The Importance of Being PI3K in the RAS Signaling Network

Ras proteins are essential mediators of a multitude of cellular processes, and its deregulation is frequently associated with cancer appearance, progression, and metastasis. Ras-driven cancers are usually aggressive and difficult to treat. Although the recent Food and Drug Administration (FDA) approval of the first Ras G12C inhibitor is an important milestone, only a small percentage of patients will benefit from it. A better understanding of the context in which Ras operates in different tumor types and the outcomes mediated by each effector pathway may help to identify additional strategies and targets to treat Ras-driven tumors. Evidence emerging in recent years suggests that both oncogenic Ras signaling in tumor cells and non-oncogenic Ras signaling in stromal cells play an essential role in cancer. PI3K is one of the main Ras effectors, regulating important cellular processes such as cell viability or resistance to therapy or angiogenesis upon oncogenic Ras activation. In this review, we will summarize recent advances in the understanding of Ras-dependent activation of PI3K both in physiological conditions and cancer, with a focus on how this signaling pathway contributes to the formation of a tumor stroma that promotes tumor cell proliferation, migration, and spread.

Spatiotemporal Control of Actomyosin Contractility by MRCKβ Signaling Drives Phagocytosis

Phagocytosis requires myosin-generated contractile force to regulate actin dynamics. However, little is known about the molecular mechanisms that guide this complex morphodynamic process. Here we show that particle binding to Mer Tyrosine Kinase (MerTK), a widely expressed phagocytic receptor, stimulates phosphorylation of the Cdc42 GEF Dbl3 in the retinal pigment epithelium (RPE), triggering activation of MRCKβ and its co-effector N-WASP that cooperate to deform the membrane into cups. Continued MRCKβ activity then drives recruitment of a mechanosensing bridge enabling transmission of the cytoskeletal force required for cup closure and particle internalization. MRCKβ is also required for Fc receptor-mediated phagocytosis by macrophages. In vivo, MRCKβ is essential for RPE phagocytosis of photoreceptor debris and, hence, retinal integrity. MerTK-independent activation of MRCKβ signaling by a phosphomimetic Dbl3 mutant rescues phagocytosis in retinitis pigmentosa RPE cells lacking functional MerTK. Thus, conserved MRCKβ signaling controls spatiotemporal regulation of actomyosin contractility to guide actomyosin dynamics-driven phagocytosis and represents the principle phagocytic effector pathway downstream of MerTK.

Changes in cAMP effector predominance are associated with increased oxytocin receptor expression in twin but not infection-associated or idiopathic preterm labour

We previously reported that at term pregnancy, a decline in myometrial protein kinase A (PKA) activity leads to an exchange protein activated by cyclic AMP (Epac1)-dependent increase in oxytocin receptor (OTR) expression, promoting the onset of labour. Here, we studied the changes in the cyclic adenosine monophosphate (cAMP) effector system present in different phenotypes of preterm labour (PTL). Myometrial biopsies obtained from women with phenotypically distinct forms of PTL and the levels of PKA and OTR were examined. Although we found similar changes in the cAMP effector pathway in all forms of PTL, only in the case of twin PTL (T-PTL) was myometrial OTR levels increased in association with these results. Although there were several changes in the mRNA levels of components of the cAMP synthetic pathway, the total myometrial cAMP levels did not change with the onset of any subtype of PTL. With regards to the expression of cAMP-responsive genes, we found that the mRNA levels of 4 of the 5 cAMP-down-regulated genes were increased in T-PTL, similar to our findings in term labour. These data signify that although changes in the cAMP effector system were common to all forms of PTL, only in T-PTL were OTR levels increased. Similarly, the mRNA levels of cAMP-repressed genes were only increased in T-PTL supporting the concept that the decline in PKA levels influences myometrial function driving the onset of T-PTL.

The level of oncogenic Ras controls the malignant transformation of Lkb1 mutant tissue in vivo

The genetic and metabolic heterogeneity of RAS-driven cancers has confounded therapeutic strategies in the clinic. To address this, rapid and genetically tractable animal models are needed that recapitulate the heterogeneity of RAS-driven cancers in vivo. Here, we generate a Drosophila melanogaster model of Ras/Lkb1mutant carcinoma. We show that low-level expression of oncogenic Ras (RasLo) promotes the survival of Lkb1 mutant tissue, but results in autonomous cell cycle arrest and non-autonomous overgrowth of wild-type tissue. In contrast, high-level expression of oncogenic Ras (RasHi) transforms Lkb1 mutant tissue resulting in lethal malignant tumors. Using simultaneous multiview light-sheet microcopy, we have characterized invasion phenotypes of Ras/Lkb1 tumors in living larvae. Our molecular analysis reveals sustained activation of the AMPK pathway in malignant Ras/Lkb1 tumors, and demonstrate the genetic and pharmacologic dependence of these tumors on CaMK-activated Ampk. We further show that LKB1 mutant human lung adenocarcinoma patients with high levels of oncogenic KRAS exhibit worse overall survival and increased AMPK activation. Our results suggest that high levels of oncogenic KRAS is a driving event in the malignant transformation of LKB1 mutant tissue, and uncover a novel vulnerability that may be used to target this aggressive genetic subset of RAS-driven tumors.One Sentence SummaryA multivariable Ras-driven Drosophila model reveals a novel LKB1 mutant lung adenocarcinoma patient subpopulation and targetable effector pathway.

Genome editing in mammals using CRISPR type I-D nuclease

SUMMARYAdoption of the CRISPR-Cas system has revolutionized genome engineering in recent years; however, application of genome editing with CRISPR type I—the most abundant CRISPR system in bacteria—has been less developed. Type I systems in which Cas3 nuclease degrades the target DNA are known; in contrast, for the sub-type CRISPR type I-D (TiD), which lacks a typical Cas3 nuclease in its cascade, the mechanism of target DNA degradation remains unknown. Here, we found that Cas10d—a nuclease in TiD—is multi-functional in PAM recognition, stabilization and target DNA degradation. TiD can be used for targeted mutagenesis of genomic DNA in human cells, directing both bi-directional long-range deletions and short insertions/deletions. TiD off-target effects, which were dependent on the mismatch position in the protospacer of TiD, were also identified. Our findings suggest TiD as a unique effector pathway in CRISPR that can be repurposed for genome engineering in eukaryotic cells.

Airway brush cells generate cysteinyl leukotrienes through the ATP sensor P2Y2

Chemosensory epithelial cells (EpCs) are specialized cells that promote innate type 2 immunity and protective neurally mediated reflexes in the airway. Their effector programs and modes of activation are not fully understood. Here, we define the transcriptional signature of two choline acetyltransferase–expressing nasal EpC populations. They are found in the respiratory and olfactory mucosa and express key chemosensory cell genes including the transcription factor Pou2f3, the cation channel Trpm5, and the cytokine Il25. Moreover, these cells share a core transcriptional signature with chemosensory cells from intestine, trachea and thymus, and cluster with tracheal brush cells (BrCs) independently from other respiratory EpCs, indicating that they are part of the brush/tuft cell family. Both nasal BrC subsets express high levels of transcripts encoding cysteinyl leukotriene (CysLT) biosynthetic enzymes. In response to ionophore, unfractionated nasal BrCs generate CysLTs at levels exceeding that of the adjacent hematopoietic cells isolated from naïve mucosa. Among activating receptors, BrCs express the purinergic receptor P2Y2. Accordingly, the epithelial stress signal ATP and aeroallergens that elicit ATP release trigger BrC CysLT generation, which is mediated by the P2Y2 receptor. ATP- and aeroallergen-elicited CysLT generation in the nasal lavage is reduced in mice lacking Pou2f3, a requisite transcription factor for BrC development. Last, aeroallergen-induced airway eosinophilia is reduced in BrC-deficient mice. These results identify a previously undescribed BrC sensor and effector pathway leading to generation of lipid mediators in response to luminal signals. Further, they suggest that BrC sensing of local damage may provide an important sentinel immune function.

Combinatorial Genetic Control of Rpd3S through histone H3K4 and H3K36 Methylation in Budding Yeast

Much of euchromatin regulation occurs through reversible methylation of histone H3 lysine-4 and lysine-36 (H3K4me and H3K36me). Using the budding yeast Saccharomyces cerevisiae, we previously found that levels of H3K4me modulated temperature sensitive alleles of the transcriptional elongation complex Spt6-Spn1 through an unknown H3K4me effector pathway. Here we identify the Rpd3S histone deacetylase complex as the H3K4me effector underlying these Spt6-Spn1 genetic interactions. Exploiting these Spt6-Spn1 genetic interactions, we show that H3K4me and H3K36me collaboratively impact Rpd3S function in an opposing manner. H3K36me is deposited by the histone methyltransferase Set2 and is known to promote Rpd3S function at RNA PolII transcribed open reading frames. Using genetic epistasis experiments, we find that mutations perturbing the Set2-H3K36me-Rpd3S pathway suppress the growth defects caused by temperature sensitive alleles of SPT6 and SPN1, illuminating that this pathway antagonizes Spt6-Spn1. Using these sensitive genetic assays, we also identify a role for H3K4me in antagonizing Rpd3S that functions through the Rpd3S subunit Rco1, which is known to bind H3 N-terminal tails in a manner that is prevented by H3K4me. Further genetic experiments reveal that the H3K4 and H3K36 demethylases JHD2 and RPH1 mediate this combinatorial control of Rpd3S. Finally, our studies also show that the Rpd3L complex, which acts at promoter-proximal regions of PolII transcribed genes, counters Rpd3S for genetic modulation of Spt6-Spn1, and that these two Rpd3 complexes balance the activities of each other. Our findings present the first evidence that H3K4me and H3K36me act combinatorially to control Rpd3S.