Immune checkpoint therapy with anti-CTLA-4 and anti-PD-1/PD-L1 has revolutionized the treatment of many solid tumors. However, the clinical efficacy of immune checkpoint therapy is limited to a ...subset of patients with specific tumor types
. Multiple clinical trials with combinatorial immune checkpoint strategies are ongoing; however, the mechanistic rationale for tumor-specific targeting of immune checkpoints is elusive. To garner an insight into tumor-specific immunomodulatory targets, we analyzed 94 patients representing five different cancer types, including those that respond relatively well to immune checkpoint therapy and those that do not, such as glioblastoma multiforme, prostate cancer and colorectal cancer. Through mass cytometry and single-cell RNA sequencing, we identified a unique population of CD73
macrophages in glioblastoma multiforme that persists after anti-PD-1 treatment. To test if targeting CD73 would be important for a successful combination strategy in glioblastoma multiforme, we performed reverse translational studies using CD73
mice. We found that the absence of CD73 improved survival in a murine model of glioblastoma multiforme treated with anti-CTLA-4 and anti-PD-1. Our data identified CD73 as a specific immunotherapeutic target to improve antitumor immune responses to immune checkpoint therapy in glioblastoma multiforme and demonstrate that comprehensive human and reverse translational studies can be used for rational design of combinatorial immune checkpoint strategies.
Immune checkpoint therapy (ICT) can provide durable clinical responses and improve overall survival. However, only subsets of patients with specific tumor types respond to ICT. Thus, significant ...challenges remain, including understanding pathways of resistance, optimizing patient selection, improving management of immune-related adverse events, and identifying rational therapeutic combinations. These challenges will need a focused approach encompassing both clinical and basic research, with the integration of reverse translational studies. This integrated approach will lead to identification of potential targets for subsequent clinical trials, which will guide decisions as we develop novel combination strategies to maximize efficacy and minimize toxicities for patients. SIGNIFICANCE: ICTs induce durable antitumor responses for subsets of patients with cancer. Recent evidence suggests that rational combinatorial strategies can improve response by overcoming primary and adaptive resistance mechanisms, although these may carry an increased risk of immune-mediated toxicities. This review surveys the current understanding of mechanisms of response and resistance to ICTs and active areas of investigation, and proposes a path forward to improving efficacy and minimizing toxicities through better patient selection and rational combinations.
Abstract Resistance to immune checkpoint therapy (ICT) presents a growing clinical challenge. The tumor microenvironment (TME) and its components, namely tumor-associated macrophages (TAMs) and ...cancer-associated fibroblasts (CAFs), play a pivotal role in ICT resistance; however, the underlying mechanisms remain under investigation. In this study, we identify expression of TNF-Stimulated Factor 6 (TSG-6) in ICT-resistant pancreatic tumors, compared to ICT-sensitive melanoma tumors, both in mouse and human. TSG-6 is expressed by CAFs within the TME, where suppressive macrophages expressing Arg1 , Mafb , and Mrc1 , along with TSG-6 ligand Cd44 , predominate. Furthermore, TSG-6 expressing CAFs co-localize with the CD44 expressing macrophages in the TME. TSG-6 inhibition in combination with ICT improves therapy response and survival in pancreatic tumor-bearing mice by reducing macrophages expressing immunosuppressive phenotypes and increasing CD8 T cells. Overall, our findings propose TSG-6 as a therapeutic target to enhance ICT response in non-responsive tumors.
Myeloid cells are the most abundant immune components of the tumour microenvironment, where they have a variety of functions, ranging from immunosuppressive to immunostimulatory roles. The myeloid ...cell compartment comprises many different cell types, including monocytes, macrophages, dendritic cells and granulocytes, that are highly plastic and can differentiate into diverse phenotypes depending on cues received from their microenvironment. In the past few decades, we have gained a better appreciation of the complexity of myeloid cell subsets and how they are involved in tumour progression and resistance to cancer therapies, including immunotherapy. In this Review, we highlight key features of monocyte and macrophage biology that are being explored as potential targets for cancer therapies and what aspects of myeloid cells need a deeper understanding to identify rational combinatorial strategies to improve clinical outcomes of patients with cancer. We discuss therapies that aim to modulate the functional activities of myeloid cell populations, impacting their recruitment, survival and activity in the tumour microenvironment, acting at the level of cell surface receptors, signalling pathways, epigenetic machinery and metabolic regulators. We also describe advances in the development of genetically engineered myeloid cells for cancer therapy.
Immune checkpoint therapy (ICT) has dramatically altered clinical outcomes for cancer patients and conferred durable clinical benefits, including cure in a subset of patients. Varying response rates ...across tumor types and the need for predictive biomarkers to optimize patient selection to maximize efficacy and minimize toxicities prompted efforts to unravel immune and non-immune factors regulating the responses to ICT. This review highlights the biology of anti-tumor immunity underlying response and resistance to ICT, discusses efforts to address the current challenges with ICT, and outlines strategies to guide the development of subsequent clinical trials and combinatorial efforts with ICT.
Immune checkpoint therapy provides clinical benefits to many patients across different tumor types. Sharma, Goswami, and colleagues review the mechanisms of and clinical advances in immune checkpoint therapy and outline the challenges and approaches to broaden the clinical utility of immune checkpoint therapy.
Enhancer of zeste homolog 2-mediated (EZH2-mediated) epigenetic regulation of T cell differentiation and Treg function has been described previously; however, the role of EZH2 in T cell-mediated ...antitumor immunity, especially in the context of immune checkpoint therapy, is not understood. Here, we showed that genetic depletion of EZH2 in Tregs (FoxP3creEZH2fl/fl mice) leads to robust antitumor immunity. In addition, pharmacological inhibition of EZH2 in human T cells using CPI-1205 elicited phenotypic and functional alterations of the Tregs and enhanced cytotoxic activity of Teffs. We observed that ipilimumab (anti-CTLA-4) increased EZH2 expression in peripheral T cells from treated patients. We hypothesized that inhibition of EZH2 expression in T cells would increase the effectiveness of anti-CTLA-4 therapy, which we tested in murine models. Collectively, our data demonstrated that modulating EZH2 expression in T cells can improve antitumor responses elicited by anti-CTLA-4 therapy, which provides a strong rationale for a combination trial of CPI-1205 plus ipilimumab.
Quorum sensing (QS) in rhizobia regulates diverse processes determining the success and efficiency of association with the legume host. Despite the notable importance of QS as well as the well-known ...underlying variability in the genomic and metabolic components thereof, its study in rhizobia is largely restricted to few laboratory strains. In this work, QS phenomenon in the rhizobia nodulating pigeon pea– one of the most important legume crops of the global-south, is characterized. Using 16S rRNA and recombinaseA sequencing analysis, the selected QS-positive and host-beneficial isolates were identified to be taxonomically affiliated to the genus Ensifer. Their QS components, including homologues of QS genes, and the repertoire of N-acyl homoserine lactone (AHL) autoinducers were identified. Sequences of the QS homologues showed significant variabilities ranging from 10 to >20% with the known Ensifer sequences. Autoinducer profiling using LC–MS/MS revealed the production of long and short chain AHLs variably by the isolates, including 3-oxo-C12-homoserine lactone (3-O-C12-HSL) and 3-OH-C16-HSL as their first report in Rhizobiaceae. Motility and attachment– two of the most crucial traits for effective establishment on host roots were discovered to be QS dependent in in vitro analysis and the same was confirmed using expression analysis of their regulatory genes using qRT-PCR; both revealing a QS mediated repression of motility and promotion of attachment. This study highlights that Ensifer nodulating pigeon pea, although with significant variance in the anatomy of their QS components, regulate symbiotically crucial cell-processes via QS in a scheme that is conserved in multiple genera.
Immune checkpoint therapy (ICT) can produce durable antitumor responses in metastatic urothelial carcinoma (mUCC); however, the responses are not universal. Despite multiple approvals of ICT in mUCC, ...we lack predictive biomarkers to guide patient selection. The identification of biomarkers may require interrogation of both the tumor mutational status and the immune microenvironment. Through multi-platform immuno-genomic analyses of baseline tumor tissues, we identified the mutation of AT-rich interactive domain-containing protein 1A (
) in tumor cells and expression of immune cytokine CXCL13 in the baseline tumor tissues as two predictors of clinical responses in a discovery cohort (
= 31). Further, reverse translational studies revealed that CXCL13
tumor-bearing mice were resistant to ICT, whereas
knockdown enhanced sensitivity to ICT in a murine model of bladder cancer. Next, we tested the clinical relevance of
mutation and baseline CXCL13 expression in two independent confirmatory cohorts (CheckMate275 and IMvigor210). We found that
mutation and expression of CXCL13 in the baseline tumor tissues correlated with improved overall survival (OS) in both confirmatory cohorts (CheckMate275, CXCL13 data,
= 217; ARID1A data,
= 139, and IMvigor210, CXCL13 data,
= 348; ARID1A data,
= 275). We then interrogated CXCL13 expression plus
mutation as a combination biomarker in predicting response to ICT in CheckMate275 and IMvigor210. Combination of the two biomarkers in baseline tumor tissues suggested improved OS compared to either single biomarker. Cumulatively, this study revealed that the combination of CXCL13 plus ARID1A may improve prediction capability for patients receiving ICT.
BackgroundImmune checkpoint therapy (ICT) has revolutionized cancer treatment, however, has produced around 20% durable clinical response rates. 1 Multiple studies demonstrate myeloid cell abundance ...and function in the tumor microenvironment (TME) correlates with poor outcome in ICT resistant cancers. 2, 3 Despite the wealth of knowledge regarding the biology of these cells, our ability to distinguish immune-suppressive versus immune-stimulatory myeloid cells remains a major challenge and, thus, targeting myeloid cells has had limited clinical success. Our objective is to better understand the phenotype of these immunosuppressive myeloid cells to identify potential combination strategies to improve response to ICT.MethodsComparative analyses of intratumoral myeloid cell subsets was performed in orthotopic murine models of ICT sensitive (B16F10 melanoma) and resistant (MT4 pancreatic) tumors at baseline and post ICT treatment (anti-PD-1 and/or anti-CTLA-4 antibodies) using single cell RNA sequencing (scRNAseq). To determine myeloid cell evolution with tumor progression, longitudinal scRNAseq was performed on MT4 tumors.ResultsWe observed two-fold higher abundance of macrophages and neutrophils in baseline MT4 tumors as compared to B16F10 tumors from our scRNAseq analysis (figure 1A-C). Overall, we identified 4 distinct macrophage subsets and 1 neutrophil subset (figure 1B). Mac_1 and mac_2 (MT4_macrophages) were the dominant macrophage clusters in MT4 tumors and while mac_1 expressed mmp14, axl and mafb; mac_2 had high expression of vegfa, arg1, ccl24 and fn1(figure 1D). In contrast, mac_3 (B16F10_macrophages), the most abundant macrophage cluster in B16F10 tumors, expresses antigen presenting genes (cd72) and interferon-induced genes (cxcl10, isg15) (figure 1D). MT4_macrophages were significantly enriched TGF beta signaling, angiogenesis, hypoxia, glycolysis and B16F10_macrophages which enriched in oxidative phosphorylation and interferon gamma and alpha response pathways (figure 1E). The neutrophil subset was present specifically in MT4 tumors and expressed cd24a, cxcl2, il1b and cd274 (figure 1D); consistent with the suppressive tumor-associated neutrophil (TAN) phenotype. Overall, at baseline, MT4 myeloid cells possess characteristics associated with T cell inhibition whereas B16F10 myeloid cells show T cell activating phenotypes. Post ICT treatment, macrophage subsets decreased moderately, however, MT4 TAN abundance increased, indicating possible compensatory resistance mechanisms (figure 1F). Longitudinal analyses indicated that neutrophil and macrophage subsets were abundant in early stage tumors and persisted during MT4 tumor progression (figure 1G).ConclusionsDistinct myeloid subsets are present in ICT sensitive and ICT resistant tumors. Myeloid targeting prior ICT treatment might be necessary to generate an effective immune response in resistant tumors. This study provides the foundation to identify novel myeloid specific targets in resistant solid tumors.AcknowledgementsThis research is supported by Parker Institute for Cancer Immunotherapy (PICI)ReferencesPons-Tostivint E, Latouche A, Vaflard P, et al. Comparative analysis of durable responses on immune checkpoint inhibitors versus other systemic therapies: a pooled analysis of phase III Trials. JCO Precision Oncology, 2019(3): 1–10.Zhang QW, Liu L, Gong CY, et al. Prognostic significance of tumor-associated macrophages in solid tumor: a meta-analysis of the literature. PLoS One, 2012. 7(12):e50946.Wang PF, Song SY, Wang TJ, et al., Prognostic role of pretreatment circulating MDSCs in patients with solid malignancies: A meta-analysis of 40 studies. Oncoimmunology, 2018. 7(10): e1494113.Ethics ApprovalAll mice were housed in accordance with the Association for Assessment and Accreditation of Laboratory Animal Care and NIH standards. Experiments were conducted according to protocol 00000893-RN02 and approved by the University of Texas MD Anderson Cancer Center Institutional Animal Care and Use Committee.Abstract 505 Figure 1(A) Representation UMAP plot of intratumoral immune cell landscape in B16F10 and MT4 tumors. 3 tumors in each were pooled for internal control. All major immune cell subsets were identified. (B) Cluster frequency plots of each immune subset in B16F10 and MT4 tumors. (C) Characterization of immune subsets identified in figure 1A based on their marker gene expressions. (D) Heatmap of functional markers for the individual myeloid subsets providing phenotypic information. Expression levels are scaled between minimum and maximum expression for each gene across all clusters. (E) GSEA analysis of hallmark pathways enriched in mac_1 and mac_2 versus mac_3. F) Cluster frequency plots of indicated myeloid subsets after treatment with ICT antibodies obtained from scRNASeq analyses. (G) Representative UMAP plots of intratumoral immune subsets collected from Day 5,7, 10 and 15 of MT4 tumors. Highlighted regions in blue and brown indicate macrophage and neutrophil subsets, respectively.Figure omitted. See PDF
The most frequent known causes of primary cardiomyopathies are mutations in the genes encoding sarcomeric proteins. Among those are 30 single-residue mutations in TPM1, the gene encoding ...α-tropomyosin. We examined seven mutant tropomyosins, E62Q, D84N, I172T, L185R, S215L, D230N, and M281T, that were chosen based on their clinical severity and locations along the molecule. The goal of our study was to determine how the biochemical characteristics of each of these mutant proteins are altered, which in turn could provide a structural rationale for treatment of the cardiomyopathies they produce. Measurements of Ca2+ sensitivity of human β-cardiac myosin ATPase activity are consistent with the hypothesis that hypertrophic cardiomyopathies are hypersensitive to Ca2+ activation, and dilated cardiomyopathies are hyposensitive. We also report correlations between ATPase activity at maximum Ca2+ concentrations and conformational changes in TnC measured using a fluorescent probe, which provide evidence that different substitutions perturb the structure of the regulatory complex in different ways. Moreover, we observed changes in protein stability and protein-protein interactions in these mutants. Our results suggest multiple mechanistic pathways to hypertrophic and dilated cardiomyopathies. Finally, we examined a computationally designed mutant, E181K, that is hypersensitive, confirming predictions derived from in silico structural analysis.
Background: Single residue substitutions in sarcomeric proteins cause most inherited cardiomyopathies.
Results: Mutant α-tropomyosins cause multiple functional alterations in actin affinity and Ca2+ sensitivity.
Conclusion: Mutants follow distinct mechanisms to change Ca2+ sensitivity.
Significance: Fluorescence assays to measure changes in troponin C conformation may provide a simple platform for preliminary high throughput screening of modulatory small molecules to treat inherited cardiomyopathies.
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