Key residues in the VDAC2-BAK complex can be targeted to modulate apoptosis.

BAK and BAX execute intrinsic apoptosis by permeabilising the mitochondrial outer membrane. Their activity is regulated through interactions with pro-survival BCL-2 family proteins and with non-BCL-2 proteins including the mitochondrial channel protein VDAC2. VDAC2 is important for bringing both BAK and BAX to mitochondria where they execute their apoptotic function. Despite this important function in apoptosis, while interactions with pro-survival family members are well characterised and have culminated in the development of drugs that target these interfaces to induce cancer cell apoptosis, the interaction between BAK and VDAC2 remains largely undefined. Deep scanning mutagenesis coupled with cysteine linkage identified key residues in the interaction between BAK and VDAC2. Obstructive labelling of specific residues in the BH3 domain or hydrophobic groove of BAK disrupted this interaction. Conversely, mutating specific residues in a cytosol-exposed region of VDAC2 stabilised the interaction with BAK and inhibited BAK apoptotic activity. Thus, this VDAC2-BAK interaction site can potentially be targeted to either inhibit BAK-mediated apoptosis in scenarios where excessive apoptosis contributes to disease or to promote BAK-mediated apoptosis for cancer therapy.

Improved drug target deconvolution with PISA-DIA using an extended, overlapping temperature gradient.

Thermal proteome profiling (TPP) is a powerful tool for drug target deconvolution. Recently, data-independent acquisition mass spectrometry (DIA-MS) approaches have demonstrated significant improvements to depth and missingness in proteome data, but traditional TPP (a.k.a. CEllular Thermal Shift Assay "CETSA") workflows typically employ multiplexing reagents reliant on data-dependent acquisition (DDA). Herein, we introduce a new experimental design for the Proteome Integral Solubility Alteration via label-free DIA approach (PISA-DIA). We highlight the proteome coverage and sensitivity achieved by using multiple overlapping thermal gradients alongside DIA-MS, which maximizes efficiencies in PISA sample concatenation and safeguards against missing protein targets that exist at high melting temperatures. We demonstrate our extended PISA-DIA design has superior proteome coverage as compared to using tandem-mass tags (TMT) necessitating DDA-MS analysis. Importantly, we demonstrate our PISA-DIA approach has the quantitative and statistical rigor using A-1331852, a specific inhibitor of BCL-xL. Due to the high melt temperature of this protein target, we utilized our extended multiple gradient PISA-DIA workflow to identify BCL-xL. We assert our novel overlapping gradient PISA-DIA-MS approach is ideal for unbiased drug target deconvolution, spanning a large temperature range whilst minimizing target dropout between gradients, increasing the likelihood of resolving the protein targets of novel compounds.

The VEGFR/PDGFR tyrosine kinase inhibitor, ABT-869, blocks necroptosis by targeting RIPK1 kinase.

Necroptosis is a mode of programmed, lytic cell death that is executed by the mixed lineage kinase domain-like (MLKL) pseudokinase following activation by the upstream kinases, receptor-interacting serine/threonine protein kinase (RIPK)-1 and RIPK3. Dysregulated necroptosis has been implicated in the pathophysiology of many human diseases, including inflammatory and degenerative conditions, infectious diseases and cancers, provoking interest in pharmacological targeting of the pathway. To identify small molecules impacting on the necroptotic machinery, we performed a phenotypic screen using a mouse cell line expressing an MLKL mutant that kills cells in the absence of upstream death or pathogen detector receptor activation. This screen identified the vascular endothelial growth factor receptor (VEGFR) and platelet-derived growth factor receptor (PDGFR) tyrosine kinase inhibitor, ABT-869 (Linifanib), as a small molecule inhibitor of necroptosis. We applied a suite of cellular, biochemical and biophysical analyses to pinpoint the apical necroptotic kinase, RIPK1, as the target of ABT-869 inhibition. Our study adds to the repertoire of established protein kinase inhibitors that additionally target RIPK1 and raises the prospect that serendipitous targeting of necroptosis signalling may contribute to their clinical efficacy in some settings.

Mitochondrial E3 ubiquitin ligase MARCHF5 controls BAK apoptotic activity independently of BH3-only proteins.

Intrinsic apoptosis is principally governed by the BCL-2 family of proteins, but some non-BCL-2 proteins are also critical to control this process. To identify novel apoptosis regulators, we performed a genome-wide CRISPR-Cas9 library screen, and it identified the mitochondrial E3 ubiquitin ligase MARCHF5/MITOL/RNF153 as an important regulator of BAK apoptotic function. Deleting MARCHF5 in diverse cell lines dependent on BAK conferred profound resistance to BH3-mimetic drugs. The loss of MARCHF5 or its E3 ubiquitin ligase activity surprisingly drove BAK to adopt an activated conformation, with resistance to BH3-mimetics afforded by the formation of inhibitory complexes with pro-survival proteins MCL-1 and BCL-XL. Importantly, these changes to BAK conformation and pro-survival association occurred independently of BH3-only proteins and influence on pro-survival proteins. This study identifies a new mechanism by which MARCHF5 regulates apoptotic cell death by restraining BAK activating conformation change and provides new insight into how cancer cells respond to BH3-mimetic drugs. These data also highlight the emerging role of ubiquitin signalling in apoptosis that may be exploited therapeutically.

The Lck inhibitor, AMG-47a, blocks necroptosis and implicates RIPK1 in signalling downstream of MLKL.

Necroptosis is a form of caspase-independent programmed cell death that arises from disruption of cell membranes by the mixed lineage kinase domain-like (MLKL) pseudokinase after its activation by the upstream kinases, receptor interacting protein kinase (RIPK)-1 and RIPK3, within a complex known as the necrosome. Dysregulated necroptosis has been implicated in numerous inflammatory pathologies. As such, new small molecule necroptosis inhibitors are of great interest, particularly ones that operate downstream of MLKL activation, where the pathway is less well defined. To better understand the mechanisms involved in necroptosis downstream of MLKL activation, and potentially uncover new targets for inhibition, we screened known kinase inhibitors against an activated mouse MLKL mutant, leading us to identify the lymphocyte-specific protein tyrosine kinase (Lck) inhibitor AMG-47a as an inhibitor of necroptosis. We show that AMG-47a interacts with both RIPK1 and RIPK3, that its ability to protect from cell death is dependent on the strength of the necroptotic stimulus, and that it blocks necroptosis most effectively in human cells. Moreover, in human cell lines, we demonstrate that AMG-47a can protect against cell death caused by forced dimerisation of MLKL truncation mutants in the absence of any upstream signalling, validating that it targets a process downstream of MLKL activation. Surprisingly, however, we also found that the cell death driven by activated MLKL in this model was completely dependent on the presence of RIPK1, and to a lesser extent RIPK3, although it was not affected by known inhibitors of these kinases. Together, these results suggest an additional role for RIPK1, or the necrosome, in mediating human necroptosis after MLKL is phosphorylated by RIPK3 and provide further insight into reported differences in the progression of necroptosis between mouse and human cells.

Too much death can kill you: inhibiting intrinsic apoptosis to treat disease.

Apoptotic cell death is implicated in both physiological and pathological processes. Since many types of cancerous cells intrinsically evade apoptotic elimination, induction of apoptosis has become an attractive and often necessary cancer therapeutic approach. Conversely, some cells are extremely sensitive to apoptotic stimuli leading to neurodegenerative disease and immune pathologies. However, due to several challenges, pharmacological inhibition of apoptosis is still only a recently emerging strategy to combat pathological cell loss. Here, we describe several key steps in the intrinsic (mitochondrial) apoptosis pathway that represent potential targets for inhibitors in disease contexts. We also discuss the mechanisms of action, advantages and limitations of small-molecule and peptide-based inhibitors that have been developed to date. These inhibitors serve as important research tools to dissect apoptotic signalling and may foster new treatments to reduce unwanted cell loss.

Potent Inhibition of Necroptosis by Simultaneously Targeting Multiple Effectors of the Pathway.

Necroptosis is an inflammatory form of programmed cell death that has been implicated in various human diseases. Compound 2 is a more potent analogue of the published compound 1 and inhibits necroptosis in human and murine cells at nanomolar concentrations. Several target engagement strategies were employed, including cellular thermal shift assays (CETSA) and diazirine-mediated photoaffinity labeling via a bifunctional photoaffinity probe derived from compound 2. These target engagement studies demonstrate that compound 2 binds to all three necroptotic effector proteins (mixed lineage kinase domain-like protein (MLKL), receptor-interacting serine/threonine protein kinase 1 (RIPK1) and receptor-interacting serine/threonine protein kinase 3 (RIPK3)) at different levels in vitro and in cells. Compound 2 also shows efficacy in vivo in a murine model of systemic inflammatory response syndrome (SIRS).

MARCH5 requires MTCH2 to coordinate proteasomal turnover of the MCL1:NOXA complex.

MCL1, a BCL2 relative, is critical for the survival of many cells. Its turnover is often tightly controlled through both ubiquitin-dependent and -independent mechanisms of proteasomal degradation. Several cell stress signals, including DNA damage and cell cycle arrest, are known to elicit distinct E3 ligases to ubiquitinate and degrade MCL1. Another trigger that drives MCL1 degradation is engagement by NOXA, one of its BH3-only protein ligands, but the mechanism responsible has remained unclear. From an unbiased genome-wide CRISPR-Cas9 screen, we discovered that the ubiquitin E3 ligase MARCH5, the ubiquitin E2 conjugating enzyme UBE2K, and the mitochondrial outer membrane protein MTCH2 co-operate to mark MCL1 for degradation by the proteasome-specifically when MCL1 is engaged by NOXA. This mechanism of degradation also required the MCL1 transmembrane domain and distinct MCL1 lysine residues to proceed, suggesting that the components likely act on the MCL1:NOXA complex by associating with it in a specific orientation within the mitochondrial outer membrane. MTCH2 has not previously been reported to regulate protein stability, but is known to influence the mitochondrial localization of certain key apoptosis regulators and to impact metabolism. We have now pinpointed an essential but previously unappreciated role for MTCH2 in turnover of the MCL1:NOXA complex by MARCH5, further strengthening its links to BCL2-regulated apoptosis.

A small molecule interacts with VDAC2 to block mouse BAK-driven apoptosis.

Activating the intrinsic apoptosis pathway with small molecules is now a clinically validated approach to cancer therapy. In contrast, blocking apoptosis to prevent the death of healthy cells in disease settings has not been achieved. Caspases have been favored, but they act too late in apoptosis to provide long-term protection. The critical step in committing a cell to death is activation of BAK or BAX, pro-death BCL-2 proteins mediating mitochondrial damage. Apoptosis cannot proceed in their absence. Here we show that WEHI-9625, a novel tricyclic sulfone small molecule, binds to VDAC2 and promotes its ability to inhibit apoptosis driven by mouse BAK. In contrast to caspase inhibitors, WEHI-9625 blocks apoptosis before mitochondrial damage, preserving cellular function and long-term clonogenic potential. Our findings expand on the key role of VDAC2 in regulating apoptosis and demonstrate that blocking apoptosis at an early stage is both advantageous and pharmacologically tractable.

VDAC2 enables BAX to mediate apoptosis and limit tumor development.

Intrinsic apoptosis is critical to prevent tumor formation and is engaged by many anti-cancer agents to eliminate tumor cells. BAX and BAK, the two essential mediators of apoptosis, are thought to be regulated through similar mechanisms and act redundantly to drive apoptotic cell death. From an unbiased genome-wide CRISPR/Cas9 screen, we identified VDAC2 (voltage-dependent anion channel 2) as important for BAX, but not BAK, to function. Genetic deletion of VDAC2 abrogated the association of BAX and BAK with mitochondrial complexes containing VDAC1, VDAC2, and VDAC3, but only inhibited BAX apoptotic function. Deleting VDAC2 phenocopied the loss of BAX in impairing both the killing of tumor cells by anti-cancer agents and the ability to suppress tumor formation. Together, our studies show that efficient BAX-mediated apoptosis depends on VDAC2, and reveal a striking difference in how BAX and BAK are functionally impacted by their interactions with VDAC2.

BAK/BAX macropores facilitate mitochondrial herniation and mtDNA efflux during apoptosis.

Mitochondrial apoptosis is mediated by BAK and BAX, two proteins that induce mitochondrial outer membrane permeabilization, leading to cytochrome c release and activation of apoptotic caspases. In the absence of active caspases, mitochondrial DNA (mtDNA) triggers the innate immune cGAS/STING pathway, causing dying cells to secrete type I interferon. How cGAS gains access to mtDNA remains unclear. We used live-cell lattice light-sheet microscopy to examine the mitochondrial network in mouse embryonic fibroblasts. We found that after BAK/BAX activation and cytochrome c loss, the mitochondrial network broke down and large BAK/BAX pores appeared in the outer membrane. These BAK/BAX macropores allowed the inner mitochondrial membrane to herniate into the cytosol, carrying with it mitochondrial matrix components, including the mitochondrial genome. Apoptotic caspases did not prevent herniation but dismantled the dying cell to suppress mtDNA-induced innate immune signaling.

BAK/BAX-Mediated Apoptosis Is a Myc-Induced Roadblock to Reprogramming.

Despite intensive efforts to optimize the process, reprogramming differentiated cells to induced pluripotent stem cells (iPSCs) remains inefficient. The most common combination of transcription factors employed comprises OCT4, KLF4, SOX2, and MYC (OKSM). If MYC is omitted (OKS), reprogramming efficiency is reduced further. Cells must overcome several obstacles to reach the pluripotent state, one of which is apoptosis. To directly determine how extensively apoptosis limits reprogramming, we exploited mouse embryonic fibroblasts (MEFs) lacking the two essential mediators of apoptosis, BAK and BAX. Our results show that reprogramming is enhanced in MEFs deficient in BAK and BAX, but only when MYC is part of the reprogramming cocktail. Thus, the propensity for Myc overexpression to elicit apoptosis creates a significant roadblock to reprogramming under OKSM conditions. Our results suggest that blocking apoptosis during reprogramming may enhance the derivation of iPSCs for research and therapeutic purposes.

Conversion of Bim-BH3 from Activator to Inhibitor of Bak through Structure-Based Design.

Certain BH3-only proteins transiently bind and activate Bak and Bax, initiating their oligomerization and the permeabilization of the mitochondrial outer membrane, a pivotal step in the mitochondrial pathway to apoptosis. Here we describe the first crystal structures of an activator BH3 peptide bound to Bak and illustrate their use in the design of BH3 derivatives capable of inhibiting human Bak on mitochondria. These BH3 derivatives compete for the activation site at the canonical groove, are the first engineered inhibitors of Bak activation, and support the role of key conformational transitions associated with Bak activation.

SMYD2 lysine methyltransferase regulates leukemia cell growth and regeneration after genotoxic stress.

The molecular determinants governing escape of Acute Myeloid Leukemia (AML) cells from DNA damaging therapy remain poorly defined and account for therapy failures. To isolate genes responsible for leukemia cells regeneration following multiple challenges with irradiation we performed a genome-wide shRNA screen. Some of the isolated hits are known players in the DNA damage response (e.g. p53, CHK2), whereas other, e.g. SMYD2 lysine methyltransferase (KMT), remains uncharacterized in the AML context. Here we report that SMYD2 knockdown confers relative resistance to human AML cells against multiple classes of DNA damaging agents. Induction of the transient quiescence state upon SMYD2 downregulation correlated with the resistance. We revealed that diminished SMYD2 expression resulted in the upregulation of the related methyltransferase SET7/9, suggesting compensatory relationships. Indeed, pharmacological targeting of SET7/9 with (R)-PFI2 inhibitor preferentially inhibited the growth of cells expressing low levels of SMYD2.Finally, decreased expression of SMYD2 in AML patients correlated with the reduced sensitivity to therapy and lower probability to achieve complete remission. We propose that the interplay between SMYD2 and SET7/9 levels shifts leukemia cells from growth to quiescence state that is associated with the higher resistance to DNA damaging agents and rationalize SET7/9 pharmacological targeting in AML.

Apoptotic caspases suppress mtDNA-induced STING-mediated type I IFN production.

Activated caspases are a hallmark of apoptosis induced by the intrinsic pathway, but they are dispensable for cell death and the apoptotic clearance of cells in vivo. This has led to the suggestion that caspases are activated not just to kill but to prevent dying cells from triggering a host immune response. Here, we show that the caspase cascade suppresses type I interferon (IFN) production by cells undergoing Bak/Bax-mediated apoptosis. Bak and Bax trigger the release of mitochondrial DNA. This is recognized by the cGAS/STING-dependent DNA sensing pathway, which initiates IFN production. Activated caspases attenuate this response. Pharmacological caspase inhibition or genetic deletion of caspase-9, Apaf-1, or caspase-3/7 causes dying cells to secrete IFN-ß. In vivo, this precipitates an elevation in IFN-ß levels and consequent hematopoietic stem cell dysfunction, which is corrected by loss of Bak and Bax. Thus, the apoptotic caspase cascade functions to render mitochondrial apoptosis immunologically silent.

Enhanced stability of Mcl1, a prosurvival Bcl2 relative, blunts stress-induced apoptosis, causes male sterility, and promotes tumorigenesis.

The B-cell CLL/lymphoma 2 (Bcl2) relative Myeloid cell leukemia sequence 1 (Mcl1) is essential for cell survival during development and for tissue homeostasis throughout life. Unlike Bcl2, Mcl1 turns over rapidly, but the physiological significance of its turnover has been unclear. We have gained insight into the roles of Mcl1 turnover in vivo by analyzing mice harboring a modified allele of Mcl1 that serendipitously proved to encode an abnormally stabilized form of Mcl1 due to a 13-aa N-terminal extension. Although the mice developed normally and appeared unremarkable, the homozygous males unexpectedly proved infertile due to defective spermatogenesis, which was evoked by enhanced Mcl1 prosurvival activity. Under unstressed conditions, the modified Mcl1 is present at levels comparable to the native protein, but it is markedly stabilized in cells subjected to stresses, such as protein synthesis inhibition or UV irradiation. Strikingly, the modified Mcl1 allele could genetically complement the loss of Bcl2, because introduction of even a single allele significantly ameliorated the severe polycystic kidney disease and consequent runting caused by Bcl2 loss. Significantly, the development of c-MYC-induced acute myeloid leukemia was also accelerated in mice harboring that Mcl1 allele. Our collective findings reveal that, under certain circumstances, the N terminus of Mcl1 regulates its degradation; that some cell types require degradation of Mcl1 to induce apoptosis; and, most importantly, that rapid turnover of Mcl1 can serve as a tumor-suppressive mechanism.

Discovery of potent and selective benzothiazole hydrazone inhibitors of Bcl-XL.

Developing potent molecules that inhibit Bcl-2 family mediated apoptosis affords opportunities to treat cancers via reactivation of the cell death machinery. We describe the hit-to-lead development of selective Bcl-XL inhibitors originating from a high-throughput screening campaign. Small structural changes to the hit compound increased binding affinity more than 300-fold (to IC50 < 20 nM). This molecular series exhibits drug-like characteristics, low molecular weights (Mw < 450), and unprecedented selectivity for Bcl-XL. Surface plasmon resonance experiments afford strong evidence of binding affinity within the hydrophobic groove of Bcl-XL. Biological experiments using engineered Mcl-1 deficient mouse embryonic fibroblasts (MEFs, reliant only on Bcl-XL for survival) and Bax/Bak deficient MEFs (insensitive to selective activation of Bcl-2-driven apoptosis) support a mechanism-based induction of apoptosis. This manuscript describes the first series of selective small-molecule inhibitors of Bcl-XL and provides promising leads for the development of efficacious therapeutics against solid tumors and chemoresistant cancer cell lines.

The dendritic cell receptor Clec9A binds damaged cells via exposed actin filaments.

The immune system must distinguish viable cells from cells damaged by physical and infective processes. The damaged cell-recognition molecule Clec9A is expressed on the surface of the mouse and human dendritic cell subsets specialized for the uptake and processing of material from dead cells. Clec9A recognizes a conserved component within nucleated and nonnucleated cells, exposed when cell membranes are damaged. We have identified this Clec9A ligand as a filamentous form of actin in association with particular actin-binding domains of cytoskeletal proteins. We have determined the crystal structure of the human CLEC9A C-type lectin domain and propose a functional dimeric structure with conserved tryptophans in the ligand recognition site. Mutation of these residues ablated CLEC9A binding to damaged cells and to the isolated ligand complexes. We propose that Clec9A provides targeted recruitment of the adaptive immune system during infection and can also be utilized to enhance immune responses generated by vaccines.

A novel BH3 ligand that selectively targets Mcl-1 reveals that apoptosis can proceed without Mcl-1 degradation.

Like Bcl-2, Mcl-1 is an important survival factor for many cancers, its expression contributing to chemoresistance and disease relapse. However, unlike other prosurvival Bcl-2-like proteins, Mcl-1 stability is acutely regulated. For example, the Bcl-2 homology 3 (BH3)-only protein Noxa, which preferentially binds to Mcl-1, also targets it for proteasomal degradation. In this paper, we describe the discovery and characterization of a novel BH3-like ligand derived from Bim, Bim(S)2A, which is highly selective for Mcl-1. Unlike Noxa, Bim(S)2A is unable to trigger Mcl-1 degradation, yet, like Noxa, Bim(S)2A promotes cell killing only when Bcl-x(L) is absent or neutralized. Furthermore, killing by endogenous Bim is not associated with Mcl-1 degradation. Thus, functional inactivation of Mcl-1 does not always require its elimination. Rather, it can be efficiently antagonized by a BH3-like ligand tightly engaging its binding groove, which is confirmed here with a structural study. Our data have important implications for the discovery of compounds that might kill cells whose survival depends on Mcl-1.