Impaired binding affinity of YTHDC1 with METTL3/METTL14 results in R-loop accumulation in myelodysplastic neoplasms with DDX41 mutation.

DEAD box helicase 41 (DDX41) mutations are the most prevalent predisposition to familial myelodysplastic syndrome (MDS). However, the precise roles of these variants in the pathogenesis of MDS have yet to be elucidated. Here, we discovered a novel mechanism by which DDX41 contributes to R-loop-induced DNA damage responses (DDR) in cooperation with the m6A-METTL complex (MAC) and YTHDC1 using DDX41 knockout (KO) and DDX41 knock-in (KI, R525H, Y259C) cell lines as well as primary samples from MDS patients. Compared to wild type (WT), DDX41 KO and KI led to increased levels of m6A RNA methylated R-loop. Interestingly, we found that DDX41 regulates m6A/R-loop levels by interacting with MAC components. Further, DDX41 promoted the recruitment of YTHDC1 to R-loops by promoting the binding between METTL3 and YTHDC1, which was dysregulated in DDX41-deficient cells, contributing to genomic instability. Collectively, we demonstrated that DDX41 plays a key role in the physiological control of R-loops in cooperation with MAC and YTHDC1. These findings provide novel insights into how defects in DDX41 influence MDS pathogenesis and suggest potential therapeutic targets for the treatment of MDS.

Single-cell RNA Sequencing Reveals Novel Cellular Factors for Response to Immunosuppressive Therapy in Aplastic Anemia.

Aplastic anemia (AA) is a lethal hematological disorder; however, its pathogenesis is not fully understood. Although immunosuppressive therapy (IST) is a major treatment option for AA, one-third of patients do not respond to IST and its resistance mechanism remains elusive. To understand AA pathogenesis and IST resistance, we performed single-cell RNA sequencing (scRNA-seq) of bone marrow (BM) from healthy controls and patients with AA at diagnosis. We found that CD34+ early-stage erythroid precursor cells and PROM1+ hematopoietic stem cells were significantly depleted in AA, which suggests that the depletion of CD34+ early-stage erythroid precursor cells and PROM1+ hematopoietic stem cells might be one of the major mechanisms for AA pathogenesis related with BM-cell hypoplasia. More importantly, we observed the significant enrichment of CD8+ T cells and T cell-activating intercellular interactions in IST responders, indicating the association between the expansion and activation of T cells and the positive response of IST in AA. Taken together, our findings represent a valuable resource offering novel insights into the cellular heterogeneity in the BM of AA and reveal potential biomarkers for IST, building the foundation for future precision therapies in AA.

Targeting FLT3-TAZ signaling to suppress drug resistance in blast phase chronic myeloid leukemia.

BACKGROUND: Although the development of BCR::ABL1 tyrosine kinase inhibitors (TKIs) rendered chronic myeloid leukemia (CML) a manageable condition, acquisition of drug resistance during blast phase (BP) progression remains a critical challenge. Here, we reposition FLT3, one of the most frequently mutated drivers of acute myeloid leukemia (AML), as a prognostic marker and therapeutic target of BP-CML. METHODS: We generated FLT3 expressing BCR::ABL1 TKI-resistant CML cells and enrolled phase-specific CML patient cohort to obtain unpaired and paired serial specimens and verify the role of FLT3 signaling in BP-CML patients. We performed multi-omics approaches in animal and patient studies to demonstrate the clinical feasibility of FLT3 as a viable target of BP-CML by establishing the (1) molecular mechanisms of FLT3-driven drug resistance, (2) diagnostic methods of FLT3 protein expression and localization, (3) association between FLT3 signaling and CML prognosis, and (4) therapeutic strategies to tackle FLT3+ CML patients. RESULTS: We reposition the significance of FLT3 in the acquisition of drug resistance in BP-CML, thereby, newly classify a FLT3+ BP-CML subgroup. Mechanistically, FLT3 expression in CML cells activated the FLT3-JAK-STAT3-TAZ-TEAD-CD36 signaling pathway, which conferred resistance to a wide range of BCR::ABL1 TKIs that was independent of recurrent BCR::ABL1 mutations. Notably, FLT3+ BP-CML patients had significantly less favorable prognosis than FLT3- patients. Remarkably, we demonstrate that repurposing FLT3 inhibitors combined with BCR::ABL1 targeted therapies or the single treatment with ponatinib alone can overcome drug resistance and promote BP-CML cell death in patient-derived FLT3+ BCR::ABL1 cells and mouse xenograft models. CONCLUSION: Here, we reposition FLT3 as a critical determinant of CML progression via FLT3-JAK-STAT3-TAZ-TEAD-CD36 signaling pathway that promotes TKI resistance and predicts worse prognosis in BP-CML patients. Our findings open novel therapeutic opportunities that exploit the undescribed link between distinct types of malignancies.

The effect of oral bacterial infection on DNA damage response in host cells.

Maintaining and transferring intact genomes from one generation to another plays a pivotal role in all living organisms. DNA damage caused by numerous endogenous and exogenous factors must be adequately repaired, as unrepaired and accumulated DNA mutations can cause severe deleterious effects, such as cell death and cancer. To prevent adverse consequences, cells have established DNA damage response mechanisms that address different forms of DNA damage, including DNA double-strand breaks, mismatches, nucleotide excision, and base excision. Among several sources of exogenous DNA damage, bacterial infections cause inflammation in the host, generating reactive oxygen species (ROS) and causing oxidative DNA damage. Recent studies have revealed the importance of the oral microbiome in inflammation and several systemic host diseases. Dysbiosis of oral bacteria can induce chronic inflammation, which enhances ROS-induced DNA damage, and improperly repaired damage can lead to carcinogenesis. This review describes the various DNA repair pathways that are affected by chronic inflammation and the discovery of the DNA damage response induced by oral bacteria such as Porphyromonas gingivalis and Fusobacterium nucleatum.

Thrap3 promotes nonalcoholic fatty liver disease by suppressing AMPK-mediated autophagy.

Autophagy functions in cellular quality control and metabolic regulation. Dysregulation of autophagy is one of the major pathogenic factors contributing to the progression of nonalcoholic fatty liver disease (NAFLD). Autophagy is involved in the breakdown of intracellular lipids and the maintenance of healthy mitochondria in NAFLD. However, the mechanisms underlying autophagy dysregulation in NAFLD remain unclear. Here, we demonstrate that the hepatic expression level of Thrap3 was significantly increased in NAFLD conditions. Liver-specific Thrap3 knockout improved lipid accumulation and metabolic properties in a high-fat diet (HFD)-induced NAFLD model. Furthermore, Thrap3 deficiency enhanced autophagy and mitochondrial function. Interestingly, Thrap3 knockout increased the cytosolic translocation of AMPK from the nucleus and enhanced its activation through physical interaction. The translocation of AMPK was regulated by direct binding with AMPK and the C-terminal domain of Thrap3. Our results indicate a role for Thrap3 in NAFLD progression and suggest that Thrap3 is a potential target for NAFLD treatment.

MSH2-MSH3 promotes DNA end resection during homologous recombination and blocks polymerase theta-mediated end-joining through interaction with SMARCAD1 and EXO1.

DNA double-strand break (DSB) repair via homologous recombination is initiated by end resection. The extent of DNA end resection determines the choice of the DSB repair pathway. Nucleases for end resection have been extensively studied. However, it is still unclear how the potential DNA structures generated by the initial short resection by MRE11-RAD50-NBS1 are recognized and recruit proteins, such as EXO1, to DSB sites to facilitate long-range resection. We found that the MSH2-MSH3 mismatch repair complex is recruited to DSB sites through interaction with the chromatin remodeling protein SMARCAD1. MSH2-MSH3 facilitates the recruitment of EXO1 for long-range resection and enhances its enzymatic activity. MSH2-MSH3 also inhibits access of POLθ, which promotes polymerase theta-mediated end-joining (TMEJ). Collectively, we present a direct role of MSH2-MSH3 in the initial stages of DSB repair by promoting end resection and influencing the DSB repair pathway by favoring homologous recombination over TMEJ.

PWWP2B promotes DNA end resection and homologous recombination.

Genome instability is one of the leading causes of gastric cancers. However, the mutational landscape of driver genes in gastric cancer is poorly understood. Here, we investigate somatic mutations in 25 Korean gastric adenocarcinoma patients using whole-exome sequencing and show that PWWP2B is one of the most frequently mutated genes. PWWP2B mutation correlates with lower cancer patient survival. We find that PWWP2B has a role in DNA double-strand break repair. As a nuclear protein, PWWP2B moves to sites of DNA damage through its interaction with UHRF1. Depletion of PWWP2B enhances cellular sensitivity to ionizing radiation (IR) and impairs IR-induced foci formation of RAD51. PWWP2B interacts with MRE11 and participates in homologous recombination via promoting DNA end-resection. Taken together, our data show that PWWP2B facilitates the recruitment of DNA repair machinery to sites of DNA damage and promotes HR-mediated DNA double-strand break repair. Impaired PWWP2B function might thus cause genome instability and promote gastric cancer development.

BAP1 promotes the repair of UV-induced DNA damage via PARP1-mediated recruitment to damage sites and control of activity and stability.

BRCA1-associated protein-1 (BAP1) is a ubiquitin C-terminal hydrolase domain-containing deubiquitinase with tumor suppressor activity. The gene encoding BAP1 is mutated in various human cancers, with particularly high frequency in kidney and skin cancers, and BAP1 is involved in many cancer-related cellular functions, such as DNA repair and genome stability. Although BAP1 stimulates DNA double-strand break repair, whether it functions in nucleotide excision repair (NER) is unknown. Here, we show that BAP1 promotes the repair of ultraviolet (UV)-induced DNA damage via its deubiquitination activity in various cell types, including primary melanocytes. Poly(ADP-ribose) polymerase 1 (PARP1) interacts with and recruits BAP1 to damage sites, with BAP1 recruitment peaking after the DDB2 and XPC damage sensors. BAP1 recruitment also requires histone H2A monoubiquitinated at Lys119, which accumulates at damage sites. PARP1 transiently poly(ADP-ribosyl)ates (PARylates) BAP1 at multiple sites after UV damage and stimulates the deubiquitination activity of BAP1 both intrinsically and via PARylation. PARP1 also promotes BAP1 stability via crosstalk between PARylation and ubiquitination. Many PARylation sites in BAP1 are mutated in various human cancers, among which the glutamic acid (Glu) residue at position 31, with particularly frequent mutation in kidney cancer, plays a critical role in BAP1 stabilization and promotes UV-induced DNA damage repair. Glu31 also participates in reducing the viability of kidney cancer cells. This study therefore reveals that BAP1 functions in the NER pathway and that PARP1 plays a role as a novel factor that regulates BAP1 enzymatic activity, protein stability, and recruitment to damage sites. This activity of BAP1 in NER, along with its cancer cell viability-reducing activity, may account for its tumor suppressor function.

Reciprocal interactions among Cobll1, PACSIN2, and SH3BP1 regulate drug resistance in chronic myeloid leukemia.

Cobll1 affects blast crisis (BC) progression and tyrosine kinase inhibitor (TKI) resistance in chronic myeloid leukemia (CML). PACSIN2, a novel Cobll1 binding protein, activates TKI-induced apoptosis in K562 cells, and this activation is suppressed by Cobll1 through the interaction between PACSIN2 and Cobll1. PACSIN2 also binds and inhibits SH3BP1 which activates the downstream Rac1 pathway and induces TKI resistance. PACSIN2 competitively interacts with Cobll1 or SH3BP1 with a higher affinity for Cobll1. Cobll1 preferentially binds to PACSIN2, releasing SH3BP1 to promote the SH3BP1/Rac1 pathway and suppress TKI-mediated apoptosis and eventually leading to TKI resistance. Similar interactions among Cobll1, PACSIN2, and SH3BP1 control hematopoiesis during vertebrate embryogenesis. Clinical analysis showed that most patients with CML have Cobll1 and SH3BP1 expression at the BC phase and BC patients with Cobll1 and SH3BP1 expression showed severe progression with a higher blast percentage than those without any Cobll1, PACSIN2, or SH3BP1 expression. Our study details the molecular mechanism of the Cobll1/PACSIN2/SH3BP1 pathway in regulating drug resistance and BC progression in CML.

Comparison of 3-month cytogenetic and molecular assays for early assessment of long-term clinical impact after BCR-ABL1 tyrosine kinase inhibitor treatment in chronic myeloid leukemia.

To compare the clinical significance of 3-month cytogenetic and molecular monitoring, we analyzed 1,410 paired cytogenetic and molecular data from 705 chronic-phase chronic myeloid leukemia patients. Based on early cytogenetic response (ECyR, Ph+≤35 %) and molecular response (EMR, BCR-ABL1IS≤10 %) at 3 months, the patients were divided into four groups (group 1: ECyR + EMR, n = 560; group 2: no ECyR + EMR, n = 27; group 3: ECyR + no EMR, n = 55; group 4: no ECyR + no EMR, n = 63). By 10 years, major molecular response (MMR), deep molecular response (MR4.5), overall survival (OS), and progression-free survival (PFS) rates were significantly high in group 1 (P < 0.001). Comparing groups 2 and 3, the MMR (P = 0.096), MR4.5 (P = 0.945), OS (P = 0.832), and PFS (P = 0.627) rates tended to be higher in group 2, although not significantly. Thus, the cytogenetic assay can not only be useful but its addition may also provide a more precise prediction of MR4.5.

Anatomical connections between the inferior fibers of the musculus orbicularis oculi and musculus levator labii superioris alaeque nasi / Conexiones anatómicas entre las fibras inferiores del músculo orbicular del ojo y levantador nasolabial

SUMMARY: This study aimed to determine the anatomical connections between the inferior fibers of M. orbicularis oculi (OOc inf.) and the M. levator labii superioris alaeque nasi (LLSAN), providing anatomical variations of the connecting fibers. This study examined the OOc and LLSAN of 44 specimens from 22 embalmed adult Korean cadavers. Connecting fibers between the OOc inf. and LLSAN were observed in 29.5 % of the specimens. Connecting patterns of the OOc inf. to the LLSAN were classified into three categories according to the directions of the connecting fibers; Type I (13.6 %), in which some of the OOc inf. coursed medially to blend with the lateral originating fibers of the LLSAN. Type II (11.4 %), in which some of the OOc inf. descended vertically to blend with the LLSAN. Type III (4.5 %), in which both types I and II were found simultaneously. Some of the OOc inf. coursed medially to blend with the lateral originating fibers of the LLSAN, and some of the OOc inf. descended vertically to blend with the LLSAN. These findings provide greater knowledge of the connecting fibers between the OOc inf. and LLSAN, thereby helping us understand complicated expressions, inject BoNT-A into related wrinkles, and analyze EMG activities.

RESUMEN: Este estudio tuvo como objetivo determinar las conexiones anatómicas entre las fibras inferiores del músculo orbicular del ojo (MOO inf.) y el levantador nasolabial (Musculus levator nasiolabialis; M. levator labii superioris alaeque nasi) (LNL), proporcionando variaciones anatómicas de las fibras conectoras. Se examinó el orbicularis oculi (MOO) y LNL de 44 especímenes de 22 cadáveres coreanos adultos embalsamados. Se observaron fibras conectoras entre los MOO inf. y LNL en el 29,5 % de los especímenes. Patrones de conexión del OOc inf. a LNL se clasificaron en tres categorías según las direcciones de las fibras conectoras; Tipo I (13,6 %), en el que algunos de los MOO inf. cursaban medialmente para mezclarse con las fibras de origen lateral del LNL. Tipo II (11,4 %), en el que algunos de los MOO inf. descendían verticalmente para mezclarse con el LNL. Tipo III (4,5 %), en el que se encontraron simultáneamente los tipos I y II. Algunos de los MOO inf. cursaron medialmente para mezclarse con las fibras de origen lateral de la LNL y algunas de las MOO inf. descendían verticalmente para mezclarse con el LNL. Estos hallazgos aportan un mayor conocimiento de las fibras conectoras entre los MOO inf. y LNL, lo que nos ayuda a comprender expresiones complicadas, inyectar BoNT-A en las arrugas relacionadas y analizar las actividades de EMG.

Thrap3 promotes R-loop resolution via interaction with methylated DDX5.

Transcription-replication conflicts lead to DNA damage and genomic instability, which are closely related to human diseases. A major source of these conflicts is the formation of R-loops, which consist of an RNA-DNA hybrid and a displaced single-stranded DNA. Although these structures have been studied, many aspects of R-loop biology and R-loop-mediated genome instability remain unclear. Here, we demonstrate that thyroid hormone receptor-associated protein 3 (Thrap3) plays a critical role in regulating R-loop resolution. In cancer cells, Thrap3 interacts with DEAD-box helicase 5 (DDX5) and localizes to R-loops. Arginine-mediated methylation of DDX5 is required for its interaction with Thrap3, and the Thrap3-DDX5 axis induces the recruitment of 5'-3' exoribonuclease 2 (XRN2) into R-loops. Loss of Thrap3 increases R-loop accumulation and DNA damage. These findings suggest that Thrap3 mediates resistance to cell death by preventing R-loop accumulation in cancer cells.

RNF126 is a positive regulator of TRAF3 ubiquitination.

Ubiquitination and deubiquitination of signaling molecules are critical regulatory mechanisms in various biological contexts such as inflammatory signaling and the DNA damage response. Thus, finely tuned regulation of protein ubiquitination is essential for maintaining cellular homeostasis. Here, we showed that the RING finger protein RNF126 interacts with TRAF3 and promotes its K63-linked polyubiquitination, which is a crucial step in the TRAF3-dependent antiviral response. We found that RNF126 also interacts with OTUB1, a deubiquitinating enzyme that negatively regulates K63-linked ubiquitination of TRAF3. RNF126 promotes ubiquitination of OTUB1, leading to reduced deubiquitinating activity toward TRAF3. Moreover, RNF126 promotes ubiquitination of OTUB1 on cysteine 91, which is reportedly required for its catalytic activity. Taken together, our results suggest that RNF126 positively regulates the antiviral response by directly promoting K63-linked polyubiquitination of TRAF3 and by reducing OTUB1 activity.

Melittin-loaded Iron Oxide Nanoparticles Prevent Intracranial Arterial Dolichoectasia Development through Inhibition of Macrophage-mediated Inflammation.

Rationale: In intracranial arterial dolichoectasia (IADE) development, the feedback loop between inflammatory cytokines and macrophages involves TNF-α and NF-κB signaling pathways and leads to subsequent MMP-9 activation and extracellular matrix (ECM) degeneration. In this proof-of-concept study, melittin-loaded L-arginine-coated iron oxide nanoparticle (MeLioN) was proposed as the protective measure of IADE formation for this macrophage-mediated inflammation and ECM degeneration. Methods: IADE was created in 8-week-old C57BL/6J male mice by inducing hypertension and elastase injection into a basal cistern. Melittin was loaded on the surface of ION as a core-shell structure (hydrodynamic size, 202.4 nm; polydispersity index, 0.158). Treatment of MeLioN (2.5 mg/kg, five doses) started after the IADE induction, and the brain was harvested in the third week. In the healthy control, disease control, and MeLioN-treated group, the morphologic changes of the cerebral arterial wall were measured by diameter, thickness, and ECM composition. The expression level of MMP-9, CD68, MCP-1, TNF-α, and NF-κB was assessed from immunohistochemistry, polymerase chain reaction, and Western blot assay. Results: MeLioN prevented morphologic changes of cerebral arterial wall related to IADE formation by restoring ECM alterations and suppressing MMP-9 expression. MeLioN inhibited MCP-1 expression and reduced CD68-positive macrophage recruitments into cerebral arterial walls. MeLioN blocked TNF-α activation and NF-κB signaling pathway. In the Sylvian cistern, co-localization was found between the CD68-positive macrophage infiltrations and the MeLioN distributions detected on Prussian Blue and T2* gradient-echo MRI, suggesting the role of macrophage harboring MeLioN. Conclusions: The macrophage infiltration into the arterial wall plays a critical role in the MMP-9 secretion. MeLioN, designed for ION-mediated melittin delivery, effectively prevents IADE formation by suppressing macrophage-mediated inflammations and MMP activity. MeLioN can be a promising strategy preventing IADE development in high-risk populations.

USP39 promotes non-homologous end-joining repair by poly(ADP-ribose)-induced liquid demixing.

Mutual crosstalk among poly(ADP-ribose) (PAR), activated PAR polymerase 1 (PARP1) metabolites, and DNA repair machinery has emerged as a key regulatory mechanism of the DNA damage response (DDR). However, there is no conclusive evidence of how PAR precisely controls DDR. Herein, six deubiquitinating enzymes (DUBs) associated with PAR-coupled DDR were identified, and the role of USP39, an inactive DUB involved in spliceosome assembly, was characterized. USP39 rapidly localizes to DNA lesions in a PAR-dependent manner, where it regulates non-homologous end-joining (NHEJ) via a tripartite RG motif located in the N-terminus comprising 46 amino acids (N46). Furthermore, USP39 acts as a molecular trigger for liquid demixing in a PAR-coupled N46-dependent manner, thereby directly interacting with the XRCC4/LIG4 complex during NHEJ. In parallel, the USP39-associated spliceosome complex controls homologous recombination repair in a PAR-independent manner. These findings provide mechanistic insights into how PAR chains precisely control DNA repair processes in the DDR.

TonEBP recognizes R-loops and initiates m6A RNA methylation for R-loop resolution.

R-loops are three-stranded, RNA-DNA hybrid, nucleic acid structures produced due to inappropriate processing of newly transcribed RNA or transcription-replication collision (TRC). Although R-loops are important for many cellular processes, their accumulation causes genomic instability and malignant diseases, so these structures are tightly regulated. It was recently reported that R-loop accumulation is resolved by methyltransferase-like 3 (METTL3)-mediated m6A RNA methylation under physiological conditions. However, it remains unclear how R-loops in the genome are recognized and induce resolution signals. Here, we demonstrate that tonicity-responsive enhancer binding protein (TonEBP) recognizes R-loops generated by DNA damaging agents such as ultraviolet (UV) or camptothecin (CPT). Single-molecule imaging and biochemical assays reveal that TonEBP preferentially binds a R-loop via both 3D collision and 1D diffusion along DNA in vitro. In addition, we find that TonEBP recruits METTL3 to R-loops through the Rel homology domain (RHD) for m6A RNA methylation. We also show that TonEBP recruits RNaseH1 to R-loops through a METTL3 interaction. Consistent with this, TonEBP or METTL3 depletion increases R-loops and reduces cell survival in the presence of UV or CPT. Collectively, our results reveal an R-loop resolution pathway by TonEBP and m6A RNA methylation by METTL3 and provide new insights into R-loop resolution processes.

TRAIP regulates Histone H2B monoubiquitination in DNA damage response pathways.

Histone H2B monoubiquitination has been shown to play critical roles in diverse cellular processes including DNA damage response. Although recent data indicate that H2B monoubiquitination is strongly connected with tumor progression and regulation, the implications of this modification in lung adenocarcinoma are relatively unknown. In the present study, we demonstrated the clinical implication of H2B monoubiquitination and the potential role of tumor necrosis factor receptor­associated factor­interacting protein (TRAIP) in regulating its modification in lung adenocarcinoma. Immunohistochemical analysis showed that H2B monoubiquitination was significantly downregulated in 68 human lung adenocarcinoma patient samples compared to their normal adjacent tissues. Depletion of TRAIP by specific siRNA treatment markedly decreased ionizing radiation (IR)­induced H2B monoubiquitination. In addition, deletion mutants without RING domain or C­terminus of TRAIP diminished the ability to induce H2B monoubiquitination at lysine 120. Notably, the nuclear expression of TRAIP was positively related with H2B monoubiquitination levels in patients with lung adenocarcinoma. Furthermore, statistical analysis indicated that low levels of both TRAIP and H2B monoubiquitination, not each alone, in patients with lung adenocarcinoma were strongly correlated with poor survival. Taken together, these results suggest that TRAIP is a novel regulator of H2B monoubiquitination in DNA damage response and cancer development in lung adenocarcinoma.

GCA links TRAF6-ULK1-dependent autophagy activation in resistant chronic myeloid leukemia.

Imatinib is the first molecularly targeted compound for chronic myeloid leukemia (CML) capable to inhibit BCR-ABL kinase activity. However, recent clinical evidence indicates that a substantial proportion of CML patients exhibit BCR-ABL-dependent or independent resistance to imatinib. Despite the importance of imatinib resistance in CML, the underlying molecular mechanisms of this resistance are largely unknown. Here, we identified GCA (grancalcin) as a critical regulator of imatinib resistance in chronic phase CML via activation of autophagy. Mechanistically, we demonstrated that GCA activates TRAF6 ubiquitin ligase activity to induce Lys63 ubiquitination of ULK1, a crucial regulator of autophagy, resulting in its stabilization and activation. We also highlighted the role of GCA-TRAF6-ULK1 autophagy regulatory axis in imatinib resistance. Our findings represent the basis for novel therapeutic strategies against CML.Abbreviation: ACTB/ß-actin: actin beta; ADM: adrenomedullin; AMBRA1: autophagy and beclin 1 regulator 1; AMPK: AMP-activated protein kinase; ANXA5: annexin A5; CP: cytogenetic response; CML: chronic myeloid leukemia; CUL3: cullin 3; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GCA: grancalcin; Dx: at diagnosis; E-64-d: (2S,3S)-trans-Epoxysuccinyl-L-leucylamido-3-methylbutane ethyl ester; IMres: Imatinib resistance; KLHL20: Kelch-like protein 20; LRMP: lymphoid-restricted membrane protein; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MMR: major molecular response; NH4Cl: ammonium chloride; PBMCs: peripheral blood mononuclear cells; PTPRC: protein tyrosine phosphatase, receptor type, C; SQSTM1/p62: sequestosome 1; SYK: spleen associated tyrosine kinase; TAP1: transporter 1, ATP binding cassette subfamily B member; TKIs: ABL-specific tyrosine kinase inhibitors; TLR9: toll- like receptor 9; TRAF6: TNF receptor associated factor 6; ULK1: unc-51 like autophagy activating kinase 1.

INC280 inhibits Wnt/ß-catenin and EMT signaling pathways and its induce apoptosis in diffuse gastric cancer positive for c-MET amplification.

OBJECTIVE: Gastric cancer is more open related to genetic predisposition. In our RNA sequencing study on gastric cancer patients, Runt-related transcription factor-3 (RUNX3) expression was significantly down-regulated in gastric cancer. We showed that decreased levels of RUNX3 are significantly associated with c-MET (r = - 0.4216, P = 0.0130). In addition, c-MET expression is a candidate for targeted therapy in gastric cancer. Therefore, in the present study, the anti-cancer effects of the c-MET inhibitor on gastric cancer cells from positive or negative for c-MET amplification were evaluated. RESULTS: INC280 treatment inhibits growth of a c-MET-amplified MKN45 (RUNX3-positive) and SNU620 (RUNX3-negative) diffuse type cells. Then, INC280 showed the highest inhibition and apoptotic rates with the lowest IC50s in MKN45 cells but not in c-MET-reduced MKN28 (intestinal type) cells. We also showed that INC280 inhibits the WNT signaling pathway and SNAIL expression in MKN45 cells. The data indicate that INC280 could be used as therapeutic agents for the prevention or treatment of diffuse gastric cancer positive for c-MET amplification.

Hypomorphic Mutations in TONSL Cause SPONASTRIME Dysplasia.

SPONASTRIME dysplasia is a rare, recessive skeletal dysplasia characterized by short stature, facial dysmorphism, and aberrant radiographic findings of the spine and long bone metaphysis. No causative genetic alterations for SPONASTRIME dysplasia have yet been determined. Using whole-exome sequencing (WES), we identified bi-allelic TONSL mutations in 10 of 13 individuals with SPONASTRIME dysplasia. TONSL is a multi-domain scaffold protein that interacts with DNA replication and repair factors and which plays critical roles in resistance to replication stress and the maintenance of genome integrity. We show here that cellular defects in dermal fibroblasts from affected individuals are complemented by the expression of wild-type TONSL. In addition, in vitro cell-based assays and in silico analyses of TONSL structure support the pathogenicity of those TONSL variants. Intriguingly, a knock-in (KI) Tonsl mouse model leads to embryonic lethality, implying the physiological importance of TONSL. Overall, these findings indicate that genetic variants resulting in reduced function of TONSL cause SPONASTRIME dysplasia and highlight the importance of TONSL in embryonic development and postnatal growth.