Cytokine Signaling Pathways Research Articles

Unlocking JAKi Potential: Redefining Axial Spondyloarthritis Treatment Paradigms

Axial spondyloarthritis (axSpA) is a chronic inflammatory disease that primarily affects the axial skeleton, encompassing both radiographic and non-radiographic forms. The disease is characterized by chronic back pain and spinal stiffness, which are the most common symptoms. However, it often includes peripheral manifestations and extra-musculoskeletal symptoms as well, making the clinical presentation quite diverse. JAK inhibitors work by blocking the intracellular signaling pathways of several proinflammatory cytokines, which play a crucial role in the pathogenesis of inflammatory diseases. Given their proven efficacy in managing rheumatoid arthritis and their ability to mediate the signaling of cytokines involved in the pathogenesis of axSpA, JAK inhibitors have been the subject of numerous clinical trials in this context. The Assessment of SpondyloArthritis international Society and the European Alliance of Associations for Rheumatology (ASAS-EULAR) joint recommendations provide comprehensive guidance for the management of axSpA in 2022. The guideline introduced the use of Janus kinase inhibitors (JAKi), as a completely new drug class in the treatment landscape, with efficacy demonstrated in radiographic axSpA. JAKi was recommended, as a means to treatment intensification, in axSpA patients with persistently high disease activity despite conventional treatment. Since then, more trial data results have been released, showing efficacy in non-radiographic axSpA population as well. In this lecture, Professor Östör will delve into the critical role of shared decision-making in the management of axial spondyloarthritis. He will emphasize the importance of tailoring treatment plans to individual patient preferences and clinical profiles, a practice that is increasingly recognized as vital in achieving optimal outcomes. By examining current international guidelines and the latest study data, Professor Östör will provide a comprehensive overview of the benefit-risk profile of Upadacitinib in the treatment of axSpA. Professor Östör will also share his clinical experience with Upadacitinib, discussing various case studies to illustrate its application in real-world settings. This will include an exploration of how patient preferences and clinical profiles can influence treatment decisions, ensuring a personalized approach to managing this complex disease.

Cytokine Modification of Adoptive Chimeric Antigen Receptor Immunotherapy for Glioblastoma.

Chimeric antigen receptor (CAR) cell-based therapies have demonstrated limited success in solid tumors, including glioblastoma (GBM). GBMs exhibit high heterogeneity and create an immunosuppressive tumor microenvironment (TME). In addition, other challenges exist for CAR therapy, including trafficking and infiltration into the tumor site, proliferation, persistence of CARs once in the tumor, and reduced functionality, such as suboptimal cytokine production. Cytokine modification is of interest, as one can enhance therapy efficacy and minimize off-target toxicity by directly combining CAR therapy with cytokines, antibodies, or oncolytic viruses that alter cytokine response pathways. Alternatively, one can genetically modify CAR T-cells or CAR NK-cells to secrete cytokines or express cytokines or cytokine receptors. Finally, CARs can be genetically altered to augment or suppress intracellular cytokine signaling pathways for a more direct approach. Codelivery of cytokines with CARs is the most straightforward method, but it has associated toxicity. Alternatively, combining CAR therapy with antibodies (e.g., anti-IL-6, anti-PD1, and anti-VEGF) or oncolytic viruses has enhanced CAR cell infiltration into GBM tumors and provided proinflammatory signals to the TME. CAR T- or NK-cells secreting cytokines (e.g., IL-12, IL-15, and IL-18) have shown improved efficacy within multiple GBM subtypes. Likewise, expressing cytokine-modulating receptors in CAR cells that promote or inhibit cytokine signaling has enhanced their activity. Finally, gene editing approaches are actively being pursued to directly influence immune signaling pathways in CAR cells. In this review, we summarize these cytokine modification methods and highlight any existing gaps in the hope of catalyzing an improved generation of CAR-based therapies for glioblastoma.

Genome-wide selection signatures address trait specific candidate genes in cattle indigenous to arid regions of India

The peculiarity of Indian cattle lies in milk quality, resistance to diseases and stressors as well as adaptability. The investigation addressed selection signatures in Gir and Tharparkar cattle, belonging to arid ecotypes of India. Double digest restriction-site associated DNA sequencing (ddRAD-seq) yielded nearly 26 million high-quality reads from unrelated seven Gir and seven Tharparkar cows. In all, 19,127 high-quality SNPs were processed for selection signature analysis. An approach involving within-population composite likelihood ratio (CLR) statistics and between-population F ST statistics was used to capture selection signatures within and between the breeds, respectively. A total of 191 selection signatures were addressed using CLR and F ST approaches. Selection signatures overlapping 86 and 73 genes were detected as Gir- and Tharparkar-specific, respectively. Notably, genes related to production (CACNA1D, GHRHR), reproduction (ESR1, RBMS3), immunity (NOSTRIN, IL12B) and adaptation (ADAM22, ASL) were annotated to selection signatures. Gene pathway analysis revealed genes in insulin/IGF pathway for milk production, gonadotropin releasing hormone pathway for reproduction, Wnt signalling pathway and chemokine and cytokine signalling pathway for adaptation. This is the first study where selection signatures are identified using ddRAD-seq in indicine cattle breeds. The study shall help in conservation and leveraging genetic improvements in Gir and Tharparkar cattle.

Resident immune responses to spinal cord injury: role of astrocytes and microglia.

Spinal cord injury can be traumatic or non-traumatic in origin, with the latter rising in incidence and prevalence with the aging demographics of our society. Moreover, as the global population ages, individuals with co-existent degenerative spinal pathology comprise a growing number of traumatic spinal cord injury cases, especially involving the cervical spinal cord. This makes recovery and treatment approaches particularly challenging as age and comorbidities may limit regenerative capacity. For these reasons, it is critical to better understand the complex milieu of spinal cord injury lesion pathobiology and the ensuing inflammatory response. This review discusses microglia-specific purinergic and cytokine signaling pathways, as well as microglial modulation of synaptic stability and plasticity after injury. Further, we evaluate the role of astrocytes in neurotransmission and calcium signaling, as well as their border-forming response to neural lesions. Both the inflammatory and reparative roles of these cells have eluded our complete understanding and remain key therapeutic targets due to their extensive structural and functional roles in the nervous system. Recent advances have shed light on the roles of glia in neurotransmission and reparative injury responses that will change how interventions are directed. Understanding key processes and existing knowledge gaps will allow future research to effectively target these cells and harness their regenerative potential.

Dehydrocorydaline alleviates sleep deprivation-induced persistent postoperative pain in adolescent mice through inhibiting microglial P2Y12 receptor expression in the spinal cord.

During adolescence, a second period of central nervous system (CNS) plasticity that follows the fetal period, which involves sleep deprivation (SD), becomes apparent. SD during adolescence may result in abnormal development of neural circuits, causing imbalance in neuronal excitation and inhibition, which not only results in pain, but increases the chances of developing emotion disorders in adulthood, such as anxiety and depression. The quantity of surgeries during adolescence is also consistently on the rise, yet the impact and underlying mechanism of preoperative SD on postoperative pain remain unexplored. This study demonstrates that preoperative SD induces upregulation of the P2Y12 receptor, which is exclusively expressed on spinal microglia, and phosphorylation of its downstream signaling pathway p38Mitogen-activated protein/Nuclear transcription factor-κB (p38MAPK/NF-κB)in spinal microglia, thereby promoting microglia activation and microglial transformation into the proinflammatory M1 phenotype, resulting in increased expression of proinflammatory cytokines that exacerbate persisting postoperative incisional pain in adolescent mice. Both intrathecal minocycline (a microglia activation inhibitor) and MRS2395 (a P2Y12 receptor blocker) effectively suppressed microglial activation and proinflammatory cytokine expression. Interestingly, supplementation with dehydrocorydaline (DHC), an extract of Rhizoma Corydalis, inhibited the P2Y12/p38MAPK/NF-κB signaling pathway, microglia activation, and expression of pro-inflammatory cytokines in the model mice. Taken together, the results indicate that the P2Y12 receptor and microglial activation are important factors in persistent postoperative pain caused by preoperative SD in adolescent mice and that DHC has analgesic effects by acting on these targets.

Abstract C174: MerTK as a putative biomarker for treatment response in triple negative breast cancer

Abstract Triple Negative Breast Cancer (TNBC) is characterized by the lack of estrogen, progesterone, and HER2 receptors. TNBC is a highly invasive subtype of breast cancer, with limited targeted therapies. Current therapeutic options for TNBC patients include surgery, chemotherapy, and radiation but disease recurrence is common. Anti-PD-1 and anti-PD-L1 therapeutic antibodies have been approved for the treatment of TNBC in combination with chemotherapy, but biomarkers for response and new therapeutic targets are still needed. The receptor tyrosine kinase MerTK has been associated with many types of cancer and is highly expressed in TNBC. To investigate the role of MerTK in TNBC, we created a syngeneic cell line (4T1) overexpressing MerTK to evaluate tumor growth, the tumor immune microenvironment (TIME), and response to immune checkpoint inhibitors (ICIs) in vivo. First, we tested the effect of MerTK on tumor growth in vivo by inoculating the 4T1 cell line, which has very low MerTK expression, and two 4T1 MerTK-overexpressing clones into eight Balb/c mice per group subcutaneously on the flank. We measured tumor growth over 27 days and found that MerTK-overexpressing tumors exhibited a statistically significant delay in tumor growth when compared to the parental 4T1 tumors. This indicates that MerTK may be inducing a hotter TIME in MerTK overexpressing tumors, as this slowed growth was not observed in athymic nude mice. To test this hypothesis, we evaluated the TIME of these tumors by flow cytometry, which revealed robust expansion of macrophage, natural killer cell and T cell compartments, and reduction in tumor-infiltrating myeloid derived suppressor cells. These results suggest that MerTK is playing a yet unidentified role in promoting infiltration of anti-tumor immune cells into the TIME and excluding pro-tumor immune cells. Further, we saw robust changes in genes associated with cytokine signaling pathways including CD38, CCL2 and CCL5 by Nanostring nCounter analysis in MerTK-overexpressing 4T1 cells compared to parental 4T1 cells. Finally, there is evidence that hotter tumors respond well to ICI therapy, and we hypothesized that MerTK-overexpressing tumors would exhibit a significant response to ICI when compared to parental 4T1 tumors. Indeed, our data showed a robust and durable response marked by a significant reduction in tumor growth in the MerTK-overexpressing tumors compared to parental 4T1 tumors in Balb/c mice when treated with either anti-PD-L1 or anti-CTLA-4 antibodies. Together, these results suggest that MerTK overexpression could serve as a predictive biomarker for response to immune therapy in patients with TNBC. Citation Format: Bridget E Mehall, Mari Iida, Kourtney L Kostecki, Jillian M Adams, Deric L Wheeler. MerTK as a putative biomarker for treatment response in triple negative breast cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C174.

PIM kinases regulate early human Th17 cell differentiation

The serine/threonine-specific Moloney murine leukemia virus (PIM) kinase family (i.e., PIM1, PIM2, and PIM3) has been extensively studied in tumorigenesis. PIM kinases are downstream of several cytokine signaling pathways that drive immune-mediated diseases. Uncontrolled T helper 17 (Th17) cell activation has been associated with the pathogenesis of autoimmunity. However, the detailed molecular function of PIMs in human Th17 cell regulation has yet to be studied. In the present study, we comprehensively investigated how the three PIMs simultaneously alter transcriptional gene regulation during early human Th17 cell differentiation. By combining PIM triple knockdown with bulk and scRNA-seq approaches, we found that PIM deficiency promotes the early expression of key Th17-related genes while suppressing Th1-lineage genes. Further, PIMs modulate Th cell signaling, potentially via STAT1 and STAT3. Overall, our study highlights the inhibitory role of PIMs in human Th17 cell differentiation, thereby suggesting their association with autoimmune phenotypes.

Single-cell transcriptomics of peripheral blood mononuclear cells indicates impaired immune and inflammatory responses in alcohol-associated hepatitis

Alcohol-associated hepatitis (AH) is often diagnosed at advanced stages, and severe AH usually carries poor prognosis and high short-term mortality. In addition, it is challenging to understand the molecular mechanisms of immune dysregulation and inflammation in AH due to the cellular complexity and heterogeneity. Using single-cell RNA sequencing, previous studies found that AH causes dysfunctional innate immune response in monocytes, involving activation of pattern recognition receptors (PRRs) and cytokine signaling pathways. To better understand the coordinated systemic immune response in AH patients, we performed combined single-cell transcriptome, cell-surface protein, and lymphocyte antigen receptor analysis of peripheral blood mononuclear cell (PBMC) samples. Our results showed inflammatory cytokines and chemokines were highly expressed in AH, including IL-2, IL-32, CXC3R1 and CXCL16 in monocytes and NK cells, whereas HLA-DR genes were reduced in monocytes. In addition, we also found altered differentiation of T-helper cells (TH1 and TH17), which could further lead to neutrophil recruitment and macrophage activation. Lastly, our results also suggest impaired NK-cell activation and differentiation in AH with reduced gene expression of KLRC2 and increased gene expression of KLRG1. Our findings indicate different mechanisms may be involved in impaired immune and inflammatory responses for the cellular subtypes of the PBMCs in AH.

Leaves and pseudostems extract of Curcuma longa attenuates immunoglobulin E/bovine serum albumin-stimulated bone marrow-derived cultured mast cell activation and passive cutaneous anaphylaxis in BALB/c mice

Ethnopharmacological relevanceCurcuma longa, known as turmeric, is an herbaceous perennial plant belonging to the genus Curcuma. It is dispersed throughout tropical and subtropical regions worldwide. Since ancient times, turmeric has been used as an ethnomedicinal plant in the Ayurvedic system, particularly in Asian countries. Rhizomes of turmeric possess several pharmacological properties that give high value as a medicinal remedy for treating a range of conditions, including inflammation, pain, allergies, and digestive issues. Moreover, turmeric leaves and pseudostems also contain a variety of health-enhancing secondary metabolites, such as curcumin, flavonoids, and other phenolic compounds, which exhibit anti-inflammatory, antitumor, antibacterial, and antioxidant properties. Aim of the studyAllergic diseases are a group of immune-mediated disorders mainly caused by an immunoglobulin E (IgE)-dependent immunological response to an innocuous allergen. Therefore, this study aimed to investigate the effect of leaves and pseudostems extract of turmeric (TLSWE-8510) on IgE/bovine serum albumin (BSA)-stimulated allergic responses in mouse bone marrow-derived cultured mast cells (BMCMCs) and passive cutaneous anaphylaxis (PCA) in BALB/c mice. Materials and methodsThe effect of TLSWE-8510 on mast cell degranulation has been evaluated by investigating the release of β-hexosaminidase and histamine in IgE/BSA-stimulated BMCMCs. Additionally, anti-allergic properties of TLSWE-8510 on IgE/BSA-stimulated BMCMCs were investigated using suppression of nuclear factor-kappa B (NF-κB), and spleen tyrosine kinase (Syk)-linker for T-cell activation (LAT)-extracellular-signal-regulated kinase (ERK)-GRB2 associated binding protein 2 (Gab2) signaling pathway and downregulation of allergy-related cytokines and chemokines expression. Furthermore, in vivo, studies were conducted using IgE-mediated PCA in BALB/c mice. ResultsTLSWE-8510 treatment significantly inhibited the degranulation of IgE/BSA-stimulated BMCMCs by inhibiting the release of β-hexosaminidase and histamine dose-dependently. Additionally, TLSWE-8510 reduced the expression of high-affinity IgE receptors (Fc epsilon receptor I-FcεRI) on the surface of BMCMCs and the binding of IgE to FcεRI. Besides, the expression of cytokines and chemokines is triggered by IgE/BSA stimulation via activating the allergy-related signaling pathways. TLSWE-8510 dose-dependently downregulated the mRNA expression and the production of allergy-related cytokines (interleukin (IL)-1β, IL-3, IL-4, IL-5, IL-6, IL-13, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ), and chemokines (thymus and activation-regulated chemokine (TARC), and regulated upon activation, normal T cell expressed and secreted (RANTES)) by regulating the phosphorylation of downstream signaling molecules, NF-κB, and Syk, LAT, ERK and Gab2 in IgE/BSA-stimulated BMCMCs. Moreover, PCA reaction in IgE/BSA-stimulated BALB/c mice ears was effectively decreased by TLSWE-8510 treatment in a dose-dependent manner. ConclusionsThese results collectively demonstrated that TLSWE-8510 suppressed mast cell degranulation by inhibiting the release of chemical mediators related to allergies. TLSWE-8510 downregulated the allergy-related cytokines and chemokines expression and phosphorylation of downstream signaling molecules in IgE/BSA-stimulated BMCMCs. Furthermore, in vivo studies with IgE-mediated PCA reaction in the BALB/c mice ears were attenuated by TLSWE-8510 treatment. These findings revealed that TLSWE-8510 has the potential as a therapeutic agent for the treatment of allergic diseases.

Transcriptomic Analyses Indicate That Pre-Leukemic B Cells from People with Monoclonal B Lymphocytosis Are Surprisingly Much Less Active Than Normal B Cells from People with Monoclonal B Lymphocytosis and from Healthy Donors

Introduction. Chronic lymphocytic leukemia (CLL) follows a stepwise developmental process, beginning in a specific group of normal B lymphocytes that undergo clonal expansion. These clonal expansions, known as monoclonal B lymphocytosis (MBL), are considered to be essential precursors of CLL based on immunologic, genetic, and epidemiologic studies. Since MBL represents the overabundance of a single CD5 + B cell, we tested how different the transcriptomes of MBL cells were from normal B cells (NBC) in people with MBL (NBC-MBL) and NBC in healthy donors (NBC-HD), expecting to identify activations pathways in the MBL cells that would explain clonal expansion. Methods. We collected and sorted specific cell populations from 13 patients with monoclonal B lymphocytosis (MBL): CD5 +CD19 +CD20 dimIgL-restricted MBL cells and CD5 -CD19 +CD20 + NBC. Additionally, we obtained CD5 -CD19 +CD20 + NBC from 13 healthy donors (NBC-HD). RNA was extracted and sequenced using SMART-Seq v4 Ultra Low Input and HiSeq platform. DESeq2 was used to analyze RNA-seq data. To identify genes with differential expression between these populations, the transcriptomes of MBL cells were compared to NBC from HD, employing a cutoff of P<0.05 and Log2fc ratio >± 1.5. We also used paired sample t-tests to compare 13 MBL cells to NBC from the same MBL patients. Significantly differentially expressed genes (DEG) resulting from these analyses were then subjected to Ingenuity Pathway Analysis (IPA). Results. When comparing the gene profiles of 13 MBL B cells to 13 NBC-HD cells, we identified 2695 DEG. Next, we compared gene profiles between MBL B cells and NBC-MBL cells obtained from 13 MBL patients, identifying 4821 genes. Using IPA, down-regulated B Cell Receptor Signaling, Cytokine Signaling, and Tumor Microenvironment Pathway were identified in both analyses. Upon overlapping the genes from these two analyses, we found that 803 genes passed the cutoff in both analyses. IPA analysis showed that 14 signaling pathways, including IL-33, IL-17, and HIF1a, were significantly downregulated in MBL cells. Moreover, the negative signaling pathway (Role of p14/p19ARF in Tumor Suppression) was upregulated in MBL cells. PDCD1 expression was significant higher in MBL (Log2Fc 6.8, P<0.0001) and 11 PDCD1 target genes were significantly regulated in MBL cells. IL-6 (Log2Fc -5.4, P<0.0001) expression was significantly lower in MBL and 47 IL-6 target genes were significantly regulated in MBL cells. Surprisingly, genes related to cell activation, proliferation and movement were largely downregulated in MBL cells. There were 2052 genes unique to the analysis MBL vs NBC-HD, IPA analysis showed several T-cell related signaling pathways e.g., T cell Exhaustion Signaling Pathway and T Lymphocyte Apoptosis were upregulated. In the 4018 genes unique in the analysis MBL vs NB-MBL. IPA results showed all the signaling pathways were downregulated in MBL. Discussion. Surprisingly, our transcriptomic analyses indicate that MBL cells are much less active than NBC from people with MBL and from HD. Specifically, we found that many cytokine signaling pathways and genes related with cell activation, proliferation and movement are downregulated in MBL cells. Also, PDCD1 is highly expressed in MBL cells, and PDCD1 target genes are also significantly regulated in MBL cells. This is consistent with the immunosuppressed features of MBL cells since interaction between PD-1 on B cells and its ligands in the microenvironment negatively influence the survival, proliferation, and immune evasion of CLL and MBL cells. Collectively, although functional studies are needed, these transcriptomic data strongly suggest that MBL cells are not in an activated state and are less active than even the NBC from the same person with MBL.

Enhanced T Cell Function of PTPN2 Deleted CAR-T Cells Comes at a Cost: PTPN2 Knockout CAR-Ts Secrete More Cytokines and Demonstrate Increased Cytotoxicity, but Exhibit More Severe CRS and Icans in a Non-Human Primate Model

Background: Despite the success of CD19 CAR-T cells (CAR-Ts) in inducing remission, relapse remains a major issue. Previous studies have shown that upregulation of negative T cell regulators can contribute to CAR-T failure, and that deletion of these regulators can enhance CAR-T efficacy. A potent negative regulator is PTPN2, which inhibits T cell function by modulating T cell receptor and cytokine signaling pathways. While murine models have shown that PTPN2-knockout T cells improve tumor control, these models fail to reliably predict clinical efficacy or toxicity. To improve this predictability, we investigated PTPN2 KO CAR-Ts in primary human cells and in a NHP model of B-cell directed CAR-T therapy. Methods: CRISPR/Cas9-mediated PTPN2 deleted (‘KO‘) human CD19 CAR-Ts were assessed for phenotype/function using cytokine secretion and cytotoxicity assays. PTPN2 KO NHP CD20 CAR-Ts were administered to lymphodepleted NHPs using our established model and a dose escalation with Level 1: 6x10 4, Level 2: 6x10 5, Level 3: 3x10 6, and Level 4: 6x10 6 CAR-T/kg, followed by assessment for CAR-T expansion, B cell aplasia, CRS and ICANS. Results: PTPN2 KO was successfully achieved in human CAR-Ts (83.8 + 5.2% deletion). IFNγ production was significantly higher in PTPN2 KO CD19 CAR-Ts vs control cells (35.1 + 0.97% vs 23.4 + 0.98%, p=0.001). In cytotoxicity assays targeting the B-ALL cell line NALM6, PTPN2 KO CD19 CAR-Ts demonstrated significantly increased killing vs control cells (for example, at E:T ratio 1:1 59.6 + 9.8% vs 42.5 + 12.9% p = 0.03). We evaluated the proliferation, efficacy, and toxicity of PTPN2 KO CD20 CAR-Ts in 5 NHPs. The CAR-T PTPN2 KO rate ranged from 63-91%. During dose escalation, no expansion of CAR-Ts was observed at dose levels 1 and 2 (n=1 for each, Fig 1). However, all animals infused at dose level 3 (n=3, 3x10 6 CAR-Ts/kg) demonstrated CAR-T expansion (max expansion 18.6 + 6.8% CAR-Ts) and B cell aplasia, and all animals developed laboratory and clinical signs of CRS and ICANS ( Fig 1). One animal at dose level 3 experienced CRS on day +4 that was severe enough to require tocilizumab, with symptoms subsequently improving. On day +6, this animal also developed ICANS, including ataxia and muscle weakness, requiring 1 dose of Dexamethasone (1mg/kg), after which symptoms resolved. One animal received dose level 4 (6x10 6 CAR-Ts/kg). This recipient displayed earlier and higher expansion of CAR-Ts (day 3: 16.7%, day 7: 56.3% CAR-Ts, Fig.1), and developed severe CRS (with elevated CRP, LDH (not shown) and Ferritin, Fig.1) and ICANS that were unresponsive to treatment with 2 doses of tocilizumab and multiple doses of dexamethasone, with this recipient meeting humane euthanasia endpoints. Importantly, previous animals (n=5) receiving 6-12 x 10 6PTPN2 expressing (WT) CAR-Ts/kg, did not develop severe CRS nor ICANS (and did not require Toci or Dex). This suggests that PTPN2 KO CAR-Ts are associated with augmented toxicities. An extensive terminal analysis of the Dose Level 4 animal was performed; pathology demonstrated widespread cerebral edema and CAR-T CNS infiltration. Because the CAR-T infusion contained both PTPN2 KO and PTPN2 WT CAR-Ts, an assessment could be made for enrichment of PTPN2 KO CAR-Ts in the blood and brain parenchyma. Compared to the infused product (63% KO CAR-Ts, Fig.2), there was enrichment for KO CAR-Ts in the blood (72% KO CAR-Ts) and the brain parenchyma (84% KO CAR-Ts, Fig 2). This suggests increased expansion and CNS infiltration of PTPN2 KO versus PTPN2 WT CAR-Ts. Flow cytometry analysis of the PTPN2 KO CAR-Ts from the blood at maximum expansion revealed high expression of the proliferation and activation markers Ki67 and GZMB (84.6%, 74.5% of all CAR-Ts). Of note, at Dose Level 3, neither CAR-T persistence (15 + 2 days vs 20 + 5 days) nor duration of B cell aplasia (41+ 5 days vs 37 + 3 days) were increased with PTPN2 KO CAR-Ts vs historic controls infused with PTPN2 WT CAR-Ts. Conclusion: We demonstrate that PTPN2 KO CAR-T cells possess increased effector function, proliferation, and CNS infiltration versus PTPN2 WT CAR-Ts. However, this enhanced efficacy was associated with increased severity of CRS and ICANS, emphasizing that careful attention to the dose of PTPN2 KO CAR-Ts would be important. These results also highlight the utility of evaluating gene-modified CAR-Ts in clinically relevant animal models, to thoroughly assess their efficacy and toxicity profiles.

Plm-102, a Next Generation FLT3 Inhibitor, Shows Potent Anti-Leukemic Activity on Resistance to Gilteritinib in FLT3 Mutated Acute Myeloid Leukemia

Introduction: Mutations in the FMS-like tyrosine kinase 3 (FLT3) gene occur in approximately 25 ~ 30% of all acute myeloid leukemia (AML) cases, with FLT3-ITD mutations having a poor prognosis. In 2022, the FDA granted approval to Gilteritinib, a 2nd generation FLT3 inhibitor, for the treatment of relapsed or refractory AML. Although Gilteritinib has demonstrated efficacy in R/R AML patients, its response duration is limited due to the development of secondary resistance. Herein, we report a next-generation drug candidate, PLM-102, which has shown promising pre-clinical results to overcome the current unmet needs of Gilteritinib resistance, including FLT3-ITD-TKD and Ras mutations in FLT3-mutated AML. Aims: The main objective of this study is to investigate the effect of PLM-102 in in-vitro and in-vivo acquired Gilteritinib-resistant models and to elucidate the underlying signaling pathways. Methods: To establish Gilteritinib-resistant cell lines, we exposed the AML cells to FLT3 ligand at 10 ng/ml and Gilteritinib at concentrations ranging from 10 nM to 100 nM for four months. Subsequently, for the following four months, we treated the cells with Gilteritinib alone at 100 nM, omitting FLT3 ligand from the treatment. Whole Exome Sequencing (WES) and mRNA sequencing were performed on primary FLT3 mutated AML cells and Gilteritinib resistance FLT3 mutated AML cells. Subsequently, cell viability assays were carried out on both parent FLT3-mutated AML cells and Gilteritinib-resistant FLT3-mutated AML cells. To assess the activities of PLM-102 at the on-target and off-target resistant mechanisms, immunoblot studies were conducted. An in vivo study was performed using a Gilteritinib-resistant MOLM-14 AML xenograft mouse model. Results: We conducted Whole Exome Sequencing on the Gilteritinib-resistant MOLM-13 and MOLM-14 cell lines. The analysis revealed the presence of KRAS G13D and FLT3-ITD-C828G mutations in the Gilteritinib-resistant MOLM-13 cell line, and NRAS G13D and FLT3-ITD-G697R mutations in the Gilteritinib-resistant MOLM-14 cell line. In the evaluation of the efficacies of Gilteritinib and PLM-102 in Gilteritinib-resistant MOLM-14 cell lines. The GI 50 values for Gilteritinib and PLM-102 were determined to be 427 nM and 6.17 nM, respectively. In the studies with a xenograft model with Gilteritinib-resistant MOLM-14 cells (20 mg/kg, p.o.), the Tumor Growth Inhibition (TGI, %) values were determined as 71.9% vs 17.1% for PLM-102 vs Gilteritinib, respectively. These results demonstrated the superior in-vivo efficacies of PLM-102 in Gilteritinib-resistant cells. mRNA sequencing was performed on both MOLM-14 cells and Gilteritinib-resistance MOLM-14 cells, resulting in an upregulation of the cytokine-mediated signaling pathway, including chemokines, in the Gilteritinib-resistant MOLM-14 cells. Additionally, we conducted mRNA sequencing on Gilteritinib-resistant MOLM-14 cells treated with and without PLM-102 at a concentration of 10 nM. Interestingly, in the group treated with PLM-102 at 10 nM, we observed a notable decrease in the cytokine-mediated signaling pathway, including chemokines. These findings provide valuable insights into the potential mechanism by which PLM-102 exerts its anti-leukemic effect in Gilteritinib-resistant cells by modulating the cytokine-mediated signaling pathway. In Gilteritinib-resistant MOLM-14 cells harboring the NRAS G13D mutation, we confirmed hyperactivation of the MEK-ERK signaling pathway. The significantly improved effects in the combination of PLM-102 and Trametinib, a MEK inhibitor, were observed through cell viability assays and a xenograft model, further supporting their potential as a synergistic therapeutic approach. Currently, the GLP toxicological studies of PLM-102 has successfully completed, and the Investigational New Drug (IND) for human clinical studies will be submitted near future. Conclusions: Our results have confirmed that RAS-MEK-ERK signaling pathway and cytokine signaling pathway were activated by Gilteritinib resistance. Notably, PLM-102 exhibits exceptional anti-leukemic activity in Gilteritinib-resistant cell lines through the unique mechanism of inhibiting the RAS-MEK-ERK-cytokine signaling pathway. These results highlight the potential of PLM-102 as a third-generation inhibitor with favorable efficacies in patients with resistance to current FLT3 inhibitors.

Small molecule. Big biology. Dual phosphatase inhibitor enters the immunotherapy fray.

The advent and clinical success of immune checkpoint inhibitors Ipilimumab, Nivolumab and Pembrolizumab has had a seismic impact on our drug discovery focus and rationale. Novel extrinsic targets that enhance immune responses to cancer are actively being pursued, while tumor intrinsic targets that render cancer cells more sensitive to the immune system have joined traditional intrinsic targets (e.g. directly cytotoxic) in the drug discovery pipeline. The phosphatase PTPN2 (TC-PTP) and its paralog PTPN1 (PTP-1B) are negative regulators of several cytokine signaling pathways and Tcell receptor (TCR) signaling. In a recent publication, Baumgartner etal. demonstrate the pre-clinical efficacy of a first-in-class dual PTPN1/N2 active site inhibitor (ABBV-CLS-484/AC484) in cancer models.

Therapeutic effect of yinchenhao decoction on cholelithiasis via mucin in the gallbladder and intestine

Cholelithiasis is a common and frequently occurring disease worldwide that belongs to the category of jaundice in traditional Chinese medicine. Yinchenhao decoction (YD) consists of Artemisia capillaris Thunb., Gardenia jasminoides J.Ellis, and Rheum palmatum L., and is traditionally used to treat jaundice, which has a significant therapeutic effect on cholelithiasis. Our study aimed to investigate the pathological mechanism of cholelithiasis and the therapeutic mechanism of YD via mucin in the gallbladder and intestine. YD was prepared and analyzed using HPLC. The supersaturation stability experiment was designed by the solvent-shift method. The cell transport experiment was conducted by coculture monolayers. The animal experiment was performed using a cholelithiasis model with a high-cholesterol diet. The related indicators were detected by automatic biochemical analyzer, PCR, western blot, or ELISA. Statistics were analyzed using χ2-tests and t-tests. As the results, in cholelithiasis, MUC5AC highly expressed in the gallbladder shortened cholesterol supersaturation and promoted cholesterol crystallization via the inflammatory cytokine signaling pathway; MUC2 highly expressed in the small intestine prolonged cholesterol supersaturation and promoted cholesterol absorption via the inflammatory cytokine signaling pathway. YD inhibited mucin expression in the gallbladder and intestine in a concentration-dependent manner for cholelithiasis treatment by inhibiting the inflammatory cytokine signaling pathway, which was attributed to the active components, including chlorogenic acid, geniposide, and rhein.

Transcriptome Changes in Glioma Cells upon Infection with the Oncolytic Virus VV-GMCSF-Lact.

Oncolytic virotherapy is a rapidly evolving approach that aims to selectively kill cancer cells. We designed a promising recombinant vaccinia virus, VV-GMCSF-Lact, for the treatment of solid tumors, including glioma. We assessed how VV-GMCSF-Lact affects human cells using immortalized and patient-derived glioma cultures and a non-malignant brain cell culture. Studying transcriptome changes in cells 12 h or 24 h after VV-GMCSF-Lact infection, we detected the common activation of histone genes. Additionally, genes associated with the interferon-gamma response, NF-kappa B signaling pathway, and inflammation mediated by chemokine and cytokine signaling pathways showed increased expression. By contrast, genes involved in cell cycle progression, including spindle organization, sister chromatid segregation, and the G2/M checkpoint, were downregulated following virus infection. The upregulation of genes responsible for Golgi vesicles, protein transport, and secretion correlated with reduced sensitivity to the cytotoxic effect of VV-GMCSF-Lact. Higher expression of genes encoding proteins, which participate in the maturation of pol II nuclear transcripts and mRNA splicing, was associated with an increased sensitivity to viral cytotoxicity. Genes whose expression correlates with the sensitivity of cells to the virus are important for increasing the effectiveness of cancer virotherapy. Overall, the results highlight molecular markers, biological pathways, and gene networks influencing the response of glioma cells to VV-GMCSF-Lact.

Abstract 15132: Proteomic Signatures of Human Immunodeficiency Virus-Associated Subclinical Left Atrial Dilation

Introduction: Persons living with HIV (PLWH) receiving combination antiretroviral therapy (cART) are at higher risk of heart failure (HF) and preceding cardiac abnormalities, including left atrial (LA) dilation, compared to persons without HIV (PWOH). Mechanisms of this excess risk are unclear. We assessed whether plasma proteomic signatures of immune activation are cross-sectionally associated with LA volume index (LAVi) and augmented among PLWH. Methods: We performed Olink proteomics on plasma obtained concurrently with cardiac magnetic resonance among PLWH and PWOH. Proteins were analyzed individually and as clusters agnostically defined using weighted gene co-expression network analysis. Associations with HIV serostatus and LAVi were estimated using multivariable linear regression with robust variance. We explored protein relationships using annotated enrichment analysis. Results: Among 352 participants (age 55±6 years; 25% female; 70% Black), mean LAVi was 29±9 mL/m 2 and 60% were PLWH (88% on cART; 73% with undetectable plasma HIV RNA). Of 2594 proteins, 104 were independently associated with LAVi (false discovery rate, FDR<0.05). We identified 49 of these as candidate contributors to the association between HIV seropositivity and higher LAVi (36 when excluding PLWH with detectable HIV RNA), enriched in T-cell activation and leukocyte cell adhesion regulators. We identified one protein cluster positively associated with LAVi and HIV seropositivity regardless of HIV suppression status, enriched in TNF signaling, ephrin signaling and extracellular matrix (ECM) organization. Conclusion: Proteomic signatures that may in part account for HIV-associated elevation in LAVi are enriched in pathways of immune activation, cytokine signaling and ECM organization, even among virally suppressed PLWH on cART. These findings support a link between persistent immune activation and subclinical LA dilation which may portend future risk for HF among PLWH.

Cooperative Networks between MYC and XPO1 Associated with Decreased T-Cell Presence and a Depleted Tumor Microenvironment May be Addressed By the Synergistic Combination of AZD4573 and Selinexor

Background: Genomically-targeted Diffuse Large B-cell (DLBCL) treatments for refractory or relapsing patients remain a clinical need alongside the growing success of Chimeric Antigen T-cells (CAR-T), which have ushered in durable responses in 40-70% of patients. However, recent studies have identified factors indicative of an inferior CAR-T response. Recent tumor Ecotype studies from Kotlov and Steen reveal that a Depleted or Cold tumor microenvironment (TME) is associated with poor CAR-T response. Notable features of these environments include a decreased presence of native T-cells alongside oncogenic alterations associated with their exhaustion, namely MYC and XPO1. Few precision treatments exist given the difficulty targeting MYC, but two avenues have shown recent success: inhibition of the downstream oncogene CDK9 via AZD4573 and upstream inhibition of a MYC propagator, the nuclear exportin XPO1 via Selinexor. Herein, we report the first observations of synergy between AZD4573 and Selinexor within DLBCL cell lines and data supporting that these genes may serve as accessible markers for inferior tumor immune microenvironments. Methods: We combined progression vs. response results from four previous CAR-T genomics studies, noting MYC status (Jaeger 2020, Jain 2021, Sworder 2021, and Shouval 2022). We next analyzed a 418-patient de-novo DLBCL patient cohort (Xu-Monette 2020) and 48-patient TCGA cohort after GEDIT immune deconvolution for differential expression and component enrichment between patients expressing high levels of MYC and XPO1 (+1 Standard Deviation) compared to normal counterparts. Significantly expressed genes were evaluated via gene set enrichment analyses. We next assayed AZD4573 and Selinexor vs. 7 DLBCL cell lines, 5 of which harbor MYC alterations, across a series of 9 concentrations (78nM-20µM) across 3-9 replicates. We followed these results by combining treatments in the MYC-altered Ly3, DHL6, and DHL4 cell lines, analyzing results using the BLISS synergy model. Results: Integrative analyses support that MYC alterations are associated with significantly reduced CAR-T outcomes. Of the 93 MYC-altered patients within the total 246-patient cohort, 65.59% of them experienced progression during CAR-T treatment compared to just 50.33% of WT CAR-T patients (p = 0.0242). Differential gene expression analyses between de-novo patients expressing high MYC or XPO1 revealed losses in Cytokine Signaling pathways (FDR = 6.96E-18) and gains in Cell Cycle modulators (FDR = 2.525E-20). Importantly, CD8A expression was significantly lower in these patients (FDR < 0.0001) alongside other key reductions of CDKN1A and TNFAIP3. After immune deconvolution, both High-MYC and High-XPO1 patients displayed significant losses of CD8 T-cell association, respectively (FDR = 0.0005, FDR = 0.0396). Notably, High-MYC patients without a CD8 T-cell presence experienced an inferior survival prognosis compared to High-MYC counterparts with a CD8 presence (p = 0.0272). High MYC or XPO1 patients that experienced a failure event before 24-months (EFS24) had significantly lower activated CD4 T-cell presence compared to normal counterparts as well (FDR = 0.0095, FDR = 0.0069). Next, AZD4573 and Selinexor assays vs. DLBCL cell lines demonstrated dose and MYC-dependent cell inhibition, with ED50 values ranging between 58.1 nM to 8.07 µM. Lastly, the molecules were combined vs. Ly3, DHL6 and DHL4 cell lines, resulting in synergistic reduction of cell viability and BLISS synergy scores of 33.76, and 11.86, and 5.04, respectively. Conclusions: Our results support that the cooperation between MYC and XPO1 is associated with T-cell reductions characteristic of a Depleted or Cold TME, a key issue for CAR-T success. We applied the targeted therapies AZD4573 and Selinexor vs. cell line models to address this pathway, with both displaying anti-tumor effects as single agents. Most importantly, we observed synergistic activity in the MYC-harboring Ly3 and DHL6 cell lines, and in the WT DHL4 cell line when these molecules were combined. These results are supported by integrative analyses that highlight the importance of targeting aberrant MYC signaling via CDK9 and XPO1 inhibition, revealing a potential avenue to address inferior response rates faced by patients harboring MYC-positive, Depleted tumor microenvironments that would otherwise face an inferior CAR-T prognosis.

Genome-Wide DNA Methylation Analysis Reveals Epigenetic Influence on Intracellular and Cytokine Signaling Pathways in Waldenström Macroglobulinemia Compared to IgM-MGUS

Introduction: Waldenström macroglobulinemia (WM) is a rare B-cell lymphoma that may be preceded by an asymptomatic IgM-monoclonal gammopathy of undetermined significance (MGUS). The mechanisms underlying the progression from IgM-MGUS to WM remain to be fully elucidated; however, the stepwise accumulation of genomic abnormalities has been suggested as a potential driver. Currently, the role of epigenetic regulation of DNA remains underexplored in this disease spectrum. Aberrant DNA methylation is a hallmark of oncogenesis that plays an important role in hematologic malignancies. The aim of this study was to assess differential DNA methylation in WM compared to IgM-MGUS and identify hyper- and hypomethylated pathways that may alter key cellular functions. Methods: This study included 15 patients with WM and 6 with IgM-MGUS. Bone marrow samples were prospectively collected and CD19+ and/or CD138+ B cells were positively selected. Subsequently, genome-wide DNA methylation analysis was performed. CpG methylation ratios were segmented into 200-bp regions and differentially methylated regions (DMRs) comparing WM to IgM-MGUS were identified. DMRs with a Q-value <0.05 and absolute methylation difference of ≥20% were considered significant and utilized for analysis. DMRs were annotated according to genomic regions of interest (promotors, 3'UTR, 5'UTR, introns, exons, CpG islands, CpG shores). Next, genes with significant promotor hyper- or hypomethylation were assessed using the Ingenuity Pathway Analysis. Pathways with a corrected p-value<0.05 were considered statistically significant and a Z-value was calculated based on the degree of differential methylation. Results: Genome-wide DNA methylation analysis revealed a significant number of DMRs (n= 15691, hyper: 8248; hypo: 7443) in WM compared to IgM-MGUS. Assessing genomic regions, promotors (hyper: 1379; hypo: 2403), introns (hyper: 1548; hypo: 2292), CpG shores (hyper: 587; hypo: 849) and 3'UTR (hyper: 66; hypo: 193) regions were observed to have a significantly greater number of hypomethylated DMRs (all comparisons, p<0.001) in WM compared to IgM-MGUS (Figure A). While exons (hyper: 1686; hypo: 1378), CpG islands (hyper: 2128; hypo: 82), and 5'UTR (hyper: 854; hypo: 246) regions had a significantly greater number of hypermethylated DMRs (all comparisons, p<0.001) (Figure A). Next, promotor methylation-based pathway analysis revealed differential methylation of multiple cellular pathways important in WM pathogenesis (Figure B). These included pathways involved in intracellular signaling and the regulation of cellular proliferation, such as the STAT3 and CXCR4 pathways which were hypomethylated, and the PTEN signaling pathway which was hypermethylated. Of interest, we have previously demonstrated the transcription factor, STAT3, to have an increased expression in WM and to be implicated in IgM secretion (Elsawa, 2011). Next, multiple cytokine signaling pathways were observed to be hypomethylated in WM compared to IgM-MGUS including members of the interleukin (IL) family (IL-1, IL-8, IL-15, IL-23), VEGF, and TGF-β. Of relevance, previous studies have demonstrated the elevation of IL-8, IL-15, and increased expression of angiogenic cytokines, including VEGF, in patients with WM (Lewicki, 2010; Anagnostopoulos, 2007). Furthermore, our group has also shown miRNA-based epigenetic regulation of IL-8 and IL-15 in WM compared to IgM-MGUS (Chohan, 2023). Lastly, several pathways critical in the regulation of the cell structure and the extracellular matrix (ECM) were found to be hypomethylated including the FGF, actin cytoskeleton, PAK, and FAK signaling pathways. Of note, the FGF/FGFR axis has been demonstrated to be dysregulated in WM, and its targeting has been shown to halt WM cell growth (Sacco, 2021). Conclusions: This study demonstrates differential DNA methylation of key genomic regions in WM compared to IgM-MGUS. Promotor methylation-based analysis reveals hypomethylation of several important cellular pathways involved in cytokine signaling, cell structure/ECM, and intracellular signaling. These results underscore the influence of epigenetics in the IgM-gammopathy disease spectrum and suggest the potential role of aberrant DNA methylation underlying the malignant progression from IgM-MGUS to WM.

Comparative genomic hybridisation and transcriptome microarray analysis in triple negative breast cancer: An INDIAN study.

Triple-negative breast cancer (TNBC), which accounts for approximately 15–20% of all breast cancers, defined as lack of expression of estrogen receptor, progesterone receptor and Her-2 neu receptors. TNBC has two subtypes basal like and non-basal like, the former characterised by aggressive biology with limited therapeutic options. This study explored molecular markers involved in pathogenesis of TNBC and investigated novel potential diagnostic and therapeutic targets by CGH array and transcriptome array. aCGH analysis in TNBC demonstrated genes amplified were 3888, number of pathway hits was 1554 and major pathways amplified was found to be WNT signalling pathway and Cadherin signalling pathway. Among all metastatic sites and remission, activation of WNT signalling pathway is commonly observed. TNBC exhibited 1486 copy number variations (CNVR) which is approximately 250 times higher than controls. More than 20 CNVR was observed in all chromosomes and more than 80 CNVR was observed in Chr 1 to Chr 4, Chr 7, Chr 11 and Chr X. Common CNVR associated with amplified regions in Chr 22, Chr 14, Chr 8 and Chr 2 was observed in TNBC and CNVR associated with Chr 22q11.22–23, Chr 6p21.32–33, Chr11q12.2, Chr14q32.22–23, Chr 8p11.22–23, was observed in metastatic disease. In transcriptome array analysis a total of 11,359 differentially expressed genes with fold change 2.0 were in observed in TNBC comprise of 7639 upregulated genes and 3720 downregulated genes. Further, with fold change 10, 1526 upregulated genes and 839 down regulated genes were identified. Panther pathway analysis identified the main pathways of upregulated genes were Wnt signalling pathway, Integrin signalling pathway and Cadherin signalling pathway. The main pathways of down regulated genes were Inflammation mediated by chemokine and cytokine signalling pathways. PPI network shows that COL12A1, COL6A3, FN1, MMP3, WNT5A were key upregulated genes and ITGB7, PTPRC, ITGA4, LCK and CD247 were key down regulated genes. Cytoscape analysis followed by multiple list comparator tool identified top 5 significant hub genes were FN1, MMP3, COLL11A1, COL12A1 and COL3A1. The significant pathway genes obtained by CGH array and transcriptome array when compared, exhibited 5 common genes COL4A1, FN1, COL6A3, COL5A2 and PCDH7. These genes were not overexpressed in Controls and therefore involved in pathogenesis of TNBC. Expressions of these genes were validated by studying protein expression by immunohistochemistry. FN1 and COL6A3 protein over expression predicted worse DFS in TNBC and can be considered as therapeutic targets at diagnosis to reduce the disease metastases. These findings provide new insights into the pathogenesis of TNBC and guide for selection of targets related to diagnosis, prognosis and prediction of treatment in TNBC.

Janus Kinase Inhibitors Differentially Inhibit Specific Cytokine Signals in the Mesenteric Lymph Node Cells of Inflammatory Bowel Disease Patients.

Janus kinase [JAK] inhibitors [JAKinibs] are effective small molecule therapies for treating Crohn's disease [CD] and ulcerative colitis [UC], collectively known as inflammatory bowel disease [IBD]. By preventing JAKs from phosphorylating signal transducer and activator of transcription proteins, JAKinibs disrupt cytokine signalling pathways that promote inflammation. Despite considerable overlap in the JAKs they target, first- and second-generation JAKinibs display different clinical efficacies in CD and UC. We conducted a comparative phosflow study of four JAKinibs [filgotinib, upadacitinib, tofacitinib, and deucravacitinib] to observe subtle mechanistic differences that may dictate their clinical behaviour. Resected mesenteric lymph node [MLN] cells from 19 patients [9 CD, 10 UC] were analysed by flow cytometry in the presence or absence of different cytokine stimuli and titrated JAKinibs. We found a higher potency of the JAK 1/3-preferential inhibitor, tofacitinib, for JAK 3-dependent cytokine signalling pathways in comparison to filgotinib, but a higher potency of the JAK 1-preferential inhibitors, filgotinib and upadacitinib, for JAK 3-independent cytokine signalling pathways. Deucravacitinib, a TYK2-preferential inhibitor, demonstrated a much narrower selectivity by inhibiting only IL-10 and IFN-β pathways, albeit more potently than the other JAKinibs. Additionally, we found some differences in the sensitivity of immune cells from CD versus UC, and patients with versus without a CD-associated NOD2 polymorphism, to phosphorylate signal transducer and activator of transcriptions in response to specific cytokine stimulation. Despite their similarities, differences exist in the relative potencies of different JAKinibs against distinct cytokine families, to explain their clinical efficacy.