Upfront allogeneic hematopoietic stem cell transplantation for adult T-cell acute lymphoblastic leukemia/lymphoma in first complete remission: a single-center study.

BACKGROUND: Real-world data on outcomes of upfront allogeneic hematopoietic stem cell transplantation (allo-HCT) for adult T-cell acute lymphoblastic leukemia/lymphoma (T-ALL) patients in first complete remission (CR1) is still lacking. METHODS: A single center retrospective study was conducted from 94 consecutive patients received their first allo-HCT between 2010 and 2021, which include 76 patients received upfront allo-HCT and 18 patients received allo-HCT in non-upfront settings. RESULTS: There were no significant differences in most variables. In the upfront allo-HCT group, 52 (68%) patients achieved CR1 with one cycle of induction regimen. 24 (32%) patients achieved CR1 with more than one cycle. In the non-upfront group, there were 14 patients with active disease and 4 patients in second CR before transplant. The majority of patients received antithymocyte globulin-based graft-versus-host disease prophylaxis. Median follow-up time was 51 months for both groups. 5-year overall survival (OS) was 54% in the upfront allo-HCT group. While, in the non-upfront group, 5-year OS were 19% (P = 0.013). 5-year progression free survival in the upfront group was higher than that in the non-upfront group (50% versus 20%, P = 0.02). 5-year cumulative incidence relapse rate was significantly higher in non-upfront group (64% vs. 32%, P = 0.006). While, there was no difference in the 5-year non-relapse mortality (NRM) rate (19% versus 16%, P = 0.56). The most common cause of death was disease progression. In multivariable analysis, non-upfront allo-HCT (hazard ratios (HR) 2.14, P = 0.03) and HCT-CI (≥ 2) (HR 6.07, P = 0.002) were identified to be associated with worse OS. Non-upfront allo-HCT and HCT-CI (≥ 2) were also found to be independent risk factors for higher relapse rate. While, haploidentical-HCT was found to be associated with increased NRM. CONCLUSIONS: Our study indicated that allo-HCT remains an important curative treatment for adult patients with T-ALL, especially when it was performed in the upfront setting.

Simultaneous occurrence of two distinct histotypes of ovarian endometriosis-associated cancer in bilateral ovaries: implications for monoclonal histogenesis from a case report.

Background: Transformation of endometriosis to malignancy is a rare occurrence. Clear cell ovarian cancer and endometrioid ovarian cancer are the two histotypes most consistently linked to endometriosis. The exact pathways leading to malignant transformation of endometriosis remain elusive. Case presentation: A 41-year-old woman presented to our hospital with a ten days history of abdominal pain which was not responsive to medication. Pathological examination revealed an unexpected finding of bilateral endometriosis associated with distinct malignancies: a clear cell carcinoma in the right ovary and a well-differentiated endometrioid carcinoma in the left ovary. Molecular analysis indicated a shared somatic driver mutation in ING1 in the eutopic endometrium and the bilateral ovaries while simultaneously exhibiting specific genetic alterations unique to each carcinoma. Notably, several common mutation sites were also identified, including previously reported common oncogenes (KRAS, PIK3CA, ARID1A). This finding prompts the hypothesis of a possible monoclonal origin of the two tumours. Conclusion: This case represents an exceedingly rare occurrence of two different histotypes of ovarian endometriosis-associated cancer manifesting simultaneously in bilateral ovaries. Based on genetic analysis, we hypothesize that these malignancies may have a monoclonal origin, providing insights into understanding the different biological mechanisms underlying carcinogenesis.

Targeted dosing of anti-thymocyte globulin in adult unmanipulated haploidentical peripheral blood stem cell transplantation: A single-arm, phase 2 trial.

Anti-thymocyte globulin (ATG) is widely used in allogeneic hematopoietic stem cell transplantation to prevent severe graft-versus-host disease (GVHD) and graft failure. However, overexposure to ATG may increase cytomegalovirus (CMV), Epstein-Barr virus (EBV) reactivation, non-relapse mortality, and disease recurrence. To investigate the optimal dosing of ATG, we established a targeted dosing strategy based on ATG concentration monitoring for haploidentical peripheral blood stem cell transplantation (haplo-PBSCT). The aim of this phase 2 trial is to evaluate the safety and efficacy of the ATG-targeted dosing strategy in adult unmanipulated haplo-PBSCT. ATG was administered for 4 days (-5 days to -2 days) during conditioning. The ATG doses on -3 days and -2 days were adjusted by our dosing strategy to achieve the optimal ATG exposure. The primary endpoint was CMV reactivation on +180 days. Between December 2020 and January 2022, 66 haplo-PBSCT patients were enrolled and 63 of them were evaluable with a median follow-up of 632 days. The cumulative incidence of CMV reactivation was 36.7% and that of EBV was 58.7%. The 1-year disease-free survival was 82.5%, overall survival was 92.1%, and CD4+ T-cell reconstruction on +100 days was 76.8%. The most common severe regimen-associated toxicities (> grade 3) were infections (51.5%) and gastrointestinal toxicity (25.5%). A total of 102 haplo-PBSCT patients who received the conventional fixed ATG dose (cumulative 10 mg/kg) comprised historical control. The outcomes in historical control were inferior to those of phase 2 trial cohort (CMV reactivation: 70.8%, p < .001; EBV reactivation: 76.0%, p = .024; CD4 + T-cell reconstruction: 54.1%, p = .040). In conclusion, ATG-targeted dosing strategy reduced CMV/EBV reactivation and improved survival without increasing GVHD after haplo-PBSCT. These advantages may be associated with accelerated immune reconstitution.

Survival outcomes of abdominal radical hysterectomy, laparoscopic radical hysterectomy, robot-assisted radical hysterectomy and vaginal radical hysterectomy approaches for early-stage cervical cancer: a retrospective study.

BACKGROUND: This study compared the survival outcomes of abdominal radical hysterectomy (ARH) (N = 32), laparoscopic radical hysterectomy (LRH) (N = 61), robot-assisted radical hysterectomy (RRH) (N = 100) and vaginal radical hysterectomy (VRH) (N = 45) approaches for early-stage cervical cancer to identify the surgical approach that provides the best survival. METHODS: Disease-free survival (DFS) and overall survival (OS) were calculated using the Kaplan-Meier method, and survival curves were compared using the log-rank test. RESULTS: The volume of intraoperative blood loss was greater in the ARH group than in the LRH group, the RRH group or the VRH group [(712.50 ± 407.59) vs. (224.43 ± 191.89), (109.80 ± 92.98) and (216.67 ± 176.78) ml, respectively; P < 0.001]. Total 5-year OS was significantly different among the four groups (ARH, 96.88%; LRH, 82.45%; RRH, 94.18%; VRH, 91.49%; P = 0.015). However, no significant difference in 5-year DFS was observed among the four groups (ARH, 96.88%; LRH, 81.99%; RRH, 91.38%; VRH, 87.27%; P = 0.061). CONCLUSION: This retrospective study demonstrated that ARH and RRH achieved higher 5-year OS rates than LRH for early-stage cervical cancer.

High Risk of Recurrence of Malignancy Noted in Four-day rATG Regimen After Allogeneic PBSCT From Matched Sibling Donors.

Hitherto, the optimal timing of rabbit anti-thymocyte globulin (rATG) in matched sibling donor peripheral blood stem cell transplantation (MSD-PBSCT) remains to be elucidated. We wanted to evaluate the effect of a new timing strategy of rATG in MSD-PBSCT patients on transplantation outcomes. In this prospective single-arm phase 2 clinical trial, 45 consecutive MSD-PBSCT patients were enrolled from February 1, 2019, to January 31, 2021. The rATG was administered intravenously at a total dose of 5 mg/kg from day -5 to day -2 before graft infusion (4d-ATG group). Thirty-seven MSD-PBSCT patients receiving rATG at the same total dose of 5 mg/kg from day -5 to day -4 before graft infusion from December 1, 2014, to January 31, 2019 served as historical control (2d-ATG group). No graft failure occurred in either group. In the 4d-ATG group, median timing to neutrophil and platelet engraftment was 12 days. The cumulative incidences (CI) of grade 2-4 and grade 3-4 acute graft-versus-host disease (GVHD) at day 100 were 37.8% and 4.4%. The 2-year CIs of severe chronic GVHD and extensive chronic GVHD were 2.2% and 9.6%. The rates of cytomegalovirus (CMV) and Epstein-Barr virus (EBV) reactivation at day 180 were 24.4% and 37.8%, respectively. No patients died of non-relapse causes. Twenty-one patients relapsed at a median of 203 days after transplantation, and the 2-year cumulative incidence of relapse (CIR) was 51.4%. The 2-year probabilities of overall survival (OS), disease-free survival (DFS), and GVHD-free and relapse-free survival (GRFS) were 72.4%, 48.6%, and 40.8%, respectively. There were no significant differences between the 4d-ATG group and 2d-ATG group with regard to timing of neutrophil and platelet engraftment, incidences of CMV reactivation, EBV reactivation, acute GVHD, chronic GVHD, probabilities of OS, and GRFS. The 2-year CIR was significantly increased, and the 2-year DFS was significantly reduced in 4d-ATG group compared with the control group (CIR: 51.4% versus 13.5%, P < .001; DFS: 48.6% versus 75.7%, P = .014). High CIR and worse DFS were noted in MSD-PBSCT receiving 4d-ATG regimen compared with historical control (2d-ATG regimen). Inappropriate rATG timing may increase the risk of relapse after MSD-PBSCT in patients with hematologic malignancies. © 2023 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.

Increased risk of nonrelapse mortality post T-cell-replete haploidentical stem cell transplantation in patients with recurrence of acute graft-versus-host disease.

Acute graft-versus-host disease (aGVHD) causes significant morbidity and mortality. While most studies focus on classic or late aGVHD, some patients with previous aGVHD achieve complete remission and later develop another episode of aGVHD. Data on recurrence of aGVHD (RaGVHD) are lacking. This study aimed to identify the incidence, risk factors, and impacts of RaGVHD after T-cell-replete haploidentical hematopoietic cell transplantation (haplo-HCT) without posttransplantation cyclophosphamide. We evaluated patients with RaGVHD after haplo-HCT between 2017 and 2019 and compared their outcomes to those of patients with no aGVHD and those of patients with one episode of de novo aGVHD. Of 199 patients included in the analysis, 45 experienced 50 cases of RaGVHD with a 1-year cumulative incidence of 19.0% (95% CI: 14.5-24.6). Grade III-IV aGVHD was more common in RaGVHD than in previous aGVHD (22.2% vs. 4.4%, p = 0.01). Female donor to male recipient was strongly associated with RaGVHD (HR: 2.5, p = 0.009). The most common death in patients with RaGVHD was GVHD-related, which was different from controls who mostly died from relapse (p = 0.008). RaGVHD was an independent risk factor for chronic GVHD (HR: 2.6, p = 0.006) and nonrelapse mortality (HR: 2.4, p = 0.019) and a significant predictor of lower GVHD relapse-free survival (HR: 1.9, p = 0.020) and cGVHD relapse-free survival (HR: 2.1, p = 0.007). In conclusion, clinical manifestations and negative impacts of RaGVHD needs to be recognized independently.

Optimal Active Anti-Thymocyte Globulin Exposure Associated with Minimum Risk of Virus Reactivation and Comparable Acute Graft-Versus-Host Disease Under Adult Myeloablative Haploidentical Peripheral Blood Stem Cell Transplantation.

Anti-thymocyte globulin (ATG) is often included in the conditioning regimen to prevent graft-versus-host disease (GVHD) in allogeneic hematopoietic cell transplantation (allo-HCT). However, the risk of virus reactivation increases significantly. We conducted a single-center prospective study to identify the optimal ATG exposure that ensures engraftment, effectively prevents acute GVHD, and reduces the risk of virus reactivation without increasing relapse of malignant diseases in haploidentical peripheral blood stem cell transplantation (haplo-PBSCT). From September 2018 to June 2020, 106 patients (median age, 32 years) with malignant hematological diseases who received haplo-PBSCT for the first time were enrolled. All patients received 10 mg/kg rabbit ATG (thymoglobulin) divided for 4 days (days -5 to -2). Pre-transplant, post-transplant, and total areas under the concentration-time curve (AUCs) of active ATG were calculated. Total AUC of active ATG was shown to be the best predictor for virus reactivation and acute GVHD of grades II to IV or grades III and IV. The optimal total AUC range of active ATG was 100 to 148.5 UE/mL/day. The median time was 14 versus 13 days (P = .184) for neutrophil engraftment and 13 versus 13 days (P = .263) for platelet engraftment in the optimal and non-optimal AUC groups, respectively. The optimal AUC group showed a lower cumulative incidence of cytomegalovirus (CMV) reactivation and persistent CMV viremia than the non-optimal AUC group: 60.6% (95% confidence interval [CI], 48.3%-73.1%) versus 77.1% (95% CI, 64.5%-87.7%; P = .016) and 31.5% (95% CI, 21.2%-45.3%) versus 56.3% (95% CI, 42.9%-70.4%; P = .007), respectively. The cumulative incidence of persistent Epstein-Barr virus (EBV) viremia in the optimal AUC group was significantly lower than the non-optimal total AUC group: 33.1% (95% CI, 22.5%-46.8%) versus 52.6% (95% CI, 39.3%-67.2%; P = .048). However, there was no difference in EBV reactivation (P = .752). Similar outcomes were observed for grade II to IV and grade III and IV acute GVHD between the two groups: 48.6% (95% CI, 36.8%-62.0%) versus 37.0% (95% CI, 24.8%-52.5%; P = .113) and 10.4% (95% CI, 4.8%-21.7%) versus 4.2% (95% CI, 1.0%-15.6%; P = .234, respectively. Relapse, non-relapse mortality, and disease-free survival demonstrated no significant differences between the two groups. But, overall survival at 2 years tended to increase in the optimal AUC group: 75.7% (95% CI, 62.4%-84.8%) versus 57.8% (95% CI, 42.4%-70.4%; P = .061). These data support an optimal active ATG exposure of 110 to 148.5 UE/mL/day in haplo-PBSCT. Individualized dosing of ATG in allo-HCT might reduce the risk of virus reactivation and effectively prevent acute GVHD simultaneously.

Structure Guided Design of Bacteriophage Qß Mutants as Next Generation Carriers for Conjugate Vaccines.

Vaccines are critical tools to treat and prevent diseases. For an effective conjugate vaccine, the carrier is crucial, but few carriers are available for clinical applications. In addition, a drawback of current protein carriers is that high levels of antibodies against the carrier are induced by the conjugate vaccine, which are known to interfere with the immune responses against the target antigen. To overcome these challenges, we obtained the near atomic resolution crystal structure of an emerging protein carrier, i.e., the bacteriophage Qß virus like particle. On the basis of the detailed structural information, novel mutants of bacteriophage Qß (mQß) have been designed, which upon conjugation with tumor associated carbohydrate antigens (TACAs), a class of important tumor antigens, elicited powerful anti-TACA IgG responses and yet produced lower levels of anticarrier antibodies as compared to those from the wild type Qß-TACA conjugates. In a therapeutic model against an aggressive breast cancer in mice, 100% unimmunized mice succumbed to tumors in just 12 days even with chemotherapy. In contrast, 80% of mice immunized with the mQß-TACA conjugate were completely free from tumors. Besides TACAs, to aid in the development of vaccines to protect against COVID-19, the mQß based conjugate vaccine has been shown to induce high levels of IgG antibodies against peptide antigens from the SARS-CoV-2 virus, demonstrating its generality. Thus, mQß is a promising next-generation carrier platform for conjugate vaccines, and structure-based rational design is a powerful strategy to develop new vaccine carriers.

Elevated REG3α predicts refractory aGVHD in patients who received steroids-ruxolitinib as first-line therapy.

We started a single-arm, phase II, open-label, prospective clinical trial using steroids-ruxolitinib as the first-line therapy for intermediate- to high-risk aGVHD (NCT04397367). Here, we report the association of a biomarker panel (sST2, REG3α, sTNFR1, IL-6 and IL-8) with responses to GVHD therapy. The novel first-line therapy for 39 patients with newly diagnosed aGVHD consisted of 1 mg/kg methylprednisolone and 5 mg/day ruxolitinib. The serum concentrations of the biomarkers were prospectively detected at planned time points. Of the 39 patients, the complete response rate at day 28 was 82.05%. In patients who achieved CR, the concentrations of REG3α (P14 = 0.01; P28 = 0.10) and sTNFR1 (P14 = 0.42; P28 = 0.04) declined at day 14 and day 28 compared with the pre-enrolment levels. In refractory patients, the levels of REG3α at day 14 were higher than those pre-enrolment (P = 0.04). REG3α (P = 0.02) was elevated in the refractory patients compared with the patients achieving CR at day 14 after enrolment, while there was no significant difference in the levels of sST2, sTNFR1 or IL-6. Elevated REG3α levels may predict refractory aGVHD after novel first-line therapy with steroids-ruxolitinib.

Increased risk of cardio-cerebrovascular disease after hematopoietic cell transplantation in patients with previous history.

BACKGROUND: The impacts of previous cardio-cerebrovascular disease (pre-CCVD) on the outcomes of hematopoietic cell transplantation (HCT) are not well described. Patients with pre-CCVD may often be poor candidates for HCT. This study aimed to investigate the impact of pre-CCVD on transplant outcomes. METHODS: A retrospective study was conducted between patients with and without pre-CCVD who consecutively received allogeneic or autologous HCT between November 2013 and January 2020 with a matching of age and disease status. The cardiovascular complications and HCT outcomes of the two groups were evaluated and compared. The primary endpoints were post-transplant cardio-cerebrovascular disease (post-CCVD) and non-relapse mortality (NRM). We used a multivariable Cox proportional hazard model and the Fine-Gray competing risk regressions for analyses to estimate the hazard ratios (HRs). RESULTS: The outcomes of 23 HCT recipients with pre-CCVD were compared with those of 107 patients in the control group. No significant differences were noted in terms of engraftment, overall survival (OS) (67.00% vs. 67.90%, P = 0.983), or relapse (29.78% vs. 28.26%, P = 0.561) between the pre-CCVD group and the control group. The cumulative incidences of 2-year NRM were similar between patients with pre-CCVD and the controls (14.68% vs. 17.08%, P = 0.670). However, pre-CCVD was associated with an increased incidence of post-CCVD (HR: 12.50, 95% confidence interval [CI]: 3.88-40.30, P < 0.001), which was an independent risk factor for increased NRM (HR: 10.29, 95% CI: 3.84-27.62, P < 0.001) and inferior OS (HR: 10.29, 95% CI: 3.84-27.62, P < 0.001). CONCLUSIONS: These findings suggest that the existence of pre-CCVD before transplantation might not result in increased mortality directly but superpose the toxicity of the transplantation procedure, leading to a risk of post-CCVD. Post-CCVD was a powerful predictor for high NRM and inferior OS. Further risk stratification of pre-CCVD is needed to reduce NRM in various transplantation settings.

[Molecular Features and Clinical Significance of Acute Myeloid Leukemia in Elderly Patients].

OBJECTIVE: To study the molecular characteristics and clinical significance of elderly patients with acute myeloid leukemia (AML). METHODS: Dideoxy sequencing was used to analyze the mutation spectrum and clinical significance of 51 hematopathy-related genes in 52 patients with newly diagnosed elderly AML. The efficacy of 39 patients receiving DCAG chemotherapy was also analyzed. RESULTS: The mutational frequency was high in elderly AML patients (98.1%, 51/52), and there were some coexistence or mutual exclusion between different mutations. Both the number of mutations and the incidence of epigenetic mutations DNMT3A, TET2 (P<0.01), as well as FLT3-ITD (P<0.05) increased with age. c-KIT mutations were most common in favorable-risk AML (P<0.01), while NPM1 and DNMT3A were common in intermediate-risk AML (P<0.05), especially in AML with normal karyotype. The complete remission rate of elderly AML patients receiving DCAG chemotherapy was 71.8% (28/39). CONCLUSION: Elderly AML patients have specific molecular characteristics, and the incidence of methylation-related gene mutations is very high, showing a certain significance for clinical diagnosis and treatment.

Ruxolitinib Combined with Corticosteroids as First-Line Therapy for Acute Graft-versus-Host Disease in Haploidentical Peripheral Blood Stem Cell Transplantation Recipients.

Corticosteroids are commonly used as first-line treatment for acute graft-versus-host disease (aGVHD); however, they are effective in only approximately one-half of patients. This study prospectively evaluated the use of ruxolitinib combined with 1 mg/kg methylprednisolone in the initial treatment of aGVHD. A total of 32 patients were enrolled. aGVHD involved the skin (53.1%), gastrointestinal tract (68.8%), and liver (6.0%). The complete response rate at day +28 was 96.9%. The 1-year and 2-year cumulative incidence rates of chronic GVHD were 9.4% and 13.8%, respectively. The 1- year cumulative incidence of nonrelapse mortality was 8.7%, and the Kaplan-Meier curve estimated 1-year overall survival after transplantation at 73.4%. This prospective study suggests that patients with aGVHD show a high response rate to ruxolitinib (5 mg/day) combined with 1 mg/kg/day methylprednisolone. This novel regimen was seen to spare steroid exposure, alleviate toxicity, and improve long-term survival.

Identification of Pirin as a Molecular Target of the CCG-1423/CCG-203971 Series of Antifibrotic and Antimetastatic Compounds.

A series of compounds (including CCG-1423 and CCG-203971) discovered through an MRTF/SRF-dependent luciferase screen has shown remarkable efficacy in a variety of in vitro and in vivo models, including significant reduction of melanoma metastasis and bleomycin- induced fibrosis. Although these compounds are efficacious in these disease models, the molecular target is unknown. Here, we describe affinity isolation-based target identification efforts which yielded pirin, an iron-dependent cotranscription factor, as a target of this series of compounds. Using biophysical techniques including isothermal titration calorimetry and X-ray crystallography, we verify that pirin binds these compounds in vitro. We also show with genetic approaches that pirin modulates MRTF- dependent luciferase reporter activity. Finally, using both siRNA and a previously validated pirin inhibitor, we show a role for pirin in TGF-ß- induced gene expression in primary dermal fibroblasts. A recently developed analog, CCG-257081, which co crystallizes with pirin, is also effective in the prevention of bleomycin-induced dermal fibrosis.

Predictive Value of Pin1 in Cervical Low-Grade Squamous Intraepithelial Lesions and Inhibition of Pin1 Exerts Potent Anticancer Activity against Human Cervical Cancer.

Many oncogenes are involved in the progression from low-grade squamous intraepithelial lesions (LSILs) to high-grade squamous intraepithelial lesions (HSILs); which greatly increases the risk of cervical cancer (CC). Thus, a reliable biomarker for risk classification of LSILs is urgently needed. The prolyl isomerase Pin1 is overexpressed in many cancers and contributes significantly to tumour initiation and progression. Therefore, it is important to assess the effects of cancer therapies that target Pin1. In our study, we demonstrated that Pin1 may serve as a biomarker for LSIL disease progression and may constitute a novel therapeutic target for CC. We used a the novel Pin1 inhibitor KPT-6566, which is able to covalently bind to Pin1 and selectively target it for degradation. The results of our investigation revealed that the downregulation of Pin1 by shRNA or KPT-6566 inhibited the growth of human cervical cancer cells (CCCs). We also discovered that the use of KPT-6566 is a novel approach to enhance the therapeutic efficacy of cisplatin (DDP) against CCCs in vitro and in vivo. We showed that KPT-6566-mediated inhibition of Pin1 blocked multiple cancer-driving pathways simultaneously in CCCs. Furthermore, targeted Pin1 treatment suppressed the metastasis and invasion of human CCCs, and downregulation of Pin1 reversed the epithelial-mesenchymal transition (EMT) of CCCs via the c-Jun/slug pathway. Collectively, we showed that Pin1 may be a marker for the risk of progression to HSIL and that inhibition of Pin1 has anticancer effects against CC.

Engineering the hCRBPII Domain-Swapped Dimer into a New Class of Protein Switches.

Protein conformational switches or allosteric proteins play a key role in the regulation of many essential biological pathways. Nonetheless, the implementation of protein conformational switches in protein design applications has proven challenging, with only a few known examples that are not derivatives of naturally occurring allosteric systems. We have discovered that the domain-swapped (DS) dimer of hCRBPII undergoes a large and robust conformational change upon retinal binding, making it a potentially powerful template for the design of protein conformational switches. Atomic resolution structures of the apo- and holo-forms illuminate a simple, mechanical movement involving sterically driven torsion angle flipping of two residues that drive the motion. We further demonstrate that the conformational "readout" can be altered by addition of cross-domain disulfide bonds, also visualized at atomic resolution. Finally, as a proof of principle, we have created an allosteric metal binding site in the DS dimer, where ligand binding results in a reversible 5-fold loss of metal binding affinity. The high resolution structure of the metal-bound variant illustrates a well-formed metal binding site at the interface of the two domains of the DS dimer and confirms the design strategy for allosteric regulation.

Estrogen receptor ß inhibits breast cancer cells migration and invasion through CLDN6-mediated autophagy.

BACKGROUND: Estrogen receptor ß (ERß) has been reported to play an anti-cancer role in breast cancer, but the regulatory mechanism by which ERß exerts this effect is not clear. Claudin-6 (CLDN6), a tight junction protein, acts as a tumor suppressor gene in breast cancer. Our previous studies have found that 17ß-estradiol (E2) induces CLDN6 expression and inhibits MCF-7 cell migration and invasion, but the underlying molecular mechanisms are still unclear. In this study, we aimed to investigate the role of ERß in this process and the regulatory mechanisms involved. METHODS: Polymerase chain reaction (PCR) and western blot were used to characterize the effect of E2 on the expression of CLDN6 in breast cancer cells. Chromatin immunoprecipitation (ChIP) assays were carried out to confirm the interaction between ERß and CLDN6. Dual luciferase reporter assays were used to detect the regulatory role of ERß on the promoter activity of CLDN6. Wound healing and Transwell assays were used to examine the migration and invasion of breast cancer cells. Western blot, immunofluorescence and transmission electron microscopy (TEM) were performed to detect autophagy. Xenograft mouse models were used to explore the regulatory effect of the CLDN6-beclin1 axis on breast cancer metastasis. Immunohistochemistry (IHC) was used to detect ERß/CLDN6/beclin1 expression in breast cancer patient samples. RESULTS: Here, E2 upregulated the expression of CLDN6, which was mediated by ERß. ERß regulated CLDN6 expression at the transcriptional level. ERß inhibited the migration and invasion of breast cancer cells through CLDN6. Interestingly, this effect was associated with CLDN6-induced autophagy. CLDN6 positively regulated the expression of beclin1, which is a key regulator of autophagy. Beclin1 knockdown reversed CLDN6-induced autophagy and the inhibitory effect of CLDN6 on breast cancer metastasis. Moreover, ERß and CLDN6 were positively correlated, and the expression of CLDN6 was positively correlated with beclin1 in breast cancer tissues. CONCLUSION: Overall, this is the first study to demonstrate that the inhibitory effect of ERß on the migration and invasion of breast cancer cells was mediated by CLDN6, which induced the beclin1-dependent autophagic cascade.

ERα is required for suppressing OCT4-induced proliferation of breast cancer cells via DNMT1/ISL1/ERK axis.

OBJECTIVE: POU5F1 (OCT4) is implicated in cancer stem cell self-renewal. Currently, some studies have shown that OCT4 has a dual function in suppressing or promoting cancer progression. However, the precise molecular mechanism of OCT4 in breast cancer progression remains unclear. MATERIALS AND METHODS: RT-PCR and Western blot were utilized to investigate OCT4 expression in breast cancer tissues and cells. Cell proliferation assays and mouse models were applied to determine the effects of OCT4 on breast cancer cell proliferation. DNMT1 inhibitors, ChIP, CoIP, IHC and ERα inhibitors were used to explore the molecular mechanism of OCT4 in breast cancer. RESULTS: OCT4 was down-regulated in breast cancer tissues, and the overexpression of OCT4 promoted MDA-MB-231 cell proliferation and inhibited the proliferation of MCF-7 cells in vitro and in vivo, respectively. Two DNMT1 inhibitors (5-aza-dC and zebularine) suppressed OCT4-induced MDA-MB-231 cell proliferation through Ras/Raf1/ERK inactivation by targeting ISL1, which is the downstream of DNMT1. In contrast, OCT4 interacted with ERα, decreased DNMT1 expression and inactivated the Ras/Raf1/ERK signalling pathway in MCF-7 cells. Moreover, ERα inhibitor (AZD9496) reversed the suppression of OCT4-induced proliferation in MCF-7 cells via the activation of ERK signalling pathway. CONCLUSIONS: OCT4 is dependent on ERα to suppress the proliferation of breast cancer cells through DNMT1/ISL1/ERK axis.

OCT4 maintains self-renewal and reverses senescence in human hair follicle mesenchymal stem cells through the downregulation of p21 by DNA methyltransferases.

BACKGROUND: Self-renewal is dependent on an intrinsic gene regulatory network centered on OCT4 and on an atypical cell cycle G1/S transition, which is also regulated by OCT4. p21, a gene negatively associated with self-renewal and a senescence marker, is a member of the universal cyclin-dependent kinase inhibitors (CDKIs) and plays critical roles in the regulation of the G1/S transition. The expression of p21 can be regulated by OCT4-targeted DNA methyltransferases (DNMTs), which play distinct roles in gene regulation and maintaining pluripotency properties. The aim of this study was to determine the role of OCT4 in the regulation of self-renewal and senescence in human hair follicle mesenchymal stem cells (hHFMSCs) and to characterize the molecular mechanisms involved. METHODS: A lentiviral vector was used to ectopically express OCT4. The influences of OCT4 on the self-renewal and senescence of hHFMSCs were investigated. Next-generation sequencing (NGS) was performed to identify the downstream genes of OCT4 in this process. Methylation-specific PCR (MSP) analysis was performed to measure the methylation level of the p21 promoter region. p21 was overexpressed in hHFMSCsOCT4 to test its downstream effect on OCT4. The regulatory effect of OCT4 on DNMTs was examined by ChIP assay. 5-aza-dC/zebularine was used to inhibit the expression of DNMTs, and then self-renewal properties and senescence in hHFMSCs were detected. RESULTS: The overexpression of OCT4 promoted proliferation, cell cycle progression, and osteogenic differentiation capacity of hHFMSCs. The cell senescence of hHFMSCs was markedly suppressed due to the ectopic expression of OCT4. Through NGS, we identified 2466 differentially expressed genes (DEGs) between hHFMSCsOCT4 and hHFMSCsEGFP, including p21, which was downregulated. The overexpression of p21 abrogated the proliferation and osteogenic differentiation capacity of hHFMSCsOCT4 and promoted cell senescence. OCT4 enhanced the transcription of DNMT genes, leading to an elevation in the methylation of the p21 promoter. The inhibition of DNMTs reversed the OCT4-induced p21 reduction, depleted the self-renewal of hHFMSCsOCT4, and triggered cell senescence. CONCLUSIONS: OCT4 maintains the self-renewal ability of hHFMSCs and reverses senescence by suppressing the expression of p21 through the upregulation of DNMTs.

Genome-wide DNA methylation drives human embryonic stem cell erythropoiesis by remodeling gene expression dynamics.

AIM: To investigate the role of DNA methylation during erythrocyte production by human embryonic stem cells (hESCs). METHODS: We employed an erythroid differentiation model from hESCs, and then tracked the genome-wide DNA methylation maps and gene expression patterns through an Infinium HumanMethylation450K BeadChip and an Ilumina Human HT-12 v4 Expression Beadchip, respectively. RESULTS: A negative correlation between DNA methylation and gene expression was substantially enriched during the later differentiation stage and was present in both the promoter and the gene body. Moreover, erythropoietic genes with differentially methylated CpG sites that were primarily enriched in nonisland regions were upregulated, and demethylation of their gene bodies was associated with the presence of enhancers and DNase I hypersensitive sites. Finally, the components of JAK-STAT-NF-κB signaling were DNA hypomethylated and upregulated, which targets the key genes for erythropoiesis. CONCLUSION: Erythroid lineage commitment by hESCs requires genome-wide DNA methylation modifications to remodel gene expression dynamics.