Afadin couples RAS GTPases to the polarity rheostat Scribble

AFDN/Afadin is required for establishment and maintenance of cell-cell contacts and is a unique effector of RAS GTPases. The biological consequences of RAS signalling to AFDN are unknown. Here, we use proximity-based proteomics to generate an interaction map for the long and short isoforms of AFDN, identifying the polarity protein SCRIB/Scribble as the top hit. We reveal that the first PDZ domain of SCRIB and the AFDN FHA domain mediate a direct but non-canonical interaction between these important adhesion and polarity proteins. Further, the dual RA domains of AFDN have broad specificity for RAS and RAP GTPases, and KRAS co-localizes with and promotes AFDN-SCRIB complex formation. Knockout of AFDN or SCRIB in MCF7 epithelial cells disrupts MAPK and PI3K activation and inhibits cell motility in a growth factor-dependent manner. These data have important implications for understanding why cells with activated RAS have reduced cell contacts and polarity defects, and finally begin to characterize AFDN as a RAS effector.

Pharmacological Targeting of PI3K-Dependent Central Tolerance Mechanisms in Refractory Pre-Germinal Center B-Cell Malignancies

Rationale: About 75% of newly formed B-cells are autoreactive and express potentially harmful autoantibodies (Wardemann 2003). Hence, a powerful mechanism, termed central tolerance, is in place to eliminate millions of newly formed autoreactive B-cells every day. Results: B-ALL, mantle cell lymphoma (MCL) and unmutated chronic lymphocytic leukemia (U-CLL) originate from early, pre-germinal center (pre-GC) stages of B-cell development that are subject to negative B-cell selection and central tolerance mechanisms. While designed to eliminate autoreactive clones during early B-cell development, we recently discovered that B-ALL, MCL and U-CLL fully retained sensitivity to central tolerance mechanisms, which are triggered by persistent PI3K-hyperactivation. PI3K-signaling code to distinguish between normal and pathological signaling. Studying short transient pulses and chronic activation of PI3K-signaling, we discovered that pre-GC B-cells have evolved a "PI3K-signaling code" to distinguish between normal B-cell activation by antigen and pathological signaling: thereby, antigen encounter induces a short transient pulse of PI3K-activation which promotes survival and proliferation. Conversely, persistent activation of PI3K-activation reflects pathological signaling, either from an autoreactive B-cell receptor (BCR) or a transforming oncogene. Pre-GC B-cell malignancies are exempt from oncogenic PI3K-lesions. PI3K-lesions in cancer result in permanent hyperactivation as in autoreactive B-cells. The PI3K pathway is targeted by oncogenic lesions in ~25% of human cancer. The phosphatases PTEN, SHIP1 and PP2A function as negative regulators of PI3K signaling and are frequently mutated in a broad range of cancers and also occur in some GC- and post-GC lymphomas (e.g. Burkitt's, DLBCL). However, our analysis in six clinical cohorts revealed that pre-GC B-cell malignancies, including B-ALL, MCL and U-CLL critically depend on PTEN, SHIP1 and PP2A function and do not tolerate persistent hyperactivation of PI3K-signaling for more than three hours. Loss-of-function mutations of these phosphatases and activating PI3K lesions were not detected in large clinical cohorts of patients with B-ALL, MCL and CLL. Likewise, phosphorylation of AKT-S473, reflecting PI3K signaling strength, is elevated throughout multiple cancer types including post-GC DLBCL, but not in B-ALL and MCL. This is in line with previous work demonstrating that inherited mutations that cause PI3K-activation predispose to various cancers but cause profound defects in human B-lymphopoiesis (Fruman 2014). Pharmacological targeting of PI3K-dependent central tolerance mechanisms. We tested the hypothesis that PI3K-hyperactivation represents a unique vulnerability in pre-GC B-cell tumors including B-ALL, MCL and U-CLL. Sensitivity to PI3K-hyperactivation of reflects their pre-GC origin and central tolerance mechanisms during early B-cell development that are designed to eliminate autoreactive B-cells based on hyperactive PI3K-signaling. For this reason, we tested pharmacological PI3K-hyperactivation as a novel strategy to selectively target pre-GC B-cell malignancies. To this end, we tested 144 compounds for their ability to engage PI3K-dependent central tolerance mechanism in B-ALL, MCL and CLL. Small molecule inhibitors of SHIP1 (3AC, K118), PTEN (SF-1670), PP2A (LB-100) and a direct PI3K-agonist (SC79) achieved strong phosphorylation of known PI3K-substrates (AKT, S6K) in vitro and prolonged overall survival in NSG mice transplanted with refractory B-ALL and MCL PDX in vivo. Conclusions and future directions: Current treatment regimens (kinase-inhibitor paradigm) use agents that apply selective pressure in one direction (e.g., PI3K-inhibitors; BCR-ABL1, SYK- or BTK-inhibitors). Here, we are pursuing a new concept (central tolerance paradigm) based on sequential treatment regimens that alternate between kinase-inhibitors (e.g., dasatinib, ibrutinib, idelalisib) and PI3K-hyperactivation (3AC, K118, LB100). By sequentially applying selective pressures in opposite directions, our approach will subvert clonal evolution and selection for drug-resistant mutants. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

The biomechanical context influences the output signaling, independently of PIK3CA mutations in breast cancer cells.

Mechanical stresses including tensile and compressive stresses are ubiquitous in na-ture, and are now well-recognized as being inherent to the development of most cancers. They are integrated by mechanotransduction in cells. Tensile stress is largely associated with YAP/TAZ pathway activation. However, less is known about signaling induced by compressive stress, the latter arising from extracellular matrix remodeling and local tumor growth. In the present study, we used transcriptomic data obtained after unidirectional compression of wild-type and mutant PIK3CA breast cancer cells from Kim et al., 2019. We analyzed in an unbiased manner signatures of cell signaling activation including phosphoinositide 3-kinases (PI3Ks) activity pathway in response to compressive stress. Because we found that PI3K activation occurred upon compression, we studied PI3K isoform-specific pathways using known transcriptional targets of PI3Kα inhibitor (BYL719) or of PI3Kβ inhibitor (AZD8186). Our study provides transcriptomic evidences for the role of PI3K pathway in compression-induced mechanotransduction, through the roles of isoform-specific class I PI3Ks and independently of PIK3CA alterations. In a compressive environment, the canonical pathways (YAP/TAZ and Piezo) was not increased, while other targetable mechanisms, such as PI3K signal or autophagy, may provide a proliferative advantage and increased cell resistance to chemotherapies.

Impact of isoflavone genistein on psoriasis in in vivo and in vitro investigations

Genistein is applied worldwide as an alternative medicament for psoriasis (Ps) because of its anti-inflammatory activity and perceived beneficial impact on the skin. Hereby, we report our in vivo and in vitro investigations to supplement scientific research in this area. The reduction of clinical and biochemical scores in mild to moderate Ps patients taking genistein, its safety, good tolerability with no serious adverse events or discontinuations of treatment, no dose-limiting toxicities, negligible changes in pharmacodynamic parameters and remarkable serum interleukin level alterations were documented in this study. A certain regression of the Ps phenotype was visible, based on photo-documented Ps lesion evaluation. Through in vitro experiments, we found that genistein reduced IL-17A and TNF-α induced MAPK, NF-κB, and PI3K activation in normal human epidermal keratinocytes. Moreover, at the mRNA level of genes associated with the early inflammatory response characteristic for Ps (CAMP, CCL20, DEFB4A, PIK3CA, S100A7, and S100A9) and key cellular signalling (MTORC1 and TFEB), we showed that this isoflavone attenuated the increased response of IL-17A- and TNF-α-related pathways. This allows us to conclude that genistein is a good candidate for Ps treatment, being attractive for co-pharmacotherapy with other drugs.

p52Shc regulates the sustainability of ERK activation in a RAF-independent manner

p52SHC (SHC) and GRB2 are adaptor proteins involved in the RAS/MAPK (ERK) pathway mediating signals from cell-surface receptors to various cytoplasmic proteins. To further examine their roles in signal transduction, we studied the translocation of fluorescently-labeled SHC and GRB2 to the cell surface, caused by the activation of ERBB receptors by heregulin (HRG). We simultaneously evaluated activated ERK translocation to the nucleus. Unexpectedly, the translocation dynamics of SHC were sustained when those of GRB2 were transient. The sustained localization of SHC positively correlated with the sustained nuclear localization of ERK, which became more transient after SHC knockdown. SHC-mediated PI3K activation was required to maintain the sustainability of the ERK translocation regulating MEK but not RAF. In cells overexpressing ERBB1, SHC translocation became transient, and the HRG-induced cell fate shifted from a differentiation to a proliferation bias. Our results indicate that SHC and GRB2 functions are not redundant, but that SHC plays the critical role in the temporal regulation of ERK activation.

The gut microbiota metabolite urolithin A inhibits NF-κB activation in LPS stimulated BMDMs

Inflammation is a natural defense process of the innate immune system, associated with the release of proinflammatory cytokines such as interleukin-1β, interleukin-6, interleukin-12 and TNFα; and enzymes including iNOS through the activation and nuclear translocation of NF-κB p65 due to the phosphorylation of IκBα. Regulation of intracellular Ca2+ is considered a promising strategy for the prevention of reactive oxygen species (ROS) production and accumulation of DNA double strand breaks (DSBs) that occurs in inflammatory-associated-diseases. Among the metabolites of ellagitannins that are produced in the gut microbiome, urolithin A (UA) has received an increasing attention as a novel candidate with anti-inflammatory and anti-oxidant effects. Here, we investigated the effect of UA on the suppression of pro-inflammatory molecules and NF-κB activation by targeting TLR4 signalling pathway. We also identified the influence of UA on Ca2+ entry, ROS production and DSBs availability in murine bone-marrow-derived macrophages challenged with lipopolysaccharides (LPS). We found that UA inhibits IκBα phosphorylation and supresses MAPK and PI3K activation. In addition, UA was able to reduce calcium entry, ROS production and DSBs availability. In conclusion, we suggest that urolithin A is a promising therapeutic agent for treating inflammatory diseases through suppression of NF-κB and preserving DNA through maintaining intracellular calcium and ROS homeostasis.

Lessons, Challenges and Future Therapeutic Opportunities for PI3K Inhibition in CLL

Chronic lymphocytic leukemia (CLL) shows constitutive phosphatidylinositol 3-kinase (PI3K) activation resulting from aberrant regulation of the B-cell receptor (BCR) signaling. PI3K inhibitors have been evaluated in CLL therapy, bringing a new treatment opportunity for patients with this disease. Despite the proven therapeutic efficacy, the use of approved PI3K inhibitors is limited by severe immune-mediated toxicities and given the availability of other more tolerable agents. This article reviews the relevance of PI3K signaling and pharmacologic inhibition in CLL. Data on efficacy and toxicity of PI3K inhibitors are also presented, as well as strategies for overcoming barriers for their clinical use in CLL treatment.