Protein Phosphatase 2A Activity Research Articles

ALA Upregulates MdPTPA Expression to Increase the PP2A Activity and Promote Stomatal Opening in Apple Leaves

5-Aminolevulinic acid (ALA) is a new natural plant growth regulator that inhibits abscisic acid (ABA)-induced stomatal closure. Studies have shown that protein phosphatase 2 A (PP2A) is involved in ALA-ABA antagonistically regulating stomatal movement; however, the molecular mechanisms underlying remain unclear. Here, we report that ALA promoted MdPP2A activity and the MdPP2AC expression in the epidermis of apple (Malus × domestica Borkh. cv. Fuji) leaves. Y2H (Yeast two hybrid), BiFC (Bimolecular fluorescence complement), and FLC (Firefly luciferase complementation imaging assay) analysis showed that MdPP2AC interacted with MdPTPA, a phosphortyrosyl phosphatase activator. Furthermore, the transient overexpression or interference-expression of MdPTPA transgenic apple leaves were developed. The results showed that overexpression of MdPTPA promoted stomatal opening by reducing Ca2+ and H2O2 but increasing flavonols in guard cells. Conversely, when the MdPTPA was silenced in transient transgenic apple leaves, the Ca2+, H2O2 and flavonols in guard cells and stomatal movement were completely conversed. In the transgenic apple leaves, exogenous ALA stimulated PP2A but repressed SnRK2.6 activity, while the responses are the same as that in the wild type. Therefore, we propose that MdPTPA, which increases the PP2A activity, mediates ALA signaling to promote stomatal opening in apple leaves.

Stathmin expression alters the antiproliferative effect of eribulin in leiomyosarcoma cells

Uterine leiomyosarcoma is an aggressive soft tissue tumor. Stathmin, a phosphoprotein that modulates microtubule dynamics, is highly expressed in many malignancies including leiomyosarcoma. The microtubule-depolymerizing agent eribulin has been recently approved for treating malignant soft tissue tumors. Although eribulin inhibits microtubule polymerization, little is known about the relationship between eribulin treatment and stathmin dynamics. In this study, we explored the role of stathmin expression in the action of eribulin in leiomyosarcoma cells. Eribulin induced phosphorylation of stathmin and reduced expression of subunits A and C of protein phosphatase 2A (PP2A) in a leiomyosarcoma cell line. The PP2A activator FTY720 reduced levels of phosphorylated stathmin. Eribulin decreased stathmin protein levels without affecting stathmin mRNA expression. Furthermore, stathmin knockdown attenuated the inhibitory effects of eribulin on cell viability, whereas stathmin overexpression enhanced the anti-proliferative effect of eribulin. Eribulin-resistant leiomyosarcoma cell lines had enhanced expression of the class Ⅰ β-tubulin TUBB1, multi-drug resistance 1 protein MDR1 and breast cancer-resistance protein BCRP, and decreased expression of stathmin. Taken together, these results suggest that stathmin expression modulates the pharmacological efficacy of eribulin in uterine leiomyosarcoma cells.

PP2A-B55 and its adapter proteins IER2 and IER5 regulate the activity of RB family proteins and the expression of cell cycle-related genes.

The retinoblastoma (RB) tumour suppressor protein regulates cell proliferation, motility, differentiation and apoptosis. The phosphorylation state of RB is modulated by kinases and phosphatases, and RB exhibits phosphorylation-sensitive interactions with E2F family transcription factors. Here, we characterize RB dephosphorylation by protein phosphatase 2A (PP2A). The growth factor-inducible immediate early response (IER) proteins IER2 and IER5 possess an adapter-like function in which IER proteins bind to both PP2A and its target proteins and enhance PP2A activity towards the proteins. IER2 interacts with RB and facilitates dephosphorylation of RB at T821/T826 by PP2A. In IER2 knockdown cells, elevated phosphorylation of RB resulted in reduced binding of RB to the promoters and derepression of cyclin D1 and p21. IER5 binds to both RB and RB-like 1 (p107/RBL1), enhances dephosphorylation of these proteins by PP2A and represses the expression of various cell cycle-related genes. However, IER2-regulated dephosphorylation at T821/T826 is not necessary for the repression function of RB in cell mobility-related gene expression. Our data identify PP2A adapter proteins as critical regulators of RB family proteins and suggest that the phosphorylation status of RB differentially affects gene expression.

GSK3β is a critical, druggable component of the network regulating the active NOTCH1 protein and cell viability in CLL

NOTCH1 alterations have been associated with chronic lymphocytic leukemia (CLL), but the molecular mechanisms underlying NOTCH1 activation in CLL cells are not completely understood. Here, we show that GSK3β downregulates the constitutive levels of the active NOTCH1 intracellular domain (N1-ICD) in CLL cells. Indeed, GSK3β silencing by small interfering RNA increases N1-ICD levels, whereas expression of an active GSK3β mutant reduces them. Additionally, the GSK3β inhibitor SB216763 enhances N1-ICD stability at a concentration at which it also increases CLL cell viability. We also show that N1-ICD is physically associated with GSK3β in CLL cells. SB216763 reduces GSK3β/N1-ICD interactions and the levels of ubiquitinated N1-ICD, indicating a reduction in N1-ICD proteasomal degradation when GSK3β is less active. We then modulated the activity of two upstream regulators of GSK3β and examined the impact on N1-ICD levels and CLL cell viability. Specifically, we inhibited AKT that is a negative regulator of GSK3β and is constitutively active in CLL cells. Furthermore, we activated the protein phosphatase 2 A (PP2A) that is a positive regulator of GSK3β, and has an impaired activity in CLL. Results show that either AKT inhibition or PP2A activation reduce N1-ICD expression and CLL cell viability in vitro, through mechanisms mediated by GSK3β activity. Notably, for PP2A activation, we used the highly specific activator DT-061, that also reduces leukemic burden in peripheral blood, spleen and bone marrow in the Eµ-TCL1 adoptive transfer model of CLL, with a concomitant decrease in N1-ICD expression. Overall, we identify in GSK3β a key component of the network regulating N1-ICD stability in CLL, and in AKT and PP2A new druggable targets for disrupting NOTCH1 signaling with therapeutic potential.

Adaptation of protein phosphatases in Oryza sativa and Cucumis sativus to microcystins.

Microcystins (MCs) in irrigation water could inhibit crop growth and yield. Protein phosphatases (PPs) play an important role in regulating physiological mechanisms in plants to adapt abiotic stresses. To clarify the adaptation mechanism in plants to MCs stress, we compared PPs in rice and cucumber leaves by analyzing PPs total activity, protein phosphatase-2A (PP2A) activity and expression, as well as related growth and gas exchange parameters. After 7-day exposure of MCs (5 ~ 100µg/L) and 7-day recovery without MCs, rice showed higher tolerance to MCs by analyzing dry weight and gas exchange parameters. Both crops may regulate PPs activity to adapt MCs stress by increasing the expression of genes encoding PPs. Among them, PP2A activity in two crops showed more sensitivity to MCs than total PPs activity. In addition, the higher expressions of PP2A catalytic and regulatory subunits and lower decrease PP2A activity were observed in rice leaves compared to cucumber. All results suggest that the expression levels of PP2A subunits could play a role in maintaining the activity of PP2A to regulating plant tolerance to MCs stress.

NNMT contributes to high metastasis of triple negative breast cancer by enhancing PP2A/MEK/ERK/c-Jun/ABCA1 pathway mediated membrane fluidity

Elucidating the mechanism for high metastasis capacity of triple negative breast cancers (TNBC) is crucial to improve treatment outcomes of TNBC. We have recently reported that nicotinamide N-methyltransferase (NNMT) is overexpressed in breast cancer, especially in TNBC, and predicts poor survival of patients undergoing chemotherapy. Here, we aimed to determine the function and mechanism of NNMT on metastasis of TNBC. Additionally, analysis of public datasets indicated that NNMT is involved in cholesterol metabolism. In vitro, NNMT overexpression promoted migration and invasion of TNBCs by reducing cholesterol levels in the cytoplasm and cell membrane. Mechanistically, NNMT activated MEK/ERK/c-Jun/ABCA1 pathway by repressing protein phosphatase 2A (PP2A) activity leading to cholesterol efflux and membrane fluidity enhancement, thereby promoting the epithelial-mesenchymal transition (EMT) of TNBCs. In vivo, the metastasis capacity of TNBCs was weakened by targeting NNMT. Collectively, our findings suggest a new molecular mechanism involving NNMT in metastasis and poor survival of TNBC mediated by PP2A and affecting cholesterol metabolism.

Development of actionable targets of multi-kinase inhibitors (AToMI) screening platform to dissect kinase targets of staurosporines in glioblastoma cells

Therapeutic resistance to kinase inhibitors constitutes a major unresolved clinical challenge in cancer and especially in glioblastoma. Multi-kinase inhibitors may be used for simultaneous targeting of multiple target kinases and thereby potentially overcome kinase inhibitor resistance. However, in most cases the identification of the target kinases mediating therapeutic effects of multi-kinase inhibitors has been challenging. To tackle this important problem, we developed an actionable targets of multi-kinase inhibitors (AToMI) strategy and used it for characterization of glioblastoma target kinases of staurosporine derivatives displaying synergy with protein phosphatase 2A (PP2A) reactivation. AToMI consists of interchangeable modules combining drug-kinase interaction assay, siRNA high-throughput screening, bioinformatics analysis, and validation screening with more selective target kinase inhibitors. As a result, AToMI analysis revealed AKT and mitochondrial pyruvate dehydrogenase kinase PDK1 and PDK4 as kinase targets of staurosporine derivatives UCN-01, CEP-701, and K252a that synergized with PP2A activation across heterogeneous glioblastoma cells. Based on these proof-of-principle results, we propose that the application and further development of AToMI for clinically applicable multi-kinase inhibitors could provide significant benefits in overcoming the challenge of lack of knowledge of the target specificity of multi-kinase inhibitors.

The Mechanism of TNF-α-Mediated Accumulation of Phosphorylated Tau Protein and Its Modulation by Propofol in Primary Mouse Hippocampal Neurons: Role of Mitophagy, NLRP3, and p62/Keap1/Nrf2 Pathway.

Background Neuroinflammation-induced phosphorylated Tau (p-Tau) deposition in central nervous system contributes to neurodegenerative disorders. Propofol possesses neuroprotective properties. We investigated its impacts on tumor necrosis factor-α (TNF-α)-mediated p-Tau deposition in neurons. Methods Mouse hippocampal neurons were exposed to propofol followed by TNF-α. Cell viability, p-Tau, mitophagy, reactive oxygen species (ROS), NOD-like receptor protein 3 (NLRP3), antioxidant enzymes, and p62/Keap1/Nrf2 pathway were investigated. Results TNF-α promoted p-Tau accumulation in a concentration- and time-dependent manner. TNF-α (20 ng/mL, 4 h) inhibited mitophagy while increased ROS accumulation and NLRP3 activation. It also induced glycogen synthase kinase-3β (GSK3β) while inhibited protein phosphatase 2A (PP2A) phosphorylation. All these effects were attenuated by 25 μM propofol. In addition, TNF-α-induced p-Tau accumulation was attenuated by ROS scavenger, NLRP3 inhibitor, GSK3β inhibitor, or PP2A activator. Besides, compared with control neurons, 100 μM propofol decreased p-Tau accumulation. It also decreased ROS and NLRP3 activation, modulated GSK3β/PP2A phosphorylation, leaving mitophagy unchanged. Further, 100 μM propofol induced p62 expression, reduced Keap1 expression, triggered the nuclear translocation of Nrf2, and upregulated superoxide dismutase (SOD) and heme oxygenase-1 (HO-1) expression, which was abolished by p62 knockdown, Keap1 overexpression, or Nrf2 inhibitor. Consistently, the inhibitory effect of 100 μM propofol on ROS and p-Tau accumulation was mitigated by p62 knockdown, Keap1 overexpression, or Nrf2 inhibitor. Conclusions In hippocampal neurons, TNF-α inhibited mitophagy, caused oxidative stress and NLRP3 activation, leading to GSK3β/PP2A-dependent Tau phosphorylation. Propofol may reduce p-Tau accumulation by reversing mitophagy and oxidative stress-related events. Besides, propofol may reduce p-Tau accumulation by modulating SOD and HO-1 expression through p62/Keap1/Nrf2 pathway.

Impaired myocellular Ca2+ cycling in protein phosphatase PP2A-B56α KO mice is normalized by β-adrenergic stimulation

The activity of protein phosphatase 2A (PP2A) is determined by the expression and localization of the regulatory B-subunits. PP2A-B56α is the dominant isoform of the B′-family in the heart. Its role in regulating the cardiac response to β-adrenergic stimulation is not yet fully understood. We therefore generated mice deficient in B56α to test the functional cardiac effects in response to catecholamine administration versus corresponding WT mice. We found the decrease in basal PP2A activity in hearts of KO mice was accompanied by a counter-regulatory increase in the expression of B′ subunits (β and γ) and higher phosphorylation of sarcoplasmic reticulum Ca2+ regulatory and myofilament proteins. The higher phosphorylation levels were associated with enhanced intraventricular pressure and relaxation in catheterized KO mice. In contrast, at the cellular level, we detected depressed Ca2+ transient and sarcomere shortening parameters in KO mice at basal conditions. Consistently, the peak amplitude of the L-type Ca2+ current was reduced and the inactivation kinetics of ICaL were prolonged in KO cardiomyocytes. However, we show β-adrenergic stimulation resulted in a comparable peak amplitude of Ca2+ transients and myocellular contraction between KO and WT cardiomyocytes. Therefore, we propose higher isoprenaline-induced Ca2+ spark frequencies might facilitate the normalized Ca2+ signaling in KO cardiomyocytes. In addition, the application of isoprenaline was associated with unchanged L-type Ca2+ current parameters between both groups. Our data suggest an important influence of PP2A-B56α on the regulation of Ca2+ signaling and contractility in response to β-adrenergic stimulation in the myocardium.

Abstract P1067: IRS Mediated Recruitment Of PI3Kgamma By Insulin Inhibits Beta-adrenergic Receptor Function

Insulin (INS) treatment results in impaired response to β-agonist isoproterenol (ISO) associated with phosphorylated β2-adrenergic receptor (β2AR) and impaired cAMP response. Although INS does not mediate dissociation of G-protein Gβγ subunits, yet surprisingly recruits phosphoinositide 3-kinase γ (PI3Kγ) to the plasma membrane. Correspondingly, knock-down of PI3Kγ (PI3Kγ KD) in HEK 293 or ablation in primary adult cardiomyocytes and fibroblasts (isolated from PI3Kγ knockout mice) abrogated β2AR phosphorylation reflecting a key role for PI3Kγ in retaining β2AR phosphorylation. Also, adult cardiomyocytes isolated from C57Bl6 mice showed significant loss of in vitro ISO-stimulated cardiomyocyte contraction upon INS pre-treatment which was remarkably preserved in the PI3Kγ knockout cardiomyocytes despite INS. As PI3Kγ is traditionally recruited to the βAR complex by the Gβγ subunits, we hypothesized that INS-mediates Gβγ-independent recruitment of PI3Kγ to the βAR complex underlying its dysfunction. INS stimulation is known to recruit insulin receptor substrate (IRS 1 & 2) to the receptor complex, and therefore, we tested whether PI3Kγ interacts with IRS 1/2 leading to Gβγ-independent recruitment of PI3Kγ to the β2AR complex. Immunoprecipitation PI3Kγ following INS showed that PI3Kγ interacts with IRS2 recruiting PI3Kγ to the β2AR complex bypassing the traditional Gβγ-dependent pathway. Since PI3Kγ KD resulted in loss of INS-mediated β2AR phosphorylation, we tested whether PI3Kγ inhibits protein phosphatase 2A (PP2A) function impairing β2AR de-phosphorylation. INS treatment resulted in significant loss of β2AR-associated PP2A activity which was rescued in PI3Kγ KD cells showing that PI3Kγ impairs PP2A function. Furthermore, our studies show that PI3Kγ phosphorylates the endogenous inhibitor of PP2A, I2PP2A which then robustly binds and inhibits PP2A activity impairing β2AR dephosphorylation. Consistently, CRISPR ablation of I2PP2A relieves this inhibition on PP2A leading to increased PP2A activity. This reduces accumulation of phosphorylated βARs despite the presence of PI3Kγ showing an underappreciated regulation of PP2A by insulin through non-canonical recruitment of PI3Kγ that impairs β2AR function.

PP2A regulates metastasis and vasculogenic mimicry formation via PI3K/AKT/ZEB1 axis in non-small cell lung cancers

Studies have shown that inhibition of PI3K/AKT signaling is a key strategy for the treatment of tyrosine kinase inhibitor resistance in non-small cell lung cancer (NSCLC). Vasculogenic mimicry (VM) not only accelerates tumor progression but also increases drug-induced resistance. As a tumor suppressor, protein phosphatase 2A (PP2A) is a ubiquitous conserved serine/threonine phosphatase. While its effects and mechanisms on VM formation and invasion in NSCLC remain unclear. The present study aimed to investigate the role of PP2A in VM formation and elucidate the underlying mechanisms. Results showed that PP2A could significantly inhibit VM formation and VM-dependent behavior, including invasion and migration both in vitro and in vivo. Activation of PP2A with FTY720 or Ad-PP2A reduced phosphorylated AKT and inhibited ZEB1 transcription, thereby further downregulating the expression of MMP-2, VE-cadherin, and VEGFR-2, whereas inhibition of PP2A with okadaic acid (OA) or Ad-dn-PP2A exerted the opposite effect. Furthermore, PP2A inhibited tumor growth and VM formation in the xenograft tumor model. PI3K inhibitor BENC-511 could potentiate activation of PP2A, leading to inhibition of p-AKT/ZEB1 and VM formation in vitro and in vivo. This study indicated that PP2A could regulate VM formation in NSCLC through the PI3K/AKT/ZEB1 axis. PP2A reactivation or combination with PI3K inhibitor might be a more effective treatment against advanced NSCLC by inhibiting VM formation.

Synaptotagmin 1-mediated cell membrane penetration and dopamine release enhancement by latroeggtoxin-VI

Latroeggtoxin-VI (LETX-VI), a proteinaceous neurotoxin mined from the egg transcriptome of spider L. tredecimguttatus, was previously found to promote the release of dopamine from PC12 cells. However, the relevant molecular mechanism has not been fully clear. Here LETX-VI was demonstrated to rapidly penetrate the plasma membrane of PC12 cells via the vesicle exocytosis/endocytosis cycle, during which vesicular transmembrane protein synaptotagmin 1 (Syt1) functions as a receptor, with its vesicle luminal domain interacting with the C-terminal region of LETX-VI. The C-terminal sequence of LETX-VI is the functional region for both entering cells and promoting dopamine release. After gaining entry into the PC12 cells, LETX-VI down-regulated the phosphorylation levels of Syt1 at T201 and T195, thereby facilitating vesicle fusion with plasma membrane and thus promoting dopamine release. The relevant mechanism analysis indicated that LETX-VI has a protein phosphatase 2A (PP2A) activator activity. The present work has not only probed into the Syt1-mediated action mechanism of LETX-VI, but also revealed the structure-function relationship of the toxin, thus suggesting its potential applications in the drug transmembrane delivery and treatment of the diseases related to dopamine release and PP2A activity deficiency.

SET improved oocyte maturation by serine/threonine protein phosphatase 2A and inhibited oocyte apoptosis in mouse oocytes

SET is a multifunctional protein involved in a variety of molecular processes such as cell apoptosis and cell-cycle regulation. In ovaries SET is predominantly expressed in theca cells and oocytes. In polycystic ovary syndrome (PCOS) patients the expression of SET was increased than healthy people. The current study was designed to determine whether SET plays a role in oocyte maturation and apoptosis, which may provide clues for the underlying pathological mechanism of follicular development in PCOS patients. Oocytes at germinal vesicle (GV) stage were collected from 6-week-old female ICR mice ovaries. The expression of SET was manipulated by AdCMV-SET and AdH1-SiRNA/SET adenoviruses. SET overexpression improved oocyte maturation whereas SET knockdown inhibited oocyte maturation. Moreover, SET negatively regulated serine/threonine protein phosphatase 2A (PP2A) activity in oocytes. Treatment with PP2A inhibitor okadaic acid (OA) promoted oocyte maturation. Furthermore, PP2A knockdown confirmed the role of PP2A in oocyte maturation, and OA was able to block the AdH1-SiRNA/SET-mediated inhibition on oocyte maturation. The central role of PP2A in SET-mediated regulation of oocyte maturation was confirmed by the finding that SET increased the expression of bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) and PP2A inhibited their expressions. Besides, SET inhibited oocyte apoptosis through decreasing the expression of caspase 3 and caspases 8, while PP2A had no effect on oocyte apoptosis. SET promoted oocyte maturation by inhibiting PP2A activity and inhibited oocyte apoptosis in mouse in-vitro cultured oocytes, which may provide a pathologic pathway leading to impaired oocyte developmental competence in PCOS.

Abstract 3341: Synthetic lethality by targeting ribonucleotide reductase in PP2A deficient uterine serous carcinoma

Abstract Uterine serous carcinoma (USC) is a highly aggressive endometrial cancer subtype with limited therapeutic options and a lack of targeted therapies. While mutations to PPP2R1A, encoding the predominant protein phosphatase 2A (PP2A) scaffolding protein Aα, occur in 30-40% of cases, the clinical actionability of these mutations has not been studied. Here, we show that mutation to Aα results in synthetic lethality to treatment with inhibitors of ribonucleotide reductase (RNR), and multiple models of Aα mutant uterine serous tumors were sensitive to Clofarabine, an RNR inhibitor in vivo. Aα mutant cells displayed impaired checkpoint signaling upon RNRi treatment, and subsequently accumulated more DNA damage than wild type cells. This was PP2A dependent as complete inhibition of PP2A activity using LB-100, a catalytic site inhibitor, sensitized wild type USC cells to RNRi. Analysis of TCGA data indicated that inactivation of PP2A, through loss of PP2A subunit expression, was prevalent in USC, with 88% of USC patients harboring loss of at least one PP2A gene. In contrast, loss of PP2A subunit expression was rare in uterine endometrioid carcinomas. While RNR inhibitors are not routinely used for uterine cancers, we identified a cohort of patients with recurrent disease treated with gemcitabine at MSKCC as a second or later line therapy. In a retrospective analysis of this cohort there was a trend for improved outcomes in USC patients treated with RNRi gemcitabine compared to patients with endometrioid histology. Overall, our data provide experimental evidence to support the use of ribonucleotide reductase inhibitors for the treatment of USC. Citation Format: Caitlin M. O'Connor, Sarah E. Taylor, Kathryn M. Miller, Lauren Hurst, Terrance J. Haanen, Tahra K. Suhan, Kaitlin P. Zawacki, Fallon K. Noto, Jonida Trako, Arathi Mohan, Jaya Sangodkar, Dmitriy Zamarin, Analisa DiFeo, Goutham Narla. Synthetic lethality by targeting ribonucleotide reductase in PP2A deficient uterine serous carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3341.

Abstract 3340: Small molecule mediated stabilization of PP2A modulates the homologous recombination pathway and potentiates DNA damage-induced cell death

Abstract High Grade Serous Carcinoma (HGSC) is the most lethal ovarian cancer subtype and accounts for approximately 60% of all ovarian tumors. Despite recent advances in drug development and increased understanding of genetic alterations that drive HGSC progression, mortality has not declined, highlighting the need for novel therapies. PARP inhibitors (PARPi) have become the mainstay of HGSC targeted therapy research given that these tumors are driven by a high degree of genomic instability resulting from the combination of fast DNA replication rates and numerous defects in the DNA-damage response (DDR) pathway. Nonetheless, only ~25% of these patients initially respond to treatment and a significant percentage eventually relapses with resistant disease. Here, we discovered that a Small Molecule Activator of Protein Phosphatase 2A (PP2A) (SMAP-061) induces apoptosis in both established and patient-derived HGSC cell lines as well as in genetically distinct Patient-Derived Xenograft (PDX) mouse models. Interestingly, we also uncovered that several genes that make-up the heterotrimer PP2A tumor suppressor protein are heterozygously lost in more than 95% of HGSC tumors, second only to p53. Mechanistically, we show that stabilization of PP2A protein by SMAP-061 inhibits the Homologous Recombination (HR) pathway via the direct inhibition of RAD51, ultimately leading to chronic accumulation of DNA damage and thus programmed cell death. Furthermore, we found that SMAP-061’s ability to inhibit HR potentiated the effects of PARP inhibition and resulted in synergistic cell death in both HR proficient and deficient models. These studies emphasize the potential of PP2A activators to expand the patient population that can benefit from PARPi therapies and possibly overcome PARPi resistance. In sum, our data highlights a new role of PP2A in regulating the DDR pathway in HGSC and supports the use of SMAPs in both HR proficient and deficient HGSC tumors. Citation Format: Rita A. Avelar, Amy Armstrong, Goutham Narla, Analisa DiFeo. Small molecule mediated stabilization of PP2A modulates the homologous recombination pathway and potentiates DNA damage-induced cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3340.

Chemogenetic profiling reveals PP2A‐independent cytotoxicity of proposed PP2A activators iHAP1 and DT‐061

Protein phosphatase 2A (PP2A) is an abundant phosphoprotein phosphatase that acts as a tumor suppressor. For this reason, compounds able to activate PP2A are attractive anticancer agents. The compounds iHAP1 and DT‐061 have recently been reported to selectively stabilize specific PP2A‐B56 complexes to mediate cell killing. We were unable to detect direct effects of iHAP1 and DT‐061 on PP2A‐B56 activity in biochemical assays and composition of holoenzymes. Therefore, we undertook genome‐wide CRISPR‐Cas9 synthetic lethality screens to uncover biological pathways affected by these compounds. We found that knockout of mitotic regulators is synthetic lethal with iHAP1 while knockout of endoplasmic reticulum (ER) and Golgi components is synthetic lethal with DT‐061. Indeed we showed that iHAP1 directly blocks microtubule assembly both in vitro and in vivo and thus acts as a microtubule poison. In contrast, DT‐061 disrupts both the Golgi apparatus and the ER and lipid synthesis associated with these structures. Our work provides insight into the biological pathways perturbed by iHAP1 and DT‐061 causing cellular toxicity and argues that these compounds cannot be used for dissecting PP2A‐B56 biology.

Sensitization of FOLFOX-resistant colorectal cancer cells via the modulation of a novel pathway involving protein phosphatase 2A

SummaryThe treatment of colorectal cancer (CRC) with FOLFOX shows some efficacy, but these tumors quickly develop resistance to this treatment. We have observed increased phosphorylation of AKT1/mTOR/4EBP1 and levels of p21 in FOLFOX-resistant CRC cells. We have identified a small molecule, NSC49L, that stimulates protein phosphatase 2A (PP2A) activity, downregulates the AKT1/mTOR/4EBP1-axis, and inhibits p21 translation. We have provided evidence that NSC49L- and TRAIL-mediated sensitization is synergistically induced in p21-knockdown CRC cells, which is reversed in p21-overexpressing cells. p21 binds with procaspase 3 and prevents the activation of caspase 3. We have shown that TRAIL induces apoptosis through the activation of caspase 3 by NSC49L-mediated downregulation of p21 translation, and thereby cleavage of procaspase 3 into caspase 3. NSC49L does not affect global protein synthesis. These studies provide a mechanistic understanding of NSC49L as a PP2A agonist, and how its combination with TRAIL sensitizes FOLFOX-resistant CRC cells.

Synergistic antitumor effects of compound-composed optimal formula from Aidi injection on hepatocellular carcinoma and colorectal cancer

BackgroundTraditional Chinese medicine formula (TCMF) possesses unique advantages in the prevention and treatment of malignant tumors such as hepatocellular carcinoma (HCC) and colorectal cancer (CRC). However, the unclear chemical composition and mechanism lead to its unstable efficacy and adverse reactions occurring frequently, especially injection. We previously proposed the research idea and strategy for compound-composed Chinese medicine formula (CCMF). PurposeA demonstration study was performed through screening of the compound-composed optimal formula (COF) from Aidi injection, confirmation of the synergistic effect, and exploration of the related mechanism in the treatment of HCC and CRC. MethodThe feedback system control (FSC) technique was applied to screening of COF. CCK-8 and calcein-AM/PI assays were performed to evaluate cell proliferation. Cell apoptosis was assessed using flow cytometry and DAPI staining. JC-1 probe and mitochondrial staining were employed to detect mitochondrial membrane potential (MMP) and the release of cytochrome c into cytoplasm, respective. Quantitative proteomics, drug affinity responsive target stability (DARTS) assay, bioinformatics, and molecular docking were carried out to explore the targets of the compounds and the synergistic mechanism involved. ResultsCOF was obtained from Aidi injection, which comprises cantharidin (CAN): calycosin-7-O-β-D-glucoside (CAG): ginsenoside Rc: ginsenoside Rd = 1:12:12:8 (molar ratio). The monarch drug CAN in combination with minister medicines consisting of CAG, Rc and Rd (abbr. TD) displayed evidently synergistic effect, which inhibited cell viability, increased dead cell number, induced apoptosis, reduced MMP, promoted cytochrome c leakage of HCC and CRC cells, and suppressed the increases of tumor volume and weight in HCC and CRC bearing nude mice. TD probably antagonized CAN enhanced activity of the ubiquitin proteasome system (UPS) to depress the degradation of cytotoxic proteins through binding to ubiquitin proteasome, thus exerting the synergistic effect with CAN activated protein phosphatase 2A (PP2A) to activate the mitochondrial apoptosis pathway. In addition, the CAN enhanced protein expression of UPS was also observed for the first time. ConclusionCAN and TD exert synergism through activation of PP2A and inhibition of UPS. It makes sense to elucidate the scientific nature of the compatibility theory of TCMF based on CCMF, which will be an important research direction of the modernization of traditional Chinese medicines.

Potential of phenothiazines to synergistically block calmodulin and reactivate PP2A in cancer cells.

Phenothiazines (PTZ) were developed as inhibitors of monoamine neurotransmitter receptors, notably dopamine receptors. Because of this activity they have been used for decades as antipsychotic drugs. In addition, they possess significant anti-cancer properties and several attempts for their repurposing were made. However, their incompletely understood polypharmacology is challenging. Here we examined the potential of the PTZ fluphenazine (Flu) and its mustard derivative (Flu-M) to synergistically act on two cancer associated targets, calmodulin (CaM) and the tumor suppressor protein phosphatase 2A (PP2A). Both proteins are known to modulate the Ras- and MAPK-pathway, cell viability and features of cancer cell stemness. Consistently, we show that the combination of a CaM inhibitor and the PP2A activator DT-061 synergistically inhibited the 3D-spheroid formation of MDA-MB-231 (K-Ras-G13D), NCI-H358 (K-Ras-G12C) and A375 (B-raf-V600E) cancer cells, and increased apoptosis in MDA-MB-231. We reasoned that these activities remain combined in PTZ, which were the starting point for PP2A activator development, while several PTZ are known CaM inhibitors. We show that both Flu and Flu-M retained CaM inhibitory activity in vitro and in cells, with a higher potency of the mustard derivative in cells. In line with the CaM dependence of Ras plasma membrane organization, the mustard derivative potently reduced the functional membrane organization of oncogenic Ras, while DT-061 had a negligible effect. Like DT-061, both PTZ potently decreased c-MYC levels, a hallmark of PP2A activation. Benchmarking against the KRAS-G12C specific inhibitor AMG-510 in MIA PaCa-2 cells revealed a higher potency of Flu-M than combinations of DT-061 and a CaM inhibitor on MAPK-output and a strong effect on cell proliferation. While our study is limited, our results suggest that improved PTZ derivatives that retain both, their CaM inhibitory and PP2A activating properties, but have lost their neurological side-effects, may be interesting to pursue further as anti-cancer agents.

Synthesis of PP2A-Activating PF-543 Derivatives and Investigation of Their Inhibitory Effects on Pancreatic Cancer Cells.

Sphingosine kinase (SK) is involved in the growth of cells, including cancer cells. However, which of its two isotypes—SK1 and SK2—is more favorable for cancer growth remains unclear. Although PF-543 strongly and selectively inhibits SK1, its anticancer effect is not high, and the underlying reason remains difficult to explain. We previously determined that the tail group of PF-543 is responsible for its low metabolic stability (MS). In this study, compounds containing aromatic or aliphatic tails in the triazole group were synthesized, and changes in the SK-inhibitory effect and anticancer activity of PF-543 were assessed using pancreatic cancer cells. The compounds with aliphatic tails showed high inhibitory effects on pancreatic cancer cells but slightly lower selectivity for SK1. A compound with an introduced aliphatic tail activated protein phosphatase 2A (PP2A), showing an effect similar to that of FTY720. Molecular docking analysis revealed that the PP2A-binding form of this newly synthesized compound was different from that noted in the case of FTY720. This compound also improved the MS of PF-543. These results indicate that the tail structure of PF-543 influences MS.