclax and extend the utility to additional indications. Here, we review recent progress in direct and selective targeting of Bcl-2 family proteins for cancer therapy and the search for rationale combinations.

Dysregulation of apoptosis in cancer and role of Bcl-2 family proteins

Apoptosis is a highly regulated form of programmed cell death and is crucial for the regulation of many processes including organ development, immune cell regulation, and the elimination of infected, mutated or damaged cells. Indeed, between 50 and 70 billion cells die each day due to apoptosis in the average human adult [1]. Dysregulation of apoptosis is a hallmark of cancer, contributing to the accumulation of neoplastic cells by slowing the rate of cell turnover. Chemotherapeutic drugs of diverse structure and specificity eliminate target cells by activating common apoptotic pathways [2]. Hence, enabling apoptosis is an important strategy for both the prevention and treatment of cancer.

At least two major apoptotic pathways exist, the extrinsic and intrinsic pathways. Extrinsic apoptosis is triggered via binding of tumor necrosis factor (TNF) family ligands (such as FAS ligand) to their cognate death receptors on the cell surface [3]. In contrast, the intrinsic pathway is triggered by internal cellular stresses, including DNA damage, loss of survival signals such as growth factor signaling, and oxidative stress. Initiation of the intrinsic pathway leads to mitochondrial outer membrane permeabilization (MOMP), which allows the release of cytochrome c. In turn, cytochrome c binds to apoptosis protease-activating factor 1 (APAF 1), which binds procaspase 9, generating an intracellular ‘apoptosome’ that activates caspase 9. Among other proteins, mitochondrial disruption also releases second mitochondria-derived activator of caspase (SMAC; also known as DIABLO), which releases caspase 3 from X-linked inhibitor of apoptosis (XIAP)-mediated inhibition. Both the extrinsic and intrinsic apoptosis pathways converge on caspase 3 and caspase 7, which drive the terminal events of apoptosis [4] (Figure 1). Cross-talk between the extrinsic and intrinsic pathways has been described, such that in some cells, apoptosis induction by TNF-family death receptors is dependent on mitochondrial cell death mechanisms [5,6].

Apoptotic pathways and the role of Bcl-2 family proteins.

Figure 1.
Apoptotic pathways and the role of Bcl-2 family proteins.

Apoptosis can be initiated by external signals such as TNF receptor ligands binding to their cognate receptors (extrinsic apoptosis) or via cellular stresses such as DNA damage (intrinsic apoptosis). Upon cellular stresses, BH3 proteins bind to and activate pro-apoptotic proteins Bax and Bak, which oligomerize in the mitochondrial membranes and release apoptogenic proteins from the mitochondria, including cytochrome c. Cytochrome c then interacts with the apoptosome and initiates caspase activation that results in apoptosis. The extrinsic pathway can also activate the mitochondrial pathway through activation of Bid.

Figure 1.
Apoptotic pathways and the role of Bcl-2 family proteins.

Apoptosis can be initiated by external signals such as TNF receptor ligands binding to their cognate receptors (extrinsic apoptosis) or via cellular stresses such as DNA damage (intrinsic apoptosis). Upon cellular stresses, BH3 proteins bind to and activate pro-apoptotic proteins Bax and Bak, which oligomerize in the mitochondrial membranes and release apoptogenic proteins from the mitochondria, including cytochrome c. Cytochrome c then interacts with the apoptosome and initiates caspase activation that results in apoptosis. The extrinsic pathway can also activate the mitochondrial pathway through activation of Bid.

Activation of intrinsic apoptosis is exquisitely regulated by pro- and anti-apoptotic members of the B-cell lymphoma 2 (Bcl-2) family of proteins. Bcl-2 was originally discovered due to its involvement in follicular lymphoma (FL) where BCL2 is overexpressed due to t(14;18) chromosomal translocations [7,8]. Early studies demonstrated that Bcl-2 could promote the survival of IL-3-dependent hematopoietic cells after cytokine withdrawal [9]. Since it had previously been recognized that factor withdrawal in these cells induced apoptosis, the fact that Bcl-2 could protect cells from death suggested that Bcl-2 was an anti-apoptotic protein. More than 20 different human Bcl-2-related proteins have since been identified that either promote or inhibit death via the intrinsic pathway [10]. Many of these proteins interact with each other in a complex network to determine whether cells live or die.

The anti-apoptotic Bcl-2 family proteins resident within membranes of the mitochondria, nuclear envelope, and endoplasmic reticulum contain four Bcl-2 homology domains (BH1–4) and include in humans Bcl-2, Bcl-XL (also known as BCL-2L1), Bcl-W (also known as BCL-2L2), myeloid cell leukemia 1 (Mcl-1), Bcl-B, and Bfl-1 (also known as A1 in the mouse). The pro-apoptotic Bcl-2 proteins can be subdivided into two families, those that contain multiple BH domains (e.g. Bax and Bak) and the so-called BH3-only proteins (e.g. Bid, Bik, Noxa, Puma, Bad, Bim, and others). All of the BH3-only proteins are capable of antagonizing anti-apoptotic Bcl-2 family proteins, but some (e.g. Bid, Bim, and Puma) also serve as agonists of multi-BH domain pro-apoptotic proteins Bax and Bak (Figure 1). Many details concerning the mechanisms of Bcl-2 family protein remain to be elucidated, but a simplistic view is that BH3-only agonists of Bax and Bak stimulate oligomerization of these proteins in the outer mitochondrial membrane, leading to MOMP. Anti-apoptotic proteins, such as Bcl-2, sequester these BH3-only agonists of Bax and Bak to preserve mitochondrial integrity. Simultaneously, various BH3-only proteins compete for binding to anti-apoptotic Bcl-2 family members, releasing Bax/Bak agonists and also releasing Bax/Bak from direct interactions with anti-apoptotic Bcl-2 family members.

The balance between the anti-apoptotic and pro-apoptotic Bcl-2 family proteins and the respective availability of their interaction sites determines how ‘primed’ a cell is to undergo apoptosis, and in turn whether MOMP will occur (reviewed in ref. [11]). BH3 profiling is a method that exposes permeabilized cells to BH3 domain-containing peptides, derived from BH3-only proteins, to quantitatively determine how ‘primed’ cells are for MOMP [12]. This method has shown a correlation between the amount of BH3 required to induce MOMP in cancer cells ex vivo and clinical exposures to chemotherapy in vivo [13]. BH3 profiling can determine between three major states of malignant cells, those that have functional BAX/BAK but are not significantly sequestering pro-apoptotic proteins (apoptosis competent, but unprimed); those that lack functional BAX/BAK (apoptotically incompetent), and those that respond to both activator and sensitizer peptides (primed for apoptosis) [12]. Cancer cells avoid mitochondria-mediated apoptosis through multiple mechanisms that ultimately affect mitochondrial priming and ability to respond to external stress signals.

Alterations in BCL-2 family gene expression in hematological malignancies

The intrinsic pathway is commonly dysregulated in hematological malignancies with multiple mechanisms identified in various types of leukemia and lymphoma. The BCL-2 gene is highly expressed in chronic lymphocytic leukemia (CLL), FL, mantle cell lymphoma (MCL), and Waldenstrom macroglobulinemia (WM). In contrast, the levels of BCL-2 expression are more variable in multiple myeloma (MM), diffuse large B-cell lymphoma (DLBCL), B-lineage acute lymphoblastic leukemia (ALL), acute and chronic myelogenous leukemias (AML and CML). In most cases, the basis for high levels of BCL-2 gene expression is epigenetic, with the exception of lymphomas that harbor t(14;18) chromosomal translocations that fuse the BCL-2 gene locus with the immunoglobulin heavy-chain locus (IgH), lymphomas that have BCL-2 gene amplification, and occasional CLL cells where the BCL-2 gene is fused to immunoglobulin light-chain loci [7,8,14]. Interestingly, in the majority of CLLs, the mechanism of BCL-2 gene dysregulation has been attributed to somatic loss of genes encoding microRNAs that suppress BCL-2 expression (miR15 and 16) [15,16].

In addition to Bcl-2, other anti-apoptotic Bcl-2 family proteins are also overexpressed in cancer. For example, BCL-XL (BCL2L1) and MCL-1 (BCL2L3) gene loci are frequently amplified in solid tumors [17]. In hematologic malignancies, BCL-XL and MCL-1 expression have been reported to be up-regulated by CD40 ligation and various lymphokines [1820].

The balance of pro- and anti-apoptotic proteins can also be disrupted through down-regulation or loss of pro-apoptotic proteins. Homozygous gene mutations that inactivate the pro-apoptotic BAX gene have been identified in both hematologic and solid tumors [21]. Less frequently, genetic loss [22] or epigenetic silencing [23] of the BIM locus is observed in some B-cell malignancies, and these can often impair the normal apoptosis responses to stress signals. Loss of TP53 through either gene deletion (loss of the long arm of chromosome 17; del (17p)) or mutation of the TP53 gene, or both is a common mechanism for cancers to avoid apoptosis. Loss of TP53 is associated with poor prognosis and resistance to (or propensity to early relapse after) DNA-damaging chemotherapy regimens in CLL, myeloma, aggressive lymphomas, and B-lineage ALL. Failure of TP53 pathway signaling diminishes the induction of pro-apoptotic BCL-2 family genes in response to cellular stress, particularly DNA damage. As BCL-2-mediated resistance to intrinsic apoptosis is a hallmark of malignancy, targeting the anti-apoptotic Bcl-2 proteins is an attractive therapeutic strategy in cancer.

Development of BH3 mimetics and venetoclax

While the role of Bcl-2 family members in protecting cancer cells from apoptosis was discovered over 30 years ago, targeting of anti-apoptotic Bcl2 family members with small molecules became feasible only in the mid-1990s with the introduction of advanced technologies for structure-based drug discovery and design [24]. The structural understanding of how BH3-only proteins bind with high affinity in a hydrophobic groove on pro-survival Bcl-2 family proteins drove the development of BH3-mimetic small molecules. The interaction surface of BH3-only proteins was reduced to an amphipathic α-helical peptide of ∼16 amino acids, making contacts with pro-survival Bcl-2 family proteins at several sites and inducing apoptosis when introduced into cancer cells. Altogether, this protein–protein interaction represents a formidable drug discovery target. Though many approaches were attempted, the only method to yield promising results so far utilized nuclear magnetic resonance(NMR)-based technologies to interrogate chemical compound interactions with the BH3-binding site on pro-survival Bcl-2 family proteins [24].

Among the first-generation molecule reported was ABT-737, which binds Bcl-2, Bcl-XL, and Bcl-W with high affinity and mimics the action of the BH3-only protein BAD [25]. Although ABT-737 demonstrated single-agent antitumor activity in preclinical models of lymphoma and primary patient-derived CLL samples, it lacked sufficient pharmacokinetic properties for clinical development [2527]. A second-generation molecule ABT-263, also known as navitoclax, was generated with oral bioavailability. Navitoclax binds with high affinity to Bcl-2 and Bcl-XL, with lower affinity to Bcl-W and with very low affinity to Mcl-1 [28]. In preclinical studies, navitoclax inhibited tumor growth when used as a monotherapy in small cell lung carcinoma (SCLC) xenograft models [28,29] and when used in combination with standard-of-care agents in both solid and hematological tumor cell lines [28,30,31], which provided a rationale for its clinical investigation.

In Phase I studies, navitoclax showed promising activity against B-cell malignancies [32], including relapsed or refractory CLL, both as monotherapy [33] and in combination with fludarabine, cyclophosphamide, and rituximab, or with bendamustine and rituximab [34]. Results of a Phase II study showed that navitoclax was also effective as a first-line treatment in CLL when combined with rituximab [34]. These data with navitoclax were preceded 25 years earlier by attempts to silence BCL2 gene expression in CLL using oligonucleotide therapeutics that target the mRNA and cause its degradation [35]. The 18-mer Oblimersen sodium (Genasense) generated a significant increase in complete remission (CR) rates and duration of remission (DoR) compared with chemotherapy alone (fludarabine + cyclophosphamide) in patients with relapsed or refractory CLL [36], results which clinically validated the Bcl-2 target for this indication but lacking sufficient disease-free survival benefit to gain regulatory approval. During the course of preclinical development, navitoclax was found to cause temporary yet substantial decreases in platelet counts in animal studies [37,38]. This was subsequently determined to result from on-target inhibition of a critical function of Bcl-XL in maintaining the lifespan of circulating platelets [37,38]. In the clinic, thrombocytopenia was found to be a major dose-limiting toxicity for navitoclax, particularly in the single-agent setting. Although thrombocytopenia limited the use of navitoclax in patients, the observed efficacy provided seminal proof of concept for Bcl-2 family inhibition and highlighted the therapeutic potential of selective Bcl-2 family inhibitors.

The first potent Bcl-2-selective BH3-mimetic to be reported was venetoclax (previously known as ABT-199 and GDC-0199) (Figure 2) [39]. Like navitoclax, its ability to induce apoptosis is dependent on the presence of Bax and Bak, a criterion for mechanism-based cytotoxicity. Unlike navitoclax, venetoclax has strong affinity only for Bcl-2, with >100-fold less affinity for Bcl-XL or Bcl-W [39]. Venetoclax binds in two hydrophobic pockets, termed P2 and P4, and provides a hydrogen bond between the indole nitrogen of venetoclax and Asp103 of Bcl-2 [39]. This electrostatic interaction was a key to provide selectivity over Bcl-XL. Consistent with this, CLL cells were highly sensitive to venetoclax (median LC50 3 nM in cell culture), whereas platelets were relatively resistant (median LC50 >5000 nM) in vitro [39,40]. Indeed, venetoclax seemed more potent than navitoclax against CLL, suggesting that Bcl-2 inhibition rather than combined Bcl-2 and Bcl-XL inhibition was responsible for the activity observed for navitoclax in patients. These observations are also consistent with reports that CLL B-cells constitutively express very high levels of Bcl-2 but generally not Bcl-X [41]. Moreover, in most CLL cells, homozyzous mutations or deletions that inactivate genes encoding Bcl-2-silencing microRNAs (e.g. miR15–16) have been discovered [15,16], thus accounting for the high levels of Bcl-2 mRNA and protein production in this type of leukemia.

Chemical structure of venetoclax and binding mode to Bcl-2 (left).

Figure 2.
Chemical structure of venetoclax and binding mode to Bcl-2 (left).

Chemical structure of venetoclax (right). The 2.07A crystal structure of venetoclax in complex with Bcl-2 (PDB code: 4MAN). The protein is shown with a protein solvent accessible surface, protein carbon atoms are colored gray, ligand carbon atoms are green, oxygen atoms are red, nitrogen atoms are blue, and sulfur atoms are yellow. Two orange arrows indicate the two hydrophobic pockets P2 and P4 where high-affinity binding of pro-apoptotic BH3 peptides is mediated. A black arrow points to the Asp103, and the hydrogen bond between the indole nitrogen of venetoclax and Asp103 of Bcl-2 is depicted by a dashed red line.

Figure 2.
Chemical structure of venetoclax and binding mode to Bcl-2 (left).

Chemical structure of venetoclax (right). The 2.07A crystal structure of venetoclax in complex with Bcl-2 (PDB code: 4MAN). The protein is shown with a protein solvent accessible surface, protein carbon atoms are colored gray, ligand carbon atoms are green, oxygen atoms are red, nitrogen atoms are blue, and sulfur atoms are yellow. Two orange arrows indicate the two hydrophobic pockets P2 and P4 where high-affinity binding of pro-apoptotic BH3 peptides is mediated. A black arrow points to the Asp103, and the hydrogen bond between the indole nitrogen of venetoclax and Asp103 of Bcl-2 is depicted by a dashed red line.

Clinical experience and approval of venetoclax

Based on the observations of cytotoxicity in primary samples of human CLL, a first in human clinical study of venetoclax in patients with relapsed or refractory CLL was initiated in June of 2011 (ClinicalTrials.gov identifier: NCT01328626) and has provided encouraging data that support the concept of venetoclax monotherapy. In the Phase I study enrolling 116 heavily pretreated and refractory CLL and small lymphocytic lymphoma (SLL) patients, 79% of all patients achieved an objective response, regardless of the dose of venetoclax; complete remissions were observed in 20% of patients and 5% of all patients were negative for minimal residual disease (MRD) [42]. This trial established a daily continuous dose of 400 mg as the recommended Phase II dose for venetoclax.

The most important safety finding in the Phase I trial was tumor lysis syndrome (TLS), which was observed in patients with high tumor burden. Among the patients treated in the early CLL trials of venetoclax, two deaths and an episode of acute renal failure attributable to TLS occurred [42]. To avoid TLS, the dosing schedule was modified to include a 5-week gradual ramp-up period starting at 20 mg/day in the first week, followed by 50, 100, and 200–400 mg/day in weekly steps and strict prophylaxis and monitoring for biochemical evidence of tumor lysis. Since using this ramp-up schedule, no clinically significant TLS events have been reported and the overall risk of TLS has been effectively mitigated [43].

Mild gastrointestinal side effects and neutropenia were also common toxicities attributed to venetoclax [42,44,45]. Although nearly half of all patients experienced mild nausea and/or vomiting, discontinuation due to gastrointestinal effects was rare in both the Phase I and Phase II trials. It is currently unknown if the mechanism of gastrointestinal toxicity is an on-target effect of Bcl-2 inhibition, or due to chemical properties of the molecule or its formulation. Grade 4 neutropenia has been observed in 23–28% of patients in Phase I and Phase II trials. Grade 4 neutropenia was most common in patients who entered the trials with pre-existing neutropenia and was effectively treated with intermittent use of granulocyte colony-stimulating factors or by dose reduction in venetoclax. Febrile neutropenia occurred in 5–6% of patients with CLL in the Phase I and Phase II trials. Serious infections (≥grade 3) were infrequent, occurring in 17–20% of patients in Phase I and Phase II trials (<1% fatal), but infection prophylaxis was not mandated in these trials.

The Phase II pivotal trial was conducted on exclusively patients with relapsed or refractory CLL who had a deletion of the 17p chromosomal region [del (17p), a high-risk population; NCT01889186] [44]. Similar to the Phase I study, objective responses were observed in 79% of patients, regardless of TP53 mutation or functional status, and included CRs (8%). MRD negativity was achieved in 18 out of 45 patients assessed for PB MRD [44]. Based on these positive results, early in 2016 venetoclax received its first successful US Food and Drug Administration (FDA) accelerated approval for the treatment of previously treated CLL patients with 17p deletion.

Recently, venetoclax as monotherapy has also been shown to be effective in CLL patients who relapsed after or were refractory to the kinase inhibitors, ibrutinib or idelalisib [46]. Venetoclax has also shown single-agent activity in heavily pretreated patients with relapsed or refractory myeloma with an overall response rate (ORR) of 40% in the subgroup containing t(11;14) [47]. Venetoclax monotherapy was also investigated in a Phase I study in patients with relapsed/refractory non-Hodgkin's lymphoma (NHL) [48]. Of the total 106 patients enrolled, 44% achieved an objective response (CR or partial response (PR)). Responses were seen in all histologies, with the highest response rate in MCL (ORR, 75% [CR, 21%]), similar to responses observed in CLL, suggesting the promise of Bcl-2 inhibitor for the treatment of this typically challenging disorder [48]. Significant antitumor activity was also observed in FL (ORR, 38% [CR, 14%]), DLBCL (ORR, 18% [CR, 12%]), DLBCL-RT (Richter's transformation) (ORR, 43% [no CRs]), marginal zone lymphoma (MZL; ORR, 67% [no CRs]), and WM (ORR, 100% all PRs) [48]. Venetoclax has also shown promising activity as a single agent in a Phase II study in patients with relapsed/refractory AML or previously untreated patients deemed unfit for intensive chemotherapy [49].

Mechanisms of resistance

Though monotherapy activity has been impressive for some types of hematological malignancies, the full potential of venetoclax and similar Bcl-2 inhibitors is likely to be realized in combination with other drugs that modulate directly or indirectly mitochondrial apoptosis — often through effects on Bcl-2 family proteins. In this regard, monotherapy with venetoclax for many types of leukemia and lymphoma results only rarely in CRs and patients often progress under treatment. For example, in the Phase I study in relapsed or refractory CLL, disease progression occurred in 41 patients (35%), including RT in 18 (16%) [42]. Monotherapy responses are relatively infrequent in FL and DLBCL, and the doses required to achieve responses are typically higher (600–1200 mg/day) than required for CLL [48]. Therefore, to deepen and broaden responses to venetoclax and identify optimal combination partners, biomarkers that can predict the sensitivity to venetoclax and an understanding of resistance mechanisms are needed.

One of the main determinants of resistance to venetoclax in multiple hematological malignancies is up-regulation of other anti-apoptotic Bcl-2 family proteins, including Bcl-XL, Bfl-1, and Mcl-1, which bind and sequester the pro-apoptotic BH3 proteins such as Bim (Figure 3a). Several mechanisms have been reported to regulate expression of these proteins across hematological malignancies (Figure 3b).

Mechanisms of resistance to venetoclax.

Figure 3.
Mechanisms of resistance to venetoclax.

Sensitivity to venetoclax is determined by multiple factors including the balance of pro-apoptotic BH3-containing proteins that are available to neutralize Bcl-2 and other anti-apoptotic members of the Bcl-2 family such as Bcl-XL, Mcl-1, and Bfl-1. Overexpression of anti-apoptotic Bcl-2-like molecules that are not inhibited by venetoclax can sequester Bim and other BH3-only pro-apoptotic proteins, preventing activation of Bax/Bak and impairing induction of apoptosis (a). Multiple mechanisms for up-regulating expression of anti-apoptotic Bcl-2 proteins that are not inhibited by venetoclax have been delineated, including silencing of miR377 (causing elevated Bcl-XL expression), activation of NF-κB via cytokines and chemokines, and activation of AKT and MAPK signaling pathways through BCR activation, and activation of STAT-family transcription factors by lymphokines. Activated AKT has also been shown to phosphorylate Bim, resulting in proteasomal degradation, and to phosphorylate Bad, resulting in its sequestration by 14-3-3 (not shown). Phosphorylation of Bcl-2 through MAPK signaling has also been reported to cause venetoclax resistance (b).

Figure 3.
Mechanisms of resistance to venetoclax.

Sensitivity to venetoclax is determined by multiple factors including the balance of pro-apoptotic BH3-containing proteins that are available to neutralize Bcl-2 and other anti-apoptotic members of the Bcl-2 family such as Bcl-XL, Mcl-1, and Bfl-1. Overexpression of anti-apoptotic Bcl-2-like molecules that are not inhibited by venetoclax can sequester Bim and other BH3-only pro-apoptotic proteins, preventing activation of Bax/Bak and impairing induction of apoptosis (a). Multiple mechanisms for up-regulating expression of anti-apoptotic Bcl-2 proteins that are not inhibited by venetoclax have been delineated, including silencing of miR377 (causing elevated Bcl-XL expression), activation of NF-κB via cytokines and chemokines, and activation of AKT and MAPK signaling pathways through BCR activation, and activation of STAT-family transcription factors by lymphokines. Activated AKT has also been shown to phosphorylate Bim, resulting in proteasomal degradation, and to phosphorylate Bad, resulting in its sequestration by 14-3-3 (not shown). Phosphorylation of Bcl-2 through MAPK signaling has also been reported to cause venetoclax resistance (b).

Silencing of microRNA (miR) 377 has emerged as a possible mechanism of Bcl-XL up-regulation and resistance to venetoclax in CLL [50]. Significantly higher Bcl-XL and lower miR377 expression have also been observed in CLL cells from patients with prior exposure to chemotherapy [50].

Expression of Mcl-1 has been associated with resistance to venetoclax in preclinical models of multiple hematological cancers. For example, in patient-derived CLL cells, the ratio of Mcl-1 : Bcl-2 protein levels plus phosphorylated Bcl-2 was found to predict the cytotoxic potential of venetoclax in culture [51]. Phosphorylation of Bcl-2 has also been mechanistically associated with resistance to venetoclax in CLL by preventing venetoclax from displacing Bax and Bim from Bcl-2, thereby blocking induction of the mitochondrial pathway for apoptosis [51].

In MM, sensitivity to venetoclax both in vitro and in vivo correlated with high Bcl-2 and low Bcl-XL or Mcl-1 expression [52]. In another study using primary patient FL samples, sensitivity to venetoclax was associated with the Bcl-2 : Bim ratio with cells expressing higher levels of Bim being more susceptible [53]. In this regard, certain protein kinase signal transduction pathways are known to impact Bim protein degradation rates via phosphorylation of this pro-apoptotic BH3-only protein [54]. In MM, venetoclax-resistant cells had increased phospho-ERK and phospho-Bim, suggesting that this MAPK pathway may contribute to resistance [53]. Additionally, AKT pathway activation has been associated preclinically with resistance to venetoclax in MM. DLBCL cell lines chronically exposed to venetoclax displayed substantially increased AKT activation and up-regulation of Mcl-1 and Bcl-XL [55]. Furthermore, in preclinical studies, AKT has been reported to impact apoptosis pathways at multiple points by phosphorylating various substrates, including pro-apoptotic BH3-only member Bad, causing its sequestration by 14-3-3 family proteins [56].

Nodal CLL has typically been more refractory to venetoclax [42], as well as FL, and one suggestion for this resistance has been up-regulation of Bcl-XL due to factors such as chemokines, cytokines, and lymphokines present in the microenvironment of nodes. Indeed, up-regulation of Bcl-XL and Bfl-1 in CLL has been associated with resistance to ABT-737 [18]. Like CLL, nodal microenvironment has been reported to mediate NF-κB activation and consequent Bcl-XL up-regulation in MCL, conferring resistance to venetoclax [57].

Rational combination strategies to overcome resistance to venetoclax

The balance of pro- and anti-apoptotic proteins in a given cancer cell dictates sensitivity to molecules such as venetoclax and navitoclax, as well as standard-of-care chemotherapeutics. Therefore, rational choices for combinations partners can be found among therapeutic agents that enhance mitochondrial pathway apoptosis by suppressing expression or activity of anti-apoptotic Bcl-2 family members or by restoring expression of pro-apoptotic family members. Preclinical evidence suggests that resistance mechanisms to venetoclax could be overcome by a diversity of drug combination partners, including monoclonal anti-CD20 antibodies, DNA-damaging chemotherapy, proteasome inhibitors, B-cell receptor (BCR) signaling inhibitors (e.g. BTK inhibitors), and selective Mcl-1 inhibitors.

Anti-CD20 antibodies

Anti-CD20 antibodies are promising combination partners to enhance efficacy of venetoclax and overcome resistance in B-cell malignancies. Combining obinutuzumab or rituximab with venetoclax demonstrated that CD40-induced resistance to this compound could be counteracted in CLL cells in culture [58]. Similarly, in MCL, resistance to venetoclax due to CD40-induced proliferation and mitochondrial priming loss could be overcome by glyco-engineered anti-CD20 antibody obinutuzumab, which reduces Bcl-XL expression through NF-κB inhibition [59]. Moreover, rituximab was shown to reduce Mcl-1 protein expression in circulating CLL B-cells in patients [60], which would be expected to remove a barrier to venetoclax-induced apoptosis.

Several clinical trials have begun to evaluate the combinations of venetoclax and anti-CD20 antibodies, rituxan or obinutuzumab, in patients with CLL, FL, DLBCL, and NHL (Table 1). Recently, a Phase I study reported the encouraging outcome of venetoclax plus rituximab in 49 relapsed/refractory CLL. The ORR was 86%, including 51% complete responses. Importantly, 80% (20/25) for the patients who were complete responders were MRD-negative in their blood marrow, with 11 of 13 responders remaining in ongoing remission after a median of 9.7 months off venetoclax; another 2 with MRD-positive complete responses progressed after 24 months off therapy and re-attained response after reiteration of venetoclax [61]. Efficacy results for venetoclax and obinutuzumab in a Phase Ib study have recently been reported (NCT02242942) [62]. The combination demonstrated an increased response rate with an ORR of 100% and a 92% MRD negativity rate in the peripheral blood 3 months after treatment completion. Venetoclax combinations with rituximab and bendamustine in FL had a 68% ORR, including 50% complete responses. ORR was 33% in the venetoclax plus rituximab group [63].

Table 1
Combination clinical trials involving venetoclax
Combination mechanism Therapeutic combination Heme indications Clinicaltrials.gov identifiers 
Anti-CD20 antibodies Venetoclax + rituxumab (+bendamustine) NHL (MALT, DLBCL, FL); CLL NCT01594229 NCT02187861 NCT02005471 NCT01671904 
Venetoclax + obinutuzumab NHL (FL, DLBCL); CLL NCT02877550 NCT02242942 NCT01685892 NCT02339181 NCT02987400 
Venetoclax + obinutuzumab + endamustine NHL (FL); CLL NCT03113422 NCT02401503 NCT01671904 
Obinutuzumab or rituximab + bendamustine + cyclophosphamide + fludarabine + ibrutinib CLL NCT02950051 
Venetoclax + CHOP + rituxan or obinutuzumab NHL (DLBCL, FL, LBL, MALT, WM) NCT02055820 
Venetoclax + rituxumab + EPOCH CLL, NHL (SLL) NCT03054896 
Venetoclax + rituxumab + DA-EPOCH NHL (DLBCL) NCT03036904 
Venetoclax + rituxumab + carboplatin + etoposide + ifosfamide NHL (DLBCL) NCT03064867 
BCR inhibitor Venetoclax + ibrutinib CLL, NHL (DLBCL, MCL, SLL) NCT03045328 NCT02756897 NCT02910583 NCT0312887 NCT02419560 NCT02956382 NCT02471391 
BCR inhibitor + anti-CD20 antibodies Venetoclax + ibrutinib + rituximab NHL (DLBCL) NCT03136497 
Venetoclax + ibrutinib + obinutuzumab NHL (MCL); CLL NCT02558816 NCT02758665 NCT02427451 
Venetoclax + rituximab or venetoclax + obinutuzumab ± ibrutinib CLL NCT02950051 
Proteosome inhibitors Venetoclax + carfilzomib + dex MM NCT02899052 
Venetoclax + bortezomib + dex MM NCT01794507 NCT02755597 
MEK kinase inhibitor Venetoclax + cobimetinib AML NCT02670044 
MDM2 inhibitor Venetoclax + idasanutlin AML NCT02670044 
Venetoclax + idasanutlin + rituxan or obinutuzumab NHL (DLBCL, FL) NCT03135262 
Epigenetic regulators Venetoclax + 5-azacitidine AML NCT02993523 NCT02203773 
Venetoclax + cytarabine AML NCT03069352 NCT02287233 
Venetoclax + ABB075 (Bet-i) AML; NHL; MM NCT02391480 
Combination mechanism Therapeutic combination Heme indications Clinicaltrials.gov identifiers 
Anti-CD20 antibodies Venetoclax + rituxumab (+bendamustine) NHL (MALT, DLBCL, FL); CLL NCT01594229 NCT02187861 NCT02005471 NCT01671904 
Venetoclax + obinutuzumab NHL (FL, DLBCL); CLL NCT02877550 NCT02242942 NCT01685892 NCT02339181 NCT02987400 
Venetoclax + obinutuzumab + endamustine NHL (FL); CLL NCT03113422 NCT02401503 NCT01671904 
Obinutuzumab or rituximab + bendamustine + cyclophosphamide + fludarabine + ibrutinib CLL NCT02950051 
Venetoclax + CHOP + rituxan or obinutuzumab NHL (DLBCL, FL, LBL, MALT, WM) NCT02055820 
Venetoclax + rituxumab + EPOCH CLL, NHL (SLL) NCT03054896 
Venetoclax + rituxumab + DA-EPOCH NHL (DLBCL) NCT03036904 
Venetoclax + rituxumab + carboplatin + etoposide + ifosfamide NHL (DLBCL) NCT03064867 
BCR inhibitor Venetoclax + ibrutinib CLL, NHL (DLBCL, MCL, SLL) NCT03045328 NCT02756897 NCT02910583 NCT0312887 NCT02419560 NCT02956382 NCT02471391 
BCR inhibitor + anti-CD20 antibodies Venetoclax + ibrutinib + rituximab NHL (DLBCL) NCT03136497 
Venetoclax + ibrutinib + obinutuzumab NHL (MCL); CLL NCT02558816 NCT02758665 NCT02427451 
Venetoclax + rituximab or venetoclax + obinutuzumab ± ibrutinib CLL NCT02950051 
Proteosome inhibitors Venetoclax + carfilzomib + dex MM NCT02899052 
Venetoclax + bortezomib + dex MM NCT01794507 NCT02755597 
MEK kinase inhibitor Venetoclax + cobimetinib AML NCT02670044 
MDM2 inhibitor Venetoclax + idasanutlin AML NCT02670044 
Venetoclax + idasanutlin + rituxan or obinutuzumab NHL (DLBCL, FL) NCT03135262 
Epigenetic regulators Venetoclax + 5-azacitidine AML NCT02993523 NCT02203773 
Venetoclax + cytarabine AML NCT03069352 NCT02287233 
Venetoclax + ABB075 (Bet-i) AML; NHL; MM NCT02391480 

BCR signaling kinase inhibitors

Another important combination strategy for overcoming resistance to venetoclax includes combinations with BCR signaling inhibitors. In primary CLL cells, sustained stimulation of BCR results in significant resistance to venetoclax due to up-regulation of Mcl-1. Spleen tyrosine kinase (SYK), BTK, and PI3Kdelta inhibitors can overcome the resistance to venetoclax by down-regulating Mcl-1 [64]. Other studies have shown that venetoclax resistance in CLL induced by environmental signals that result in overexpression of Bcl-XL, Mcl-1, and Bfl-1/A1 could be overcome by sunitinib, ibrutinib, and idelalisib [65]. Ibrutinib has also been shown to effectively down-regulate overexpressed Mcl-1 and Bcl-XL in CLL [66] and to synergize with venetoclax in vitro in MCL [57,67]. In addition, since nodal CLL is more refractory to venetoclax [33], and ibrutinib is very effective in mobilizing CLL cells from the lymph node, it has been hypothesized that the combination of venetoclax plus ibrutinib may be particularly effective [68]. Clinical trials are in progress to investigate the efficacy of ibrutinib plus venetoclax in B-cell lymphoid malignancies (Table 1). Indeed, in a Phase Ib/2 study of obinutuzmab, ibrutinib, and venetoclax in relapsed/refractory CLL, all six patients available for assessment-achieved objective responses: five PR, including one MRD-negative in the peripheral blood, one MRD-negative in both the peripheral blood and the bone marrow, and one CR with MRD-negative peripheral blood and bone marrow [69]. The combination of ibrutinib and venetoclax has also shown promising activity, including CR in R/R MCL patients [70].

The dual PI3K/mTOR inhibitor NVP-BEZ235 or idelalisib has been shown to overcome resistance to venetoclax in DLBCL by reducing Mcl-1 levels, inducing release of Bim from Mcl-1 and Bcl-XL thereby inducing apoptosis by Bax activation [55]. BEZ235 has also been shown to synergize with venetoclax in DLBCL cells by inducing the accumulation of BAD and BIM [71]. In addition, a dual SYK/JAK inhibitor, cerdulatinib, was shown to induce apoptosis of CLL cells following inhibition of the BCR/IL-4 signaling pathways and could thus overcome nurse-like cell- or anti-IgM/CD40L + IL-4-mediated protection in primary CLL patient samples. The combination of cerdulatinib with venetoclax synergized to augment apoptosis in CLL samples induced by IL-4 [72]. Finally, CC-115, an mTOR and DNA-dependent protein kinase (DNA-PK), blocked cell proliferation induced by CD40 + interleukin-21 stimulation and reverted CD40-mediated resistance to venetoclax in CLL cell lines and primary patient CLL samples.

Cyclin-dependent kinase inhibitors and MEK inhibitors

In addition to BCR pathway inhibitors, other kinase inhibitors have also been shown to down-regulate Mcl-1 and overcome resistance to venetoclax. The pan-CDK inhibitor, dinaciclib, which exhibits significant single-agent activity in relapsed/refractory CLL [73], decreases Mcl-1 levels via inhibition of cyclin E/CDK2, promoting its ubiquitination and degradation [74]. Inhibition of Cyclin E/Cdk2 releases Bim from Mcl-1, resulting in synergistic killing of CLL cell lines and primary patient samples in combination with ABT-737 or venetoclax [74].

MEK inhibitors have also been previously shown to efficiently down-regulate Mcl-1 in AML cell lines and primary AML cells and synergize in vivo with ABT-737 [75]. Based on these data, a Phase I/II combination clinical trial of the MEK inhibitor cobimetinib (Cotellic) plus venetoclax has been initiated in patients with relapsed or refractor AML (NCT02670044).

Mdm2-antagonizing p53 activators

Loss of the tumor suppressor p53 has been associated with resistance to apoptosis, particularly when induced by DNA-damaging chemotherapeutic drugs [76]. Among the important mechanisms by which p53 contributes to apoptosis is by directly stimulating the transcription of several pro-apoptotic BCL2 family genes, including NOXA, BID, PUMA, and BAX [7780]. In some malignancies, genes encoding p53 remain intact, but p53 protein is degraded because of overexpression of the E3 ligase, Mdm2 (murine double minute homolog 2) [81,82]. Small molecules that bind Mdm2, release p53, and thereby allow p53 protein accumulation are in clinical development currently. Indeed, venetoclax is being evaluated currently in combination with the Mdm2 inhibitor, idasanutlin, in elderly AML patients unfit for chemotherapy (NCT02670044) and in relapsed/refractory NHL (NCT03135262).

In this regard, combining Mdm2 inhibitors with Bcl-2 antagonists shows preclinical synergy in mouse models of AML, MM, and CML [8385]. Because MDM2 inhibitors restore function of wild-type p53, patients must have functional TP53 — which is true for most patients with AML and low-grade NHLs. The combination of idasanutlin and venetoclax has recently been studied in TP53 wild-type AML cell lines that are relatively resistant to venetoclax and idasanutlin, including OCI-AML3 cells that are characterized by high levels of basal Mcl-1 expression, in vitro and in vivo [86,87]. The combination was synergistic both in culture and in mouse xenograft studies. Inhibition of Mcl-1 by the combination treatment was confirmed to contribute to the superior activity of the regimen [86,87]. Mechanistically, idasanutlin-mediated p53 activation was shown to reduce phosphorylation of ERK2 and cause destabilization of Mcl-1 protein [87].

Epigenetic regulators

Hypomethylating agents, such as azacitidine, have been shown preclinically to synergize with Bcl-2 inhibitors, including ABT-737 and venetoclax. The combination of azacitidine and ABT-737 synergistically induced p53-independent mitochondria-mediated apoptosis in primary AML cells which was accompanied by azacitidine-induced down-regulation of Mcl-1 [88]. Additional studies demonstrated that ABT-737, and to a lesser degree venetoclax, sensitized most myeloid cell lines and primary patient samples to azacitidine [89]. Azacitidine also been shown to restore response to venetoclax in CLL patient samples by increasing levels of miR-377 and, as a consequence, decreasing levels of Bcl-XL. Taken together, these findings suggest the opportunity to extend the use of hypomethylating agents in combination with venetoclax for the treatment of AML and CLL [90,91]. The combination of venetoclax with azacitidine or decitabine is being explored in a Phase 1b trial in patients 65 years of age or older with newly diagnosed AML unfit for intensive chemotherapy (NCT02203773). To date, promising results have been reported with an ORR of 76% in the first 39 patients and a median time to complete response was 29.5 days [92]. In addition, a Phase III study in treatment naive elderly AML patients has been initiated to test the combination of azacitidine and venetoclax (NCT02993523).

In addition to the promising results with hypomethylating agents, expression of anti-apoptotic BCL2 family members has also shown to be subject to epigenetic regulation by bromodomain and extra-terminal domain (BET) inhibitors, such as JQ1. Using the Eμ-Myc model of BCL, JQ1 was shown to induce of p53-independent apoptosis by enhancing the expression of pro-apoptotic BH3-only protein Bim and down-regulating Bcl-2 and Bcl-XL to directly engage the mitochondrial apoptotic pathway [93]. Bim knockout or bcl-2 overexpression inhibited apoptosis induction by JQ1 and acquired resistance following in vivo exposure was associated with strong bcl-2 up-regulation [93]. Additional preclinical data are emerging that suggest synergy with Bcl-2 inhibition and BET inhibitors. For example, JQ1 synergized with venetoclax in vitro with ‘double hit’ BCLs that harbor genomic alterations in both c-myc and bcl-2 [94]. More recently, in vitro drug screening in primary leukemia specimens that were derived from patients with high risk of relapse or relapse and cell lines revealed synergistic activity between venetoclax and JQ1. Notably, this drug synergism was confirmed in vivo using the T-ALL cell line and patient-derived xenograft models. Moreover, the therapeutic benefit of this drug combination was proposed to be mediated by an acute induction of the pro-apoptotic factor Bim and concomitant reduction in Bcl-2 upon BET inhibition, ultimately resulting in an enhanced binding of Bim to Bcl-2 [95]. Finally, ABBV-075, a potent BET inhibitor, was shown to induce mitochondrial-mediated apoptosis in AML, NHL, and MM cell lines and patient samples. Thus, induction of apoptosis was accompanied by down-regulation of Bcl-XL and Bcl-2 levels, and in some cell lines, increases in Bim and Puma were also observed [96]. ABBV-075 showed strong synergy with venetoclax in AML lines both in vitro and in vivo [96].

Selective BCL-XL and MCL-1 inhibitors

Since up-regulation of Bcl-XL and Mcl-1 is a major contributing factor to resistance to both the Bcl-2-selective inhibitor venetoclax, the development of selective and potent inhibitors of these anti-apoptotic Bcl-2 family members would potentially provide therapies that could directly overcome resistance. Given the on-target toxicity of platelet depletion seen with navitoclax due to Bcl-XL inhibition, it remains to be determined whether compounds that target only Bcl-XL will have less toxicity in humans. Bcl-XL selective inhibitors, A-1155463 and A-1331852, in combination with docetaxel have shown efficacy in a range of solid tumors while avoiding neutropenia [97], but presumably the thrombocytopenia risk remains.

Several Mcl-1 inhibitors have been reported in the literature and in patents [98]. AMG176 is the first putative Mcl-1 inhibitor to reach clinical evaluation (NCT02675452), although no clinical data have been reported yet. AMG176 is reported to have submicromolar activity in vitro and to induce robust tumor growth inhibition in the xenograft MM model OPM2. The preclinical compound S63845 has been reported to bind human Mcl-1 with a Kd of 0.19 nM (surface plasmon resonance) and mouse Mcl-1 with an ∼6-fold lower affinity, with no appreciable binding to Bcl-2 or Bcl-XL [99]. S63845 potently killed Mcl-1-dependent cancer cells, including MM, leukemia, and lymphoma cells, by activating the Bax/Bak-dependent mitochondrial apoptotic pathway. S63845 had potent antitumor activity in a xenograft model of AML (e.g. MV11). Sensitivity correlated inversely with expression levels of Bcl-XL [99]. The safety of Mcl-1-selective compounds remains to be defined. In mice, knocking out the mcl-1 gene perturbs hematopoiesis, thus raising the specter of possible bone marrow toxicity with attendant cytopenias.

An alternative to selective inhibition of particular Bcl-2 family proteins is to aim for broad-spectrum pan-inhibitors of several (or all) six anti-apoptotic members of the family. By building on natural products that interact with the BH3-binding site on anti-apoptotic Bcl-2 family members, potent compounds with activity against several pro-survival Bcl-2 family proteins have been generated. Sabutoclax (BI-97CI) is an optically pure apogossypol derivative with submicromolar affinities for several Bcl-2 family proteins including Bcl-2, Mcl-1, and Bfl-1 [100]. Sabutoclax killed cells in a Bax- and Bak-dependent manner, and was effective in DLBCL cell lines that were resistant to ABT-737 [100]. These data suggest that such pan-Bcl-2 family inhibitors may provide a therapeutic advantage in cancers that have up-regulated multiple anti-apoptotic Bcl-2 family proteins; however, the trade-off may well be unmanageable toxicity, as was observed with parent gossypol inhibitors [101].

Conclusions and outlook

Overexpression of BCL2 is a key factor mediating resistance to chemotherapy and an important survival mechanism for hematological malignancies. After over 30 years of research, the first BH3-mimetic targeting Bcl-2 protein, venetoclax, has received regulatory approval with impressive single-agent activity in relapsed CLL with 17p del. The use of venetoclax will likely expand to additional types of leukemia and lymphoma in the coming years. As one would predict, resistance mechanisms are emerging and include high levels of Bcl-XL and Mcl-1, two anti-apoptotic proteins of the Bcl-2 family not inhibited by venetoclax. Progress has been made targeting selectively Bcl-XL and Mcl-1 inhibitors which are now in clinical development. In addition, several clinical and preclinical combinations have been identified that have the potential to deepen and broaden responses to venetoclax, often by down-regulating expression of pro-survival members and enhancing expression of pro-apoptotic members of the Bcl-2 family. Finally, while high levels of Bcl-2 expression are often necessary for predicting venetoclax sensitivity, they are not generally sufficient. Therefore, continued efforts to identify biomarkers of response are much needed.

Abbreviations

     
  • ALL

    acute lymphoblastic leukemia

  •  
  • AML

    acute myelogenous leukemia

  •  
  • BAD

    Bcl-2 associated death promoter

  •  
  • Bcl-2

    B-cell lymphoma 2

  •  
  • BCR

    B-cell receptor

  •  
  • BET

    bromodomain and extra-terminal domain

  •  
  • BH

    Bcl-2 homology

  •  
  • BIM

    Bcl-2-like protein 11

  •  
  • BTK

    Bruton's tyrosine kinase

  •  
  • CLL

    chronic lymphocytic leukemia

  •  
  • CML

    chronic myelogenous leukemia

  •  
  • CR

    complete remission

  •  
  • DLBCL

    diffuse large B-cell lymphoma

  •  
  • FL

    follicular lymphoma

  •  
  • IL-3

    interleukin-3

  •  
  • MCL

    mantle cell lymphoma

  •  
  • Mcl-1

    myeloid cell leukemia 1

  •  
  • MDM2

    murine double minute homolog 2

  •  
  • miR

    microRNA

  •  
  • MM

    multiple myeloma

  •  
  • MOMP

    mitochondrial outer membrane permeabilization

  •  
  • MRD

    minimal residual disease

  •  
  • mTOR

    mammalian target of rapamycin

  •  
  • NHL

    non-Hodgkin's lymphoma

  •  
  • ORR

    overall response rate

  •  
  • PR

    partial response

  •  
  • RT

    Richter's transformation

  •  
  • SLL

    small lymphocytic lymphoma

  •  
  • SYK

    Spleen tyrosine kinase

  •  
  • TLS

    tumor lysis syndrome

  •  
  • TNF

    tumor necrosis factor

  •  
  • WM

    Waldenstrom macroglobulinemia

Competing Interests

A.R.-B. and J.C.R. are employees of F. Hoffmann-La Roche, Ltd.

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