Molecular patterns of response and treatment failure after frontline venetoclax combinations in older patients with AML.

The BCL-2 inhibitor venetoclax combined with hypomethylating agents or low-dose cytarabine represents an important new therapy for older or unfit patients with acute myeloid leukemia (AML). We analyzed 81 patients receiving these venetoclax-based combinations to identify molecular correlates of durable remission, response followed by relapse (adaptive resistance), or refractory disease (primary resistance). High response rates and durable remissions were typically associated with NPM1 or IDH2 mutations, with prolonged molecular remissions prevalent for NPM1 mutations. Primary and adaptive resistance to venetoclax-based combinations was most commonly characterized by acquisition or enrichment of clones activating signaling pathways such as FLT3 or RAS or biallelically perturbing TP53. Single-cell studies highlighted the polyclonal nature of intratumoral resistance mechanisms in some cases. Among cases that were primary refractory, we identified heterogeneous and sometimes divergent interval changes in leukemic clones within a single cycle of therapy, highlighting the dynamic and rapid occurrence of therapeutic selection in AML. In functional studies, FLT3 internal tandem duplication gain or TP53 loss conferred cross-resistance to both venetoclax and cytotoxic-based therapies. Collectively, we highlight molecular determinants of outcome with clinical relevance to patients with AML receiving venetoclax-based combination therapies.

Genetic effects of vascular endothelial growth factor and its receptor 2 on feather maturity in three chicken breeds.

1. The goal of the current study was to evaluate the genetic effects of the vascular endothelial growth factor (VEGF) and its receptor (VEGFR-2) on feather maturity in the Qingyuan partridge chicken, Guangxi sanhuang chicken and Princess chicken. 2. Both SSCP-PCR and qPCR were employed to detect the polymorphism and gene expression of the VEGF and VEGFR-2 genes. 3. Four SNPs were identified in the VEGFR-2 gene. Exon10-A69G was associated with feather maturity (P < 0.01). Princess chickens with the genotype EF had higher feather maturity scores (P < 0.01). Higher expression levels of VEGF and VEGFR-2 were detected in the immature feather group of Qingyuan partridge chickens, especially in the skin. 4. The VEGF and VEGFR-2 genes play critical roles in feather maturity. In addition, exon10-A69G and genotype EF in the Princess chicken could potentially be utilised as genetic markers to improve efficiency in breeding.

Enhancing venetoclax activity in acute myeloid leukemia by co-targeting MCL1.

Targeted therapies are frequently combined with standard cytotoxic drugs to enhance clinical response. Targeting the B-cell lymphoma 2 (BCL-2) family of proteins is an attractive option to combat chemoresistance in leukemia. Preclinical and clinical studies indicate modest single-agent activity with selective BCL-2 inhibitors (for example, venetoclax). We show that venetoclax synergizes with cytarabine and idarubicin to increase antileukemic efficacy in a TP53-dependent manner. Although TP53 deficiency impaired sensitivity to combined venetoclax and chemotherapy, higher-dose idarubicin was able to suppress MCL1 and induce cell death independently of TP53. Consistent with an MCL1-specific effect, cell death from high-dose idarubicin was dependent on pro-apoptotic Bak. Combining higher-dose idarubicin with venetoclax was able to partially overcome resistance in Bak-deficient cells. Using inducible vectors and venetoclax to differentially target anti-apoptotic BCL-2 family members, BCL-2 and MCL1 emerged as critical and complementary proteins regulating cell survival in acute myeloid leukemia. Dual targeting of BCL-2 and MCL1, but not either alone, prolonged survival of leukemia-bearing mice. In conclusion, our findings support the further investigation of venetoclax in combination with standard chemotherapy, including intensified doses of idarubicin. Venetoclax should also be investigated in combination with direct inhibitors of MCL1 as a chemotherapy-free approach in the future.

BET inhibition represses miR17-92 to drive BIM-initiated apoptosis of normal and transformed hematopoietic cells.

The BET (bromodomain and extraterminal domain) bromodomain-containing proteins, such as BRD4, are highly promising targets for treating lymphoid and myeloid malignancies. They act to modulate the expression of multiple genes that control diverse cellular processes including proliferation, survival and differentiation that are consequentially disrupted by small-molecule BET bromodomain inhibitors such as JQ1. By assessing the impact of these inhibitors on normal mouse hematopoietic cells or their transformed counterparts, we establish definitively that their cytotoxic action in vitro and in vivo relies predominantly on the activation of BAX/BAK-dependent mitochondrial (intrinsic) apoptosis. In large part, this is triggered by marked upregulation of the BH3-only protein BIM when the BET inhibitors suppress miR-17-92, a key post-transcriptional repressor of BIM expression. Thus, our study strongly suggests that mutations that permit the evasion of apoptosis (for example, BCL2 overexpression, BIM inactivation) are likely to blunt the activity of the BET bromodomain inhibitors and should be anticipated when therapy resistance develops. Strikingly, we also found that certain normal hematopoietic cells, especially those of lymphoid origin, are as prone to apoptosis induced by the BET inhibitors as their transformed counterparts, indicating that their susceptibility to BET inhibitors did not arise from oncogenic transformation.

HSP90 activity is required for MLKL oligomerisation and membrane translocation and the induction of necroptotic cell death.

Necroptosis is a caspase-independent form of regulated cell death that has been implicated in the development of a range of inflammatory, autoimmune and neurodegenerative diseases. The pseudokinase, Mixed Lineage Kinase Domain-Like (MLKL), is the most terminal known obligatory effector in the necroptosis pathway, and is activated following phosphorylation by Receptor Interacting Protein Kinase-3 (RIPK3). Activated MLKL translocates to membranes, leading to membrane destabilisation and subsequent cell death. However, the molecular interactions governing the processes downstream of RIPK3 activation remain poorly defined. Using a phenotypic screen, we identified seven heat-shock protein 90 (HSP90) inhibitors that inhibited necroptosis in both wild-type fibroblasts and fibroblasts expressing an activated mutant of MLKL. We observed a modest reduction in MLKL protein levels in human and murine cells following HSP90 inhibition, which was only apparent after 15 h of treatment. The delayed reduction in MLKL protein abundance was unlikely to completely account for defective necroptosis, and, consistent with this, we also found inhibition of HSP90 blocked membrane translocation of activated MLKL. Together, these findings implicate HSP90 as a modulator of necroptosis at the level of MLKL, a function that complements HSP90's previously demonstrated modulation of the upstream necroptosis effector kinases, RIPK1 and RIPK3.

A phase I study of two dosing schedules of volasertib (BI 6727), an intravenous polo-like kinase inhibitor, in patients with advanced solid malignancies.

BACKGROUND: Polo-like kinase 1 (Plk1) has an important role in mitosis. Volasertib (BI 6727), a potent and selective cell cycle kinase inhibitor, induces mitotic arrest and apoptosis by targeting Plk; this phase I study sought to determine its maximum tolerated dose (MTD) in Asian patients with advanced solid tumours. METHODS: Patients were enrolled simultaneously into two 3-week schedules of volasertib: a 2-h infusion on day 1 (schedule A) or days 1 and 8 (schedule B). Dose escalation followed a 3+3 design. The MTD was determined based on dose-limiting toxicities (DLT) in the first treatment course. RESULTS: Among 59 treated patients, the most common first course DLTs were reversible thrombocytopenia, neutropenia and febrile neutropenia; MTDs were 300 mg for schedule A and 150 mg for schedule B. Volasertib exhibited multi-exponential pharmacokinetics (PK), a long terminal half-life of ∼135 h, a large volume of distribution (>3000 l), and a moderate clearance. Partial responses were observed in two pre-treated patients (ureteral cancer; melanoma). Volasertib was generally well tolerated, with an adverse event profile consistent with its antimitotic mode of action and a favourable PK profile. CONCLUSIONS: These data support further development of volasertib and a harmonised dosing for Asian and Caucasian patients.

Both leukaemic and normal peripheral B lymphoid cells are highly sensitive to the selective pharmacological inhibition of prosurvival Bcl-2 with ABT-199.

Overexpression of the prosurvival protein Bcl-2 marks many B-lymphoid malignancies and contributes to resistance to many commonly used chemotherapeutic agents. The first effective BH3 mimetic inhibitors of Bcl-2, ABT-737 and navitoclax, also target Bcl-xL, causing dose-limiting thrombocytopenia. This prompted the development of the Bcl-2-selective antagonist, ABT-199. Here we show that in lymphoid cells, ABT-199 specifically causes Bax/Bak-mediated apoptosis that is triggered principally by the initiator BH3-only protein Bim. As expected, malignant cells isolated from patients with chronic lymphocytic leukaemia are highly sensitive to ABT-199. However, we found that normal, untransformed mature B cells are also highly sensitive to ABT-199, both in vitro and in vivo. By contrast, the B-cell precursors are largely spared, as are cells of myeloid origin. These results pinpoint the probable impact of the pharmacological inhibition of Bcl-2 by ABT-199 on the normal mature haemopoietic cell lineages in patients, and have implications for monitoring during ABT-199 therapy as well as for the clinical utility of this very promising targeted agent.

The restricted binding repertoire of Bcl-B leaves Bim as the universal BH3-only prosurvival Bcl-2 protein antagonist.

B-cell lymphoma-2 (Bcl-2) proteins mediate intrinsic-, or mitochondrial-, initiated apoptosis. We have investigated the structure and function of the least characterized Bcl-2 family member, Bcl-B, solving the crystal structure of a Bcl-B:Bim complex to 1.9 Å resolution. Bcl-B is distinguished from other Bcl-2 family members through an insertion of an unstructured loop between helices α5 and α6. Probing Bcl-B interactions with Bcl-2 homology (BH)3 motifs using a combination of biophysical- and cell-based assays revealed a unique BH3-only protein binding profile. Bcl-B has high-affinity interactions with Bim and Bik only. Our results not only delineate the mode of action of Bcl-B but also complete our understanding of the specific interactions between BH3-only proteins and their prosurvival Bcl-2 counterparts. Notably, we conclude that Bim is the universal prosurvival antagonist as no other BH3-only protein binds all six prosurvival proteins and that Mcl-1 and Bcl-x(L) form a distinct prosurvival dyad.

Translation inhibitors induce cell death by multiple mechanisms and Mcl-1 reduction is only a minor contributor.

There is significant interest in treating cancers by blocking protein synthesis, to which hematological malignancies seem particularly sensitive. The translation elongation inhibitor homoharringtonine (Omacetaxine mepesuccinate) is undergoing clinical trials for chronic myeloid leukemia, whereas the translation initiation inhibitor silvestrol has shown promise in mouse models of cancer. Precisely how these compounds induce cell death is unclear, but reduction in Mcl-1, a labile pro-survival Bcl-2 family member, has been proposed to constitute the critical event. Moreover, the contribution of translation inhibitors to neutropenia and lymphopenia has not been precisely defined. Herein, we demonstrate that primary B cells and neutrophils are highly sensitive to translation inhibitors, which trigger the Bax/Bak-mediated apoptotic pathway. However, contrary to expectations, reduction of Mcl-1 did not significantly enhance cytotoxicity of these compounds, suggesting that it does not have a principal role and cautions that strong correlations do not always signify causality. On the other hand, the killing of T lymphocytes was less dependent on Bax and Bak, indicating that translation inhibitors can also induce cell death via alternative mechanisms. Indeed, loss of clonogenic survival proved to be independent of the Bax/Bak-mediated apoptosis altogether. Our findings warn of potential toxicity as these translation inhibitors are cytotoxic to many differentiated non-cycling cells.

Transgenic, inducible RNAi in megakaryocytes and platelets in mice.

BACKGROUND: RNA interference (RNAi) is a powerful tool for suppressing gene function. The tetracycline (tet)-regulated expression system has recently been adapted to allow inducible RNAi in mice, however its efficiency in a particular cell type in vivo depends on a transgenic tet transactivator expression pattern and is often highly variable. OBJECTIVE: We aimed to establish a transgenic strategy that allows efficient and inducible gene knockdown in particular hematopoietic lineages in mice. METHODS AND RESULTS: Using a tet-regulated reporter gene strategy, we found that transgenic mice expressing the rtTA (tet-on) transactivator under control of the cytomegalovirus (CMV) promoter (CMV-rtTA mice) display inducible reporter gene expression with unusual and near-complete efficiency in megakaryocytes and platelets. To test whether the CMV-rtTA transgene can drive inducible and efficient gene knockdown within this lineage, we generated a novel mouse strain harboring a tet-regulated short hairpin RNA (shRNA) targeting Bcl-x(L) , a pro-survival Bcl-2 family member known to be essential for maintaining platelet survival. Doxycycline treatment of adult mice carrying both transgenes induces shRNA expression, depletes Bcl-x(L) in megakaryocytes and triggers severe thrombocytopenia, whereas doxycycline withdrawal shuts off shRNA expression, normalizes Bcl-x(L) levels and restores platelet numbers. These effects are akin to those observed with drugs that target Bcl-x(L) , clearly demonstrating that this transgenic system allows efficient and inducible inhibition of genes in megakaryocytes and platelets. CONCLUSIONS: We have established a novel transgenic strategy for inducible gene knockdown in megakaryocytes and platelets that will be useful for characterizing genes involved in platelet production and function in adult mice.

Apoptosis and non-inflammatory phagocytosis can be induced by mitochondrial damage without caspases.

A central issue regarding vertebrate apoptosis is whether caspase activity is essential, particularly for its crucial biological outcome: non-inflammatory clearance of the dying cell. Caspase-9 is required for the proteolytic cascade unleashed by the mitochondrial outer membrane permeabilization (MOMP) regulated by the Bcl-2 protein family. However, despite the severely blunted apoptosis in cells from Casp9(-/-) mice, some organs with copious apoptosis, such as the thymus, appear unaffected. To address this paradox, we investigated how caspase-9 loss affects apoptosis and clearance of mouse fibroblasts and thymocytes. Although Casp9(-/-) cells were initially refractory to apoptotic insults, they eventually succumbed to slower caspase-independent cell death. Furthermore, in gamma-irradiated mice, the dying Casp9(-/-) thymocytes were efficiently cleared, without apparent inflammation. Notably, MOMP proceeded normally, and the impaired mitochondrial function, revealed by diminished mitochondrial membrane potential (DeltaPsi(m)), committed cells to die, as judged by loss of clonogenicity. Upon the eventual full collapse of DeltaPsi(m), presumably reflecting failure of respiration, intact dying Casp9(-/-) cells unexpectedly exposed the prototypic 'eat-me' signal phosphatidylserine, which allowed their recognition and engulfment by phagocytes without overt inflammation. Hence, caspase-9-induced proteolysis accelerates apoptosis, but impaired mitochondrial integrity apparently triggers a default caspase-independent program of cell death and non-inflammatory clearance. Thus, caspases appear dispensable for some essential biological functions of apoptosis.

The BH3 mimetic compound, ABT-737, synergizes with a range of cytotoxic chemotherapy agents in chronic lymphocytic leukemia.

As chronic lymphocytic leukemia (CLL) is characterized by overexpression of pro-survival BCL2, compounds that mimic its physiological antagonists, the BH3-only proteins, may have a role in treatment of this disease. ABT-737 is a BH3 mimetic compound that selectively targets BCL2 and BCLX(L). In the present work, we report that ABT-737 is highly effective (LC(50)<50 nM) as a single agent against most (21/30) primary CLL samples, but that a sizable minority is relatively insensitive. In vitro sensitivity to ABT-737 could not be simply predicted by the patients' clinical features, including response to prior therapy or known prognostic markers (CD38 expression, 17p deletion), or the relative expression of BCL2 family proteins (BCL2, MCL1, BAX, BIM). Strikingly, co-incubation with cytotoxic agents (dexamethasone, etoposide, fludarabine, doxorubicin) sensitized most CLL samples to ABT-737, but this could not be predicted by responses to either ABT-737 or the cytotoxic agent alone. Of 17 samples least sensitive to ABT-737, 13 were sensitized by co-treatment with at least one cytotoxic agent. These data indicate that combination of ABT-737 with a second anti-leukemic agent would improve response rates and suggest a potential role for combination therapies that include BH3 mimetics for the treatment of this disease.

Bax activation by Bim?

The mechanism by which the cell death mediator Bax becomes activated to cause mitochondrial damage, a key step for the intrinsic pathway to apoptosis, remain highly contentious. Although some data support a role for certain BH3-only proteins, such as Bim or tBid, to directly activate Bax, others have led to the conclusion that BH3-only proteins act indirectly by antagonizing the prosurvival Bcl-2 proteins, thereby allowing Bax activation to proceed. A recent paper in Nature by Gavathiotis et al. provides the first biophysical evidence for a direct interaction between a BH3 domain, that of Bim, with Bax. Here, we review these intriguing observations and discuss their implications for our understanding of how the BH3-only proteins initiate apoptosis.

Virally mediated inhibition of Bax in leukocytes promotes dissemination of murine cytomegalovirus.

The evolutionary survival of viruses relies on their ability to disseminate infectious progeny to sites of transmission. The capacity to subvert apoptosis is thought to be crucial for ensuring efficient viral replication in permissive cells, but its role in viral dissemination in vivo has not been considered. We show here that the murine cytomegalovirus (MCMV) m38.5 protein specifically counters the action of Bax. As predicted from our biochemical data, the capacity of m38.5 to inhibit apoptosis is only apparent in cells unable to activate Bak. Deletion of m38.5 resulted in an attenuated growth of MCMV in vitro. In vivo replication of the Deltam38.5 virus was not significantly impaired in visceral organs. However, m38.5 played a central role in protecting leukocytes from Bax-mediated apoptosis, thereby promoting viral dissemination to the salivary glands, the principal site of transmission. These results establish that in vivo MCMV replication induces the activation of Bax in leukocytes, but not other permissive cells, and that MCMV interferes with this process to attain maximum dissemination.

EGL-1 BH3 mutants reveal the importance of protein levels and target affinity for cell-killing potency.

Studies of the cell death pathway in the nematode Caenorhabditis elegans provided the first evidence of the evolutionary conservation of apoptosis signalling. Here we show that the worm Bcl-2 homology domain-3 (BH3)-only protein EGL-1 binds mammalian pro-survival proteins very poorly, but can be converted into a high-affinity ligand for Bcl-2 and Bcl-x(L) by subtle mutation of the cysteine residue at position 62 within the BH3 domain. A 100-fold increase in affinity was observed following a single atom change (cysteine to serine substitution), and a further 10-fold increase by replacement with glycine. The low affinity of wild-type EGL-1 for mammalian pro-survival proteins and its poor expression correlates with its weak killing activity in mammalian cells whereas the high-affinity C62G mutant is a very potent killer of cells lacking Mcl-1. Cell killing by the C62S mutant with intermediate affinity only occurs when this EGL-1 BH3 domain is placed in a more stable context, namely that of Bim(S), which allows higher expression, though the kinetics of cell death now vary depending on whether Mcl-1 is neutralized by Noxa or genetically deleted. These results demonstrate how levels of BH3-only proteins, target affinity and the spectrum of neutralization of pro-survival proteins all contribute to killing activity.

Vaccinia virus anti-apoptotic F1L is a novel Bcl-2-like domain-swapped dimer that binds a highly selective subset of BH3-containing death ligands.

Apoptosis is an important part of the host's defense mechanism for eliminating invading pathogens. Some viruses express proteins homologous in sequence and function to mammalian pro-survival Bcl-2 proteins. Anti-apoptotic F1L expressed by vaccinia virus is essential for survival of infected cells, but it bears no discernable sequence homology to proteins other than its immediate orthologues in related pox viruses. Here we report that the crystal structure of F1L reveals a Bcl-2-like fold with an unusual N-terminal extension. The protein forms a novel domain-swapped dimer in which the alpha1 helix is the exchanged domain. Binding studies reveal an atypical BH3-binding profile, with sub-micromolar affinity only for the BH3 peptide of pro-apoptotic Bim and low micromolar affinity for the BH3 peptides of Bak and Bax. This binding interaction is sensitive to F1L mutations within the predicted canonical BH3-binding groove, suggesting parallels between how vaccinia virus F1L and myxoma virus M11L bind BH3 domains. Structural comparison of F1L with other Bcl-2 family members reveals a novel sequence signature that redefines the BH4 domain as a structural motif present in both pro- and anti-apoptotic Bcl-2 members, including viral Bcl-2-like proteins.

BH3-only proteins and their roles in programmed cell death.

The Bcl-2 family of proteins controls the mitochondrial pathway to apoptosis. It consists of pro-survival and pro-apoptotic members, and their interactions decide whether apoptogenic factor confined to the mitochondrial intermembrane space can leak to the cytosol. Despite the intense efforts to understand the molecular mechanisms that lead to the permeabilization of the mitochondrial membrane, this particular issue remains a matter of intense controversy. It is well accepted that pro-apoptotic Bax and Bak are directly responsible for the damage to the mitochondria, but pro-survival family members prevent them from doing so. It is also accepted that stress signals activate selected Bcl-2 homology (BH)3-only proteins. But do these BH3-only proteins bind and activate Bax and Bak directly, or do they inhibit the pro-survival family members?

Bim, Bad and Bmf: intrinsically unstructured BH3-only proteins that undergo a localized conformational change upon binding to prosurvival Bcl-2 targets.

All BH3-only proteins, key initiators of programmed cell death, interact tightly with multiple binding partners and have sequences of low complexity, properties that are the hallmark of intrinsically unstructured proteins (IUPs). We show, using spectroscopic methods, that the BH3-only proteins Bim, Bad and Bmf are unstructured in the absence of binding partners. Detailed sequence analyses are consistent with this observation and suggest that most BH3-only proteins are unstructured. When Bim binds and inactivates prosurvival proteins, most residues remain disordered, only the BH3 element becomes structured, and the short alpha-helical molecular recognition element can be considered to behave as a 'bead on a string'. Coupled folding and binding is typical of many IUPs that have important signaling roles, such as BH3-only proteins, as the inherent structural plasticity favors interaction with multiple targets. This understanding offers promise for the development of BH3 mimetics, as multiple modes of binding are tolerated.