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Supplementary Data from Systemic Immune Dysfunction in Cancer Patients Driven by IL6 Induction of LAG3 in Peripheral CD8+ T Cells

Many cancer patients do not develop a durable response to the current standard-of-care immunotherapies, despite substantial advances in targeting immune inhibitory receptors. A potential compounding issue, which may serve as an unappreciated, dominant resistance mechanism, is an inherent systemic immune dysfunction that is often associated with advanced cancer. Minimal response to inhibitory receptor (IR) blockade therapy and increased disease burden have been associated with peripheral CD8+ T-cell dysfunction, characterized by suboptimal T-cell proliferation and chronic expression of IRs (e.g., PD1 and LAG3). Here, we demonstrated that approximately a third of cancer patients analyzed in this study have peripheral CD8+ T cells that expressed robust intracellular LAG3 (LAG3IC), but not surface LAG3 (LAG3SUR) due to a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) cleavage. This is associated with poor disease prognosis and decreased CD8+ T-cell function, which could be partially reversed by anti-LAG3. Systemic immune dysfunction was restricted to CD8+ T cells, including, in some cases, a high percentage of peripheral naïve CD8+ T cells, and was driven by the cytokine IL6 via STAT3. These data suggest that additional studies are warranted to determine if the combination of increased LAG3IC in peripheral CD8+ T cells and elevated systemic IL6 can serve as predictive biomarkers and identify which cancer patients may benefit from LAG3 blockade.

Supplementary Table 1

Supplementary Figure 1: FGF2 treatment induces branching morphogenesis of primary epithelial cells in 3D culture. Supplementary Figure 2: Basal Expression Levels of TWIST1 in KRAS mutant and MET Amplified/Mutant NSCLC Cell Lines. Supplementary Figure 3: Harmine treatment induces Oncogene-Induced Senescence (OIS) in EGFR and MET-mutant NSCLC cell lines. Supplementary Figure 4: Harmine induces apoptosis in oncogene-driven NSCLC cell lines. Supplementary Figure 5: Harmine treatment promotes TWIST1 degradation and decreases TWIST1 protein stability. Supplementary Figure 6: Silencing of E2A induces apoptosis and phenocopies silencing of TWIST1. Supplementary Figure 7: Overexpression of TWIST1 or its binding partner, E2A, rescues harmine induced growth inhibition. Supplementary Figure 8: Treatment with harmine decreases tumor growth in a KRAS mutant Patient-Derived Xenograph (PDX) model and degrades Twist1 and induces apoptosis in transgenic mouse model of Kras mutant lung cancer. Supplementary Table 1: Rank list of compounds from Connectivity mapping (CMAP) analysis Supplementary Table 2: List of primers used for qRT-PCR Supplementary Table 3: List of primers used for Taqman qRT-PCR Supplementary Table 4: List of antibodies used in current study Supplementary Table 5: Sequences for TWIST1/TCF3 shRNA (5’ â€" 3’) in pKLO.1 Supplementary Table 6: ORFs obtained from Johns Hopkins University HiT Center Supplementary Table 7: Source of Plasmids utilized

Supplementary Figure legends and methods

TWIST1, an epithelial–mesenchymal transition (EMT) transcription factor, is critical for oncogene-driven non–small cell lung cancer (NSCLC) tumorigenesis. Given the potential of TWIST1 as a therapeutic target, a chemical–bioinformatic approach using connectivity mapping (CMAP) analysis was used to identify TWIST1 inhibitors. Characterization of the top ranked candidates from the unbiased screen revealed that harmine, a harmala alkaloid, inhibited multiple TWIST1 functions, including single-cell dissemination, suppression of normal branching in 3D epithelial culture, and proliferation of oncogene driver-defined NSCLC cells. Harmine treatment phenocopied genetic loss of TWIST1 by inducing oncogene-induced senescence or apoptosis. Mechanistic investigation revealed that harmine targeted the TWIST1 pathway through its promotion of TWIST1 protein degradation. As dimerization is critical for TWIST1 function and stability, the effect of harmine on specific TWIST1 dimers was examined. TWIST1 and its dimer partners, the E2A proteins, which were found to be required for TWIST1-mediated functions, regulated the stability of the other heterodimeric partner posttranslationally. Harmine preferentially promoted degradation of the TWIST1-E2A heterodimer compared with the TWIST-TWIST1 homodimer, and targeting the TWIST1-E2A heterodimer was required for harmine cytotoxicity. Finally, harmine had activity in both transgenic and patient-derived xenograft mouse models of KRAS-mutant NSCLC. These studies identified harmine as a first-in-class TWIST1 inhibitor with marked anti-tumor activity in oncogene-driven NSCLC including EGFR mutant, KRAS mutant and MET altered NSCLC.Implications: TWIST1 is required for oncogene-driven NSCLC tumorigenesis and EMT; thus, harmine and its analogues/derivatives represent a novel therapeutic strategy to treat oncogene-driven NSCLC as well as other solid tumor malignancies. Mol Cancer Res; 15(12); 1764–76. ©2017 AACR.

Many cancer patients do not develop a durable response to the current standard-of-care immunotherapies, despite substantial advances in targeting immune inhibitory receptors. A potential compounding issue, which may serve as an unappreciated, dominant resistance mechanism, is an inherent systemic immune dysfunction that is often associated with advanced cancer. Minimal response to inhibitory receptor (IR) blockade therapy and increased disease burden have been associated with peripheral CD8+ T-cell dysfunction, characterized by suboptimal T-cell proliferation and chronic expression of IRs (e.g., PD1 and LAG3). Here, we demonstrated that approximately a third of cancer patients analyzed in this study have peripheral CD8+ T cells that expressed robust intracellular LAG3 (LAG3IC), but not surface LAG3 (LAG3SUR) due to a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) cleavage. This is associated with poor disease prognosis and decreased CD8+ T-cell function, which could be partially reversed by anti-LAG3. Systemic immune dysfunction was restricted to CD8+ T cells, including, in some cases, a high percentage of peripheral naïve CD8+ T cells, and was driven by the cytokine IL6 via STAT3. These data suggest that additional studies are warranted to determine if the combination of increased LAG3IC in peripheral CD8+ T cells and elevated systemic IL6 can serve as predictive biomarkers and identify which cancer patients may benefit from LAG3 blockade.

The success of immunotherapy in oncology underscores the vital role of the immune system in cancer development. Regulatory T cells (Tregs) maintain a fine balance between autoimmunity and immune suppression. They have multiple roles in the tumor microenvironment (TME) but act particularly in suppressing T cell activation. This review focuses on the detrimental and sometimes beneficial roles of Tregs in tumors, our current understanding of recruitment and stabilization of Tregs within the TME, and current Treg-targeted therapeutics. Research identifying subpopulations of Tregs and their respective functions and interactions within the complex networks of the TME will be crucial to develop the next generation of immunotherapies. Through these advances, Treg-targeted immunotherapy could have important implications for the future of oncology.

The coinhibitory receptor lymphocyte activation gene 3 (LAG-3) is an immune checkpoint molecule that negatively regulates T cell activation, proliferation, and homeostasis. Blockade or deletion of LAG-3 in autoimmune-prone backgrounds or induced-disease models has been shown to exacerbate disease. We observed significantly fewer LAG-3+ CD4 and CD8 T cells from subjects with relapsing-remitting multiple sclerosis (RRMS) and type 1 diabetes. Low LAG-3 protein expression was linked to alterations in mRNA expression and not cell surface cleavage. Functional studies inhibiting LAG-3 suggest that in subjects with RRMS, LAG-3 retains its ability to suppress T cell proliferation. However, LAG-3 expression was associated with the expression of markers of apoptosis, indicating a role for low LAG-3 in T cell resistance to cell death. In T cells from subjects with RRMS, we observed a global dysregulation of LAG-3 expression stemming from decreased transcription and persisting after T cell stimulation. These findings further support the potential clinical benefits of a LAG-3 agonist in the treatment of human autoimmunity.

534 Background: HCC is an aggressive cancer as a sequela of cirrhosis. For pts with no extrahepatic metastases and well-compensated liver function, Y90 radioembolization is a therapeutic option. However, high-risk patients with macrovascular invasion (MVI) or multifocal disease treated with Y90 alone have a median time to progression of less than 6 months. Pembro is an anti-PD-1 monoclonal antibody that is FDA-approved for advanced HCC pts who have progressed on sorafenib. Given pre-clinical evidence that radiotherapy can increase PD-L1 expression and enhance tumoral T-cell recruitment, this study explored the safety and efficacy of pembro with Y90 radioembolization in pts with poor prognosis HCC. Methods: GI15-225 was a multi-center, single-arm study in poor prognosis HCC pts, defined as having multifocal disease, MVI, or diffuse disease. Eligible pts had disease amenable to 1-2 embolization procedures, Child Pugh A/B7 cirrhosis, no prior Y90 treatment; previous locoregional therapy and resection were allowed. Pts with extrahepatic mets were excluded. Treatment consisted of pembro 200mg every 3 weeks with standard dose Y90 performed 7-10 days after first dose of pembro. The primary objective was to estimate the progression free survival (PFS) rate at 6 months per RECIST 1.1; secondary endpoints included safety, time to progression (TTP), overall response rate (ORR) and overall survival (OS). Imaging was performed every 9 weeks. Results: A total of 29 pts were enrolled 10/23/17 to 11/24/20. Median age 66 years, 89% male, 7% Child’s Pugh B, and 47% with MVI. 27 pts were evaluable for primary endpoint having received Y90 and at least one dose of pembro. The 6-month PFS rate was 57.7% (95% CI 36.9 – 76.6). Median PFS was 9.95 months (95% CI 4.14 – 15.24) and median TTP was 9.95 months (95% 4.14 – 18.56). Median OS was 27.30 months (95% CI 10.15 – 39.52). The ORR was 30.8% (Table). Three patients (11%) have ongoing treatment response with one of these patients off treatment for 16 weeks. Most common treatment related grade 3-4 AEs were decreased lymphocytes (n=5), increased bilirubin (n=3), hypertension (n=3), ascites (n=2), and AST/ALT elevation (n=2). One pt experienced grade 5 toxicity of hepatic failure after receiving one dose of pembro and Y90 that was attributed to Y90 and disease progression. Conclusions: Concurrent administration of pembro with Y90 in pts with poor prognosis HCC demonstrated promising clinical activity with median TTP and OS that exceeds historical data with three patients having ongoing response. With a 6-month PFS rate of 57.7%, median OS of 27.30 months and an acceptable toxicity profile, the combination of checkpoint blockade and Y90 deserves further evaluation in larger randomized clinical trials. Clinical trial information: NCT03099564 .[Table: see text]

While there have been extensive analyses characterizing cellular and humoral responses across the severity spectrum in COVID-19, predictors of outcomes within severe COVID-19 remain to be comprehensively elucidated. Recently, we identified divergent monocyte states as predictors of outcomes within severe COVID-19, but corresponding humoral profiles of risk have not been delineated. Furthermore, the nature of antibodies (Abs) directed against viral antigens beyond the spike protein or endemic coronavirus antigens and their associations with disease severity and outcomes remain poorly defined. We performed deep molecular profiling of Abs directed against a wide range of antigenic specificities in severe COVID-19 patients admitted to the ICU. The profiles consisted of canonical (S, RBD, N) and non-canonical (orf3a, orf8, nsp3, nps13 and M) antigenic specificities. Notably, multivariate machine learning (ML) models, generated using profiles of Abs directed against canonical or non-canonical antigens, were equally discriminative of recovery and mortality COVID-19 outcomes. In both ML models, survivors were associated with increased virus-specific IgA and IgG3 antibodies and with higher antigen-specific antibody galactosylation. Intriguingly, pre-pandemic healthy controls had cross-reactive Abs directed against nsp13 which is a conserved protein in other alpha and beta coronaviruses. Notably, higher levels of nsp13-specific IgA antibodies were associated with recovery in severe COVID-19. In keeping with these findings, a model built on Ab profiles for endemic coronavirus antigens was also predictive of COVID-19 outcome bifurcation, with higher levels of IgA and IgG3 antibodies against OC43 S and NL63 S being associated with survival. Our results suggest the importance of Abs targeting non-canonical SARS-CoV-2 antigens as well as those directed against endemic coronaviruses in favorable outcomes of severe COVID-19.

Despite extensive analyses, there remains an urgent need to delineate immune cell states that contribute to mortality in critically ill Coronavirus disease 2019 (COVID-19) patients. Here, we present high-dimensional profiling of blood and respiratory samples in severe COVID-19 patients to examine the association between cell-linked molecular features and mortality outcomes. Peripheral transcriptional profiles by single-cell RNAseq based deconvolution of immune states are associated with COVID-19 mortality. Further, persistently high levels of an interferon signaling module in monocytes over time leads to subsequent concerted upregulation of inflammatory cytokines. SARS-CoV-2 infected myeloid cells in the lower respiratory tract upregulate CXCL10, leading to a higher risk of death. Our analysis suggests a pivotal role for viral infected myeloid cells and protracted interferon signaling in severe COVID-19., Graphical Abstract, Cillo et al identify transcriptional profiles in peripheral blood that are associated with mortality in critically ill COVID-19 patients. Inflammatory monocyte signatures are correlated with CXCL10 in plasma and precede upregulation of inflammatory cytokines in blood. SARS-CoV-2-infected macrophages in the respiratory tract expressed CXCL10, linking peripheral and lung immune profiles.