Acute Myeloid Leukemia with Myelodysplasia - Related Changes after Isolated Myeloid Sarcoma.

BACKGROUND: As a tumor mass, a myeloid sarcoma consists of myeloid blasts and presents at an anatomical site other than the bone marrow. In about one quarter of cases, myeloid sarcoma happens without an underlying acute myeloid leukemia or other myeloid neoplasm, and it may precede or coincide with AML or form acute blastic transformation of MDSs, MPNs, or MDS/MPNs. METHODS: Herein, we described a rare case of acute myeloid leukemia with myelodysplasia-related changes (AML-MRC), with WT1 mutation and high expression of TP53 after isolated myeloid sarcoma of lymph nodes showing a higher proportion of blasts, dysplasia of both megakaryocytes and granulocytes. CONCLUSIONS: The case highlights the importance of a bone marrow examination, including morphology, immunophenotyping, cytogenetic, and molecular examination in all cases to exclude the possibility of myeloid sarcoma, especially the morphological feature of bone marrow dysplasia in the early stage before AML.

The single-cell chromatin landscape in gonadal cell lineage specification.

Gonad development includes sex determination and divergent maturation of the testes and ovaries. Recent advances in measuring gene expression in single cells are providing new insights into this complex process. However, the underlying epigenetic regulatory mechanisms remain unclear. Here, we profiled chromatin accessibility in mouse gonadal cells of both sexes from embryonic day 11.5 to 14.5 using single-cell assay for transposase accessible chromatin by sequencing (scATAC-seq). Our results showed that individual cell types can be inferred by the chromatin landscape, and that cells can be temporally ordered along developmental trajectories. Integrative analysis of transcriptomic and chromatin-accessibility maps identified multiple putative regulatory elements proximal to key gonadal genes Nr5a1, Sox9 and Wt1. We also uncover cell type-specific regulatory factors underlying cell type specification. Overall, our results provide a better understanding of the epigenetic landscape associated with the progressive restriction of cell fates in the gonad.

Homeobox regulator Wilms Tumour 1 is displaced by androgen receptor at cis-regulatory elements in the endometrium of PCOS patients.

Decidualisation, the process whereby endometrial stromal cells undergo morphological and functional transformation in preparation for trophoblast invasion, is often disrupted in women with polycystic ovary syndrome (PCOS) resulting in complications with pregnancy and/or infertility. The transcription factor Wilms tumour suppressor 1 (WT1) is a key regulator of the decidualization process, which is reduced in patients with PCOS, a complex condition characterized by increased expression of androgen receptor in endometrial cells and high presence of circulating androgens. Using genome-wide chromatin immunoprecipitation approaches on primary human endometrial stromal cells, we identify key genes regulated by WT1 during decidualization, including homeobox transcription factors which are important for regulating cell differentiation. Furthermore, we found that AR in PCOS patients binds to the same DNA regions as WT1 in samples from healthy endometrium, suggesting dysregulation of genes important to decidualisation pathways in PCOS endometrium due to competitive binding between WT1 and AR. Integrating RNA-seq and H3K4me3 and H3K27ac ChIP-seq metadata with our WT1/AR data, we identified a number of key genes involved in immune response and angiogenesis pathways that are dysregulated in PCOS patients. This is likely due to epigenetic alterations at distal enhancer regions allowing AR to recruit cofactors such as MAGEA11, and demonstrates the consequences of AR disruption of WT1 in PCOS endometrium.

Not too much, not too little-just the right amount: the story of YAP in the podocyte.

Chen et al. identify dysregulation of the transcriptional activator Yes-associated protein in the podocytes of diabetic mouse and human kidneys. Podocyte Yes-associated protein deficiency led to downregulation of the key transcription factor Wilms' tumor 1, and worsened podocyte injury in a mouse model of diabetic kidney injury. Yes-associated protein may therefore play a critical role in diabetic podocyte injury via regulation of Wilms' tumor 1 expression.

A simple protocol to establish a conditionally immortalized mouse podocyte cell line.

Podocytes are specialized terminally differentiated cells in the glomerulus that are the primary target cells in many glomerular diseases. However, the current podocyte cell lines suffer from prolonged in vitro differentiation and limited survival time, which impede research progress. Therefore, it is necessary to establish a cell line that exhibits superior performance and characteristics. We propose a simple protocol to obtain an immortalized mouse podocyte cell (MPC) line from suckling mouse kidneys. Primary podocytes were cultured in vitro and infected with the SV40 tsA58 gene to obtain immortalized MPCs. The podocytes were characterized using Western blotting and quantitative real-time PCR. Podocyte injury was examined using the Cell Counting Kit-8 assay and flow cytometry. First, we successfully isolated an MPC line and identified 39 °C as the optimal differentiation temperature. Compared to undifferentiated MPCs, the expression of WT1 and synaptopodin was upregulated in differentiated MPCs. Second, the MPCs ceased proliferating at a nonpermissive temperature after day 4, and podocyte-specific proteins were expressed normally after at least 15 passages. Finally, podocyte injury models were induced to simulate podocyte injury in vitro. In summary, we provide a simple and popularized protocol to establish a conditionally immortalized MPC, which is a powerful tool for the study of podocytes.

P62 promotes FSH-induced antral follicle formation by directing degradation of ubiquitinated WT1.

In females, the pathophysiological mechanism of poor ovarian response (POR) is not fully understood. Considering the expression level of p62 was significantly reduced in the granulosa cells (GCs) of POR patients, this study focused on identifying the role of the selective autophagy receptor p62 in conducting the effect of follicle-stimulating hormone (FSH) on antral follicles (AFs) formation in female mice. The results showed that p62 in GCs was FSH responsive and that its level increased to a peak and then decreased time-dependently either in ovaries or in GCs after gonadotropin induction in vivo. GC-specific deletion of p62 resulted in subfertility, a significantly reduced number of AFs and irregular estrous cycles, which were same as pathophysiological symptom of POR. By conducting mass spectrum analysis, we found the ubiquitination of proteins was decreased, and autophagic flux was blocked in GCs. Specifically, the level of nonubiquitinated Wilms tumor 1 homolog (WT1), a transcription factor and negative controller of GC differentiation, increased steadily. Co-IP results showed that p62 deletion increased the level of ubiquitin-specific peptidase 5 (USP5), which blocked the ubiquitination of WT1. Furthermore, a joint analysis of RNA-seq and the spatial transcriptome sequencing data showed the expression of steroid metabolic genes and FSH receptors pivotal for GCs differentiation decreased unanimously. Accordingly, the accumulation of WT1 in GCs deficient of p62 decreased steroid hormone levels and reduced FSH responsiveness, while the availability of p62 in GCs simultaneously ensured the degradation of WT1 through the ubiquitin‒proteasome system and autophagolysosomal system. Therefore, p62 in GCs participates in GC differentiation and AF formation in FSH induction by dynamically controlling the degradation of WT1. The findings of the study contributes to further study the pathology of POR.

Overexpression of WT1 in all molecular subtypes of breast cancer and its impact on survival: exploring oncogenic and tumor suppressor roles of distinct WT1 isoforms.

BACKGROUND: Breast cancer is a highly heterogeneous solid tumor, posing challenges in developing targeted therapies effective for all mammary carcinoma subtypes. WT1 emerges as a promising target for breast cancer therapy due to its potential oncogenic role in various cancer types. Previous works have yielded inconsistent results. Therefore, further studies are needed to clarify the behavior of this complex gene in breast cancer. METHODS AND RESULTS: In this study, we examined WT1 expression in both Formalin Fixed Paraffin Embedded breast tumors (n = 41) and healthy adjacent tissues (n = 41) samples from newly diagnosed cases of ductal invasive breast cancer. The fold change in gene expression between the tumor and healthy tissue was determined by calculating 2-∆∆Ct. Disease-free survival analysis was computed using the Kaplan-Meier method. To identify the expression levels of different WT1 isoforms, we explored the ISOexpresso database. Relative quantification of the WT1 gene revealed an overexpression of WT1 in most cases. The percentage of patients surviving free of disease at 8 years of follow-up was lower in the group overexpressing WT1 compared to the group with down-regulated WT1. CONCLUSIONS: Interestingly, this overexpression was observed in all molecular subtypes of invasive breast cancer, underscoring the significance of WT1 as a potential target in all these subtypes. The observed WT1 down-expression in a few cases of invasive breast cancer, associated with better survival outcomes, may correspond to the down-regulation of a particular WT1-KTS (-) isoform: the WT1 A isoform (EX5-/KTS-). The co-expression of this WT1 oncogenic isoform with a regulated WT1- tumor suppressor isoform, such as the major WT1 F isoform (EX5-/KTS +), could also explain such survival outcomes. Due to its capacity to adopt dual roles, it becomes imperative to conduct individual molecular expression profiling of the WT1 gene. Such an approach holds great promise in the development of personalized treatment strategies for breast cancer.

Comprehensive Transcriptomic Analysis of EWSR1::WT1 Targets Identifies CDK4/6 Inhibitors as an Effective Therapy for Desmoplastic Small Round Cell Tumors.

Desmoplastic small round cell tumors (DSRCT) are a type of aggressive, pediatric sarcoma characterized by the EWSR1::WT1 fusion oncogene. Targeted therapies for DSRCT have not been developed, and standard multimodal therapy is insufficient, leading to a 5-year survival rate of only 15% to 25%. Here, we depleted EWSR1::WT1 in DSRCT and established its essentiality in vivo. Transcriptomic analysis revealed that EWSR1::WT1 induces unique transcriptional alterations compared with WT1 and other fusion oncoproteins and that EWSR1::WT1 binding directly mediates gene upregulation. The E-KTS isoform of EWSR1::WT1 played a dominant role in transcription, and it bound to the CCND1 promoter and stimulated DSRCT growth through the cyclin D-CDK4/6-RB axis. Treatment with the CDK4/6 inhibitor palbociclib successfully reduced growth in two DSRCT xenograft models. As palbociclib has been approved by the FDA for the treatment of breast cancer, these findings demonstrate the sensitivity of DSRCT to palbociclib and support immediate clinical investigation of palbociclib for treating this aggressive pediatric cancer. SIGNIFICANCE: EWSR1::WT1 is essential for desmoplastic small round cell tumors and upregulates the cyclin D-CDK4/6-RB axis that can be targeted with palbociclib, providing a targeted therapeutic strategy for treating this deadly tumor type.

[Gene Profile and Clinical Significance of Concomitant Mutations in CN-AML Patients with CEBPA Mutation].

OBJECTIVE: To analyze the occurrence of concomitant gene mutations in cytogenetically normal acute myeloid leukemia (CN-AML) patients with CEBPA mutation and its impact on the clinical characteristics and prognosis of the patients. METHODS: 151 newly diagnosed patients with CN-AML in the Second Hospital of Shanxi Medical University from June 2013 to June 2020 were analyzed retrospectively. 34 common genetic mutations associated with hematologic malignancies were detected by next-generation sequencing technology. The occurrence of concomitant gene mutations in patients with CEBPA positive and negative groups was compared, and the correlation between concomitant mutations in different functional groups and the clinical characteristics and prognosis of CN-AML patients with CEBPA mutation was analyzed. RESULTS: In 151 patients with CN-AML, 55 (36.42%) were positive for CEBPA mutation (including 36 cases of CEBPAdm and 19 cases of CEBPAsm), of which 41 (74.55%) had co-mutations with other genes. The main mutated genes were GATA2 (25.45%, 14/55), TET2 (21.82%, 12/55), FLT3 (20.00%, 11/55), NRAS (12.73%, 7/55) and WT1 (9.09%, 9/55), etc. Some cases had two or more concomitant gene mutations. Grouping the mutant genes according to their functions showed that CEBPA+ group had lower mutation rates of histone methylation (P =0.002) and chromatin modification genes (P =0.002, P =0.033), and higher mutation rates of transcription factors (P =0.037) than CEBPA- group. In 55 patients with CEBPA+ CN-AML, the platelet count at diagnosis in signaling pathway gene mutation-positive group was lower than that in the mutation-negative group (P =0.005), the proportion of bone marrow blasts in transcription factor mutation-positive group was higher than that in the mutation-negative group (P =0.003), and the onset age in DNA methylation gene mutation-positive group and chromatin modifier mutation-positive group was older than that in the mutation-negative group, respectively (P =0.002, P =0.008). DFS of CEBPA+ CN-AML patients in signaling pathway gene mutation group was shorter than that in signaling pathway gene mutation-negative group (median DFS: 12 months vs not reached) (P =0.034). Compared with DNA methylation gene mutation-negative group, CEBPA+ CN-AML patients with DNA methylation gene mutation had lower CR rate (P =0.025) significantly shorter OS and DFS (median OS: 20 months vs not reached, P =0.006; median DFS: 15 months vs not reached, P =0.049). OS in patients with histone methylation gene mutation was significantly shorter than that in the histone methylation gene mutation-negative group (median OS: 12 months vs 40 months) (P =0.008). Multivariate analysis of prognostic factors showed that the proportion of bone marrow blasts (P =0.046), concomitant DNA methylation gene mutation (P =0.006) and histone methylation gene mutation (P =0.036) were independent risk factors affecting the prognosis. CONCLUSION: CN-AML patients with CEBPA mutation have specific concomitant gene profile, and the concomitant mutations of different functional genes have a certain impact on the clinical characteristics and prognosis of the patients.

New-onset diabetes after kidney transplantation: Assessing urinary Wilm's tumor-1 protein to predict renal allograft dysfunction.

PURPOSE: New-onset diabetes after transplantation (NODAT) is a frequent metabolic complication associated with podocyte damage and renal allograft dysfunction. Thus, Wilm's tumor-1 (WT-1) protein, as a podocyte marker, holds promise as an option to evaluate renal allograft dysfunction in NODAT. Therefore, the study aimed to investigate urinary WT-1 levels in NODAT patients during the first year after kidney transplantation (KTx). MATERIALS AND METHODS: KTx patients were categorized into non-NODAT and NODAT groups. Fasting blood glucose, glycated hemoglobin (HbA1c), urinary albumin/creatinine ratio (ACR), serum creatinine, estimated glomerular filtration rate (eGFR), and urinary WT-1 were measured at 3, 6, 9, and 12-months post-KTx. RESULTS: The NODAT group manifested elevated levels of blood glucose and HbA1c during the first year post-KTx. Also, exhibited elevations in ACR and serum creatinine levels at 6, 9, and 12-months post-KTx when compared to non-NODAT group. Conversely, eGFR values in the NODAT group demonstrated significant declines at 3, 6, and 9-months post-KTx relative to non-NODAT. Furthermore, NODAT group exhibited a median annual eGFR of 47 â€‹mL/min/1.73 â€‹m2. Urinary WT-1 levels at 3, 6, 9, and 12-months post-KTx were significantly higher in the NODAT group compared to non-NODAT. Additionally, noteworthy positive correlations were identified between urinary WT-1 and HbA1c levels, along with significant negative correlations between urinary WT-1 and eGFR at the 3, 6, 9, and 12-months post-KTx. CONCLUSION: The increased urinary WT-1 levels from 3-months post-KTx in NODAT patients may indicate the first sign of podocyte injury, predicting a renal allograft dysfunction in these patients.

Adjuvant Wilms' tumour 1-specific dendritic cell immunotherapy complementing conventional therapy for paediatric patients with high-grade glioma and diffuse intrinsic pontine glioma: protocol of a monocentric phase I/II clinical trial in Belgium.

INTRODUCTION: Diffuse intrinsic pontine glioma (DIPG) and paediatric high-grade glioma (pHGG) are aggressive glial tumours, for which conventional treatment modalities fall short. Dendritic cell (DC)-based immunotherapy is being investigated as a promising and safe adjuvant therapy. The Wilms' tumour protein (WT1) is a potent target for this type of antigen-specific immunotherapy and is overexpressed in DIPG and pHGG. Based on this, we designed a non-randomised phase I/II trial, assessing the feasibility and safety of WT1 mRNA-loaded DC (WT1/DC) immunotherapy in combination with conventional treatment in pHGG and DIPG. METHODS AND ANALYSIS: 10 paediatric patients with newly diagnosed or pretreated HGG or DIPG were treated according to the trial protocol. The trial protocol consists of leukapheresis of mononuclear cells, the manufacturing of autologous WT1/DC vaccines and the combination of WT1/DC-vaccine immunotherapy with conventional antiglioma treatment. In newly diagnosed patients, this comprises chemoradiation (oral temozolomide 90 mg/m2 daily+radiotherapy 54 Gy in 1.8 Gy fractions) followed by three induction WT1/DC vaccines (8-10×106 cells/vaccine) given on a weekly basis and a chemoimmunotherapy booster phase consisting of six 28-day cycles of oral temozolomide (150-200 mg/m2 on days 1-5) and a WT1/DC vaccine on day 21. In pretreated patients, the induction and booster phase are combined with best possible antiglioma treatment at hand. Primary objectives are to assess the feasibility of the production of mRNA-electroporated WT1/DC vaccines in this patient population and to assess the safety and feasibility of combining conventional antiglioma treatment with the proposed immunotherapy. Secondary objectives are to investigate in vivo immunogenicity of WT1/DC vaccination and to assess disease-specific and general quality of life. ETHICS AND DISSEMINATION: The ethics committee of the Antwerp University Hospital and the University of Antwerp granted ethics approval. Results of the clinical trial will be shared through publication in a peer-reviewed journal and presentations at conferences. TRIAL REGISTRATION NUMBER: NCT04911621.

Decade-long WT1-specific CTLs induced by WT1 peptide vaccination.

INTRODUCTION: The peptide-based cancer vaccine targeting Wilms' tumor 1 (WT1) is a promising immunotherapeutic strategy for hematological malignancies. It remains unclear how long and to what extent the WT1-specific CD8 + cytotoxic T cell (CTL) persist after WT1 peptide vaccination. METHODS: The WT1 peptide vaccine was administered with written consent to a patient with CML in the chronic phase who did not respond well to imatinib, and the patient was followed for 12 years after vaccination. Immune monitoring was performed by specific amplification of WT1-specific CTLs using a mixed lymphocyte peptide culture. T-cell receptors (TCRs) of amplified WT1-specific CTLs were analyzed using next-generation sequencing. This study was approved by the Institutional Review Board of our institution. RESULT: WT1-specific CTLs, which were initially detected during WT1 peptide vaccination, persisted at a frequency of less than 5 cells per 1,000,000 CD8 + T cells for more than 10 years. TCR repertoire analysis confirmed the diversity of WT1-specific CTLs 11 years after vaccination. CTLs exhibited WT1 peptide-specific cytotoxicity in vitro. CONCLUSION: The WT1 peptide vaccine induced an immune response that persists for more than 10 years, even after cessation of vaccination in the CML patient.

Inhibition of transcriptional coactivator YAP Impairs the expression and function of transcription factor WT1 in diabetic podocyte injury.

Podocyte injury and loss are hallmarks of diabetic nephropathy (DN). However, the molecular mechanisms underlying these phenomena remain poorly understood. YAP (Yes-associated protein) is an important transcriptional coactivator that binds with various other transcription factors, including the TEAD family members (nuclear effectors of the Hippo pathway), that regulate cell proliferation, differentiation, and apoptosis. The present study found an increase in YAP phosphorylation at S127 of YAP and a reduction of nuclear YAP localization in podocytes of diabetic mouse and human kidneys, suggesting dysregulation of YAP may play a role in diabetic podocyte injury. Tamoxifen-inducible podocyte-specific Yap gene knockout mice (YappodKO) exhibited accelerated and worsened diabetic kidney injury. YAP inactivation decreased transcription factor WT1 expression with subsequent reduction of Tead1 and other well-known targets of WT1 in diabetic podocytes. Thus, our study not only sheds light on the pathophysiological roles of the Hippo pathway in diabetic podocyte injury but may also lead to the development of new therapeutic strategies to prevent and/or treat DN by targeting the Hippo signaling pathway.

Prognostic impact of peripheral blood WT1 mRNA dynamics in patients with acute myeloid leukemia treated with venetoclax combination therapy.

BACKGROUND: Wilms' tumor gene 1 (WT1) mRNA quantification is a useful marker of measurable residual disease in acute myeloid leukemia (AML). However, whether monitoring the WT1 mRNA levels may predict the outcome of venetoclax (VEN) combination therapy in AML is not reported. This study aims to elucidate whether WT1 mRNA dynamics could predict long-term prognosis. METHODS: 33 patients with untreated or relapsed/refractory AML evaluated for peripheral blood WT1 dynamics in VEN combination therapy were analyzed. RESULTS: The median age was 73 years (range 39-87). Azacitidine was combined with VEN in 91% of patients. Overall, the median overall survival (OS) was 334 days (95% CI 210-482), and the complete remission (CR) plus CR with incomplete hematologic recovery rate was 59%. A 1-log reduction of WT1 mRNA values by the end of cycle 2 of treatment was associated with significantly better OS and event-free survival (EFS) (median OS 482 days vs. 237 days, p = 0.049; median EFS 270 days vs. 125 days, p = 0.02). The negativity of post-treatment WT1 mRNA value during the treatment was associated with significantly better OS and EFS (median OS 482 days vs. 256 days, p = 0.02; median EFS not reached vs. 150 days, p = 0.005). Multivariate analysis confirmed the significance of these two parameters as strong EFS predictors (HR 0.26, p = 0.024 and HR 0.15, p = 0.013, respectively). The increase in WT1 mRNA values was correlated with relapse. CONCLUSION: This study demonstrates that WT1 mRNA dynamics can be a useful marker for assessing long-term prognosis of VEN combination therapy for AML.

WT1 and TP53 as valuable diagnostic biomarkers for relapse after hematopoietic stem cell transplantation in acute myeloid leukemia.

BACKGROUND: Relapse following hematopoietic stem cell transplantation (HSCT) occurs relatively frequently and is a significant risk factor for mortality in patients with acute myeloid leukemia (AML). Early diagnosis is, therefore, of utmost importance and can provide valuable guidance for appropriate and timely intervention. Here, the diagnostic value of two molecular markers, Wilms tumor 1 (WT1) and tumor suppressor protein p53 (TP53), were studied. METHODS AND RESULTS: Twenty AML patients undergoing HSCT participated in this investigation. Some had relapsed following HSCT, while others were in remission. Peripheral blood (PB) and bone marrow (BM) samples were collected following relapse and remission. WT1 and TP53 messenger RNA (mRNA) expression was evaluated using reverse transcription-quantitative polymerase chain reaction (RT‒qPCR). The diagnostic value of genes was evaluated by utilizing receiver-operating characteristic (ROC) curve analysis. ROC analysis showed WT1 and TP53 as diagnostic markers for relapse after HSCT in AML patients. The mRNA expression level of WT1 was elevated in individuals who experienced relapse compared to those in a state of remission (p value < 0.01). Conversely, the expression level of TP53 mRNA was lower in individuals who had relapsed compared to those in remission (p value < 0.01). CONCLUSIONS: WT1 and TP53 possess the potential to serve as invaluable biomarkers in the identification of molecular relapse after HSCT in patients with AML. Further studies for a definitive conclusion are recommended.

Dendritic cell vaccination in combination with erlotinib in a patient with inoperable lung adenocarcinoma: a case report.

BACKGROUND: Satisfactory treatment for patients with unresectable advanced lung cancer has not yet been established. We report a case of unresectable advanced lung cancer (stage IIIb: T2aN3M0) treated with a total of 15 doses of dendritic cells pulsed with a Wilms' tumor 1 and mucin 1 vaccine in combination with erlotinib, a small molecule epidermal growth factor receptor tyrosine kinase inhibitor, for more than 699 days without recurrence or metastasis. CASE PRESENTATION: A 63-year-old Korean woman was diagnosed with lung adenocarcinoma by pathology and computed tomography. The adenocarcinoma showed an epidermal growth factor receptor (EGFR) mutation, no anaplastic lymphoma kinase expression, and less than 1% expression of programmed death ligand 1. She received erlotinib alone for approximately 1 month. She then received erlotinib and the dendritic cells pulsed with Wilms' tumor 1 and mucin 1 vaccine. The diameter of the erythema at the vaccinated sites was 30 mm at 48 hours after the first vaccination. Moreover, it was maintained at more than 20 mm during the periods of vaccination. These results suggested the induction of antitumor immunity by the vaccine. Remarkably, the tumor size decreased significantly to 12 mm, a 65.7% reduction, after combined therapy with eight doses of the dendritic cells pulsed with Wilms' tumor 1 and mucin 1 vaccine and erlotinib for 237 days based on fluorodeoxyglucose uptake by positron emission tomography/computed tomography and computed tomography. Interestingly, after 321 days of combination therapy, the clinical findings improved, and no tumor was detected based on computed tomography. Validation of the tumor's disappearance persisted for at least 587 days after treatment initiation, without any indication of recurrence or metastasis. CONCLUSION: Standard anticancer therapy combined with the dendritic cells pulsed with Wilms' tumor 1 and mucin 1 vaccine may have therapeutic effects for such patients with unresectable lung adenocarcinoma.

Wilms' tumor 1 (WT1) antigen is overexpressed in Kaposi Sarcoma and is regulated by KSHV vFLIP.

In people living with HIV, Kaposi Sarcoma (KS), a vascular neoplasm caused by KS herpesvirus (KSHV/HHV-8), remains one of the most common malignancies worldwide. Individuals living with HIV, receiving otherwise effective antiretroviral therapy, may present with extensive disease requiring chemotherapy. Hence, new therapeutic approaches are needed. The Wilms' tumor 1 (WT1) protein is overexpressed and associated with poor prognosis in several hematologic and solid malignancies and has shown promise as an immunotherapeutic target. We found that WT1 was overexpressed in >90% of a total 333 KS biopsies, as determined by immunohistochemistry and image analysis. Our largest cohort from ACTG, consisting of 294 cases was further analyzed demonstrating higher WT1 expression was associated with more advanced histopathologic subtypes. There was a positive correlation between the proportion of infected cells within KS tissues, assessed by expression of the KSHV-encoded latency-associated nuclear antigen (LANA), and WT1 positivity. Areas with high WT1 expression showed sparse T-cell infiltrates, consistent with an immune evasive tumor microenvironment. We show that major oncogenic isoforms of WT1 are overexpressed in primary KS tissue and observed WT1 upregulation upon de novo infection of endothelial cells with KSHV. KSHV latent viral FLICE-inhibitory protein (vFLIP) upregulated total and major isoforms of WT1, but upregulation was not seen after expression of mutant vFLIP that is unable to bind IKKÆ´ and induce NFκB. siRNA targeting of WT1 in latent KSHV infection resulted in decreased total cell number and pAKT, BCL2 and LANA protein expression. Finally, we show that ESK-1, a T cell receptor-like monoclonal antibody that recognizes WT1 peptides presented on MHC HLA-A0201, demonstrates increased binding to endothelial cells after KSHV infection or induction of vFLIP expression. We propose that oncogenic isoforms of WT1 are upregulated by KSHV to promote tumorigenesis and immunotherapy directed against WT1 may be an approach for KS treatment.

Rreb1 is a key transcription factor in Sertoli cell maturation and function and spermatogenesis in mouse.

Spermatogenesis is a developmental process driven by interactions between germ cells and Sertoli cells. This process depends on appropriate gene expression, which might be regulated by transcription factors. This study focused on Rreb1, a zinc finger transcription factor, and explored its function and molecular mechanisms in spermatogenesis in a mouse model. Our results showed that RREB1 was predominantly expressed in the Sertoli cells of the testis. The decreased expression of RREB1 following injection of siRNA caused impaired Sertoli cell development, which was characterized using a defective blood-testis barrier structure and decreased expression of Sertoli cell functional maturity markers; its essential trigger might be SMAD3 destabilization. The decreased expression of RREB1 in mature Sertoli cells influenced the cell structure and function, which resulted in abnormal spermatogenesis, manifested as oligoasthenoteratozoospermia, and we believe RREB1 plays this role by regulating the transcription of Fshr and Wt1. RREB1 has been reported to activate Fshr transcription, and we demonstrated that the knockdown of Rreb1 caused a reduction in follicle-stimulating hormone receptor (FSHR) in the testis, which could be the cause of the increased sperm malformation. Furthermore, we confirmed that RREB1 directly activates Wt1 promoter activity, and RREB1 downregulation induced the decreased expression of Wt1 and its downstream polarity-associated genes Par6b and E-cadherin, which caused increased germ-cell death and reduced sperm number and motility. In conclusion, RREB1 is a key transcription factor essential for Sertoli cell development and function and is required for normal spermatogenesis.