PAX6 promotes neuroendocrine phenotypes of prostate cancer via enhancing MET/STAT5A-mediated chromatin accessibility.

BACKGROUND: Neuroendocrine prostate cancer (NEPC) is a lethal subset of prostate cancer which is characterized by neuroendocrine differentiation and loss of androgen receptor (AR) signaling. Growing evidence reveals that cell lineage plasticity is crucial in the failure of NEPC therapies. Although studies suggest the involvement of the neural transcription factor PAX6 in drug resistance, its specific role in NEPC remains unclear. METHODS: The expression of PAX6 in NEPC was identified via bioinformatics and immunohistochemistry. CCK8 assay, colony formation assay, tumorsphere formation assay and apoptosis assay were used to illustrate the key role of PAX6 in the progression of in vitro. ChIP and Dual-luciferase reporter assays were conducted to confirm the binding sequences of AR in the promoter region of PAX6, as well as the binding sequences of PAX6 in the promoter regions of STAT5A and MET. For in vivo validation, the xenograft model representing NEPC subtype underwent pathological analysis to verify the significant role of PAX6 in disease progression. Complementary diagnoses were established through public clinical datasets and transcriptome sequencing of specific cell lines. ATAC-seq was used to detect the chromatin accessibility of specific cell lines. RESULTS: PAX6 expression was significantly elevated in NEPC and negatively regulated by AR signaling. Activation of PAX6 in non-NEPC cells led to NE trans-differentiation, while knock-down of PAX6 in NEPC cells inhibited the development and progression of NEPC. Importantly, loss of AR resulted in an enhanced expression of PAX6, which reprogramed the lineage plasticity of prostate cancer cells to develop NE phenotypes through the MET/STAT5A signaling pathway. Through ATAC-seq, we found that a high expression level of PAX6 elicited enhanced chromatin accessibility, mainly through attenuation of H4K20me3, which typically causes chromatin silence in cancer cells. CONCLUSION: This study reveals a novel neural transcription factor PAX6 could drive NEPC progression and suggest that it might serve as a potential therapeutic target for the management of NEPC.

E3 ubiquitin ligase RNF128 negatively regulates the IL-3/STAT5 signaling pathway by facilitating K27-linked polyubiquitination of IL-3Rα.

IL-3/STAT5 signaling pathway is crucial for the development and activation of immune cells, contributing to the cellular response to infections and inflammatory stimuli. Dysregulation of the IL-3/STAT5 signaling have been associated with inflammatory and autoimmune diseases characterized by inflammatory cell infiltration and organ damage. IL-3 receptor α (IL-3Rα) specifically binds to IL-3 and initiates intracellular signaling, resulting in the phosphorylation of STAT5. However, the regulatory mechanisms of IL-3Rα remain unclear. Here, we identified the E3 ubiquitin ligase RNF128 as a negative regulator of IL-3/STAT5 signaling by targeting IL-3Rα for lysosomal degradation. RNF128 was shown to selectively bind to IL-3Rα, without interacting with the common beta chain IL-3Rß, which shares the subunit with GM-CSF. The deficiency of Rnf128 had no effect on GM-CSF-induced phosphorylation of Stat5, but it resulted in heightened Il-3-triggered activation of Stat5 and increased transcription of the Id1, Pim1, and Cd69 genes. Furthermore, we found that RNF128 promoted the K27-linked polyubiquitination of IL-3Rα in a ligase activity-dependent manner, ultimately facilitating its degradation through the lysosomal pathway. RNF128 inhibited the activation and chemotaxis of macrophages in response to LPS stimulation, thereby attenuating excessive inflammatory responses. Collectively, these results reveal that RNF128 negatively regulates the IL-3/STAT5 signaling pathway by facilitating K27-linked polyubiquitination of IL-3Rα. This study uncovers E3 ubiquitin ligase RNF128 as a novel regulator of the IL-3/STAT5 signaling pathway, providing potential molecular targets for the treatment of inflammatory diseases.

Hyperactive STAT5 hijacks T cell receptor signaling and drives immature T cell acute lymphoblastic leukemia.

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive immature T cell cancer. Mutations in IL7R have been analyzed genetically, but downstream effector functions such as STAT5A and STAT5B hyperactivation are poorly understood. Here, we studied the most frequent and clinically challenging STAT5BN642H driver in T cell development and immature T cell cancer onset and compared it with STAT5A hyperactive variants in transgenic mice. Enhanced STAT5 activity caused disrupted T cell development and promoted an early T cell progenitor-ALL phenotype, with upregulation of genes involved in T cell receptor (TCR) signaling, even in absence of surface TCR. Importantly, TCR pathway genes were overexpressed in human T-ALL and mature T cell cancers and activation of TCR pathway kinases was STAT5 dependent. We confirmed STAT5 binding to these genes using ChIP-Seq analysis in human T-ALL cells, which were sensitive to pharmacologic inhibition by dual STAT3/5 degraders or ZAP70 tyrosine kinase blockers in vitro and in vivo. We provide genetic and biochemical proof that STAT5A and STAT5B hyperactivation can initiate T-ALL through TCR pathway hijacking and suggest similar mechanisms for other T cell cancers. Thus, STAT5 or TCR component blockade are targeted therapy options, particularly in patients with chemoresistant clones carrying STAT5BN642H.

Stat5b/Ezh2 axis governs high PD-L1 expressing tolerogenic dendritic cell subset in autoimmune diabetes.

Dendritic cells (DCs) are specialized antigen-presenting cells that play an important role in inducing and maintaining immune tolerance. The altered distribution and/or function of DCs contributes to defective tolerance in autoimmune diseases such as type 1 diabetes (T1D). In human T1D and in NOD mouse models, DCs share some defects and are often described as less tolerogenic and excessively immunogenic. In the NOD mouse model, the autoimmune response is associated with a defect in the Stat5b signaling pathway. We have reported that expressing a constitutively active form of Stat5b in DCs of transgenic NOD mice (NOD.Stat5b-CA), re-established their tolerogenic function, restored autoimmune tolerance and conferred protection from diabetes. However, the role and molecular mechanisms of Stat5b signaling in regulating splenic conventional DCs tolerogenic signature remained unclear. In this study, we reported that, compared to immunogenic splenic DCs of NOD, splenic DCs of NOD.Stat5b-CA mice exhibited a tolerogenic profile marked by elevated PD-L1 and PD-L2 expression, reduced pro-inflammatory cytokine production, increased frequency of the cDC2 subset and decreased frequency of the cDC1 subset. This tolerogenic profile was associated with increased Ezh2 and IRF4 but decreased IRF8 expression. We also found an upregulation of PD-L1 in the cDC1 subset and high PD-L1 and PD-L2 expression in cDC2 of NOD.Stat5b-CA mice. Mechanistically, we demonstrated that Ezh2 plays an important role in the maintenance of high PD-L1 expression in cDC1 and cDC2 subsets and that Ezh2 inhibition resulted in PD-L1 but not PD-L2 downregulation which was more drastic in the cDC2 subset. Additionally, Ezh2 inhibition severely reduced the cDC2 subset and increased the cDC1 subset and Stat5b-CA.DC pro-inflammatory cytokine production. Together our data suggest that the Stat5b-Ezh2 axis is critical for the maintenance of tolerogenic high PD-L1-expressing cDC2 and autoimmune tolerance in NOD.Stat5b-CA mice.

Involvement of the JAK-STAT pathway in the molecular landscape of tyrosine kinase fusion-negative hypereosinophilic syndromes: A nationwide CEREO study.

We investigated using a custom NGS panel of 149 genes the mutational landscape of 64 consecutive adult patients with tyrosine kinase fusion-negative hypereosinophilia (HE)/hypereosinophilic syndrome (HES) harboring features suggestive of myeloid neoplasm. At least one mutation was reported in 50/64 (78%) patients (compared to 8/44 (18%) patients with idiopathic HE/HES/HEUS used as controls; p < .001). Thirty-five patients (54%) had at least one mutation involving the JAK-STAT pathway, including STAT5B (n = 18, among which the hotspot N642H, n = 13), JAK1 (indels in exon 13, n = 5; V658F/L, n = 2), and JAK2 (V617F, n = 6; indels in exon 13, n = 2). Other previously undescribed somatic mutations were also found in JAK2, JAK1, STAT5B, and STAT5A, including three patients who shared the same STAT5A V707fs mutation and features consistent with primary polycythemia. Nearly all JAK-STAT mutations were preceded by (or associated with) myelodysplasia-related gene mutations, especially in RNA-splicing genes or chromatin modifiers. In multivariate analysis, neurologic involvement (hazard ratio [HR] 4.95 [1.87-13.13]; p = .001), anemia (HR 5.50 [2.24-13.49]; p < .001), and the presence of a high-risk mutation (as per the molecular international prognosis scoring system: HR 6.87 [2.39-19.72]; p < .001) were independently associated with impaired overall survival. While corticosteroids were ineffective in all treated JAK-STAT-mutated patients, ruxolitinib showed positive hematological responses including in STAT5A-mutated patients. These findings emphasize the usefulness of NGS for the workup of tyrosine kinase fusion-negative HE/HES patients and support the use of JAK inhibitors in this setting. Updated classifications could consider patients with JAK-STAT mutations and eosinophilia as a new "gene mutated-entity" that could be differentiated from CEL, NOS, and idiopathic HES.

Ponatinib and STAT5 Inhibitor Pimozide Combined Synergistic Treatment Applications Potentially Overcome Drug Resistance via Regulating the Cytokine Expressional Network in Chronic Myeloid Leukemia Cells.

Chronic myeloid leukemia (CML) is a clonal myeloproliferative hematological disease characterized by the chimeric breakpoint-cluster region/Abelson kinase1 (BCR::ABL1) oncoprotein; playing a pivotal role in CML molecular pathology, diagnosis, treatment, and possible resistance arising from the success and tolerance of tyrosine kinase inhibitor (TKI)-based therapy. The transcription factor STAT5 constitutive signaling, which is influenced by the cytokine signaling network, triggers BCR::ABL1-based CML pathogenesis and is also relevant to acquired TKI resistance. The unsuccessful therapeutic approaches targeting BCR::ABL1, in particular third-line therapy with ponatinib, still need to be further developed with alternative combination strategies to overcome drug resistance. As treatment with the STAT5 inhibitor pimozide in combination with ponatinib resulted in an efficient and synergistic therapeutic approach in TKI-resistant CML cells, this study focused on identifying the underlying amplification of ponatinib response mechanisms by determining different cytokine expression profiles in parental and ponatinib-resistant CML cells, in vitro. The results showed that expression of interleukin (IL) 1B, IL9, and IL12A-B was increased by 2-fold, while IL18 was downregulated by 2-fold in the ponatinib-resistant cells compared to sensitive ones. Importantly, ponatinib treatment upregulated the expression of 21 of the 23 interferon and IL genes in the ponatinib-resistant cells, while treatment with pimozide or a combination dose resulted in a reduction in the expression of 19 different cytokine genes, such as for example, inflammatory cytokines, IL1A-B and IL6 or cytokine genes associated with supporting tumor progression, leukemia stem cell growth or poor survival, such as IL3, IL8, IL9, IL10, IL12, or IL15. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis results showed that the genes were mainly enriched in the regulation of receptor signaling through the Janus kinase/signal transducer and activator of transcription pathway, cytokine-cytokine receptor interaction, and hematopoietic cell lineage. Protein-protein interaction analysis showed that IL2, IL6, IL15, IFNG, and others appeared in the top lists of pathways, indicating their high centrality and importance in the network. Therefore, pimozide could be a promising agent to support TKI therapies in ponatinib resistance. This research would help to clarify the role of cytokines in ponatinib resistance and advance the development of new therapeutics to utilize the STAT5 inhibitor pimozide in combination with TKIs.

PRL-mediated STAT5B/ARRB2 pathway promotes the progression of prostate cancer through the activation of MAPK signaling.

Previous study showed that higher expression of prolactin (PRL) was found in CRPC samples compared with hormone-naive prostate cancer (HNPC) and benign prostatic hyperplasia (BPH) samples. We further investigate the function of PRL in prostate cancer (PCa) and explored its downstream effects. We found heterogeneous expression of the PRLR in clinical prostate samples. The VCaP and 22Rv1 cells exhibited PRLR expression. Among the downstream proteins, STAT5B was the dominant subtype in clinical samples and cell lines. Human recombinant PRL stimulation of PCa cells with PRLR expression resulted in increased phosphorylation of STAT5B(pSTAT5B) and progression of PCa in vitro and in vivo, and STAT5B knockdown can suppress the malignant behavior of PCa. To understand the mechanism further, we performed Bioinformatic analysis, ChIP qPCR, and luciferase reporter gene assay. The results revealed that ARRB2 was the transcription target gene of STAT5B, and higher expression of ARRB2 was related to higher aggression and poorer prognosis of PCa. Additionally, Gene set enrichment analysis indicated that higher expression of ARRB2 was significantly enriched in the MAPK signaling pathway. Immunohistochemistry (IHC) demonstrated elevated pSTAT5B, ARRB2, and pERK1/2 expression levels in CRPC tissues compared to HNPC and BPH. Mechanically, ARRB2 enhanced the activation of the MAPK pathway by binding to ERK1/2, thereby promoting the phosphorylation of ERK1/2 (pERK1/2). In conclusion, our study demonstrated that PRL stimulation can promote the progression of PCa through STAT5B/ARRB2 pathway and activation of MAPK signaling, which can be suppressed by intervention targeting STAT5B. Blockade of the STAT5B can be a potential therapeutic target for PCa.

The novel TERF2::PDGFRB fusion gene enhances tumorigenesis via PDGFRB/STAT5 signalling pathways and sensitivity to TKI in ph-like ALL.

Patients with Philadelphia chromosome-like acute lymphoblastic leukaemia (Ph-like ALL) often face a grim prognosis, with PDGFRB gene fusions being commonly detected in this subgroup. Our study has unveiled a newfound fusion gene, TERF2::PDGFRB, and we have found that patients carrying this fusion gene exhibit sensitivity to dasatinib. Ba/F3 cells harbouring the TERF2::PDGFRB fusion display IL-3-independent cell proliferation through activation of the p-PDGFRB and p-STAT5 signalling pathways. These cells exhibit reduced apoptosis and demonstrate sensitivity to imatinib in vitro. When transfused into mice, Ba/F3 cells with the TERF2::PDGFRB fusion gene induce tumorigenesis and a shortened lifespan in cell-derived graft models, but this outcome can be improved with imatinib treatment. In summary, we have identified the novel TERF2::PDGFRB fusion gene, which exhibits oncogenic potential both in vitro and in vivo, making it a potential therapeutic target for tyrosine kinase inhibitors (TKIs).

IKZF3/Aiolos H195Y mutation identified in a mouse model of B cell leukemia results in altered DNA binding and altered STAT5-dependent gene expression.

Precursor B cell acute lymphoblastic leukemia (Pre-B-ALL) arises from developing B cells and frequently involves mutations in genes encoding transcription factors. In this study, we investigated the function of mutations in the transcription factor IKZF3 (Aiolos), R137* and H195Y, discovered in a mouse model of pre-B-ALL. R137* IKZF3 mutation resulted in a truncated protein, while electrophoretic mobility shift assay showed that H195Y IKZF3 mutation resulted in a protein with altered DNA binding. 38B9 pre-B cell lines were generated expressing WT and H195Y IKZF3 proteins. Anti-IKZF3 ChIP-seq showed that H195Y IKZF3 interacted with a larger number of sites that were different than WT IKZF3. Treatment with interleukin-7 induced changes in gene expression in 38B9 cells expressing WT IKZF3, but did not induce any changes in gene expression in cells expressing H195Y IKZF3. Anti-STAT5 ChIP-seq showed that expression of H195Y IKZF3 resulted in redistribution of STAT5 binding sites in the genome. H195Y IKZF3 binding sites overlapped with a subset of STAT5 binding sites, including in the promoter of the Cish gene. These findings suggest that H195Y mutation of IKZF3 results in altered DNA binding specificity and altered binding of STAT5 to target genes.

Growth hormone directly stimulates GATA2 expression.

OBJECTIVE: GATA2 is a key transcription factor involved in the differentiation and determination of thyrotrophs and gonadotrophs in pituitary and hematopoietic development. However, studies on the upstream ligands of the GATA2 signal transduction pathway have been limited. To identify upstream ligands, we examined growth hormone (GH) as a plausible stimulator. DESIGN: We evaluated GH-induced GATA2 expression in murine TtT/GF thyrotrophic pituitary tumor cells and its direct impact on the GHR/JAK/STAT5 pathway using a combination of a reporter assay, real-time quantitative polymerase chain reaction, and western blotting. RESULTS: GATA2 expression increased with activated STAT5B in a dose-dependent manner and was inhibited by a STAT5 specific inhibitor. Moreover, we found functional STAT5B binding site consensus sequences at -359 bp in the GATA2 promoter region. CONCLUSION: These findings suggest that GH directly stimulates GATA2 via the GHR/JAK/STAT pathway and participates in various developmental phenomena mediated by GATA2.

Impact of STAT5A-CNVs on growth traits in Chinese beef cattle breeds.

CNVs, which are a type of structural variation, make a substantial impact on diverse characteristics in multiple species. Q-PCR and data association analysis were used for STAT5A gene copy in this study. This study aimed to investigate the copy number variation (CNV) of the STAT5A gene in seven Chinese cattle breeds, namely Qinchuan cattle, Xianan cattle, Yunling cattle, Ji'an cattle, Jiaxian Red cattle, Qaidam cattle, and Guyuan yellow cattle. Blood samples were collected for CNV typing, and the correlation between CNV type and growth traits was analyzed using SPSS 23.0 software and ANOVA. The findings revealed variations in the distribution of different copy number types among the different cattle breeds. Furthermore, association analysis demonstrated a positive impact of CNV in the STAT5A gene on cattle growth: in the JX, individuals with duplication types exhibited superior performance in terms of rump length (P < 0.05). Conversely, normal GY cattle demonstrated better body height and abdomen circumference (P < 0.05), while QD cattle exhibited a significant correlation between weight and body length with normal individuals (P < 0.05). Moreover, QC bovine duplication individuals outperformed other types, with copy number variation significantly associated with chest depth, chest width, and body length (P < 0.05). The results validate the correlation between copy number variation (CNV) of the STAT5A gene and growth characteristics in five different cattle breeds, providing a reliable benchmark for the purpose of cattle breeding.

STAT5-mediated transcription of miR-33-5p in Mycoplasma gallisepticum-infected DF-1 cells.

RESEARCH HIGHLIGHTS: MG-HS regulates the expression of transcription factor STAT5.Transcription factor STAT5 can target miR-33-5p promoter element.MG-influenced STAT5 regulates miR-33-5p and its target gene expression.

A novel EGFR variant EGFRx maintains glioblastoma stem cells through STAT5.

BACKGROUND: Glioblastomas are universally lethal brain tumors containing tumor-propagating glioblastoma stem cells (GSCs). EGFR gene amplification or mutation is frequently detected in GBMs and is associated with poor prognosis. However, EGFR variants in GSCs and their role in the maintenance of GSCs and progression of GBM are unclear. METHODS: EGFR variants were detected through bioinformatic HISAT-StringTie-Ballgown pipeline and verified through 5' RACE, RT-PCR, ribonuclease protection, and northern blotting assays. EGFRx function was investigated through neurosphere, cell viability, intracranial xenograft and RNA-seq assays. EGFRx-STAT5 signaling was investigated through western blotting, coimmunoprecipitation, immunofluorescence, luciferase reporter, RT-PCR and CUT&Tag assays. RESULTS: We identified a novel EGFR variant (EGFRx), that is specifically expressed in GSCs. Unlike the EGFRvIII variant, which lacks exons 2-7, EGFRx is characterized by the absence of exons 2-14, and encodes an EGFR protein that does not possess the entire extracellular ligand-binding domain. We observed that EGFRx exhibits significant glycosylation, is required for GSC self-renewal, proliferation, and tumorigenesis, and highly active in glioblastomas compared to normal brain tissue. Mechanistically, EGFRx constitutively and specifically activates STAT5 in GSCs through spontaneous asymmetric dimerization of the kinase domain. CONCLUSIONS: EGFRx plays essential roles in the maintenance of the GSC phenotype through constitutive activation of STAT5 and promotes GBM progression, suggesting that EGFRx-STAT5 signaling represents a promising therapeutic target for GBM.

Recombinant Human Insulin-Like Growth Factor-1 Treatment of Severe Growth Failure in Three Siblings with STAT5B Deficiency.

INTRODUCTION: Patients with homozygous recessive mutations in STAT5B have severe progressive postnatal growth failure and insulin-like growth factor-I (IGF-I) deficiency associated with immunodeficiency and increased risk of autoimmune and pulmonary conditions. This report describes the efficacy and safety of recombinant human IGF-1 (rhIGF-1) in treating severe growth failure due to STAT5B deficiency. CASE PRESENTATION: Three siblings (P1, 4.4 year-old female; P2, 2.3 year-old male; and P3, 7 month-old female) with severe short stature (height SDS [HtSDS] -6.5, -4.9, -5.3, respectively) were referred to the Center for Growth Disorders at Cincinnati Children's Hospital Medical Center. All three had a homozygous mutation (p.Trp631*) in STAT5B. Baseline IGF-I was 14.7, 14.1, and 10.8 ng/mL, respectively (all < -2.5 SDS for age and sex), and IGFBP-3 was 796, 603, and 475 ng/mL, respectively (all < -3 SDS for age and sex). The siblings were started on rhIGF-1 at 40 µg/kg/dose twice daily subcutaneously (SQ), gradually increased to 110-120 µg/kg/dose SQ twice daily as tolerated. HtSDS and height velocity (HV) were monitored over time. RESULTS: Six years of growth data was utilized to quantify growth response in the two older siblings and 5 years of data in the youngest. Pre-treatment HVs were, respectively, 3.0 (P1), 3.0 (P2), and 5.2 (P3) cm/year. With rhIGF-1 therapy, HVs increased to 5.2-6.0, 4.8-7.1, and 5.5-7.4 cm/year, respectively, in the first 3 years of treatment, before they decreased to 4.7, 3.8, and 4.3 cm/year, respectively, at a COVID-19 pandemic delayed follow-up visit and with decreased treatment adherence. ΔHtSDS for P1 and P2 was +2.21 and +0.93, respectively, over 6 years, but -0.62 for P3 after 5 years and in the setting of severe local lipohypertrophy and suboptimal weight gain. P3 also experienced hypoglycemia that limited our ability to maintain target rhIGF-1 dosing. CONCLUSION: The response to rhIGF-1 therapy is less than observed with rhIGF-1 therapy for patients previously described with severe primary IGF-I deficiency, including patients with documented defects in the growth hormone receptor, but may still provide patients with STAT5B deficiency with an opportunity to prevent worsening growth failure.

ALKBH5 protects against stroke by reducing endoplasmic reticulum stress-dependent inflammation injury via the STAT5/PERK/EIF2α/CHOP signaling pathway in an m6A-YTHDF1-dependent manner.

BACKGROUND: Endoplasmic reticulum (ER) stress causes neuroinflammation and neuronal apoptosis during ischemic stroke progression. This study has investigated the role of ALKBH5 in ER stress during ischemic stroke progression. METHODS: In vivo and in vitro models of ischemic stroke were established by middle cerebral artery occlusion (MCAO) and OGD/R treatment, respectively. Cerebral infarct size was detected using triphenyltetrazolium chloride staining (TTC), and pathological changes were examined using histological staining. The levels of inflammatory factors were analyzed using Enzyme-linked immunosorbent assay. Cell counting kit-8 assay and flow cytometry were used to measure cell viability and apoptosis, respectively. The global m6A level was detected using the commercial kit, and STAT5 mRNA m6A level was determined using methylated RNA binding protein immunoprecipitation (Me-RIP). ALKBH5, YTHDF1, and STAT5 interactions were analyzed using RIP and RNA pull-down assays. RESULTS: ALKBH5 was upregulated in MCAO animals and OGD/R cell models. ALKBH5 knockdown exacerbated ER stress, neuroinflammation, and neuronal apoptosis in brain tissues and neuronal cells. ALKBH5 inhibited STAT5 mRNA stability and expression in an m6A-YTHDF1-dependent manner. STAT5 promoted ER stress by activating the PERK/eIF2/CHOP signaling pathway. Furthermore, STAT5 knockdown reversed the effects of ALKBH5 knockdown on OGD/R-induced ER stress and neuroinflammation in HT22 cells. CONCLUSION: ALKBH5 knockdown exacerbated ischemic stroke by increasing ER stress-dependent neuroinflammation and neuronal apoptosis via the STAT5/PERK/EIF2α/CHOP signaling pathway in an m6A-YTHDF1-dependent manner.

A newly identified 45-kDa JAK2 variant with an altered kinase domain structure represents a novel mode of JAK2 kinase inhibitor resistance.

Tyrosine-protein kinase (janus kinase; JAK)-signal transducer and activator of transcription (STAT) signaling plays a pivotal role in the development of myeloproliferative neoplasms (MPNs). Treatment with the potent JAK1/JAK2-specific inhibitor, ruxolitinib, significantly reduces tumor burden; however, ruxolitinib treatment does not fully eradicate the malignant clone. As the molecular basis for the disease persistence is not well understood, we set out to gain new insights by generating ruxolitinib-resistant cell lines. Surprisingly, these cells harbor a 45 kDa JAK2 variant (FERM-JAK2) consisting of the N-terminal FERM domain directly fused to the C-terminal kinase domain in 80% of sublines resistant to ruxolitinib. At the molecular level, FERM-JAK2 is able to directly bind and activate STAT5 in the absence of cytokine receptors. Furthermore, phosphorylation of activation-loop tyrosines is dispensable for FERM-JAK2-mediated STAT5 activation and cellular transformation, in contrast to JAK2-V617F. As a result, FERM-JAK2 is highly resistant to several ATP-competitive JAK2 inhibitors, whereas it is particularly sensitive to HSP90 inhibition. A murine model of FERM-JAK2 leukemogenesis showed an accelerated MPN phenotype with pronounced splenomegaly. Notably, most current protocols for the monitoring of emerging JAK variants are unable to detect FERM-JAK2, highlighting the urgent need for implementing next-generation sequencing approaches in MPN patients receiving ruxolitinib.

Plasma growth hormone pulses induce male-biased pulsatile chromatin opening and epigenetic regulation in adult mouse liver.

Sex differences in plasma growth hormone (GH) profiles, pulsatile in males and persistent in females, regulate sex differences in hepatic STAT5 activation linked to sex differences in gene expression and liver disease susceptibility, but little is understood about the fundamental underlying, GH pattern-dependent regulatory mechanisms. Here, DNase-I hypersensitivity site (DHS) analysis of liver chromatin accessibility in a cohort of 18 individual male mice established that the endogenous male rhythm of plasma GH pulse-stimulated liver STAT5 activation induces dynamic, repeated cycles of chromatin opening and closing at several thousand liver DHS and comprises a novel mechanism conferring male bias to liver chromatin accessibility. Strikingly, a single physiological replacement dose of GH given to hypophysectomized male mice restored, within 30 min, liver STAT5 activity and chromatin accessibility at 83% of the dynamic, pituitary hormone-dependent male-biased DHS. Sex-dependent transcription factor binding patterns and chromatin state analysis identified key genomic and epigenetic features distinguishing this dynamic, STAT5-driven mechanism of male-biased chromatin opening from a second GH-dependent mechanism operative at static male-biased DHS, which are constitutively open in male liver. Dynamic but not static male-biased DHS adopt a bivalent-like epigenetic state in female liver, as do static female-biased DHS in male liver, albeit using distinct repressive histone marks in each sex, namely, H3K9me3 at male-biased DHS in female liver and H3K27me3 at female-biased DHS in male liver. Moreover, sex-biased H3K36me3 marks are uniquely enriched at static sex-biased DHS, which may serve to keep these sex-dependent hepatocyte enhancers free of H3K27me3 repressive marks and thus constitutively open. Pulsatile chromatin opening stimulated by endogenous, physiological hormone pulses is thus one of two distinct GH-determined mechanisms for establishing widespread sex differences in hepatic chromatin accessibility and epigenetic regulation, both closely linked to sex-biased gene transcription and the sexual dimorphism of liver function.

SOCS2 inhibits hepatoblastoma metastasis via downregulation of the JAK2/STAT5 signal pathway.

Metastasis of hepatoblastoma (HB) is a key factor that impairs the prognosis and treatment of children. The suppressor of cytokine signaling 2 (SOCS2) is a classical negative feedback protein that regulates cytokine signal transduction and has been known to be downregulated in several tumor, but the molecular mechanisms of its involvement in HB metastasis are unknown. We found that SOCS2 was a gene down-regulated in hepatoblastoma and associated with HB metastasis through bioinformatics. The qRT-PCR, Western blot and IHC showed that SOCS2 was significantly lower in HB tissues. Clinicopathological correlation analysis revealed that low expression of SOCS2 was significantly correlated with tumor metastasis (P = 0.046) and vascular invasion (P = 0.028), associated with poor prognosis. Overexpression of SOCS2 inhibited the migration and invasion of hepatoblastoma cells, while knockdown of SOCS2 expression promoted these malignant phenotypes. In vivo studies revealed overexpression of SOCS2 inhibited the formation of lung metastasis. Up-regulation of SOCS2 in HB cell inhibited EMT and JAK2/STAT5. Conversely, down-regulation of SOCS2 promoted EMT and JAK2/STAT5. The addition of the JAK2 inhibitor Fedratinib partially reversed the effects of si-SOCS2 on HB cells. SOCS2 may inhibit the migration and invasion of HB cells by inhibiting the JAK2/STAT5 signaling pathway. These results may provide guiding significance for the clinical treatment of HB.

Notch3 restricts metastasis of breast cancers through regulation of the JAK/STAT5A signaling pathway.

PURPOSE: To explore the potential role of signal transducer and activator of transcription 5A (STAT5A) in the metastasis of breast cancer, and its mechanism of regulation underlying. METHODS AND RESULTS: TCGA datasets were used to evaluate the expression of STAT5A in normal and different cancerous tissues through TIMER2.0, indicating that STAT5A level was decreased in breast cancer tissues compared with normal ones. Gene Set Enrichment Analysis predicted that STAT5A was associated with the activation of immune cells and cell cycle process. We further demonstrated that the infiltration of immune cells was positively associated with STAT5A level. Influorescence staining revealed the expression and distribution of F-actin was regulated by STAT5A, while colony formation assay, wound healing and transwell assays predicted the inhibitory role of STAT5A in the colony formation, migratory and invasive abilities in breast cancer cells. In addition, overexpression of the Notch3 intracellular domain (N3ICD), the active form of Notch3, resulted in the increased expression of STAT5A. Conversely, silencing of Notch3 expression by siNotch3 decreased STAT5A expression, supporting that STAT5A expression is positively associated with Notch3 in human breast cancer cell lines and breast cancer tissues. Mechanistically, chromatin immunoprecipitation showed that Notch3 was directly bound to the STAT5A promoter and induced the expression of STAT5A. Moreover, overexpressing STAT5A partially reversed the enhanced mobility of breast cancer cells following Notch3 silencing. Low expression of Notch3 and STAT5A predicted poorer prognosis of patients with breast cancer. CONCLUSION: The present study demonstrates that Notch3 inhibits metastasis in breast cancer through inducing transcriptionally STAT5A, which was associated with tumor-infiltrating immune cells, providing a novel strategy to treat breast cancer.

Distinct use of super-enhancer elements controls cell type-specific CD25 transcription and function.

The IL-2 receptor α chain (IL-2Rα/CD25) is constitutively expressed on double-negative (DN2/DN3 thymocytes and regulatory T cells (Tregs) but induced by IL-2 on T and natural killer (NK) cells, with Il2ra expression regulated by a STAT5-dependent super-enhancer. We investigated CD25 regulation and function using a series of mice with deletions spanning STAT5-binding elements. Deleting the upstream super-enhancer region mainly affected constitutive CD25 expression on DN2/DN3 thymocytes and Tregs, with these mice developing autoimmune alopecia, whereas deleting an intronic region decreased IL-2-induced CD25 on peripheral T and NK cells. Thus, distinct super-enhancer elements preferentially control constitutive versus inducible expression in a cell type-specific manner. The mediator-1 coactivator colocalized with specific STAT5-binding sites. Moreover, both upstream and intronic regions had extensive chromatin interactions, and deletion of either region altered the super-enhancer structure in mature T cells. These results demonstrate differential functions for distinct super-enhancer elements, thereby indicating previously unknown ways to manipulate CD25 expression in a cell type-specific fashion.