Catherine Chang

The MCL1 inhibitor S63845 is tolerable and effective in diverse cancer models.

Avoidance of apoptosis is critical for the development and sustained growth of tumours. The pro-survival protein myeloid cell leukemia 1 (MCL1) is overexpressed in many cancers, but the development of small molecules targeting this protein that are amenable for clinical testing has been challenging. Here we describe S63845, a small molecule that specifically binds with high affinity to the BH3-binding groove of MCL1. Our mechanistic studies demonstrate that S63845 potently kills MCL1-dependent cancer cells, including multiple myeloma, leukaemia and lymphoma cells, by activating the BAX/BAK-dependent mitochondrial apoptotic pathway. In vivo, S63845 shows potent anti-tumour activity with an acceptable safety margin as a single agent in several cancers. Moreover, MCL1 inhibition, either alone or in combination with other anti-cancer drugs, proved effective against several solid cancer-derived cell lines. These results point towards MCL1 as a target for the treatment of a wide range of tumours.

Prostacyclin signaling boosts NADPH oxidase 4 in the endothelium promoting cytoprotection and angiogenesis.

AIMS Prostacyclin (PGI2) that is released from the vascular endothelium plays an important role in vasodilatation and thrombo-resistance, and it has long been suspected to protect cell survival. How it does so has never been clear. Recently, it has been shown that the NADPH oxidase 4 (Nox4) improves endothelial cell functions and promotes angiogenesis in vivo, but it was not known how to boost Nox4 therapeutically to exploit its protective functions in the vasculature. Here, we identified such a stimulus. RESULTS The selective and stable prostacyclin receptor (IP-R) agonist cicaprost increases the expression of Nox4 in human endothelial cells of several types, including endothelial progenitor cells. The elevation of cellular cyclic-AMP increased Nox4 expression and H2O2 production and prevented endothelial cell apoptosis. We delineate the intracellular signaling that promotes cytoprotection: Cicaprost acts via the IP-R/protein kinase A (PKA)/cyclic adenosine monophosphate (cAMP) response element binding (CREB) protein pathway. Importantly, the up-regulation of Nox4 by cicaprost also enhanced endothelial cell proliferation, migration, and angiogenesis, with all effects being substantially decreased by Nox4 gene silencing. Finally, cicaprost enhanced the growth of blood vessels into subcutaneous sponges implanted in mice, an effect that was also blocked by Nox4 gene silencing. INNOVATION The prostacyclin analogue cicaprost induces Nox4 via IP receptor-cAMP/PKA/CREB pathway. The activation of this pathway protects endothelial cells and enhances pro-angiogenic activity both in vitro and in vivo. CONCLUSION Prostacyclin promotes the up-regulation of Nox4 in endothelial cells, which opens up a novel strategy that protects and enhances endothelial cell functions in cardiovascular disease, such as repair after myocardial infarction or other ischemic conditions.

Cytoprotection by Natural and Synthetic Polyphenols in the Heart: Novel Mechanisms and Perspectives

While many naturally occurring polyphenols have been shown to have therapeutic benefits against myocardial injury following ischemia-reperfusion in various experimental models, our studies have demonstrated that synthetic flavonoids may also have potent cardiac cytoprotective actions. Together with the results reported in the literature, we suggest that synthetic polyphenols may be an ideal replacement for natural compounds in the development of myocardial protective drugs. Polyphenols exert myocardial protective effects via antioxidant activities, preservation of nitric oxide, antiinflammatory activities and modulation of matrix metalloproteinases. Recent studies have identified some novel mechanisms that may also contribute to polyphenol-induced myocardial protection, including prevention of mitochondrial dysfunction, pharmacological preconditioning, and modulation of the function of enzymes involved in epigenetic modifications such as histone acetyltransferases. In addition to the protective effects against acute myocardial injury, there has been experimental evidence showing that polyphenols may also modulate the development of cardiac hypertrophy, ventricular remodeling and fibrosis after myocardial infarction.

MCL-1 is required throughout B-cell development and its loss sensitizes specific B-cell subsets to inhibition of BCL-2 or BCL-XL

Pro-survival BCL-2 family members protect cells from programmed cell death that can be induced by multiple internal or external cues. Within the haematopoietic lineages, the BCL-2 family members BCL-2, BCL-XL and MCL-1 are known to support cell survival but the individual and overlapping roles of these pro-survival BCL-2 proteins for the persistence of individual leukocyte subsets in vivo has not yet been determined. By combining inducible knockout mouse models with the BH3-mimetic compound ABT-737, which inhibits BCL-2, BCL-XL and BCL-W, we found that dependency on MCL-1, BCL-XL or BCL-2 expression changes during B-cell development. We show that BCL-XL expression promotes survival of immature B cells, expression of BCL-2 is important for survival of mature B cells and long-lived plasma cells (PC), and expression of MCL-1 is important for survival throughout B-cell development. These data were confirmed with novel highly specific BH3-mimetic compounds that target either BCL-2, BCL-XL or MCL-1. In addition, we observed that combined inhibition of these pro-survival proteins acts in concert to delete specific B-cell subsets. Reduced expression of MCL-1 further sensitized immature as well as transitional B cells and splenic PC to loss of BCL-XL expression. More markedly, loss of MCL-1 greatly sensitizes PC populations to BCL-2 inhibition using ABT-737, even though the total wild-type PC pool in the spleen is not significantly affected by this drug and the bone marrow (BM) PC population only slightly. Combined loss or inhibition of MCL-1 and BCL-2 reduced the numbers of established PC >100-fold within days. Our data suggest that combination treatment targeting these pro-survival proteins could be advantageous for treatment of antibody-mediated autoimmune diseases and B-cell malignancies.

Coordinated repression of BIM and PUMA by Epstein–Barr virus latent genes maintains the survival of Burkitt lymphoma cells

While the association of Epstein–Barr virus (EBV) with Burkitt lymphoma (BL) has long been recognised, the precise role of the virus in BL pathogenesis is not fully resolved. EBV can be lost spontaneously from some BL cell lines, and these EBV-loss lymphoma cells reportedly have a survival disadvantage. Here we have generated an extensive panel of EBV-loss clones from multiple BL backgrounds and examined their phenotype comparing them to their isogenic EBV-positive counterparts. We report that, while loss of EBV from BL cells is rare, it is consistently associated with an enhanced predisposition to undergo apoptosis and reduced tumorigenicity in vivo. Importantly, reinfection of EBV-loss clones with EBV, but surprisingly not transduction with individual BL-associated latent viral genes, restored protection from apoptosis. Expression profiling and functional analysis of apoptosis-related proteins and transcripts in BL cells revealed that EBV inhibits the upregulation of the proapoptotic BH3-only proteins, BIM and PUMA. We conclude that latent EBV genes cooperatively enhance the survival of BL cells by suppression of the intrinsic apoptosis pathway signalling via inhibition of the potent apoptosis initiators, BIM and PUMA.

ASCIZ/ATMIN is dispensable for ATM signaling in response to replication stress

Highlights • ASCIZ/ATMIN is not required for ATM activation by replication stress in MEFs.• ATM activation is normal in human ASCIZ/ATMIN KO cells.• ASCIZ/ATMIN is dispensable for aphidicolin-induced 53BP1 focus formation.

Recombinant expression of Proteorhodopsin and biofilm regulators in Escherichia coli for nanoparticle binding and removal in a wastewater treatment model

The small size of nanoparticles is both an advantage and a problem. Their high surface-area-to-volume ratio enables novel medical, industrial, and commercial applications. However, their small size also allows them to evade conventional filtration during water treatment, posing health risks to humans, plants, and aquatic life. This project aims to remove nanoparticles during wastewater treatment using genetically modified Escherichia coli in two ways: 1) binding citrate-capped nanoparticles with the membrane protein Proteorhodopsin, and 2) trapping nanoparticles using Escherichia coli biofilm produced by overexpressing two regulators: OmpR234 and CsgD. We demonstrate experimentally that Escherichia coli expressing Proteorhodopsin binds to 60 nm citrate-capped silver nanoparticles. We also successfully upregulate biofilm production and show that Escherichia coli biofilms are able to trap 30 nm gold particles. Finally, both Proteorhodopsin and biofilm approaches are able to bind and remove nanoparticles in simulated wastewater treatment tanks. We envision integrating our trapping system in both rural and urban wastewater treatment plants to efficiently capture all nanoparticles before treated water is released into the environment. Financial Disclosure This work was funded by the Taipei American School. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests The authors have declared that no competing interests exist. Ethics Statement N/A Data Availability Yes – all data are fully available without restriction. Sequences for the plasmids used in this study are available through the Registry of Standard Biological Parts. Links to raw data are included in Supplementary Information.

BAX requires VDAC2 to mediate apoptosis and to limit tumor development

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.

Mutant TRP53 exerts a target gene-selective dominant-negative effect to drive tumor development

Mutations in Trp53, prevalent in human cancer, are reported to drive tumorigenesis through dominant-negative effects (DNEs) over wild-type TRP53 function as well as neomorphic gain-of-function (GOF) activity. We show that five TRP53 mutants do not accelerate lymphomagenesis on a TRP53-deficient background but strongly synergize with c-MYC overexpression in a manner that distinguishes the hot spot Trp53 mutations. RNA sequencing revealed that the mutant TRP53 DNE does not globally repress wild-type TRP53 function but disproportionately impacts a subset of wild-type TRP53 target genes. Accordingly, TRP53 mutant proteins impair pathways for DNA repair, proliferation, and metabolism in premalignant cells. This reveals that, in our studies of lymphomagenesis, mutant TRP53 drives tumorigenesis primarily through the DNE, which modulates wild-type TRP53 function in a manner advantageous for neoplastic transformation.

Humanized Mcl-1 mice enable accurate preclinical evaluation of MCL-1 inhibitors destined for clinical use

Myeloid cell leukemia-1 (MCL-1) is a prosurvival B-cell lymphoma 2 (BCL-2) family member required for the sustained growth of many cancers. Recently, a highly specific MCL-1 inhibitor, S63845, showing sixfold higher affinity to human compared with mouse MCL-1, has been described. To accurately test efficacy and tolerability of this BH3-mimetic (BH3-only protein mimetic) drug in preclinical cancer models, we developed a humanized Mcl-1 (huMcl-1) mouse strain in which MCL-1 was replaced with its human homolog. huMcl-1 mice are phenotypically indistinguishable from wild-type mice but are more sensitive to the MCL-1 inhibitor S63845. Importantly, nontransformed cells and lymphomas from huMcl-1;Eµ-Myc mice are more sensitive to S63845 in vitro than their control counterparts. When huMcl-1;Eµ-Myc lymphoma cells were transplanted into huMcl-1 mice, treatment with S63845 alone or alongside cyclophosphamide led to long-term remission in ∼60% or almost 100% of mice, respectively. These results demonstrate the potential of our huMcl-1 mouse model for testing MCL-1 inhibitors, allowing precise predictions of efficacy and tolerability for clinical translation.