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Histone deacetylase inhibitors (HDACi) are established anticancer drugs, especially in hematological cancers. This study aimed to design, synthesize, and evaluate a set of HDACi featuring a pentyloxyamide connecting unit linker region and substituted phenylthiazole cap groups. A structural optimization program yielded HDACi with nanomolar inhibitory activity against histone deacetylase class I/IIb enzymes. The novel inhibitors ( 4d and 4m ) showed superior antileukemic activity compared to several approved HDACi. Furthermore, 4d and 4m displayed synergistic activity when combined with chemotherapeutics, decitabine, and clofarabine. In vitro pharmacokinetic studies showed the most promising profile for 4d with intermediate microsomal stability, excellent plasma stability, and concentration-independent plasma protein binding. Additionally, 4d demonstrated comparable in vivo pharmacokinetics to vorinostat. When administered in vivo, 4d effectively inhibited the proliferation of leukemia cells without causing toxicity. Furthermore, the binding modes of 4d and 4m to the catalytic domain 2 of HDAC6 from Danio rerio were determined by X-ray crystallography.

Combining multiple drugs broadens the window of therapeutic opportunities and is crucial for diseases that are currently lacking fully curative treatments. A powerful emerging tool for selecting effective drugs and combinations is the high-throughput drug screening (HTP). The histone deacetylase inhibitor (HDACi) givinostat (ITF2357) has been shown to act effectively against CRLF2-rearranged pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL), a subtype characterized by poor outcome and enriched in children with Down Syndrome, very fragile patients with a high susceptibility to treatment-related toxicity. The aim of this study is to investigate possible synergies with givinostat for these difficult-to-treat patients by performing HTP screening with a library of 174 drugs, either approved or in preclinical studies. By applying this approach to the CRLF2-r MHH-CALL-4 cell line, we identified 19 compounds with higher sensitivity in combination with givinostat compared to the single treatments. Next, the synergy between givinostat and the promising candidates was further validated in CRLF2r cell lines with a broad matrix of concentrations. The combinations with trametinib (MEKi) or venetoclax (BCL2i) were found to be the most effective and with the greatest synergy across three metrics (ZIP, HAS, Bliss). Their efficacy was confirmed in primary blasts treated ex vivo at concentration ranges with a safe profile on healthy cells. Finally, we described givinostat-induced modifications in gene expression of MAPK and BCL-2 family members, supporting the observed synergistic interactions. Overall, our study represents a model of drug repurposing strategy using HTP screening for identifying synergistic, efficient, and safe drug combinations., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors. Published by Elsevier Ltd.)

Although a great cure rate has been achieved for pediatric BCP-ALL, approximately 15% of patients do not respond to conventional chemotherapy and experience disease relapse. A major effort to improve the cure rates by treatment intensification would result in an undesirable increase in treatment-related toxicity and mortality, raising the need to identify novel therapeutic approaches. High-throughput (HTP) drug screening enables the profiling of patients' responses in vitro and allows the repurposing of compounds currently used for other diseases, which can be immediately available for clinical application. The aim of this study was to apply HTP drug screening to identify potentially effective compounds for the treatment of pediatric BCP-ALL patients with poor prognosis, such as patients with Down Syndrome (DS) or carrying rearrangements involving PAX5 or KMT2A/MLL genes. Patient-derived Xenografts (PDX) samples from 34 BCP-ALL patients (9 DS CRLF2r, 15 PAX5r, 10 MLLr), 7 human BCP-ALL cell lines and 14 hematopoietic healthy donor samples were screened on a semi-automated HTP drug screening platform using a 174 compound library (FDA/EMA-approved or in preclinical studies). We identified 9 compounds active against BCP-ALL (ABT-199/venetoclax, AUY922/luminespib, dexamethasone, EC144, JQ1, NVP-HSP990, paclitaxel, PF-04929113 and vincristine), but sparing normal cells. Ex vivo validations confirmed that the BCL2 inhibitor venetoclax exerts an anti-leukemic effect against all three ALL subgroups at nanomolar concentrations. Overall, this study points out the benefit of HTP screening application for drug repurposing to allow the identification of effective and clinically translatable therapeutic agents for difficult-to-treat childhood BCP-ALL subgroups., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Inc.)

Somatic loss of function mutations in cohesin genes are frequently associated with various cancer types, while cohesin disruption in the germline causes cohesinopathies such as Cornelia-de-Lange syndrome (CdLS). Here, we present the discovery of a recurrent heterozygous RAD21 germline aberration at amino acid position 298 (p.P298S/A) identified in three children with lymphoblastic leukemia or lymphoma in a total dataset of 482 pediatric cancer patients. While RAD21 p.P298S/A did not disrupt the formation of the cohesin complex, it altered RAD21 gene expression, DNA damage response and primary patient fibroblasts showed increased G2/M arrest after irradiation and Mitomycin-C treatment. Subsequent single-cell RNA-sequencing analysis of healthy human bone marrow confirmed the upregulation of distinct cohesin gene patterns during hematopoiesis, highlighting the importance of RAD21 expression within proliferating B- and T-cells. Our clinical and functional data therefore suggest that RAD21 germline variants can predispose to childhood lymphoblastic leukemia or lymphoma without displaying a CdLS phenotype.

Sister chromatid cohesion, which depends on cohesin, is essential for the faithful segregation of replicated chromosomes. Here, we report that splicing complex Prp19 is essential for cohesion in both G2 and mitosis, and consequently for the proper progression of the cell through mitosis. Inactivation of splicing factors SF3a120 and U2AF65 induces similar cohesion defects to Prp19 complex inactivation. Our data indicate that these splicing factors are all required for the accumulation of cohesion factor Sororin, by facilitating the proper splicing of its pre-mRNA. Finally, we show that ectopic expression of Sororin corrects defective cohesion caused by Prp19 complex inactivation. We propose that the Prp19 complex and the splicing machinery contribute to the establishment of cohesion by promoting Sororin accumulation during S phase, and are, therefore, essential to the maintenance of genome stability., (© 2014 The Authors.)

The primary objective of this study was to assess the biological effects of a new dentine substitute based on Ca₃SiO₅ (Biodentine™) for use in pulp-capping treatment, on pseudo-odontoblastic (MDPC-23) and pulp (Od-21) cells. The secondary objective was to evaluate the effects of Biodentine and mineral trioxide aggregate (MTA) on gene expression in cultured spheroids. We used the acid phosphatase assay to compare the biocompatibility of Biodentine and MTA. Cell differentiation was investigated by RT-qPCR. We investigated the expression of genes involved in odontogenic differentiation (Runx2), matrix secretion (Col1a1, Spp1) and mineralisation (Alp). ANOVA and PLSD tests were used for data analysis. MDPC-23 cells cultured in the presence of MTA had higher levels of viability than those cultured in the presence of Biodentine and control cells on day 7 (P = 0.0065 and P = 0.0126, respectively). For Od-21 cells, proliferation rates on day 7 were significantly lower in the presence of Biodentine or MTA than for control (P < 0.0001). Col1a1 expression levels were slightly lower in cells cultured in the presence of MTA than in those cultured in the presence of Biodentine and in control cells. Biodentine and MTA may modify the proliferation of pulp cell lines. Their effects may fluctuate over time, depending on the cell line considered. The observed similarity between Biodentine and MTA validates the indication for direct pulp-capping claimed by the manufacturers.

The ITI (inter-trypsine inhibitor) gene family includes five genes (ITIH1 to ITIH5) that encode proteins involved in the dynamics of the extracellular matrix (ECM). ITIH5 was found inactivated by partial deletion in a case of congenital uterovaginal aplasia, a human rare disease also called Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome. The aim of the present study was to analyze the expression of ITIH5 in the uterus in adult life and during embryogenesis in order to establish the involvement of this gene in both normal and pathological conditions of uterus development. This was achieved in mice by reverse transcription-quantitative PCR, whole-mount hybridization, and Western blot analysis. Itih5 expression was much stronger in female genital tract primordia (Müllerian ducts) and derivatives than elsewhere in the body. This gene was strongly expressed during pregnancy and development of the female genital tract, indicating that the encoded protein probably had an important function in the uterus during these periods. Two different specific isoforms of the protein were detected in Müllerian derivatives during embryogenesis and in adults. Although ITIH genes are expected to be predominantly expressed in the liver, ITIH5 is mainly expressed in the uterus during development and adult life. This tends to indicate an additional and specific role of this gene in the female reproductive tract, and furthermore reinforces ITIH5 as a putative candidate gene for MRKH syndrome.

Background: Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome is characterized by congenital aplasia of the uterus and the upper part of the vagina in women showing normal development of secondary sexual characteristics and a normal 46, XX karyotype. The uterovaginal aplasia is either isolated (type I) or more frequently associated with other malformations (type II or Müllerian Renal Cervico-thoracic Somite (MURCS) association), some of which belong to the malformation spectrum of DiGeorge phenotype (DGS). Its etiology remains poorly understood. Thus the phenotypic manifestations of MRKH and DGS overlap suggesting a possible genetic link. This would potentially have clinical consequences., Methods: We searched DiGeorge critical chromosomal regions for chromosomal anomalies in a cohort of 57 subjects with uterovaginal aplasia (55 women and 2 aborted fetuses). For this candidate locus approach, we used a multiplex ligation-dependent probe amplification (MLPA) assay based on a kit designed for investigation of the chromosomal regions known to be involved in DGS.The deletions detected were validated by Duplex PCR/liquid chromatography (DP/LC) and/or array-CGH analysis., Results: We found deletions in four probands within the four chromosomal loci 4q34-qter, 8p23.1, 10p14 and 22q11.2 implicated in almost all cases of DGS syndrome., Conclusion: Uterovaginal aplasia appears to be an additional feature of the broad spectrum of the DGS phenotype. The DiGeorge critical chromosomal regions may be candidate loci for a subset of MRKH syndrome (MURCS association) individuals. However, the genes mapping at the sites of these deletions involved in uterovaginal anomalies remain to be determined. These findings have consequences for clinical investigations, the care of patients and their relatives, and genetic counseling.

The mammalian female reproductive tract develops from the Mullerian ducts which differentiate, in a cranial to caudal direction, into oviducts, uterine horns, cervix and the anterior vagina. The developmental processes taking place during this organogenesis are notably under the control of steroid hormones, such as members of the Wnt and Hox families, which regulate key developmental genes. At later stages, steroid hormones also participate in the development of the female genital tract. Chemical compounds homologous to steroids can thus act as agonists or antagonists in fetuses exposed to them. These so-called endocrine disruptors are nowadays found in increasing amounts in the environment and may therefore have a particular impact on such developing organs. Epidemiological studies have revealed that endocrine disruptors have had drastic effects on female health and fertility during the last decades. Furthermore, these adverse effects might be transmitted to subsequent generations through epigenetic modifications. Given the potential hazard of inherited epigenetic marks altering reproduction and/or human health, such molecular mechanisms must be urgently investigated. This review aims to summarize the cellular and molecular mechanisms involved in female genital tract development, to highlight key genes involved in this process and to present epigenetic mechanisms triggered by endocrine disruptors and their consequences in regard to female reproductive tract development.

The Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome is characterized by congenital aplasia of the uterus and the upper part (two-third) of the vagina. It may be isolated (type I) or associated with other malformations (type II or MURCS association). These latter involve the upper urinary tract, the skeleton and, to a lesser extent, the otologic sphere or the heart. The incidence of MRKH syndrome has been estimated as 1 in 4500 women. The prime feature is a primary amenorrhea in women presenting otherwise with normal development of secondary sexual characteristics and normal external genitalia. However, the vagina is reduced to a vaginal dimple with variable depth. The ovaries are normal and functional as well as the endocrine status. Karyotype is 46,XX, with no visible chromosome modification. The phenotypic manifestations of MRKH syndrome overlap with various other syndromes or malformations and thus require accurate delineation as well as differential diagnosis. For a long time, the syndrome has been considered as a sporadic anomaly, but increasing familial cases now support the hypothesis of a genetic cause currently under investigation. The syndrome appears to be transmitted as an autosomal dominant trait with incomplete penetrance and variable expressivity.

Background: Postcholecystectomy complex bile duct injuries involving the hilar confluence, which are often associated with vascular injuries and liver atrophy, remain a considerable surgical challenge. The aim of this study is to report our experience of major hepatectomy with long-term outcome in these patients., Methods: From January 1987 to January 2002, 18 patients underwent a major hepatectomy for complex bile duct injuries. The hilar confluence was involved in all cases and was associated with vascular injuries in 13 (72%), including arterial injuries in 11, and partial liver atrophy in 15 (83%). The average time interval between the initial cholecystectomy and hepatectomy was 43 +/- 63 months and 16 (88%) patients had previously undergone an average of 2 (range 1-3) surgical repairs., Results: Major liver resection included a right hepatectomy in 14 (78%) patients, a left hepatectomy in 3, and a left trisectionectomy in one. There was no postoperative mortality, but severe postoperative morbidity was experienced in 11 (61%) patients, including biliary fistula in 7 (39%), prolonged ascites in 8 (44%) and hemorrhage requiring reoperation in one. With a median follow-up time of 8 years (range 3 to 12), 17 (94%) patients have excellent or good results, including 13 patients without symptoms., Conclusion: This study shows that salvage major hepatectomy is an efficient treatment for patients with complex hilar bile duct injuries and should be considered before liver transplantation or recourse to metallic stents.

Clusterin is a usually secreted glycoprotein with chaperone properties. Recently, it has been suggested that clusterin isoforms reside in the nuclear and cytosolic compartments of human cell types, where they can influence various cellular programs including DNA repair, transcription and apoptosis. Several mechanisms have been proposed to explain this atypical location, including alternative transcription initiation and alternative splicing. However, none of these have been unequivocally established as occurring in live cells. Here we provide direct experimental evidence that in live intact cells, under certain stress conditions, clusterin can evade the secretion pathway and reach the cytosol. This was demonstrated using several complementary approaches. Flow cytometry and selective permeabilization of U251 cell membranes with digitonin allowed detection of cytosolic clusterin in stressed U251 cells. In addition, a stringent enzymatic assay reliant upon the exclusively cytosolic deubiquitinase enzymes confirmed that clusterin synthesized with its hydrophobic secretion signal sequence can reach the cytosol of U251 cells. The retrotranslocation of clusterin is likely to occur through a mechanism similar to the endoplasmic reticulum (ER)-associated protein degradation pathway and involves passage through the Golgi apparatus. We also report that the ER-associated ubiquitin ligase Hrd1/synoviolin can interact with, and ubiquitinate clusterin. The possible biological functions of these novel behaviours of clusterin are discussed.

Terminal deletions of the long arm of chromosome 4 are associated with a recognizable phenotype consisting of dysmorphic facial features, cleft palate, upper and lower limb malformations, cardiac defects and growth and mental retardation. Here we report on two female patients, a mother and her daughter, carrying the same 4q34-->qter deletion but presenting with a different phenotype. The mother's presentation is consistent with previous findings in patients with terminal deletions of the long arm of chromosome 4. However, she presented at the age of 54years with bilateral serous carcinoma of the Fallopian tubes, a rare gynaecologic cancer that might be attributed to the haploinsufficiency of the tumor suppressor gene FAT. The daughter presented isolated congenital aplasia of the uterus and vagina, the prime feature of the MRKH syndrome. This has not been described before in association with a 46,XX,del(4)(q34qter).

In mammalian somatic cells, the post-transcriptional control of cytokine or proto-oncogene expression is often achieved by factors binding to sequence elements in the 3' untranslated region (3'UTR). The most studied are the AU-rich elements (ARE) that have been divided into three classes. Here, we show that in mammalian cells, the presence of the class III c-jun ARE in the 3'UTR of a reporter mRNA enhanced reporter protein expression. In contrast, the presence of a class II ARE in the 3'UTR decreased reporter protein expression. CUG-BP1/CELF1 is able to bind c-jun ARE. Protein expression was enhanced similarly to what was observed for c-jun ARE when the reporter mRNA contained a synthetic CUG-BP1/CELF1-binding site, or when this protein was tethered to the 3'UTR of a reporter mRNA. These results reveal an unexpected complexity of ARE-mediated post-transcriptional regulations, and indicate a function for CUG-BP1/CELF1 in class III ARE directed regulations.

While studies have highlighted the role of HOXA9-13 and PBX1 homeobox genes during the development of the female genital tract, the molecular mechanisms triggered by these genes are incompletely elucidated. In several developmental pathways, PBX1 binds to MEINOX family members in the cytoplasm to be imported into the nucleus where they associate with HOX proteins to form a higher complex that modulates gene expression. This concept has been challenged by a recent report showing that in some cell cultures, PBX1 nuclear localization might be regulated independently of MEINOX proteins (Kilstrup-Nielsen et al., 2003). Our work gives the first illustration of this alternative mechanism in an organogenesis process. Indeed, we show that PBX1 is mostly cytoplasmic in epithelial endometrial cells of the developing female genital tract despite the nuclear localization of MEIS1. We thus provide evidence for a control of PBX1 intracellular distribution which is independent of MEINOX proteins, but is cell cycle correlated.

Biliary papillomatosis is a papillary adenomatosis of the biliary mucosa of the extra- and the intrahepatic biliary tree. It is a rare neoplasm difficult to manage, characterized by extensive lesions and a great potential for malignant transformation. We report a case of a 75 year-old man, who presented with malignant papillomatosis of the common bile duct without involvement of the intrahepatic biliary ducts. Duodenopancreatectomy enabled the diagnosis of papillomatosis lined 5.5 cm of the common bile duct which displayed an invasive 1.5 cm papillary carcinoma located in the distal portion of the choledocus. Immunohistochemistry showed strong expression of p53 in the distally located invasive carcinoma and in distant dysplastic lesions. MUC5AC was exclusively detected in both malignant and dysplastic lesions without detection of MUC1 or MUC2. Detection of p53 expression on biliary brush samples could be interesting for the follow-up and the prediction of malignant progression in multifocal biliary papillomatosis.

Arterial pseudo-aneurysms complicating pancreaticoduodenectomy are rare but have a poor prognosis. They usually result from arterial erosion due to pancreatic fistula. The authors report a pseudo-aneurysm with an uncommon localization (first jejunal artery), diagnosed after a negative first arteriography, and successfully treated by radiological embolization. Special features of pseudo-aneurysms complicating pancreaticoduodenectomy are reviewed.

Objective: To compare the clinical presentation and results of treatment of postcholecystectomy bile duct injuries in patients with and without arterial injuries., Summary Background Data: Incidence and impact of arterial injuries in patients with a postcholecystectomy biliary injury are unknown, although they are claimed to increase the risk of septic complications, difficulty of biliary repair and risk of recurrent stricture., Methods: Fifty-five patients referred for postcholecystectomy biliary strictures and who underwent surgical repair were prospectively evaluated by celiac and superior mesenteric angiography. Circumstance and presenting symptoms of the biliary injury in patients with and without vascular injury as well as intra- and postoperative outcome in the 43 patients who underwent a Hepp-Couinaud biliary repair were compared., Results: Incidence of vascular injury was 47%, the most frequent of which was right-sided hepatic artery disruptions (36%). Indication of cholecystectomy (cholecystitis, 42 vs. 45%), technique of resection (laparoscopy, 80 vs. 79%) as well as delay of recognition and presenting symptom of the biliary injury were comparable in patients with and without vascular injury. Among patients undergoing a biliary repair, the level of the biliary injury (Bismuth's type III or IV 63% vs. 54%), duration of surgery, and incidence of postoperative complications (21 vs. 21%) were also comparable in patients with and without arterial injury. One patient in each group experienced recurrent biliary stricture., Conclusions: The discovery of a disruption of the right branch of the hepatic artery should not affect management of the biliary stricture when if a Hepp-Couinaud repair is performed.

We report herein a modification of the standard wirsungojejunostomy in patients with chronic pancreatitis, which includes concomitant duodenum-preserving resection of the head of the pancreas as described by Frey. This technique is safe and seems to provide more effective long-term pain relief than standard wirsungo-jejunal anastomosis.

Background: Percutaneous drainage (PD) of complex postoperative abscesses associated with a variety of factors such as multiple location or enteric fistula remains a matter of debate. Accordingly, this retrospective study was designed to determine the predictive factors for failure of PD of postoperative abscess, in order to better select the patients who may benefit from PD., Methods: From 1992 to 2000, the data of 73 patients who underwent computed tomography (CT)-guided PD for postoperative intra-abdominal abscess, were reviewed. PD was considered as failure when clinical sepsis persisted or subsequent surgery was needed. The possible association between failure of PD and 27 patient-, abscess-, surgical-, and drainage-related variables were assessed using univariate and multivariate analysis., Results: Successful PD was achieved in 59 of 73 (81%) patients. The overall mortality was 3% but no patient died after salvage surgery. Multivariate analysis showed that only an abscess diameter of less than 5 cm (P = 0.042) and absence of antibiotic therapy (P = 0.01) were significant predictive variables for failure of PD., Conclusions: CT-guided PD associated with antibiotic therapy could be attempted as the initial treatment of postoperative abdominal abscesses even in complex cases such as loculated abscess or abscess associated with enteric fistula.