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In cancer, oncogene activation is partly mediated by acquired superenhancers, which therefore represent potential targets for inhibition. Superenhancers are enriched for BRD4 and Mediator, and both BRD4 and the Mediator MED12 subunit are disproportionally required for expression of superenhancer-associated genes in stem cells. Here we show that depletion of Mediator kinase module subunit MED12 or MED13 together with MED13L can be used to reduce expression of cancer-acquired superenhancer genes, such as the MYC gene, in colon cancer cells, with a concomitant decrease in proliferation. Whereas depletion of MED12 or MED13/MED13L caused a disproportional decrease of superenhancer gene expression, this was not seen with depletion of the kinases cyclin-dependent kinase 9 (CDK8) and CDK19. MED12-MED13/MED13L-dependent superenhancer genes were coregulated by β-catenin, which has previously been shown to associate with MED12. Importantly, β-catenin depletion caused reduced binding of MED12 at the MYC superenhancer. The effect of MED12 or MED13/MED13L depletion on cancer-acquired superenhancer gene expression was more specific than and partially distinct from that of BRD4 depletion, with the most efficient inhibition seen with combined targeting. These results identify a requirement of MED12 and MED13/MED13L for expression of acquired superenhancer genes in colon cancer, implicating these Mediator subunits as potential therapeutic targets for colon cancer, alone or together with BRD4.

Growth factors secreted by stromal fibroblasts regulate the intestinal epithelium. Stroma-derived Epidermal growth factor (EGF) family ligands are implicated in epithelial regeneration and tumorigenesis, but their specific contributions and associated mechanisms remain unclear. Here, we use primary intestinal organoids modeling homeostatic, injured, and tumorigenic epithelium to assess how fibroblast-derived EGF family ligands Neuregulin-1 (NRG1) and Epiregulin (EREG) regulate the intestinal epithelium. NRG1 was expressed exclusively in the stroma, robustly increased crypt budding and protected intestinal epithelial organoids from radiation-induced damage. NRG1 also induced regenerative features in the epithelium including a fetal-like transcriptome, suppression of the Lgr5+ stem cell pool, and remodeling of the epithelial actin cytoskeleton. Intriguingly, unlike EGF and EREG, NRG1 failed to support the growth of pre-tumorigenic intestinal organoids lacking the tumor suppressor Apc, commonly mutated in human colorectal cancer (CRC). Interestingly, high expression of stromal NRG1 was associated with improved survival in CRC cohorts, suggesting a tumor suppressive function. Our results highlight the power of stromal NRG1 in transcriptional reprogramming and protection of the intestinal epithelium from radiation injury without promoting tumorigenesis.

Statement of Translational relevance: LKB1 inactivation in NSCLC is clinically relevant as it leads to resistance to immune checkpoint inhibitors. The findings of this study provide evidence that this immune evasion is due to subsequent inactivation of AMPK and attenuation of antigen presentation and suggest that restoration of AMPK activity could increase the number of patients benefiting from immunotherapy. Furthermore, the study expands the correlation between immune evasion and LKB1 mutations to include a larger number of NSCLC without detectable LKB1 mutation.

Intestinal epithelial organoids recapitulate many of the in vivo features of the intestinal epithelium, thus representing excellent research models. Morphology of the organoids based on light microscope images is used as a proxy to assess the biological state of the intestinal epithelium. Currently, organoid classification is manual and therefore subjective and time-consuming, hampering large-scale quantitative analyses. Here we describe Tellu, an object detector algorithm trained to classify cultured intestinal organoids. Tellu was trained by manual annotation of >20000 intestinal organoids to identify cystic non-budding organoids, early organoids, late organoids, and spheroids. Tellu can also be used to quantify relative organoid size. Tellu classifies intestinal organoids to these four subclasses with accuracy comparable to trained scientists but significantly faster and without bias. Tellu is provided as an open, user-friendly online tool to benefit the increasing number of investigations using organoids through fast and unbiased organoid morphology and size analysis.

<p>Supplementary Figures S1-S6</p>

<div>AbstractPurpose:<p>Mutations in <i>STK11</i> (<i>LKB1</i>) occur in 17% of lung adenocarcinoma (LUAD) and drive a suppressive (cold) tumor immune microenvironment (TIME) and resistance to immunotherapy. The mechanisms underpinning the establishment and maintenance of a cold TIME in <i>LKB1</i>-mutant LUAD remain poorly understood. In this study, we investigated the role of the LKB1 substrate AMPK in immune evasion in human non—small cell lung cancer (NSCLC) and mouse models and explored the mechanisms involved.</p>Experimental Design:<p>We addressed the role of AMPK in immune evasion in NSCLC by correlating AMPK phosphorylation and immune-suppressive signatures and by deleting <i>AMPKα1</i> (<i>Prkaa1</i>) and <i>AMPKα2</i> (<i>Prkaa2</i>) in a <i>Kras^G12D</i>-driven LUAD. Furthermore, we dissected the molecular mechanisms involved in immune evasion by comparing gene-expression signatures, AMPK activity, and immune infiltration in mouse and human LUAD and gain or loss-of-function experiments with LKB1- or AMPK-deficient cell lines.</p>Results:<p>Inactivation of both AMPK<i>α</i>1 and AMPK<i>α</i>2 together with Kras activation accelerated tumorigenesis and led to tumors with reduced infiltration of CD8⁺/CD4⁺ T cells and gene signatures associated with a suppressive TIME. These signatures recapitulate those in <i>Lkb1</i>-deleted murine LUAD and in <i>LKB1-</i>deficient human NSCLC. Interestingly, a similar signature is noted in human NSCLC with low AMPK activity. In mechanistic studies, we find that compromised LKB1 and AMPK activity leads to attenuated antigen presentation in both LUAD mouse models and human NSCLC.</p>Conclusions:<p>The results provide evidence that the immune evasion noted in <i>LKB1</i>-inactivated lung cancer is due to subsequent inactivation of AMPK and attenuation of antigen presentation.</p></div>