Abrogation Of Signaling Research Articles

Nuclear Factor κB Signaling Deficiency in CD11c-Expressing Phagocytes Mediates Early Inflammatory Responses and Enhances Mycobacterium tuberculosis Control.

Early innate immune responses play an important role in determining the protective outcome of Mycobacterium tuberculosis (Mtb) infection. Nuclear factor κB (NF-κB) signaling in immune cells regulates the expression of key downstream effector molecules that mount early antimycobacterial responses. Using conditional knockout mice, we studied the effect of abrogation of NF-κB signaling in different myeloid cell types and its impact on Mtb infection. Our results show that the absence of IKK2-mediated signaling in all myeloid cells resulted in increased susceptibility to Mtb infection. In contrast, the absence of IKK2-mediated signaling in CD11c+ myeloid cells induced early proinflammatory cytokine responses, enhanced the recruitment of myeloid cells, and mediated early resistance to Mtb. Abrogation of IKK2 in MRP8-expressing neutrophils did not affect disease pathology or Mtb control. Thus, we describe an early immunoregulatory role for NF-κB signaling in CD11c-expressing phagocytes and a later protective role for NF-κB in LysM-expressing cells during Mtb infection.

Romidepsin and Afatinib Abrogate Jak–Signal Transducer and Activator of Transcription Signaling and Elicit Synergistic Antitumor Effects in Cutaneous T-Cell Lymphoma

Cutaneous T-cell lymphomas (CTCL) are mature lymphoid neoplasias resulting from the malignant transformation of skin-resident T cells. A distinctive clinical feature of CTCL is their sensitivity to treatment with histone deacetylase (HDAC) inhibitors. However, responses to HDAC inhibitor therapy are universally transient and non-curative, highlighting the need for effective and durable drug combinations. Here we demonstrate that the combination of romidepsin, a selective class I HDAC inhibitor, with afatinib, an epidermal growth factor receptor (EGFR) family inhibitor, induces strongly synergistic antitumor effects in CTCL models in vitro and in vivo via abrogation of JAK-STAT signalling. These results support a previously unrecognized potential role for HDAC inhibitor plus afatinib combination in the treatment of CTCL.

TGF-β broadly modifies rather than specifically suppresses reactivated memory CD8 T cells in a dose-dependent manner.

Transforming growth factor β (TGF-β) directly acts on naive, effector, and memory T cells to control cell fate decisions, which was shown using genetic abrogation of TGF-β signaling. TGF-β availability is altered by infections and cancer; however, the dose-dependent effects of TGF-β on memory CD8 T cell (Tmem) reactivation are still poorly defined. We examined how activation and TGF-β signals interact to shape the functional outcome of Tmem reactivation. We found that TGF-β could suppress cytotoxicity in a manner that was inversely proportional to the strength of the activating TCR or proinflammatory signals. In contrast, even high doses of TGF-β had a comparatively modest effect on IFN-γ expression in the context of weak and strong reactivation signals. Since CD8 Tmem may not always receive TGF-β signals concurrently with reactivation, we also explored whether the temporal order of reactivation versus TGF-β signals is of importance. We found that exposure to TGF-β before or after an activation event were both sufficient to reduce cytotoxic effector function. Concurrent ATAC-seq and RNA-seq analysis revealed that TGF-β altered ~10% of the regulatory elements induced by reactivation and also elicited transcriptional changes indicative of broadly modulated functional properties. We confirmed some changes on the protein level and found that TGF-β-induced expression of CCR8 was inversely proportional to the strength of the reactivating TCR signal. Together, our data suggest that TGF-β is not simply suppressing CD8 Tmem but modifies functional and chemotactic properties in context of their reactivation signals and in a dose-dependent manner.

OR20-03 Leptin-mediated Regulation Of Gene Regulatory Networks In Gonadotropes

Abstract Disclosure: J. Banik: None. A. Lagasse: None. A. Haney: None. S. Byrum: None. U. Boehm: None. G.V. Childs: None. M.C. MacNicol: None. A.M. MacNicol: None. A.K. Odle: None. The female reproductive system is energetically expensive and requires tight control of the hypothalamic-pituitary-gonadal (HPG) axis to promote gonadal priming and ovulation. Thus, a crosstalk between the metabolic and reproductive systems exists to regulate the rate-limiting steps of reproduction. Leptin is an adipokine hormone that is produced and secreted proportionally from white fat cells, and leptin receptors (Lepr) are present in the hypothalamus, pituitary, and gonads. Leptin binding to hypothalamic LepR functions as a sensor of nutritional status and, thus, orchestrates energy expenditure during reproductive events. We hypothesized that leptin is crucial to influence fertility in females via differential gene regulation during the estrus cycle in pituitary gonadotropes. To test this hypothesis, we utilized Cre-LoxP technology to selectively ablate leptin receptors within female gonadotropes (CreGnRHR+/- Leprfl/fl TdTomato+/+, referred to as “Gon-Lepr-null”) and performed single-cell RNA-sequencing (scRNA-seq) to identify leptin-dependent targets within the gonadotrope transcriptome. We observed a significant decrease in cell percentage within the gonadotrope cluster, and differential analysis reported a significant decrease in Fshb expression in the Lepr-null mutant gonadotropes. Moreover, the lack of leptin signaling in gonadotropes also caused a shift in the lactotrope-specific transcript signature in the Gon-Lepr-null mutants. We employed SCENIC, a computational method for simultaneous gene regulatory network reconstruction and cell-state identification from single-cell RNA-seq data, to delineate altered upstream regulators contributing to the reduction of Fshb expression in gonadotropes and the shift in the lactotrope transcriptome signature in Gon-Lepr-null mutants versus control animals. Regulons are co-regulated gene sets that share binding sites for a common transcription factor driver, and we identified 381 and 399 regulons specific to controls and mutants, respectively. Examination of the gonadotrope regulons revealed Gata2, Nhlh2, NeuroD1, and Sox11 to be drivers of Fshb expression in control animals only. Differential expression analysis showed significant downregulation of Gata2 and Sox11 in Lepr-null mutant gonadotropes, suggesting Fshb is a leptin-dependent target of Gata2 and Sox11. Moreover, Gata2, Nhlh2, NeuroD1, and Sox11 target 117 genes in controls and 263 genes in mutants, implying a more restricted ability of Gata2, Nhlh2, NeuroD1, and/or Sox11 to target gene expression in the presence of leptin signaling in controls, as an abrogation of leptin signaling leads to an expansion of the regulatory network in Lepr-null mutant gonadotropes. Overall, our analysis reveals how leptin signaling modulates the gonadotrope transcriptome to optimize pituitary reproductive functions at the molecular level. Presentation: Saturday, June 17, 2023

Regulation of human and mouse bystander T cell activation responses by PD-1.

Bystander activation of memory T cells occurs via cytokine signaling alone in the absence of T cell receptor (TCR) signaling and provides a means of amplifying T cell effector responses in an antigen-nonspecific manner. While the role of Programmed Cell Death Protein 1 (PD-1) on antigen-specific T cell responses is extensively characterized, its role in bystander T cell responses is less clear. We examined the role of the PD-1 pathway during human and mouse non-antigen-specific memory T cell bystander activation and observed that PD-1+ T cells demonstrated less activation and proliferation than activated PD-1- populations in vitro. Higher activation and proliferative responses were also observed in the PD-1- memory population in both mice and patients with cancer receiving high-dose IL-2, mirroring the in vitro phenotypes. This inhibitory effect of PD-1 could be reversed by PD-1 blockade in vivo or observed using memory T cells from PD-1-/- mice. Interestingly, increased activation through abrogation of PD-1 signaling in bystander-activated T cells also resulted in increased apoptosis due to activation-induced cell death (AICD) and eventual T cell loss in vivo. These results demonstrate that the PD-1/PD-Ligand 1 (PD-L1) pathway inhibited bystander-activated memory T cell responses but also protected cells from AICD.

The MRN complex maintains the biliary-derived hepatocytes in liver regeneration through ATR-Chk1 pathway

When the proliferation of residual hepatocytes is prohibited, biliary epithelial cells (BECs) transdifferentiate into nascent hepatocytes to accomplish liver regeneration. Despite significant interest in transdifferentiation, little is known about the maintenance of nascent hepatocytes in post-injured environments. Here, we perform an N-ethyl-N-nitrosourea (ENU) forward genetic screen and identify a mutant containing a nonsense mutation in the gene nibrin (nbn), which encodes a component of the Mre11-Rad50-Nbn (MRN) complex that activates DNA damage response (DDR). The regenerated hepatocytes cannot be maintained and exhibit apoptosis in the mutant. Mechanistically, the nbn mutation results in the abrogation of ATR-Chk1 signaling and accumulations of DNA damage in nascent hepatocytes, which eventually induces p53-mediated apoptosis. Furthermore, loss of rad50 or mre11a shows similar phenotypes. This study reveals that the activation of DDR by the MRN complex is essential for the survival of BEC-derived hepatocytes, addressing how to maintain nascent hepatocytes in the post-injured environments.

Abstract 1090: Dominant-negative TP53 variants confer radiation resistance in endometrial cancer

Abstract Introduction: High-grade endometrial cancers (EC) belonging to the “serous-like” cluster are known to portend a worse clinical outcome. The direct contribution of commonly identified TP53 variants on radiotherapy (RT) response has not been demonstrated in EC. Methods: To assess the impact of TP53 variants, allelic frequency, and p53/p21 signaling on radio-resistance in vitro, we utilized a high-throughput radiation assay and intron targeting CRISPR-Cas9 knockout approach in two low copy-number/TP53 wild-type EC cell lines (JHUEM1/JHUEM2). This approach facilitated TP53 cDNA complementation in a TP53-null background without significant impact from copy-number burden. The effect on radiation response was determined using high-throughput methodology and area under the curve (AUC) calculation along 5 dose-points. All experiments were carried out using 2 monoclonal isolates for each guide RNA (4 total replicates) and 2 non-targeting controls. Results: Knockout (KO) of wild-type TP53 resulted in loss of downstream p21 signaling and increased radio-resistance, demonstrating the role of p53 signaling on RT response. Complementation with five common TP53 missense variants (Y220C, R273C, R273H, R248Q, R248W) into the KO cell lines led to increased p53 accumulation without an effect on p21 signaling or RT response. Complementation into the WT cell lines, mimicking tumors with low allelic frequency/heterozygous mutations, led to abrogation of p21 signaling and increased radio-resistance, establishing their gain-of-function/dominant-negative impact. Conclusions: Our findings directly implicate TP53 status on radiotherapy response in EC, providing biologic rationale for the clinical observation that these patients may benefit from chemotherapy. Additionally, our findings demonstrate that dominant-negative variant alleles similarly impact RT response by abrogating p21 signaling. Citation Format: Aaron Petty, Arda Durmaz, Brian Yard, Jacob Scott, Roberto Vargas. Dominant-negative TP53 variants confer radiation resistance in endometrial cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1090.

Resveratrol-induced SIRT1 activation inhibits glycolysis-fueled angiogenesis under rheumatoid arthritis conditions independent of HIF-1α.

This study investigated the impacts of SIRT1 activation on rheumatoid arthritis (RA)-related angiogenesis. HUVECs were cultured by different human serum. Intracellular metabolites were quantified by UPLC-MS. Next, HUVECs and rat vascular epithelial cells under different inflammatory conditions were treated by a SIRT1 agonist resveratrol (RSV). Cytokines and biochemical indicators were detected by corresponding kits. Protein and mRNA expression levels were assessed by immunoblotting and PCR methods, respectively. Angiogenesis capabilities were evaluated by migration, wound-healing and tube-formation experiments. To down-regulate certain signals, gene-specific siRNA were applied. Metabolomics study revealed the accelerated glycolysis in RA serum-treated HUVECs. It led to ATP accumulation, but did not affect GTP levels. RSV inhibited pro-angiogenesis cytokines production and glycolysis in both the cells, and impaired the angiogenesis potentials. These effects were mimicked by an energy metabolism interrupter bikini in lipopolysaccharide (LPS)-primed HUVECs, largely independent of HIF-1α. Both RSV and bikinin can inhibit the activation of the GTP-dependent pathway Rho/ROCK and reduce VEGF production. Abrogation of RhoA signaling reinforced HIF-1α silencing-brought changes in LPS-stimulated HUVECs, and overshadowed the anti-angiogenesis potentials of RSV. Glycolysis provides additional energy to sustain Rho/ROCK activation in RA subjects, which promotes VEGF-driven angiogenesis and can be inhibited by SIRT1 activation.

Abstract 2659: Anti-HER3 antibody, HMBD-001, in combination with an EGFR inhibitor effectively inhibits tumor growth in biomarker-selected pre-clinical models of squamous cell carcinomas

Abstract Squamous cell carcinomas (SCC) originate from stratified squamous epithelium in diverse anatomical locations that share common histological features and genetic mutations. Non-melanoma skin cancer, head and neck cancer (HNSCC), esophageal cancer (ESCC), and non-small cell lung cancer (sqNSCLC) make up the majority of SCC cases and together represent the largest subtype of cancer. There is a significant unmet need for effective treatment options post-progression of standard-of-care therapies. Alterations in receptor tyrosine kinase signaling pathways are commonly found in SCCs, e.g. EGFR is expressed in 90% of HNSCC, 76% of ESCC and 82% of sqNSCLC. However, the combination of EGFR targeting antibody cetuximab with chemotherapy confers limited clinical benefit and even initially sensitive tumors often develop resistance. Studies in SCC have implicated HER3, a key dimerization partner of HER family members, as one likely cause of treatment failure, through HER3 heterodimer activation of PI3K/AKT and MAPK/ERK pathways. In this study, we investigated the potential of combining HER3 and EGFR inhibitors to improve efficacy and overcome resistance. Previous attempts to target HER3 have shown limited clinical efficacy due to suboptimal HER3 inhibition, especially the lack of efficacy in ligand-independent activation mechanisms. They also lacked biomarker stratification for patients where HER3 inhibition would confer benefit. HMBD-001 is a clinical-stage anti-HER3 antibody which is being evaluated in a first-in-human, open-label, multi-center, dose escalation and expansion Phase I/IIa trial in patients with HER3 expressing advanced solid tumors (NCT05057013). HMBD-001 was rationally developed to uniquely block the HER3 heterodimerization interface to potently inhibit all HER3 dimer formation, including ligand-dependent and independent HER3 dimerization. Here, we show that dual blockade of EGFR and HER3 using cetuximab and HMBD-001 robustly inhibits tumor growth and is superior to either monotherapy. In multiple cell and patient derived SCC xenograft models, including models of HNSCC, ESCC and sqNSCLC, selected based on a novel gene signature that is robustly predictive of response, HMBD-001 in combination with cetuximab demonstrated up to 100% tumor growth inhibition and the abrogation of PI3K signalling. This combination was well tolerated with no relapse observed, even after a prolonged period of over 100 days in selected models. This study lays the foundation for testing HMBD-001 in combination with EGFR inhibitors in clinical settings. Better combination strategies of SoC and targeted therapeutics alongside a potent anti-HER3 mAb, in biomarker-selected patient populations, have the potential to meaningfully improve patient outcomes and prolong clinical benefit. Citation Format: Weiyi Toy, Dipti Thakkar, Khang Luu, Bui Ngoc Linh Chi, Jason Lai, Debleena Ray, Namita Gandhi, Alejandro Mas, Konrad Paszkiewicz, Piers Ingram, Jerome Boyd-Kirkup. Anti-HER3 antibody, HMBD-001, in combination with an EGFR inhibitor effectively inhibits tumor growth in biomarker-selected pre-clinical models of squamous cell carcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2659.

Nanoceria Ameliorates Fibrosis, Inflammation, and Cellular Stress in Experimental Chronic Pancreatitis

Chronic pancreatitis (CP) is an inflammatory, irreversible disorder of the pancreas which leads to organ atrophy and poses high risk for the development of pancreatic cancer. Given the lack of clinically approved therapy, we explored the pharmacological potential of the nanoparticles of cerium oxide (nanoceria, NC) against animal models of CP. Nanoceria ameliorated the features of CP as evident from biochemical parameters. It inhibited the inflammatory cytokines and chemokines by abrogation of macrophage signaling. Further, NC attenuated the fibrogenesis by inhibition of TGF-β signaling, endoplasmic reticulum stress, and epithelial-to-mesenchymal transition. Our findings reveal the anti-CP potential of the novel redox regenerative nanoceria against two models of CP.

Leishmania donovani induces CD300a expression to dampen effector properties of CD11c+ dendritic and antigen activated CD8+ T cells

CD8+ T cells are an important regiment of adaptive immunity that play a decisive role in elimination of many species of Leishmania parasite from the host. In visceral leishmaniasis, caused by L. donovani, the loss of CD8+ T cells function has been found associated with augmented pathogenesis. The factors determining CD8+ T cells activation and function against Leishmania antigens are largely unknown. In this study, we investigated the role of an immune inhibitory receptor, CD300a, on the effector properties of dendritic cells and CD8+ T cells. We observed that the Leishmania regulates the effectors function of CD8+ T cells by increasing CD300a expression on CD11c+ dendritic cells. The abrogation of CD300a signaling in parasites infected animals induced CD8+ T cell abilities to produce IFN-γ, TNF-α and also helped them to acquire desired multifunctionality. The CD300a receptor blocking also enhanced the number of CD8+ T cells memory phenotypes at the early days of infection, suggesting its potential beneficial role in vaccine induced immunity. We also observed significantly enhanced levels of pro-inflammatory cytokines in the spleen of CD300a blocked infected animals with concomitant reduced spleen parasite load. Additionally, the abrogation of CD300a signals in the infected animals helped in establishing Th1 type protective humoral immunity with significantly elevated levels of IgG2a antibodies. Since CD8+ T cells are an important determinant of vaccine induced immunity against leishmaniasis, the findings corroborate the potential of CD300a in vaccine induced immunity and thus require further attention.

A Critical Role of the IL-22-IL-22 Binding Protein Axis in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) ranks among the five most common cancer entities worldwide and leads to hundred-thousands of deaths every year. Despite some groundbreaking therapeutical revelations during the last years, the overall prognosis remains poor. Although the immune system fights malignant transformations with a robust anti-tumor response, certain immune mediators have also been shown to promote cancer development. For example, interleukin (IL)-22 has been associated with HCC progression and worsened prognosis in multiple studies. However, the underlying mechanisms of the pathological role of IL-22-signaling as well as the role of its natural antagonist IL-22 binding protein (IL-22BP) in HCC remain elusive. Here, we corroborate the pathogenic role of IL-22 in HCC by taking advantage of two mouse models. Moreover, we observed a protective role of IL-22BP during liver carcinogenesis. While IL-22 was mainly produced by CD4+ T cells in HCC, IL-22BP was abundantly expressed by neutrophils during liver carcinogenesis. Hepatocytes could be identified as a major target of this pathological IL-22-signaling. Moreover, abrogation of IL-22 signaling in hepatocytes in IL22ra1flox/flox × AlbCre+ mice reduced STEAP4 expression-a known oncogene-in HCC in vivo. Likewise, STEAP4 expression correlated with IL22 levels in human HCC samples, but not in healthy liver specimens. In conclusion, these data encourage the development of therapeutical approaches that target the IL-22-IL-22BP axis in HCC.

Brucein D imparts a growth inhibitory effect in multiple myeloma cells by abrogating the Akt-driven signaling pathway.

The Akt signaling pathway is an oncogenic cascade activated in the bone marrow microenvironment of multiple myeloma (MM) cells and contributes to their uncontrolled proliferation. Abrogation of Akt signaling has been presentedas one of the prime therapeutic targets in the treatment of MM. In the present report, we have investigated the effect of Brucein D (BD) on Akt-driven signaling events in MM cells. BD (300 nM) substantially inhibited cell viability and imparted growth-inhibitory effects in U266 cells as evidenced by cell viability assays and flow cytometric analysis. Effect of BD on cell viability was evaluated by MTT assay. Apoptotic cells and cell cycle arrest by BD were analyzed by flow cytometer. The results of the TUNEL assay and western blotting showed that BD induces apoptosis of MM cells by activating caspase-8 and 9 with subsequent reduction in the expression of antiapoptotic proteins (Bcl-2, Bcl-xl, survivin, cyclin D1, COX-2, VEGF, MMP-9). Analysis of activated kinases by Phospho-Kinase Array Kit revealed that Akt, p70S6K, HSP60, p53, and WNK1 were strongly expressed in untreated cells and BD treatment reversed this effect. Using transfection experiments, AKT depletion led to a decrease in phosphorylation of Akt, mTOR, p70S6K, and WNK. However, Akt overexpression led to increase in phosphorylation of these proteins. Depletion of Akt potentiated the apoptosis-inducing effect of BD whereas overexpression displayed resistance to BD-induced apoptosis suggesting the role of Akt in chemoresistance. Taken together, BD mitigates Akt-dependent signaling pathways in MM cells to impart its anticancer activity.

Microglial STAT1-sufficiency is required for resistance to toxoplasmic encephalitis.

Toxoplasma gondii is a ubiquitous intracellular protozoan parasite that establishes a life-long chronic infection largely restricted to the central nervous system (CNS). Constant immune pressure, notably IFN-γ-STAT1 signaling, is required for preventing fatal pathology during T. gondii infection. Here, we report that abrogation of STAT1 signaling in microglia, the resident immune cells of the CNS, is sufficient to induce a loss of parasite control in the CNS and susceptibility to toxoplasmic encephalitis during the early stages of chronic infection. Using a microglia-specific genetic labeling and targeting system that discriminates microglia from blood-derived myeloid cells that infiltrate the brain during infection, we find that, contrary to previous in vitro reports, microglia do not express inducible nitric-oxide synthase (iNOS) during T. gondii infection in vivo. Instead, transcriptomic analyses of microglia reveal that STAT1 regulates both (i) a transcriptional shift from homeostatic to "disease-associated microglia" (DAM) phenotype conserved across several neuroinflammatory models, including T. gondii infection, and (ii) the expression of anti-parasitic cytosolic molecules that are required for eliminating T. gondii in a cell-intrinsic manner. Further, genetic deletion of Stat1 from microglia during T. gondii challenge leads to fatal pathology despite largely equivalent or enhanced immune effector functions displayed by brain-infiltrating immune populations. Finally, we show that microglial STAT1-deficiency results in the overrepresentation of the highly replicative, lytic tachyzoite form of T. gondii, relative to its quiescent, semi-dormant bradyzoite form typical of chronic CNS infection. Our data suggest an overall protective role of CNS-resident microglia against T. gondii infection, illuminating (i) general mechanisms of CNS-specific immunity to infection (ii) and a clear role for IFN-STAT1 signaling in regulating a microglial activation phenotype observed across diverse neuroinflammatory disease states.

Activation of non-classical NMDA receptors by glycine impairs barrier function of brain endothelial cells

Blood–brain barrier (BBB) integrity is necessary to maintain homeostasis of the central nervous system (CNS). NMDA receptor (NMDAR) function and expression have been implicated in BBB integrity. However, as evidenced in neuroinflammatory conditions, BBB disruption contributes to immune cell infiltration and propagation of inflammatory pathways. Currently, our understanding of the pathophysiological role of NMDAR signaling on endothelial cells remains incomplete. Thus, we investigated NMDAR function on primary mouse brain microvascular endothelial cells (MBMECs). We detected glycine-responsive NMDAR channels, composed of functional GluN1, GluN2A and GluN3A subunits. Importantly, application of glycine alone, but not glutamate, was sufficient to induce NMDAR-mediated currents and an increase in intracellular Ca2+ concentrations. Functionally, glycine-mediated NMDAR activation leads to loss of BBB integrity and changes in actin distribution. Treatment of oocytes that express NMDARs composed of different subunits, with GluN1 and GluN3A binding site inhibitors, resulted in abrogation of NMDAR signaling as measured by two-electrode voltage clamp (TEVC). This effect was only detected in the presence of the GluN2A subunits, suggesting the latter as prerequisite for pharmacological modulation of NMDARs on brain endothelial cells. Taken together, our findings argue for a novel role of glycine as NMDAR ligand on endothelial cells shaping BBB integrity.

Cell-matrix interface regulates dormancy in human colon cancer stem cells.

Cancer relapse after chemotherapy remains a main cause of cancer-related death. Although the relapse is thought to result from the propagation of resident cancer stem cells1, a lack of experimental platforms that enable the prospective analysis of cancer stem cell dynamics with sufficient spatiotemporal resolution has hindered the testing of this hypothesis. Here we develop a live genetic lineage-tracing system that allows the longitudinal tracking of individual cells in xenotransplanted human colorectal cancer organoids, and identify LGR5+ cancer stem cells that exhibit a dormant behaviour in a chemo-naive state. Dormant LGR5+ cells are marked by the expression of p27, and intravital imaging provides direct evidence of the persistence of LGR5+p27+ cells during chemotherapy, followed by clonal expansion. Transcriptome analysis reveals that COL17A1-a cell-adhesion molecule that strengthens hemidesmosomes-is upregulated in dormant LGR5+p27+ cells. Organoids in which COL17A1 is knocked out lose the dormant LGR5+p27+ subpopulation and become sensitive to chemotherapy, which suggests that the cell-matrix interface has a role in the maintenance of dormancy. Chemotherapy disrupts COL17A1 and breaks the dormancy in LGR5+p27+ cells through FAK-YAP activation. Abrogation of YAP signalling prevents chemoresistant cells from exiting dormancy and delays the regrowth of tumours, highlighting the therapeutic potential of YAP inhibition in preventing cancer relapse. These results offer a viable therapeutic approach to overcome the refractoriness of human colorectal cancer to conventional chemotherapy.

Abstract 1105: MET or SHP2 inhibition enhances targeted therapies and delays the emergence of resistance in oncogene-driven NSCLC

Abstract Background: Although advancements have been made in available targeted therapies for non-small cell lung cancer (NSCLC), most patients have an incomplete response and eventually acquire resistance. It is well known that MET signaling is a key pathway that mediates osimertinib resistance. Concurrently targeting multiple proteins in critical signaling pathways, including the MET receptor or SHP2, a key node downstream of receptor tyrosine kinases, may prevent or delay resistance. Here, we explore the promising concept of combining upfront targeted therapies with a selective MET inhibitor or SHP2 inhibitor as promising therapeutic strategies for oncogene-driven NSCLC. Methods: All cell lines were obtained from commercial vendors. Drugs were synthesized at Merck KGaA or purchased from commercial vendors. Cell viability upon inhibitor treatment was investigated by Resazurin Cell Viability Assay (R&D Systems), and the effect of the inhibitors on colony forming ability was assessed using colony formation assays. Western blot was conducted to analyze pathway activity. The emergence of resistance was investigated by measuring the cell surface confluence using the IncuCyte S3 system (Essen Bioscience). Results: The lung cancer cell lines used were NCI-H358 (KRAS G12C), HCC-827 (EGFR L858R), NCI-H2228 (ALK fusion), HCC-78 (ROS1 fusion), and NCI-H1781 (HER2 exon 20 insertion), with their respective targeted therapies sotorasib, osimertinib, alectinib, entrectinib, and poziotinib. The antiproliferative effects of targeted therapies combined with a MET or SHP2 inhibitor were assessed using combination dose matrices in 6-day viability assays. While MET inhibition did not further sensitize the already sensitive cells to the targeted therapies, synergism occurred when the drugs were combined with the SHP2 inhibitor. Similar results were obtained by colony formation assays lasting 2 weeks. Furthermore, the combination of the targeted therapies with the SHP2 inhibitor led to stronger ERK signaling abrogation than with single therapies. In a long-term proliferation assay, the combined inhibition of EGFR and MET receptor delayed the emergence of osimertinib resistance in the EGFR mutant HCC827 cells, which have MET protein overexpression and phosphorylation. Conclusions: Our data show that combining targeted therapies with a SHP2 inhibitor synergistically decreases the viability of oncogene-driven NSCLC cell lines, indicating that initial combination therapy may be an appealing approach to improve patient outcomes. The observed delay in the emergence of osimertinib resistance, from combining a MET inhibitor with osimertinib, provides preclinical evidence to support further investigating MET inhibition upfront to improve the long-term therapeutic efficacy of EGFR inhibitors. Citation Format: Nadine Reischmann, Lorenz Pudelko, Christopher Stroh, Nina Linde, Doreen Musch, Marina Keil, Linda Pudelko, Christina Esdar, Andree Blaukat, Karl M. Schumacher, Niki Karachaliou. MET or SHP2 inhibition enhances targeted therapies and delays the emergence of resistance in oncogene-driven NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1105.

Abrogation of Notch Signaling in Embryonic TECs Impacts Postnatal mTEC Homeostasis and Thymic Involution.

Notch signaling is crucial for fate specification and maturation of thymus-seeding progenitors along the T-cell lineage. Recent studies have extended the role of Notch signaling to thymic epithelial cells (TECs), showing that Notch regulates TEC progenitor maintenance and emergence of medullary TECs (mTECs) in fetal thymopoiesis. Based on immunohistochemistry studies of spatiotemporal regulation of Notch activation in the postnatal thymus, we show that in vivo Notch activation is not confined to fetal TECs. Rather, Notch signaling, likely mediated through the Notch1 receptor, is induced in postnatal cortical and medullary TECs, and increases significantly with age in the latter, in both humans and mice, suggesting a conserved role for Notch signaling in TEC homeostasis during thymus aging. To investigate the functional impact of Notch activation in postnatal TEC biology, we used a mouse model in which RPBJκ, the transcriptional effector of canonical Notch signaling, is deleted in epithelial cells, including TECs, under the control of the transcription factor Foxn1. Immunohistochemistry and flow cytometry analyses revealed no significant differences in TEC composition in mutant (RPBJκ-KOTEC) and wild-type (WT) littermate mice at early postnatal ages. However, a significant reduction of the medullary region was observed in mutant compared to WT older thymi, which was accompanied by an accelerated decrease of postnatal mTEC numbers. Also, we found that organization and integrity of the postnatal thymic medulla critically depends on activation of the canonical Notch signaling pathway, as abrogation of Notch signaling in TECs led to the disruption of the medullary thymic microenvironment and to an accelerated thymus atrophy. These features paralleled a significant increase in the proportion of intrathymic non-T lineage cells, mostly B cells, and a slight decrease of DP thymocyte numbers compatible with a compromised thymic function in mutant mice. Therefore, impaired Notch signaling induced in embryonic development impacts postnatal TECs and leads to an accelerated mTEC degeneration and a premature thymus involution. Collectively, our data have uncovered a new role for Notch1 signaling in the control of adult mTEC homeostasis, and point toward Notch signaling manipulation as a novel strategy for thymus regeneration and functional recovery from immunosenescence.

Dendritic Inhibition by Shh Signaling-Dependent Stellate Cell Pool Is Critical for Motor Learning.

Cerebellar inhibitory interneurons are important regulators of neural circuit activity for diverse motor and nonmotor functions. The molecular layer interneurons (MLIs), consisting of basket cells (BCs) and stellate cells (SCs), provide dendritic and somatic inhibitory synapses onto Purkinje cells, respectively. They are sequentially generated in an inside-out pattern from Pax2+ immature interneurons, which migrate from the prospective white matter to the ML of the cortex. However, little is known about how MLI subtype identities and pool sizes are determined, nor are their contributions to motor learning well understood. Here, we show that GABAergic progenitors fated to generate both BCs and SCs respond to the Sonic hedgehog (Shh) signal. Conditional abrogation of Shh signaling of either sex inhibited proliferation of GABAergic progenitors and reduced the number of Pax2+ cells, whereas persistent Shh pathway activation increased their numbers. These changes, however, did not affect early born BC numbers but selectively altered the SC pool size. Moreover, genetic depletion of GABAergic progenitors when BCs are actively generated also resulted in a specific reduction of SCs, suggesting that the specification of MLI subtypes is independent of Shh signaling and their birth order and likely occurs after Pax2+ cells settle into their laminar positions in an inside-out sequence. Mutant mice with reduced SC numbers displayed decreased dendritic inhibitory synapses and neurotransmission onto Purkinje cells, resulting in an impaired acquisition of eyeblink conditioning. These findings also reveal an essential role of Shh signaling-dependent SCs in regulating inhibitory dendritic synapses and motor learning.SIGNIFICANCE STATEMENT The cerebellar circuit that enables fine motor learning involves MLIs of BCs and SCs, which provide dendritic and somatic inhibitory synapses onto Purkinje cells. Little is known about how their identities and numbers are determined, nor are their specific contributions to motor learning well understood. We show that MLI subtypes are specified independent of Shh signaling and their birth orders but appear to occur in their terminal laminar positions according to the inside-out sequence. This finding challenges the current view that MLI subtypes are specified sequentially at the progenitor level. We also demonstrate that dendritic inhibition by Shh signaling-dependent SC pool is necessary for motor learning.

Epithelial cells release IL-36α in extracellular vesicles following mechanical damage

The epithelium is an integral part of barrier tissues, and plays a critical role in the initiation of the innate immune responses. The pro-inflammatory cytokine IL-36α has been previously reported to be strongly expressed during oral mucosal wound healing, but regulation of IL-36α expression and secretion in the oral mucosa are not well known. The objective of this study was to determine the types of stimuli that lead to expression and secretion of IL-36α in epithelial cells. Maxillary tissues from C57BL/6J mice during wound healing were utilized to identify endogenous expression of IL-36α, β, and γ in oral epithelial tissue. Immortalized HaCaT cells and primary normal human oral keratinocytes were subjected to Escherichia coli derived lipopolysaccharide (LPS), Poly(I:C), heat killed Candida albicans (HKCa), and mechanical damage. IL-36α and IL-1β levels in supernatant were assessed by sandwich ELISA, and expression of pro-inflammatory cytokines and IL-36 family genes were assessed by quantitative real-time PCR in HaCaT cells. Migration ability of keratinocytes was assessed with or without functional IL-36 signaling. IL-36α but not IL-36β or γ levels in the oral epithelium were elevated during wound healing. Treatment of epithelial cells with LPS, Poly(I:C), HKCa and mechanical damage revealed little to no soluble IL-36α in the media supernatant. However, sonication of the supernatant to disrupt the membranes of extracellular vesicles revealed a dose-dependent increase in IL-36α for each of the tested conditions. IL-1 superfamily genes were upregulated following mechanical damage in keratinocytes. Abrogation of IL-36 signaling led to severe inhibition of migration. Our data show for the first time that IL-36α is released primarily in extracellular vesicles by oral keratinocytes. Additionally, we show that IL-36α – but not IL-36β or γ – is upregulated in keratinocytes following mechanical damage, and that IL-36 signaling is important for keratinocyte migration.