STAT5 Activation Research Articles

Rescue of NLRC5 expression restores antigen processing machinery in head and neck cancer cells lacking functional STAT1 and p53

The antigen processing machinery (APM) components needed for a tumor cell to present an antigen to a T cell are expressed at low levels in solid tumors, constituting an important mechanism of immune escape. More than most other solid tumors, head and neck squamous cell carcinoma (HNSCC) cells tend to have low APM expression, rendering them insensitive to immune checkpoint blockade and most other forms of immunotherapy. In HNSCC, this APM deficiency is largely driven by high levels of EGFR and SHP2, leading to low expression and activation of STAT1; however, recent studies suggest that p53, which is often mutated in HNSCCs, may also play a role. In the current study, we aimed to investigate the extent to which STAT1 and p53 individually regulate APM component expression in HNSCC cells. We found that in cells lacking functional p53, APM expression could still be induced by interferon-gamma or DNA-damaging chemotherapy (cisplatin) as long as STAT1 expression remained intact; when both transcription factors were knocked down, APM component expression was abolished. When we bypassed these deficient pathways by rescuing the expression of NLRC5, APM expression was also restored. These results suggest that dual loss of functional STAT1 and p53 may render HNSCC cells incapable of processing and presenting antigens, but rescue of downstream NLRC5 expression may be an attractive strategy for restoring sensitivity to T cell-based immunotherapy.

Gut instinct: harnessing the power of probiotics to tame pathogenic signaling pathways in ulcerative colitis.

Ulcerative colitis (UC) is a chronic inflammatory bowel disease (IBD) marked by persistent inflammation of the mucosal lining of the large intestine, leading to debilitating symptoms and reduced quality of life. Emerging evidence suggests that an imbalance of the gut microbiota plays a crucial role in UC pathogenesis, and various signaling pathways are implicated in the dysregulated immune response. Probiotics are live microorganisms that confer health benefits to the host, have attracted significant attention for their potential to restore gut microbial balance and ameliorate inflammation in UC. Recent studies have elucidated the mechanisms by which probiotics modulate these signaling pathways, often by producing anti-inflammatory molecules and promoting regulatory immune cell function. For example, probiotics can inhibit the nuclear factor-κB (NF-κB) pathway by stabilizing Inhibitor of kappa B alpha (IκBα), dampening the production of proinflammatory cytokines. Similarly, probiotics can modulate the Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway, suppressing the activation of STAT1 and STAT3 and thus reducing the inflammatory response. A better understanding of the underlying mechanisms of probiotics in modulating pathogenic signaling pathways in UC will pave the way for developing more effective probiotic-based therapies. In this review, we explore the mechanistic role of probiotics in the attenuation of pathogenic signaling pathways, including NF-κB, JAK/STAT, mitogen-activated protein kinases (MAPKs), Wnt/β-catenin, the nucleotide-binding domain (NOD)-, leucine-rich repeat (LRR)- and pyrin domain-containing protein 3 (NLRP3) inflammasome, Toll-like receptors (TLRs), interleukin-23 (IL-23)/IL-17 signaling pathway in UC.

HFcγRIIa: a double-edged sword in osteoclastogenesis and bone balance in transgenic mice.

Rheumatoid arthritis (RA) is a chronic autoimmune disease accompanied by local and systemic bone loss. FcγRs, especially FcγRIIa (hFcγRIIa), have been implicated in the pathogenesis of RA. However, the contribution of hFcγRIIa to bone loss has not been fully elucidated. In the present study, we demonstrated the double-edged sword role of hFcγRIIa on osteoclast differentiation through investigations involving hFcγRIIa-transgenic (hFcγRIIa-Tg) mice. Our findings reveal that hFcγRIIa-Tg mice, previously shown to exhibit heightened susceptibility to collagen-induced arthritis (CIA), displayed increased osteoporosis during CIA or at advanced ages (40 weeks), accompanied by heightened in vivo osteoclast differentiation. Notably, bone marrow cells from hFcγRIIa-Tg mice exhibited enhanced efficiency in differentiating into osteoclasts and bone resorption in vitro compared to wild-type mice when stimulated with receptor activators of NF-κB ligand (RANKL). Additionally, hFcγRIIa-Tg mice exhibited augmented sensitivity to RANKL-induced bone loss in vivo, highlighting the osteoclast-promoting role of hFcγRIIa. Mechanistically, bone marrow cells from hFcγRIIa-Tg mice displayed heightened Syk self-activation, leading to mTOR-pS6 pathway activation, thereby promoting RANKL-driven osteoclast differentiation. Intriguingly, while hFcγRIIa crosslinking hindered RANKL-induced osteoclast differentiation, it activated the kinase cAbl, subsequently triggering STAT5 activation and inhibiting the expression of osteoclast-associated genes. This study provides novel insights into hFcγRIIa-mediated osteoclast biology, suggesting promising therapeutic targets for managing bone remodeling disorders.

Type II mode of JAK2 inhibition and destabilization are potential therapeutic approaches against the ruxolitinib resistance driven myeloproliferative neoplasms.

Ruxolitinib has been approved by the US FDA for the treatment of myeloproliferative neoplasms such as polycythemia vera and primary myelofibrosis. Ruxolitinib will remain a main stay in the treatment of MPN patients due to its effective therapeutic benefits. However, there have been instances of ruxolitinib resistance in MPN patients. As JAK2 is a direct target of ruxolitinib, we generated ruxolitinib-resistant clones to find out the mechanism of resistance. Cell-based screening strategy was used to detect the ruxolitinib-resistant mutations in JAK2. The Sanger sequencing method was used to detect the point mutations in JAK2. Mutations were re-introduced using the site-directed mutagenesis method and stably expressed in Ba/F3 cells. Drug sensitivities against the JAK2 inhibitors were measured using an MTS-based assay. JAK2 and STAT5 activation levels and total proteins were measured using immunoblotting. Computational docking studies were performed using the Glide module of Schrodinger Maestro software. In this study, we have recovered seven residues in the kinase domain of JAK2 that affect ruxolitinib sensitivity. All these mutations confer cross-resistance across the panel of JAK2 kinase inhibitors except JAK2-L983F. JAK2-L983F reduces the sensitivity towards ruxolitinib. However, it is sensitive towards fedratinib indicating that our screen identifies the drug-specific resistance profiles. All the ruxolitinib-resistant JAK2 variants displayed sensitivity towards type II JAK2 inhibitor CHZ-868. In this study, we also found that JAK1-L1010F (homologous JAK2-L983F) is highly resistant towards ruxolitinib suggesting the possibility of JAK1 escape mutations in JAK2-driven MPNs and JAK1 mutated ALL. Finally, our study also shows that HSP90 inhibitors are potent against ruxolitinib-resistant variants through the JAK2 degradation and provides the rationale for clinical evaluation of potent HSP90 inhibitors in genetic resistance driven by JAK2 inhibitors. Our study identifies JAK1 and JAK2 resistance variants against the type I JAK2 inhibitors ruxolitinib, fedratinib, and lestaurtinib. The sensitivity of these resistant variants towards the type II JAK2 inhibitor CHZ-868 indicates that this mode of type II JAK2 inhibition is a potential therapeutic approach against ruxolitinib refractory leukemia. This also proposes the development of potent and specific type II JAK2 inhibitors using ruxolitinib-resistance variants as a prototype.

PDK3 drives colorectal carcinogenesis and immune evasion and is a therapeutic target for boosting immunotherapy.

Pyruvate Dehydrogenase Kinase 3 (PDK3) has emerged as a significant player in various cancer types, yet its specific impact on cancers including colon cancer remains ambiguous. Through pan-cancer analysis using TCGA data, we found that the expression of PDK3 and the composition of the immune microenvironment for different tumors were highly heterogeneous across tumors. PDK3 is highly expressed in colorectal cancer and may promote tumor proliferation by activating PI3K-AKT signaling. In addition, we found that PDK3 was able to inhibit tumor antigen presentation signals to suppress immune killing. High PDK3 expression predicts less CD8+ T cell infiltration and effector function. Moreover, inhibition of PDK3 expression bolstered CD8+ T cell-mediated cytotoxicity CD8+ T cell infiltration and activation in vivo. Notably, PDK3 was found to facilitate STAT1 activation and elevate programmed death-ligand 1 (PD-L1) expression in colon cancer cells. Importantly, PDK3 inhibition combination with PD-1 blockade significantly activates the infiltrated CD8+ T cells to suppress tumor growth and improves the survival benefit in several murine tumor models. In summary, these findings underscore PDK3's role in fueling colon cancer growth by orchestrating PI3K-AKT signaling and PD-L1 expression and dampening CD8+ T cell function.

Sebacic acid, a royal jelly-containing fatty acid, decreases LPS-induced IL-6 mRNA expression in differentiated human THP-1 macrophage-like cells.

Macrophages produce many inflammatory mediators, such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), in innate immune responses. However, excess production of these mediators by activated macrophages triggers deleterious effects, leading to disorders associated with inflammation. Royal jelly (RJ), a milky-white substance secreted by worker bees, contains unique fatty acids, including 10-hydroxy-2-decenoic acid (10H2DA) and sebacic acid (SA). 10H2DA has been reported to have various biological functions, such as anti-inflammation. However, the anti-inflammatory effect of SA is not fully understood. In this study, we investigated the effects of SA on lipopolysaccharide (LPS)-induced cytokine expression using differentiated human THP-1 macrophage-like cells. SA dose-dependently decreased LPS-induced mRNA expression of IL-6, but not TNF-α and IL-1β. SA suppressed the phosphorylation of signal transducers and activators of transcription 1 (STAT1) and STAT3, but hardly affected the activation of JNK, p38, or NF-κB. In addition, SA decreased LPS-induced interferon-β (IFN-β) expression, and the addition of IFN-β restored the inhibition by SA of LPS-induced STAT activation and IL-6 expression. Furthermore, SA suppressed LPS-induced nuclear translocation of interferon regulatory factor 3 (IRF3), a transcription factor responsible for IFN-β expression. Taken together, we conclude that SA selectively decreases LPS-induced expression of IL-6 mRNA through inhibition of the IRF3/IFN-β/STAT axis.

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.

Phospholipid scramblase 1 is involved in immunogenic cell death and contributes to dendritic cell–based vaccine efficiency to elicit antitumor immune response in vitro

Background aimsWhole tumor cell lysates (TCLs) obtained from cancer cells previously killed by treatments able to promote immunogenic cell death (ICD) can be efficiently used as a source of tumor-associated antigens for the development of highly efficient dendritic cell (DC)-based vaccines. Herein, the potential role of the interferon (IFN)-inducible protein phospholipid scramblase 1 (PLSCR1) in influencing immunogenic features of dying cancer cells and in enhancing DC-based vaccine efficiency was investigated. MethodsPLSCR1 expression was evaluated in different mantle-cell lymphoma (MCL) cell lines following ICD induction by 9-cis-retinoic acid (RA)/IFN-α combination, and commercial kinase inhibitor was used to identify the signaling pathway involved in its upregulation. A Mino cell line ectopically expressing PLSCR1 was generated to investigate the potential involvement of this protein in modulating ICD features. Whole TCLs obtained from Mino overexpressing PLSCR1 were used for DC loading, and loaded DCs were employed for generation of tumor antigen-specific cytotoxic T lymphocytes. ResultsThe ICD inducer RA/IFN-α combination promoted PLSCR1 expression through STAT1 activation. PLSCR1 upregulation favored pro-apoptotic effects of RA/IFN-α treatment and enhanced the exposure of calreticulin on cell surface. Moreover, DCs loaded with TCLs obtained from Mino ectopically expressing PLSCR1 elicited in vitro greater T-cell–mediated antitumor responses compared with DCs loaded with TCLs derived from Mino infected with empty vector or the parental cell line. Conversely, PLSCR1 knock-down inhibited the stimulating activity of DCs loaded with RA/IFN-α–treated TCLs to elicit cyclin D1 peptide-specific cytotoxic T lymphocytes. ConclusionsOur results indicate that PLSCR1 improved ICD-associated calreticulin exposure induced by RA/IFN-α and was clearly involved in DC-based vaccine efficiency as well, suggesting a potential contribution in the control of pathways associated to DC activation, possibly including those involved in antigen uptake and concomitant antitumor immune response activation.

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.

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.

Nonself RNA rewires IFN-β signaling: A mathematical model of the innate immune response.

Type I interferons (IFNs) are key coordinators of the innate immune response to viral infection, which, through activation of the transcriptional regulators STAT1 and STAT2 (STAT1/2) in bystander cells, induce the expression of IFN-stimulated genes (ISGs). Here, we showed that in cells transfected with poly(I:C), an analog of viral RNA, the transcriptional activity of STAT1/2 was terminated because of depletion of the interferon-β (IFN-β) receptor, IFNAR. Activation of RNase L and PKR, products of two ISGs, not only hindered the replenishment of IFNAR but also suppressed negative regulators of IRF3 and NF-κB, consequently promoting IFNB transcription. We incorporated these findings into a mathematical model of innate immunity. By coupling signaling through the IRF3-NF-κB and STAT1/2 pathways with the activities of RNase L and PKR, the model explains how poly(I:C) switches the transcriptional program from being STAT1/2 induced to being IRF3 and NF-κB induced, which converts IFN-β-responding cells to IFN-β-secreting cells.

Medrysone promotes corneal injury repair by promoting M2-like polarization of macrophages

BackgroundAccumulated evidence suggests that M2-like polarized macrophages plays an important role in reducing inflammation, promoting and accelerating wound healing process and tissue repair. Thus, M2-like TAMs (Tumour-associated macrophages) was an appealing target for therapy intervention.MethodFlow cytometry and RT-PCR assay were used to detect the polarization of macrophages induced by Medrysone, and the rat corneal mechanical injury model was established to evaluate the efficacy of Medrysone in cornel repair.ResultsHere we found that Medrysone enhanced IL-4 induced M2 polarization of macrophages, as illustrated by increased expression of CD206, up-regulation of M2 marker mRNAs. Medrysone promoted VEGF and CCL2 secretion in IL-4 induced M2-like polarization. IL-4 triggered STAT6 activation was further enhanced by Medrysone and silencing of STAT6 partially abrogated the stimulatory effect of Medrysone. Medrysone improved migration-promoting feature of M2-like macrophages, as indicated by increased migration of endothelial cells. Further, Medrysone promoted corneal injury repair by inducing M2 polarization of macrophages in vivo.ConclusionOur study suggest that Medrysone promotes corneal injury repair by inducing the M2 polarization of macrophages, providing a theoretical basis for the application of Medrysone in the treatment of corneal injury.

Distinct Role of STAT3 and STAT5 Factors in MDS and AML

Introduction: Myelodysplastic Syndrome (MDS) refers to a heterogeneous group of clonal hematologic disorders characterized by hematopoietic dysfunctions and impaired differentiation in the hematopoietic stem and progenitor cells compartment. MDS is commonly referred to as a pre-leukemic stage due to the increased risk of progression to Acute Myeloid Leukemia (AML). AML is an aggressive hematologic malignancy characterized by the accumulation of immature myeloblasts in the bone marrow and the peripheral blood. Signal transducers and activators of transcription 3 and 5 (STAT3 and STAT5) are important regulators of several cellular processes including cell proliferation and survival. Abnormal STAT3 and STAT5 signaling has been implicated in various solid and hematologic malignancies, including MDS and AML, which renders them appealing targets for the development of novel therapeutic strategies. Materials: STAT3, STAT5A or STAT5B knock-downs have been generated in two AML (MOLM-13 and Kasumi-1) and two MDS (MDS92 and MDS-L, gift of Prof. Kaoru Tohyama) cell lines, using shRNAs through lenti-viral delivery. The 4 biological replicates with the highest levels of knock-downs for STAT5A, STAT5B or STAT3 have been selected for each cell line and mRNA sequencing has been performed. To define the genome-wide binding of STAT5A, STAT5B and STAT3 in the two conditions, CUT&Tag has been performed in wild type MOLM-13, Kasumi-1 and MDS-L cells. Additionally, ATAC sequencing has been carried out in the same cell lines to investigate the chromatin conformation changes between the two conditions. Furthermore, STAT3, STAT5A and STAT5B knock-downs have been conducted using shRNAs through lenti-viral delivery in CD34+ enriched cells, isolated from bone marrow mononuclear cells of 14 high-risk MDS patients and 14 secondary (MDS-related) AML patients. The samples have been processed for mRNA-Sequencing to model the leukemic transformation in primary human cells. Results: To elucidate the role of STAT3 and STAT5 in MDS to AML transformation, changes in STAT3 and STAT5 target gene networks have been identified. mRNA sequencing data from STAT3, STAT5A or STAT5B knock-downs in MDS and AML cell lines revealed a distinct role of the three factors in each state. The differential binding of each factor, explored through CUT&Tag, and the gene expression profiles in MDS and AML reciprocally reveal a different function of each factor, including pathways involved in cell cycle and apoptosis. Also, the direct and indirect target genes of STAT3, STAT5A and STAT5B in each condition, as well as a possible interplay between STAT3 and STAT5 activation in AML has been uncovered. The exploration of the chromatin accessibility landscapes between the MDS and AML conditions through ATAC sequencing has provided an insight into the chromatin conformation states, unveiling potential leukemia promoting networks, as well as the involvement of STAT3 and STAT5 pathways in the malignant transformation. Furthermore, a potential cross talk of STAT3 and STAT5 factors with the IKAROS family members, has been linked to AML transformation, as significant differential regulation has been detected between MDS and AML conditions after knock-down of STAT5A and STAT5B. The results are currently validated in primary hematopoietic cells of MDS and AML patients after STAT3, STAT5A or STAT5B knock-downs. Conclusions: Our results show a different role between the two STAT5 factors (STAT5A and STAT5B) in MDS and AML conditions and delineate the role of STAT5A, STAT5B and STAT3 factors and their target gene networks in leukemic transformation, thus providing novel targets for therapeutic management of MSD and AML. Funding: This project is funded by the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 813091 (ARCH)

Anti-Inflammatory Response to Anti-Folate and Vitamin D Analog Combination in Large Granular Lymphocyte Leukemia

Large granular lymphocyte (LGL) leukemia is a rare chronic hematological disorder characterized by clonal expansion of circulating cytotoxic T or natural killer (NK) cells. The disease is associated with a number of comorbidities including neutropenia, anemia, and autoimmune diseases. There is no standard of care or curative therapy for LGL leukemia. Instead, it is treated when symptoms arise with a variety of immunosuppressive therapies, chief among them, the anti-folate, methotrexate (MTX). However, a significant proportion of patients remain refractory to treatment, and little characterization has been done regarding MTX's mechanism in vitro. Thus, there is a need to identify new therapeutic approaches such as stronger anti-folates or combination of MTX with existing therapies. Pralatrexate (PDX) is a more potent analog of MTX, not yet utilized in LGL leukemia. It is approved for treatment of relapsed or refractory peripheral T cell lymphomas and may offer a promising new avenue for therapy. Vitamin D is a natural supplement that we have shown to impact key LGL leukemia signaling pathways in vitro by decreasing cytokine levels and STAT1 and STAT3 signaling, all of which are typically elevated in LGL leukemia. Treatment with vitamin D also increases levels of vitamin D receptor (VDR), a known transcriptional controller of reduced folate carrier (RFC), the method of cellular entry for both MTX and PDX. Therefore, we hypothesized that treatment with EB1089, a synthetic vitamin D analog, would sensitize leukemic LGLs to anti-folates, PDX and MTX, by leading to a VDR-mediated increase in RFC thus allowing increased MTX/PDX uptake and therapeutic efficacy. In this study, single MTX treatment of LGL leukemia model cell lines (NKL and TL-1) demonstrated strong decreases in viability at 72h time points (EC50 NKL 10.5nM, TL-1 15.4nM). Single agent PDX effects on viability were even stronger (4-11 fold lower EC50) (Figure A). Key LGL leukemia signaling pathways like STAT phosphorylation were lowered as early as 24h after high dose treatment with MTX (10 µM) in TL-1 cells, and VDR was also lowered by MTX single high dose treatment. When MTX or PDX was given in combination with EB1089, critical inflammatory signaling, such as phosphorylation of STAT1 and STAT3, was attenuated, while VDR was stabilized from anti-folate mediated decrease. Dose dependent changes to STAT3 regulation, RFC, VDR, and PTPN2, a phosphatase specific to STAT1 and STAT3, occurred as early as 6h post combinatorial treatment with a range of MTX dosages (1, 10, 100 nM) in the NK cell line model. A similar anti-inflammatory response in PTPN2, STAT1, and STAT3 levels was observed at 24h following co-treatment in TL-1 cells (Figure B). Additionally, treatment with a combination of EB1089 and PDX (0.1, 1, 10 nM; 10% of the MTX doses) led to anti-inflammatory response with decreases in STAT1 activation and increases in RFC in the TL-1 cell line. In short term assays, there were no measurable changes in anti-folate mediated cell killing when the drugs were combined across cell lines and different dosages of the combination. Taken together, these data suggest a strong single agent potency for PDX and an anti-inflammatory benefit to MTX/PDX and EB1089 combination. Continued mechanistic study of the anti-folate/vitamin D receptor agonist pairing as well as further investigation into PDX's impact on LGLs will be crucial to follow up investigations.

Cooperation between SF3B1 and JAK2V617F Mutations Accelerates Fibrotic Progression in Myeloproliferative Neoplasms By Enhancing STAT1 Signaling

Myeloproliferative neoplasms (MPN) are heterogenous clonal hematologic neoplasms where current therapies show limited disease-modification. We previously reported that the splicing factor, SF3B1, is mutated in 5-10% of MPN correlating with myelofibrotic progression in essential thrombocythemia. This adverse phenotype contrasts with SF3B1 mutationsin myelodysplasia where it is associated with milder disease. Moreover, SF3B1 K666 is the dominant hotspot in MPN in contrast to K700E in MDS (abstract #185043). The mechanism by which SF3B1 mutations accelerate myelofibrotic progression in JAK2V617F-mutated MPN is not understood and will be a key step towards the development of disease-modifying therapies. To address this, we applied two complimentary single-cell multiomic methods to study 15 individuals (healthy donor [HD, n=5], JAK2V617F-single mutated [J+, n=5] and JAK2V617F- SF3B1K666double mutated MPN cases [JS+, n=5]). First, we characterised the cellular landscape of JS+ MPN using CITE-seq/10X genomics platform. Then, we analyzed SF3B1-mutant ( SF3B1mut) disease-driving hematopoietic stem and progenitor cells (HSPCs) and their associated aberrant splicing signatures using TARGET-seq, which permitted single-cell genotyping with allelic resolution and intra-patient comparison of mutant versus wild-type (wt) cells. In an analysis of 109,498 cells, we observed abnormally expanded erythroid (ERP), megakaryocyte (MK) and unexpectedly, eosinophil-basophil-mast cell (EBM) progenitors in JS+ as compared with J+ or HD HSPCs (Fig 1A). Two HSC clusters were present, one of which (HSC_mpp2) principally comprised of JS+ cells and had a distinct molecular program (Fig 1A); upregulation in MK (eg. PF4, VWF) and EBM lineage signature genes (eg. TPSB2), interferon signaling genes but downregulation in apoptosis genes and canonical stemness markers (eg. CD133). These signatures suggest the JS+ driven HSC cluster may be MK/EBM-transcriptionally primed and may therefore, promote expansion of these abnormal progenitors which are implicated in driving fibrosis development. Through genotyping of >5000 HSPCs, we resolved the clonal architecture of JS+ MPN, observing that in most cases double mutant cells were present and clonally dominant, with mutant SF3B1 the initiating event. Mutations arose from the HSC level and genotypes were equally distributed in the HSPC compartment. Highlighting the importance of single-cell analysis, and not apparent at the bulk level, JAK2V617F and SF3B1K666mutationsresided in separate clones for one case with a WHO fibrosis score 0 in contrast to the other 4 cases with double mutant cells and fibrosis scores 1-3. This reinforces the cooperative effect of thesemutationsto drive fibrosis. We performed full-length transcriptomics of select HSPCs to study SF3B1mut-specific aberrant splicing events (ASE). In a global splicing analysis, more events were detected in JAK2V617F- SF3B1K666 double mutant than single mutant HSPCs. Retained intron (RI) events were the most prevalent ASE, followed by skipped exon and alternative 3' splice site events. We identified previously described SF3B1mut-specific ASE ( MAP3K7, ERGIC3 and SEPTIN6) confirming the validity of our dataset to study new ASE. Inter- and intra-patient comparison of SF3B1mut versus SF3B1wt cells uncovered a number of novel biologically relevant ASE, including two RI events in STAT1. These RI events were reduced in SF3B1mut HSPCs with the predicted consequence of increased STAT1 expression and activation, of direct relevance to MPN pathobiology since STAT1 is known to play a key role in enhancing megakaryopoiesis in MPN (Fig 1B). We validated these events using long-read Nanopore sequencing of single-cell cDNA libraries and have developed a cell model system with transient expression of SF3B1K666 mutation versus SF3B1wt in HEK293T to corroborate STAT1 ASE occurring as a direct consequence of mutant SF3B1. In further support of upregulated STAT1 activation, we observed upregulation of STAT1 downstream targets in abnormally expanded JS+ HSPCs. In conclusion, this analysis resolved the clonal architecture and hematopoietic cellular composition of JS+ MPN, identifying abnormally expanded and transcriptionally primed HSPC populations with abnormal splicing of STAT1 enhancing JAK-STAT signaling, uncovering the mechanism by which these mutations promote an accelerated fibrotic phenotype.

Effectiveness and Safety of Hetrombopag in the Management of Radiotherapy-Induced Thrombocytopenia in Patients with Gynecological Malignancies

Background: Radiotherapy is a cornerstone of treatment strategies for gynecological malignancies, However, while effective against neoplastic cells, pelvic radiotherapy frequently incurs collateral damage to normal tissues, especially to the bone marrow. This unintended consequence often precipitates thrombocytopenia, a condition characterized by diminished platelet levels, thereby elevating the risk of bleeding and posing hurdles for the continuity of anticancer regimens. Addressing this clinical challenge, hetrombopag, an innovative, orally administered non-peptide thrombopoietin receptor agonist, presents promise. It works by kindling megakaryocyte proliferation and differentiation, thereby enhancing platelet production via the activation of thrombopoietin receptor dependent STAT and MAPK signaling pathways. Previous studies have documented its efficacy in expediting platelet recovery in patients with chemotherapy-induced thrombocytopenia, thereby curtailing the duration of the condition and ensuring the execution of planned chemotherapy cycles. This study aims to investigate the therapeutic effectiveness and safety profile of hetrombopag in the treatment of radiotherapy-induced thrombocytopenia within a real-world context. Methods: This retrospective study encompassed gynecological cancer patients who developed thrombocytopenia (platelet counts<100×10 9/L) following radiotherapy and subsequently underwent treatment with hetrombopag from January 2022 to June 2023. The key inclusion criteria included an age of ≥18 years, the development of thrombocytopenia after radiotherapy or a combination of radiotherapy and other anticancer interventions, the administration of hetrombopag as monotherapy or as part of a combined regimen with other platelet-boosting agents, and the availability of follow-up data. The primary efficacy outcome was the proportion of patients exhibiting a platelet response (an elevation in platelet counts to ≥75×10 9/L) within 14 days. Results: Our cohort consisted of 28 gynecological cancer patients, encompassing 21 (75.0%) cases of cervical cancer, 3 (10.7%) of endometrial cancer, 1 (3.6%) of ovarian cancer, and 3 (10.7%) of other gynecological malignancies. The patients' median age was 58 years (range: 29-76 years), with 50% in clinical stage III/IV. Among them, 19 had received radiotherapy, 8 had undergone concurrent chemoradiotherapy, and 1 had received radiotherapy plus targeted therapy and immunotherapy. The baseline platelet count prior to hetrombopag treatment was 54.1±16.5×10 9/L. Hetrombopag was administered at a dose of 2.5mg/day as monotherapy in 32.1% of patients and combined with recombinant human thrombopoietin or recombinant human interleukin 11 in 67.9% of patients. The observed platelet response rate was 53.6% within 7 days and increased to 75.0% by the 14-day mark. The median time to platelet response was 7 days (95% confidence interval: 5-14). Following the introduction of hetrombopag as monotherapy or part of a combination regimen, we noted a steady uptick in patients' platelet counts. The mean platelet counts on days 3, 5, 7, 9, 11, and 14 were 62.3, 74.9, 78.7, 88.3, 87.9, and 147.9 ×10 9/L, respectively (Figure). Throughout the treatment period, no adverse events related to hetrombopag were reported. Conclusion: In conclusion, our findings provide preliminary evidence for the effectiveness and safety of hetrombopag, administered either as monotherapy or in combination, for managing radiotherapy-induced thrombocytopenia. Given the retrospective design of this study, further corroboration is needed through large-scale, prospective, randomized, and controlled clinical trials.

An Atypical Somatic Calr Variant Acquired in Cis to a Germline Calr variant Induces Constitutive Signaling of the Thrombopoietin Receptor and Results in Essential Thrombocythemia

Acquired mutations in the gene encoding the endoplasmic reticulum (ER) chaperone calreticulin (CALR) are responsible for ~ 30% of essential thrombocythemia (ET) and myelofibrosis (MF) cases. Two CALR mutations, a deletion of 52 base pairs (del52) and an insertion of 5 base pairs (ins5) are the most frequent in ET and MF patients. All CALR mutations result in a +1-frameshift leading to 1) the replacement of the negatively-charged wild-type (WT) C-terminus that binds calcium in the ER by a positively-charged mutant tail including 44 residues that are common to all CALR mutant proteins and 2) the loss of the ER retention KDEL; together leading to the deregulation of calcium homeostasis. The CALR mutants are also secreted and bind to MPL in the ER but induce its constitutive dimerization and downstream signaling pathways at the cell surface. A 16-year-old girl presented with a platelet count of 797 x 10 3/mL rising to 1,228 x 10 3/mL within 2 years. After Informed Consent, we detected two atypical CALR variants in her blood cells by NGS analysis: V1 (c.1122-1125del) at 47% variant allele frequency (VAF) and V2 (c.1227-1231del) in cis of the V1 leading to V1-V2 (c.1122-1125del ; c.1227-1231del) at 5% VAF. The presence of V1 in blood and nails from the propositus and her healthy mother confirmed its germline origin. The V1-V2 was only found in the propositus at 5% in isolated monocytes and at very low levels in NK and B cells, confirming its somatic acquisition. The germline V1 induces a frameshift resulting in the replacement of the CALR WT C-terminus by the positively charged 44 residue-long C-terminal mutant tail present in del52 and ins5 and the loss of KDEL together with an insertion of 10 amino acids not present in CALR del52 nor ins5. In contrast, the V1-V2 generates a similar but shortened C-terminus containing the Arg-rich MPL binding site and KDEL (Fig. 1A). To investigate the functional impact of these CALR variants, we cloned V1 and V1-V2 with a N-terminal hemagglutinin (HA) tag as well as CALR WT and del52 as controls in a retroviral plasmid. Viral particles were produced to transduce Ba/F3 cell lines expressing MPL. Structural modeling using AlphaFold was used to investigate the role of KDEL restoration in V1-V2 in producing an oncogenic protein leading to ET. Both CALR WT and V1-V2 variants that contain the KDEL motif were detected to a similar extent in Ba/F3-MPL cell lysates. In contrast, del52 and V1, that lost the ER-retention KDEL motif, were barely detected in cell lysates. Unexpectedly, despite the presence of the KDEL motif, V1-V2 was found at a similar level than del52 at the Ba/F3-MPL cell surface. In contrast, V1 was barely detectable at the cell surface, like CALR WT. In order to investigate the role of the KDEL in V1-V2 cell surface expression, we generated a CALR WT lacking the KDEL and added the KDEL motif to del52. The presence of the KDEL motif stabilized the intracellular expression of del52 without affecting cell surface localization while the removal of the KDEL motif in CALR WT appeared to destabilize the protein and induce a slight leak to the cell surface. Structural modeling using AlphaFold predicted that the KDEL in V1-V2 structure is accessible to KDEL receptors, suggesting that the motif may be masked by the interaction of V1-V2 with MPL while trafficking to the cell surface. Indeed, cell-surface expression of V1-V2 was decreased in Ba/F3 that do not express MPL. We show that V1-V2, but not V1, constitutively activates the phosphorylation of STATs, ERK and AKT in Ba/F3-MPL. TPO further increases activation of STATs but not of AKT and ERK by V1-V2 and del52. Coherently, V1-V2, but not V1, sustained oncogenic MPL stimulation able to sustain cell autonomous Ba/F3-MPL cell proliferation (Fig. 1B). Both del52 and V1-V2 up-regulated BCL-2 mRNA. Dose-response cytotoxicity assays with the BCL-2 inhibitor venetoclax showed that these two mutants are sensitive to lower doses compared to CALR WT- or V1-expressing Ba/F3-MPL cells. This is the first report of a germline loss-of-function CALR variant (V1) expressed as an unstable protein and that might predispose to the acquisition of a somatic gain-of-function CALR variant (V1-V2) responsible for ET. The presence of KDEL in V1-V2 leads to stabilized CALR expression that does not prevent cell surface localization. Moreover, it leads to induction of MPL signaling and cytokine-independent cell growth.

Peimisine ameliorates DSS-induced colitis by suppressing Jak-Stat activation and alleviating gut microbiota dysbiosis in mice.

Inflammatory cytokine secretion and gut microbiota dysbiosis play crucial roles in ulcerative colitis. In this research, the protective effects of peimisine on colitis mice were investigated. The protective effects were evaluated by the disease activity index, colonic length, hematoxylin-eosin, and AB/PAS Staining. The protective mechanisms were analyzed by ELISA, Western-blot, immunohistochemistry staining, immunofluorescence staining, and 16S rRNA gene analysis. The results showed that peimisine treatment could reduce the disease activity index, prevent colonic shortening, and alleviate colon tissue damage. Peimisine treatment also decreased the levels of MCP-1, IL-1β, IL-6, IFN-γ, TNF-α and affected macrophage polarization and Th17/Treg cell balance by downregulating the expression of jak1/2, p-jak1/2, stat1/3, and p-stat1/3. Moreover, peimisine treatment significantly increased the abundances of beneficial microbes (e.g. Ruminococcaceae UCG-014 and Lachnospiraceae_NK4A136_group) and decreased the abundances of harmful microbes (e.g. Bacteroides and Escherichia). Peimisine can ameliorate colitis by inhibiting Jak-Stat signaling pathway, reversing gut microbiota alterations, suppressing macrophage M1 polarization, maintaining the Th17/Treg cell balance, and reducing sustained inflammatory cytokines-related inflammatory injury.

STEM-15. STAT5-OLIG2 TRANSCRIPTIONAL COMPLEX MEDIATES GLIOMA STEM CELL SELF RENEWAL AND INVASION

Abstract Glioblastoma (GBM) is the most common and lethal primary brain tumor in adults, despite current treatment options. One of the major contributing factors to this dismal outcome is a subset of cells called glioma stem cells (GSCs) that are highly tumorigenic, invasive, and resistant to conventional treatments. Thus, studies that focus on the properties of GSC biology are needed to develop novel therapies to improve overall survival. Here we report a novel role of STAT5 in GSC stemness and survival. In GBM tumors, STAT5 has been shown to be active in the infiltrative, residual population of cells remaining post-surgery. Knockdown of STAT5 results in the decrease in GSC stemness and therapeutic resistance. Interestingly, we find that expression of OLIG2, a key cell fate factor important for the tumorigenic potential of GSCs, affects STAT5 activation with a positive correlation between OLIG2 and STAT5 mRNA expression in GBM tumors. Furthermore, we find that in GSCs expressing EGFRvIII, STAT5 interacts with OLIG2 and pharmacological inhibition of STAT5 activation or knockdown of STAT5 expression results in decrease of OLIG2-mediated GSC invasion. Together, these data suggest a novel transcriptional complex, STAT5-OLIG2, in the potential role for GSC survival, maintenance, and invasion.

CSIG-31. JAK/STAT PATHWAY CO-OPERATES WITH CIC-FUSIONS TO DRIVE CIC- REARRANGED SARCOMAS

Abstract BACKGROUND CIC-rearranged sarcoma (CRS) is a rare disease driven by a specific fusion protein involving the CIC gene. The native CIC protein is a transcriptional repressor of the (RTK)/Ras/ERK signaling pathway, which is one of the most tumorigenic pathways in cancer. The most common rearrangement is with the double homeobox 4 (DUX4) transcription factor (CIC-DUX4), and others, such as CIC-NUTM1 fusions, have been identified in a subset of pediatric primitive neuroectodermal tumors. However, the molecular mechanisms by which CIC-fusions drive CRS remain unknown. HYPOTHESIS Preliminary data shows that CIC-DUX4/NUTM1 fusions activate JAK (cytokine receptors) and its downstream effector STAT1/3 (signal transducers and transcription factors). We hypothesize that the JAK/STAT1/3 signal transduction pathway cooperates with CIC-fusions to induce the expression of oncogenic transcription factors ETV1/4/5 and drive these sarcomas. METHODS AND RESULTS JAK1 is a potential target for CIC-fusions - We show high levels of JAK1/STAT1/3 activation in patient-derived CRS cell lines (NCC-SCC-89/C) as compared to other sarcoma cell lines without the fusion. JAK1 inhibition using Solicitinub and Ruxolitinib reduced phosphorylation of STAT1/3 as well as protein and mRNA expression of ETV1/4/5 and cell proliferation. JAK1/STAT1/3 inhibition in patient-derived CIC-DUX4 xenograft models- To evaluate the pre-clinical effect of targeting the JAK1/STAT1/3 pathway, A CRS cell line (NCC-SCC-89/C) were grafted into NSG mice. Ruxolitinib treatment significantly reduced tumor volume, STAT activation, and ETV1/4/5. CONCLUSION We show that the JAK1/STAT1/3 pathway plays a critical role in cooperating with CIC-fusions to drive CRS, providing insight into potential therapeutic avenues for these aggressive tumors.