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MYC is a prolific proto-oncogene driving the malignant behaviors of numerous common cancers, yet potent and selective cell-permeable inhibitors of MYC remain elusive. In order to ultimately realize the goal of therapeutic MYC inhibition in cancer, we have initiated discovery chemistry efforts aimed at inhibiting MYC translation. Here we describe a series of conformationally stabilized synthetic antisense oligonucleotides designed to target MYC mRNA (MYCASOs). To support bioactivity, we designed and synthesized this focused library of MYCASOs incorporating locked nucleic acid (LNA) bases at the 5'- and 3'-ends, a phosphorothioate backbone, and internal DNA bases. Treatment of MYC-expressing cancer cells with MYCASOs leads to a potent decrease in MYC mRNA and protein levels. Cleaved MYC mRNA in MYCASO-treated cells is detected with a sensitive 5' Rapid Amplification of cDNA Ends (RACE) assay. MYCASO treatment of cancer cell lines leads to significant inhibition of cellular proliferation while specifically perturbing MYC-driven gene expression signatures. In a MYC-induced model of hepatocellular carcinoma, MYCASO treatment decreases MYC protein levels within tumors, decreases tumor burden, and improves overall survival. MYCASOs represent a new chemical tool for in vitro and in vivo modulation of MYC activity, and promising therapeutic agents for MYC-addicted tumors.

Single-cell technologies have described heterogeneity across tissues, but the spatial distribution and forces that drive single-cell phenotypes have not been well defined. Combining single-cell RNA and protein analytics in studying the role of stromal cancer-associated fibroblasts (CAFs) in modulating heterogeneity in pancreatic cancer (pancreatic ductal adenocarcinoma [PDAC]) model systems, we have identified significant single-cell population shifts toward invasive epithelial-to-mesenchymal transition (EMT) and proliferative (PRO) phenotypes linked with mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription 3 (STAT3) signaling. Using high-content digital imaging of RNA in situ hybridization in 195 PDAC tumors, we quantified these EMT and PRO subpopulations in 319,626 individual cancer cells that can be classified within the context of distinct tumor gland "units." Tumor gland typing provided an additional layer of intratumoral heterogeneity that was associated with differences in stromal abundance and clinical outcomes. This demonstrates the impact of the stroma in shaping tumor architecture by altering inherent patterns of tumor glands in human PDAC.

Aberrant expression of viral-like repeat elements is a common feature of epithelial cancers, and the substantial diversity of repeat species provides a distinct view of the cancer transcriptome. Repeatome profiling across ovarian, pancreatic, and colorectal cell lines identifies distinct clustering independent of tissue origin that is seen with coding gene analysis. Deeper analysis of ovarian cancer cell lines demonstrated that human satellite II (HSATII) satellite repeat expression was highly associated with epithelial-mesenchymal transition (EMT) and anticorrelated with IFN-response genes indicative of a more aggressive phenotype. SATII expression--and its correlation with EMT and anticorrelation with IFN-response genes-- was also found in ovarian cancer RNA-Seq data and was associated with significantly shorter survival in a second independent cohort of patients with ovarian cancer. Repeat RNAs were enriched in tumor-derived extracellular vesicles capable of stimulating monocyte-derived macrophages, demonstrating a mechanism that alters the tumor microenvironment with these viral-like sequences. Targeting of HSATII with antisense locked nucleic acids stimulated IFN response and induced MHC I expression in ovarian cancer cell lines, highlighting a potential strategy of modulating the repeatome to reestablish antitumor cell immune surveillance., Introduction Repetitive elements make up approximately 50% of the human genome (1, 2), and their aberrant expression has been described across a wide range ofcancers (3-5). Repeat suppression is achieved [...]

A subset of patients with metastatic melanoma have sustained remissions following treatment with immune checkpoint inhibitors. However, analyses of pretreatment tumor biopsies for markers predictive of response, including PD-1 ligand (PD-L1) expression and mutational burden, are insufficiently precise to guide treatment selection, and clinical radiographic evidence of response on therapy may be delayed, leading to some patients receiving potentially ineffective but toxic therapy. Here, we developed a molecular signature of melanoma circulating tumor cells (CTCs) to quantify early tumor response using blood-based monitoring. A quantitative 19-gene digital RNA signature (CTC score) applied to microfluidically enriched CTCs robustly distinguishes melanoma cells, within a background of blood cells in reconstituted and in patient-derived (n = 42) blood specimens. In a prospective cohort of 49 patients treated with immune checkpoint inhibitors, a decrease in CTC score within 7 weeks of therapy correlates with marked improvement in progression-free survival [hazard ratio (HR), 0.17; P = 0.008] and overall survival (HR, 0.12; P = 0.04). Thus, digital quantitation of melanoma CTC-derived transcripts enables serial noninvasive monitoring of tumor burden, supporting the rational application of immune checkpoint inhibition therapies. [ABSTRACT FROM AUTHOR]

Immune exclusion inhibits anti-tumor immunity and response to immunotherapy, but its mechanisms remain poorly defined. Here, we demonstrate that Trophoblast Cell-Surface Antigen 2 (TROP2), a key target of emerging anti-cancer Antibody Drug Conjugates (ADCs), controls barrier-mediated immune exclusion in Triple-Negative Breast Cancer (TNBC) through Claudin 7 association and tight junction regulation. TROP2 expression is inversely correlated with T cell infiltration and strongly associated with outcomes in TNBC. Loss-of-function and reconstitution experiments demonstrate TROP2 is sufficient to drive tumor progression in vivo in a CD8 T cell-dependent manner, while its loss deregulates expression and localization of multiple tight junction proteins, enabling T cell infiltration. Employing a humanized TROP2 syngeneic TNBC model, we show that TROP2 targeting via hRS7, the antibody component of Sacituzumab govitecan (SG), enhances the anti-PD1 response associated with improved T cell accessibility and effector function. Correspondingly, TROP2 expression is highly associated with lack of response to anti-PD1 therapy in human breast cancer. Thus, TROP2 controls an immune exclusion program that can be targeted to enhance immunotherapy response., Competing Interests: D.T.T. has received consulting fees from ROME Therapeutics, Sonata Therapeutics, Tekla Capital, and abrdn. D.T.T. is a founder and has equity in ROME Therapeutics, PanTher Therapeutics and TellBio, Inc., which is not related to this work. D.T.T. is on the advisory board for ImproveBio, Inc. D.T.T. has received honorariums from AstraZeneca, Moderna, and Ikena Oncology that are not related to this work. D.T.T. receives research support from ACD-Biotechne, AVA LifeScience GmbH, Incyte Pharmaceuticals, and Sanofi, which was not used in this work. D.T.T.’s interests were reviewed and are managed by Massachusetts General Hospital and Mass General Brigham in accordance with their conflict of interest policies. S.J.I.: Institutional support from Genentech and Astra Zeneca. L.M.S.: Consultant/advisory board: Novartis, Daiichi Pharma, Astra Zeneca, Eli Lilly, Precede, Seagen; Institutional research support: Merck, Genentech, Gilead, Eli Lilly. A.B.: Consultant/advisory board: Pfizer, Novartis, Genentech, Merck, Radius Health; Immunomedics/Gilead, Sanofi, Daiichi Pharma/Astra Zeneca, Phillips, Eli Lilly, Foundation Medicine; Contracted Research/Grant (to institution): Genentech, Novartis, Pfizer, Merck, Sanofi, Radius Health, Immunomedics/Gilead, Daiichi Pharma/Astra Zeneca, Eli Lilly. L.W.E.: Consultant to Mersana, Inc.; Consultant to Kisoji Biotech; Consultant to Astra Zeneca; Consultant to Gilead; Sponsored Research Agreement with Sanofi. The other authors declare no competing interests.

Purpose: Dysregulation of viral-like repeat RNAs are a common feature across many malignancies that are linked with immunological response, but the characterization of these in hepatocellular carcinoma (HCC) is understudied. In this study, we performed RNA in situ hybridization (RNA-ISH) of different repeat RNAs, immunohistochemistry (IHC) for immune cell subpopulations, and spatial transcriptomics to understand the relationship of HCC repeat expression, immune response, and clinical outcomes., Experimental Design: RNA-ISH for LINE1, HERV-K, HERV-H, and HSATII repeats and IHC for T-cell, Treg, B-cell, macrophage, and immune checkpoint markers were performed on 43 resected HCC specimens. Spatial transcriptomics on tumor and vessel regions of interest was performed on 28 specimens from the same cohort., Results: High HERV-K and high LINE1 expression were both associated with worse overall survival. There was a positive correlation between LINE1 expression and FOXP3 T-regulatory cells (r = 0.51 p < 0.001) as well as expression of the TIM3 immune checkpoint (r = 0.34, p = 0.03). Spatial transcriptomic profiling of HERV-K high and LINE-1 high tumors identified elevated expression of multiple genes previously associated with epithelial mesenchymal transition, cellular proliferation, and worse overall prognosis in HCC including SSX1, MAGEC2, and SPINK1., Conclusion: Repeat RNAs may serve as useful prognostic biomarkers in HCC and may also serve as novel therapeutic targets. Additional study is needed to understand the mechanisms by which repeat RNAs impact HCC tumorigenesis., Competing Interests: COMPETING INTERESTS STATEMENT DTT has received consulting fees from ROME Therapeutics, Sonata Therapeutics, and Tekla Capital. DTT is a founder and has equity in ROME Therapeutics, PanTher Therapeutics and TellBio, Inc., which is not related to this work. DTT is on the advisory board for ImproveBio, Inc. DTT has received honorariums from Moderna and Ikena Oncology that are not related to this work. DTT receives research support from ACD-Biotechne, AVA LifeScience GmbH, and Incyte Pharmaceuticals, which was not used in this work. DTT’s interests were reviewed and are managed by Massachusetts General Hospital and Mass General Brigham in accordance with their conflict-of-interest policies.

Immune checkpoint inhibitors (ICIs) are widely used anti-cancer therapies that can cause morbid and potentially fatal immune-related adverse events (irAEs). ICI-related myocarditis (irMyocarditis) is uncommon but has the highest mortality of any irAE. The pathogenesis of irMyocarditis and its relationship to anti-tumor immunity remain poorly understood. We sought to define immune responses in heart, tumor, and blood during irMyocarditis and identify biomarkers of clinical severity by leveraging single-cell (sc)RNA-seq coupled with T cell receptor (TCR) sequencing, microscopy, and proteomics analysis of 28 irMyocarditis patients and 23 controls. Our analysis of 284,360 cells from heart and blood specimens identified cytotoxic T cells, inflammatory macrophages, conventional dendritic cells (cDCs), and fibroblasts enriched in irMyocarditis heart tissue. Additionally, potentially targetable, pro-inflammatory transcriptional programs were upregulated across multiple cell types. TCR clones enriched in heart and paired tumor tissue were largely non-overlapping, suggesting distinct T cell responses within these tissues. We also identify the presence of cardiac-expanded TCRs in a circulating, cycling CD8 T cell population as a novel peripheral biomarker of fatality. Collectively, these findings highlight critical biology driving irMyocarditis and putative biomarkers for therapeutic intervention., Competing Interests: Conflict of Interest S.M.B has been a paid consultant to Two River Consulting and Third Rock Ventures. He has equity positions in Kronos Bio, 76Bio, and Allogene Therapeutics. D.A.Z. has been a paid consultant to Bristol Myers Squibb, Freeline Therapeutics, and Intrinsic Imaging. L.Z. has received consulting fees from Bristol Myers Squibb and Merck. R.J.S has been a paid consultant to Bristol Myers Squibb, Merck, Pfizer, Marengo Therapeutics, Novartis, Eisai, Iovance, OncoSec, and AstraZeneca and has received research funding from Merck. T.G.N has been a paid consultant to Bristol Myers Squibb, Genentech, CRC Oncology, Roche, Sanofi and Parexel Imaging Pharmaceuticals and has received grant funding from Astra Zeneca and Bristol Myers Squibb related to the cardiac effects of immune checkpoint inhibitors. K.L.R has served as an advisory board to SAGA Diagnostics and received speaker’s fees from CMEOutfitters and Medscape as well as research funding from Bristol Myers Squibb. A.C.V. has been a paid consultant to Bristol Myers Squibb.

The organization of immune cells in human tumors is not well understood. Immunogenic tumors harbor spatially-localized multicellular 'immunity hubs' defined by expression of the T cell-attracting chemokines CXCL10/CXCL11 and abundant T cells. Here, we examined immunity hubs in human pre-immunotherapy lung cancer specimens, and found that they were associated with beneficial responses to PD-1-blockade. Immunity hubs were enriched for many interferon-stimulated genes, T cells in multiple differentiation states, and CXCL9/10/11 + macrophages that preferentially interact with CD8 T cells. Critically, we discovered the stem-immunity hub, a subtype of immunity hub strongly associated with favorable PD-1-blockade outcomes, distinct from mature tertiary lymphoid structures, and enriched for stem-like TCF7+PD-1+ CD8 T cells and activated CCR7 + LAMP3 + dendritic cells, as well as chemokines that organize these cells. These results elucidate the spatial organization of the human intratumoral immune response and its relevance to patient immunotherapy outcomes.

We compared the use of linear discriminant analysis and an artificial neural network for classifying optical spectral measurements of oral sites. Consistent with studies of optical spectroscopy for diagnosis of other forms of cancer, our results suggest that a non-linear classifier may be warranted. Our study also demonstrated that the classifiers were better able to distinguish between sites that were more histopathologically distinct.