An open question in aggressive cancers such as melanoma is how malignant cells can shift the immune system to pro-tumourigenic functions. Here we identify Midkine (MDK) as a melanoma-secreted driver of an "inflamed", but immune evasive, microenvironment that defines poor patient prognosis and resistance to immune checkpoint blockade. Mechanistically, MDK was found to control the transcriptome of melanoma cells allowing for a coordinated activation of NF-B and downregulation of interferon-associated pathways. The resulting MDK-modulated secretome educated macrophages towards tolerant phenotypes that promoted CD8 + -T cell dysfunction. In contrast, genetic targeting of MDK sensitized melanoma cells to anti-PD1/PDL1 treatment. Emphasizing the translational relevance of these findings, the expression profile of MDK-depleted tumours was enriched in key indicators of good response to immune checkpoint blockers in independent patient cohorts. Together, these data reveal that MDK acts as an internal modulator of autocrine and paracrine signals that maintain immune suppression in aggressive melanomas.

Secreted extracellular vesicles (EVs) influence the tumor microenvironment and promote distal metastasis. Here, we analyzed the involvement of melanoma-secreted EVs in lymph node pre-metastatic niche formation in murine models. We found that small EVs (sEVs) derived from metastatic melanoma cell lines were enriched in nerve growth factor receptor (NGFR, p75NTR), spread through the lymphatic system and were taken up by lymphatic endothelial cells, reinforcing lymph node metastasis. Remarkably, sEVs enhanced lymphangiogenesis and tumor cell adhesion by inducing ERK kinase, nuclear factor (NF)-κB activation and intracellular adhesion molecule (ICAM)-1 expression in lymphatic endothelial cells. Importantly, ablation or inhibition of NGFR in sEVs reversed the lymphangiogenic phenotype, decreased lymph node metastasis and extended survival in pre-clinical models. Furthermore, NGFR expression was augmented in human lymph node metastases relative to that in matched primary tumors, and the frequency of NGFR + metastatic melanoma cells in lymph nodes correlated with patient survival. In summary, we found that NGFR is secreted in melanoma-derived sEVs, reinforcing lymph node pre-metastatic niche formation and metastasis.

Highlights d Combined Egfr/Raf1 ablation results in complete regression of a subset of PDACs d Mouse mutant Kras/Trp53-induced PDACs display distinct transcriptional profiles d PDAC transcriptional profiles determine their response to Egfr/Raf1 ablation d EGFR/c-RAF inhibition also prevents proliferation of PDXderived tumor cells

Lymphatic vessels are essential for skin fluid homeostasis and immune cell trafficking. Whether the lymphatic vasculature is associated with hair follicle regeneration is, however, unknown. Here, using steady and live imaging approaches in mouse skin, we show that lymphatic vessels distribute to the anterior permanent region of individual hair follicles, starting from development through all cycle stages and interconnecting neighboring follicles at the bulge level, in a stem cell‐dependent manner. Lymphatic vessels further connect hair follicles in triads and dynamically flow across the skin. At the onset of the physiological stem cell activation, or upon pharmacological or genetic induction of hair follicle growth, lymphatic vessels transiently expand their caliber suggesting an increased tissue drainage capacity. Interestingly, the physiological caliber increase is associated with a distinct gene expression correlated with lymphatic vessel reorganization. Using mouse genetics, we show that lymphatic vessel depletion blocks hair follicle growth. Our findings point toward the lymphatic vasculature being important for hair follicle development, cycling, and organization, and define lymphatic vessels as stem cell niche components, coordinating connections at tissue‐level, thus provide insight into their functional contribution to skin regeneration.

Pluripotent stem cells (PSCs) transition between cell states in vitro and reflect developmental changes in the early embryo. PSCs can be stabilized in the naïve state by blocking extracellular differentiation stimuli, particularly FGF-MEK signaling. Here, we report that multiple features of the naïve state in human and mouse PSCs can be recapitulated without affecting FGF-MEK-signaling or global DNA methylation. Mechanistically, chemical inhibition of CDK8 and CDK19 kinases removes their ability to repress the Mediator complex at enhancers. Thus CDK8/19 inhibition increases Mediator-driven recruitment of RNA Pol II to promoters and enhancers. This efficiently stabilizes the naïve transcriptional program and confers resistance to enhancer perturbation by BRD4 inhibition.Moreover, naïve pluripotency during embryonic development coincides with a reduction in CDK8/19. We conclude that global hyperactivation of enhancers drives naïve pluripotency, and this can be achieved in vitro by inhibiting CDK8/19 kinase activity. These principles may apply to other contexts of cellular plasticity. RESULTS Inhibition of Mediator kinase stabilizes mouse naïve pluripotencyGFP knock-in reporters at key stem cell marker genes such as Nanog represent well-established and precise indicators of the naïve (GFP high ) and primed states (GFP low ) 18,22,29 . For example, in 2i-naïve state, Nanog promoter activity is enhanced, yielding a characteristically homogenous Nanog-GFP high cell expression pattern and uniform dome-shaped colonies (Fig. 1A-C, and Extended Data Fig. 1A). In contrast, the Nanog promoter is metastable in primed state PSCs, reversibly oscillating between high and low activity, presenting a heterogeneous Nanog-GFP expression pattern and flattened diffuse colonies, indicative of a general underlying switch in transcriptional program 18,20,23,29,30 . The BRD4 inhibitor JQ1 destabilizes enhancers and resulted in colony flattening and GFP low status (Fig. 1A), as reported [26][27][28] . In this experimental setting, we tested the effect of manipulating the transcriptional cyclin-dependent kinases (CDK7, CDK8/19 and CDK9) with a panel of small molecule inhibitors. Several potent Lynch et al., submitted 19 19 and structurally-unrelated CDK8/19 inhibitors had a positive effect, inducing the formation of homogenous dome-shaped colonies, and upregulating both the Nanog-GFP reporter and endogenous Nanog expression, similar to PSC in the 2i-naïve state (Fig. 1A-E; Extended Data Fig. 1A; Supplementary Table 1), while inhibition of CDK7 or CDK9 did not. Potency and selectivity of CDK8/19inhibitors, commercially available or developed in-house, were assessed by multiple methods: (i) selectivity was suggested by a KinomeScan panel of 456 kinases; (ii) Lanthascreen assays demonstrated inhibitory activity at nanomolar concentrations against pure recombinant CDK8/CCNC and CDK19/CCNC; (iii) luciferase reporter cell assays (TOP-FLASH); and (iv) potent inhibition of STAT1-Ser727 phosphorylation in human PSCs, a well-documented CDK8 t...