Expression Of Interferon Regulatory Factor Research Articles

NEURAL INFLAMMATION CONTRIBUTES TO VOLTAGE-GATED POTASSIUM CHANNELS DYSFUNCTION IN LUMBAR DORSAL ROOT GANGLIA IN CHRONIC HEART FAILURE RATS

Objective: Skeletal muscle afferent sensitization causes excessive sympatho-excitation and exercise intolerance during physical activity in the chronic heart failure (CHF) state. Our recent study reported that downregulated voltage-gated potassium (Kv) channels in lumbar dorsal root ganglia (DRGs) contribute to muscle afferent sensitization in CHF rats. However, the cellular mechanisms underlying downregulated Kv channels in CHF remain unknown. We hypothesized that chronic macrophage activation contributes to Kv channels dysfunction in the lumbar DRGs in CHF. Design and method: Myocardial infarction (MI) was produced by left coronary artery ligation in rats. Molecular experiments were carried out to assess macrophage activation, cytokines and Kv channel expression in L4-L6 DRGs post MI. In vitro co-culture of lipopolysaccharide (LPS)-pretreated RAW264.7 (activated M1 macrophage) with 50B11 DRG neurons were performed. Patch clamp experiments were performed to assess Kv channel activity in acutely isolated muscle afferent DRG neurons. Chronic oral administration of minocycline (Mino, 20 mg/kg/day) was used to suppress macrophage activation in L4-L6 DRGs. Results: Compared to sham rats, male MI rats exhibited a robust increase in the number of Iba1-positive macrophages and the protein expression of Interferon regulatory factor 8 (IRF8, a pro-inflammatory macrophage marker) in L4-L6 DRGs at 8 weeks post MI, which was largely prevented by Mino (Figure 1). SEM showed macrophage infiltration into lumbar DRGs 8 weeks post MI. PCR data suggested that the mRNA expressions of pro-inflammatory cytokines including IL1beta and IL6 in lumbar DRGs were significantly elevated in male rats at 8-weeks post MI compared to sham rats. Protein expression of Kv channels including Kv1.4, 3.4, 4.2 and 4.3, was significantly downregulated in male rat L4-L6 DRGs at 8 weeks post MI. This was largely attenuated by Mino. Patch clamp data demonstrated decreased Ito current in isolated male rat muscle afferent DRG neurons 8 weeks post MI, also attenuated by Mino. Moreover, LPS-pretreated BV2 cells or RAW264.7 (LPS was washed out 4 hours after exposure, co-cultured with 50B11 DRG neurons significantly downregulated several Kv isoforms in DRG neurons. Conclusions: Macrophage activation contributes to Kv channels dysfunction in lumbar DRGs in CHF male rats.

Fish ubiquitin-specific protease 8 (USP8) inhibits IFN production through autophagy-lysosomal dependent degradation of IRF7

Interferon regulatory factor 7 (IRF7) is considered the master regulator of virus-induced interferon (IFN) production. However, to avoid an autoimmune response, the expression of IRF7 must be tightly controlled. In this study, we report that zebrafish ubiquitin-specific protease 8 (USP8) promotes IRF7 degradation through an autophagy-lysosome-dependent pathway to inhibit IFN production. First, zebrafish usp8 is induced upon spring viremia of carp virus (SVCV) infection and polyinosinic/polycytidylic acid (poly I:C) stimulation. Second, overexpression of USP8 suppresses SVCV or poly I:C-mediated IFN expression. Mechanistically, USP8 interacts with IRF7 and promotes its degradation via an autophagy-lysosome-dependent pathway. Finally, USP8 significantly suppresses cellular antiviral responses and enhances SVCV proliferation. In summary, our discoveries offer a perspective on the role of zebrafish USP8 and provide additional understanding of the regulation of IRF7 in host antiviral immune response.

Interferon alpha promotes caspase-8 dependent ultraviolet light-mediated keratinocyte apoptosis via interferon regulatory factor 1.

Ultraviolet (UV) light is a known trigger of both cutaneous and systemic disease manifestations in lupus patients. Lupus skin has elevated expression of type I interferons (IFNs) that promote increased keratinocyte (KC) death after UV exposure. The mechanisms by which KC cell death is increased by type I IFNs are unknown. Here, we examine the specific cell death pathways that are activated in KCs by type I IFN priming and UVB exposure using a variety of pharmacological and genetic approaches. Mice that overexpress Ifnk in the epidermis were exposed to UVB light and cell death was measured. RNA-sequencing from IFN-treated KCs was analyzed to identify candidate genes for further analysis that could drive enhanced cell death responses after UVB exposure. We identify enhanced activation of caspase-8 dependent apoptosis, but not other cell death pathways, in type I IFN and UVB-exposed KCs. In vivo, overexpression of epidermal Ifnk resulted in increased apoptosis in murine skin after UVB treatment. This increase in KC apoptosis was not dependent on known death ligands but rather dependent on type I IFN-upregulation of interferon regulatory factor 1 (IRF1). These data suggest that enhanced sensitivity to UV light exhibited by lupus patients results from type I IFN priming of KCs that drives IRF1 expression resulting in caspase-8 activation and increased apoptosis after minimal exposures to UVB.

Stenotrophomonas maltophilia contributes to smoking-related emphysema through IRF1-triggered PANoptosis of alveolar epithelial cells

Cigarette smoke (CS), the main source of indoor air pollution and the primary risk factor for respiratory diseases, contains chemicals that can perturb microbiota through antibiotic effects. Although smoking induces a disturbance of microbiota in the lower respiratory tract, whether and how it contributes to initiation or promotion of emphysema are not well clarified. Here, we demonstrated an aberrant microbiome in lung tissue of patients with smoking-related COPD. We found that Stenotrophomonas maltophilia (S. maltophilia) was expanded in lung tissue of patients with smoking-related COPD. We revealed that S. maltophilia drives PANoptosis in alveolar epithelial cells and represses formation of alveolar organoids through IRF1 (interferon regulatory factor 1). Mechanistically, IRF1 accelerated transcription of ZBP1 (Z-DNA Binding Protein 1) in S. maltophilia-infected alveolar epithelial cells. Elevated ZBP1 served as a component of the PANoptosome, which triggered PANoptosis in these cells. By using of alveolar organoids infected by S. maltophilia, we found that targeting of IRF1 mitigated S. maltophilia-induced injury of these organoids. Moreover, the expansion of S. maltophilia and the expression of IRF1 negatively correlated with the progression of emphysema. Thus, the present study provides insights into the mechanism of lung dysbiosis in smoking-related COPD, and presents a potential target for mitigation of COPD progression.

An IRF2-Expressing Oncolytic Virus Changes the Susceptibility of Tumor Cells to Antitumor T Cells and Promotes Tumor Clearance.

IFN regulatory factor 1 (IRF1) can promote antitumor immunity. However, we have shown previously that in the tumor cell, IRF1 can promote tumor growth, and IRF1-deficient tumor cells exhibit severely restricted tumor growth in several syngeneic mouse tumor models. Here, we investigate the potential of functionally modulating IRF1 to reduce tumor progression and prolong survival. Using inducible IRF1 expression, we established that it is possible to regulate IRF1 expression to modulate tumor progression in established B16-F10 tumors. Expression of IRF2, which is a functional antagonist of IRF1, downregulated IFNγ-induced expression of inhibitory ligands, upregulated MHC-related molecules, and slowed tumor growth and extended survival. We characterized the functional domain(s) of IRF2 needed for this antitumor activity, showing that a full-length IRF2 was required for its antitumor functions. Finally, using an oncolytic vaccinia virus as a delivery platform, we showed that IRF2-expressing vaccinia virus suppressed tumor progression and prolonged survival in multiple tumor models. These results suggest the potency of targeting IRF1 and using IRF2 to modulate immunotherapy.

Abstract 4773: Interferon regulatory factor 8 (IRF8) as a biomarker for early detection and prevention of axillary lymph node-positive breast cancer

Abstract Breast cancer (BC) remains a leading health concern worldwide and is often undetected until the cancer metastasizes. Early detection can limit the need for aggressive treatments and improve prognosis. We analyzed early immune and molecular markers in benign breast disease (BBD) biopsies prior to BC development to identify women at elevated risk of developing node-positive invasive BC, which might direct surveillance and prevention efforts aimed at reducing incidence of this disease. Utilizing the Mayo Clinic BBD cohort, we compared women who developed node-positive BC following their BBD biopsy (cases; n=42) with those who remained cancer-free (controls; n=37), matched on patient age and biopsy date. We used NanoString nCounter system to identify differentially expressed genes (DEGs) between cases and controls. We also developed a multiplex immunofluorescence (mIF) approach using Opal system by Akoya Biosciences for comprehensive marker detection in FFPE tissue, enabling correlation of cells expressing DEGs with innate and adaptive immune cells. Digitized images were segmented, and cell phenotyping, and spatial relationships were compared between cases and controls. Our findings revealed significantly increased expression levels of IRF8 (interferon regulatory factor 8, a factor involved in immune cell differentiation) in controls as compared to cases and found that increased IRF8 expression in cases is correlated with delayed cancer onset. Additionally, controls exhibited higher frequencies of CD4+, CD8+, CD68+, CD20+ and CD11c+ immune cells, and lower frequency of Ki67+ staining, with a notable increase of CD11c+/CD68+ cells, a marker for M1 type macrophages, indicating a pro-inflammatory, antitumorigenic immune microenvironment polarization. Furthermore, our mIF analyses offer new insights into spatial biomarker localization, enhancing prognostic accuracy. Our findings suggest that decreased numbers of immune cells in BBD biopsies may be associated with increased risk of developing axillary node-positive BC. This research underscores the importance of dissecting molecular and immune interactions in BBD for assessing the risk of this disease. Our results have the potential to improve individualized risk assessment, leading to more targeted surveillance and informed screening, potentially reducing BC incidence and mortality through early intervention. This study not only contributes to our understanding of BC pathogenesis but also opens avenues for developing novel preventive strategies. Citation Format: Matilde Rossi, Nicole Cruz-Reyes, Melody L. Stallings Mann, Bryan M. McCauley, Tanya L. Hoskin, Robert A. Vierkant, Stacey J. Winham, Amy C. Degnim, Mark E. Sherman, Derek C. Radisky. Interferon regulatory factor 8 (IRF8) as a biomarker for early detection and prevention of axillary lymph node-positive breast cancer [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 4773.

Interferon Regulatory Factor 4 (IRF4) Plays a Key Role in Osteoblast Differentiation of Postmenopausal Osteoporosis.

Postmenopausal osteoporosis (PMOP) is a prevalent disease, which features decreased bone mass, bone weakness and deteriorated bone microstructure in postmenopausal women. Although many factors have been revealed to contribute to the occurrence of PMOP, its mechanism remains undefined. This work aimed to identify significant changes in gene expression during PMOP formation and to examine the most valuable differential genes in postmenopausal osteoporosis versus the control group. The GSE68303 dataset that contains 12 ovariectomize (OVX) experimental and 11 sham groups was downloaded and analyzed. The results indicated that interferon regulatory factor 4 (IRF4) might be a hub gene in the development of postmenopausal osteoporosis. Western blot and immunohistochemistry were carried out to evaluate IRF4 levels in thoracic vertebra extracts from OVX and Sham mice. To assess IRF4's impact on osteogenic differentiation in postmenopausal bone marrow mesenchymal stem cells (BM-MSCs), IRF4 overexpression (OV-IRF4) and knockdown (Sh-IRF4) plasmids were constructed. The results showed that comparing with the sham group, bone samples from the OVX group showed higher IRF4 expression. Alkaline phosphatase (ALP) staining revealed that IRF4 overexpression significantly inhibited ALP activity, while IRF4 knockdown promoted ALP activity in BM-MSCs. Simvastatin-treated OVX mice showed increased total bone volume/total tissue volume (BV/TV) and elevated Runx2 expression by immunohistochemical staining compared with the OVX group. This study demonstrated that IRF4 is associated with OVX induced osteoporosis, it can regulate bone stability by inhibiting the osteogenic differentiation BM-MSCs. This study may help enhance our understanding of the molecular mechanism of PMOP formation, providing new insights into estrogen defiance induced osteoporosis.

IRF4-mediated Treg phenotype switching can aggravate hyperoxia-induced alveolar epithelial cell injury

Bronchopulmonary dysplasia (BPD) is characterized by alveolar dysplasia, and evidence indicates that interferon regulatory factor 4 (IRF4) is involved in the pathogenesis of various inflammatory lung diseases. Nonetheless, the significance and mechanism of IRF4 in BPD remain unelucidated. Consequently, we established a mouse model of BPD through hyperoxia exposure, and ELISA was employed to measure interleukin-17 A (IL-17 A) and interleukin-6 (IL-6) expression levels in lung tissues. Western blotting was adopted to determine the expression of IRF4, surfactant protein C (SP-C), and podoplanin (T1α) in lung tissues. Flow cytometry was utilized for analyzing the percentages of FOXP3+ regulatory T cells (Tregs) and FOXP3+RORγt+ Tregs in CD4+ T cells in lung tissues to clarify the underlying mechanism. Our findings revealed that BPD mice exhibited disordered lung tissue structure, elevated IRF4 expression, decreased SP-C and T1α expression, increased IL-17 A and IL-6 levels, reduced proportion of FOXP3+ Tregs, and increased proportion of FOXP3+RORγt+ Tregs. For the purpose of further elucidating the effect of IRF4 on Treg phenotype switching induced by hyperoxia in lung tissues, we exposed neonatal mice with IRF4 knockout to hyperoxia. These mice exhibited regular lung tissue structure, increased proportion of FOXP3+ Tregs, reduced proportion of FOXP3+RORγt+ Tregs, elevated SP-C and T1α expression, and decreased IL-17 A and IL-6 levels. In conclusion, our findings demonstrate that IRF4-mediated Treg phenotype switching in lung tissues exacerbates alveolar epithelial cell injury under hyperoxia exposure.

The RNA m6A reader IGF2BP3 regulates NFAT1/IRF1 axis-mediated anti-tumor activity in gastric cancer

N6-methyladenosine (m6A) and its associated reader protein insulin like growth factor 2 mRNA binding protein 3 (IGF2BP3) are involved in tumor initiation and progression via regulating RNA metabolism. This study aims to investigate the biological function and clinical significance of IGF2BP3 in gastric cancer (GC). The clinical significance of IGF2BP3 was evaluated using tumor related databases and clinical tissues. The biological role and molecular mechanism of IGF2BP3 in GC progression were investigated by multi-omics analysis including Ribosome sequence (Ribo-seq), RNA sequence (RNA-seq) and m6A sequence (m6A-seq) combined with gain- and loss- of function experiments. IGF2BP3 expression is significantly elevated in GC tissues and associated with poor prognosis of GC patients. Knockdown of IGF2BP3 significantly weakens the migration and clonogenic ability, promotes the apoptosis, inhibits translation, and suppresses in vitro growth and progression of GC cells. Mechanistically, IGF2BP3 regulates the mRNA stability and translation of the nuclear factor of activated T cells 1(NFAT1) in a m6A dependent manner. Then NFAT1 induced by IGF2BP3 acts as a transcription factor (TF) to negatively regulates the promoter activities of interferon regulatory factor 1 (IRF1) to inhibit its expression. Inhibition of IGF2BP3-induced expression of IRF1 activates interferon (IFN) signaling pathway and then exerts its anti-tumor effect. Elevated IGF2BP3 promotes in vivo and in vitro GC progression via regulation of NFAT1/IRF1 pathways. Targeted inhibition of IGF2BP3 might be a potential therapeutic approach for GC treatment.

Correlation between methylation of interferon regulatory factor 6 gene promoter in renal tissues and overall survival of patients with Kidney renal clear cell carcinoma

To assess the prognostic value of methylation of interferon regulatory factor 6 (IRF6) gene promoter in patients diagnosed with Kidney renal clear cell carcinoma (KIRC). The primary lesions of fifty KIRC patients who were diagnosed at the First Affiliated Hospital of Nanjing Medical University from January 2016 to January 2020 were collected. The expression of IRF6 protein was determined with an immunohistochemical method. The correlation between the level of IRF6 expression and survival and/or metastasis status was analyzed. The mRNA and protein levels of the IRF6 in KIRC and normal renal tissues were compared by using bioinformatic tools. The difference in the methylation rate of the IRF6 gene promoter between tumor and adjacent tissues was analyzed by searching the online databases. Statistical analysis was carried out for the methylation status of the IRF6 gene promoter region to select those negatively correlated with the overall survival (OS) among the patients. In vitro experiments were conducted with cell lines to verify the correlation between the status of promoter methylation and transcription level of the IRF6 gene. The mRNA and protein levels of the IRF6 gene in KIRC tissues were significantly lower than those of the normal controls, and this was more prominent in patients who had died or developed metastasis. The extent of IRF6 gene promoter methylation in the KIRC tissues was much higher compared with that of the adjacent normal renal tissues. There was a significant negative correlation between the methylation of the IRF6 gene promoter and mRNA level of the IRF6 (R = -0.52). The higher methylation degree in the IRF6 gene promoter regions cg12034118 and cg16030177, the shorter the OS and worse prognosis in the patients. Only twenty CpG sites in cg12034118 were confirmed to be highly methylated in KIRC cell lines. The transcription level of the IRF6 gene was upregulated in a time- and dose-dependent manner after the treatment with demethylation reagent 5-azadeoxycytidine. The methylation of IRF6 gene promoter in the renal tissues of KIRC patients is closely correlated with the OS. Cg12034118 may provide a promising biomarker for laboratory detection, and its high methylation rate has certain reference value for the prognosis.

A suitable and efficient optimization system for the culture of Chlamydia trachomatis in adult inclusion conjunctivitis.

The prevalence of Chlamydia trachomatis infection in the genitourinary tract is increasing, with an annual rise of 9 million cases. Individuals afflicted with these infections are at a heightened risk of developing adult inclusive conjunctivitis (AIC), which is commonly recognized as the ocular manifestation of this sexually transmitted infection. Despite its significant clinical implications, the lack of distinctive symptoms and the overlap with other ocular conditions often lead to underdiagnosis or misdiagnosis of AIC associated with C. trachomatis infection. Here, we established six distinct C. trachomatis culture cell lines, specifically highlighting the MA104 N*V cell line that exhibited diminished expression of interferon regulatory factor 3 (IRF3) and signal transducer and activator of transcription 1 (STAT1), resulting in reduced interferons. Infected MA104 N*V cells displayed the highest count of intracytoplasmic inclusions detected through immunofluorescence staining, peaking at 48 h postinfection. Subsequently, MA104 N*V cells were employed for clinical screening in adult patients diagnosed with AIC. Among the evaluated cohort of 20 patients, quantitative PCR (qPCR) testing revealed positive results in seven individuals, indicating the presence of C. trachomatis infection. Furthermore, the MA104 N*V cell cultures derived from these infected patients demonstrated successful cultivation and replication of the pathogen, confirming its viability and infectivity. Molecular genotyping identified four distinct urogenital serovars, with serovar D being the most prevalent (4/7), followed by E (1/7), F (1/7), and Ia (1/7). This novel cellular model contributes to studies on C. trachomatis pathogenesis, molecular mechanisms, and host-pathogen interactions both in vitro and in vivo. It also aids in acquiring clinically relevant strains critical for advancing diagnostics, treatments, and vaccines against C. trachomatis.

IFNT-induced IRF1 enhances bovine endometrial receptivity by transactivating LIFR

Interferon-τ (IFN-τ) participates in the establishment of endometrial receptivity in ruminants. However, the precise mechanisms by which IFN-τ establishes bovine endometrial receptivity remain largely unknown. Interferon regulatory factor 1 (IRF1) is a classical interferon-stimulated gene (ISG) induced by type I interferon, including IFN-τ. Leukemia inhibitory factor receptor (LIFR) is a transmembrane receptor for leukemia inhibitory factor (LIF), which is a key factor in regulating embryo implantation in mammals. This study aimed to investigate the roles of IRF1 and LIFR in the regulation of bovine endometrial receptivity by IFN-τ. In vivo, we found IRF1 and LIFR were upregulated in the bovine endometrial luminal epithelium on Day 18 of pregnancy compared to Day 18 of the estrous cycle. In vitro, IFN-τ could upregulate IRF1, LIFR, and endometrial receptivity markers (LIF, HOXA10, ITGAV, and ITGB3) expression, downregulate E-cadherin expression and reduce the quantity of microvilli of bovine endometrial epithelial cells (bEECs). Overexpression of IRF1 had similar effects to IFN-τ on endometrial receptivity, and interference of LIFR could block these effects, suggesting the positive effects of IRF1 on endometrial receptivity were mediated by LIFR. Dual luciferase reporter assay verified that IRF1 could transactivate LIFR transcription by binding to its promoter. In conclusion, IFN-τ can induce IRF1 expression in bovine endometrial epithelial cells, and IRF1 upregulates LIFR expression by binding to LIFR promoter, contributing to the enhancement of bovine endometrial receptivity.

Amino Acids Fueling Fibroblast-Like Synoviocyte Activation and Arthritis By Regulating Chemokine Expression and Leukocyte Migration.

We analyzed the impact of amino acid (AA) availability on the inflammatory response in arthritis. We stimulated rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLSs) with tumor necrosis factor (TNF) in the presence or absence of proteinogenic AAs and measured their response by QuantSeq 3' messenger RNA sequencing, quantitative polymerase chain reaction, and enzyme-linked immunosorbent assay. Signal transduction events were determined by Western blot. We performed K/BxN serum transfer arthritis in mice receiving a normal and a low-protein diet and analyzed arthritis clinically and histologically. Deprivation of AAs decreased the expression of a specific subset of genes, including the chemokines CXCL10, CCL2, and CCL5 in TNF-stimulated FLSs. Mechanistically, the presence of AAs was required for the TNF-induced activation of an interferon regulatory factor 1 (IRF1)-STAT1 signaling circuit that drives the expression of chemotactic factors. The expression of IRF1 and the IRF1-dependent gene set in FLSs was highly correlated with the presence of inflammatory cells in human RA, emphasizing the important role of this AA-dependent pathway in inflammatory cell recruitment to the synovial tissue. Finally, we show that mice receiving a low-protein diet expressed less IRF1 in the inflamed synovium and consequently developed reduced clinical and histologic signs of arthritis. AA deprivation reduces the severity of arthritis by suppressing the expression of IRF1-STAT1-driven chemokines, which are crucial for leukocyte recruitment to the arthritic joint. Overall, our study provides novel insights into critical determinants of inflammatory arthritis and may pave the way for dietary intervention trials in RA.

The neuroprotective effect of near infrared light therapy in aged mice with postoperative neurocognitive disorder by upregulating IRF7

BackgroundPostoperative neurocognitive disorder (PND) is a common central nervous system complication after undergoing surgery and anesthesia especially in elderly patients, while the therapeutic options are very limited. This study was carried out to investigate the beneficial effects of transcranial near infrared light (NIRL) which was employed to the treatment of PND and propose the involved mechanisms. MethodsThe PND mice were established through left carotid artery exposure under isoflurane anesthesia and received transcranial NIRL treatment. Behavioral testing was performed to evaluate the cognitive function of PND mice after transcranial NIRL therapy. Changes in the transcriptomic profiles of prefrontal cortex (PFC) and hippocampus (HP) were identified by next generation sequencing (NGS), and the molecular mechanisms involved were examined by both in vivo mouse model and in vitro cell culture studies. ResultsWe found that transcranial NIRL therapy effectively ameliorated learning and memory deficit induced by anesthesia and surgery in aged mice. Specifically, we identified down-regulation of interferon regulatory factor 7 (IRF7) in the brains of PND mice that was mechanistically associated with increased pro-inflammatory M1 phenotype of microglia and elevated neuroinflammatory. NIRL treatment produced protective effects through the upregulation of IRF7 expression and reversing microglial phenotypes from pro-inflammatory to neuroprotective, resulting in reduced brain damage and improved cognitive function in PND mice. ConclusionOur results indicate that transcranial NIRL is an effective and safe therapy for PND via alleviating neuroinflammation, and IRF7 plays a key transcription factor in regulating the M1-to-M2 switch of microglia.

Inhibition of Rho-Associated Coiled-Coil Containing Protein Kinases with Belumosudil Mesylate Shows Anti-Tumor and Immune Modulatory Properties in Models of Multiple Myeloma

Background: Myeloma patient outcomes have continuously improved with the introduction of proteasome inhibitors (PIs), immunomodulatory drugs (IMiDs), monoclonal antibodies (mAbs) recognizing cell surface proteins and, most recently, B-cell maturation antigen (BCMA)-targeted bispecifics and chimeric antigen receptor (CAR)-guided T-cells. However, the prognosis in triple-class refractory (TCR) disease that is refractory to PIs, IMiDs, and CD38 mAbs remains poor and, since BCMA-targeted CAR-Ts and bispecifics are not curative, quadruple-class refractory (QCR) disease that progresses during or after these therapies is an emerging challenge, indicating a persistent need for novel agents. Rho-associated coiled-coil containing protein kinases (ROCK1 and ROCK2) may represent novel potential targets since ROCK1 has been associated with a plasma cell leukemia-like transcriptional profile and as a myeloma dependency. Moreover, ROCK2 mediates expression of Interferon regulatory factor 4 (IRF4) and c-MYC, as well as Th17 responses that may contribute to an immune suppressive microenvironment. Methods: To examine the central hypothesis that targeting ROCK1/2 could be a novel and effective approach to myeloma therapy, we performed pre-clinical studies with the ROCK1/2 inhibitor belumosudil mesylate using drug-naïve and drug-resistant myeloma cell lines in vitro. Also, in vivo studies with immune correlates were performed in the Vk*MYC immune-competent syngeneic myeloma model, which has been reported to have a 67% positive predictive value for clinical efficacy in humans. Results: Belumosudil mesylate as a single agent reduced the viability of a panel of myeloma cell lines representing different molecular subtypes in a time- and concentration-dependent manner, with median inhibitory concentrations from 618 to 1,114 nM at 72 hours. These represent clinically relevant concentrations as the mean steady state C max for belumosudil mesylate given with food in studies of chronic graft-versus-host disease patients was 4.36 mM. Staining of myeloma cells with Annexin V and propidium iodide (PI) demonstrated that belumosudil induced appearance first of Annexin +/PI - (early apoptotic) cells, followed later by Annexin +/PI + (late apoptotic) and Annexin -/PI + (necrotic) cells, in a concentration-dependent manner. Studies of myeloma resistance mechanisms showed belumosudil largely overcame adhesion-mediated drug resistance, and was active against MM.1S and RPMI 8226 cells that are considered daratumumab-resistant. Moreover, belumosudil showed equal or even, in some cases, higher potency against bortezomib-, carfilzomib-, dexamethasone-, iberdomide-, lenalidomide-, melphalan-, and mezigdomide-resistant cell lines compared to their drug-naïve counterparts. Belumosudil induced in vitro killing of myeloma cells in a time- and dose-dependent manner independent of the presence of natural killer (NK) cells. Notably, when used in combination with the CD38 mAb isatuximab and in the presence of NK cells, belumosudil enhanced myeloma cell killing and prevented isatuximab-induced loss of CD38 expression. Mechanistically, belumosudil induced a reduction in c-MYC, IRF4, and phospho-STAT3 levels, consistent with its action on the IRF4/c-MYC axis. Dosing of belumosudil in vivo at 50 mg/kg twice weekly for three weeks in Vk*MYC mice with paraprotein levels consistent with active myeloma produced a consistent decline in disease burden as determined by serum protein electrophoresis monitoring ( A) compared to vehicle controls. NanoString analysis of tumor samples from these mice ( B) found down-regulation of XBP1 (X-box binding protein 1) and SLAMF7 (Signaling lymphocytic activation molecule family member 7), consistent with the anti-tumor efficacy of belumosudil mesylate. In addition, up-regulation was seen of c- JUN and c- FOS, consistent with promotion of T-cell activation and clonal expansion, as well as effector differentiation of antigen-activated CD8 + T cells. Conclusions: These pre-clinical in vitro and in vivo data support the hypothesis that targeting of ROCK1 and ROCK2 with belumosudil mesylate may be a promising strategy for relapsed/refractory myeloma and provide a rationale for its translation to the clinic. Currently, clinical trials of belumosudil are planned.

Tuning the Innate Immune Multiple Myeloma Microenvironment By Modulating IRF4

The tumor microenvironment (TME) has emerged as a key contributor to cancer progression in numerous solid tumors as well as blood cancers that evolve in the bone marrow (BM). In the context of multiple myeloma (MM), survival of malignant plasma cells is supported by interactions with the BM microenvironment, which is comprised of various components including cells, extracellular matrix, and soluble factors. Therefore, the development of strategies that specifically target the MM BM microenvironment is crucial for improving current disease prognosis. Within the BM niche, M2-like myeloma-associated macrophages (MAMs) play a significant role in cancer cell survival and progression and have been implicated in producing an immune-suppressive TME by generating inflammatory mediators, growth factors, cytokines, chemokines, etc. Macrophages exhibit plasticity with regard to their polarization state, which enable them to dynamically respond and adapt to different environmental stimuli in order to fulfill important immune functions. Thus, the reprogramming of M2-like, pro-tumoral MAMs toward a M1-like, anti-tumoral phenotype represents a promising therapeutic strategy. In MM, inflammatory and anti-viral pathways promote disease progression. Interferon regulatory factor 4 (IRF4) is a transcription factor that has previously been shown to promote MM progression through enhanced survival of malignant plasma cells. Under physiological conditions, IRF4 also regulates macrophage polarization, where its expression drives alternative activation of macrophages primarily to M2-like (anti-inflammatory) phenotypes. However, the extent to which IRF4 activation or direct inhibition governs the polarization status and pro-tumoral activity of macrophages in the MM microenvironment has not been fully elucidated. In this study, we sought to further explore the role of IRF4 in macrophage polarization and investigated the potential of modulating IRF4 expression to reprogram myeloma-associated macrophages (MAMs) as a novel therapeutic strategy. First, we developed myeloma-tailored macrophage polarization models using primary human peripheral blood-derived monocytes and the THP1 monocyte cell line. Our M1 and M2 polarization models utilize a customized cocktail of myeloid growth factors and cytokines, supplemented with interleukin (IL)-6 to mimic the IL-6-enriched MM microenvironment. Characterization of M1 (CD80, TNFα, and CXCL10) and M2 (MRC1, FN1, and CCL22) markers confirmed the presence of distinct macrophage subpopulations, as well as upregulated expression of IRF4 in M2-like progeny and IRF5 in M1-like macrophages. To investigate the extent to which IRF4 activation or direct inhibition governs macrophage polarization and function, overexpression and knockdown experiments on macrophages were performed in vitro, along with whole transcriptome sequencing of IRF4-overexpressing THP1 monocytes compared to vector-transduced controls. Gene expression analyses revealed that genetic down-modulation of IRF4 in M2-like macrophages shifts the polarization status toward a hybrid phenotype that displays enhanced M1-like properties, while still retaining M2-like characteristics. This shift in polarization state was further amplified when monocytes were transduced to overexpress IRF4 prior to differentiation into M2-like macrophages and subsequent IRF4 knockdown, suggesting that IRF4 plays a key regulatory role in tuning macrophage polarization. Taken together, our preclinical findings provide groundwork for future studies on MAMs and suggest that an IRF4-mediated macrophage reprogramming strategy is a promising therapeutic approach for patients with MM and a variety of other malignancies typified by an anti-inflammatory TME.

IL-33 Suppresses the Progression of Atherosclerosis via the ERK1/2-IRF1-VCAM-1 Pathway.

This study was designed to explore the effects of interleukin 33 (IL-33) on the progression of atherosclerosis and the possible mechanism. The adhesion assay was performed on isolated peripheral blood mononuclear cells (PBMCs) and human umbilical vein endothelial cells (HUVEC). The expression of proteins and messenger RNA (mRNA) were detected by western blot and quantitative real-time polymerase chain reaction (PCR), including intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and P-selectin. The effect of IL-33 on the interaction of growth stimulation expressed gene 2 (ST2) with myeloid differentiation factor 88 (MyD88) and interleukin-1 receptor-associated kinase (IRAK) 1/4 were investigated using co-immunoprecipitation assay. An apolipoprotein (Apo) E-/- mice model was used to confirm the effect of IL-33 on atherosclerosis progression. Area of plaques was recorded by hematoxylin-eosin (H&E) staining. The severity of atherosclerosis plaque was evaluated using immunohistochemistry assay, and lipid accumulation was measured by an oil red O staining. In contrast, western blot was performed to detect the expression levels of VCAM-1, extracellular signal-regulated kinase (ERK) 1/2, and interferon regulatory factor 1 (IRF1). Our study observed that IL-33 suppressed cell adhesion and the expression of VCAM-1 in tumor necrosis factor-α (TNF-α) exposed HUVEC. Moreover, the addition of IL-33 significantly inhibited the expression of IRF1 and the binding level of IRF1 to VCAM-1 and also promoted the phosphorylation level of IRAK1/4 and ERK1/2 compared to TNF-α-stimulated HUVEC. The ST2 neutralizing antibody or ERK pathway inhibitor SCH772984 reversed the regulatory effects of IL-33 on HUVEC, suggesting that IL-33 suppressed IRF1 and VCAM-1 dependent on binding to ST2 and activating the ERK1/2 signaling pathway. Further investigation in vivo confirmed that IL-33 decreased the expressions of IRF1 and VCAM-1 by activating the phosphorylation of ERK1/2 in the thoracic aorta of Apo E-/- mice. In conclusion, our results demonstrated that IL-33 plays a protective role in the progression of atherosclerosis by inhibiting cell adhesion via the ERK1/2-IRF1-VCAM-1 pathway. This study may provide a potential therapeutic way to prevent the development of atherosclerosis.

ONO-7018, a First-in-Class MALT1 Inhibitor, Provides Novel Therapeutic Strategies for B Cell Malignancies: Overcoming BTK Inhibitor Acquired Resistance and Enhancing the Antitumor Effect of BTK Inhibitors

Introduction: B cell receptor (BCR) signaling plays a crucial role in promoting survival and growth in B cell malignancies, and several compounds targeting the signaling pathways have been approved. Bruton's tyrosine kinase (BTK) is a key molecule in BCR signaling pathways and BTK inhibitors are one of the essential treatment options for chronic lymphocytic leukemia (CLL) and certain subtypes of B cell Non-Hodgkin Lymphoma. However, there are issues that acquired resistance mutations in the BTK kinase domain and gain of function mutation of its downstream molecules, which lead to BTK inhibitor resistance and limit its efficacy. For example, BTK C481S mutation is well known as a resistant mutation to covalent BTK inhibitors and several mutations of BTK (such as T474I and L528W mutation) have recently been reported in relapsed or refractory CLL patients with acquired resistance to pirtobrutinib, a non-covalent BTK inhibitor. In addition, there are unmet medical needs to improve the duration of response of current standard therapies including BTK inhibitors for B cell malignancies. Therefore, therapeutic strategies to overcome the BTK inhibitor resistance and novel combination therapies to enhance the efficacy of BTK inhibitors are required. Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) is known as a key growth and survival regulator in BCR signaling and an emerging therapeutic target. ONO-7018 (formerly known as CTX-177), a potent and selective MALT1 inhibitor, demonstrated preclinical efficacy in several lymphoma models. Aim: Firstly, to explore the potential of ONO-7018 to overcome BTK inhibitor acquired resistance, we tested the antitumor effects of ONO-7018 on cell lines harboring BTK inhibitor-resistant mutations. Secondly, we examined the combination antitumor efficacy of ONO-7018 and tirabrutinib, a second generation BTK inhibitor in vitro and in vivo. Methods: Diffuse large B-cell lymphoma (DLBCL) cell lines harboring BTK inhibitor-resistant mutations (BTK T474I, C481S, or L528: PLCG2 R665W) were generated by continuous passage with BTK inhibitor or CRISPR-Cas9 gene editing followed by single cell cloning. In in vivo study, human DLBCL cell line, TMD8 cells were subcutaneously implanted in immune deficient mice. ONO-7018 and tirabrutinib were orally administered to the mice twice a day. Results: Anti-proliferative activities of ONO-7018 on these BTK inhibitor resistant cell lines were evaluated in vitro. ONO-7018 inhibited the proliferation of these cells with comparable IC 50 values to the parent cell line. Anti-proliferative activities of ONO-7018 alone or in combination with tirabrutinib on DLBCL and mantle cell lymphoma (MCL) cell lines were evaluated in vitro. ONO-7018 exhibited synergistic effects with tirabrutinib in both DLBCL and MCL cell lines. In addition, combination treatment of ONO-7018 with tirabrutinib strongly inhibited the expression of interferon regulatory factor 4, which is a downstream signal molecule in BCR signaling pathways, compared to their single treatment. We then evaluated the antitumor effects of ONO-7018 in combination with tirabrutinib in the xenograft model. Combination treatment of ONO-7018 with tirabrutinib showed dramatic antitumor effect with tumor regression. These results suggest that ONO-7018 could demonstrate antitumor effect on patients with B cell malignancies harboring BTK inhibitor acquired resistant mutations and synergistic antitumor effect with BTK inhibitors in clinic. Conclusion: ONO-7018 would provide novel therapeutic strategies to overcome the BTK inhibitor acquired resistance and enhance the antitumor effect of BTK inhibitors in clinic. Phase 1 study of ONO-7018 (NCT05515406) is currently ongoing.

IRF1 regulates self-renewal and stress responsiveness to support hematopoietic stem cell maintenance.

Hematopoietic stem cells (HSCs) are tightly controlled to maintain a balance between blood cell production and self-renewal. While inflammation-related signaling is a critical regulator of HSC activity, the underlying mechanisms and the precise functions of specific factors under steady-state and stress conditions remain incompletely understood. We investigated the role of interferon regulatory factor 1 (IRF1), a transcription factor that is affected by multiple inflammatory stimuli, in HSC regulation. Our findings demonstrate that the loss of IRF1 from mouse HSCs significantly impairs self-renewal, increases stress-induced proliferation, and confers resistance to apoptosis. In addition, given the frequent abnormal expression of IRF1 in leukemia, we explored the potential of IRF1 expression level as a stratification marker for human acute myeloid leukemia. We show that IRF1-based stratification identifies distinct cancer-related signatures in patient subgroups. These findings establish IRF1 as a pivotal HSC controller and provide previously unknown insights into HSC regulation, with potential implications to IRF1 functions in the context of leukemia.

Analysis of structure–activity relationship of indol-3-yl-N-phenylcarbamic amides as potent STING inhibitors

A structure–activity relationship (SAR) study of stimulator of interferon gene (STING) inhibition was performed using a series of indol-3-yl-N-phenylcarbamic amides and indol-2-yl-N-phenylcarbamic amides. Among these analogs, compounds 10, 13, 15, 19, and 21 inhibited the phosphorylation of STING and interferon regulatory factor 3 (IRF3) to a greater extent than the reference compound, H-151. All five analogs showed stronger STING inhibition than H-151 on the 2′,3′-cyclic GMP-AMP-induced expression of interferon regulatory factors (IRFs) in a STINGR232 knock-in THP-1 reporter cell line. The half-maximal inhibitory concentration of the most potent compound, 21, was 11.5 nM. The molecular docking analysis of compound 21 and STING combined with the SAR study suggested that the meta- and para-positions of the benzene ring of the phenylcarbamic amide moiety could be structurally modified by introducing halides or alkyl substituents.