Constitutive Activation Of STAT3 Research Articles

Abstract 5287: Ex vivo pharmacologic inhibition of STAT3 effectively targets tumor and tumor associated suppressor cells in high-grade glioma 3D spheroids

Abstract Gliomas, a type of brain tumor originating from glial cells, comprise a heterogenous group of neoplasms. High-grade gliomas, in particular, present significant therapeutic challenges in the clinic. Unlike other solid tumors, the heterogenous glioma microenvironment is heavily enriched with tumor associated suppressor cells including myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs), which can account for 30-40% of the cellular composition1. These pro-tumorigenic myeloid cells are thought to contribute to the resistance of glioma to immunotherapy. Efforts to target the suppressive nature of these dominating cell types are in development to yield more therapeutic options for patients with these devastating diseases. Constitutive activation of STAT3 in gliomas has been associated with increased tumor progression and reduced immune response. We have observed in other cancer models that a phenotypic switch occurs from STAT3 pathway activation in 3D spheroid culture to STAT3 inactivation when cultured in 2D. Therefore, we sought to determine the role of STAT3 activation and its role in the interplay between glioma and their immune suppressive environment using a 3D spheroid model. We modified our commercially available test platform, 3D-Predict™ Glioma, such that it is conducive to monitoring immune cell function. Newly diagnosed treatment naive primary samples from HGG patients were dissociated and cryogenically preserved and banked. Samples were profiled for the abundance of tumor associated suppressive myeloid cells using flow cytometry. We determined that relative proportions of tumor suppressor cells were detectable and varied across HGG primary patient samples with TAMs being the predominate immune cell type identified. Pro-tumorigenic TAM markers including CD163 and CD206 were correlated with temozolomide response and were associated with poor patient prognosis. When we evaluated the role of p-STAT in HGG primary samples, p-STAT was detected at baseline and abundance was related back to the presence of tumor associated immune suppressor cells. The impact of STAT3 signaling blockade by the inhibitor WP1066 was determined ex vivo, and efficacy was related back to MGMT promotor methylation status and patient clinical outcome. We detected shifts in cytokine signatures following treatment of HGG 3D spheroid models with WP1066 which are known to target tumor associated suppressor cell functions. This work demonstrates the critical role suppressive immune cells play in HGG clinical response. Given p-STAT can be altered in 2D culture compared to 3D culture, ex vivo therapeutic response profiling of HGG samples is necessary using the more physiologically relevant spheroid culture model. 1.Hambardzumyan et al. The role of microglia and macrophages in glioma maintenance and progression. Nat. Neurosci. 19, 20-27 (2016). Citation Format: Katy A. Lassahn, Ashley K. Elrod, Teresa M. DesRochers, Kathryn M. Appleton. Ex vivo pharmacologic inhibition of STAT3 effectively targets tumor and tumor associated suppressor cells in high-grade glioma 3D spheroids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5287.

Targeting STAT3 Enzyme for Cancer Treatment.

A category of cytoplasmic transcription factors called STATs mediates intracellular signaling, which is frequently generated at receptors on cell surfaces and subsequently sent to the nucleus. STAT3 is a member of a responsible for a variety of human tumor forms, including lymphomas, hematological malignancies, leukemias, multiple myeloma and several solid tumor types. Numerous investigations have demonstrated constitutive STAT3 activation lead to cancer development such as breast, head and neck, lung, colorectal, ovarian, gastric, hepatocellular, and prostate cancers. It's possible to get a hold of the book here. Tumor cells undergo apoptosis when STAT3 activation is suppressed. This review highlights the STAT3 activation and inhibition which can be used for further studies.

Dependence of peripheral T-cell lymphoma on constitutively activated JAK3: Implication for JAK3 inhibition as a therapeutic approach.

Peripheral T-cell lymphoma (PTCL) is a clinically heterogeneous group that represents 10%-15% of all lymphomas. Despite improved genetic and molecular understanding, treatment outcomes for PTCL have not shown significant improvement. Although Janus kinase-2 (JAK2) plays an important role in myeloproliferative neoplasms, the critical role of JAK isoforms in mediating prosurvival signaling in PTCL cells is not well defined. Immunohistochemical analysis of PTCL tumors (n=96) revealed high levels of constitutively active JAK3 (pJAK3) that significantly (p<0.04) correlated with the activation state of its canonical substrate STAT3. Furthermore, constitutive activation of JAK3 and STAT3 positively correlated, at least in part, with an oncogenic tyrosine phosphatase PTPN11. Pharmacological inhibition of JAK3 but not JAK1/JAK2 significantly (p<0.001) decreased PTCL proliferation, survival and STAT3 activation. A sharp contrast was observed in the pJAK3 positivity between ALK+ (85.7%) versus ALK-negative (10.0%) in human PTCL tumors and PTCL cell lines. Moreover, JAK3 and ALK reciprocally interacted in PTCL cells, forming a complex to possibly regulate STAT3 signaling. Finally, combined inhibition of JAK3 (by WHI-P154) and ALK (by crizotinib or alectinib) significantly (p<0.01) decreased the survival of PTCL cells as compared to either agent alone by inhibiting STAT3 downstream signaling. Collectively, our findings establish that JAK3 is a therapeutic target for a subset of PTCL, and provide rationale for the clinical evaluation of JAK3 inhibitors combined with ALK-targeted therapy in PTCL.

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

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. 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. 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. 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.

STAT3/LKB1 controls metastatic prostate cancer by regulating mTORC1/CREB pathway

Prostate cancer (PCa) is a common and fatal type of cancer in men. Metastatic PCa (mPCa) is a major factor contributing to its lethality, although the mechanisms remain poorly understood. PTEN is one of the most frequently deleted genes in mPCa. Here we show a frequent genomic co-deletion of PTEN and STAT3 in liquid biopsies of patients with mPCa. Loss of Stat3 in a Pten-null mouse prostate model leads to a reduction of LKB1/pAMPK with simultaneous activation of mTOR/CREB, resulting in metastatic disease. However, constitutive activation of Stat3 led to high LKB1/pAMPK levels and suppressed mTORC1/CREB pathway, preventing mPCa development. Metformin, one of the most widely prescribed therapeutics against type 2 diabetes, inhibits mTORC1 in liver and requires LKB1 to mediate glucose homeostasis. We find that metformin treatment of STAT3/AR-expressing PCa xenografts resulted in significantly reduced tumor growth accompanied by diminished mTORC1/CREB, AR and PSA levels. PCa xenografts with deletion of STAT3/AR nearly completely abrogated mTORC1/CREB inhibition mediated by metformin. Moreover, metformin treatment of PCa patients with high Gleason grade and type 2 diabetes resulted in undetectable mTORC1 levels and upregulated STAT3 expression. Furthermore, PCa patients with high CREB expression have worse clinical outcomes and a significantly increased risk of PCa relapse and metastatic recurrence. In summary, we have shown that STAT3 controls mPCa via LKB1/pAMPK/mTORC1/CREB signaling, which we have identified as a promising novel downstream target for the treatment of lethal mPCa.

Guggulsterone Induces Apoptosis in Multiple Myeloma Cells by Targeting High Mobility Group Box 1 via Janus Activated Kinase/Signal Transducer and Activator of Transcription Pathway.

Multiple myeloma (MM) is a hematological disorder characterized by the abnormal expansion of plasma cells in the bone marrow. Despite great advances over the past three decades in discovering the efficacious therapies for MM, the disease remains incurable for most patients owing to emergence of drug-resistant cancerous cells. Guggulsterone (GS), a phytosteroid, extracted from the gum resin of guggul plant, has displayed various anticancer activities in vitro and in vivo; however, the molecular mechanisms of its anticancer activity have not been evaluated in MM cells. Therefore, in this study, we investigated the anticancer activity of GS in various MM cell lines (U266, MM.1S, and RPMI 8226) and the mechanisms involved. GS treatment of MM cells caused inhibition of cell proliferation and induction of apoptotic cell death as indicated by increased Bax protein expression, activation of caspases, and cleavage of poly (ADP-ribose) polymerase. This was associated with the downregulation of various proliferative and antiapoptotic gene products, including cyclin D, Bcl-2, Bcl-xL, and X-linked inhibitor of apoptosis protein. GS also suppressed the constitutive and interleukin 6-induced activation of STAT3. Interestingly, the inhibition of Janus activated kinase or STAT3 activity by the specific inhibitors or by siRNA knockdown of STAT3 resulted in the downregulation of HMGB1, suggesting an association between GS, STAT3, and HMGB1. Finally, GS potentiated the anticancer effects of bortezomib (BTZ) in MM cells. Herein, we demonstrated that GS could be a potential therapeutic agent for the treatment of MM, possibly alone or in combination with BTZ.

Pharmacologic Inhibitors of STAT3 or BCL6 Transcriptional Function Sensitize Lymphoma Cells to the Novel PD-1 Cis-Targeted PD1-IL2v Immunocytokine in a Murine Model

Although immune-based therapies like immune checkpoint inhibitors or CAR-T cells have led to dramatic advances in treating relapsed and refractory lymphomas, most patients still do not derive a durable response. Lymphoma cells, while being immunogenic, can have intrinsic mechanisms of resistance to immune-based killing and may also establish an immuno-suppressive microenvironment in which immune effector cells have diminished activity. This immune resistance may be a direct result of the inappropriate and constitutive activation of transcription factors whose physiologic function is to promote survival and resistance to immune-based killing. Two transcription factors that play such a role in lymphomas are STAT3 and BCL6. We therefore wished to test the hypothesis that drugs that block the transcriptional function of these proteins would enhance the efficacy of an immune-based therapy. We chose to use the A20 murine lymphoma system, which grows as an aggressive tumor in syngeneic Balb/c mice, and is generally resistant to immune checkpoint inhibitors such as PD-1 antagonists. A20 cells are characterized by constitutive STAT3 activation and BCL6 expression. To enhance the translational relevance of this study, we used inhibitors of STAT3 or BCL6 transcriptional function that could rapidly be introduced into clinical trials. We identified drugs that inhibit the transcriptional effects of these proteins based on a computational search of drugs that are known to be safe in humans, and which lead to gene expression changes opposite to that driven by each transcription factor. Using this approach, we identified atovaquone as a STAT3 inhibitor and disulfiram as a BCL6 inhibitor. The activity of each drug was then confirmed by in vitro testing. Furthermore, the resultant changes in gene expression in A20 cells would be predicted to make these cells more immunogenic. To stimulate the endogenous anti-tumor immune response, we utilized a novel murine specific PD-1 cis-targeted PD1-IL2v immunocytokine. muPD1-IL2v is, a fusion protein consisting of a PD-1 antibody and a C-terminally fused variant form of the cytokine IL-2 (IL2v) that does not bind to CD25. CD25 is the alpha chain that confers high affinity to the IL-2 receptor and which is highly expressed on immunosuppressive T regulatory cells (Tregs). This design enables muPD1-IL2v to preferentially activate PD-1 expressing antigen specific T effector cells by removing the competitive advantage of immunosuppressive T regulatory cells. At the same time, the PD-1 targeting allows the preferential delivery of IL-2v to PD-1+ tumor specific lymphocytes while bypassing the binding to Tregs. We then tested the activity of atovaquone or disulfiram in combination with muPD1-IL2v in an immunocompetent model with A20 lymphoma cells. As a single agent, muPD1-IL2v had a modest effect in delaying the growth of these tumors. Similarly, atovaquone or disulfiram each showed only a small change in the rate of growth of A20 tumors. However, when used in combination with muPD1-IL2v, each of these transcriptional inhibitors led to a significant fraction of animals having complete and long term regression of these tumors. Furthermore, tumors resected from animals treated with atovaquone showed evidence of on-target inhibition of STAT3 function, and tumors resected from animals treated with disulfiram showed evidence for on-target effects on BCL6 target gene expression. These findings suggest that inhibition of oncogenic transcription factors like STAT3 and BCL6, in conjunction with novel immune-targeted therapeutics like PD1-IL2v, have the potential for long term disease control in lymphomas. This research collaboration was supported through the imCORE network.

An OGT-STAT5 Axis in Regulatory T Cells Controls Energy and Iron Metabolism.

The immunosuppressive regulatory T (Treg) cells exert emerging effects on adipose tissue homeostasis and systemic metabolism. However, the metabolic regulation and effector mechanisms of Treg cells in coping with obesogenic insults are not fully understood. We have previously established an indispensable role of the O-linked N-Acetylglucosamine (O-GlcNAc) signaling in maintaining Treg cell identity and promoting Treg suppressor function, via STAT5 O-GlcNAcylation and activation. Here, we investigate the O-GlcNAc transferase (OGT)-STAT5 axis in driving the immunomodulatory function of Treg cells for metabolic homeostasis. Treg cell-specific OGT deficiency renders mice more vulnerable to high-fat diet (HFD)-induced adiposity and insulin resistance. Conversely, constitutive STAT5 activation in Treg cells confers protection against adipose tissue expansion and impaired glucose and insulin metabolism upon HFD feeding, in part by suppressing adipose lipid uptake and redistributing systemic iron storage. Treg cell function can be augmented by targeting the OGT-STAT5 axis to combat obesity and related metabolic disorders.

Abstract 5476: Novel azetidine-based STAT3 inhibitors induce misfolded protein response, endoplasmic reticulum stress and mitophagy, and inhibit breast tumor growth in vivo

Abstract Anticancer drugs that directly target Signal Transducer and Activator of Transcription (Stat)3 have remained elusive. We present azetidine-based small molecules as a new class of potent Stat3 inhibitors. Compounds H172 and H182 have potency (IC50) of 0.38-0.98 µM, H105 and H120 have IC50 of 1.75-2.07 µM against Stat3 DNA-binding activity in vitro, compared to the IC50 of &amp;gt;17 µM against Stat1 or Stat5 activity. Treatment of the triple-negative breast cancer (TNBC), MDA-MB-231 and MDA-MB-468 cells with the new compounds inhibited constitutive Stat3 activation, promoted Stat3 ubiquitination, blocked anchorage-dependent and independent growth, with potency, EC50 of 1.0-1.9 μM, and induced apoptosis of these cells. By contrast, pYStat1, pYStat5, pS727Stat3, pSrc, pJanus kinase, or the induction of other signaling events are not inhibited in compound-treated cells, and normal or tumor cells that do not harbor aberrantly-active Stat3 are only weakly inhibited. In vivo delivery of H120 or H182 as a single agent inhibited growth of human TNBC xenografts, and of H278 (HCl salt of H182) combined with radiation abrogated tumor growth and improved survival in mouse TNBC syngeneic models. These studies provide novel mechanism of disruption of aberrantly-active Stat3 and its dynamics in showing that the azetidine-based Stat3 inhibitors trigger Stat3 ubiquitination, and tumor cell death. H182 and H172 are new entities for further therapeutic development against TNBC and other cancers that harbor aberrantly-active Stat3. Citation Format: Peibin Yue, Francisco Lopez-Tapia, Yinsong Zhu, Nagendra Verma, Felix Alonso-Valenteen, Simoun Mikhael, Lali Medina-Kauwe, Marcus Tius, James Turkson. Novel azetidine-based STAT3 inhibitors induce misfolded protein response, endoplasmic reticulum stress and mitophagy, and inhibit breast tumor growth in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5476.

Combination of a STAT3 inhibitor with anti-PD-1 immunotherapy is an effective treatment regimen for a vemurafenib-resistant melanoma

Patients with BRAFV600E-mutant melanoma are effectively treated with the BRAF-inhibiting drug, vemurafenib, but soon develop drug resistance, limiting vemurafenib’s therapeutic efficacy. Constitutive activation of STAT3 in cancer cells and immune cells in the tumor microenvironment (TME) is a crucial contributor to the development of drug resistance and immune evasion in most cancers. Here, we investigated the antitumor efficacy and TME remodeling by APTSTAT3-9R, a cell-permeable STAT3 inhibitory peptide, as a strategy to treat vemurafenib-resistant melanoma. We found that vemurafenib-resistant melanoma remodels into immunosuppressive TME by increasing the expression of specific chemokines to facilitate the infiltration of immunosuppressive immune cells, such as myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs). Intratumoral treatment of APTSTAT3-9R led to a reduction in the population of MDSCs and TAMs, while increasing infiltration of cytotoxic T lymphocytes in the TME. Moreover, combination therapy with APTSTAT3-9R and an anti-PD-1 antibody enhanced significant suppression of tumor growth by decreasing infiltration of these immunosuppressive immune cells while increasing the infiltration and cytotoxicity of CD8+ T cells. These findings suggest that combined blockade of STAT3 and PD-1 signaling pathways may be an effective treatment option for overcoming poor therapeutic outcomes associated with drug-resistant BRAF-mutant melanoma.

Cytotoxic granzyme C-expressing ILC1s contribute to antitumor immunity and neonatal autoimmunity.

Innate lymphocytes are integral components of the cellular immune system that can coordinate host defense against a multitude of challenges and trigger immunopathology when dysregulated. Natural killer (NK) cells and innate lymphoid cells (ILCs) are innate immune effectors postulated to functionally mirror conventional cytotoxic T lymphocytes and helper T cells, respectively. Here, we showed that the cytolytic molecule granzyme C was expressed in cells with the phenotype of type 1 ILCs (ILC1s) in mouse liver and salivary gland. Cell fate-mapping and transfer studies revealed that granzyme C-expressing innate lymphocytes could be derived from ILC progenitors and did not interconvert with NK cells, ILC2s, or ILC3s. Granzyme C defined a maturation state of ILC1s. These granzyme C-expressing ILC1s required the transcription factors T-bet and, to a lesser extent, Eomes and support from transforming growth factor-β (TGF-β) signaling for their maintenance in the salivary gland. In a transgenic mouse breast cancer model, depleting ILC1s caused accelerated tumor growth. ILC1s gained granzyme C expression following interleukin-15 (IL-15) stimulation, which enabled perforin-mediated cytotoxicity. Constitutive activation of STAT5, a transcription factor regulated by IL-15, in granzyme C-expressing ILC1s triggered lethal perforin-dependent autoimmunity in neonatal mice. Thus, granzyme C marks a cytotoxic effector state of ILC1s, broadening their function beyond "helper-like" lymphocytes.

Targeting STAT3 Signaling in COL1+ Fibroblasts Controls Colitis-Associated Cancer in Mice.

Simple SummaryColitis-associated cancer is a colorectal cancer entity with poor prognosis and limited therapeutic options typically occurring as long-term complications of inflammatory bowel diseases. Connective tissue cells such as cancer-associated fibroblasts are part of the tumor microenvironment that can influence cancer development. The aim of this study was to determine the role of STAT3 activation in a frequent subset of fibroblasts during the development of inflammation-associated colorectal cancer in vivo. Our work highlights the functional role of cancer-associated fibroblasts in colitis-associated cancer, suggesting that strategies targeting the activation of that cell type could evolve as promising therapeutic option in inflammation-associated colorectal cancer and possibly additional entities.Colorectal cancer (CRC) is a common disease and has limited treatment options. The importance of cancer-associated fibroblasts (CAFs) within the tumor microenvironment (TME) in CRC has been increasingly recognized. However, the role of CAF subsets in CRC is hardly understood and opposing functions of type I (COL1+) vs. type VI (COL6+) collagen-expressing subsets were reported before with respect to NFκB-related signaling. Here, we have focused on COL1+ fibroblasts, which represent a frequent CAF population in CRC and studied their role upon STAT3 activation in vivo. Using a dual strategy with a conditional gain-of-function and a conditional loss-of-function approach in an in vivo model of colitis-associated cancer, tumor development was evaluated by different readouts, including advanced imaging methodologies, e.g., light sheet microscopy and CT-scan. Our data demonstrate that the inhibition of STAT3 activation in COL1+ fibroblasts reduces tumor burden, whereas the constitutive activation of STAT3 promotes the development of inflammation-driven CRC. In addition, our work characterizes the co-expression and distribution of type I and type VI collagen by CAFs in inflammation-associated colorectal cancer using reporter mice. This work indicates a critical contribution of STAT3 signaling in COL1+ CAFs, suggesting that the blockade of STAT3 activation in type I collagen-expressing fibroblasts could serve as promising therapeutic targets in colitis-associated CRC. In combination with previous work by others and us, our current findings highlight the context-dependent roles of COL1+ CAFs and COL6+ CAFs that might be variable according to the specific pathway activated.

Minecoside promotes apoptotic progression through STAT3 inactivation in breast cancer cells.

Breast cancer is one of the most common malignant tumors in women worldwide, and is a major cause of mortality and morbidity in cancer patients. Constitutive activation of STAT3 has been found in a variety of malignant tumors, including breast cancer. Since STAT3 activation is capable of regulating various important features of tumor cells, identification of a novel STAT3 inhibitor is considered a potential strategy for treating breast cancer. The aim of the present study was to examine whether minecoside (MIN), an active compound extracted from Veronica peregrina L., exerts an antitumor effect by inhibiting STAT3 signaling pathway in MDA-MB-231 cells. The results revealed that MIN inhibited the constitutive STAT3 activation in a dose- and time-dependent manner. MIN also blocked the nuclear translocation of STAT3 and suppressed STAT3-DNA binding. In addition, MIN downregulated the STAT3-mediated expression of proteins such as Bcl-xL, Bcl-2, CXCR4, VEGF, and cyclin D1. Subsequently, MIN promoted the caspase-dependent apoptosis in MDA-MB-231 cells. Overall, results of the present study provide evidence that MIN exerted anticancer activity via inhibition of the STAT3 signaling pathway. Further studies using animal models are required to determine the potential of this molecule as an anticancer drug.

Gene therapy with SOCS1 induces potent preclinical antitumor activities in oral squamous cell carcinoma.

Constitutive activation of STAT3 promotes oncogenesis and growth of oral squamous cell carcinoma (OSCC). We investigated the mechanism of action of suppressor of cytokine signaling 1 (SOCS1), an endogenous inhibitor of JAK, as gene therapy for OSCC. Antitumor effect of SOCS1 was compared to JAK inhibitor I by cell proliferation assay, cell cycle analysis, and apoptosis analysis in vitro. In addition, antitumor effect was evaluated using xenograft mouse models in vivo. JAK inhibitor I inhibited the proliferation of KOSC2 cl3-43 or T3M-1 clone2 OSCC cell lines in vitro. While JAK inhibitor I arrested both cell lines at the G2/M phase, induction of apoptosis was observed in T3M-1 clone2 cells, but not KOSC2-cl3-43 cells. An adenoviral vector expressing SOCS1 (AdSOCS1) significantly decreased the proliferation of both OSCC cell lines and induced G2/M phase cell cycle arrest and apoptosis, suggesting that induction of apoptosis of KOSC2 cl3-43 cells by AdSOCS1 is regulated by the JAK/STAT independent pathway. Overexpression of SOCS1 inhibited activation of the JAK/STAT and p44/42 MAPK pathways, while JAK inhibitor I inhibited activation of the JAK/STAT pathway only. Consistently, expression of Mcl-1 was decreased by overexpression of SOCS1, but not JAK inhibitor I. Additionally, KOSC2 cl3-43 or T3M-1 clone2 OSCC cells were subcutaneously implanted in the flanks of two xenograft mouse models. As compared to a control adenovirus vector (AdLacZ), intratumor injection of AdSOCS1 significantly decreased the tumor volume and induced apoptosis in vivo. SOCS1 gene therapy may be a beneficial approach for the treatment of OSCC.

MiR-181a is a novel player in the STAT3-mediated survival network of TCR\u03b1\u03b2+ CD8+ T large granular lymphocyte leukemia

T-LGL cells arise as a consequence of chronic antigenic stimulation and inflammation and thrive because of constitutive activation of the STAT3 and ERK pathway. Notably, in 40% of patients, constitutive STAT3 activation is due to STAT3 activating mutations, whereas in 60% this is unknown. As miRNAs are amongst the most potent regulators in health and disease, we hypothesized that aberrant miRNA expression could contribute to dysregulation of these pathways. miRNA sequencing in T-LGL leukemia cases and aged-matched healthy control TEMRA cells revealed overexpression of miR-181a. Furthermore, geneset enrichment analysis (GSEA) of downregulated targets of miR-181a implicated involvement in regulating STAT3 and ERK1/2 pathways. Flow cytometric analyses showed increased SOCS3+ and DUSP6+ T-LGL cells upon miR-181a inhibition. In addition, miR-181a-transfected human CD8+ T cells showed increased basal STAT3 and ERK1/2 phosphorylation. By using TL1, a human T-LGL cell line, we could show that miR-181a is an actor in T-LGL leukemia, driving STAT3 activation by SOCS3 inhibition and ERK1/2 phosphorylation by DUSP6 inhibition and verified this mechanism in an independent cell line. In addition, miR-181a inhibition resulted in a higher sensitivity to FAS-mediated apoptosis. Collectively, our data show that miR-181a could be the missing link to explain why STAT3-unmutated patients show hyperactive STAT3.

Selective Targeting of Splicing Factor Mutant Hematopoietic Stem and Progenitor Cells Via STAT3 Inhibition

Myelodysplastic syndrome (MDS) is a bone marrow failure disorder driven by dysfunction of hematopoietic stem and progenitor cells (HSPCs). Patient sequencing studies over the last decade have revealed that mutations in splicing machinery predominate in MDS, thus selective targeting of these cells is therapeutically attractive. STAT3 inhibition has been explored previously as a means to eradicate HSPCs in MDS. While efficacy was demonstrated in a subset of samples, the underlying mechanism for this selectivity remains unknown. We examined RNAseq of MDS CD34+ HSPCs with splicing factor mutations versus wildtype, finding alternative splicing and differential expression of STAT3 pathway components. Functionally, we explored if STAT3 signaling represents a novel vulnerability in SF3B1 mutant HSPCs using a multi-model approach of in vivo zebrafish and mouse systems, and in vitro assays of CRISPR-engineered human leukemia K562 cells and primary MDS samples. Utilizing the small molecule STAT3 inhibitor STATTIC, we found that human cells carrying MDS-associated SF3B1 point mutations had heightened sensitivity to STAT3 inhibition compared to wildtype controls. To evaluate the activity of STAT3 inhibition in vivo, we utilized an Mx1-cre conditional knock-in mouse model of mutant SF3B1 (Sf3b1+/K700E). We demonstrated that in vivo STATTIC treatment selectively depleted Sf3b1 mutant cells over wildtype in vivo. RNAseq of sf3b1 homozygous mutantzebrafish cells revealed conserved dysregulation of STAT3 pathway splicing and target expression. Diminishing Stat3 (via morpholino knockdown, stable mutants, or STATTIC treatment) decreased HSPCs in sf3b1 heterozygotes but not wildtype embryos, demonstrating synthetic lethality between Sf3b1 and Stat3. Our data indicate that SF3B1 heterozygosity, regardless of the type of mutation, confers a heightened sensitivity to STAT3 inhibition in zebrafish, mouse, and human HSPCs. Critically, our data indicate that SF3B1-mutant cells can be selectively killed in vivo while sparing wildtype cells. We sought to rescue HSPCs in sf3b1 homozygous mutant zebrafish, however overexpression of ligands Osm and Il6 or wildtype Stat3 was insufficient. Instead, overexpression of constitutively-active Stat3 partially restored HSPCs, indicating that functional Stat3 signaling downstream of Sf3b1 is critical for HSPC formation. To investigate the specificity of the synthetic lethality for SF3B1, we assessed the STAT3 synthetic lethal interaction with other mutated splicing factors in MDS. Similar to SF3B1, we demonstrated STAT3 synthetic lethality with U2AF1 and SRSF2 heterozygosity in zebrafish and human cells. RNA-sequencing analysis of STATTIC-treated K562 cells revealed an exacerbation of splicing alterations upon STAT3 inhibition that was more pronounced in SF3B1+/K666N cells compared to wildtype. Even more strikingly, we demonstrated that constitutive activation of STAT3 could partially reverse defective splicing in zebrafish sf3b1 homozygous mutant cells. Mechanistically, these data strongly support coordinated splicing dysfunction as the underlying cause for STAT3-SF3B1 synthetic lethality. Together, we demonstrated a conserved and selective synthetic lethal interaction between STAT3 function and splicing factor defects that represents a novel liability for mutant HSPCs with important implications for MDS treatment. DisclosuresShastri: Guidepoint: Consultancy; Kymera Therapeutics: Research Funding; Onclive: Honoraria; GLC: Consultancy. Verma: Curis: Research Funding; BMS: Research Funding; Stelexis: Consultancy, Current equity holder in publicly-traded company; Eli Lilly: Research Funding; Medpacto: Research Funding; Incyte: Research Funding; GSK: Research Funding; Novartis: Consultancy; Acceleron: Consultancy; Celgene: Consultancy; Stelexis: Current equity holder in publicly-traded company; Throws Exception: Current equity holder in publicly-traded company.

Dual recombinase action in the normal and neoplastic mammary gland epithelium

We developed a transgenic mouse line that expresses the codon-optimized Flp recombinase under the control of the MMTV promoter in luminal epithelial cells of the mammary gland. In this report, we demonstrate the versatile applicability of the new MMTV-Flp strain to manipulate genes in a temporally and spatially controlled manner in the normal mammary gland, in luminal-type mammary tumors that overexpress ERBB2, and in a new KRAS-associated mammary cancer model. Although the MMTV-Flp is expressed in a mosaic pattern in the luminal epithelium, the Flp-mediated activation of a mutant KrasG12D allele resulted in basal-like mammary tumors that progressively acquired mesenchymal features. Besides its applicability as a tool for gene activation and cell lineage tracing to validate the cellular origin of primary and metastatic tumor cells, we employed the MMTV-Flp transgene together with the tamoxifen-inducible Cre recombinase to demonstrate that the combinatorial action of both recombinases can be used to delete or to activate genes in established tumors. In a proof-of-principle experiment, we conditionally deleted the JAK1 tyrosine kinase in KRAS-transformed mammary cancer cells using the dual recombinase approach and found that lack of JAK1 was sufficient to block the constitutive activation of STAT3. The collective results from the various lines of investigation showed that it is, in principle, feasible to manipulate genes in a ligand-controlled manner in neoplastic mammary epithelial cells, even when cancer cells acquire a state of cellular plasticity that may no longer support the expression of the MMTV-Flp transgene.

Abstract 929: A novel STAT3 inhibitor directly targeting STAT3 N-terminus simultaneously abrogates the nuclear and mitochondrial STAT3 activities leading to highly potent anti-cancer efficacy on the breast cancer

Abstract STAT3 functions are largely bifurcated: (1) STAT3 activated by Y705 phosphorylation (pY705) regulates target-gene expression in nucleus. (2) STAT3 activated by S727 phosphorylation (pS727) supports a electron transport complex (ETC) in mitochondria. Constitutive activation of STAT3 has long been considered to correlate with activation of diverse oncogenic pathways, which is a promising target for anti-cancer agents, but clinical therapeutic options are still unsatisfactory. Here we discovered a highly novel small-molecule inhibitor, JW-STAT3i directly binding to STAT3 N-terminal domain: 1) Through the cancer cell line panel screening, JW-STAT3i shows strong anti-cancer potency on various cancer cell lines including the breast cancer cell lines. 2) Anti-cancer mechanism of JW-STAT3i was elucidated: Firstly, the JW-STAT3i impairs pY705-STAT3 to abrogate pro-tumorigenic transcriptional programs mediated by STAT3. Secondly, JW-STAT3i also blocks pS727-STAT3 leading to mitochondrial dysfunction and AMPK activation. Consequently, JW-STAT3i treated cancer cells undergo growth arrest and apoptosis. 3) We further validated JW-STAT3i efficacy using cancer cell line- and patient-derived xenograft models whose response is well-correlated with the predictive biomarkers identified. Accuracy of in vivo efficacy in selected breast cancer PDX models based on predictive biomarker (estimated responders and non-responders) was very high. 4) We also demonstrated that JW-STAT3i shows a strong synergy with anti-PD-L1 antibody by induction of anti-tumorigenic immune environment. In conclusion, JW-STAT3i possesses both negative and positive effects on cancer cell proliferation and anti-immunity respectively. Currently, JW-STAT3i is under preclinical development and highly expected to be a promising anticancer therapeutic option in the near future. Citation Format: Jinsuk Kang. A novel STAT3 inhibitor directly targeting STAT3 N-terminus simultaneously abrogates the nuclear and mitochondrial STAT3 activities leading to highly potent anti-cancer efficacy on the breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 929.

Abstract 1230: High-affinity azetidine-based small-molecules as a new class of direct inhibitors of STAT3 activity and breast cancer phenotype

Abstract Signal Transducer and Activator of Transcription (STAT)3 is a member of the STAT family of transcription factors that is frequently aberrantly-activated in breast cancer and many other cancers. STAT3 represents a valid target for the development of novel anticancer therapeutics. However, it has posed significant challenges to the discovery efforts to identify potent and suitable small molecule inhibitors. As a result, there is currently no anti-STAT3 drugs in clinical application to realize the potential clinical benefits of inhibiting abnormal STAT3 function. Herein is a new series of (R)-azetidine-2-carboxamide-based small molecules, exemplified by H098, H127, H152, H169, H174, H182, and H203, which selectively inhibit STAT3 DNA-binding activity, with in vitro potency (IC50) in the range of 0.28-0.66 μM, compared to potencies greater than 18 µM against the activities of the family members, STAT1 and STAT5. Equally notable is the high affinity binding to STAT3, as shown by KD of 1 pM and 3.7 nM for H169 and H174, respectively. Treatment of human triple-negative breast cancer (TNBC) cells with H182 or H169 inhibited constitutive STAT3 phosphorylation (pY705STAT3) and DNA-binding activity in both time- and dose-dependent manner. Furthermore, both inhibitors potently inhibited anchorage-dependent and independent growth, colony survival, and migration of TNBC cells in vitro, with potencies, EC50 of 1.0-1.9 μM, and the compounds also induced apoptosis of same cells. Additionally, the expression of STAT3 target genes, including c-Myc, VEGF, and survivin, were suppressed in the compound-treated TNBC cells subsequent to the inhibition of constitutive STAT3 activation, thereby validating the inhibition of constitutively-active STAT3 and its downstream targets as being responsible for the antitumor cell responses. Moreover, H169 and docetaxel combinatorial treatment led to enhanced suppression of TNBC cell viability. By contrast, treatments with the new compounds had minimal effects on the induction of STAT1, Janus kinase 2, epidermal growth factor receptor, extracellular signal-regulated kinases (Erk1/2), Src, Shc phosphatase, or Akt in TNBC cells, and they were relatively weak on normal breast epithelial, MCF-10A or breast cancer, MCF-7 cells that do not harbor constitutively-active STAT3. Significantly, H182 delivered at 5-20 mg/kg via oral gavage or intra-peritoneal route inhibited growth of TNBC tumor xenograft in mice, with no evidence of toxicity. These studies identify novel, sub-micromolar potent azetidine-based small molecule STAT3 inhibitors that have shown antitumor efficacy against human TNBC in xenograft models. H182 and H169 represent suitable novel chemical entities for developing new therapeutics against TNBC and other cancers that harbor constitutively-active STAT3. Citation Format: Peibin Yue, Francisco Lopez-Tapia, Yinsong Zhu, Christine Brotherton-Pleiss, Wenzhen Fu, Felix Alonso-Valenteen, Simoun Mikhael, Lali Medina-Kauwe, Marcus Tius, James Turkson. High-affinity azetidine-based small-molecules as a new class of direct inhibitors of STAT3 activity and breast cancer phenotype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1230.

High Expression of the Resistant Factor YY1 and Low Expression of the RKIP Chemosensitizing Factor in Multiple Myeloma: Delineation of Several Crosstalk Pathways That Regulate the Expression of These Factors

Background: We have reported that multiple myeloma (MM) expresses high levels of the transcription (oncogenic) factor YY1, which regulates cell survival, proliferation, and resistance to both immuno- and chemotherapeutic drugs.1 Inhibition of YY1 sensitizes the tumor cells to cell death/apoptosis to cytotoxic drugs. In contrast, we have also reported that MM expresses very low levels of Raf kinase inhibitor protein (RKIP) and/or high levels of inactive phosphorylated RKIP.2 Overexpression of RKIP or de-phosphorylation of phosphorylated RKIP sensitized drug-resistant MM to drug-induced apoptosisHypothesis: The inverse relationship between YY1 and RKIP expression levels suggested that the two factors may be regulated through crosstalk pathways.Methods: Analysis of the literature revealed the presence of five crosstalk pathways that link RKIP and YY1.Results: The crosstalks consisted of the followings: (1) The NF-κB/YY1/Snail/RKIP loop. In the presence of RKIP, the NF-κB pathway is inhibited and consequently, the NF-κB targets YY1 and Snail are also inhibited. In turn, inhibition of the RKIP repressor Snail resulted in the upregulation of RKIP expression, all of which led to sensitization of drug-resistant tumor cells. (2) The p38 MAPK/RKIP/GSK3β/Snail/YY1 loop. In the presence of RKIP expression, the p38 MAPK-mediated phosphorylation of GSK3β is inhibited and results in the phosphorylation of Snail and its degradation, leading to the de-repression of RKIP expression and inhibition of YY1. (3) The RKIP/Smurf-2/YY1/Snail loop. SMAD ubiquitin regulatory factor 2 (Smurf-2) is an E3 ubiquitin ligase that degrades YY1. In tumor cells, there is low expression of Smurf-2 and, thus, little inhibition of YY1 with high Snail expression in the presence of low expression of RKIP. In the presence of high levels of Smurf-2, YY1 is inhibited, resulting in the inhibition of Snail and de-repression of RKIP. (4) The RKIP/MAPK/Myc/Let-7/HMGA2/Snail/YY1 loop. Let-7/miR98 suppresses Ras and inhibits the MAPK signaling pathway. In the absence of RKIP, the Raf/MEK1-2 pathway is activated and downstream Myc is expressed. In turn, Myc-induced Lin-28 inhibits Let-7, and leads to upregulation of HMGA2. Consequently, HMGA2 activates Snail, in addition to its regulation by YY1. In contrast, in the presence of RKIP, the Raf/MEK pathway is inhibited and there is downstream inhibition of its targets Myc, Lin-28, and HMGA2, leading to an inhibition of Snail and de-repression of RKIP. (5) The RKIP/GPCR/STAT3/miR-34/YY1 loop. In tumor cells expressing low levels of RKIP, there is no efficient inhibition of GPCR and MAKP signaling pathways. Hence, there is a constitutive activation of oncogenic STAT3 that inhibits the tumor suppressor miR-34. Thus, low miR-34 levels de-repress YY1 translation and facilitate its effects on the survival and the resistance of tumor cells to cytotoxic drugs.Conclusion and Implications: The above YY1/RKIP gene signatures might predict the tumor expanding potential and overall aggressiveness and their detriments that contribute to tumor regression. Targeting YY1 inhibition or RKIP induction via interference of the above crosstalk pathways may result in synergistic activity in combination with conventional treatments.References1 Bonavida B, Kaufhold S. Prognostic significance of YY1 protein expression and mRNA levels by bioinformatics analysis in human cancers: a therapeutic target. Pharmacol Ther. 150:149-68, 2015.2 Baritaki, S, Huerta-Yepez, S, Cabrava-Haimandez, M, Sensi, M, Canevari, S, Libra, M, Penichet, M, Chen, H, Berenson, JR, Bonavida, B. Unique Pattern of Overexpression of Raf-1 Kinase Inhibitory Protein in Its Inactivated Phosphorylated Form in Human Multiple Myeloma. Forum on Immunopathological Diseases and Therapeutics, 2: 179-188, 2011. DisclosuresNo relevant conflicts of interest to declare.