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Classically, it is thought that citrate synthase only works in one direction: to catalyze the production of citrate from acetyl coenzyme A and oxaloacetate in the tricarboxylic acid (TCA) cycle. The TCA cycle can run in reverse to cleave citrate and fix carbon dioxide autotrophically, but this was thought to occur only with alternative enzymes, such as citrate lyase. Now Nunoura et al. and Mall et al. have discovered thermophilic bacteria with highly efficient and reversible citrate synthase that requires reduced ferredoxin (see the Perspective by Ragsdale). This function is undetectable by metagenomics, but classical biochemistry filled in the gaps seen between the genome sequences and the phenotypes of the organisms. The direction of catalysis depends on the availability of organic versus inorganic carbon and reflects a flexible bet-hedging strategy for survival in fluctuating environments. In evolutionary terms, this capacity might predate the classical TCA cycle and is likely to occur in a wide range of anaerobic microorganisms. Science , this issue p. 559 , p. 563 ; see also p. 517 Classical biochemistry reveals the occurrence of an unexpected capacity to reverse the tricarboxylic acid cycle in anaerobic microbes. Inorganic carbon fixation is essential to sustain life on Earth, and the reductive tricarboxylic acid (rTCA) cycle is one of the most ancient carbon fixation metabolisms. A combination of genomic, enzymatic, and metabolomic analyses of a deeply branching chemolithotrophic Thermosulfidibacter takaii ABI70S6 T revealed a previously unknown reversible TCA cycle whose direction was controlled by the available carbon source(s). Under a chemolithoautotrophic condition, a rTCA cycle occurred with the reverse reaction of citrate synthase (CS) and not with the adenosine 5′-triphosphate–dependent citrate cleavage reactions that had been regarded as essential for the conventional rTCA cycle. Phylometabolic evaluation suggests that the TCA cycle with reversible CS may represent an ancestral mode of the rTCA cycle and raises the possibility of a facultatively chemolithomixotrophic origin of life.

Wnt5b is a member of the same family of proteins as Wnt5a, the overexpression of which is associated with cancer aggressiveness. Wnt5b is also suggested to be involved in cancer progression, however, details remain unclarified. We analyzed the biochemical properties of purified Wnt5b and the mode of secretion of Wnt5b by cancer cells. Wnt5b was glycosylated at three asparagine residues and lipidated at one serine residue, and these post‐translational modifications of Wnt5b were essential for secretion. Purified Wnt5b showed Dvl2 phosphorylation and Rac activation abilities to a similar extent as Wnt5a. In cultured‐cell conditioned medium, Wnt5b was detected in supernatant or precipitation fractions that were separated by centrifugation at 100 000 g. In PANC‐1 pancreatic cancer cells, 55% of secreted endogenous Wnt5b was associated with exosomes. Exosomes from wild‐type PANC‐1 cells, but not those from Wnt5b‐knockout PANC‐1 cells, activated Wnt5b signaling in CHO cells and stimulated migration and proliferation of A549 lung adenocarcinoma cells, suggesting that endogenous, Wnt5b‐associated exosomes are active. The exosomes were taken up by CHO cells and immunoelectron microscopy revealed that Wnt5b is indeed associated with exosomes. In Caco‐2 colon cancer cells, most Wnt5b was recovered in precipitation fractions when Wnt5b was ectopically expressed (Caco‐2/Wnt5b cells). Knockdown of TSG101, an exosome marker, decreased the secretion of Wnt5b‐associated exosomes from Caco‐2/Wnt5b cells and inhibited Wnt5b‐dependent cell proliferation. Exosomes secreted from Caco‐2/Wnt5b cells stimulated migration and proliferation of A549 cells. These results suggest that Wnt5b‐associated exosomes promote cancer cell migration and proliferation in a paracrine manner. The secretory modes of Wnt5b varied among cancer cells and Wnt5b‐associated exosomes promote cancer cells migration and proliferation in a paracrine manner.

Immunomodulatory drugs (IMiDs) lenalidomide and pomalidomide show remarkable antitumor activity in multiple myeloma (MM) via directly inhibiting MM-cell growth in the bone marrow (BM) microenvironment and promoting immune effector cell function. They are known to bind to the ubiquitin 3 ligase CRBN complex and thereby triggering degradation of IKZF1/3. In this study, we demonstrate that IMiDs also directly bind and activate zeta-chain-associated protein kinase-70 (Zap-70) via its tyrosine residue phosphorylation in T cells. IMiDs also triggered phosphorylation of Zap-70 in natural killer (NK) cells. Importantly, increased granzyme-B (GZM-B) expression and NK-cell activity triggered by IMiDs is associated with Zap-70 activation and inhibited by Zap-70 knockdown (KD), independent of CRBN. We also demonstrate a second mechanism whereby IMiDs trigger GZM-B and NK cytotoxicity which is CRBN and IKZF3 mediated, and inhibited or enhanced by KD of CRBN or IKZF3, respectively, independent of Zap-70. Our studies therefore show that IMiDs can enhance NK and T-cell cytotoxicity in (1) ZAP-70-mediated CRBN independent, as well as (2) CRBN-mediated ZAP-70 independent mechanisms; and provide the framework for developing novel therapeutics to activate Zap-70 and thereby enhance T and NK anti-MM cytotoxicity.

Wnt5a, a representative Wnt ligand that activates the β-catenin-independent pathway, has been shown to promote tumorigenesis. However, it is unclear where Wnt5a is produced and how it affects colon cancer aggressiveness. In this study, we demonstrate that Wnt5a is expressed in fibroblasts near the luminal side of the tumor, and its depletion suppresses mouse colon cancer formation. To characterize the specific fibroblast subtype, a meta-analysis of human and mouse colon fibroblast single-cell RNA-seq data is performed. The results show that Wnt5a is expressed in hypoxia-induced inflammatory fibroblast (InfFib), accompanied by the activation of HIF2. Moreover, Wnt5a maintains InfFib through the suppression of angiogenesis mediated by soluble VEGF receptor1 (Flt1) secretion from endothelial cells, thereby inducing further hypoxia. InfFib also produces epiregulin, which promotes colon cancer growth. Here, we show that Wnt5a acts on endothelial cells, inducing a hypoxic environment that maintains InfFib, thereby contributing to colon cancer progression through InfFib. Wnt5a is a member of the Wnt family of proteins that mainly activates the noncanonical Wnt signaling pathway. Here, the authors discover that inflammatory fibroblasts expressing Wnt5a, which are induced by hypoxia, contribute to colon cancer progression.

Lung adenocarcinoma is the most common histological type of lung cancer and is classified into adenocarcinoma in situ (AIS), minimally invasive adenocarcinoma (MIA) and invasive adenocarcinoma (IA). Atypical adenomatous hyperplasia (AAH) lesions are possible precursors to adenocarcinoma. However, the mechanism underlying the stepwise continuum of lung adenocarcinoma is unclear. In this study, the involvement of ADP‐ribosylation factor (ARF)‐like (ARL) 4C (ARL4C), a member of the small GTP‐binding protein family, in the progression of lung adenocarcinoma and the possibility of ARL4C as a molecular target for lung cancer therapy were explored. ARL4C was frequently expressed in AAH and ARL4C expression in immortalized human small airway epithelial cells promoted cell proliferation and suppressed cell death. In addition, ARL4C was expressed with increased frequency in AIS, MIA and IA in a stage‐dependent manner, and the expression was correlated with histologic grade, fluorine‐18 fluorodeoxyglucose uptake and poor prognosis. An anti–sense oligonucleotide (ASO) against ARL4C (ARL4C ASO‐1316) inhibited RAS‐related C3 botulinum toxin substrate activity and nuclear import of Yes‐associated protein and transcriptional coactivator with PDZ‐binding motif, and suppressed in vitro proliferation and migration of lung cancer cells with KRAS or epidermal growth factor receptor (EGFR) mutations. In addition, transbronchial administration of ARL4C ASO‐1316 suppressed orthotopic tumor formation induced by these cancer cells. Thus, ARL4C is involved in the initiation of the premalignant stage and is associated with the stepwise continuum of lung adenocarcinoma. ARL4C ASO‐1316 would be useful for lung adenocarcinoma patients expressing ARL4C regardless of the KRAS or EGFR mutation. ARL4C is involved in the initiation of the premalignant stage and is associated with the stepwise continuum of lung adenocarcinoma. Transbronchial administration of ARL4C ASO suppressed orthotopic tumor formation of lung cancer cells.

IntroductionMultiple myeloma (MM) is a malignancy of plasma cells with characteristic bone disease. Despite recent great strides achieved in MM treatment owing to the implementation of new anti-MM agents, MM is still incurable and bone destruction remains a serious unmet issue in patients with MM.ApproachIn this review, we will summarize and discuss the mechanisms of the formation of bone disease in MM and the available preclinical and clinical evidence on the treatment for MM bone disease.ConclusionsMM cells produce a variety of cytokines to stimulate receptor activator of nuclear factor-κB ligand-mediated osteoclastogenesis and suppress osteoblastic differentiation from bone marrow stromal cells, leading to extensive bone destruction with rapid loss of bone. MM cells alter the microenvironment through bone destruction where they colonize, which in turn favors tumor growth and survival, thereby forming a vicious cycle between tumor progression and bone destruction. Denosumab or zoledronic acid is currently recommended to be administered at the start of treatment in newly diagnosed patients with MM with bone disease. Proteasome inhibitors and the anti-CD38 monoclonal antibody daratumumab have been demonstrated to exert bone-modifying activity in responders. Besides their anti-tumor activity, the effects of new anti-MM agents on bone metabolism should be more precisely analyzed in patients with MM. Because prognosis in patients with MM has been significantly improved owing to the implementation of new agents, the therapeutic impact of bone-modifying agents should be re-estimated in the era of these new agents.

Multiple myeloma (MM) is an incurable cancer that derives pro-survival/proliferative signals from the bone marrow (BM) niche. Novel agents targeting not only cancer cells, but also the BM-niche have shown the greatest activity in MM. Histone deacetylases (HDACs) are therapeutic targets in MM and we previously showed that HDAC3 inhibition decreases MM proliferation both alone and in co-culture with bone marrow stromal cells (BMSC). In this study, we investigate the effects of HDAC3 targeting in BMSCs. Using both BMSC lines as well as patient-derived BMSCs, we show that HDAC3 expression in BMSCs can be induced by co-culture with MM cells. Knock-out (KO), knock-down (KD), and pharmacologic inhibition of HDAC3 in BMSCs results in decreased MM cell proliferation; including in autologous cultures of patient MM cells with BMSCs. We identified both quantitative and qualitative changes in exosomes and exosomal miRNA, as well as inhibition of IL-6 trans-signaling, as molecular mechanisms mediating anti-MM activity. Furthermore, we show that HDAC3-KD in BM endothelial cells decreases neoangiogenesis, consistent with a broad effect of HDAC3 targeting in the BM-niche. Our results therefore support the clinical development of HDAC3 inhibitors based not only on their direct anti-MM effects, but also their modulation of the BM microenvironment.

Genetically modified nonhuman primates (NHP) are useful models for biomedical research. Gene editing technologies have enabled production of target-gene knock-out (KO) NHP models. Target-gene-KO/knock-in (KI) efficiency of CRISPR/Cas9 has not been extensively investigated in marmosets. In this study, optimum conditions for target gene modification efficacies of CRISPR/mRNA and CRISPR/nuclease in marmoset embryos were examined. CRISPR/nuclease was more effective than CRISPR/mRNA in avoiding mosaic genetic alteration. Furthermore, optimal conditions to generate KI marmoset embryos were investigated using CRISPR/Cas9 and 2 different lengths (36 nt and 100 nt) each of a sense or anti-sense single-strand oligonucleotide (ssODN). KIs were observed when CRISPR/nuclease and 36 nt sense or anti-sense ssODNs were injected into embryos. All embryos exhibited mosaic mutations with KI and KO, or imprecise KI, of c-kit . Although further improvement of KI strategies is required, these results indicated that CRISPR/Cas9 may be utilized to produce KO/KI marmosets via gene editing.

Hyperthermia is a unique treatment option for cancers. Multiple myeloma (MM) remains incurable and innovative therapeutic options are needed. We investigated the efficacy of hyperthermia and carfilzomib in combination against MM cells. Although MM cell lines exhibited different susceptibilities to pulsatile carfilzomib treatment, mild hyperthermia at 43℃ induced MM cell death in all cell lines in a time-dependent manner. Hyperthermia and carfilzomib cooperatively induced MM cell death even under suboptimal conditions. The pro-survival mediators PIM2 and NRF2 accumulated in MM cells due to inhibition of their proteasomal degradation by carfilzomib; however, hyperthermia acutely suppressed translation in parallel with phosphorylation of eIF2α to reduce these proteins in MM cells. Recovery of β5 subunit enzymatic activity from its immediate inhibition by carfilzomib was observed at 24 h in carfilzomib-insusceptible KMS-11, OPM-2, and RPMI8226 cells, but not in carfilzomib-sensitive MM.1S cells. However, heat treatment suppressed the recovery of β5 subunit activity in these carfilzomib-insusceptible cells. Therefore, hyperthermia re-sensitized MM cells to carfilzomib. Our results support the treatment of MM with hyperthermia in combination with carfilzomib. Further research is warranted on hyperthermia for drug-resistant extramedullary plasmacytoma.

Certain methanogens deteriorate steel surfaces through a process called microbiologically influenced corrosion (MIC). However, the mechanisms of MIC, whereby methanogens oxidize zerovalent iron (Fe 0 ), are largely unknown. In this study, Fe 0 -corroding Methanococcus maripaludis strain OS7 and its derivative (strain OS7mut1) defective in Fe 0 -corroding activity were isolated. Genomic analysis of these strains demonstrated that the strain OS7mut1 contained a 12-kb chromosomal deletion. The deleted region, termed “MIC island”, encoded the genes for the large and small subunits of a [NiFe] hydrogenase, the TatA/TatC genes necessary for the secretion of the [NiFe] hydrogenase, and a gene for the hydrogenase maturation protease. Thus, the [NiFe] hydrogenase may be secreted outside the cytoplasmic membrane, where the [NiFe] hydrogenase can make direct contact with Fe 0 , and oxidize it, generating hydrogen gas: Fe 0  + 2 H +  → Fe 2+  + H 2 . Comparative analysis of extracellular and intracellular proteomes of strain OS7 supported this hypothesis. The identification of the MIC genes enables the development of molecular tools to monitor epidemiology, and to perform surveillance and risk assessment of MIC-inducing M. maripaludis .