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Clathrin-mediated endocytosis (CME) is critical for internalisation of molecules across cell membranes. The FCH domain only 1 (FCHO1) protein is key molecule involved in the early stages of CME formation. The consequences of mutations in FCHO1 in humans were unknown. We identify ten unrelated patients with variable T and B cell lymphopenia, who are homozygous for six distinct mutations in FCHO1. We demonstrate that these mutations either lead to mislocalisation of the protein or prevent its interaction with binding partners. Live-cell imaging of cells expressing mutant variants of FCHO1 provide evidence of impaired formation of clathrin coated pits (CCP). Patient T cells are unresponsive to T cell receptor (TCR) triggering. Internalisation of the TCR receptor is severely perturbed in FCHO1-deficient Jurkat T cells but can be rescued by expression of wild-type FCHO1. Thus, we discovered a previously unrecognised critical role of FCHO1 and CME during T-cell development and function in humans. FCH domain only 1 (FCHO1) is a key molecule involved in clathrin-mediated endocytosis (CME). Here, the authors report homozygous FCHO1 mutations in individuals with variable T and B cell lymphopenia, which are associated with loss-of-function of FCHO1 and impaired formation of clathrin-coated pits in T cells.

Resistance to conventional chemotherapy remains a huge challenge in the clinical management and treatment of hepatoblastoma, the most common liver tumor of childhood. The in silico repurposing of drugs with known pharmacokinetics and safety profiles, which have been approved for other indications, represents a promising strategy to identify new drugs effective in chemoresistant hepatoblastoma. In this study, we integrated RNA sequencing-derived gene expression data into the pharmacologic perturbation prediction tool Connectivity Map (CMap) and identified the clinical widely used anthelmintic drug mebendazole as a putative drug to circumvent chemoresistance in hepatoblastoma. Consequently, we experimentally validated the efficacy of mebendazole in permanent and patient-derived xenograft cell lines that are resistant to cisplatin, the therapeutic backbone of hepatoblastoma treatment. Viability assays clearly indicated a potent reduction of tumor cell growth upon mebendazole treatment in a dose-dependent manner. In contrast, similar mebendazole concentrations had little inhibitory effect on the growth of normal neonatal and adult skin fibroblasts. The combination of mebendazole and cisplatin revealed a strong synergistic effect, which was comparable to the one seen with cisplatin and doxorubicin (cardiotoxic), the current standard treatment for high-risk hepatoblastoma patients. Moreover, mebendazole treatment resulted in reduced colony formation capability, induction of apoptosis, and cell cycle arrest of hepatoblastoma cells. Immunofluorescent staining of alpha-tubulin showed that mebendazole causes blockage of microtubule formation and subsequent RNA sequencing analyses confirmed the transcriptional downregulation of tubulins as the major mode of action of mebendazole treatment. Most importantly, mebendazole significantly reduced tumor growth in a subcutaneous xenograft transplantation mouse model. In conclusion, our results strongly support the clinical use of mebendazole as a replacement for doxorubicin in the treatment of chemoresistant hepatoblastoma. This could potentially not only improve outcome but also reduce severe long-term side effects. Citation Format: Qian Li, Salih Demir, Xuanwen Bao, Alexandra Wagner, Yanxin Fan, Stefano Cairo, Roland Kappler. In silico drug repurposing identifies mebendazole as a treatment option for chemoresistant hepatoblastoma [abstract]. In: Proceedings of the AACR Special Conference: Advances in the Pathogenesis and Molecular Therapies of Liver Cancer; 2022 May 5-8; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(17_Suppl):Abstract nr PO012.

// Jian Ma 1, * , Yanxin Fan 2, * , Tingting Feng 1, 3, * , Fangjun Chen 4 , Zhipeng Xu 1 , Suqing Li 1 , Qingfeng Lin 1 , Xiaoting He 1 , Weihong Shi 1, 5 , Yang Liu 6 , Xiufeng Cao 1 , Bin Zhu 1 and Zhihua Liu 7 1 Department of Surgical Oncology, Affiliated Nanjing First Hospital of Nanjing Medical University, Oncology Center of Nanjing Medical University, Nanjing 210008, Jiangsu Province, China 2 Jiangsu Cancer Hospital, Medical School of Nanjing University, Nanjing 210008, Jiangsu Province, China 3 Institute of Biology and Medical Sciences, Jiangsu Key Laboratory of Infection and Immunity, Soochow University, Suzhou 215123, Jiangsu Province, China 4 The Comprehensive Cancer Centre of Drum Tower Hospital, Medical School of Nanjing University, Clinical Cancer Institute of Nanjing University, Nanjing 210008, Jiangsu Province, China 5 Yancheng Vocational Institute of Health Sciences, Yancheng 224005, Jiangsu Province, China 6 Department of Pharmacy, No.401 Hospital of Chinese People's Liberation Army, Qingdao 266071, Shandong Province, China 7 State Key Laboratory of Molecular Oncology, National Cancer Center/Cancer Hospital, Chinese Academy of Medical Sciences, Beijing 100021, China * These authors have contributed equally to this work Correspondence to: Zhihua Liu, email: liuzh@pubem.cicams.ac.cn Xiufeng Cao, email: cxf551101@sina.com Bin Zhu, email: forrest771220@hotmail.com Keywords: HOTAIR, miR-125, miR-143, ESCC, HK2 Received: June 15, 2017 Accepted: August 08, 2017 Published: September 23, 2017 ABSTRACT Esophageal Squamous Cell Carcinoma (ESCC) is one of the most common malignant cancers worldwide with a high death rate worldwide. Long non-coding RNA (LncRNA) has been recently demonstrated to play a critical role in ESCC. LncRNA HOTAIR played important regulatory roles in ESCC. We highlight the molecular mechanisms by which HOTAIR could influence the expression of Hexokinase 2 (HK2) in ESCC through binding miR-125 and miR-143 directly. Taken together, this study identified a functional lncRNA HOTAIR involved with regulation of glycolysis via miRNA-125/miRNA-143-HK2 in ESCC cells. The “competitive endogenous RNA” (ceRNA) model of HOTAIR/miR-125 and miR143/HK2 interaction might serve as important targets for ESCC diagnosis and therapy.

To research the value of virtual reality (VR) technology in the preoperative planning of transtrochanteric curved varus osteotomy for avascular necrosis of the femoral head (ANFH) in adults.Between June 2018 and November 2018, 7 patients (11 hips) with ANFH, who were treated with transtrochanteric curved varus osteotomy, were enrolled in the study. There were 4 males (7 hips) and 3 females (4 hips) with an average age of 31.9 years (range, 14-46 years). Among them, 3 patients were unilateral ANFH and 4 patients were bilateral ANFH. There was 1 patient (1 hip) of traumatic ANFH, 2 patients (4 hips) of alcohol-induced ANFH, 2 patients (3 hips) of hormonal ANFH, and 2 patients (3 hips) of idiopathic ANFH. All hips were Association Research Circulation Osseous (ARCO) stage Ⅲ. There were 5 hips for Japanese Investigation Committee (JIC) type C1 and 6 hips for type C2. There were 5 hips for China-Japan Friendship Hospital (CJFH) type L1,1 for type L2, and 5 for type L3. The disease duration ranged from 5 to 12 months (mean, 8 months). Preoperative Harris score was 53.91±7.66. The neck-shaft angle ranged from 128 to 143° (mean, 133.9°). VR technology was adopted for the preoperative planning. CT data were imported into the software to construct the morphology of necrotic area, and the transrtrochanteric varus osteotomy was simulated. The varus angle was designed according to the integrity rate of femoral head. The planned varus angle was 6 to 16° (mean, 9.7°). The transtrochanteric curved varus osteotomy was performed according to the preoperative planning, and the varus angle and loading area were confirmed under fluoroscopy. If the planned varus angle was too small, it would continue to increase under the fluoroscopy until a satisfactory varus angle. Postoperative changes of the neck-shaft angle were calculated and compared with the preoperative planned varus angle (error). The hip function was assessed by using the Harris score.All incisions healed by first intention. All patients were followed up 6-11 months with an average of 8 months. The X-ray film at 2 days after operation showed that the neck-shaft angle was 112-135° (mean, 123.4°). The difference of the neck-shaft angle between pre- and post-operation was 6-16° (mean, 11.0°). Among them, the difference of the neck-shaft angle was consistent with planned varus angle in 5 hips, while the error of the remaining 6 hips was 1-4°. There was 1 patient (1 hip) of osteotomy nonunion at 4 months after operation, 1 patient (1 hip) of proximal femur fracture at 2 months after operation. The rest 5 patients (9 hips) obtained union at the osteotomy. At last follow-up, the Harris score was 82.18±16.35, showing significant difference when compared with preoperative score (VR technology is a brand-new preoperative planning method for transtrochanteric curved varus osteotomy in treating ANFH.探讨经转子间弧形内翻截骨术治疗成人股骨头缺血性坏死（avascular necrosis of the femoral head，ANFH）时，采用虚拟现实（virtual reality，VR）技术进行术前规划的应用价值。.以 2018 年 6 月—11 月拟行经转子间弧形内翻截骨术的 7 例（11 髋）ANFH 患者作为研究对象。其中，男 4 例（7 髋），女 3 例（4 髋）。年龄 14～46 岁，平均 31.9 岁。单髋 3 例，双髋 4 例。创伤性 1 例（1 髋），酒精性 2 例（4 髋），激素性 2 例（3 髋），特发性 2 例（3 髋）。ANFH 根据国际骨循环学会（ARCO）分期均为Ⅲ期；日本骨坏死研究会（JIC）分型：C1 型 5 髋、C2 型 6 髋；中日友好医院（CJFH）分型：L1 型 5 髋、L2 型 1 髋、L3 型 5 髋。病程 5～12 个月，平均 8 个月。术前 Harris 评分为（53.91±7.66）分。颈干角为 128～143°，平均 133.9°。术前采用 VR 技术进行方案设计，将 CT 数据导入软件构建坏死区域形态，模拟转子间内翻截骨，根据负重区股骨头完整率设计内翻角度。VR 术前规划内翻角度为 6～16°，平均 9.7°。术中按照术前规划进行内翻截骨，C 臂 X 线机透视确认内翻角度和负重区情况。如出现计划内翻角度偏小情况，则在透视下继续增加至获得满意位置。术后 2 d 摄 X 线片测量颈干角，计算手术前后差值，并与术前规划内翻角度比较（误差值）。随访时采用 Harris 评分评估髋关节功能。.术后切口均Ⅰ期愈合。患者均获随访，随访时间 6～11 个月，平均 8 个月。术后 2 d 复查 X 线片，颈干角为 112～135°，平均 123.4°；手术前后差值为 6～16°，平均 11.0°；其中，5 髋颈干角手术前后差值与 VR 术前规划内翻角度一致，其余 6 髋误差值为 1～4°。术后 1 例（1 髋）出现截骨处骨不连、1 例（1 髋）发生股骨近端骨折，其余 5 例（9 髋）截骨处愈合良好。末次随访时，Harris 评分为（82.18±16.35）分，与术前比较差异有统计学意义（VR 技术为经转子间弧形内翻截骨术治疗 ANFH 提供了一种新的术前规划方法。.

Clathrin-mediated endocytosis (CME) is critical for internalisation of molecules across cell membranes. The FCH domain only 1 (FCHO1) protein is key molecule involved in the early stages of CME formation. The consequences of mutations in FCHO1 in humans were unknown. We identify ten unrelated patients with variable T and B cell lymphopenia, who are homozygous for six distinct mutations in FCHO1. We demonstrate that these mutations either lead to mislocalisation of the protein or prevent its interaction with binding partners. Live-cell imaging of cells expressing mutant variants of FCHO1 provide evidence of impaired formation of clathrin coated pits (CCP). Patient T cells are unresponsive to T cell receptor (TCR) triggering. Internalisation of the TCR receptor is severely perturbed in FCHO1-deficient Jurkat T cells but can be rescued by expression of wild-type FCHO1. Thus, we discovered a previously unrecognised critical role of FCHO1 and CME during T-cell development and function in humans., FCH domain only 1 (FCHO1) is a key molecule involved in clathrin-mediated endocytosis (CME). Here, the authors report homozygous FCHO1 mutations in individuals with variable T and B cell lymphopenia, which are associated with loss-of-function of FCHO1 and impaired formation of clathrin-coated pits in T cells.

KLK13 downregulation occurs in tumour tissues in comparison with adjacent normal tissues from patients with oesophageal squamous cell carcinoma (OSCC). KLK13 mRNA levels were tested in OSCC tumour tissues and adjacent noncancerous tissues from 138 patients. In addition, the correlation between KLK13 mRNA levels and OSCC clinicopathologic features was analysed. KLK13 mRNA levels decreased notably in tumour tissues compared with those in adjacent noncancerous tissues. And decreased KLK13 mRNA levels indicated significant correlations with higher tumour grade, elevated TNM (UICC, 2009) stage classification, deeper infiltration and more lymph node metastases. And thus KLK13 may be a promising diagnostic marker. Decreased KLK13 mRNA levels also correlate with poor survival, which indicates that KLK13 mRNA expression may be a potential prognostic marker, although it could not be an independent prognostic factor by multivariate analysis. In vitro experiments of the OSCC cell lines KYSE150 and KYSE450 demonstrated that overexpression of KLK13 inhibits cell invasion and migration. Thus, KLK13 is a unique novel molecule useful for monitoring OSCC progression. Full elucidation of the role of KLK13 in OSCC may reveal avenues for investigating the molecule's functional potential as a novel therapeutic drug for targeting OSCC.

Differentiation of hematopoietic stem cells follows a hierarchical program of transcription factor regulated events1-3. Early myeloid cell differentiation is dependent on PU.1 and CEBPA (CCAAT/enhancer binding protein alpha), late myeloid differentiation is orchestrated by CEBPE (CCAAT/enhancer binding protein epsilon)4. The influence of SWI/SNF (SWItch/Sucrose Non-Fermentable) chromatin remodelling factors as novel master regulators of hematopoietic differentiation is only beginning to be explored3,5,6. Here, we identify SMARCD2 (SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily d, member 2) as a critical regulator of myeloid differentiation in humans, mice, and zebrafish. Studying patients from three unrelated pedigrees characterized by neutropenia, specific granule deficiency, myelodysplasia with excess of blast cells and various developmental aberrations, we identified three loss-of-function mutations in SMARCD2. Using mice and zebrafish as model systems, we showed that SMARCD2 controls early steps in the differentiation of myeloid-erythroid progenitor cells. In vitro, SMARCD2 interacts with the transcription factor CEBPE and controls expression of neutrophil proteins stored in specific granules. Defective expression of SMARCD2 leads to transcriptional and chromatin changes in acute myeloid leukemia (AML) human promyelocytic cells. In summary, SMARCD2 is a key factor controlling myelopoiesis and a potential tumor suppressor in leukemia.

Esophageal Squamous Cell Carcinoma (ESCC) is one of the most common malignant cancers worldwide with a high death rate worldwide. Long non-coding RNA (LncRNA) has been recently demonstrated to play a critical role in ESCC. LncRNA HOTAIR played important regulatory roles in ESCC. We highlight the molecular mechanisms by which HOTAIR could influence the expression of Hexokinase 2 (HK2) in ESCC through binding miR-125 and miR-143 directly. Taken together, this study identified a functional lncRNA HOTAIR involved with regulation of glycolysis via miRNA-125/miRNA-143-HK2 in ESCC cells. The “competitive endogenous RNA” (ceRNA) model of HOTAIR/miR-125 and miR143/HK2 interaction might serve as important targets for ESCC diagnosis and therapy.

Differentiation of hematopoietic stem cells follows a hierarchical program of transcriptional-regulated events. We here identify SMARCD2 (Swi/Snf-related matrix associated actin dependent regulator of chromatin, subfamily D, member 2) as critical regulator of myelopoiesis in humans, mice, and zebrafish. We studied four patients from three unrelated pedigrees presenting with a novel syndromatic phenotype comprising congenital neutropenia, specific granule deficiency, susceptibility to myelodysplasia with excess of blasts, and various skeletal anomalies. All patients had homozygous loss-of-function mutations in SMARCD2. In contrast to wildtype alleles, the variant alleles did not give rise to proteins with capacity to interact with the SWI/SNF subunits BRG1, BAF170, BAF155, and BAF47, as shown by co-immunoprecipitation experiments. In vitro, knockdown of SMARCD2 in promyelocytic NB4 cells, differentiated in the presence of ATRA, led to decreased expression of genes encoding the primary granule proteins cathelicidin (CAMP) and alpha-1-antitrypsin (AAT) as well as specific granule proteins matrix metalloproteinase-8 (MMP8), transcobalamin (TCN1) and lactoferrin (LTF). This phenotype is reminiscent of patients with specific granule deficiency, characterized by mutations in CEBPE, a known transcription factor controlling terminal neutrophil development. We therefore hypothesized that SMARCD2 may act via CEBPe and performed immunoprecipitation studies in transfected cells. Upon pull-down of SMARCD2, CEPBE could be detected, and vice versa, suggesting that both proteins physically interact to control transcriptional networks. To interrogate effects of SMARCD2 deficiency on global chromatin accessibility we made use of ATAC sequencing of undifferentiated and ATRA-differentiated NB4 cells and compared this data with comprehensive RNA-sequencing results. A specific subset of genes was found deregulated in both assays, affecting vesicular trafficking, migration and signalling pathways. To validate a role for SMARCD2 in hematopoiesis in vivo, we generated murine and zebrafish model systems. We generated Smarcd2-/- mice by injection of Smarcd2+/- murine ES cells into blastocysts, transfer into pseudo-pregnant mice and interbreeding of heterozygous Smarcd2+/- offsprings. The mutant allele was inherited in a Mendelian fashion but no viable mice were born. 14.5dpc embryos were characterized by anemia and reduced size compared to their littermates. Analysis of fetal liver hematopoiesis revealed a complete absence of CD11b+Gr1+ and CD11b+Ly6c+ cells, whereas the number of LSK stem cells was not affected. Futhermore, Smarcd2-/- embryos showed aberrations in erythroid cells such as extensive anisocytosis, multinucleated cells, and perturbed mitosis. In cytokine-driven colony forming unit assays, GM-CSF, M-CSF, and G-CSF induced myeloid cell differentiation was decreased. Transcriptional profiling of LSK stem cells revealed a striking dysbalance affecting genes involved in signaling pathways and host defence, including CEBPE-dependent genes. Among a total of 12362 detected genes, we found 4290 to be differentially expressed (DESeq2, FDR 1.4, FDR Disclosures Abboud: Novartis: Honoraria; MAST Therapeutics: Research Funding; Eli Lilly and Company: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees.

Mitoribosome in mammalian cells is responsible for synthesis of 13 mtDNA-encoded proteins, which are integral parts of four mitochondrial respiratory chain complexes (I, III, IV and V). ERAL1 is a nuclear-encoded GTPase important for the formation of the 28S small mitoribosomal subunit. Here, we demonstrate that knockdown of ERAL1 by RNA interference inhibits mitochondrial protein synthesis and promotes reactive oxygen species (ROS) generation, leading to autophagic vacuolization in HeLa cells. Cells that lack ERAL1 expression showed a significant conversion of LC3-I to LC3-II and an enhanced accumulation of autophagic vacuoles carrying the LC3 marker, all of which were blocked by the autophagy inhibitor 3-MA as well as by the ROS scavenger NAC. Inhibition of mitochondrial protein synthesis either by ERAL1 siRNA or chloramphenicol (CAP), a specific inhibitor of mitoribosomes, induced autophagy in HTC-116 TP53 (+/+) cells, but not in HTC-116 TP53 (-/-) cells, indicating that tumor protein 53 (TP53) is essential for the autophagy induction. The ROS elevation resulting from mitochondrial protein synthesis inhibition induced TP53 expression at transcriptional levels by enhancing TP53 promoter activity, and increased TP53 protein stability by suppressing TP53 ubiquitination through MAPK14/p38 MAPK-mediated TP53 phosphorylation. Upregulation of TP53 and its downstream target gene DRAM1, but not CDKN1A/p21, was required for the autophagy induction in ERAL1 siRNA or CAP-treated cells. Altogether, these data indicate that autophagy is induced through the ROS-TP53-DRAM1 pathway in response to mitochondrial protein synthesis inhibition.