TGF-β Activation Research Articles

Longitudinal markers of cerebral amyloid angiopathy and related inflammation in rTg-DI rats

Cerebral amyloid angiopathy (CAA) is a prevalent vascular dementia and common comorbidity of Alzheimer’s disease (AD). While it is known that vascular fibrillar amyloid β (Aβ) deposits leads to vascular deterioration and can drive parenchymal CAA related inflammation (CAA-ri), underlying mechanisms of CAA pathology remain poorly understood. Here, we conducted brain regional proteomic analysis of early and late disease stages in the rTg-DI CAA rat model to gain molecular insight to mechanisms of CAA/CAA-ri progression and identify potential brain protein markers of CAA/CAA-ri. Longitudinal brain regional proteomic analysis revealed increased differentially expressed proteins (DEP) including ANXA3, HTRA1, APOE, CST3, and CLU, shared between the cortex, hippocampus, and thalamus, at both stages of disease in rTg-DI rats. Subsequent pathway analysis indicated pathway enrichment and predicted activation of TGF-β1, which was confirmed by immunolabeling and ELISA. Further, we identified numerous CAA related DEPs associate with astrocytes (HSPB1 and MLC1) and microglia (ANXA3, SPARC, TGF-β1) not previously associated with astrocytes or microglia in other AD models, possibly indicating that they are specific to CAA-ri. Thus, the data presented here identify several potential brain protein biomarkers of CAA/CAA-ri while providing novel molecular and mechanistic insight to mechanisms of CAA and CAA-ri pathological progression and glial cell mediated responses.

Exploring alveolar recruitability using positive end-expiratory pressure in mice overexpressing TGF-β1: a structure–function analysis

Pre-injured lungs are prone to injury progression in response to mechanical ventilation. Heterogeneous ventilation due to (micro)atelectases imparts injurious strains on open alveoli (known as volutrauma). Hence, recruitment of (micro)atelectases by positive end-expiratory pressure (PEEP) is necessary to interrupt this vicious circle of injury but needs to be balanced against acinar overdistension. In this study, the lung-protective potential of alveolar recruitment was investigated and balanced against overdistension in pre-injured lungs. Mice, treated with empty vector (AdCl) or adenoviral active TGF-β1 (AdTGF-β1) were subjected to lung mechanical measurements during descending PEEP ventilation from 12 to 0 cmH2O. At each PEEP level, recruitability tests consisting of two recruitment maneuvers followed by repetitive forced oscillation perturbations to determine tissue elastance (H) and damping (G) were performed. Finally, lungs were fixed by vascular perfusion at end-expiratory airway opening pressures (Pao) of 20, 10, 5 and 2 cmH2O after a recruitment maneuver, and processed for design-based stereology to quantify derecruitment and distension. H and G were significantly elevated in AdTGF-β1 compared to AdCl across PEEP levels. H was minimized at PEEP = 5–8 cmH2O and increased at lower and higher PEEP in both groups. These findings correlated with increasing septal wall folding (= derecruitment) and reduced density of alveolar number and surface area (= distension), respectively. In AdTGF-β1 exposed mice, 27% of alveoli remained derecruited at Pao = 20 cmH2O. A further decrease in Pao down to 2 cmH2O showed derecruitment of an additional 1.1 million alveoli (48%), which was linked with an increase in alveolar size heterogeneity at Pao = 2–5 cmH2O. In AdCl, decreased Pao resulted in septal folding with virtually no alveolar collapse. In essence, in healthy mice alveoli do not derecruit at low PEEP ventilation. The potential of alveolar recruitability in AdTGF-β1 exposed mice is high. H is optimized at PEEP 5–8 cmH2O. Lower PEEP folds and larger PEEP stretches septa which results in higher H and is more pronounced in AdTGF-β1 than in AdCl. The increased alveolar size heterogeneity at Pao = 5 cmH2O argues for the use of PEEP = 8 cmH2O for lung protective mechanical ventilation in this animal model.

Abstract CT109: Livmoniplimab and budigalimab combination therapy in treating patients with metastatic ovarian granulosa cell tumors: Results from dose escalation in a first-in-human study

Abstract Background: Ovarian granulosa cell tumors (OGC) are rare, accounting for 5-7% of all ovarian cancers and fatal when metastatic. There are no approved targeted therapies for metastatic OGC. Several agents with different targets, such as pembrolizumab, nirogacestat, orteronel, and STM 434, are being evaluated but to date have not shown clinical benefit. Most OGC carry a C134W mutation in the FOXL2 gene, which manifests its protumor effects via the transforming growth factor (TGF)-β signaling pathway. Livmoniplimab (livmo) is a monoclonal antibody (mAb) that specifically binds to the TGF-β1-glycoprotein-A repetitions predominant protein complex, blocking the release of active TGF-β1. Livmo has been assessed with or without budigalimab (budi), an anti-PD-1 mAb, in the dose escalation (ESC) part of a first-in-human study in patients (pts) with advanced solid tumors, including OGC. Herein, we report results of 4 pts with OGC enrolled into the combination ESC part of this phase 1 study (NCT03821935). Methods: Pts ≥18 years with ECOG PS 0-1 were enrolled in ESC, including pts with metastatic OGC refractory to/not eligible for standard therapies. The maximum tolerated dose was not reached, and pts received the maximum administered dose of 1500 mg livmo (Q2W IV) + 500 mg budi (Q4W IV). Safety and overall response rate (ORR) per RECIST v1.1 were assessed. Results: As of 28 Sep 2023, 57 pts were enrolled in ESC, including 4 with metastatic OGC aged 50-63 years who received 3-7 prior lines of systemic therapy. In total, 98% (n=56) pts in ESC experienced TEAEs, most commonly fatigue (42%), nausea (37%), and anemia (37%). One pt with OGC was enrolled in the study with metastatic target lesions in the lung, liver, and peritoneum at screening. She had received 7 prior lines of therapy including doxorubicin, bevacizumab, anastrozole, tamoxifen, and carboplatin + paclitaxel. Upon treatment with livmo + budi, she had a deep partial response (-92% from baseline) and a complete response in the target liver lesion. The pt stayed in response for ~2.5 years before death due to an event unrelated to study drugs. Furthermore, 2 other pts with metastatic OGC had a partial response (ORR=75%) and none of the 4 pts with OGC experienced disease progression (disease control rate=100%). Conclusions: Livmo + budi had promising antitumor efficacy and a tolerable safety profile in pts with metastatic OGC, a rare TGF-β signaling-dependent tumor. All enrolled pts with OGC showed tumor shrinkage and 1 pt had durable and near-complete response. Since single-agent pembrolizumab exhibited an ORR of 0% in pts with OGC (How et al. Invest New Drugs, 2021;39:829-835), we conclude that OGC is a proof-of-mechanism indication for livmo and that targeting TGF-β may represent an effective strategy for treating pts with OGC. Citation Format: Ildefonso Rodriguez Rivera, David Sommerhalder, Mohammad Sahtout, Cristiano Ferlini, Duyen Ngo, Sujata Jha, John Engelhardt, Anthony Tolcher. Livmoniplimab and budigalimab combination therapy in treating patients with metastatic ovarian granulosa cell tumors: Results from dose escalation in a first-in-human study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT109.

TGF-β, IL-1β, IL-6 levels and TGF-β/Smad pathway reactivity regulate the link between allergic diseases, cancer risk, and metabolic dysregulations.

The risk of cancer is higher in patients with asthma compared to those with allergic rhinitis for many types of cancer, except for certain cancers where a contrasting pattern is observed. This study offers a potential explanation for these observations, proposing that the premalignant levels of circulating transforming growth factor-β (TGF-β), IL-1β, and IL-6 as well as the reactivity of the TGF-β/Smad signaling pathway at the specific cancer site, are crucial factors contributing to the observed disparities. Circulating TGF-β, IL- β and IL-6 levels also help clarify why asthma is positively associated with obesity, Type 2 diabetes, hypertension, and insulin resistance, whereas allergic rhinitis is negatively linked to these conditions. Furthermore, TGF-β/Smad pathway reactivity explains the dual impact of obesity, increasing the risk of certain types of cancer while offering protection against other types of cancer. It is suggested that the association of asthma with cancer and metabolic dysregulations is primarily linked to the subtype of neutrophilic asthma. A binary classification of TGF-β activity as either high (in the presence of IL-1β and IL-6) or low (in the presence or absence of IL-1β and IL-6) is proposed to differentiate between allergy patients prone to cancer and metabolic dysregulations and those less prone. Glycolysis and oxidative phosphorylation, the two major metabolic pathways utilized by cells for energy exploitation, potentially underlie this dichotomous classification by reprogramming metabolic pathways in immune cells.

Armored TGFβRIIDN ROR1-CAR T cells reject solid tumors and resist suppression by constitutively-expressed and treatment-induced TGFβ1

BackgroundChimeric antigen receptor (CAR) T-cell therapy target receptor tyrosine kinase-like orphan receptor 1 (ROR1) is broadly expressed in hematologic and solid tumors, however clinically-characterized ROR1-CAR T cells with single chain...

Targeting TGF-β Activation in Cutaneous T-Cell Lymphomas

Targeting TGF-β Activation in Cutaneous T-Cell Lymphomas

Noncanonical WNT5A controls the activation of latent TGF-β to drive fibroblast activation and tissue fibrosis.

Transforming growth factor β (TGF-β) signaling is a core pathway of fibrosis, but the molecular regulation of the activation of latent TGF-β remains incompletely understood. Here, we demonstrate a crucial role of WNT5A/JNK/ROCK signaling that rapidly coordinates the activation of latent TGF-β in fibrotic diseases. WNT5A was identified as a predominant noncanonical WNT ligand in fibrotic diseases such as systemic sclerosis, sclerodermatous chronic graft-versus-host disease, and idiopathic pulmonary fibrosis, stimulating fibroblast-to-myofibroblast transition and tissue fibrosis by activation of latent TGF-β. The activation of latent TGF-β requires rapid JNK- and ROCK-dependent cytoskeletal rearrangements and integrin αV (ITGAV). Conditional ablation of WNT5A or its downstream targets prevented activation of latent TGF-β, rebalanced TGF-β signaling, and ameliorated experimental fibrosis. We thus uncovered what we believe to be a novel mechanism for the aberrant activation of latent TGF-β in fibrotic diseases and provided evidence for targeting WNT5A/JNK/ROCK signaling in fibrotic diseases as a new therapeutic approach.

Identification of unique rectal cancer-specific subtypes.

Existing colorectal cancer subtyping methods were generated without much consideration of potential differences in expression profiles between colon and rectal tissues. Moreover, locally advanced rectal cancers at resection often have received neoadjuvant chemoradiotherapy which likely has a significant impact on gene expression. We collected mRNA expression profiles for rectal and colon cancer samples (n = 2121). We observed that (i) Consensus Molecular Subtyping (CMS) had a different prognosis in treatment-naïve rectal vs. colon cancers, and (ii) that neoadjuvant chemoradiotherapy exposure produced a strong shift in CMS subtypes in rectal cancers. We therefore clustered 182 untreated rectal cancers to find rectal cancer-specific subtypes (RSSs). We identified three robust subtypes. We observed that RSS1 had better, and RSS2 had worse disease-free survival. RSS1 showed high expression of MYC target genes and low activity of angiogenesis genes. RSS2 exhibited low regulatory Tcell abundance, strong EMT and angiogenesis signalling, and high activation of TGF-β, NF-κB, and TNF-α signalling. RSS3 was characterised by the deactivation of EGFR, MAPK and WNT pathways. We conclude that RSS subtyping allows for more accurate prognosis predictions in rectal cancers than CMS subtyping and provides new insight into targetable disease pathways within these subtypes.

FAT1 upregulation is correlated with an immunosuppressive tumor microenvironment and predicts unfavorable outcome of immune checkpoint therapy in non-small cell lung cancer

BackgroundPrevious studies found that FAT1 was recurrently mutated and aberrantly expressed in multiple cancers, and the loss function of FAT1 promoted the formation of cancer-initiating cells in several cancers. However, in some types of cancer, FAT1 upregulation could lead to epithelial-mesenchymal transition (EMT). The role of FAT1 in cancer progression, which appears to be cancer-type-specific, is largely unknown. MethodsQRT-PCR and immunochemistry were used to verify the expression of FAT1 in non-small cell lung cancer (NSCLC). QRT-PCR and Western blot were used to detect the influence of siFAT1 knockdown on the expression of potential targets of FAT1 in NSCLC cell lines. GEPIA, KM-plotter, CAMOIP, and ROC-Plotter were used to evaluate the association between FAT1 and clinical outcomes based on expression and clinical data from TCGA and immune checkpoint inhibitors (ICI) treated cohorts. ResultsWe found that FAT1 upregulation was associated with the activation of TGF-β and EMT signaling pathways in NSCLC. Patients with a high FAT1 expression level tend to have a poor prognosis and hard to benefit from ICI therapy. Genes involved in TGF-β/EMT signaling pathways (SERPINE1, TGFB1/2, and POSTN) were downregulated upon knockdown of FAT1. Genomic and immunologic analysis showed that high cancer-associated fibroblast (CAF) abundance, decreased CD8+ T cells infiltration, and low TMB/TNB were correlated with the upregulation of FAT1, thus promoting an immunosuppressive tumor microenvironment (TME) which influence the effect of ICI-therapy. ConclusionOur findings revealed the pattern of FAT1 upregulation in the TME of patients with NSCLC, and demonstrated its utility as a biomarker for unfavorable clinical outcomes, thereby providing a potential therapeutic target for NSCLC treatment.

Abstract 6349: 2MW4991, a novel ADCC-enhanced integrin αvβ8 blocker, exhibits high anti-tumor potency and was well tolerated in cynomolgus monkeys

Abstract Background: The three highly structurally related TGF-β isoforms (TGF-β1, TGF-β2, and TGF-β3) exert greatly different effects on cancer and immune cells under various physiological conditions. Efforts to target the TGF-β pathway have been hampered by systemic toxicity or limited efficacy due to challenges associated with the global inhibition of TGF-βs. Recently, TGF-β1 was proven to be a critical player in generating an immunosuppressive tumor microenvironment (TME). TGF-β1 is expressed as an inactive latent form (L-TGF-β1) and activated upon binding to integrins, and integrin αvβ8 is a key activator of TGF-β1 in TME. Here, an ADCC-enhanced αvβ8 blocking antibody, 2MW4991, was developed to specifically inhibit TGF-β1-mediated signaling for the treatment of αvβ8-expressing cancers. Methods: Binding ability, specificity, and affinity of antibodies were measured by Flow cytometry and Biacore T200. A blocking assay was performed using U251MG cells and TGF-β signaling reporter cells. Jurkat/h FcγRIIIa reporter system was used to assess the ADCC activity of 2MW4991. Mouse syngeneic tumor models were used to characterize the in vivo anti-tumor efficacy. A repeat dose administration study was performed in cynomolgus monkeys to evaluate the pharmacokinetic and toxicity profiles of 2MW4991. Results: ScRNA-seq analysis of kidney renal clear cell carcinoma (KIRC) and head and neck squamous cell carcinoma (HNSCC) clinical samples revealed that αvβ8 highly expresses in tumor and some myeloid-derived cells, suggesting the development of ADCC-enhanced αvβ8 blocking antibodies for the treatment of αvβ8-expressing cancers. 2MW4991 showed sub-nanomolar binding affinity and high specificity to αvβ8, with no cross-reactivity to other integrins. 2MW4991 could effectively block TGF-β1 release in a GARP/L-TGF-β1/αvβ8 reporter system. In an immune-excluded EMT6 syngeneic model, 2MW4991 promoted substantial tumor regression and reversed immune suppressive TME by significantly increasing tumor infiltration of CD8+ T and innate immune cells. As expected, TGF-β1 blockade significantly increased the sensitivity of anti-PD-1 therapy in a combination study. Furthermore, 2MW4991 displayed potent anti-tumor efficacy in the human αvβ8 knock-in syngeneic model mimicking αvβ8 expression in human cancers. In a repeat dose administration NHP study, 2MW4991 showed a good pharmacokinetic profile and was well tolerated at a high dose of 100 mg/kg, accompanied by significantly declined pharmacodynamic biomarker TGF-β1 in peripheral blood. Conclusion: Integrin αvβ8 is a main activator of TGF-β1 in the TME and overexpressed in some cancer types. 2MW4991 demonstrated potent anti-tumor activity by unleashing the immune cells suppressed by TGF-β1 in TME and Fc-mediated immune functions. These compelling preclinical results support further evaluation of 2MW4991 in clinical trials. Citation Format: Cuicui Guo, Zhen Han, Xiaowei Cen, Hai Wu, Jinchao Zhang, Xun Gui. 2MW4991, a novel ADCC-enhanced integrin αvβ8 blocker, exhibits high anti-tumor potency and was well tolerated in cynomolgus monkeys [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6349.

Abstract 5198: Non-invasive biomarkers of ECM turnover are prognostic for combinations of checkpoint inhibition immunotherapy in solid tumors

Abstract Background: Immune checkpoint inhibitors (ICIs) are investigated in many different combinations (Table 1). As only a fraction of patients responds to ICIs, prognostic biomarkers are needed to assess the likelihood of benefit. High collagen turnover in the tumor microenvironment are closely related to response to ICIs and survival outcomes. In this study, we explored the clinical utility of non-invasive biomarkers of type I collagen (reC1M), type III collagen (PRO-C3), type IV collagen (C4M), type XIX collagen (PRO-C19), type XX collagen (PRO-C20), TGF-β activity in metastatic cancer patients treated with ICIs. Methods: PRO-C3, PRO-C19, PRO-C20, TGF-β, reC1M and C4M were measured with ELISAs in pre-treatment serum from 33 patients with metastatic disease in a basket trial comprising 14 different tumor types, treated with 8 different checkpoint inhibition regimens (Table 1). The association between biomarker levels and overall survival (OS) outcome was evaluated by Kaplan-Meier analysis and Cox regression analysis after dichotomizing patients at the 75th percentile (Q4). Results: When evaluating the combined group of cancer patients, high levels (Q4) of PRO-C3 (HR=3.00, 95%CI=1.21-7.44, p=0.018), PRO-C19 (HR=2.49, 95%CI=1.04-5.99, p=0.041), PRO-C20 (HR=2.49, 95%CI=1.04-5.99, p=0.041), TGF-β (HR=2.52, 95%CI=1.05-6.04, p=0.039), reC1M (HR=2.79, 95%CI=1.16-6.67, p=0.021) and C4M (HR=2.52, 95%CI=1.05-6.04, p=0.039) were all associated with poor OS compared to low levels (Q1-Q3). The median OS was 4.7-6.1 months in biomarker-high patients compared to 8.7-11.4 in biomarker-low patients. Conclusion: Across a diverse cohort of metastatic cancer patients treated with different checkpoint inhibition regimens, non-invasive biomarkers associated with tumor fibrosis and collagen turnover (PRO-C3, PRO-C19, PRO-C20, TGF-β, reC1M and C4M) could identify cancer patients with poor prognosis. Table 1. Cancer type Number of patients Treatment Esophageal 1 Nivolumab (Nivo) +Anti-LAG3 Breast 2 Carboplatine+Gemciabine+Pembrolizumab (Pembro) Ovarian 4 Atezolizumab+BET inhibitor Parotid 1 Nivo Gastric 2 Nivo+Anti-LAG3 or Atezolizumab+CD40-agonist Bladder 3 Pembro Colorectal 5 Nivo, Pembro, Atezolizumab++bispecific antibody targeting CEA and CD3 or Atezolizumab+CD40-agonist Hepatocellular carcinoma 3 Nivo+Anti-LAG3 Colorectal 1 Atezolizumab+bispecific antibody targeting CEA and CD3 Mesothelioma 2 Pembro or Atezolizumab+CD40-agonist Neuroendocrine tumor 1 Pembro non-small cell 5 Nivo, Pembro or ipilumumab+Nivolumab Pancreas 1 Atezolizumab+CD40-agonist Unknown primary 2 Atezolizumab++bispecific antibody targeting CEA and CD3 or Pembro Citation Format: Caroline N. Bistrup, Vinicius Araujo Barbosa de Lima, Christina Jensen, Morten A. Karsdal, Inge Marie Svane, Ulrik Lassen, Nicholas Willumsen. Non-invasive biomarkers of ECM turnover are prognostic for combinations of checkpoint inhibition immunotherapy in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5198.

Abstract 6528: Integrin ɑvβ6 upregulation as a mechanism of T-cell evasion in head and neck squamous cell carcinoma

Abstract Head and neck squamous cell carcinoma (HNSCC) encompasses malignancies of the mucosal epithelium in the oral cavity, pharynx, and larynx. The lack of effective screening strategies and the typically late stage of detection are contributors to a considerable annual death toll of HNSCC. Additionally, the anatomical proximity of the malignancy to delicate structures of the head and neck leads to tremendous treatment-related morbidity. Therefore, there is great interest in developing life-saving therapies and strategies for treatment de-escalation. The immune checkpoint inhibitor pembrolizumab (αPD-1) has been approved for recurrent or metastatic HNSCC and as a frontline therapy for unresectable disease. However, only about a quarter of patients respond to αPD-1 therapy. The immunosuppressive effect of TGFβ has been widely described as relevant to the HNSCC tumor microenvironment. However, attempts to blockade TGFβ have failed mainly due to the widespread side effects of disrupting a highly promiscuous cytokine. Integrin αvβ6 is a major activator of the inert latent TGFβ into the active, immunosuppressive form. With its high relative specificity to HNSCC tissue, inhibition of αvβ6 could offer a safer approach for disrupting TGFβ to overcome resistance to immune checkpoint blockade. After screening ɑvβ6 expression levels of HNSCC cell lines via flow cytometry, cell lines FaDu and CAL27 were selected for further investigation due to high endogenous ITGB6 expression (FaDu 35.57% cells ITGB6+, CAL27 95.69% cells ITGB6+). The cell lines were transduced with a doxycycline-inducible short hairpin RNA (shRNA) knockdown of αvβ6 using a lentiviral system. Both knockdown and control shRNA cell lines were pretreated with 1 μg/mL of doxycycline for five days to induce the shRNA and subsequently co-cultured with TALL-104 T-cells. Quantitative fluorescence microscopy of the co-culture revealed that the knockdown of αvβ6 substantially increased T-cell killing over 24hrs (FaDu CTRL shRNA 6.72%±2.49 dead vs. FaDu ITGB6 shRNA 12.80%±3.16 dead, p<0.001). However, treatment of FaDu and CAL27 cell lines with active-TGFβ showed no change in PD-L1 expression via flow cytometric analysis. To determine whether the T cell evasive effect of αvβ6 is driven by other immune checkpoints on the cancer cells or by the direct effect that TGFβ has on immune cells, we show the generation of relevant mouse αvβ6 shRNA knockdown cells. These syngeneic in vivo studies will facilitate cytokine profiling to uncover the dynamics of tumor-immune cell interactions in response to αvβ6 inhibition. Citation Format: William J. MacDonald, Praveen R. Srinivasan, Maximilian Pinho-Schwermann, Shengliang Zhang, Vida Tajiknia, Connor Purcell, Jillian Strandberg, Nolan Stubbs, Wafik S. El-Deiry. Integrin ɑvβ6 upregulation as a mechanism of T-cell evasion in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6528.

Abstract 5140: ITGB6 as a predictive biomarker for overall prognosis and PD-(L)1 immune checkpoint blockade response in various cancer types

Abstract Our previous work studying the effects of integrin αvβ6 knockdown in cancer cells demonstrated αvβ6 as a method of T cell suppression in a co-culture model. ITGB6, the gene encoding the β6 subunit of αvβ6, is a potent prognostic marker across multiple cancer types. As a major activator of latent TGFβ, αvβ6, and consequently, ITGB6, has considerable therapeutic implications due to the immunosuppressive effect that activated TGFβ has on the tumor microenvironment. A Human Protein Atlas search of ITGB6 expression surveyed the upregulation of ITGB6 across cancer types. Cancer ITGB6 expression was then compared to expression in paired adjacent normal tissue using the TNMplot tool (Bartha, 2021) on a concatenated set of GEO, GTex, TCGA, and TARGET RNA-seq databases. Kaplan-Meier curves were generated using TCGA mRNA data through the cBio portal, splitting the cohorts according to an ITGB6 mRNA expression threshold of one standard deviation above the mean (z-score ± 1.0). ITGB6 expression and immune checkpoint blockade (ICB) response were evaluated with the Kaplan-Meier Plotter tool (Kovacs, 2023). ITGB6 expression levels between ICB responders and non-responders were quantified using the ROC Plotter tool (Fekete, 2023), and the figures were generated using Matplotlib in Python. Lung, bladder, head and neck, pancreatic, and cervical cancer all demonstrated high expression of ITGB6. Head and neck squamous cell carcinoma (median fold change 2.70, p<0.0001), bladder urothelial carcinoma (FC 2.41, p<0.001), cholangiocarcinoma (FC 5.33, p<0.01), and esophageal carcinoma (FC 2.72, p<0.03) showed strong upregulation of ITGB6 compared to normal tissue. Interestingly, lung adenocarcinoma (FC 0.77, p<0.04) and lung squamous cell carcinoma (FC 0.55, p<0.0001) showed downregulation of ITGB6 compared to normal tissue. Survival data revealed that ITGB6 was a potent marker of a poor prognosis in pancreatic adenocarcinoma (high ITGB6 median survival 16.79 mos, vs. low ITGB6 21.88 mos, p<0.004) and head and neck squamous cell carcinoma (high ITGB6 30.06 mos vs. low ITGB6 57.42 mos, p<0.03). However, no significant prognostic difference was observed in non-small cell lung cancers. Finally, high pan-cancer expression of ITGB6 led to poorer response to αPD-1 (high ITGB6 Hazard Ratio 1.37, p<0.05) and αPD-L1 (high ITGB6 HR 1.50, p<0.001). In vitro and in vivo experiments to validate the benefit of ITGB6 as a biomarker and as a therapeutic target are underway. Citation Format: William J. MacDonald, Praveen R. Srinivasan, Maximilian Pinho-Schwermann, Vida Tajiknia, Connor Purcell, Lindsey Carlsen, Wafik S. El-Deiry. ITGB6 as a predictive biomarker for overall prognosis and PD-(L)1 immune checkpoint blockade response in various cancer types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5140.

Abstract 710: Post-exposure suppression of radiation pneumonitis suggests non-redundant independent effects of TGF-beta and TRAIL/DR5

Abstract Radiation pneumonitis is one of the most common but challenging toxicities seen in patients that are receiving ionizing radiotherapy for thoracic malignancies. While radiation effectively eliminates cancer cells, the damage it causes to surrounding healthy lung tissue leads to an immune response that can impact treatment regimen and lead to long term fibrotic tissue formation. The mechanism of radiation pneumonitis and the inflammatory response in the lungs is poorly understood. However, it is known that the TRAIL pathway plays an important role in inflammatory and fibrotic response. Through our previous work, it was discovered that modulating the TRAIL pathway via pegylated recombinant long-acting TRAIL (TLY012) in both WT and TRAIL-/- C57Bl/6 could suppress radiation pneumonitis in mice that were exposed to 20 Gy of thoracic radiation with shielding of other organs as early two weeks post exposure. In addition to the TRAIL pathway, transforming growth factor-beta (TGF-β) is a cytokine that is known to play a key role in fibrosis. Broad spectrum integrin inhibitor GLPG0187 is known to inhibit the activation of TGF-β via binding to specific integrin receptors. In order to determine if the inhibition of TGF-β could suppress radiation pneumonitis without stimulation of the TRAIL pathway, DR5 null mice were utilized for the experiment. Male DR5-/- C57Bl/6 mice received a single 20 Gy thoracic radiation dose with shielding of other organs and were treated with 100 mg/kg of GLPG-0187 or control twice a week via IP injection with the first dose being administered 1 hour before radiation (n = 4/treatment/group). 13 days post-irradiation, lungs, sternal bone marrow, and peripheral serum were collected. Upon histological analysis, it was observed that there were thinner alveolar borders and lessened inflammation compared to the control mice. Mice were weighed every three days, and it was discovered that mice treated with GLPG-0187 maintained steady weight compared to the control group. Additional analysis including immunohistochemistry, cytokine profiling, and quantification needs to be conducted. As the TRAIL pathway could not be modulated in the DR5 null mice, these findings suggest that GLPG-0187 was able to suppress radiation pneumonitis via inhibition of TGF-β. While some rescue from radiation pneumonitis was observed via GLPG0187, it was to a lesser extent compared to treatment of WT and TRAIL-/- C57Bl/6 mice with TLY012. Future directions include investigating the synergistic effects between TLY012 and GLPG0187 in suppressing radiation pneumonitis and decreasing fibrotic response to radiation. Citation Format: Jillian Strandberg, Anna Louie, William MacDonald, Praveen Srinivasan, Leiqing Zhang, Lanlan Zhou, Seulki Lee, Wafik El-Deiry. Post-exposure suppression of radiation pneumonitis suggests non-redundant independent effects of TGF-beta and TRAIL/DR5 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 710.

Abstract 4554: Novel combination therapy for treating proteasome inhibitor-resistant multiple myeloma

Abstract Multiple myeloma (MM) is an incurable plasma cell malignancy characterized by osteolytic bone disease and immunosuppression. Proteasome inhibitors (PIs) have remarkably improved the survival of MM patients, but dose-limiting toxicities and the development of drug resistance limit their long-term utility. Elevated TGF-β levels in MM patient sera correlate with drug resistance, disease progression, metastasis and poor prognosis. Therefore, we evaluated the anti-MM therapeutic potential of a TGF-β type I receptor kinase inhibitor, Vactosertib. In vitro treatment of Vactosertib synergistically inhibited the growth of bortezomib (BTZ)-resistant MM cells in combination with either BTZ or ixazomib (IXZ) by suppressing TGF-β activation of Smad2/3 and expression of PSMB5, which encodes the proteasome 5 catalytic subunit targeted by PIs. Oral administration of Vactosertib as a single agent decreased MM progression and mortality of the mice bearing BTZ-resistant MM. Vactosertib alone also attenuated PSMB5 expression and proteasome activity, reduced the expansion of CD11b+Gr-1+ myeloid derived suppressor cells (MDSCs) in bone marrow (BM) tumor microenvironment (TME), and diminished the population of Foxp3+ regulatory T cells (Treg) in the spleen. Combination therapy of Vactosertib plus PI (BTZ or IXZ) exhibited a synergistic anti-myeloma effect when compared to either Vactosertib or PI alone by greatly prolonged survival and significant a reduction in both MDSCs and Tregs. Furthermore, therapy of BCMA+ CAR T cells in combination with Vactosertib exhibited a synergistic anti-tumor effect, compared to either CAR T cells or Vactosertib. Taken together, our data provide the rationale for clinical evaluation of Vactosertib in MM and demonstrate proof-of-concept that combination of Vactosertib and either PI or cell therapy may overcome drug resistance and enhance durable patient responses. Citation Format: Byung-Gyu Kim, Sung Hee Choi, Huong Nguyen, Fu-Sen Liang, Seong-Jin Kim, John Letterio, Alex Huang. Novel combination therapy for treating proteasome inhibitor-resistant multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4554.

Abstract 5398: Single-cell transcriptomic analysis of partial endothelial-to-mesenchymal transition in sprouting angiogenesis

Abstract Endothelial-to-mesenchymal transition (EndoMT) is a process of morphological, functional, and molecular shift of endothelial cells (EC) towards a mesenchymal identity. This process is critical for embryonic development, and knockout animals lacking copies of the individual master EndoMT transcription factors – including Slug, Snail, and Twist – exhibit significant cardiovascular defects. We previously showed that while Slug knockout animals have a modest developmental vascular phenotype, there is a striking loss of tumor angiogenesis in these animals. Based on these and other data, we hypothesize that sprouting angiogenesis involves transient acquisition of an intermediate state of the EndoMT program –i.e., partial EndoMT (pEndoMT). To test this hypothesis, we used single cell RNA sequencing at day 2 and day 6 following TGF-β activation to map the transcriptomic changes associated with complete endothelial-to-mesenchymal transition. In parallel, we sequenced EC activated to undergo sprouting angiogenesis in vitro at the same timepoints. We show that angiogenic EC transiently acquire a transcriptomic profile of the pEndoMT state, but then later reacquire the transcriptomic profile of committed EC rather than transition completely into mesenchymal cells. We further use this dataset to identify several candidate genes that appear to be regulated by Slug and associated with the pEndoMT state. Future work will use in vitro and in vivo angiogenesis models to identify how pEndoMT is regulated in angiogenesis, and how this regulation may differ in the presence of tumor stroma. Citation Format: Kapil Thapa, Christopher J. Hatch, Christopher Hughes, Jennifer S. Fang. Single-cell transcriptomic analysis of partial endothelial-to-mesenchymal transition in sprouting angiogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5398.

Nintedanib downregulates the profibrotic M2 phenotype in cultured monocyte-derived macrophages obtained from systemic sclerosis patients affected by interstitial lung disease

BackgroundSystemic sclerosis (SSc) is an autoimmune connective tissue disease characterized by vasculopathy and progressive fibrosis of skin and several internal organs, including lungs. Macrophages are the main cells involved in the immune-inflammatory damage of skin and lungs, and alternatively activated (M2) macrophages seem to have a profibrotic role through the release of profibrotic cytokines (IL10) and growth factors (TGFβ1). Nintedanib is a tyrosine kinase inhibitor targeting several fibrotic mediators and it is approved for the treatment of SSc-related interstitial lung disease (ILD). The study aimed to evaluate the effect of nintedanib in downregulating the profibrotic M2 phenotype in cultured monocyte-derived macrophages (MDMs) obtained from SSc-ILD patients.MethodsFourteen SSc patients, fulfilling the 2013 ACR/EULAR criteria for SSc, 10 SSc patients affected by ILD (SSc-ILD pts), 4 SSc patients non affected by ILD (SSc pts no-ILD), and 5 voluntary healthy subjects (HSs), were recruited at the Division of Clinical Rheumatology-University of Genova, after obtaining Ethical Committee approval and patients’ informed consent. Monocytes were isolated from peripheral blood, differentiated into MDMs, and then maintained in growth medium without any treatment (untreated cells), or treated with nintedanib (0.1 and 1µM) for 3, 16, and 24 h. Gene expression of macrophage scavenger receptors (CD204, CD163), mannose receptor-1 (CD206), Mer tyrosine kinase (MerTK), identifying M2 macrophages, together with TGFβ1 and IL10, were evaluated by quantitative real-time polymerase chain reaction. Protein synthesis was investigated by Western blotting and the level of active TGFβ1 was evaluated by ELISA. Statistical analysis was carried out using non-parametric Wilcoxon test.ResultsCultured untreated SSc-ILD MDMs showed a significant increased protein synthesis of CD206 (p < 0.05), CD204, and MerTK (p < 0.01), together with a significant upregulation of the gene expression of MerTK and TGFβ1 (p < 0.05; p < 0.01) compared to HS-MDMs. Moreover, the protein synthesis of CD206 and MerTK and the gene expression of TGFβ1 were significantly higher in cultured untreated MDMs from SSc-ILD pts compared to MDMs without ILD (p < 0.05; p < 0.01). In cultured SSc-ILD MDMs, nintedanib 0.1 and 1µM significantly downregulated the gene expression and protein synthesis of CD204, CD206, CD163 (p < 0.05), and MerTK (p < 0.01) compared to untreated cells after 24 h of treatment. Limited to MerTK and IL10, both nintedanib concentrations significantly downregulated their gene expression already after 16 h of treatment (p < 0.05). In cultured SSc-ILD MDMs, nintedanib 0.1 and 1µM significantly reduced the release of active TGFβ1 after 24 h of treatment (p < 0.05 vs. untreated cells).ConclusionsIn cultured MDMs from SSc-ILD pts, nintedanib seems to downregulate the profibrotic M2 phenotype through the significant reduction of gene expression and protein synthesis of M2 cell surface markers, together with the significant reduction of TGFβ1 release, and notably MerTK, a tyrosine kinase receptor involved in lung fibrosis.

Nickel induces epithelial-mesenchymal transition in pulmonary fibrosis in mice via activation of the oxidative stress-mediated TGF-β1/Smad signaling pathway.

Nickel (Ni) is recognized as a carcinogenic metal, and its widespread use has led to severe environmental and health problems. Although the lung is among the main organs affected by Ni, the precise mechanisms behind this effect remain poorly understood. This study aimed to elucidate the physiological mechanisms underlying Ni-induced pulmonary fibrosis (PF), using various techniques including histopathological detection, biochemical analysis, immunohistochemistry, western blotting, and quantitative real-time PCR. Mice were treated with nickel chloride (NiCl2), which induced PF (detected by Masson staining), up-regulation of α-smooth muscle actin (α-SMA), and collagen-1 mRNA and protein expression. NiCl2 was found to induce PF by: activation of the epithelial-mesenchymal transition (EMT) and the transforming growth factor-β1 (TGF-β1)/Smad signaling pathway; up-regulation of protein and mRNA expression of TGF-β1, p-Smad2, p-Smad3, vimentin, and N-cadherin; and down-regulation of protein and mRNA expression of E-cadherin. In addition, NiCl2 treatment increased malondialdehyde content while inhibiting antioxidant activity, as indicated by decreased catalase, total antioxidant capacity, and superoxide dismutase activities, and glutathione content. Co-treatment with the effective antioxidant and free radical scavenger N-acetyl cysteine (NAC) plus NiCl2 was used to study the effects of oxidative stress in NiCl2-induced PF. The addition of NAC significantly mitigated NiCl2-induced PF, and reversed activation of the TGF-β1/Smad signaling pathway and EMT. NiCl2-induced PF was therefore shown to be due to EMT activation via the TGF-β1/Smad signaling pathway, mediated by oxidative stress.

Trans-2-nonadecyl-4-(hydroxymethyl)-1,3-dioxolane (TNHD) purified from freshwater clams markedly alleviates dimethylnitrosamine-induced hepatic fibrosis.

Liver fibrosis occurs due to injury or inflammation, which results in the excessive production of collagen and the formation of fibrotic scar tissue that impairs liver function. Despite the limited treatment options available, freshwater clams may hold promise in the treatment of liver fibrosis. In this study, we demonstrated the effects of ethanol extract of freshwater clam (FCE), ethyl acetate extract of FCE (EA-FCE), and trans-2-nonadecyl-4-(hydroxymethyl)-1,3-dioxolane (TNHD) on liver fibrosis induced by dimethylnitrosamine (DMN). Administration of FCE and TNHD alleviated liver injury, including tissue damage, necrosis, inflammation scores, fibrosis scores, serum enzymes, and triglyceride levels. Furthermore, we analyzed the expression of fibrosis-related proteins, such as α-smooth muscle actin (α-SMA) and transforming growth factor (TGF-β), as well as the hydroxyproline content, which decreased after treatment with FCE and TNHD. Animal experiments revealed that FCE and TNHD can reduce liver fibrosis by inhibiting cytokines that activate stellate cells and decreasing extracellular matrix (ECM) secretion. Cell experiments have shown that TNHD inhibits the MAPK/Smad signaling pathway and TGF-β1 activation, resulting in a reduction in the expression of fibrosis-related proteins. Therefore, freshwater clam extracts, particularly TNHD, may have potential therapeutic and preventive effects for the amelioration of liver fibrosis.

Organosulfur Compounds in Colorectal Cancer Prevention and Progression.

This work represents an overview of the current investigations involving organosulfur compounds and colorectal cancer. The molecules discussed in this review have been investigated regarding their impact on colorectal cancer directly, at the in vitro, in vivo, and clinical stages. Organosulfur compounds may have indirect effects on colorectal cancer, such as due to their modulating effects on the intestinal microbiota or their positive effects on intestinal mucosal health. Here, we focus on their direct effects via the repression of multidrug resistance proteins, triggering of apoptosis (via the inhibition of histone deacetylases, increases in reactive oxygen species, p53 activation, β-catenin inhibition, damage in the mitochondrial membrane, etc.), activation of TGF-β, binding to tubulin, inhibition of angiogenesis and metastasis mechanisms, and inhibition of cancer stem cells, among others. In general, the interesting positive effects of these nutraceuticals in in vitro tests must be further analyzed with more in vivo models before conducting clinical trials.