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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.

SUMMARYIntrinsic 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 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.

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.

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.

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.

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).

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.

Summary 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., Highlights • We generated MEFS lacking two essential mediators of apoptosis, BAK and BAX • Loss of BAK and BAX significantly enhances MEF reprogramming in the presence of MYC • Thus, mitochondrial apoptosis limits reprogramming of MEFs in the presence of MYC • The integrity of the genome is not reduced in reprogrammed MEFs lacking BAK and BAX, In this article, Heath, van Delft, and colleagues show that mitochondrial apoptosis limits OKSM-mediated reprogramming of MEFs. Not only is reprogramming of MEFs lacking the two essential mediators of mitochondrial apoptosis, BAK and BAX, significantly enhanced in the presence of MYC, but reprogramming in these conditions does not compromise genome integrity.

Intrinsic apoptosis is critical for normal physiology including the prevention of tumor formation. BAX and BAK are essential for mediating this process and for the cytotoxic action of many anticancer drugs. BAX and BAK are thought to act in a functionally redundant manner and are considered to be regulated similarly. From an unbiased genome-wide CRISPR/Cas9 screen, we identified VDAC2 (voltage-dependent anion channel 2) as essential for BAX, but not BAK, to function. The genetic deletion of VDAC2 abrogated the association of BAX and BAK with mitochondrial complexes that contain VDAC1, VDAC2 and VDAC3. By disrupting its localization to mitochondria, BAX is rendered completely ineffective. Moreover, we defined an interface unique to VDAC2 that is required to drive BAX activity. Consequently, interfering with this interaction or deleting VDAC2 phenocopied the loss of BAX, including impairing the killing of tumor cells by anti-cancer agents such as the BCL-2 inhibitor venetoclax. Furthermore, the ability of BAX to prevent tumor formation was attenuated in the absence of VDAC2. Taken together, our studies show for the first time that BAX-mediated apoptosis, but not BAK-mediated apoptosis, is critically dependent on VDAC2, hence revealing the differential regulation of BAX and BAK.

SummaryActivated 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-β. In vivo, this precipitates an elevation in IFN-β 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.

The transcription factor IRF4 is essential for the survival of both normal plasma cells and the plasma cell malignancy multiple myeloma (MM). However the basis for this dependency remains uncertain, with the prevailing view that IRF4 loss in MM induces "death by a thousand cuts." Here we have explored the genetic basis for IRF4 addiction through CRISPR-Cas9 mediated deletion of IRF4 in three lenalidomide sensitive MM cells lines (MM1S, OPM2 and H929) and one resistant cell line (RPMI-8226). Loss of IRF4 resulted in marked loss of viability, irrespective of lenalidomide sensitivity, which was driven by two distinct mechanisms: 1) induction of apoptosis, which was rescued through prior deletion of apoptotic mediators BAX and BAK, and 2) proliferative arrest, which persisted despite rescue from apoptosis. RNA-sequencing following IRF4 inactivation identified a core group of 86 genes whose dysregulation was shared across all 4 MM cell lines. We identified 31 common down-regulated genes (IRF4 activated), including several with previously described roles in survival (such as KLF2, TNFRSF17/BCMA and MYB). Furthermore, MYC, a previously identified target of IRF4 known to play an important role in MM pathogenesis and survival, was down-regulated in 3 of the 4 cell lines. Most surprisingly, IRF4 inactivation resulted in 55 common up-regulated genes that included two BH3-only pro-apoptotic proteins, BMF and BCL2L11 (BIM). Up-regulation of BMF appeared more marked than BIM with an average fold change of 6.5x (range 2.7 - 10.3) vs 1.9x (range: 1.8 - 2.2) respectively. Remarkably, genetic ablation of BMF in the OPM2 cell line and BMF/BIM in the MM1S largely protected from the cell death observed following IRF4 inactivation, suggesting that IRF4 maintains MM survival through the transcriptional repression of BMF and BIM. Our results confirm the vital role of IRF4 for MM proliferation and survival, and shed further light into its critical downstream transcriptional targets. Most interestingly, we have demonstrated that the 'killer blow' appears to be the concerted effect of the BH3-only pro-apoptotic proteins BMF and BIM. Disclosures Huang: Genentech: Patents & Royalties: DCSH is an employee of the Walter and Eliza Hall Institute which receives milestone and royalty payments related to venetoclax.

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.

// Adi Zipin-Roitman 1 , Nasma Aqaqe 1 , Muhammad Yassin 1 , Shahar Biechonski 1 , Mariam Amer 1 , Mark F. van Delft 2, 3 , Olga I. Gan 2, 8 , Sean P. McDermott 2, 4 , Alla Buzina 6, 8 , Troy Ketela 6, 8 , Liran Shlush 2, 5 , Stephanie Xie 2, 8 , Veronique Voisin 6, 8 , Jason Moffat 6, 8 , Mark D. Minden 7, 9 , John E. Dick 2, 8 , Michael Milyavsky 1 1 Department of Pathology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel 2 Princess Margaret Cancer Centre, University Health Network, University of Toronto, Toronto, Canada 3 Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia 4 Leidos Biomedical Research, Washington D.C., USA 5 Department of Immunology, Weizmann Institute of Science, Rehovot, Israel 6 Donnelly Centre for Cellular and Biomedical Research, University of Toronto, Toronto, Canada 7 Princess Margaret Cancer Centre, University Health Network, University of Toronto, Toronto, Canada 8 Department of Molecular Genetics, University of Toronto, Toronto, Canada 9 Department of Medicine, University of Toronto, Toronto, Canada Correspondence to: Michael Milyavsky, email: mmilyavsky@post.tau.ac.il Keywords: SMYD2 lysine methyltransferase, AML, quiescence, chemotherapy, DNA damage Received: November 23, 2016 Accepted: January 24, 2017 Published: February 06, 2017 ABSTRACT 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.

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.

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, BimS2A, which is highly selective for Mcl-1. Unlike Noxa, BimS2A is unable to trigger Mcl-1 degradation, yet, like Noxa, BimS2A promotes cell killing only when Bcl-xL 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.

Apoptosis is held in check by prosurvival proteins of the Bcl-2 family. The distantly related BH3-only proteins bind to and antagonize them, thereby promoting apoptosis. Whereas binding of the BH3-only protein Noxa to prosurvival Mcl-1 induces Mcl-1 degradation by the proteasome, binding of another BH3-only ligand, Bim, elevates Mcl-1 protein levels. We compared the three-dimensional structures of the complexes formed between BH3 peptides of both Bim and Noxa, and we show that a discrete C-terminal sequence of the Noxa BH3 is necessary to instigate Mcl-1 degradation.

Summary Many viruses express antiapoptotic proteins to counter host defense mechanisms that would otherwise trigger the rapid clearance of infected cells. For example, adenoviruses and some γ-herpesviruses express homologs of prosurvival Bcl-2 to subvert the host's apoptotic machinery. Myxoma virus, a double-stranded DNA virus of the pox family, harbors antiapoptotic M11L, its virulence factor. Analysis of its three-dimensional structure reveals that despite lacking any primary sequence similarity to Bcl-2, it adopts a virtually identical protein fold. This allows it to associate with BH3 domains, especially those of Bax and Bak. We found that M11L acts primarily by sequestering Bax and Bak, thereby blocking the killing action of these essential cell-death mediators. These findings expand the family of protein sequences that act like Bcl-2 to block apoptosis and support the conclusion that the prosurvival action of these proteins critically depends on their ability to bind and antagonize Bax and/or Bak.

A central issue in the regulation of apoptosis by the Bcl-2 family is whether its BH3-only members initiate apoptosis by directly binding to the essential cell-death mediators Bax and Bak, or whether they can act indirectly, by engaging their pro-survival Bcl-2–like relatives. Contrary to the direct-activation model, we show that Bax and Bak can mediate apoptosis without discernable association with the putative BH3-only activators (Bim, Bid, and Puma), even in cells with no Bim or Bid and reduced Puma. Our results indicate that BH3-only proteins induce apoptosis at least primarily by engaging the multiple pro-survival relatives guarding Bax and Bak.

Commitment of cells to apoptosis is governed largely by protein-protein interactions between members of the Bcl-2 protein family. Its three sub-families have distinct roles: the BH3-only proteins trigger apoptosis by binding via their BH3 domain to pro-survival relatives, while the pro-apoptotic Bax and Bak have an essential downstream role involving disruption of organellar membranes and induction of caspase activation. The BH3-only proteins act as damage sensors, held inert until their activation by stress signals. Once activated, they were thought to bind promiscuously to pro-survival protein targets but unexpected selectivity has recently emerged from analysis of their interactions. Some BH3-only proteins also bind to Bax and Bak. Whether Bax and Bak are activated directly by these BH3-only proteins, or indirectly as a consequence of BH3-only proteins neutralizing their pro-survival targets is the subject of intense debate. Regardless of this, a detailed understanding of the interactions between family members, which are often selective, has notable implications for designing anti-cancer drugs to target the Bcl-2 family.

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.

The effects of distinct classes of peroxisome proliferator-activated receptor gamma (PPARgamma) ligands on myogenesis and MyoD gene expression were examined in mouse skeletal muscle C2C12 myoblasts. Treatment of C2C12 cells with the PPARgamma ligand, 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2), repressed morphologically defined myogenesis and reduced endogenous mRNA levels of the myogenic differentiation markers MyoD, myogenin, and alpha-actin. In contrast, two synthetic PPARgamma ligands, L-805645 and ciglitazone, exhibited no effects. In transient transfection assays, 15d-PGJ2 specifically inhibited the expression of a MyoD promoter-luciferase reporter gene (MyoDLuc) in a cell type- and promoter-specific manner, indicating that 15d-PGJ2 functions in part by repressing MyoD gene transcription. The inhibition of MyoD gene expression by 15d-PGJ2 is mediated by the distal region of the MyoD gene promoter. PPARgamma on its own also inhibited MyoDLuc expression and further augmented the 15d-PGJ2 response. In contrast, L-805645 and ciglitazone did not inhibit MyoDLuc expression on their own but did so in the presence of ectopically expressed PPARgamma. Interestingly, a transdominant inhibitor of PPARgamma (hPPARgamma2Delta500) had no effect on the 15d-PGJ2-dependent repression of MyoDLuc expression but overcame L-805645/PPARgamma-dependent repression. Finally, saturating concentrations of L-805645, which did not affect myogenesis, failed to ablate 15d-PGJ2-mediated repression of the myogenic program. Thus, distinct PPARgamma ligands may repress MyoD gene expression through PPARgamma-dependent and -independent pathways, and 15d-PGJ2 can inhibit the myogenic program independent of its cognate receptor, PPARgamma.

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.

Multiple myeloma is a malignancy of plasma cells. The abnormal clonal accumulation of plasma cells interferes with the blood cell production, thereby causing cytopenias and aberrant, excessive antibody production. Patients can also experience bone pain, neurological symptoms and renal failure. With the exception of bone marrow transplantation that is associated with significant morbidity and mortality, there is currently no cure for patients with this disease. Thus, novel therapies are desperately needed for treating patients especially those harboring disease with adverse prognostic markers or when they become refractory to current treatment regimens. Recently, we have screened a panel of well-established myeloma cell lines against multiple anti-myeloma agents, including standard-of-care agents such as bortezomib, lenalidomide, dexamethasone or melphalan, as well as novel targeted agents such as the BH3 mimetic compounds (e.g. venetoclax/ABT-199 to target BCL2) or HDAC inhibitors (e.g. panobinostat). Specifically, we sought to determine whether their cytotoxic action depends on the induction of BAX/BAK-mediated apoptosis. When activated, these two proteins drive permeabilization of the outer mitochondrial membrane. In their absence, apoptosis cannot proceed and we could readily establish if BAX/BAK-mediated apoptosis is critical by comparing the sensitivity of isogenic cell lines lacking these two cell death mediators to their wild-type parental counterparts. As anticipated, the action of the BH3 mimetic compounds such as venetoclax/ABT-199 to inhibit BCL2 or small molecule inhibitors to selectively target relatives of BCL2, such as BCLxL and MCL1, rely exclusively on BAX and BAK. In the their absence, the treated cells remained viable. While the action of some drugs such as dexamethasone is already known to be mediated in part by BAX/BAK-driven apoptosis, we were very surprised that lenalidomide, an immunomodulatory drug (IMiD), can also kill some myeloma cell lines through BAX/BAK mediated apoptosis. A more potent relative of lenalidomide, pomalidamide, does likewise. While the cytotoxic action of these IMiDs, most notably the induction of cell cycle arrest upon activation of the E3 ligase cereblon (CRBN) is well known, our data is the first to definitively demonstrate that the IMiDs can actively induce BAX/BAK-mediated apoptosis. Next we explored the mechanisms by which the IMiDs can trigger apoptosis. Most stress signals and many cytotoxic drugs activate the BH3-only proteins such as BIM, BAD, BID or PUMA to initiate apoptosis. We checked whether killing by lenalidomide might also depend on specific BH3-only proteins and found that the genetic deletion of BIM conferred almost complete resistance to treatment with lenalidomide in vitro. We are currently investigating the precise mechanism by which the IMiDs can activate BIM to drive apoptosis. While lenalidomide can trigger BAX/BAK-mediated apoptosis in some myeloma cell lines, many others were spared. We expect that by deciphering the mechanisms by which anti-myeloma agents can induce BAX/BAK-mediated apoptosis of myeloma cell lines, we can probably improve the utility of drugs such as the IMiDs and understand why they might fail in some patients. Disclosures Gong: The Walter and Eliza Hall Institute of Medical Research: Employment. Segal:The Walter and Eliza Hall Institute of Medical Research: Employment. Riffkin:The Walter and Eliza Hall Institute of Medical Research: Employment. van Delft:The Walter and Eliza Hall Institute of Medical Research: Employment. Roberts:Walter and Eliza Hall Institute of Medical Research: Employment. Huang:The Walter and Eliza Hall Institute of Medical Research: Employment.

Multiple myeloma is a clonal B-cell malignancy characterized by complex genetic aberrations. Despite recent advances in our understanding of genetic basis of this disease and the introduction of novel therapies, the outcome for many patients remains poor. For example, 20% of newly diagnosed patients with stage 3 myeloma have disease marked by high lactate dehydrogenase levels or unfavorable cytogenetic abnormalities (e.g. 17p deletion, t(4;14)). Half of these patients die within 2 years of diagnosis in spite of advanced therapies. A promising class of novel agents to treat patients with other B-cell malignancies is the BH3 mimetic compounds, which targets the pro-survival BCL2 proteins. For example, the majority of patients with CLL (chronic lymphocytic leukaemia) respond to venetoclax (ABT-199), a BCL2-selective inhibitor. Myeloma cell lines with t(11;14) translocations are also reported to be sensitive to BCL2 inhibition and venetoclax is in clinic trails for patients with relapsed or refractory multiple myeloma. We are eager to know whether the other pro-survival BCL2 proteins, namely BCLXL, BCLW, MCL1 and BCL2A1 (BFL1) play a role in maintaining the survival of myeloma cells. To determine this, we screened a large panel of immortalized (25) and low-passage (7) myeloma cell lines to killing by BH3 mimetics with varying specificities. We found that a fraction of the myeloma cell lines were rapidly killed when BCL2 (25%, 8/32) or BCLXL (25%, 8/32) were targeted. Interestingly, we also identified a distinct t(4;14) subgroup, as well as the previous recognized t(11;14) subgroup, to be highly sensitive to BCL2 inhibition. Unlike CLL, in which the majority patients respond to venetoclax, BCL2 inhibition is only likely to account for some cases. We then asked which other pro-survival BCL2 protein keeps the other myeloma cell lines alive. A prime candidate is MCL1 since normal plasma cells rely on it and previous studies had implicated MCL1 in myeloma. By targeting the expression of the pro-survival BCL2 proteins using CRISPR/Cas9 genome editing technology, we found that a significant fraction (74%, 14/19) of our cell line panel died rapidly in the absence of MCL1. The importance of MCL1 was confirmed using an orthogonal approach: we found that expression of a MCL1-selective peptidyl ligand BIM2A rapidly killed a sizeable fraction of both immortalized (17/25) and low-passage (5/7) myeloma cell lines. This activity strongly correlated with the genetic targeting of MCL1 by CRISPR/Cas9. Importantly, even cell lines that harbor the poor prognostic t(4;14) chromosomal translocation were readily killed by BIM2A and the cell lines were killed regardless of their TP53 status. Moreover, targeting MCL1 also constrained the growth of myeloma in vivo. Since the inhibition of MCL1, BCLXL or BCL2 can on their own can kill almost all the myeloma cell lines screened, we next identified the molecular mechanisms by which these BH3 mimetics act. Firstly, we tested likely candidates for their role in driving the killing induced by a specific BH3 mimetic, for example whether deleting the BH3-only protein BIM impact upon killing by venetoclax. Secondly, we undertook genome-wide recessive CRISPR/Cas9 screens for genes that are critical for the action of the BH3 mimetics. We have identified a number of hits from such screens and are in the process of validating these. By identifying the unique susceptibility of the myeloma cell lines to the BH3 mimetics and elucidating how they act to kill, our studies are critical for the clinical testing of BH3 mimetics that target BCL2 or its close pro-survival relatives in patients with multiple myeloma. Disclosures Gong: The Walter and Eliza Hall Institute of Medical Research: Employment. Segal:The Walter and Eliza Hall Institute of Medical Research: Employment. van Delft:The Walter and Eliza Hall Institute of Medical Research: Employment. Roberts:The Walter and Eliza Hall Institute of Medical Research: Employment. Huang:The Walter and Eliza Hall Institute of Medical Research: Employment.

Apoptosis in response to developmental cues and stress stimuli is mediated by caspases that are regulated by the Bcl-2 protein family. Although caspases 2 and 9 have each been proposed as the apical caspase in that pathway, neither is indispensable for the apoptosis of leukocytes or fibroblasts. To investigate whether these caspases share a redundant role in apoptosis initiation, we generated caspase-2−/−9−/− mice. Their overt phenotype, embryonic brain malformation and perinatal lethality mirrored that of caspase-9−/− mice but were not exacerbated. Analysis of adult mice reconstituted with caspase-2−/−9−/− hematopoietic cells revealed that the absence of both caspases did not influence hematopoietic development. Furthermore, lymphocytes and fibroblasts lacking both remained sensitive to diverse apoptotic stimuli. Dying caspase-2−/−9−/− lymphocytes displayed multiple hallmarks of caspase-dependent apoptosis, including the release of cytochrome c from mitochondria, and their demise was antagonized by several caspase inhibitors. These findings suggest that caspases other than caspases 2 and 9 can promote cytochrome c release and initiate Bcl-2–regulated apoptosis.

Abstract 1289 Hematopoietic stem cells due to their life-long function should protect genome integrity to avoid accumulation of genetic aberrations leading to malignant transformation or bone marrow failure. We reported recently that human HSC of cord blood origin are exquisitely sensitive to DNA damage-induced apoptosis. Thus, following 3Gy of ionizing radiation HSC show evidence of persistent DNA damage response and greater p53-dependent apoptosis in comparison with commited myeloid progenitors. To elucidate the molecular basis of these observations we carried out a genome-wide loss-of-function genetic screen using a library of 80 000 shRNA vectors targeting more than 16 000 human genes. A screen was performed on immortalized (by TLS-ERG infection) cord blood cells (TEX), which radio-sensitivity is similar to early hematopoietic cells. To find out the radio-protective hits we exposed infected cells to four rounds of 4Gy irradiation in three independent experiments. TEX cells exhibited steady increase of their proliferative potential after each irradiation exposure. Cells were gathered after each irradiation round and their DNA was subjected to sequencing to determine protective hits. Upon the analysis we recognized known regulators of DNA damage response (e.g. p53) and identified many genes that previously were not connected to genotoxic stress response. Thus, the knockdown of these genes was at least as effective as p53 knockdown in protection of TEX cells against gamma-irradiation. The validation of the chosen hits on TEX cells showed that about half of them indeed mediate the protection against irradiation. Further validation included real-time PCR of infected cells to ensure the absence of off-target effect along with Western blot analysis of affected protein. We followed up the investigation of chosen hits on primary human lineage-negative cord blood cells and observed that only few candidates were mediating the same effect on HSC/progenitors cells as on TEX cells. To further validate the effect of the most prominently effective hits we employed several shRNA vectors for the same target gene. Following this step of validation, we choose to investigate the knockdown of CHEK2, the gene with reported role in DNA damage response in several murine tissues. Our preliminary studies in long-term cytokine-supplemented cultures demonstrated that CHEK2 is involved in DNA damage response of human HSC and progenitors. The further investigation of the effect of CHEK2 knockdown on repopulating human HSC is currently underway. An integrated analysis of our observations revealed several putative CHEK2-centered molecular networks, which connect DNA damage response to HSC function. Disclosures: No relevant conflicts of interest to declare.

Lymphoid tumors often respond poorly to conventional cytotoxics, a common cause being their impaired sensitivity to apoptosis, such as that caused by Bcl-2 overexpression. A strategy to overcoming this is to use mimics of the natural antagonists of pro-survival Bcl-2, the BH3 only proteins. A promising BH3 mimetic is ABT-737, which targets Bcl-2 and closely related pro-survival proteins. We evaluated its potential utility by testing it on cell lines, clinical samples and on a relevant mouse lymphoma model. We assessed the sensitivity of B cell lymphoma cell lines and primary CLL samples to ABT-737, either alone or in combination. To ascertain its efficacy in vivo, we utilized a mouse model based on the Eμ-myc tumor that is readily transplantable and amenable to genetic manipulation. When syngeneic recipient mice were inoculated with tumors, they develop widespread lymphoma, fatal unless treated by agents such as cyclophosphamide. We found that ABT-737, on its own, was cytotoxic only to a subset of cell lines and primary CLL samples. However, it can synergize potently with agents such as dexamethasone, suggesting that this agent might be useful in combination with currently used chemotherapeutics. In the Eμ myc mouse lymphoma model, treatment with ABT-737 alone did not control the disease as multiple independently derived tumors proved refractory to treatment with this agent. However, ABT-737 was partially effective as a single agent for treating bitransgenic tumors derived from crosses of the Eμmyc and Eμ-bcl-2 transgenic mice. ABT-737 therapy prolonged the survival of recipient mice transplanted with tumors from 30 to 60 days. When combined with a low dose of cyclophosphamide (50mg/kg), long term stable remissions were achieved, which were sustained even longer than control mice treated with much higher doses of cyclophosphamide (300mg/kg). We found that ABT-737 was well tolerated as a single agent and when combined with low doses of cytotoxics such as cyclophosphamide. Thus, ABT-737 may prove to be efficacious for those tumors highly dependent on Bcl-2 for their survival. We found that despite its high affinity for Bcl-2, Bcl-xL and Bcl-w, many cell types proved refractory to ABT-737 as a single agent. We show that this resistance reflects its inability to target another pro-survival relative Mcl-1. Down-regulation of Mcl-1 by several strategies conferred sensitivity to ABT-737. Furthermore, enforced Mcl-1 expression in the Eμmyc/bcl-2 bitransgenic mouse lymphoma model conferred marked resistance as mice transplanted with such tumors died as rapidly as the untreated counterparts. However, enhanced Bcl-2 overexpression on these tumors had little impact on the in vivo response, suggesting that ABT-737 can be utilized even when Bcl-2 is markedly overexpressed. ABT-737 appears to be a promising agent for the clinic. It potently sensitizes certain lymphoid tumors to conventional cytotxics in vitro. The synergy observed between dexamethasone and ABT-737 on some lymphoid lines in culture suggests that it is attractive for clinical testing. Encouragingly, ABT-737 appeared efficacious in vivo against Bcl-2 overexpressing tumors when combined with a reduced dose of cyclophosphamide, suggesting that it will be useful for treating even those Bcl-2-overexpressing tumors that are normally highly chemoresistant.

As overactivity of pro-survival Bcl-2 proteins promotes neoplasia and enhances resistance of malignant cells to cytotoxic therapies, a promising approach for incurable lymphoid tumors is to directly target the pro-survival proteins. One approach is to mimic their physiological antagonists, the BH3-only proteins. We initially determined which of the diverse BH3-only proteins would be optimal to mimic. The interactions of the BH3 domains of this family with a groove on the Bcl-2-like proteins have been considered promiscuous. However, we found that the interactions between eight BH3 peptides and five Bcl-2-like proteins varied over 10,000 fold in affinity, and that only certain protein pairs associate inside cells (Chen et al Mol Cell;17:393–403, 2005). Bim and Puma potently engaged all the pro-survival proteins comparably. Bad, however, bound preferentially to Bcl-2, Bcl-xL and Bcl-w. Strikingly, Noxa bound only Mcl-1 and A1. In accord with their complementary binding, Bad and Noxa cooperated to induce potent killing. We next determined the mechanism of action of putative BH3 mimetic compounds. Because killing by the BH3-only proteins require the action of the essential cell death mediators, Bax and Bak, we initially screened putative BH3 mimetic compounds on cells genetically engineered to lack both Bax and Bak. Only those compounds that mimicked the BH3-only proteins (i.e, inert on BaxBak double null cells, but active in Bax or Bak expressing cells) were evaluated further. Surprisingly, among seven putative BH3 mimetics tested, we found that only the recently described ABT-737 (Abbott Laboratories; Oltersdorf et al, Nature435:677–81, 2005), required the pro-apoptotic protein Bax or Bak to induce apoptosis. The cytotoxicity of ABT-737 alone was modest in a range of hematopoietic and non-hematopoietic cell types. Further investigations revealed that, like Bad, ABT-737 only targeted Bcl-2, Bcl-xL and Bcl-w. As hematopoietic cells typically express Mcl-1, we attempted to augment the activity of ABT-737 by concomitantly neutralizing Mcl-1. Targeting of Mcl-1 by overexpressing the BH3-only protein Noxa, or Mcl-1 down-regulation by RNAi, cytokine deprivation or cytotoxic agents, allowed ABT-737 to efficiently kill diverse cell types, even when Bcl-2 was over-expressed. We conclude that ABT-737 is a highly selective and specific BH3 mimetic compound that should prove highly efficacious in tumors where Mcl-1 is low, or when combined with agents that down-regulate Mcl-1. This hypothesis is currently being tested in vitro in primary human lymphoid malignancies, and in vivo using immunocompetent murine models of lymphoma. ABT-737 also provides strong proof-of-principle that targeting pro-survival Bcl-2 proteins is feasible, but the optimal utility of such BH3 mimetics depends on a thorough understanding of the pathways to apoptosis.

SummarySince apoptosis is impaired in malignant cells overexpressing prosurvival Bcl-2 proteins, drugs mimicking their natural antagonists, BH3-only proteins, might overcome chemoresistance. Of seven putative BH3 mimetics tested, only ABT-737 triggered Bax/Bak-mediated apoptosis. Despite its high affinity for Bcl-2, Bcl-xL, and Bcl-w, many cell types proved refractory to ABT-737. We show that this resistance reflects ABT-737's inability to target another prosurvival relative, Mcl-1. Downregulation of Mcl-1 by several strategies conferred sensitivity to ABT-737. Furthermore, enforced Mcl-1 expression in a mouse lymphoma model conferred resistance. In contrast, cells overexpressing Bcl-2 remained highly sensitive to ABT-737. Hence, ABT-737 should prove efficacious in tumors with low Mcl-1 levels, or when combined with agents that inactivate Mcl-1, even to treat those tumors that overexpress Bcl-2.

SummaryThe 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.