Determining the Anticancer Activity of Sphingosine Kinase Inhibitors Containing Heteroatoms in Their Tail Structure

Sphingosine kinase (SK) enzyme, a central player of sphingolipid rheostat, catalyzes the phosphorylation of sphingosine to the bioactive lipid mediator sphingosine 1 phosphate (S1P), which regulates cancer cell proliferation, migration, differentiation, and angiogenesis through its extracellular five G protein-coupled S1P receptors (S1PR1–5). Recently, several research studies on SK inhibitors have taken place in order use them for the development of novel anticancer-targeted therapy. In this study, we designed and synthesized analog derivatives of known SK1 inhibitors, namely RB005 and PF-543, by introducing heteroatoms at their tail structure, as well as investigated their anticancer activities and pharmacokinetic parameters in vitro. Compounds 1–20 of RB005 and PF-543 derivatives containing an aliphatic chain or a tail structure of benzenesulfonyl were synthesized. All compounds of set 1 (1–10) effectively reduced cell viability in both HT29 and HCT116 cells, whereas set 2 derivatives (11–20) showed poor anticancer effect. Compound 10, having the highest cytotoxic effect (48 h, HT29 IC50 = 6.223 µM, HCT116 IC50 = 8.694 µM), induced HT29 and HCT116 cell death in a concentration-dependent manner through the mitochondrial apoptotic pathway, which was demonstrated by increased annexin V-FITC level, and increased apoptotic marker cleaved caspase-3 and cleaved PARP. Compound 10 inhibited SK1 by 20%, and, thus, the S1P level decreased by 42%. Unlike the apoptosis efficacy, the SK1 inhibitory effect and selectivity of the PF-543 derivative were superior to that of the RB005 analog. As a result, compounds with an aliphatic chain tail exhibited stronger apoptotic effects. However, this ability was not proportional to the degree of SK inhibition. Compound 10 increased the protein phosphatase 2A (PP2A) activity (1.73 fold) similar to FTY720 (1.65 fold) and RB005 (1.59 fold), whereas compounds 11 and 13 had no effect on PP2A activation. Since the PP2A activity increased in compounds with an aliphatic chain tail, it can be suggested that PP2A activation has an important effect on anticancer and SK inhibitory activities.

PP2A is a therapeutically targetable driver of cell fate decisions via a c-Myc/p21 axis in Acute Myeloid Leukemia

Dysregulated cellular differentiation is a hallmark of acute leukemogenesis. Phosphatases are widely suppressed in cancers but have not been traditionally associated with differentiation. Herein, we identified that the silencing of Protein Phosphatase 2A (PP2A) directly contributes to differentiation block in acute myeloid leukemia (AML). Gene expression and mass cytometric profiling reveal that PP2A activation modulates cell cycle and transcriptional regulators that program terminal myeloid differentiation. Using a novel pharmacological agent OSU-2S in parallel with genetic approaches, we discovered that PP2A enforces c-Myc and p21 dependent terminal differentiation, proliferation arrest and apoptosis in AML. Finally, we demonstrate that PP2A activation decreases leukemia initiating stem cells, increases leukemic blast maturation, and improves overall survival in murine Tet2-/-Flt3ITD/WT and human AML models in-vivo. Our findings identify the PP2A/c-Myc/p21 axis as a critical regulator of the differentiation/proliferation switch in AML that can be therapeutically targeted in malignancies with dysregulated maturation fate.

Deposition of Phosphorylated α-Synuclein and Activation of GSK-3β and PP2A in the PS19 Mouse Model of Tauopathy

The simultaneous accumulation of multiple pathological proteins, such as hyperphosphorylated tau (hp-tau) and phosphorylated α-synuclein (p-αSyn), has been reported in the brains of patients with various neurodegenerative diseases. We previously demonstrated that hp-tau-dependent p-αSyn accumulation was associated with the activation of GSK-3β in the brains of P301L tau transgenic mice. To confirm the effects of another mutant tau on p-αSyn accumulation in vivo, we herein examined the brains of PS19 mice that overexpress human P301S mutant tau. Immunohistochemically, hp-tau and p-αSyn aggregates were detected in the same neuronal cells in the cerebrum and brain stem of aged PS19 mice. A semiquantitative analysis showed a positive correlation between hp-tau and p-αSyn accumulation. Furthermore, an activated form of GSK-3β was detected within cells containing both hp-tau and p-αSyn aggregates in PS19 mice. Western blotting showed a decrease in inactivated PP2A levels in PS19 mice. The present results suggest that the overexpression of human P301S mutant tau induces p-αSyn accumulation that is accompanied by not only GSK-3β, but also PP2A activation in PS19 mice, and highlight the synergic effects between tau and αSyn in the pathophysiology of neurodegenerative diseases that show the codeposition of tau and αSyn.

Prematurity Alters the Feeding-Induced Activation of Signaling Components Towards AKT in Skeletal Muscle of Neonatal Piglets

Objectives Postnatal growth faltering is a common complication of premature birth. Our recent study in a neonatal pig model of prematurity showed that preterm birth reduces weight gain and protein synthesis in skeletal muscle and this is associated with blunted insulin-induced activation of signaling components downstream of AKT. However, prematurity does not affect the activation of the IR/IRS-1/PI3K axis. In this study, we aimed to identify components in the AKT signaling pathway in skeletal muscle that underlie the differential response to insulin between preterm and term pigs. Methods Cesarean-derived piglets delivered 11 d (preterm) or 2 d (term) before term were fitted with a jugular vein catheter for delivery of total parenteral nutrition. On day 3, all piglets were fasted for 4 h and then assigned randomly to fast one additional h or to receive an elemental meal by oral gavage. Macronutrient content of the elemental meal mimicked sow milk composition at day 3 of lactation. Piglets were euthanized for tissue collection in the fasted state or 60 min after feeding. The longissimus dorsi muscle was collected and subjected to Western blot and immunoprecipitation assays for analyzing upstream regulators of AKT activity. Results Phosphorylation of PDK1 and mTORC2 was lower in preterm than term pigs (P < 0.05). The abundance of phosphatase PHLPP, but not PTEN, was significant higher in preterm than term pigs (P < 0.05). Preterm pigs had lower PP2A activation (P < 0.05), but its activation was not affected by feeding, unlike term pigs where feeding inhibited PP2A activation (P < 0.05). The abundance of Ubl4A, required for insulin-induced translocation of AKT to the plasma membrane, was reduced by prematurity (P < 0.05). While AKT1 abundance was higher in preterm than term pigs (P < 0.05), the abundances of both AKT2 and AKT3 were lower in preterm than term pigs (P < 0.05). Conclusions Our results demonstrate that prematurity reduces the abundance and/or activation of positive regulators of AKT such as Ubl4A, PDK1, and mTORC2. Conversely, prematurity increased the activation of AKT inhibitors such as PHLPP and PP2A. Our findings potentially pave the way for a better understanding of the mechanisms that underlie postnatal growth faltering in premature infants. Funding Sources NIH and USDA.

Protein phosphatase 2A activation as a therapeutic strategy for managing MYC-driven cancers

The tumor suppressor protein phosphatase 2A (PP2A) is a serine/threonine phosphatase whose activity is inhibited in most human cancers. One of the best-characterized PP2A substrates is MYC proto-oncogene basic helix–loop–helix transcription factor (MYC), whose overexpression is commonly associated with aggressive forms of this disease. PP2A directly dephosphorylates MYC, resulting in its degradation. To explore the therapeutic potential of direct PP2A activation in a diverse set of MYC-driven cancers, here we used biochemical assays, recombinant cell lines, gene expression analyses, and immunohistochemistry to evaluate a series of first-in-class small-molecule activators of PP2A (SMAPs) in Burkitt lymphoma, KRAS-driven non–small cell lung cancer, and triple-negative breast cancer. In all tested models of MYC-driven cancer, the SMAP treatment rapidly and persistently inhibited MYC expression through proteasome-mediated degradation, inhibition of MYC transcriptional activity, decreased cancer cell proliferation, and tumor growth inhibition. Importantly, we generated a series of cell lines expressing PP2A-dependent phosphodegron variants of MYC and demonstrated that the antitumorigenic activity of SMAPs depends on MYC degradation. Collectively, the findings presented here indicate a pharmacologically tractable approach to drive MYC degradation by using SMAPs for the management of a broad range of MYC-driven cancers.

Protein Phosphatase 2A Activation Dependent Down Regulation of PGC1-Alpha and FOXO1 Phosphorylation with OSU-2S in Human Lymphoma Cells

Metabolic reprogramming has been recognized to provide survival advantage in cancer cells. Protein phosphatase 2A (PP2A) is a serine/threonine phosphatase that is involved in regulation of multiple cellular pathways including metabolic events. OSU-2S, is a novel PP2A activator that exhibited potent anti-cancerous properties against human and canine lymphoma cell lines and primary human and canine lymphoma patient samples. It has been shown to activate PP2A in Ramos human lymphoma cell line leading to cytotoxicity that is prevented by inhibition of PP2A with okadaic acid (OA). Peroxisome proliferative activated receptor-gamma co-activator 1 (PPARGC1, also known as PGC1a), is a transcriptional co-activator that serves as a positive regulator of mitochondrial biogenesis and respiration, gluconeogenesis as well as many other metabolic processes such as lipid and energy metabolism. FOXO1 is a transcription factor that directly binds to the promoters of PGC1a and gluconeogenic genes involved in activation of gluconeogenesis. Activated PP2A has been shown to directly interact with FOXO1 and dephosphorylate it, leading to its delayed nuclear translocation. Given the role of PP2A in dephosphorylation of pFOX01, a regulator of PGC1a gene transcription, we hypothesized that PP2A activator OSU-2S, will down regulate PGC-1a expression through PP2A dependent FOXO1 regulation. Consistent with this hypothesis OSU-2S treatment inhibited PGC1a mRNA and protein expression in Jeko, OCI-ly18 and OCI-ly19 and raji lymphoma cell lines 24 hours post treatment. OSU-2S mediated downregulation of PGC1a and mitochondria biogenesis genes (NRF1, ERR alpha and TFAM) are dependent on PP2A activation as concentrations of OA that inhibited PP2A activation abrogated OSU-2S which induced up regulation of PGC1a and mitochondria biogenesis genes . To determine if the OSU-2S mediated inhibition of PGC1a expression is associated with its PP2A dependent modulation of phosphoFOXO1(pFOXO1), we tested the effect of OSU-2S on pFOXO1. Treatment of lymphoma cells with OSU-2S induced 60-70% decrease in pFOXO1 compared to vehicle control P =0.0001)], that is correlated with the decrease in PGC1a protein expression. Importantly OA mediated inhibition of PP2A, prevented OSU-2S-induced FOXO1 dephosphorylation. These studies suggest a role of OSU-2S induced modulation of metabolic regulator PGC1a via PP2A dependent dephosphorylation of FOXO1. Importantly, OSU-2S-induced PGC1a reduction resulted in decreased mitochondrial biogenesis as evidenced by ~43 % decrease in mitochondrial mass and ATP generation that led to reduced energy production as determined by Nonyl Acridine Orange dye staining followed by flow cytometry analysis. Interestingly, OSU-2S decreased expression of genes involved in mitochondrial biogenesis including NRF1a, ERR1a and TFAM by 75, 65 and 60% respectively P<0.0001. Ongoing mechanistic studies are aimed to define the molecular basis of OSU-2S induced transcriptional regulation of PGC1a and other genes involved in mitochondrial biogenesis in human lymphoma cell lines and primary cells. (This work was supported by NIH-R01 CA197844-02. SMK is a recipient of Egyptian Cultural and Educational Bureau (ECEB) Award). SG is a recipient of Pelotonia Graduate Fellowship) Keywords: PGC1- alpha, OSU-2S, PP2A, FOXO1, metabolism, lymphoma Disclosures Muthusamy: Ohio State University: Patents & Royalties: OSU-2S.