Aberrant Activation Of STAT3 Research Articles

Regulation of MHC II by OPTN, a novel regulator of MARCH1 via mTOR/AKT2/STAT3 pathway ameliorates severe outcomes during herpesvirus infection.

Abstract Protective immune responses elicited by primary activation of CD4+ T cells via dendritic cells is an essential requirement to evade herpesvirus infections. Optineurin (OPTN), a selective autophagy adaptor protein, is a crucial host restriction factor for herpes simplex virus (HSV) infection. We observed a significant reduction in the basal level of MHCII expression in OPTN–/– DCs accompanied by a concomitant decrease in CD4+ T cell counts in OPTN–/– mouse model. We hypothesize that abrogation of antigen presentation via MHCII in DCs weakens adaptive immune responses, leading to severe HSV1 infection. In this study, we elucidate the mechanism of OPTN-mediated down-regulation of MHCII via AKT/mTOR/STAT3 pathway. Mechanistically, OPTN induces activation of mTORC2 sequentially upregulating AKT2 which adversely affects the activation of mTORC1 and STAT3 protein. This could be attributed to the dynamic interplay between AKT1 and AKT2. Our findings provide novel insights into how the absence of OPTN downregulates AKT2 and triggers STAT3 activation. Furthermore, aberrant activation of STAT3 influences transcriptional activity of MARCH1(an E3 ubiquitin ligase) via IL10. MARCH1 mediates ubiquitination of MHCII resulting in the reduction of MHCII expression in OPTN–/– and AKT2–/– DCs. This study represents the first comprehensive delineation of the signaling pathway involved in the regulation of MHCII, shedding light on the intricate interplay between OPTN, AKT, mTOR, STAT3, and MARCH1.

Bergaptol inhibits glioma cell proliferation and induces apoptosis via STAT3/Bcl-2 pathway.

Glioblastoma (GBM) is the most common primary malignant brain tumour and lacks therapeutic options with significant effects. The aberrant activation of STAT3 is a critical factor in glioma progression via activating multiple signalling pathways that promote glioma. Among them, the antiapoptotic gene Bcl-2 could be upregulated by p-STAT3, which is an important reason for the continuous proliferation of glioma. We previously reported that bergaptol, a natural furanocoumarin widely found in citrus products, exerts antineuroinflammatory effects by inhibiting the overactivation of STAT3. Here, we aimed to evaluate whether bergaptol could promote glioma apoptosis by inhibiting the STAT3/Bcl-2 pathway. This study found that bergaptol inhibited the proliferation and migration of GBM cell lines (U87 and A172) and promoted apoptosis in vitro. We also found that bergaptol significantly inhibited the STAT3/Bcl-2 pathway in GBM cells. U87 cells were implanted intracranially into nude mice to establish a glioma model, and glioma-bearing mice were treated with bergaptol (40 mg/kg). Bergaptol treatment significantly inhibited glioma growth and prolonged the glioma-bearing mice's survival time. In addition, bergaptol administration also significantly inhibited the STAT3/Bcl-2 pathway of tumour tissue in vivo. Overall, we found that bergaptol could effectively play an antiglioma role by inhibiting STAT3/Bcl-2 pathway, suggesting the potential efficacy of bergaptol in treating glioma.

Small-molecule PROTAC mediates targeted protein degradation to treat STAT3-dependent epithelial cancer.

The aberrant activation of STAT3 is associated with the etiology and progression in a variety of malignant epithelial-derived tumors, including head and neck squamous cell carcinoma (HNSCC) and colorectal cancer (CRC). Due to the lack of an enzymatic catalytic site or a ligand-binding pocket, there are no small-molecule inhibitors directly targeting STAT3 that have been approved for clinical translation. Emerging proteolysis targeting chimeric (PROTAC) technology-based approach represents a potential strategy to overcome the limitations of conventional inhibitors and inhibit activation of STAT3 and downstream genes. In this study, the heterobifunctional small-molecule-based PROTACs are successfully prepared from toosendanin (TSN), with 1 portion binding to STAT3 and the other portion binding to an E3 ubiquitin ligase. The optimized lead PROTAC (TSM-1) exhibits superior selectivity, potency, and robust antitumor effects in STAT3-dependent HNSCC and CRC - especially in clinically relevant patient-derived xenografts (PDX) and patient-derived organoids (PDO). The following mechanistic investigation identifies the reduced expression of critical downstream STAT3 effectors, through which TSM-1 promotes cell cycle arrest and apoptosis in tumor cells. These findings provide the first demonstration to our knowledge of a successful PROTAC-targeting strategy in STAT3-dependent epithelial cancer.

Leveraging Pre-Clinical Animal Model of CTCL to Explore Therapeutic Potential of a Novel STAT3 Degrader

STAT3 enhances pro-survival signaling essential for T cell expansion, but when this pathway is not appropriately regulated, aberrant STAT3 signaling contributes to T cell lymphomagenesis. Cutaneous T cell lymphoma (CTCL) is a type of mature T cell lymphoma characterized by the accumulation of malignant T cells in the skin. Our analysis, along with other recently published sequencing studies, identified upregulation of the STAT3 cytokine signaling pathway as a key driver of CTCL pathogenesis. In normal processes, STAT3 activity is transient, but dysregulation of this pathway is observed in several human malignancies, including CTCL. Aberrant STAT3 activity in malignant cells promotes transcription of target genes that promote proliferation and survival of transformed T cells. Translocations resulting in STAT3 duplications, activating mutations in the SH2 domain of STAT3 and activating mutations in kinases upstream of STAT3 all contribute to hyperactivation of this JAK/STAT signaling pathway in CTCL. STAT3 signaling in bystander cells within the tumor microenvironment may further contribute to tumor progression, although the precise role of this signaling pathway in malignant disease is context dependent. We have used conditional gene targeting to develop a fully penetrant STAT3-dependent small animal model of CTCL that recapitulates many key features of human disease. In this model, a constitutively active form of STAT3 is expressed exclusively in T lymphocytes. The autochthonous mouse model is characterized by progressive accumulation of malignant T cells in the skin and secondary lymphoid organs. Our molecular analysis of T cells from this mouse model revealed a transcriptional signature that largely mirrored the one found in malignant cells from patients. We have now taken advantage of this tractable animal model of CTCL to evaluate the therapeutic potential of a potent and selective E3 ubiquitin ligase-based novel STAT3 heterobifunctional degrader for targeting this difficult-to-treat hematologic malignancy. Weekly intravenous administration of the degrader was well tolerated and led to significant reduction of STAT3 levels in circulating and skin-resident T lymphocytes. Progression of disease in our preclinical model was notably blunted upon administration of the STAT3 degrader with dramatic reduction in T cell accumulation in the skin and lymph nodes. We will report on pre-clinical evaluation of the degrader and the impact on disease progression, T cell accumulation, activation and proliferation in vivo following weekly drug administration. Targeted protein degradation represents a novel therapeutic modality enabling direct targeting of previously undruggable oncoproteins such as STAT3. These data provide a rationale for selective STAT3 degradation as a therapeutic strategy for malignancies such as CTCL that are associated with constitutive activation of STAT3 signaling. Fig Legend:Treatment of mice exhibiting CTCL phenotypes starting at ~3mo of age. Phenotype score reflects presentation of fur loss, scaling/flaking of the skin, appearance of lesions and death. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Investigating the potential therapeutic role of targeting STAT3 for overcoming drug resistance by regulating energy metabolism in chronic myeloid leukemia cells.

Objective(s):STATs are one of the initial targets of emerging anti-cancer agents due to their regulatory roles in survival, apoptosis, drug response, and cellular metabolism in CML. Aberrant STAT3 activity promotes malignancy, and acts as a metabolic switcher in cancer cell metabolism, contributing to resistance to TKI nilotinib. To investigate the possible therapeutic effects of targeting STAT3 to overcome nilotinib resistance by evaluating various cellular responses in both sensitive and nilotinib resistant CML cells and to test the hypothesis that energy metabolism modulation could be a mechanism for re-sensitization to nilotinib in resistant cells.Materials and Methods:By using RNAi-mediated STAT3 gene silencing, cell viability and proliferation assays, apoptotic analysis, expressional regulations of STAT mRNA transcripts, STAT3 total, pTyr705, pSer727 protein expression levels, and metabolic activity as energy metabolism was determined in CML model K562 cells, in vitro.Results:Targeting STAT3 sensitized both parental and especially nilotinib resistant cells by decreasing leukemic cell survival; inducing leukemic cell apoptosis, and decreasing STAT3 mRNA and protein expression levels. Besides, cell energy phenotype was modulated by switching energy metabolism from aerobic glycolysis to mitochondrial respiration in resistant cells. RNAi-mediated STAT3 silencing accelerated the sensitization of leukemia cells to nilotinib treatment, and STAT3-dependent energy metabolism regulation could be another underlying mechanism for regaining nilotinib response.Conclusion:Targeting STAT3 is an efficient strategy for improving the development of novel CML therapeutics for regaining nilotinib response, and re-sensitization of resistant cells could be mediated by induced apoptosis and regulation in energy metabolism.

Comorbidity-associated glutamine deficiency is a predisposition to severe COVID-19.

SARS-CoV-2 vaccinations have greatly reduced COVID-19 cases, but we must continue to develop our understanding of the nature of the disease and its effects on human immunity. Previously, we suggested that a dysregulated STAT3 pathway following SARS-Co-2 infection ultimately leads to PAI-1 activation and cascades of pathologies. The major COVID-19-associated metabolic risks (old age, hypertension, cardiovascular diseases, diabetes, and obesity) share high PAI-1 levels and could predispose certain groups to severe COVID-19 complications. In this review article, we describe the common metabolic profile that is shared between all of these high-risk groups and COVID-19. This profile not only involves high levels of PAI-1 and STAT3 as previously described, but also includes low levels of glutamine and NAD+, coupled with overproduction of hyaluronan (HA). SARS-CoV-2 infection exacerbates this metabolic imbalance and predisposes these patients to the severe pathophysiologies of COVID-19, including the involvement of NETs (neutrophil extracellular traps) and HA overproduction in the lung. While hyperinflammation due to proinflammatory cytokine overproduction has been frequently documented, it is recently recognized that the immune response is markedly suppressed in some cases by the expansion and activity of MDSCs (myeloid-derived suppressor cells) and FoxP3+ Tregs (regulatory T cells). The metabolomics profiles of severe COVID-19 patients and patients with advanced cancer are similar, and in high-risk patients, SARS-CoV-2 infection leads to aberrant STAT3 activation, which promotes a cancer-like metabolism. We propose that glutamine deficiency and overproduced HA is the central metabolic characteristic of COVID-19 and its high-risk groups. We suggest the usage of glutamine supplementation and the repurposing of cancer drugs to prevent the development of severe COVID-19 pneumonia.

Bazedoxifene inhibits sustained STAT3 activation and increases survival in GBM

An important factor correlated with poor survival in glioblastoma (GBM) is the aberrant and persistent activation of STAT3, a critical transcription factor that regulates multiple genes with key roles in cell survival, proliferation, resistance to chemotherapy, and stem cell maintenance. The Interleukin-6 (IL6)-STAT3 signaling axis has been studied extensively in inflammation and cancer. However, it is not completely understood how high levels of activated STAT3 are sustained in tumors. Previously, we identified a novel mechanism of biphasic activation of STAT3 in response to gp130-linked cytokines, including IL6, in which activation of STAT3 is prolonged by circumventing the negative regulatory mechanisms induced by its initial activationTo target prolonged STAT3 activation, we used the small molecule inhibitor bazedoxifene (BZA), which blocks formation of the IL6 receptor-gp130 complex. Glioma stem-like cells (GSCs) are more tumorigenic and more resistant to therapy. STAT3 is a key driver of the expression of stem cell transcription factors, making it a therapeutically important target in GBM. We show that treating GSCs with BZA decreases their self-renewal capacity and the expression of GSC markers in vitro. Additionally, BZA crosses the blood-brain barrier and confers a survival advantage in an orthotopic syngeneic mouse model of GBM. Although IL6-STAT3 signaling is important for GSC survival, a therapeutic agent that inhibits this pathway without toxicity has yet to be identified. Our findings reveal a mechanism of sustained STAT3 signaling in GBM and reveal its role in GSC maintenance, and we identify BZA as a novel candidate for treating GBM.

Inhibition of heat shock protein (HSP) 90 reverses signal transducer and activator of transcription (STAT) 3-mediated muscle wasting in cancer cachexia mice.

Cancer cachexia is a common cause of death among cancer patients with no currently effective treatment available. In animal models, aberrant activation of STAT3 in skeletal muscle contributes to muscle wasting. However, clinically the factors regulating STAT3 activation and the molecular mechanisms involved remain incompletely understood. The expression of HSP90 and the activation of STAT3 were detected in muscle from the patients with cancer cachexia or the tumour-bearing cachectic mice. HSP90 inhibitors, including 17DMAG (alvespimycin) and PU-H71, were administered to cachexic mice and cachexia parameters, weight loss, food intake, survival rate, body composition, serum metabolites, muscle wasting pathology and catabolic activation were analysed. The co-culture of C2C12 myotube cells with C26 conditioned media was performed to investigate the pathological mechanism involved in catabolic muscle wasting. The roles of HSP90, STAT3 and FOXO1 in myotube atrophy were explored via overexpression or knockdown. An enhanced interaction between activated STAT3 and HSP90 in the skeletal muscle of cancer cachexia patients, is a crucial for the development of cachectic muscle wasting. HSP90 inhibitors 17DMAG and PU-H71 alleviated the muscle wasting in C26 and models or the myotube atrophy of C2C12 cells induced by C26 conditional medium. Prolonged STAT3 activation transactivated FOXO1 by binding directly to its promoter and triggered the muscle wasting in a FOXO1-dependent manner in muscle cells. The HSP90/STAT3/FOXO1 axis plays a critical role in cachectic muscle wasting, which might be a potential therapeutic target for the treatment of cancer cachexia.

New Transcriptional Insights into Silent and Active Corticotroph Pituitary Tumors at Single Cell Resolution

Silent pituitary corticotroph tumors derive from the Tpit (aka TBX19) pituitary lineage. Accounting for ~ 30% of corticotroph tumors, they are not infrequently clinically aggressive and invade locally into adjacent sellar structures, making complete surgical resection challenging and contributing to their higher recurrence rates. How silent and active corticotroph tumor subtypes differ is not clear although some studies reported that silent corticotroph tumors exhibit reduced PC1 expression causing impaired POMC processing. We used single cell RNAseq to compare the transcriptome between silent (n = 2) and active (n = 4) corticotroph tumors at the single cell level. We obtained an average of 265 million reads, and 24,682 genes per patient with an average of 1,240 genes expressed and 3,5442 unique molecular identifiers (UMIs) detected per cell. We further defined 5 distinct cell populations from a total of 23,269 cells, namely tumor cells (62%), stromal cells (25%), immune cells (7%), progenitor cells (5%), and a minor population of erythrocytes (1%). Tumor cells clustered in an origin-dependent manner and all expressed POMC and TBX19. However, the gene signatures of silent and active corticotroph tumors differed in 3 major aspects. Firstly, and supporting prior studies, a series of hormone processing peptidase genes, including PC1 (aka PCSK1), PDIA3, SEC11C, SPCS1 and CTSB, were reduced in silent corticotroph tumors (p=5.54e-5) compared to active corticotroph tumors. Secondly, genes involved in organization of secretory vesicles such as SCG5, TIMP1, VGF, SYT17, LGALS3 and CALY were also reduced in silent corticotroph tumors, which could further compound their inability to secrete ACTH. Thirdly, the silent corticotroph tumors exhibited several features of endothelial-to-mesenchymal transition (EMT), including increased expression of the mesenchymal genes CDH2 (aka NCAD), COL1A1, PCDH9, FGF5, ID2 (p=8.4e-3), and loss of EPCAM, which regulate cell migration and movement. Upstream analysis suggested that aberrant STAT3 activation may be related to these changes. Consequently, we noted that the stromal content was higher in silent corticotroph tumors (47.5% vs. 18.13%), concordant with the observed EMT de-differentiation of tumor cells. In summary, our scRNAseq analysis provides an unprecedented precise investigation of the transcriptomic features of thousands of heterogenous corticotroph tumor cells simultaneously. We demonstrate that although silent corticotroph tumor cells still express the pituitary lineage markers PITX1 and TBX19, they exhibit EMT, potentially affording increased migratory capacity at the cost of reduced neuroendocrine function with inability to produce and secrete mature ACTH. Our findings provide novel insights into the pathogenesis of silent versus active corticotroph tumor, but may reveal novel molecular targets for treatment of these challenging tumors.

MiR-210-5p regulates STAT3 activation by targeting STAT5A in the differentiation of dermal fibroblasts.

Elucidating the molecular mechanism of the microRNAs in skin fibrosis is critical for identifying a novel therapeutic strategy for hypertrophic scar (HS). In this study, it was shown that miR-210-5p is induced by TGFβ, and that overexpression of miR-210-5p promoted the differentiation of human dermal fibroblasts (HDFs) into myofibroblasts. STAT5A is required for TGFβ-induced STAT3 activity. Here, we show that miR-210-5p attenuated TGFβ-induced STAT3 signaling pathway by suppressing the expression of STAT5A. Taken together, the present study suggests that TGFβ-induced miR-210-5p reduced STAT5A expression, leading to aberrant activation of STAT3, and facilitate skin fibrosis in HDFs.

SH2B3, Transcribed by STAT1, Promotes Glioblastoma Progression Through Transducing IL-6/gp130 Signaling to Activate STAT3 Signaling.

Glioblastoma (GBM) is the most common and aggressive brain tumor in adults. The aberrant activation of STAT3 commonly occurs in GBM and is a key player in GBM tumorigenesis. Yet, the aberrant activation of STAT3 signaling is not fully understood. Here, we report that SH2B adaptor protein 3 (SH2B3) is highly expressed in GBM and preferentially expressed in GBM stem cells (GSCs). Moreover, SH2B3 high expression predicts worse survival of GBM patients. Targeting SH2B3 considerably impairs GBM cell proliferation, migration, and GSCs’ self-renewal in vitro as well as xenograft tumors growth in vivo. Additionally, we provide evidence suggesting that STAT1 directly binds to the promoter of SH2B3 and activates SH2B3 expression in the transcriptional level. Functionally, SH2B3 facilitates GBM progression via physically interacting with gp130 and acting as an adaptor protein to transduce IL-6/gp130/STAT3 signaling. Together, our work firstly uncovers that the STAT1/SH2B3/gp130/STAT3 signaling axis plays critical roles in promoting GBM progression and provides insight into new prognosis marker and therapeutic target in GBM.

The Important Role of STAT3 in Chronic Lymphocytic Leukaemia Biology.

Signal transducer and activator of transcription (STAT) proteins are cytoplasmic transcription factors that transmit the signal of cytokines, hormones and growth factors. STAT proteins control fundamental cellular processes including survival, proliferation and differentiation. Inappropriate activation of STATs might contribute to cellular transformation and leukaemogenesis. About 70% of all solid and haematological tumours exhibit aberrant STAT3 expression and/or activation, highlighting its essential role in tumourigenesis. Aberrant STAT3 activation has been found in several solid tumours and haematologic malignancies. Importantly, constitutive activation of STAT proteins has been found in several leukaemias including acute myeloid leukaemia, acute promyelocytic leukaemia, acute lymphoblastic leukaemia, chronic myeloid leukaemia and chronic lymphocytic leukaemia (CLL). Constitutively activated STAT3 plays an important role in CLL biology. CLL cells harbour constitutive phosphorylation on S727 and acetylation on K685 and transient phosphorylation on Y705 residues. Moreover, STAT3 messenger RNA expression is significantly higher in CLL cells compared to healthy B-lymphocytes. Interestingly, STAT3 inhibition was disclosed as an important by-product of ibrutinib treatment in CLL patients. The purpose of this review is to describe the consequences of STAT3 dysregulation in CLL cells. Here, we discuss aberrantly modified processes by STAT3 activation in CLL cells such as proliferation, apoptosis, B cell receptor signalling, cytokine secretion, immune checkpoint regulation, microRNA regulation, free fatty acid metabolism and electron transport chain in the mitochondria.

Leukemia Inhibitory Factor Signaling Enhances Production of Galactose-Deficient IgA1 in IgA Nephropathy

Objectives: IgA nephropathy (IgAN) is thought to involve an autoimmune process wherein galactose-deficient IgA1 (Gd-IgA1), recognized as autoantigen by autoantibodies, forms pathogenic immune complexes. Mounting evidence has implicated abnormal activation of some protein-tyrosine kinases (PTKs) in IgAN. Furthermore, genome-wide association studies (GWAS) of IgAN provided insight into disease pathobiology and genetics. A GWAS locus on chromosome 22q12 contains genes encoding leukemia inhibitory factor (LIF) and oncostatin M, interleukin (IL)-6-related cytokines implicated in mucosal immunity and inflammation. We have previously shown that IL-6 mediates overproduction of Gd-IgA1 through aberrant STAT3 activation. Here, we show that LIF enhanced production of Gd-IgA1 in IgA1-secreting cells of patients with IgAN and provide initial analyses of LIF signaling. Methods: We characterized LIF signaling that is involved in the overproduction of Gd-IgA1, using IgA1-secreting cell lines derived from peripheral blood of patients with IgAN and healthy controls (HC). We used global PTK activity profiling, immunoblotting, lectin ELISA, and siRNA knock-down. Results: LIF stimulation did not significantly affect production of total IgA1 in IgA1-secreting cells from patients with IgAN or HC. However, LIF increased production of Gd-IgA1, but only in the cells from patients with IgAN. LIF stimulation enhanced phosphorylation of STAT1 in IgA1-secreting cells from patients with IgAN to a higher degree than in the cells from HC. siRNA knock-down of STAT1 blocked LIF-mediated overproduction of Gd-IgA1. Unexpectedly, this abnormal phosphorylation of STAT1 in IgA1-secreting cells from patients with IgAN was not mediated by JAK, but rather involved activation of Src-family PTKs (SFKs). Conclusion: Abnormal LIF/STAT1 signaling represents another pathway potentially leading to overproduction of Gd-IgA1 in IgAN, providing possible explanation for the phenotype associated with chromosome 22q12 GWAS locus. Abnormal LIF/STAT1 signaling and the associated SFKs may represent potential diagnostic and/or therapeutic targets in IgAN.

The IκB Kinase Inhibitor ACHP Targets the STAT3 Signaling Pathway in Human Non-Small Cell Lung Carcinoma Cells.

STAT3 is an oncogenic transcription factor that regulates the expression of genes which are involved in malignant transformation. Aberrant activation of STAT3 has been observed in a wide range of human malignancies and its role in negative prognosis is well-documented. In this report, we performed high-throughput virtual screening in search of STAT3 signaling inhibitors using a cheminformatics platform and identified 2-Amino-6-[2-(Cyclopropylmethoxy)-6-Hydroxyphenyl]-4-Piperidin-4-yl Nicotinonitrile (ACHP) as the inhibitor of the STAT3 signaling pathway. The predicted hit was evaluated in non-small cell lung cancer (NSCLC) cell lines for its STAT3 inhibitory activity. In vitro experiments suggested that ACHP decreased the cell viability and inhibited the phosphorylation of STAT3 on Tyr705 of NSCLC cells. In addition, ACHP imparted inhibitory activity on the constitutive activation of upstream protein tyrosine kinases, including JAK1, JAK2, and Src. ACHP decreased the nuclear translocation of STAT3 and downregulated its DNA binding ability. Apoptosis was evidenced by cleavage of caspase-3 and PARP with the subsequent decline in antiapoptotic proteins, including Bcl-2, Bcl-xl, and survivin. Overall, we report that ACHP can act as a potent STAT3 signaling inhibitor in NSCLC cell lines.

Abstract C054: Discovery of KYM-003, a potent and selective STAT3 degrader with antitumor activity in heme malignancies

Abstract Purpose: Targeted protein degradation is a novel therapeutic modality that holds the promise to target previously “undruggable” proteins by harnessing the innate ubiquitin proteasome system in cells. In this study we describe the discovery and characterization of KYM-003, a novel, selective degrader of the oncogenic transcription factor STAT3 that shows potent antitumor activity in vitro and in vivo in STAT3-dependent models. Introduction: STAT3 is a transcription factor downstream of several signaling events including the IL-6-JAK pathway. Activating mutations and aberrant STAT3 activation are found in numerous cancers and STAT3 activation has been directly linked to the promotion of cancer cell survival, proliferation and immune evasion, making it a highly attractive target for oncology. Potent and selective agents specifically and directly targeting STAT3 have remained elusive, however. Herein we report the discovery of a potent and selective STAT3 degrader, KYM-003, which displays strong anti-tumor activity in models of heme malignancies. Methods: The binary binding of KYM-003 to STAT3 and the E3 ligase were characterized in fluorescence polarization assays. An AlphaLISA immunoassay was used to measure the formation of the E3-KYM-003-STAT3 ternary complex. Ubiquitination of endogenous STAT3 was evaluated in human cell lysates in the presence of KYM-003. STAT3 levels in multiple primary cells, cell lines, and xenograft tumor tissues were quantified by immunoassays. STAT3 signaling was monitored through its target gene levels by RT-qPCR. Cell viability was monitored by CellTiter-Glo. Tumor xenograft studies were conducted by implanting human cancer cell lines into immune-compromised mice and assessment of tumor volume changes. Key data: KYM-003 degrades STAT3 via an E3 ligase-dependent mechanism. KYM-003 potently binds to and demonstrated strong cooperativity in promoting the formation of the STAT3-KYM-003-E3 ligase ternary complex. In a biochemical assay with A549 cell lysates, KYM-003 potently induced ubiquitination of endogenous STAT3. KYM-003 robustly degraded STAT3 in numerous primary cells or cell lines with DC50 < 100 nM. Degradation was highly selective for STAT3 vs >10,000 other detected proteins (including all other STAT family members) in A549 and hPBMCs. Degradation of STAT3 by KYM-003 led to strong downregulation of STAT3 targets gene expression, such as SOCS3, MYC, and PIM1 Importantly, total STAT3 and pSTAT3 levels in tumors were reduced by >90% for at least 24 hours after a single dose of KYM-003 and repeated dosing of KYM-003 showed dose-dependent antitumor activity in xenograft models of heme maliganancies. Conclusion: KYM-003 is a potent and selective STAT3 degrader that demonstrated strong anti-tumor activity in heme cancer models. These data support STAT3 degraders as a promising new therapeutic opportunity. Citation Format: Fred Csibi, Nan Ji, Bin Yang, Karen Yuan, Michele Mayo, Haojing Rong, Scott Rusin, Kirti Sharma, Christine Loh, Henry Li, Sharon Townson, Hari Kamadurai, Jesse Chen, Duncan Walker, Jared Gollob, Nello Mainolfi. Discovery of KYM-003, a potent and selective STAT3 degrader with antitumor activity in heme malignancies [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C054. doi:10.1158/1535-7163.TARG-19-C054

Tumor Suppressor Function of Dnmt3b in Hematologic Malignancies Critically Depends on Its Catalytic Activity

DNA methylation regulates gene transcription and it is involved in various physiological processes in mammals including development and hematopoiesis. It is catalyzed by several DNA methyltransferases, including DNMT1, DNMT3A and DNMT3B that not only mediate methylation but also interact with a number of repressive proteins, thereby contributing to transcriptional repression in methylation-independent manner. Dnmt3b is critical gene in mouse embryogenesis whose loss results in embryonic lethality around E13.5. It also functions as a tumor suppressor in lymphoid and myeloid malignancies in mice. The extent to which Dnmt3b's catalytic activity is involved in these processes is unclear. To address the role of catalytic activity in embryogenesis and tumorigenesis, we generated mice carrying a catalytically inactive allele of Dnmt3b (Dnmt3bCI) by introducing a double amino acid substitution in enzyme active center (P656V; C657D) in endogenous Dnmt3b locus. By interbreeding Dnmt3b+/CI mice we found that homozygous mutant Dnmt3bCI/CImice survived embryonic development and had a long life span. Such results demonstrated that catalytic activity of Dnmt3b is, surprisingly, dispensable for both pre- and post-natal development. The presence of catalytically inactive Dnmt3b rescued most methylation changes observed in the absence of Dnmt3b in embryogenesis suggesting that Dnmt3b plays a role of accessory protein for methylation mediated by other Dnmts. In contrast, the absence of Dnmt3b's catalytic activity promoted MYC/Ras-induced transformation of mouse embryonic fibroblasts in vitro, and accelerated both MYC-induced T-cell lymphomagenesis and MLL-AF9-induced acute myeloid leukemia. Global gene expression and methylation profiling revealed a number of deregulated events specific for MYC;Dnmt3bCI/CI, resulting in aberrant activation of IL7-R and STAT3 and downregulation of putative tumor suppressor genes including Cdkn2c and Dusp6. Altogether, our data suggest that Dnmt3b's methyltransferase activity is dispensable for mouse development but critical to prevent hematologic malignancies by controlling events involved in cellular transformation. Disclosures No relevant conflicts of interest to declare.

STAT3 Dysregulation in Mature T and NK Cell Lymphomas.

T cell lymphomas comprise a distinct class of non-Hodgkin’s lymphomas, which include mature T and natural killer (NK) cell neoplasms. While each malignancy within this group is characterized by unique clinicopathologic features, dysregulation in the Janus tyrosine family of kinases/Signal transducer and activator of transcription (JAK/STAT) signaling pathway, specifically aberrant STAT3 activation, is a common feature among these lymphomas. The mechanisms driving dysregulation vary among T cell lymphoma subtypes and include activating mutations in upstream kinases or STAT3 itself, formation of oncogenic kinases which drive STAT3 activation, loss of negative regulators of STAT3, and the induction of a pro-tumorigenic inflammatory microenvironment. Constitutive STAT3 activation has been associated with the expression of targets able to increase pro-survival signals and provide malignant fitness. Patients with dysregulated STAT3 signaling tend to have inferior clinical outcomes, which underscores the importance of STAT3 signaling in malignant progression. Targeting of STAT3 has shown promising results in pre-clinical studies in T cell lymphoma lines, ex-vivo primary malignant patient cells, and in mouse models of disease. However, targeting this pleotropic pathway in patients has proven difficult. Here we review the recent contributions to our understanding of the role of STAT3 in T cell lymphomagenesis, mechanisms driving STAT3 activation in T cell lymphomas, and current efforts at targeting STAT3 signaling in T cell malignancies.

Abstract 2365: Selective Stat3 inhibition in the tumor microenvironment restricts gastrointestinal tumor growth

Abstract The transcription factor Stat3 plays essential roles in biological processes involving development, immunity and inflammation. Stat3 is known to regulate the expression of a large repertoire of genes which when deregulated, as in the context of tumorigenesis, can lead to the development of some of the well-known hallmarks of cancer, such as resistance to cell death and sustained proliferation. Indeed the aberrant activation of Stat3 has been characterized across a number of different cancers including gastrointestinal cancers, presenting Stat3 and its signalling pathway a promising therapeutic target. Although intrinsic Stat3 signalling in tumor cells is well characterized, the effect(s) of Stat3 signalling among the infiltrating cells of the tumor microenvironment is still poorly understood. With this project we aim to elucidate the role of Stat3 in the non-tumoral compartment, and explore the therapeutic value of Stat3 inhibition for gastric and colon cancer. We generated the CAGsrtTA3;STAT3.1348 (shStat3) mouse that utilizes short-hairpin RNAi technology allowing for conditional and reversible Stat3 reduction. To study the effects of systemic Stat3 inhibition, the shStat3 was crossed with the gp130F/F mutant mouse that spontaneously develops gastric cancer. To assess the effects of Stat3 suppression in the non-tumoral compartment alone, the shStat3 mice were sub-cutaneously injected with MC38 murine colon cancer cells. Tumors and other tissues of these mice were excised and analysed. We found that shStat3-mediated systemic Stat3 suppression resulted in significant tumor reduction in the gp130F/F gastric cancer mice. Similarly, isolated Stat3 reduction in the non-tumoral compartment of the shStat3 mice, restricted the growth of Stat3-proficient MC38 tumor allografts. Immunophenotyping of excised tumors highlighted an increase of a monocytic Ly6C+Ly6G- myeloid population. Our data provides evidence for the therapeutic value of specific Stat3 targeting in gastrointestinal cancers. Interestingly, we have shown that Stat3 suppression in the non-tumoral compartment alone was enough to result in significant tumor reduction, indicating a prominent role of Stat3 in creating a pro-tumorigenic microenvironment. We have also identified a monocytic population as candidate cell type that drives this Stat3 suppression mediated anti-tumor effect. Citation Format: Mariah Grace Alorro, Frederick Masson, Moritz Eissmann, Matthias Ernst. Selective Stat3 inhibition in the tumor microenvironment restricts gastrointestinal tumor growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2365.

PB1819 STAT3 PROTEIN AS A PROGNOSTIC FACTOR OF DIFFUSE LARGE B‐CELL LYMPHOMA

Background:Diffuse large B‐cell lymphoma (DLBCL) is the most common type of non‐Hodgkin's lymphoma (NHL) in adults (30% to 40%). Nosology combines a heterogeneous group of lymphoid neoplasias with various morphological, immunophenotypic, genetic characteristics, as well as variable clinical course and response to therapy.The R‐CHOP regimen is considered the standard therapeutic option for diffuse large B‐cell lymphoma. Although combination chemotherapy has improved the outcome, many patients do not achieve complete remission, 5‐year overall survival (OS) for DLBCL patients ranges from 30% to 50%, the disease remains resistant to treatment or recurs in its early stages.The development of International Prognostic Indices (IPI, aaIPI and others) has provided with a wide range of criteria for predicting the evolution of aggressive lymphomas. However, they do not always allow evaluating the individual risk of treatment failure. In this regard, the search for additional criteria that determine the prognosis of the disease is of a large interest.STAT3 protein may be one of the promising biomarkers in DLBCL. This protein is an important member of the Janus kinase (JAK) and signal transducer and activator (JAK/STAT) pathway participators. Aberrant activation of STAT3 has been found in several tumors including DLBCL. The value of protein in this nosology has not been studied enough, the question of its prognostic role remains open.Aims:To determine the prognostic value of the expression of STAT3 protein in patients with DLBCL.Methods:The cohort consisted of 50 patients with de novo DLBCL treated with the standard R‐CHOP regimen from 2014 to 2017. Immunohistochemical studies used 2 μm thick tissue samples of the tumor, the antibody used STAT3 (pSTAT3‐Tyr705). Based on the literature data, the threshold value for the pSTAT3 marker were 66%. Patients were divided into two groups relative to the number of STAT3 positive tumor cells. The first group included 16 patients (32%) with a low degree of expression of the marker (less than 66% of the cells), the second ‐ 34 subjects (68%) with overexpression of the protein (66% and above). Overall and non‐progressive survival were analyzed by Kaplan–Meier method, with difference compared by the log‐rank test.Results:For cases of low STAT3 expression the OS was significantly higher than cases of high STAT3 expression (93,8% versus 41.2%, (p = 0.04)). The PFS for low and high STAT3 expression group was 50% and 38%, respectively, with statistical significance (p = 0.047). At the same time, the median survival without progression in the second group was 8 months, in the first group ‐ 26 months.Summary/Conclusion:Among the patients with DLBCL treated with the R‐CHOP protocol, overexpression of STAT3 protein is associated with an unfavorable course of the disease. The expression level of this marker can act as an additional prognostic criterion that determines the sensitivity of the tumor to standard therapy and allows personalizing the treatment tactics.

Three Different Jak2/Stat3-Related Pathways Favor the Survival of Chronic Lymphocytic Leukemia Neoplastic Clone

INTRODUCTIONChronic lymphoproliferative disorders are characterized by the expansion of malignant lymphocytes, the most common form being Chronic Lymphocytic Leukemia (CLL). Besides intrinsic abnormalities, the acquisition of the transformed phenotype and the diffusion of disease are related to the favorable cross-talking tumor cell-microenvironment. Furthermore, it has been demonstrated that CLL cells own an amplified mitochondrial respiration leading to an increased Reactive Oxygen Species (ROS) production and intrinsic oxidative stress. Several molecules released by microenvironmental partners signal through JAK-STAT pathway. The deregulation of JAK2/STAT3 axis may lead to aberrant activation of STAT3 and, as a result, to tumor development in hematopoietic cells. We previously analyzed STAT3 and JAK2 expression, phosphorylation and localization in normal and leukemic B cells, demonstrating an abnormal activation of this pathway in the neoplastic clone with respect to normal B lymphocytes. We focused on STAT3 constitutive phosphorylation at serine (Ser) 727 residue since it has been recently observed that the presence of mt-STAT3 Ser727 promotes mitochondrial respiration and, generally, cell viability. We hypothesized the involvement of JAK2/STAT3 axis in 3 different pathways: (i) canonical “IL-6 related pathway”; (ii) JAK2/STAT3 - BCR/Lyn crosstalk; (iii) STAT3 effects on mitochondrial regulation.METHODSSTAT3 expression and phosphorylation were evaluated by Western Blotting (WB) and Flow Cytometry (FC). Purified cells (2x106 cells/ml) were cultured, and treated with the JAK2 inhibitor AG490 (10, 50 and 100μM) and the STAT3 inhibitor Stattic (5, 7.5, and 10μM) for 24, 48 and 72h. Experiments with AG490 and Stattic were performed with/without MSCs and with/without Ibrutinib (2.5μM) and Venetoclax (1nM). CLL and normal B cell viability was tested with Annexin V/PI test by FC. P-STAT3 Ser727 expression has been correlated with p44/42, SAPK-JNK, NF-kB p65 and p38 MAPK activation by Reverse Phase Protein Microarrays (RPPA) in 57 therapy-free patients presenting good (mutated IGHV, absence of CD38 or normal karyotype) and poor (unmutated IGHV, CD38 expression or 17p deletion) prognostic factors.RESULTSWe found that STAT3 was highly expressed in malignant B cells with respect to normal B lymphocytes. We demonstrated that STAT3 and JAK2 were similarly overexpressed in both good and poor prognosis CLL patients. However, a significant correlation between STAT3 expression levels and their overall survival was observed. Considering STAT3 over-expression and its correlation with the clinical outcome in CLL and, according to our hypothesis, we moved forward with the analysis of the three different JAK2/STAT3 pathways. We demonstrated that AG490 and Stattic were able to induce a dose-dependent apoptosis in CLL cells, also bypassing environmental protection. AG490, targeting JAK2, inhibited the phosphorylation of SHP-1 at Ser591, activating the phosphatase. In turn, SHP-1 activation led to Lyn Tyr396 dephosphorylation/inactivation. Treatment with Stattic did not affect Lyn and SHP-1 phosphorylation since this inhibitor acts downstream to AG490. In fact, simultaneous administration of Ibrutinib led to an increase of apoptosis only in Stattic, but not in AG490 treated cells. This confirms a possible dual role of JAK2 inhibition. Venetoclax/AG490 and Venetoclax/Stattic co-treatment did not show an increased cell death rate, consistent with a downstream effect of Venetoclax to both AG490 and Stattic. RPPA analysis demonstrated a correlation between STAT3, Ser727 constitutively phosphorylated in CLL, and P-p44/42 Thr202/Tyr204, P-SAPK-JNK Thr-183/Tyr185, NF-kB p65 Ser-536 and p38 MAPK Thr-180/Tyr-182 expression. All these proteins are described as involved in an alternative pathway that allows STAT3 Ser727 to exert a pro-survival effect thorough the regulation of mitochondrial activity.CONCLUSIONSThe analysis of JAK2 and STAT3 expression, activation and inhibition lets us to highlight the importance of JAK2/STAT3 axis in the maintenance of 3 different survival pathways in CLL B cells. Furthermore, the correlation we demonstrated with clinical outcome of patients and the strengthening of Ibrutinib effect when we targeted this axis could represent a starting point for the development of new therapeutic strategies in CLL. DisclosuresTrentin:Abbvie: Honoraria; Gilead: Research Funding; Janssen: Research Funding; Roche: Membership on an entity's Board of Directors or advisory committees.