Negative regulation of SH2B3 by SMYD5 controls epithelial-mesenchymal transition in lung cancer.

The main cause of death in lung cancer patients is metastasis. Thus, efforts to suppress micrometastasis or distant metastasis in lung cancer, identify therapeutic targets and develop related drugs are ongoing. In this study, we identified SMYD5 as a novel metastasis regulator in lung cancer and found that SMYD5 was overexpressed in lung cancer based on both RNA-sequencing (RNA-seq) analysis results derived from the TCGA portal and immunohistochemical analysis results; knockdown of SMYD5 inhibited cell migration and invasion by changing EMT (epithelial-mesenchymal transition) markers and MMP9 expression in NCI-H1299 and H1703 cell lines. Additionally, SMYD5 knockdown increased SH2B3 expression by decreasing the level of H4K20 trimethylation. Furthermore, in an in vitro EMT system using TGF-ß treatment, SMYD5 knockdown resulted in reduced cell migration and invasion in the highly invasive NCI-H1299 and H1703 cell lines. Based on these findings, we propose that SMYD5 could serve as a potential therapeutic target for lung cancer treatment and that cotreatment with an SMYD5 inhibitor and chemotherapy may enhance the therapeutic effect of lung cancer treatment.

TAZ deficiency exacerbates psoriatic pathogenesis by increasing the histamine-releasing factor.

BACKGROUND: Transcriptional coactivator with PDZ-biding motif (TAZ) is widely expressed in most tissues and interacts with several transcription factors to regulate cell proliferation, differentiation, and death, thereby influencing organ development and size control. However, very little is known about the function of TAZ in the immune system and its association with inflammatory skin diseases, so we investigated the role of TAZ in the pathogenesis of psoriasis. RESULTS: Interestingly, TAZ was expressed in mast cells associated, particularly in lysosomes, and co-localized with histamine-releasing factor (HRF). TAZ deficiency promoted mast cell maturation and increased HRF expression and secretion by mast cells. The upregulation of HRF in TAZ deficiency was not due to increased transcription but to protein stabilization, and TAZ restoration into TAZ-deficient cells reduced HRF protein. Interestingly, imiquimod (IMQ)-induced psoriasis, in which HRF serves as a major pro-inflammatory factor, was more severe in TAZ KO mice than in WT control. HRF expression and secretion were increased by IMQ treatment and were more pronounced in TAZ KO mice treated with IMQ. CONCLUSIONS: Thus, as HRF expression was stabilized in TAZ KO mice, psoriatic pathogenesis progressed more rapidly, indicating that TAZ plays an important role in preventing psoriasis by regulating HRF protein stability.

Comprehensive insights into AML relapse: genetic mutations, clonal evolution, and clinical outcomes.

INTRODUCTION: Acute myeloid leukemia (AML) is a complex hematologic malignancy characterized by uncontrolled proliferation of myeloid precursor cells within bone marrow. Despite advances in understanding of its molecular underpinnings, AML remains a therapeutic challenge due to its high relapse rate and clonal evolution. METHODS: In this retrospective study, we analyzed data from 24 AML patients diagnosed at a single institution between January 2017 and August 2023. Comprehensive genetic analyses, including chromosomal karyotyping, next-generation sequencing, and gene fusion assays, were performed on bone marrow samples obtained at initial diagnosis and relapse. Clinical data, treatment regimens, and patient outcomes were also documented. RESULTS: Mutations in core genes of FLT3, NPM1, DNMT3A, and IDH2 were frequently discovered in diagnostic sample and remained in relapse sample. FLT3-ITD, TP53, KIT, RUNX1, and WT1 mutation were acquired at relapse in one patient each. Gene fusion assays revealed stable patterns, while chromosomal karyotype analyses indicated a greater diversity of mutations in relapsed patients. Clonal evolution patterns varied, with some cases showing linear or branching evolution and others exhibiting no substantial change in core mutations between diagnosis and relapse. CONCLUSIONS: Our study integrates karyotype, gene rearrangements, and gene mutation results to provide a further understanding of AML heterogeneity and evolution. We demonstrate the clinical relevance of specific mutations and clonal evolution patterns, emphasizing the need for personalized therapies and measurable residual disease monitoring in AML management. By bridging the gap between genetics and clinical outcome, we move closer to tailored AML therapies and improved patient prognoses.

Clinical outcomes of etoposide and cytarabine as consolidation in elderly patients with primary CNS lymphoma.

BACKGROUND: A consolidation strategy has not been established for transplant-ineligible elderly patients with primary central nervous system lymphoma (PCNSL). In this study, we aimed to retrospectively evaluate the clinical outcomes of etoposide and cytarabine (EA) as consolidation chemotherapy for transplant-ineligible patients with PCNSL following high-dose methotrexate (MTX)-based induction chemotherapy. MATERIALS AND METHODS: Between 2015 and 2021, newly diagnosed transplant-ineligible patients with PCNSL with diffuse large B-cell lymphoma were consecutively enrolled. All enrolled patients were over 60 years old and received EA consolidation after achieving a complete or partial response following induction chemotherapy. RESULTS: Of the 85 patients who achieved a complete or partial response to MTX-based induction chemotherapy, 51 received EA consolidation chemotherapy. Among the 25 (49.0%, 25/51) patients in partial remission before EA consolidation, 56% (n = 14) achieved complete remission after EA consolidation. The median overall survival and progression-free survival were 43 and 13 months, respectively. Hematological toxicities were most common, and all patients experienced grade 4 neutropenia and thrombocytopenia. Forty-eight patients experienced febrile neutropenia during consolidation chemotherapy, and 4 patients died owing to treatment-related complications. CONCLUSION: EA consolidation chemotherapy for transplant-ineligible, elderly patients with PCNSL improved response rates but showed a high relapse rate and short progression-free survival. The incidences of treatment-related mortality caused by hematologic toxicities and severe infections were very high, even after dose modification. Therefore, the use of EA consolidation should be reconsidered in elderly patients with PCNSL.

3D Amplified Single-Cell RNA and Protein Imaging Identifies Oncogenic Transcript Subtypes in B-Cell Acute Lymphoblastic Leukemia.

Simultaneous in situ detection of transcript and protein markers at the single-cell level is essential for gaining a better understanding of tumor heterogeneity and for predicting and monitoring treatment responses. However, the limited accessibility to advanced 3D imaging techniques has hindered their rapid implementation. Here, we present a 3D single-cell imaging technique, termed 3D digital rolling circle amplification (4DRCA), capable of the multiplexed and amplified simultaneous digital quantification of single-cell RNAs and proteins using standard fluorescence microscopy and off-the-shelf reagents. We generated spectrally distinguishable DNA amplicons from molecular markers through an integrative protocol combining single-cell RNA and protein assays and directly enumerated the amplicons by leveraging an open-source algorithm for 3D deconvolution with a custom-built automatic gating algorithm. With 4DRCA, we were able to simultaneously quantify surface protein markers and cytokine transcripts in T-lymphocytes. We also show that 4DRCA can distinguish BCR-ABL1 fusion transcript positive B-cell acute lymphoblastic leukemia cells with or without CD19 protein expression. The accessibility and extensibility of 4DRCA render it broadly applicable to other cell-based diagnostic workflows, enabling sensitive and accurate single-cell RNA and protein profiling.

Chemically-defined and scalable culture system for intestinal stem cells derived from human intestinal organoids.

Three-dimensional human intestinal organoids (hIO) are widely used as a platform for biological and biomedical research. However, reproducibility and challenges for large-scale expansion limit their applicability. Here, we establish a human intestinal stem cell (ISC) culture method expanded under feeder-free and fully defined conditions through selective enrichment of ISC populations (ISC3D-hIO) within hIO derived from human pluripotent stem cells. The intrinsic self-organisation property of ISC3D-hIO, combined with air-liquid interface culture in a minimally defined medium, forces ISC3D-hIO to differentiate into the intestinal epithelium with cellular diversity, villus-like structure, and barrier integrity. Notably, ISC3D-hIO is an ideal cell source for gene editing to study ISC biology and transplantation for intestinal diseases. We demonstrate the intestinal epithelium differentiated from ISC3D-hIO as a model system to study severe acute respiratory syndrome coronavirus 2 viral infection. ISC3D-hIO culture technology provides a biological tool for use in regenerative medicine and disease modelling.

Clearing soluble MIC reverses the impaired function of natural killer cells from patients with multiple myeloma.

BACKGROUND: Major histocompatibility complex (MHC) class I chain-related protein (MIC) is a stress-induced ligand released from multiple myeloma (MM) cells during progression, and soluble MIC impairs natural killer group 2D (NKG2D) activating receptor-mediated recognition and function of natural killer (NK) cells. However, whether clearing soluble MIC with a monoclonal antibody (mAb) can restore NK cell activity of MM patients remains undetermined. METHODS: We analyzed The Cancer Genome Atlas (TCGA) Multiple Myeloma Research Foundation (MMRF) CoMMpass data set to examine the prognostic significance of MIC expression in MM. We examined the level of soluble MIC in paired peripheral blood (PB) and bone marrow (BM) plasma of patients with MM at diagnosis by ELISA. We evaluated the correlation between the level of soluble MIC and immunophenotype of NK cells from MM patients by multicolor flow cytometry. We also generated MIC-overexpressing MM cell line and characterized the cytotoxic function of patient NK cells in the presence of soluble MIC, and examined the impact of clearing soluble MIC with a humanized mAb (huB10G5). RESULTS: We characterize the importance of MICA in MM by revealing the significantly better overall survival of patients with high MICA expression from TCGA MMRF CoMMpass data set. The level of soluble MICA is more highly elevated in MM than in precursor stages, and the concentration of soluble MICA is higher in BM plasma than in PB. The concentration of soluble MICA in BM was correlated with myeloma burden, while it was negatively correlated with the frequency of NKG2D+ NK cells in diagnostic BM aspirates of MM patients. Soluble MICA downregulated NKG2D expression and decreased cytotoxicity of MM patient NK cells ex vivo, which were reversed by a humanized soluble MIC-clearing mAb (huB10G5) with enhanced degranulation of NK cells. CONCLUSIONS: Our findings indicate targeting soluble MIC with huB10G5 might be a viable therapeutic approach to promote NKG2D-dependent cellular immunotherapy outcome in MM.

Efficient and reproducible generation of human induced pluripotent stem cell-derived expandable liver organoids for disease modeling.

Genetic liver disease modeling is difficult because it is challenging to access patient tissue samples and to develop practical and relevant model systems. Previously, we developed novel proliferative and functional liver organoids from pluripotent stem cells; however, the protocol requires improvement for standardization and reproducible mass production. Here, we improved the method such that it is suitable for scalable expansion and relatively homogenous production, resulting in an efficient and reproducible process. Moreover, three medium components critical for long-term expansion were defined. Detailed transcriptome analysis revealed that fibroblast growth factor signaling, the essential pathway for hepatocyte proliferation during liver regeneration, was mainly enriched in proliferative liver organoids. Short hairpin RNA-mediated knockdown of FGFR4 impaired the generation and proliferation of organoids. Finally, glycogen storage disease type Ia (GSD1a) patient-specific liver organoids were efficiently and reproducibly generated using the new protocol. They well maintained disease-specific phenotypes such as higher lipid and glycogen accumulation in the liver organoids and lactate secretion into the medium consistent with the main pathologic characteristics of patients with GSD1a. Therefore, our newly established liver organoid platform can provide scalable and practical personalized disease models and help to find new therapies for incurable liver diseases including genetic liver diseases.

Recent advances in cellular immunotherapy for lymphoid malignancies.

Cellular immunotherapy with chimeric antigen receptor (CAR) T-cells has revolutionized the treatment of lymphoid malignancies. This review addresses the need for CAR expression in our endogenous T-cells to kill tumor cells with a focus on the basic principles of T-cell receptor recognition of major histocompatibility complex-peptide complexes. We review the factors associated with CAR T-cell outcomes and recent efforts to employ CAR T-cells in earlier lines of therapy. We also discuss the value of bispecific T-cell engagers as off-the-shelf products with better toxicity profiles. Finally, natural killer cells are discussed as an important cellular immunotherapy platform with the potential to broaden immunotherapeutic applications beyond lymphoid malignancies.

A bivalent form of a RBD-specific synthetic antibody effectively neutralizes SARS-CoV-2 variants.

Coronavirus Disease 2019 (COVID-19) pandemic is severely impacting the world, and tremendous efforts have been made to deal with it. Despite many advances in vaccines and therapeutics, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants remains an intractable challenge. We present a bivalent Receptor Binding Domain (RBD)-specific synthetic antibody, specific for the RBD of wild-type (lineage A), developed from a non-antibody protein scaffold composed of LRR (Leucine-rich repeat) modules through phage display. We further reinforced the unique feature of the synthetic antibody by constructing a tandem dimeric form. The resulting bivalent form showed a broader neutralizing activity against the variants. The in vivo neutralizing efficacy of the bivalent synthetic antibody was confirmed using a human ACE2-expressing mouse model that significantly alleviated viral titer and lung infection. The present approach can be used to develop a synthetic antibody showing a broader neutralizing activity against a multitude of SARS-CoV-2 variants.

A natural variation in the RNA polymerase of severe fever with thrombocytopenia syndrome virus enhances viral replication and in vivo virulence.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne disease with high mortality in Eastern Asia. The disease is caused by the SFTS virus (SFTSV), also known as Dabie bandavirus, which has a segmented RNA genome consisting of L, M, and S segments. Previous studies have suggested differential viral virulence depending on the genotypes of SFTSV; however, the critical viral factor involved in the differential viral virulence is unknown. Here, we found a significant difference in viral replication in vitro and virulence in vivo between two Korean isolates belonging to the F and B genotypes, respectively. By generating viral reassortants using the two viral strains, we demonstrated that the L segment, which encodes viral RNA-dependent RNA polymerase (RdRp), is responsible for the enhanced viral replication and virulence. Comparison of amino acid sequences and viral replication rates revealed a point variation, E251K, on the surface of RdRp to be the most significant determinant for the enhanced viral replication rate and in vivo virulence. The effect of the variation was further confirmed using recombinant SFTSV generated by reverse genetic engineering. Therefore, our results indicate that natural variations affecting the viral replicase activity could significantly contribute to the viral virulence of SFTSV.

Structure-based drug discovery of a corticotropin-releasing hormone receptor 1 antagonist using an X-ray free-electron laser.

Thus far, attempts to develop drugs that target corticotropin-releasing hormone receptor 1 (CRF1R), a drug target in stress-related therapy, have been unsuccessful. Studies have focused on using high-resolution G protein-coupled receptor (GPCR) structures to develop drugs. X-ray free-electron lasers (XFELs), which prevent radiation damage and provide access to high-resolution compositions, have helped accelerate GPCR structural studies. We elucidated the crystal structure of CRF1R complexed with a BMK-I-152 antagonist at 2.75 Å using fixed-target serial femtosecond crystallography. The results revealed that two unique hydrogen bonds are present in the hydrogen bond network, the stalk region forms an alpha helix and the hydrophobic network contains an antagonist binding site. We then developed two antagonists-BMK-C203 and BMK-C205-and determined the CRF1R/BMK-C203 and CRF1R/BMK-C205 complex structures at 2.6 and 2.2 Å, respectively. BMK-C205 exerted significant antidepressant effects in mice and, thus, may be utilized to effectively identify structure-based drugs against CRF1R.

Utility of Plasma Microbial Cell-Free DNA Whole-Genome Sequencing for Diagnosis of Invasive Aspergillosis in Patients With Hematologic Malignancy or COVID-19.

BACKGROUND: We evaluated the clinical accuracy and utility of whole-genome sequencing (WGS) of plasma microbial cell-free DNA (cfDNA) as a novel noninvasive method in diagnosing invasive aspergillosis (IA) in patients with hematologic malignancy (HM) or coronavirus disease 2019 (COVID-19). METHODS: Adults with HM or COVID-19 and suspected IA were recruited. IA cases were retrospectively diagnosed according to EORTC/MSG definitions and ECMM/ISHAM criteria for HM and COVID-19 patients, respectively. The results of cfDNA WGS were compared with the conventional diagnosis. RESULTS: Microbial cfDNA WGS was performed 53 times from 41 participants (19 from HM, 16 from COVID-19, and 7 from the control group). In participants with HM, Aspergillus cfDNA was detected in 100% of proven IA and 91.7% of probable IA cases. In participants with COVID-19, 50.0% of probable IA were positive for Aspergillus in cfDNA WGS. Concordance between Aspergillus cfDNA detection and proven/probable IA conventional diagnosis was significantly higher in participants with HM than in those with COVID-19. IA diagnosed using EORTC/MGS definitions showed significantly high concordance between Aspergillus cfDNA detection and proven/probable IA. CONCLUSIONS: Aspergillus cfDNA detection strongly correlated with proven/probable IA diagnosed using EORTC/MSG definitions and could be used as an additional diagnostic tool for IA.

Anticancer Effects of Gut Microbiota-Derived Short-Chain Fatty Acids in Cancers.

Short-chain fatty acids (SCFAs), such as butyrate, propionate, and acetate produced by the gut microbiota have been implicated in physiological responses (defense mechanisms, immune responses, and cell metabolism) in the human body. In several types of cancers, SCFAs, especially butyrate, suppress tumor growth and cancer cell metastasis via the regulation of the cell cycle, autophagy, cancer-related signaling pathways, and cancer cell metabolism. In addition, combination treatment with SCFAs and anticancer drugs exhibits synergistic effects, increasing anticancer treatment efficiency and attenuating anticancer drug resistance. Therefore, in this review, we point out the importance of SCFAs and the mechanisms underlying their effects in cancer treatment and suggest using SCFA-producing microbes and SCFAs to increase therapeutic efficacy in several types of cancers.

Epigenetic regulation of DHRS2 by SUV420H2 inhibits cell apoptosis in renal cell carcinoma.

Renal cell carcinoma (RCC), also known as kidney cancer, is a common malignant tumor of the urinary system. While surgical treatment is essential, novel therapeutic targets and corresponding drugs for RCC are still needed due to the high relapse rate and low five-year survival rate. In this study, we found that SUV420H2 is overexpressed in renal cancers and that high SUV420H2 expression is associated with a poor prognosis, as evidenced by RCC RNA-seq results derived from the TCGA. SUV420H2 knockdown using siRNA led to growth suppression and cell apoptosis in the A498 cell line. Furthermore, we identified DHRS2 as a direct target of SUV420H2 in the apoptosis process through a ChIP assay with a histone 4 lysine 20 (H4K20) trimethylation antibody. Rescue experiments showed that cotreatment with siSUV420H2 and siDHRS2 attenuated cell growth suppression induced by SUV420H2 knockdown only. Additionally, treatment with the SUV420H2 inhibitor A-196 induced cell apoptosis via upregulation of DHRS2. Taken together, our findings suggest that SUV420H2 may be a potential therapeutic target for the treatment of renal cancer.

Oligomeric states of ASC specks regulate inflammatory responses by inflammasome in the extracellular space.

Inflammasomes are multi-protein complexes and play a crucial role in host defense against pathogens. Downstream inflammatory responses through inflammasomes are known to be related to the oligomerization degree of ASC specks, but the detailed mechanism still remains unexplored. Here, we demonstrate that oligomerization degrees of ASC specks regulate the caspase-1 activation in the extracellular space. A protein binder specific for a pyrin domain (PYD) of ASC (ASCPYD) was developed, and structural analysis revealed that the protein binder effectively inhibits the interaction between PYDs, disassembling ASC specks into low oligomeric states. ASC specks with a low oligomerization degree were shown to enhance the activation of caspase-1 by recruiting and processing more premature caspase-1 through interactions between CARD of caspase-1 (caspase-1CARD) and CARD of ASC (ASCCARD). These findings can provide insight into controlling the inflammasome-mediated inflammatory process as well as the development of inflammasome-targeting drugs.

Epigenetic regulation of SMAD3 by histone methyltransferase SMYD2 promotes lung cancer metastasis.

Epigenetic alterations, especially histone methylation, are key factors in cell migration and invasion in cancer metastasis. However, in lung cancer metastasis, the mechanism by which histone methylation regulates metastasis has not been fully elucidated. Here, we found that the histone methyltransferase SMYD2 is overexpressed in lung cancer and that knockdown of SMYD2 could reduce the rates of cell migration and invasion in lung cancer cell lines via direct downregulation of SMAD3 via SMYD2-mediated epigenetic regulation. Furthermore, using an in vitro epithelial-mesenchymal transition (EMT) system with a Transwell system, we generated highly invasive H1299 (In-H1299) cell lines and observed the suppression of metastatic features by SMYD2 knockdown. Finally, two types of in vivo studies revealed that the formation of metastatic tumors by shSMYD2 was significantly suppressed. Thus, we suggest that SMYD2 is a potential metastasis regulator and that the development of SMYD2-specific inhibitors may help to increase the efficacy of lung cancer treatment.

Novel bispecific human antibody platform specifically targeting a fully open spike conformation potently neutralizes multiple SARS-CoV-2 variants.

Rapid emergence of new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has prompted an urgent need for the development of broadly applicable and potently neutralizing antibody platform against the SARS-CoV-2, which can be used for combatting the coronavirus disease 2019 (COVID-19). In this study, based on a noncompeting pair of phage display-derived human monoclonal antibodies (mAbs) specific to the receptor-binding domain (RBD) of SARS-CoV-2 isolated from human synthetic antibody library, we generated K202.B, a novel engineered bispecific antibody with an immunoglobulin G4-single-chain variable fragment design, with sub- or low nanomolar antigen-binding avidity. Compared with the parental mAbs or mAb cocktail, the K202.B antibody showed superior neutralizing potential against a variety of SARS-CoV-2 variants in vitro. Furthermore, structural analysis of bispecific antibody-antigen complexes using cryo-electron microscopy revealed the mode of action of K202.B complexed with a fully open three-RBD-up conformation of SARS-CoV-2 trimeric spike proteins by simultaneously interconnecting two independent epitopes of the SARS-CoV-2 RBD via inter-protomer interactions. Intravenous monotherapy using K202.B exhibited potent neutralizing activity in SARS-CoV-2 wild-type- and B.1.617.2 variant-infected mouse models, without significant toxicity in vivo. The results indicate that this novel approach of development of immunoglobulin G4-based bispecific antibody from an established human recombinant antibody library is likely to be an effective strategy for the rapid development of bispecific antibodies, and timely management against fast-evolving SARS-CoV-2 variants.