Magnolol and Honokiol Inhibited the Function and Expression of BCRP with Mechanism Exploration

Breast cancer resistance protein (BCRP), one of the ATP-binding cassette (ABC) transporters, was associated with the multidrug resistance (MDR) of chemotherapy. Magnolol (MN) and honokiol (HK) are major bioactive polyphenols of Magnolia officinalis. This study investigated the effects of MN and HK on the function and expression of BCRP for the purpose of developing BCRP inhibitor to overcome MDR. Cell lines including MDCKII-BCRP and MDCKII-WT were used for evaluating the function and expression of BCRP. The results showed that MN (100–12.5 µM) and HK (100–12.5 µM) significantly decreased the function of BCRP by 80~12% and 67~14%, respectively. In addition, MN and HK were verified as substrates of BCRP. Furthermore, MN and HK reduced the protein expression of BCRP, and inhibited the phosphorylation of epidermal growth factor receptor (EGFR) and phosphatidylinositol 3-kinase (PI3K). In conclusion, both MN and HK decreased the function and expression of BCRP via EGFR/PI3K signaling pathway. Therefore, both compounds were promising candidates for reversing the MDR of chemotherapy.

TBX2 controls a proproliferative gene expression program in melanoma

Senescence shapes embryonic development, plays a key role in aging, and is a critical barrier to cancer initiation, yet how senescence is regulated remains incompletely understood. TBX2 is an antisenescence T-box family transcription repressor implicated in embryonic development and cancer. However, the repertoire of TBX2 target genes, its cooperating partners, and how TBX2 promotes proliferation and senescence bypass are poorly understood. Here, using melanoma as a model, we show that TBX2 lies downstream from PI3K signaling and that TBX2 binds and is required for expression of E2F1, a key antisenescence cell cycle regulator. Remarkably, TBX2 binding in vivo is associated with CACGTG E-boxes, present in genes down-regulated by TBX2 depletion, more frequently than the consensus T-element DNA binding motif that is restricted to Tbx2 repressed genes. TBX2 is revealed to interact with a wide range of transcription factors and cofactors, including key components of the BCOR/PRC1.1 complex that are recruited by TBX2 to the E2F1 locus. Our results provide key insights into how PI3K signaling modulates TBX2 function in cancer to drive proliferation.

Evodiamine Inhibits Gastric Cancer Cell Proliferation via PTEN-Mediated EGF/PI3K Signaling Pathway

Aims. In this study, the pharmacological effects and potential molecular mechanisms of evodiamine in treating gastric cancer (GC) were investigated. Methods. GC cells lines of AGS and BGC-823 were treated with evodiamine at various concentrations for different times (24, 48, and 72 h). Inhibition of the proliferation of AGS and BGC-823 cells was assessed using a CCK-8 assay. The morphology of gastric cancer cells was detected by high-content screening (HCS). The apoptosis-inducing effect of evodiamine on AGS and BGC-823 cells was detected by flow cytometric analysis. Cell migration and invasion were detected by Transwell assay. The relative mRNA and protein expression levels of PTEN-mediated EGF/PI3K signaling pathways were investigated via RT-qPCR or western blotting, respectively. Results. Evodiamine substantially inhibited AGS and BGC-823 cells proliferation in a dose- and time-dependent manner. Flow cytometric analysis revealed that evodiamine could induce apoptosis of AGS and BGC-823 cells in a dose-dependent manner. In addition, evodiamine inhibited AGS and BGC-823 cell migration and invasion. Mechanistically, the results demonstrated that evodiamine promoted the relative mRNA and protein expression of PTEN and decreased expression of EGF, EGFR, PI3K, AKT, p-AKT, and mTOR. Most importantly, evodiamine could effectively increase the mRNA and protein expression of PTEN and decrease the protein expression of EGF/PI3K pathway, indicating that evodiamine downregulated EGF/PI3K through the activation of PTEN pathway. Conclusion. Evodiamine inhibited the directional migration and invasion of GC cells by inhibiting PTEN-mediated EGF/PI3K signaling pathway. These findings revealed that evodiamine might serve as a potential candidate for the treatment or prevention of GC.

Comprehensive targeted next-generation sequencing in patients with slow-flow vascular malformations

Recent studies have shown that the PI3K signaling pathway plays an important role in the pathogenesis of slow-flow vascular malformations (SFVMs). Analysis of genetic mutations has advanced our understanding of the mechanisms involved in SFVM pathogenesis and may identify new therapeutic targets. We screened for somatic variants in a cohort of patients with SFVMs using targeted next-generation sequencing. Targeted next-generation sequencing of 29 candidate genes associated with vascular anomalies or with the PI3K signaling pathway was performed on affected tissues from patients with SFVMs. Fifty-nine patients with SFVMs (venous malformations n = 21, lymphatic malformations n = 27, lymphatic venous malformations n = 1, and Klippel–Trenaunay syndrome n = 10) were included in the study. TEK and PIK3CA were the most commonly mutated genes in the study. We detected eight TEK pathogenic variants in 10 samples (16.9%) and three PIK3CA pathogenic variants in 28 samples (47.5%). We also identified a pathogenic variant in RASA1 in one sample. In total, 38 of 59 patients (64.4%) with SFVMs harbored pathogenic variants in these three genes involved in the PI3K signaling pathway. Inhibitors of this pathway may prove useful as molecular targeted therapies for SFVMs.

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.

Identification of a Conserved Intracellular Loop (CIL) Structure That Scaffolds PIP3 to Amplify Oncogenic Signaling during Malignant B-Cell Transformation

Background: Within seconds of antigen-encounter, B-cell receptor (BCR) signaling induces dramatic changes of cell membrane lipid composition, including >40-fold increases of local PIP3-concentrations within lipid rafts. While several structural elements, including pleckstrin homology (PH) domains have been identified as PIP3-binding proteins, the underlying mechanisms that amplify BCR-signaling to assemble large signaling complexes within lipid rafts within 15 to 30 seconds, remained elusive. To understand the mechanistic and biophysical requirements for PIP3 accumulation during normal B-cell activation and acute oncogenic transformation, we identified PIP3-interacting proteins by cell-surface proteomic analyses. Results: In addition to proteins known to bind PIP3 with their PH-domains, we identified the short 133 aa protein IFITM3 (interferon-inducible transmembrane protein 3) as a top-ranking PIP3 scaffold. This was unexpected because IFITM3 was previously identified as endosomal protein that blocks viral infection by stiffening endosomal membranes to firmly contain viral cargo. Previous studies revealed that polymorphisms that lead to the expression of truncated IFITM3 are associated with increased susceptibility to viral infections, including SARS-CoV2. Among known cell membrane lipids, PIP3 has the highest negative charge. Instead of a PH-domain, IFITM3 laterally sequestered PIP3 through electrostatic interactions with two basic lysine residues (K83 and K104) located at the membrane-solution interface. Together with three other basic lysine and arginine residues K83 and K104 form a conserved intracellular loop (CIL), which enable IFITM3 to efficiently capture two PIP3 molecules. Bivalent PIP3-binding of the IFITM3-CIL enables a crosslinking mechanism that results in dramatic amplification of B-cell activation signals and clustering of large signaling complexes within lipid rafts. In normal resting B-cells, Ifitm3 was minimally expressed and mainly localized in endosomes. However, B-cell activation and oncogenic kinases induced phosphorylation at IFITM3-Y20, resulting in translocation of IFITM3 from endosomes and massive accumulation at the cell surface. Ifitm3ˉ /ˉ naïve B-cells developed at normal numbers, however, activation by antigen encounter was compromised. In Ifitm3ˉ /ˉ B-cells, lipid rafts were depleted of PIP3, resulting in defective expression of >60 lipid raft-associated surface receptors and impaired PI3K-signaling. Ifitm3ˉ /ˉ B-cells were unable to undergo affinity maturation and di not contribute to germinal center formation upon immunization. Analyses of gene expression and clinical outcome data from patients in six clinical cohorts for pediatric and adult B-ALL, mantle cell lymphoma, CLL and DLBCL, we consistently identified IFITM3 as a top-ranking predictor of poor clinical outcome. Inducible activation of BCR-ABL1 and NRAS G12D rapidly induced development of B-ALL but failed to transform and initiate B-ALL from Ifitm3ˉ /ˉ B-cell precursors. Conversely, the phospho-mimetic IFITM3-Y20E mutation, mimicking phosphorylation of the IFITM3 N-terminus at Y20 induced constitutive membrane localization of IFITM3, spontaneous aggregation of large oncogenic signaling complexes and readily initiated transformation in a genetic model of pre-malignant B-cells. Conclusions: We conclude that phosphorylation of IFITM3 upon B-cell activation induces a dynamic switch from antiviral effector functions in endosomes to oncogenic signal-amplification at the cell-surface. IFITM3-dependent amplification of PI3K-signaling is critical to enable rapid expansion of activated B-cells. In addition, multiple oncogenes depend on IFITM3 to assemble PIP3-dependent signaling complexes and amplify PI3K-signaling for malignant transformation and initiation of B-lymphoid leukemia and lymphoma. Figure 1 Figure 1. Disclosures Weinstock: SecuraBio: Consultancy; ASELL: Consultancy; Bantam: Consultancy; Abcuro: Research Funding; Verastem: Research Funding; Daiichi Sankyo: Consultancy, Research Funding; AstraZeneca: Consultancy; Travera: Other: Founder/Equity; Ajax: Other: Founder/Equity.