Multifaceted anticancer activity of BH3 mimetics: current evidence and future prospects
Abstract
BH3 mimetics represent a novel class of anticancer agents specifically designed to target pro-survival proteins within the Bcl-2 family. Similar to endogenous BH3-only proteins, these mimetics competitively bind to the hydrophobic grooves on the surface of pro-survival Bcl-2 family members, effectively neutralizing their protective effects and facilitating apoptosis in cancer cells.
Among the identified small-molecule BH3 mimetics, ABT-737 and its analogs, as well as obatoclax and gossypol derivatives, are the most well-characterized. The anticancer potential of these compounds, whether applied as single agents or in combination with chemotherapeutic drugs, is currently under evaluation in both preclinical studies and clinical trials. Despite promising results, the precise mechanisms underlying their anticancer effects remain to be fully elucidated.
Findings from preclinical studies suggest that BH3 mimetics exhibit additional activities in cancer cells beyond apoptosis induction. These off-target effects include the induction of autophagy and necrotic cell death, as well as modulation of the cell cycle and various cell signaling pathways. A comprehensive understanding of both their role as inhibitors of pro-survival Bcl-2 proteins and their potential additional effects is crucial for optimizing their clinical implementation.
This review provides an overview of the most representative BH3 mimetic compounds, with a particular emphasis on their off-target effects. Based on current knowledge of the diverse actions of BH3 mimetics in cancer cells, this commentary discusses potential challenges and highlights the significant promise of these agents in cancer treatment.
Introduction
Cellular homeostasis relies on maintaining a delicate balance between pro-survival and pro-death members of the Bcl-2 family. These proteins play a crucial role in regulating programmed cell death and determining cellular fate. A hallmark of cancer cells is their ability to evade apoptosis, often through the aberrant expression of Bcl-2 family members. The overexpression of pro-survival Bcl-2 proteins is not only essential for cancer development but also presents a significant barrier to effective treatment.
Given this, targeting Bcl-2 proteins has emerged as a promising strategy for cancer therapy. Advances in understanding the roles, structures, and interactions of Bcl-2 family members have led to the development of novel anticancer agents known as BH3 mimetics. These small-molecule compounds selectively bind to the hydrophobic groove of pro-survival Bcl-2 proteins, thereby activating the intrinsic apoptotic pathway.
Over a decade of research on BH3 mimetics has demonstrated their high anticancer efficacy and provided strong justification for their use in cancer therapy. However, several questions remain regarding their pharmacological effects and the factors contributing to their cytotoxic activity. Although BH3 mimetics were originally designed to function as apoptosis inducers or sensitizers, recent findings suggest that their effects can vary depending on the cellular context. In certain cases, these agents may act through off-target mechanisms, triggering alternative cell death pathways and modulating multiple signaling networks.
Understanding the non-apoptotic mechanisms involved in cancer cell responses to BH3 mimetics is particularly important for developing new therapeutic strategies aimed at enhancing cancer cell death. This review aims to summarize the current knowledge on BH3 mimetics and their mechanisms of anticancer action. Key topics covered include:
1) The apoptotic and non-apoptotic functions of Bcl-2 family members and their roles in cancer development.
2) A comparison of the specificity, mechanisms of action, and anticancer activity of the most advanced and well-characterized BH3 mimetics.
3) An overview of the off-target effects of BH3 mimetics and their implications for therapeutic efficacy.
Additionally, this review discusses the challenges and future perspectives in BH3 mimetic development, highlighting the need for further research to optimize their clinical application.
Bcl-2 protein family
Role of Bcl-2 family proteins in apoptosis
Proteins of the Bcl-2 family play a crucial role in regulating the intrinsic pathway of apoptosis by controlling the release of apoptogenic factors from the mitochondria. This family consists of both pro-survival and pro-death proteins, which exert opposing effects on mitochondrial integrity.
Members of the Bcl-2 family are categorized based on the number of conserved Bcl-2 homology (BH) domains (BH1–BH4) present in their sequence. Pro-survival proteins, such as Bcl-2, Bcl-XL, and Bcl-w, typically contain all four BH domains, whereas pro-death proteins fall into two subgroups: multidomain pro-death members (Bax, Bak, and Bok), which contain three BH domains, and the BH3-only subfamily (Bid, Bad, Bim, Noxa, Puma, and Bik), which possess only the BH3 domain.
Pro-survival Bcl-2 proteins prevent apoptosis by maintaining mitochondrial membrane integrity. They achieve this by binding to and inhibiting Bax and Bak, the key effectors responsible for mitochondrial membrane permeabilization. Activation of Bax and Bak occurs in response to cellular stress and is mediated by BH3-only proteins, which act as damage sensors and are regulated through transcriptional or post-translational mechanisms. Although BH3-only proteins can have overlapping functions, their activity is typically specific to certain tissues or cellular signals.
The fate of a stressed cell is determined by the balance between pro-survival and pro-death members of the Bcl-2 family and their interactions. Pro-survival proteins such as Bcl-2, Bcl-XL, and Mcl-1 selectively bind and neutralize different pro-apoptotic proteins. Notably, the BH3-only proteins Bim, tBid, and Puma are considered potent inducers of apoptosis because they can bind to and inhibit all pro-survival members, thereby tipping the balance toward cell death.
Certain BH3-only proteins appear to have selective binding preferences for their pro-survival counterparts. For example, Noxa was initially believed to interact exclusively with Mcl-1 and A1. However, recent findings suggest that Noxa, along with other BH3-only proteins, may exhibit broader binding capabilities, also associating with Bcl-2 and Bcl-XL.
Two primary models were initially proposed to explain the pro-apoptotic function of BH3-only proteins. The **displacement model** suggests that BH3-only proteins promote apoptosis by neutralizing pro-survival Bcl-2 family members, thereby freeing Bax and Bak to initiate cell death. The **direct activation model**, on the other hand, posits that BH3-only proteins directly activate Bax and Bak, leading to mitochondrial membrane permeabilization, the release of cytochrome c, and the activation of effector caspases 3 and 7, ultimately triggering apoptosis.
More recently, a **unified model** incorporating elements of both theories has been proposed. According to this model, pro-survival Bcl-2 proteins function in two distinct ways: **Mode 1**, in which they sequester direct-activator BH3-only proteins, and **Mode 2**, in which they bind to active Bax and Bak to prevent apoptosis.
Bcl-2 family proteins are tightly regulated through both transcriptional and post-translational mechanisms. The expression levels of certain BH3-only proteins, such as Noxa and Puma, increase in response to cell death signals, often in a **p53-dependent** manner. However, other transcription factors can also contribute to their regulation. In healthy cells, Bim is sequestered by microtubules but relocates to mitochondria upon apoptotic stimulation.
Post-translational modifications further modulate the activity of Bcl-2 family members. These modifications include phosphorylation, caspase-mediated cleavage, ubiquitination, and proteasomal degradation, all of which influence the stability and function of these proteins in apoptosis regulation.
Non-apoptotic role of Bcl-2 family proteins
Emerging evidence suggests that Bcl-2 family proteins play roles beyond the regulation of apoptosis. For instance, Bad has been implicated in **cell cycle control** and **metabolic regulation**, while Bid has been identified as a **phospholipid transporter** within mitochondria. Additionally, bNIP1 appears to contribute to the **biogenesis and structural organization** of the endoplasmic reticulum (ER).
The connection between Bcl-2 proteins and the ER has been extensively studied, with substantial evidence supporting their involvement in the **regulation of intracellular Ca²⁺ homeostasis**. Several reports indicate that Bcl-2 and Bcl-XL localize to the ER, where they interact with the inositol 1,4,5-trisphosphate receptor (IP3R), influencing its activity. However, the exact mechanisms by which Bcl-2 family proteins modulate Ca²⁺ fluxes remain complex.
Anti-apoptotic proteins such as Bcl-2 and Bcl-XL appear to **promote pro-survival IP3R-mediated Ca²⁺ oscillations**, whereas Bax and Bak can destabilize the ER membrane, facilitating Ca²⁺ release and contributing to cell death pathways. An intriguing aspect of this interaction is how Bcl-2 and Bcl-XL bind to IP3R and whether **Bcl-2 inhibitors could disrupt this association**.
According to a recent report by Yang et al., the **hydrophobic groove** of anti-apoptotic proteins may serve as the binding site for IP3R. However, earlier findings suggested that this interaction was mediated by the **BH4 domain** of Bcl-2 and Bcl-XL. Further research is necessary to clarify the precise binding mechanisms and their potential implications for therapeutic interventions targeting Bcl-2 proteins.
Autophagy, a fundamental cellular process, serves as a self-degradative mechanism activated in response to various stress conditions. While generally considered a pro-survival pathway, excessive or dysregulated autophagy can paradoxically lead to cell death through a non-apoptotic mechanism termed “autophagic cell death.” However, the precise definition and independent existence of this specific cell death modality remain subjects of ongoing debate within the scientific community. Some researchers prefer the more nuanced term “death with autophagy,” emphasizing the observed correlation between autophagy and cell death without necessarily implying a direct causal relationship.
The Bcl-2 family of proteins, traditionally recognized for their roles in regulating apoptosis, has also been implicated in the modulation of autophagy, providing a crucial link between these two cellular processes. Both Bcl-2 and Bcl-XL, anti-apoptotic members of this family, have been shown to interact with Beclin-1, a key protein involved in the initiation and execution of autophagy. Beclin-1 possesses a BH3 domain, a structural motif that allows it to interact with Bcl-2 family members. Consequently, these anti-apoptotic proteins can also function as inhibitors of autophagy by sequestering Beclin-1.
Under conditions of cellular stress, such as nutrient deprivation (starvation), signaling pathways are activated that lead to the dissociation of Bcl-2/Beclin-1 complexes. Specifically, the c-Jun N-terminal kinase (JNK) pathway mediates the phosphorylation of Bcl-2, triggering its release from Beclin-1 and subsequently initiating autophagy. Similarly, phosphorylation of specific residues within Beclin-1 itself can also disrupt its interaction with Bcl-2 or Bcl-XL, leading to the induction of autophagy. This dual functionality of Bcl-2 and Bcl-XL, acting as both apoptosis regulators and autophagy modulators, can explain the frequent observation of autophagy in various models of apoptosis induction.
The precise role of autophagy in cell death, whether it promotes or hinders it, remains a complex and controversial issue. In the majority of studies, pharmacological or genetic inhibition of autophagy has been observed to accelerate cell death, suggesting a predominant pro-survival role for this process. This implies that in many situations, autophagy acts to protect cells from stress-induced damage.
However, the interplay between apoptosis and autophagy is further complicated by observations made when the mitochondrial pathway of apoptosis is disrupted. In such scenarios, overexpression of Bcl-2 or Bcl-XL fails to significantly affect autophagy. This suggests that the apparent influence of these pro-survival proteins on autophagy may be indirect, potentially mediated through their effects on apoptosis signaling.
Adding another layer of complexity, the pro-apoptotic protein Bim has also been shown to act as an inhibitor of autophagy by directly binding to Beclin-1. This observation underscores the intricate and multifaceted nature of the relationship between apoptosis and autophagy, highlighting the need for further research to fully elucidate the mechanisms governing these essential cellular processes.
For a considerable period, necrosis was perceived as an uncontrolled and haphazard process of cellular demise. However, recent advancements in cellular biology have revealed a distinct form of regulated necrosis, termed necroptosis. Necroptosis, unlike classical necrosis, is a precisely orchestrated cell death pathway, predominantly triggered by the tumor necrosis factor-alpha receptor 1 (TNFR1). Nevertheless, alternative inducers, such as the chemotherapeutic agent etoposide and demethylating compounds, have also been implicated in the activation of this specific mode of cell death.
Two distinct protein complexes have been identified as key mediators of necrotic cell death: the “necrosome” and the “ripoptosome.” According to Tenev et al., the ripoptosome-mediated necroptosis pathway operates independently of the Bcl-2 family of proteins and does not necessitate mitochondrial involvement. This perspective suggests a divergence from traditional apoptosis, where Bcl-2 family members and mitochondria play pivotal roles.
However, the precise role of mitochondria in necroptosis remains a subject of debate. Mitochondrial permeabilization has been proposed as a crucial step in apoptosis-inducing factor (AIF)-mediated necroptosis, suggesting a potential mitochondrial contribution to this process. Furthermore, both Bax and Bak, pro-apoptotic members of the Bcl-2 family, have been implicated in TNF-α/Z-VAD-fmk-induced necroptosis in mouse fibroblasts, further complicating the picture. More recently, studies have demonstrated that Smac mimetics in conjunction with dexamethasone can induce Bak-dependent necroptosis, highlighting the intricate involvement of Bcl-2 family members in this form of regulated cell death.
Beyond their well-established roles in apoptosis and potentially necroptosis, Bcl-2 and Bcl-XL have also been implicated in the regulation of cell cycle progression. Specifically, these anti-apoptotic proteins appear to play a role in delaying the transition from the G0/G1 phase to the S phase, thereby influencing cell cycle entry. Consistently, the loss of Bcl-2 expression has been associated with increased proliferation rates in breast cancer cells, reinforcing the notion that Bcl-2 can exert anti-proliferative effects.
The cell cycle regulatory functions of Bcl-2 and Bcl-XL can be counteracted by the pro-apoptotic BH3-only protein Bad, suggesting a dynamic interplay between pro-survival and pro-death members of the Bcl-2 family in cell cycle control. Notably, the cell cycle regulatory role of Bcl-2 and Bcl-XL appears to be independent of their anti-apoptotic activity. Mutational analyses have pinpointed the unstructured BH3-BH4 loop and the BH4 domain as critical regions involved in cell cycle regulation, further distinguishing this function from their apoptotic roles.
The anti-proliferative effect of Bcl-2 can be abrogated during tumor progression due to the accumulation of additional genetic alterations. Conversely, certain pro-death proteins of the Bcl-2 family have been shown to paradoxically increase proliferation rates, highlighting the complex and sometimes counterintuitive roles of these proteins in cellular regulation. This emphasizes the need for careful consideration of context-dependent effects when studying these proteins.
Role of Bcl-2 family proteins in cancer
A critical characteristic of tumor cells is their ability to evade apoptosis, the programmed cell death mechanism that normally eliminates damaged or unwanted cells. This evasion is a fundamental contributor to the uncontrolled growth and survival of malignancies. The molecular mechanisms underlying this resistance to apoptosis are multifaceted and encompass a wide range of alterations, with significant emphasis on changes in the expression and functional activity of Bcl-2 family proteins.
The discovery of Bcl-2 itself stemmed from its observed overexpression in a substantial proportion of B-cell lymphomas, highlighting its crucial role in the pathogenesis of these cancers. Furthermore, elevated levels of Bcl-2 have been detected in neoplasms originating from diverse tissue types, underscoring its broad relevance in tumorigenesis. Similarly, Bcl-XL, another prominent pro-survival member of the Bcl-2 family, has been shown to be overexpressed in a wide array of solid tumors, further reinforcing the importance of these proteins in cancer survival. Mcl-1, an additional pro-survival protein, is also frequently upregulated in numerous cancer types, notably in multiple myeloma, where it plays a particularly critical role.
As previously discussed, pro-survival proteins of the Bcl-2 family can exert an inhibitory effect on cell proliferation. This observation helps explain why overexpression of Bcl-2 or other members of this family, in isolation, may not be sufficient to induce tumorigenesis. The development of cancer typically requires the accumulation of multiple genetic alterations that drive uncontrolled growth and survival.
However, the survival of established cancer cells often becomes critically dependent on the sustained expression of anti-apoptotic proteins from the Bcl-2 family. These cancer cells become “addicted” to these survival signals. For instance, myeloma cells rapidly succumb to cell death when Mcl-1 expression is disrupted, either through genetic ablation using CRISPR-Cas9 technology or through pharmacological inhibition with Mcl-1-specific inhibitors. This dependency underscores the critical role of these proteins in maintaining the viability of cancer cells.
This heightened reliance on anti-apoptotic Bcl-2 proteins for survival is frequently a consequence of the concurrent expression of pro-apoptotic members of the same family. These pro-apoptotic proteins “prime” the cells for death, rendering them highly sensitive to apoptotic triggers. In the absence of death-inducing signals, interactions between pro-survival and pro-death proteins effectively prevent the activation of the mitochondrial apoptotic pathway. However, when the apoptotic machinery is engaged, the pro-apoptotic proteins are released, triggering mitochondrial outer membrane permeabilization and the subsequent activation of caspases, the executioner enzymes of apoptosis. This balance between pro-survival and pro-death signals dictates the fate of cancer cells, making the Bcl-2 family a crucial target for therapeutic intervention.
A critical question in the context of cancer treatment revolves around the influence of Bcl-2 family proteins on the efficacy of chemotherapy. Given that numerous antitumor agents exert their cytotoxic effects by triggering cell death through the intrinsic, or mitochondrial, pathway of apoptosis, understanding this interplay is paramount.
To address this, Letai and colleagues have developed a technique known as “BH3 profiling,” which serves as a predictive tool for assessing cellular susceptibility to apoptosis. This assay involves isolating mitochondria from cells and exposing them to a panel of different BH3 peptides, which are short sequences derived from pro-apoptotic proteins. The subsequent release of cytochrome c from the mitochondria is then measured. Cytochrome c release is a key event in the initiation of the mitochondrial apoptotic pathway, providing a direct measure of mitochondrial permeability and thus the propensity for apoptosis.
This in vitro system enables researchers to determine which specific anti-apoptotic protein within the Bcl-2 family a given tumor cell is most “addicted” to for survival. In other words, it reveals which pro-survival protein is most critical for preventing apoptosis in those specific cells. The clinical relevance of BH3 profiling is underscored by its successful application using mitochondria isolated directly from patient tumor cells, demonstrating its potential for personalized medicine.
In practical terms, BH3 profiling offers a powerful tool for the development of novel therapeutic strategies. By determining the “BH3 profile” of a particular tumor, clinicians can identify the specific pro-survival Bcl-2 family protein that is essential for the survival of that tumor. This information can then be used to guide the selection of targeted therapies, such as BH3 mimetics, which are drugs designed to specifically inhibit the function of these pro-survival proteins. This personalized approach holds the promise of improving treatment outcomes by tailoring therapy to the unique vulnerabilities of individual tumors.
Development of BH3 mimetics
Given the pivotal role of pro-survival Bcl-2 proteins in both the progression of cancer and the development of resistance to therapy, there has been a significant and sustained effort to develop therapeutic strategies aimed at modulating their activity. Following the structural elucidation of the intricate interactions between pro-apoptotic and anti-apoptotic proteins, researchers have focused on designing molecules that functionally mimic the BH3 domain, a critical binding motif.
This endeavor has led to the development of BH3 mimetic compounds, a novel class of inhibitors that target pro-survival Bcl-2 proteins. These mimetics can be broadly categorized into two main types: peptides modeled on BH3 domains and non-peptidic synthetic small molecules. The synthesis of hydrocarbon-stapled BH3 peptides (SAHBs), such as Mcl-1 BH3 SAHB, Noxa SAHB, SAHBA (based on the BH3-only protein Bid), and 072RB (based on the BH3 region of Bim), has provided valuable tools for biomedical research. These peptides serve as invaluable tools for studying the nature of protein-protein interactions and as prototypes for the development of novel therapeutics.
Building upon these foundational studies, researchers have identified a diverse array of non-peptidic synthetic small-molecule inhibitors of pro-survival Bcl-2 proteins. This identification has been accomplished through extensive screening of chemical libraries, encompassing both natural products and computer-designed molecules, to assess their binding affinities for these target proteins.
Among the most advanced and well-characterized small-molecule BH3 mimetics that have progressed into clinical trials are ABT-737 and its analogs, obatoclax, and gossypol and its derivatives. These compounds exhibit significant variations in several key aspects, including their specificity for different Bcl-2 family members, their underlying mechanisms of action, and their observed anticancer efficacy. These differences are explored in greater detail in the subsequent sections, highlighting the nuances of this therapeutic class.
Obatoclax
Obatoclax (GX15-070) is a synthetic indolyl-dipyrromethene compound developed through meticulous structure-activity relationship studies, focusing on the pyrrolic ring A of the bacterial prodiginine family. Prodiginines are a group of naturally occurring red-pigmented secondary metabolites, characterized by a common pyrrolylpyrromethene skeleton that includes a 4-methoxy, 2-2 bi-pyrrole ring system. These compounds exhibit a wide range of biological activities, including immunosuppressive and anticancer properties.
Notably, prodiginines have been identified as potent pro-apoptotic agents, exerting their effects through interactions with multiple cellular targets. Obatoclax was rationally designed based on the observation that streptorubin B, a cyclic member of the prodiginine family, enhances apoptotic signaling in cancer cells by interacting with mitochondrial Bcl-2 proteins.
Obatoclax is a pan-Bcl-2 inhibitor, meaning it can bind to all pro-survival members of the Bcl-2 family. However, it exhibits relatively low affinity, operating in the submicromolar range with a Ki value of 220 nM. The hydrophobic nature of obatoclax is believed to play a critical role in its localization to the mitochondrial membrane, facilitating its interactions with the resident Bcl-2 proteins.
As a single agent, obatoclax demonstrates significant anticancer activity in cell lines derived from patients with various hematologic malignancies and solid tumors. It has also shown promise in suppressing tumor growth in xenograft models of small cell lung cancer (SCLC), thyroid cancer, and colorectal cancer, often exhibiting additive efficacy when used in combination with cytotoxic agents.
Obatoclax has undergone numerous phase I and II clinical trials, both as a monotherapy and in combination with chemotherapy, for a range of cancers including SCLC, non-small cell lung carcinoma (NSCLC), Hodgkin’s lymphoma, mantle cell lymphoma (MCL), chronic lymphocytic leukemia (CLL), and acute myeloid leukemia (AML). However, these trials have generally yielded modest efficacy results.
Central nervous system (CNS)-related side effects, such as ataxia, somnolence, euphoria, and confusion, have been identified as the most common adverse events associated with obatoclax. The underlying mechanisms responsible for these neurological symptoms remain unclear. Currently, there are no active clinical trials investigating the use of obatoclax in hematological malignancies or solid tumors.
More recently, prodigiosin (2-methyl-3-pentyl-6-methoxyprodigiosene), a natural compound belonging to the prodiginine family, has emerged as a potential new BH3 mimetic molecule. Studies have shown that Mcl-1 is a molecular target of prodigiosin, contributing to its cytotoxic effects. Similar to obatoclax, prodigiosin binds to a specific region within the hydrophobic groove of the Mcl-1 BH3 domain, effectively antagonizing Mcl-1 function. This suggests that prodigiosin may serve as an attractive lead compound for the development of more specific and potent Bcl-2 inhibitors.
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On-target effect of BH3 mimetics
BH3 mimetics have been rationally designed to directly activate the mitochondrial pathway of apoptosis in malignant cells. Lessen et al. have proposed two key criteria for defining genuine inhibitors of Bcl-2 family proteins: high-affinity binding to their targets (in the nanomolar range) and the ability to induce Bax/Bak-dependent apoptosis.
In accordance with these criteria, ABT-737, obatoclax, and gossypol have been shown to disrupt the interactions between Bcl-2/Bcl-XL and pro-death proteins, thereby initiating the intrinsic apoptotic pathway. Furthermore, ABT-737 and obatoclax have been observed to act as “sensitizers” to apoptotic signals. These compounds displace Bim from the BH3-binding hydrophobic groove of Bcl-2, allowing Bim to activate Bax, induce mitochondrial permeabilization, and ultimately commit cancer cells to death.
Cell-based protein redistribution assays have revealed that ABT-737 preferentially disrupts Bcl-2/Bim complexes compared to Bcl-XL/Bim or Bcl-w/Bim. Mechanistic studies using mitochondria isolated from cancer cells have further demonstrated that ABT-737 induces the desequestration of Bax, Bak, and Bim from Bcl-2 and Bcl-XL, but not from Mcl-1.
Cancer cells expressing high levels of Mcl-1 have been shown to exhibit intrinsic resistance to ABT-737. However, more recent research indicates that not only Mcl-1 expression but also, critically, Noxa/Mcl-1 interactions determine cancer cell sensitivity to ABT-737. Therefore, strategies aimed at upregulating Noxa and/or inhibiting Mcl-1 may be crucial for enhancing the therapeutic efficacy of ABT-737 against cancer.
Indeed, combinations of ABT-737 or its analogs with compounds that indirectly suppress Mcl-1 activity and/or activate Noxa, such as vorinostat, vinblastine, bortezomib, and the kinase inhibitor dinaciclib, have been shown to overcome cancer cell resistance to ABT-737 and improve its therapeutic efficacy. The combination of ABT-737 with specific inhibitors of Mcl-1 also holds considerable promise.
Recently, a small-molecule Mcl-1 inhibitor, designated S63845, has been developed. This novel BH3 mimetic exhibits high specificity for Mcl-1 and demonstrates both in vitro and in vivo antitumor activity against a variety of cancer types.
It is important to acknowledge that cancer cells initially responsive to ABT-737 can develop resistance through the upregulation of Mcl-1 levels. In vitro studies have demonstrated that post-translational modifications, such as the phosphorylation of Mcl-1 at specific residues, are crucial for its stability and its ability to bind BH3-only proteins.
Specifically, it has been observed that prolonged exposure of cancer cells to ABT-737 results in the phosphorylation of Mcl-1 at Ser-64, facilitating its stabilization and interaction with Bim. Consequently, Bim, which is displaced from Bcl-2/Bcl-XL in response to ABT-737, is sequestered by Mcl-1, thereby preventing the activation of apoptosis.
The priming of Bcl-2 proteins by BH3-only proteins, such as Bim, is a critical determinant of cancer cell sensitivity to BH3 mimetics. Conversely, decreased Bim expression can lead to a poor therapeutic response. Therefore, combining BH3 mimetics with agents that induce Bim expression, such as kinase inhibitors, histone deacetylase inhibitors, or topoisomerase inhibitors, may enhance cancer cell killing and circumvent resistance mechanisms.
Overall, the mechanisms of resistance to BH3 mimetics, whether intrinsic or acquired, have significant implications for both patient selection and the development of strategies to overcome this resistance.
In a study by Vogler et al., it was shown that among six Bcl-2 inhibitors, including ABT-737, obatoclax, and gossypol, only ABT-737 failed to induce cell death in Bax/Bak double knockout cells. Apoptosis induced by obatoclax and gossypol was diminished, but not completely abolished, in the absence of Bax/Bak, suggesting that these agents may target additional cellular components to activate the mitochondrial pathway. Thus, ABT-737 appeared to be a more authentic BH3 mimetic, while obatoclax and gossypol might be better classified as putative pan-Bcl-2 inhibitors.
However, evidence suggests that ABT-737 can also induce apoptosis through mechanisms independent of the established BH3 sensitizer or effector models that modulate Bcl-2 family protein interactions.
Notably, a recent report by Shin et al. demonstrated that ABT-737′s effects on human oral cancer cells may be attributed to the regulation of Bim levels through modulation of the ERK1/2 signaling pathway, in a cell type-dependent manner. Furthermore, ABT-737 has been shown to activate JNK and its downstream target c-Jun, leading to the upregulation of Bim expression in HeLa cells. These findings indicate that the mechanisms of action of BH3 mimetics can be more complex than previously thought.
Cell cycle disturbance
Recent studies have unveiled that BH3 mimetics, in addition to their pro-apoptotic activity, also exhibit anti-proliferative effects. At growth-inhibitory doses that did not induce apoptosis, obatoclax caused an S-G2 cell-cycle block in acute myeloid leukemia (AML) cells.
In mixed-lineage leukemia-AF4 acute lymphoblastic leukemia (MLL-AF4 ALL) cell lines SEM-K2 and RS4:11, obatoclax had variable effects on the cell cycle. Obatoclax induced a time- and dose-dependent increase in the percentage of SEM-K2 cells in the S-phase, whereas the cell cycle was not altered in RS4:11 cells.
Other studies have demonstrated that obatoclax induced G1/G0-phase cell cycle arrest in colorectal, esophageal, and thyroid cancer cells. Additionally, Zhang et al. have suggested that obatoclax induced cell cycle arrest via the p38/p21(waf1/Cip1) signaling pathway. Similarly, Koehler et al. have shown that colorectal cancer cell cycle arrest by obatoclax was accompanied by a downregulation of cyclin D1 and upregulation of p27 and p21.
This effect was not antagonized by overexpression of pro-survival Bcl-2 proteins. The efficacy of obatoclax on colorectal cancer cells also included Bcl-2-dependent inhibition of cell migration. Interestingly, the same report showed that, in contrast to obatoclax, ABT-737 treatment was not sufficient to block the cell cycle or migration of colorectal cancer cells.
Conversely, exposure of apoptosis-resistant renal, lung, and prostate cancer cell lines to ABT-737 caused significant growth inhibition and a G1/S cell cycle blockade with the induction of p53-dependent cellular senescence. Moreover, ABT-737 and obatoclax reduced cell migration of hepatoblastoma and hepatocellular carcinoma.
In some recent reports, the anti-proliferative activity of BH3 mimetics was found to be associated with an increased production of reactive oxygen species (ROS). Other findings indicated that ApoG2 could potently disturb proliferation of nasopharyngeal carcinoma cells by suppressing the c-Myc signaling pathway.
Whether the inhibition of Bcl-2 protein function in cell cycle progression is involved in the anti-proliferative effects of BH3 mimetics remains an open question. It is worth noting that the effect of BH3 mimetics on the cell cycle has implications for BH3 mimetic-chemotherapy combinations because many chemotherapeutic agents are cell cycle-dependent, and the cell cycle also determines cancer cell sensitivity to therapeutic drugs. Therefore, understanding how BH3 mimetics affect the cell cycle is an important issue to evaluate.
Challenges and future perspectives in BH3 mimetic development
The past decade has witnessed remarkable progress in the development of Bcl-2 inhibitors. Numerous BH3 mimetics have been identified, and several are currently undergoing clinical evaluation. While significant effort has been dedicated to elucidating their pro-apoptotic activity in cancer cells, a comprehensive understanding of their precise mechanisms of action remains elusive. Recent studies have revealed that, beyond inducing the mitochondrial apoptotic pathway, BH3 mimetics can influence other cellular functions, including the endoplasmic reticulum (ER) stress response, autophagy, necrotic cell death, and cell proliferation.
These findings have prompted researchers to categorize BH3 mimetics as authentic or putative based on their binding affinity to Bcl-2 proteins and their off-target effects. However, it is crucial to recognize that Bcl-2 family proteins are involved in a multitude of physiological functions beyond the regulation of apoptosis. Therefore, the inhibition of both canonical and non-canonical functions of Bcl-2 family proteins by BH3 mimetics may contribute to the observed responses of cancer cells to these compounds.
This issue is particularly challenging due to the complexity of interactions within the Bcl-2 family network and the inherent heterogeneity of cancer cells. The underlying mechanisms of cancer cell response to BH3 mimetic treatment may vary depending on the specific genetic and cellular context. Importantly, both Bcl-2-dependent and independent targets of BH3 mimetics have clinical applicability. Research that provides further insights into the nature of interactions between BH3 mimetics and their targets is eagerly anticipated.
Consequently, several key questions need to be addressed to advance the development of BH3 mimetics. Firstly, what is the relationship between BH3 mimetic selectivity and therapeutic efficacy? It would be valuable to determine whether highly selective or pan-BH3 mimetics offer a greater clinical advantage. A variety of pan-Bcl-2 inhibitors, as well as selective inhibitors of Bcl-2, Bcl-XL, or Mcl-1, are currently under development. Maritoclax, MIM-1, S63485, and A-1210477 are examples of compounds designed to specifically inhibit Mcl-1. Other selective inhibitors, such as WEHI-539, A-1155463, and BXI compounds, are highly selective antagonists of Bcl-XL.
When considering selectivity, the potential in vivo toxicities of BH3 mimetics and the development of cancer resistance to these compounds are critical factors that need to be carefully evaluated.
Secondly, a critical question arises regarding the translatability of results obtained from in vitro and in vivo studies to clinical practice. Identifying patient populations that are most likely to benefit from BH3 mimetic therapy poses a significant challenge for future research. In this context, the application of validated biomarkers is of paramount importance for the success of this therapeutic strategy.
Finally, how can undesired toxicities and cancer resistance to BH3 mimetics be effectively overcome? Given this challenge, the pursuit of optimal therapeutic regimens and rational combination treatments involving BH3 mimetics is crucial from a clinical perspective. Bcl-2 inhibitors are designed to restore sensitivity to apoptosis in cancer cells and lower their apoptotic threshold. In this regard, BH3 mimetics may offer the advantage of reduced normal tissue toxicity compared to conventional anticancer therapies that interfere with general mechanisms such as DNA synthesis, mitosis, and tyrosine kinase activity.
Despite the remaining uncertainties, BH3 mimetics have emerged as promising therapeutic agents, capable of overcoming apoptotic resistance in cancers and improving patient outcomes. Their potential role in targeted cancer therapy warrants further exploration. GX15-070