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The BCL2 inhibitor venetoclax has established therapeutic roles in chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML). As BCL2 is an important determinant of survival of both myeloid progenitor and B cells, we investigated whether clinical and molecular abnormalities arise in the myeloid compartment during long-term continuous venetoclax treatment of CLL in 89 patients (87 with relapsed/refractory CLL). Over a median follow-up of 75 (range 21-98) months, persistent cytopenias (≥1 of neutropenia, thrombocytopenia, anemia) lasting ≥4 months and unrelated to CLL occurred in 25 patients (28%). Of these patients, 20 (80%) displayed clonal hematopoiesis, including 10 with therapy-related myeloid neoplasms (t-MNs). t-MNs occurred exclusively in patients previously exposed to fludarabine-alkylator combination therapy with a cumulative 5-year incidence of 10.4% after venetoclax initiation, consistent with rates reported for patients exposed to fludarabine-alkylator combination therapy without venetoclax. To determine whether the altered myelopoiesis reflected the acquisition of mutations, we analyzed samples from patients with no or minimal bone marrow CLL burden (n = 41). Mutations in the apoptosis effector BAX were identified in 32% (13/41). In cellular assays, C-terminal BAX mutants abrogated outer mitochondrial membrane localization of BAX and engendered resistance to venetoclax killing. BAX-mutated clonal hematopoiesis occurred independently of prior fludarabine-alkylator combination therapy exposure and was not associated with t-MNs. Single-cell sequencing revealed clonal co-occurrence of mutations in BAX with DNMT3A or ASXL1. We also observed simultaneous BCL2 mutations within CLL cells and BAX mutations in the myeloid compartment of the same patients, indicating lineage-specific adaptation to venetoclax therapy.

The conformational changes converting BAX from an inert cytosolic monomer into the homo-oligomers that permeabilize the mitochondrial outer membrane (MOM) are crucial steps toward apoptosis. Here, we have explored the potential role of the BAX α1-α2 loop in this process by three mutagenic approaches: replacing loop segments with cognate loop regions from closely related proteins, alanine scanning and analysis of BAX α1-α2 loop missense mutations observed in tumours. Responsiveness to a death signal, such as tBID, was reduced by mutations in the N-terminal but not C-terminal half of the loop. N-terminal loop variants, which were enriched in tumours, impaired MOM integration by allosterically reducing exposure of the BAX α9 transmembrane anchor. Most C-terminal loop variants reduced BAX stability, leading to increased BAX apoptotic function in some variants. Thus, our systematic mutagenesis suggests that the two halves of the α1-α2 loop have distinct functions. We show that the N-terminal half of the loop (its first nine residues) comprises an important allosteric regulator of BAX activation by setting the proportion of MOM-integrated BAX following a death signal. The enrichment of N-terminal loop mutations in tumours indicates that they may promote tumour cell survival and underscore the loop as a target for therapeutic manipulation of BAX function.

Selective targeting of BCL-2 with the BH3-mimetic venetoclax has been a transformative treatment for patients with various leukemias. TP-53 controls apoptosis upstream of where BCL-2 and its prosurvival relatives, such as MCL-1, act. Therefore, targeting these prosurvival proteins could trigger apoptosis across diverse blood cancers, irrespective of TP53 mutation status. Indeed, targeting BCL-2 has produced clinically relevant responses in blood cancers with aberrant TP-53. However, in our study, TP53-mutated or -deficient myeloid and lymphoid leukemias outcompeted isogenic controls with intact TP-53, unless sufficient concentrations of BH3-mimetics targeting BCL-2 or MCL-1 were applied. Strikingly, tumor cells with TP-53 dysfunction escaped and thrived over time if inhibition of BCL-2 or MCL-1 was sublethal, in part because of an increased threshold for BAX/BAK activation in these cells. Our study revealed the key role of TP-53 in shaping long-term responses to BH3-mimetic drugs and reconciled the disparate pattern of initial clinical response to venetoclax, followed by subsequent treatment failure among patients with TP53-mutant chronic lymphocytic leukemia or acute myeloid leukemia. In contrast to BH3-mimetics targeting just BCL-2 or MCL-1 at doses that are individually sublethal, a combined BH3-mimetic approach targeting both prosurvival proteins enhanced lethality and durably suppressed the leukemia burden, regardless of TP53 mutation status. Our findings highlight the importance of using sufficiently lethal treatment strategies to maximize outcomes of patients with TP53-mutant disease. In addition, our findings caution against use of sublethal BH3-mimetic drug regimens that may enhance the risk of disease progression driven by emergent TP53-mutant clones.

Ferroptosis is a recently defined form of regulated cell death, which is biochemically and morphologically distinct from traditional forms of programmed cell death such as apoptosis or necrosis. It is driven by iron, reactive oxygen species, and phospholipids that are oxidatively damaged, ultimately resulting in mitochondrial damage and breakdown of membrane integrity. Numerous cellular signaling pathways and molecules are involved in the regulation of ferroptosis, including enzymes that control the cellular redox status. Alterations in the ferroptosis-regulating network can contribute to the development of various diseases, including cancer. Evidence suggests that ferroptosis is commonly suppressed in cancer cells, allowing them to survive and progress. However, cancer cells which are resistant to common chemotherapeutic drugs seem to be highly susceptible to ferroptosis inducers, highlighting the great potential of pharmacologic modulation of ferroptosis for cancer treatment. Non-coding RNAs (ncRNAs) are considered master regulators of various cellular processes, particularly in cancer where they have been implicated in all hallmarks of cancer. Recent work also demonstrated their involvement in the molecular control of ferroptosis. Hence, ncRNA-based therapeutics represent an exciting alternative to modulate ferroptosis for cancer therapy. This review summarizes the ncRNAs implicated in the regulation of ferroptosis in cancer and highlights their underlying molecular mechanisms in the light of potential therapeutic applications.

Significance Neutralizing antibodies are important for immunity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and as therapeutics for the prevention and treatment of COVID-19. We identified high-affinity nanobodies against SARS-CoV-2 receptor-binding domain and found that nanobody cocktails consisting of two noncompeting nanobodies were able to block ACE2 engagement with RBD variants present in human populations and potently neutralize both wild-type SARS-CoV-2 and the N501Y D614G variant at low concentrations. Prophylactic administration of nanobody cocktails reduced viral loads in mice infected with the N501Y D614G SARS-CoV-2 virus, showing that nanobody cocktails are useful as prophylactic agents against SARS-CoV-2., Neutralizing antibodies are important for immunity against SARS-CoV-2 and as therapeutics for the prevention and treatment of COVID-19. Here, we identified high-affinity nanobodies from alpacas immunized with coronavirus spike and receptor-binding domains (RBD) that disrupted RBD engagement with the human receptor angiotensin-converting enzyme 2 (ACE2) and potently neutralized SARS-CoV-2. Epitope mapping, X-ray crystallography, and cryo-electron microscopy revealed two distinct antigenic sites and showed two neutralizing nanobodies from different epitope classes bound simultaneously to the spike trimer. Nanobody-Fc fusions of the four most potent nanobodies blocked ACE2 engagement with RBD variants present in human populations and potently neutralized both wild-type SARS-CoV-2 and the N501Y D614G variant at concentrations as low as 0.1 nM. Prophylactic administration of either single nanobody-Fc or as mixtures reduced viral loads by up to 104-fold in mice infected with the N501Y D614G SARS-CoV-2 virus. These results suggest a role for nanobody-Fc fusions as prophylactic agents against SARS-CoV-2.

Summary Programmed cell death contributes to host defense against pathogens. To investigate the relative importance of pyroptosis, necroptosis, and apoptosis during Salmonella infection, we infected mice and macrophages deficient for diverse combinations of caspases-1, -11, -12, and -8 and receptor interacting serine/threonine kinase 3 (RIPK3). Loss of pyroptosis, caspase-8-driven apoptosis, or necroptosis had minor impact on Salmonella control. However, combined deficiency of these cell death pathways caused loss of bacterial control in mice and their macrophages, demonstrating that host defense can employ varying components of several cell death pathways to limit intracellular infections. This flexible use of distinct cell death pathways involved extensive cross-talk between initiators and effectors of pyroptosis and apoptosis, where initiator caspases-1 and -8 also functioned as executioners when all known effectors of cell death were absent. These findings uncover a highly coordinated and flexible cell death system with in-built fail-safe processes that protect the host from intracellular infections., Graphical Abstract, Highlights • Mice lacking pyroptosis and apoptosis cannot control Salmonella infection • Macrophages lacking pyroptosis and apoptosis resist Salmonella-induced killing • Caspase-1 kills Salmonella-infected cells by activating GSDMD, BID, or other caspases • Caspase-1 and -8 act as cell death executioners when all cell death effectors are lost, The clearance of intracellular pathogens requires the killing of infected cells, but it remains unclear why host cells have so many different means of inducing programmed cell death. Doerflinger et al. demonstrate that interconnectivity between pyroptosis and apoptosis involving flexible deployment of caspases ensures control of Salmonella infection in mice.

Apoptosis-regulating BCL-2 family members, which can promote malignant transformation and resistance to therapy, have become prime therapeutic targets, as illustrated by the striking efficacy in certain lymphoid malignancies of the BCL-2-specific inhibitor venetoclax. In other lymphoid malignancies, however, such as the aggressive mantle cell lymphoma (MCL), cell survival might rely instead or also on BCL-2 relative MCL-1. We have explored MCL-1 as a target for killing MCL cells by both genetic and pharmacologic approaches. In several MCL cell lines, MCL-1 knockout with an inducible CRISPR/Cas9 system triggered spontaneous apoptosis. Accordingly, most MCL cell lines proved sensitive to the specific MCL-1 inhibitor S63845, and MCL-1 inhibition also proved efficacious in an MCL xenograft model. Furthermore, its killing efficacy rose on combination with venetoclax, the BCL-XL-specific inhibitor A-1331852, or Bruton's tyrosine kinase (BTK) inhibitor ibrutinib, which reduced pro-survival signals. We also tested the MCL-1 inhibitor in primary samples from 13 MCL patients, using CD40L-expressing feeder cells to model their microenvironmental support. Notably, all unstimulated primary MCL samples were very sensitive to S63845, but the CD40L stimulation attenuated their sensitivity. Mass cytometric analysis revealed that the stimulation likely conveyed protection by elevating BCL-XL and MCL-1. Accordingly, sensitivity of the CD40L-stimulated cells to S63845 was substantially restored by co-treatment with venetoclax, the BCL-XL-specific inhibitor or ibrutinib. Overall, our findings indicate that MCL-1 is very important for survival of MCL cells and that the MCL-1 inhibitor, both alone and together with ibrutinib, venetoclax or a BCL-XL inhibitor, offers promise for novel improved MCL therapies.

Summary: To elicit apoptosis, BAX metamorphoses from an inert cytosolic monomer into homo-oligomers that permeabilize the mitochondrial outer membrane (MOM). A long-standing puzzle is that BH3 domains apparently activate BAX by not only its canonical groove but also a proposed site involving helices α1 and α6. Our mutagenesis studies reveal that late steps like oligomerization require activation through the groove but probably not earlier steps like MOM association. Conversely, α1 or α6 obstruction and alanine mutagenesis scanning implicate these helices early in BAX activation. The α1 and α6 mutations lowered BH3 binding, altered the BAX conformation, and reduced its MOM translocation and integration; their exposure of the BAX α1-α2 loop allosterically sequestered its α9 membrane anchor in the groove. The crystal structure of an α6 mutant revealed additional allosteric effects. The results suggest that the α1 and α6 region drives MOM association and integration, whereas groove binding favors subsequent steps toward oligomerization. : BAX activation for apoptosis may require BH3 binding to its canonical groove and/or a less-characterized α1 and α6 site. Dengler et al. identify α1 and α6 mutations that impair early steps in BAX activation and reveal allosteric changes that control exposure of its membrane anchor and association with the mitochondrial outer membrane. Keywords: BCL-2 family, BAX activation, apoptosis, membrane association, oligomerization, allosteric changes, crystal structure, protein-protein association, protein conformation

Treatment response of follicular lymphomas (FL) is highly variable. We, therefore, investigated the role of FL cancer-associated fibroblasts (CAFs) on tumor cell viability, in particular in response to treatment with cytotoxic drugs. Stromal cells outgrown from FL patients were characterized and pure CAF populations were co-cultivated with FL cells. To analyze fibroblast-mediated effects, cells in co-culture were treated with ABT-737 and Bortezomib. The adherent cell population was positive for all fibroblastic markers tested and showed increased mRNA-expression of the activation marker FAP. No effect on FL cell viability was noted when co-cultivating them with CAFs. However, stromal cells protected tumor cells from apoptosis in response to cytotoxic treatment. This might be explained by mRNA-induction of ABCC1 and ABCG2 and up-regulation of BCL2L1 in FL cells. Our finding of protective mechanisms mediated by CAFs is of pivotal impact for further studies of cytotoxic agents in FL.

Aims The purpose of this clinical trial was to investigate whether cardiovascular magnetic resonance imaging (CMR) using ferumoxytol (Feraheme™, FH), an ultrasmall superparamagnetic iron oxide nanoparticle (USPIO), allows more detailed characterization of infarct pathology compared with conventional gadolinium-based necrosis/fibrosis imaging in patients with acute myocardial infarction. Methods and results Fourteen patients who had experienced an acute ST-elevation myocardial infarction were included in this study. Following coronary angiography, a first baseline study (pre-FH) was performed followed by subsequent CMR studies (post-FH) 48 h after intravenous ferumoxytol administration. The CMR studies comprised cine-CMR, T 2-weighted short tau inversion recovery spin echo imaging, T 2*-mapping, and T 1-weighted late gadolinium enhancement (LGE) imaging. The median extent of short-axis in-plane LGE was 30% [inter-quartile range (IQR) 26–40%]. The median in-plane extent of T 2-weighted ‘hypoenhancement’ in the region of myocardial infarction, which was not present prior to ferumoxytol administration in any patient, was 19% (IQR 14–22%; P < 0.001 compared with the extent of LGE). The median in-plane extent of areas showing signal void in T 2*-mapping images post-FH in the region of myocardial infarction was 16% (IQR 12–18%; P < 0.001 compared with the extent of LGE; P = 0.34 compared with the extent of T 2-weighted hypoenhancement). A substantial drop in absolute T 2*-values was observed not only in the infarct core and peri-infarct zone, but also in the remote ‘healthy’ myocardium, although there was only a minor change in the skeletal muscle. Substantial ferumoxytol uptake was detected only in cultured macrophages, but not in peripheral blood monocytes from study patients. Conclusion We could demonstrate in humans that USPIO-based contrast agents enable a more detailed characterization of myocardial infarct pathology mainly by detecting infiltrating macrophages. Considering the multi-functionality of USPIO-based particles and their superior safety profile compared with gadolinium-based compounds, these observations open up new vistas for the clinical application of USPIO.

Testicular germ cell tumors (TGCT) are considered a paradigm of chemosensitive tumors. Embryonal carcinoma cells represent the pluripotent entity of TGCTs and are characterized by expression of Oct-4, a key regulator of pluripotency and a determinant of their inherent hypersensitivity to cisplatin. However, the mechanisms underlying this Oct-4–mediated sensitivity are poorly understood. We previously showed that p53 is a major player in cisplatin hypersensitivity and therefore investigated whether Oct-4 may directly affect p53 activity. Despite a significant decrease in sensitivity, depletion of Oct-4 neither did alter cisplatin-induced transactivation of p53 target genes nor its subcellular localization. These data indicate that, rather than directly modulating p53 activity, Oct-4 provides a cellular context that augments the proapoptotic activity of p53. As mitochondrial priming by the Bcl-2 family is a known determinant of chemosensitivity, we compared the constitutive levels of these proteins in Oct-4–positive and -depleted cells. We identified Noxa as the only Bcl-2 family protein to be highly correlated with Oct-4 status and cisplatin sensitivity. Compared with differentiated cells, constitutive Noxa levels were significantly higher in Oct-4–positive cell lines and cancer patient samples. Furthermore, RNA interference–mediated knockdown of Oct-4 resulted in reduced Noxa transcript, in an almost complete loss of constitutive Noxa protein and decreased cisplatin hypersensitivity to a similar extent as did Noxa depletion. In conclusion, our study indicates that Noxa is a central determinant of hypersensitivity to cisplatin. Oct-4–dependent high constitutive levels of this BH3-only protein prime embryonal carcinoma cells to undergo rapid and massive apoptosis in response to p53 activation. Cancer Res; 73(5); 1460–9. ©2012 AACR.

Mantle cell lymphoma (MCL) is an aggressive lymphoid neoplasm with transient response to conventional chemotherapy. We here investigated the role of the Bcl-2 homology domain 3-only protein NOXA for life–death decision in MCL. Surprisingly, NOXA (PMAIP1) mRNA and NOXA protein levels were extremely discrepant in MCL cells: NOXA mRNA was found to be highly expressed whereas NOXA protein levels were low. Chronic active B-cell receptor signaling and to a minor degree cyclin D1 overexpression contributed to high NOXA mRNA expression levels in MCL cells. The phoshatidyl-inositol-3 kinase/AKT/ mammalian target of rapamycin pathway was identified as the major downstream signaling pathway involved in the maintenance ofNOXA gene expression. Interestingly, MCL cells adapt to this constitutive pro-apoptotic signal by extensive ubiquitination and rapid proteasomal degradation of NOXA protein (T½B15–30 min). In addition to the proteasome inhibitor Bortezomib, we identified the neddylation inhibitor MLN4924 and the fatty acid synthase inhibitor Orlistat as potent inducers of NOXA protein expression leading to apoptosis in MCL. All inhibitors targeted NOXA protein turnover. In contrast to Bortezomib, MLN4924 and Orlistat interfered with the ubiquitination process of NOXA protein thereby offering new strategies to kill Bortezomib-resistant MCL cells. Our data, therefore, highlight a critical role of NOXA in the balance between life and death in MCL. The discrepancy between NOXA transcript and protein levels is essential for sensitivity of MCL to ubiquitin-proteasome system inhibitors and could therefore provide a druggable Achilles’ heel of MCL cells. Cell Death and Disease (2014) 5, e1013; doi:10.1038/cddis.2013.552; published online 23 January 2014 Subject Category: Cancer

Consistent with the excellent clinical results in testicular germ cell tumors (TGCT), most cell lines derived from this cancer show an exquisite sensitivity to Cisplatin. It is well accepted that the high susceptibility of TGCT cells to apoptosis plays a central role in this hypersensitive phenotype. The role of the tumor suppressor p53 in this response, however, remains controversial. Here we show that siRNA-mediated silencing of p53 is sufficient to completely abrogate hypersensitivity not only to Cisplatin but also to non-genotoxic inducers of p53 such as the Mdm2 antagonist Nutlin-3 and the proteasome inhibitor Bortezomib. The close relationship between p53 protein levels and induction of apoptosis is lost upon short-term differentiation, indicating that this predominant pro-apoptotic function of p53 is unique in pluripotent embryonal carcinoma (EC) cells. RNA interference experiments as well as microarray analysis demonstrated a central role of the pro-apoptotic p53 target gene NOXA in the p53-dependent apoptotic response of these cells. In conclusion, our data indicate that the hypersensitivity of TGCT cells is a result of their unique sensitivity to p53 activation. Furthermore, in the very specific cellular context of germ cell-derived pluripotent EC cells, p53 function appears to be limited to induction of apoptosis. © 2011 Gutekunst et al.