Scaffolding Subunit Research Articles

Mutant PP2A Induces IGFBP2 Secretion to Promote Development of High-Grade Uterine Cancer.

Uterine serous carcinoma (USC) and uterine carcinosarcoma (UCS) tumors are uniquely aggressive, suggesting that the primary tumor is intrinsically equipped to disseminate and metastasize. Previous work identified mutational hotspots within PPP2R1A, which encodes the Aα scaffolding subunit of protein phosphatase 2A (PP2A), a heterotrimeric serine/threonine phosphatase. Two recurrent heterozygous PPP2R1A mutations, P179R and S256F, occur exclusively within high-grade subtypes of uterine cancer and can drive tumorigenesis and metastasis. Elucidation of the mechanisms by which PP2A-Aα mutants promote tumor development and progression could help identify therapeutic opportunities. Here, we showed that expression of these mutants in USC/UCS cell-lines enhanced tumor-initiating capacity, drove a hybrid epithelial-to-mesenchymal (EM) plasticity phenotype, and elevated secretion of the tumorigenic cytokine IGFBP2. Therapeutic targeting of the IGFBP2/IGF1R signaling axis using small molecules and genetic approaches resulted in marked tumor growth inhibition. Mechanistically, PP2A regulated IGFBP2 expression through the transcription factor, NF-κB, which harbors a B56 recognition motif. Collectively, these results identify a role for PP2A in regulating paracrine cancer cell signaling that can be targeted to block the initiation and metastasis of high-grade uterine cancer.

STRIPAK, a fundamental signaling hub of eukaryotic development.

SUMMARYThe striatin-interacting phosphatase and kinase (STRIPAK) complex is involved in the regulation of many developmental processes in eukaryotic microorganisms and all animals, including humans. STRIPAK is a component of protein phosphatase 2A (PP2A), a highly conserved serine-threonine phosphatase composed of catalytic subunits (PP2Ac), a scaffolding subunit (PP2AA) and various substrate-directing B regulatory subunits. In particular, the B''' regulatory subunit called striatin has evoked major interest over the last 20 years. Studies in fungal systems have contributed substantially to our current knowledge about STRIPAK composition, assembly, and cellular localization, as well as its regulatory role in autophagy and the morphology of fungal development. STRIPAK represents a signaling hub with many kinases and thus integrates upstream and downstream information from many conserved eukaryotic signaling pathways. A profound understanding of STRIPAK's regulatory role in fungi opens the gateway to understanding the multifarious functions carried out by STRIPAK in higher eukaryotes, including its contribution to malignant cell growth.

Histone deacetylase 6 inhibition promotes microtubule acetylation and facilitates autophagosome-lysosome fusion in dystrophin-deficient mdx mice.

Duchenne muscular dystrophy is a progressive muscle-wasting disease caused by mutations in the dystrophin gene. Despite progress in dystrophin-targeted gene therapies, it is still a fatal disease requiring novel therapeutics that can be used synergistically or alternatively to emerging gene therapy. Defective autophagy and disorganized microtubule networks contribute to dystrophic pathogenesis, yet the mechanisms by which microtubule alterations regulate autophagy remain elusive. The present study was designed to uncover possible mechanisms underpinning the role of microtubules in regulating autophagy in dystrophic mice. Mdx mice were also supplemented with Tubastatin A, a pharmacological inhibitor of histone deacetylase 6, and pathophysiology was assessed. Mdx mice with a genetic deletion of the Nox-2 scaffolding subunit p47phox were used to assess redox dependence on tubulin acetylation. Our data show decreased acetylation of α-tubulin with enhanced histone deacetylase 6 expression. Tubastatin A increases tubulin acetylation and Q-SNARE complex formation but does not alter microtubule organization or density, indicating improved autophagosome-lysosome fusion. Tubastatin A increases the acetylation of peroxiredoxin and protects it from hyper-oxidation, hence modulating intracellular redox status in mdx mice. Tubastatin A reduces muscle damage and enhances force production. Genetic down regulation of Nox2 activity in the mdx mice promotes autophagosome maturation but not autolysosome formation. Our data highlight that autophagy is differentially regulated by redox and acetylation in mdx mice. By improving autophagy through promoting tubulin acetylation, Tubastatin A decreases the dystrophic phenotype and improves muscle function, suggesting a great potential for clinical translation and treating dystrophic patients.

Protein phosphatase 2A regulates blood cell proliferation and differentiation in Drosophila larval lymph glands.

Protein phosphatase 2A (PP2A), one of the most abundant protein phosphatases, has divergent functions in multiple types of cells. Its inactivation has been closely associated with leukemia diseases. However, the physiological function of PP2A for hematopoiesis has been poorly understood in organisms. Drosophila hematopoiesis parallels the vertebrate counterpart in developmental and functional features but involves a much simpler hematopoietic system. Here, utilizing the Drosophila major larval hematopoietic organ lymph gland, we studied the function of PP2A for hematopoiesis in vivo. By knocking down the expression of Pp2A-29B that encodes the scaffold subunit of the PP2A holoenzyme complex, we found that PP2A silencing in the differentiating hemocytes resulted in their excessive proliferation. Furthermore, this PP2A inhibition downregulated the expression of Smoothened (Smo), a crucial component in the Hedgehog pathway, and smo overexpression was able to rescue the phenotypes of PP2A depletion, indicating that Smo functions as a downstream effector of PP2A to restrict the hemocyte proliferation. PDGF/VEGF-receptor (Pvr) overexpression also restored the Smo expression and lymph gland morphology of PP2A silencing, suggesting a PP2A-Pvr-Smo axis to regulate lymph gland growth and hemocyte proliferation. Moreover, inhibiting PP2A activity in the blood progenitor cells promoted their differentiation, but which was independent with Smo. Together, our data suggested that PP2A plays a dual role in the Drosophila lymph gland by preserving the progenitor population and restraining the hemocyte proliferation, to properly regulate the hematopoietic process.

Protein phosphatase 6 promotes stemness of colorectal cancer cells.

Colorectal cancer (CRC) remains a significant global health concern, demanding a more profound comprehension of its molecular foundations for the development of improved therapeutic strategies. This study aimed to elucidate the role of protein phosphatase 6 (PP6), a member of the type 2A protein phosphatase family, in CRC. Protein phosphatase 6 functions as a heterotrimer with a catalytic subunit (PP6c), regulatory subunits (PP6Rs; PP6R1, PP6R2, and PP6R3), and scaffold subunits (ANKRD28, ANKRD44, and ANKRD52). Elevated PP6c expression has been identified in CRC tissues compared to normal mucosa, aligning with its potential involvement in CRC pathogenesis. PP6c knockdown resulted in decreased colony-forming ability and invivo proliferation of various CRC cell lines. Transcriptome analysis revealed that PP6c knockdown resulted in altered expression of genes associated with cancer stemness. Notably, the PP6c-PP6R3 complex is a key player in regulating cancer stem cell (CSC) markers. Additionally, increased PP6c expression was observed in CSC-like cells induced by sphere formation, implicating the role of PP6c in CSC maintenance. This study highlights the role of PP6c in CRC and suggests that it is a potential therapeutic target disrupting a pathway critical for CRC progression and stem cell maintenance.

Influence of point mutations on PR65 conformational adaptability: Insights from molecular simulations and nanoaperture optical tweezers.

PR65 is the HEAT repeat scaffold subunit of the heterotrimeric protein phosphatase 2A (PP2A) and an archetypal tandem repeat protein. Its conformational mechanics plays a crucial role in PP2A function by opening/closing substrate binding/catalysis interface. Using in silico saturation mutagenesis, we identified PR65 "hinge" residues whose substitutions could alter its conformational adaptability and thereby PP2A function, and selected six mutations that were verified to be expressed and soluble. Molecular simulations and nanoaperture optical tweezers revealed consistent results on the specific effects of the mutations on the structure and dynamics of PR65. Two mutants observed in simulations to stabilize extended/open conformations exhibited higher corner frequencies and lower translational scattering in experiments, indicating a shift toward extended conformations, whereas another displayed the opposite features, confirmed by both simulations and experiments. The study highlights the power of single-molecule nanoaperture-based tweezers integrated with in silico approaches for exploring the effect of mutations on protein structure and dynamics.

Structural characterization of methylation-independent PP2A assembly guides alphafold2Multimer prediction of family-wide PP2A complexes

Dysregulation of phosphorylation-dependent signaling is a hallmark of tumorigenesis. Protein phosphatase 2 (PP2A) is an essential regulator of cell growth. One scaffold subunit (A) binds to a catalytic subunit (C) to form a core AC heterodimer, which together with one of many regulatory (B) subunits forms the active trimeric enzyme. The combinatorial number of distinct PP2A complexes is large, which results in diverse substrate specificity and subcellular localization. The detailed mechanism of PP2A assembly and regulation remains elusive and reports about an important role of methylation of the carboxy terminus of PP2A C are conflicting. A better understanding of the molecular underpinnings of PP2A assembly and regulation is critical to dissect PP2A function in physiology and disease. Here, we combined biochemical reconstitution, mass spectrometry, X-ray crystallography and functional assays to characterize the assembly of trimeric PP2A. In vitro studies demonstrated that methylation of the carboxy-terminus of PP2A C was dispensable for PP2A assembly in vitro. To corroborate these findings, we determined the X-ray crystal structure of the unmethylated PP2A Aα-B56ε-Cα trimer complex to 3.1 Å resolution. The experimental structure superimposed well with an Alphafold2Multimer prediction of the PP2A trimer. We then predicted models of all canonical PP2A complexes providing a framework for structural analysis of PP2A. In conclusion, methylation was dispensable for trimeric PP2A assembly and integrative structural biology studies of PP2A offered predictive models for all canonical PP2A complexes.

The protein phosphatase-2A subunit PR130 is involved in the formation of cytotoxic protein aggregates in pancreatic ductal adenocarcinoma cells

As a major source of cellular serine and threonine phosphatase activity, protein phosphatase-2A (PP2A) modulates signaling pathways in health and disease. PP2A complexes consist of catalytic, scaffolding, and B-type subunits. Seventeen PP2A B-type subunits direct PP2A complexes to selected substrates. It is ill-defined how PP2A B-type subunits determine the growth and drug responsiveness of tumor cells. Pancreatic ductal adenocarcinoma (PDAC) is a disease with poor prognosis. We analyzed the responses of murine and human mesenchymal and epithelial PDAC cells to the specific PP2A inhibitor phendione. We assessed protein levels by immunoblot and proteomics and cell fate by flow cytometry, confocal microscopy, and genetic manipulation. We show that murine mesenchymal PDAC cells express significantly higher levels of the PP2A B-type subunit PR130 than epithelial PDAC cells. This overexpression of PR130 is associated with a dependency of such metastasis-prone cells on the catalytic activity of PP2A. Phendione induces apoptosis and an accumulation of cytotoxic protein aggregates in murine mesenchymal and human PDAC cells. These processes occur independently of the frequently mutated tumor suppressor p53. Proteomic analyses reveal that phendione upregulates the chaperone HSP70 in mesenchymal PDAC cells. Inhibition of HSP70 promotes phendione-induced apoptosis and phendione promotes a proteasomal degradation of PR130. Genetic elimination of PR130 sensitizes murine and human PDAC cells to phendione-induced apoptosis and protein aggregate formation. These data suggest that the PP2A-PR130 complex dephosphorylates and thereby prevents the aggregation of proteins in tumor cells.Graphical

Protein phosphatase 2A modulation and connection with miRNAs and natural products.

Protein phosphatase 2A (PP2A), a heterotrimeric holoenzyme (scaffolding, catalytic, and regulatory subunits), regulates dephosphorylation for more than half of serine/threonine phosphosites and exhibits diverse cellular functions. Although several studies on natural products and miRNAs have emphasized their impacts on PP2A regulation, their connections lack systemic organization. Moreover, only part of the PP2A family has been investigated. This review focuses on the PP2A-modulating effects of natural products and miRNAs' interactions with potential PP2A targets in cancer and non-cancer cells. PP2A-modulating natural products and miRNAs were retrieved through a literature search. Utilizing the miRDB database, potential PP2A targets of these PP2A-modulating miRNAs for the whole set (17 members) of the PP2A family were retrieved. Finally, PP2A-modulating natural products and miRNAs were linked via a literature search. This review provides systemic directions for assessing natural products and miRNAs relating to the PP2A-modulating functions in cancer and disease treatments.

Abstract IA012: The role of the serine/threonine phosphatase PP2A in endometrial cancer development and progression: lessons learned from mutations and small molecules

Abstract Uterine cancer is the most common gynecologic malignancy in the United States with approximately 60,000 new diagnoses each year. While many cases of uterine endometrial carcinoma (EMCA) have favorable outcomes with recurrence-free long-term survival, outcomes for the high-grade, treatment-refractory histological subtypes, including uterine serous carcinoma (USC) and uterine carcinosarcoma (UCS), remain an important clinical problem. Uterine serous carcinomas and uterine carcinosarcomas tumors are the most aggressive subtypes of uterine cancer, displaying a high propensity to invade the peritoneal cavity and to metastasize to distant sites. Accordingly, 50-70% of high-grade tumors exhibit extrauterine spread at time of initial surgery and have limited treatment options. The inherent aggressiveness of these tumors suggests that they are intrinsically equipped to disseminate and propagate tumors at secondary sites. The molecular drivers of this phenotype, however, have yet to be completely characterized. A lack of established and validated molecular drivers of the high-grade subtypes, especially those that are responsible for the high rate of metastasis, has limited the use of targeted treatment strategies. However, we and other groups have shown that PPP2R1A, the scaffolding subunit of the tumor suppressor protein phosphatase 2A, is mutated in up to 40% of USC patients, and these recurrent mutations are found at both the primary and metastatic sites. These mutations are heterozygous, and two specific mutations: P179R and S256F, occur exclusively within these high-grade subtypes of uterine cancer (i.e., are not found in any other cancer type). PP2A, is a family of serine/threonine phosphatases, where the active heterotrimeric form of the enzyme is comprised of a catalytic (C) subunit, a scaffolding (A) subunit, and a substrate directing regulatory (B) subunit. In aggregate, PP2A is considered a tumor suppressor protein, but certain heterotrimers have tumor promoting roles. Here we present data related to the molecular and biological basis through which these recurrent pathogenic mutations drive endometrial cancer development and progression using disease relevant cellular and in vivo model systems. Additionally, building from previously published work from our group, we have developed a novel series of small molecule molecular glues (PMGs) that specifically correct the mutant heterotrimer driving tumor regressions in both cellular and patient derived xenograft models of high-grade endometrial cancer. Collectively, this data suggests that recurrent pathogenic mutations in the PPP2R1A gene drive endometrial cancer pathogenesis and these mutations can be therapeutically targeted using a series of pharmaceutically tractable small molecules. Citation Format: Goutham Narla. The role of the serine/threonine phosphatase PP2A in endometrial cancer development and progression: lessons learned from mutations and small molecules [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr IA012.

Abstract B006: Mutant PPP2R1A induces the expression and secretion of IGFBP2 to promote uterine serous carcinoma metastasis

Abstract Uterine serous carcinoma (USC) tumors demonstrate a unique aggressiveness suggesting that the primary tumor is intrinsically equipped to disseminate, metastasize and initiate tumor formation at secondary sites. Yet the drivers and pathways regulating dissemination and metastasis in USC remain incompletely understood. Previous work by our group and others identified a mutational hotspot within PPP2R1A, which encodes the Aa scaffolding subunit of protein phosphatase 2A (PP2A), a heterotrimeric serine/threonine phosphatase and known tumor suppressor. Two recurrent mutations, P179R and S256F, occur exclusively within high-grade subtypes of uterine cancer at a rate of 30-40%. We previously published that the P179R mutant drives USC tumorigenesis through the inactivation of PP2A, and genetic correction of this mutant resulted in decreased primary and metastatic tumor formation. The underlying mechanisms for these observations, however, remain unclear. To study the mechanistic basis for these observations, we generated isogenic patient-derived USC cell lines expressing the Aa – P179R and S256F mutants. Expression of these mutants enhanced the tumor-initiating capacity (TIC), a hybrid epithelial to mesenchymal (EM) plasticity profile, and the secretion of tumor-promoting factors in vitro and in vivo. Molecular analysis of these cell lines and tumors demonstrated that these mutants drove increased NF-kb phosphorylation resulting in the upregulation and secretion of the EM cytokine, IGFBP2. We found that the Aa mutant phenotype was dependent on the IGFBP2 cytokine function in vitro and in vivo. Future experiments will focus on identifying which PP2A B regulatory subunits are responsible for regulating the onset of the NF-kb transcriptional program. Cellular reprogramming through the IGFBP2-induced NF-kb signaling pathway mediated by recurrent Aa mutants may explain the intrinsically aggressive nature of USC, through enhanced dissemination and metastasis. Citation Format: Terrance James Haanen, Sophie Boock, Sarah Taylor, Caitlin O'Connor, Analisa DiFeo, Goutham Narla. Mutant PPP2R1A induces the expression and secretion of IGFBP2 to promote uterine serous carcinoma metastasis [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr B006.

Differential Synonymous Codon Selection in the B56 Gene Family of PP2A Regulatory Subunits.

Protein phosphatase 2A (PP2A) functions as a tumor suppressor and consists of a scaffolding, catalytic, and regulatory subunit. The B56 gene family of regulatory subunits impart distinct functions onto PP2A. Codon usage bias (CUB) involves the selection of synonymous codons, which can affect gene expression by modulating processes such as transcription and translation. CUB can vary along the length of a gene, and differential use of synonymous codons can be important in the divergence of gene families. The N-termini of the gene product encoded by B56α possessed high CUB, high GC content at the third codon position (GC3), and high rare codon content. In addition, differential CUB was found in the sequence encoding two B56γ N-terminal splice forms. The sequence encoding the N-termini of B56γ/γ, relative to B56δ/γ, displayed CUB, utilized more frequent codons, and had higher GC3 content. B56α mRNA had stronger than predicted secondary structure at their 5' end, and the B56δ/γ splice variants had long regions of weaker than predicted secondary structure at their 5' end. The data suggest that B56α is expressed at relatively low levels as compared to the other B56 isoforms and that the B56δ/γ splice variant is expressed more highly than B56γ/γ.

Prediction of PP2A Holoenzymes Guided By the Structural Characterization of Methylation Independent PP2A Assembly

Introduction: The dysregulation of phosphorylation-dependent signaling is a hallmark of leukemogenesis. Kinase inhibitors have revolutionized the outcomes of patients with chronic myeloid leukemia. An orthogonal, but less explored way to target phosphorylation pathways is to activate phosphatases, such as the ubiquitously expressed protein- phosphatase 2A (PP2A). PP2A is an essential serine/threonine phosphatase that regulates various cellular processes, including cell growth, division, and differentiation. Dysregulation of PP2A has been linked to leukemia. PP2A is a modular multi-subunit enzyme: One scaffold subunit (A) binds to a catalytic subunit (C) to form a core AC heterodimer, which together with one of many regulatory (B) subunits forms the active enzyme. The regulatory (B) subunits are classified into 4 distinct subfamilies without significant homology. The combinatorial assembly of these subunits generates a wide range of PP2A complexes, resulting in diverse substrate specificity and subcellular localization. The detailed mechanism of PP2A regulation remains elusive and reports about a governing role of methylation of the carboxy-terminal Leucin of PP2A C are conflicting. Understanding the underlying molecular regulation of PP2A activity is of significant interest for future therapeutic development for acute myeloid leukemia. Methods: Recombinant PP2A was expressed in insect cells, and purified by affinity purification, anion exchange chromatography and size exclusion chromatography (SEC). Extensive quality control, including liquid chromatography-mass spectrometry, SDS-PAGE gel and fast protein liquid chromatography was performed. The degree of methylation of PP2A C Leucine 309 was confirmed by mass spectrometry. We validated protein stability with differential scanning fluorimetry. Pull-down assays and size exclusion chromatography characterized complex formation. A calorimetric assay using p-nitrophenyl phosphate as a substrate functionally assessed PP2A in vitro. Structural information was obtained by X-ray crystallography and AlphaFold Multimer. Results: Using in vitro biochemistry, including pull down and size exclusion chromatography, we demonstrate that methylation of the carboxy terminus of the PP2A C subunit is dispensable for PP2A assembly in vitro. Absence of methylation of Leucine 309 was confirmed by mass-spectrometry. We show by SEC that instead PP2A complex formation is dependent on B subunit concentration. Carboxy-terminal methylation of the PP2A C subunit was also not required for functional activity in a calorimetric assay using p-nitrophenyl phosphate as a substrate. PP2A formed spontaneously in vitro and was enzymatically active independent of methylation of Leucine 309. To corroborate these findings, we determined the X-ray crystal structure of the PP2A:PPP2B56E:PPP2C complex to 2.7 Å resolution with the unmethylated Leucine 309. Interestingly our experimental structure superimposed with an AlphaFold Multimer model of the PP2A trimer. Building on this finding, we predicted the models of all 64 possible PP2A complexes providing a framework for further study and structural analysis of PP2A complexes and their activation. These studies provide key information for the structural design and development of novel drugs for hematologic malignancies by targeting of selected PP2A complexes and clarified the role of Leucine 309 methylation in PP2A activation. Conclusion: Methylation of the carboxy-terminal Leucine 309 of the PP2A C subunitis dispensable for PP2A activity in vitro. Integrative structural biology studies, combining biochemistry, X-ray crystallography and AlphaFold Multimer provide a comprehensive map of PP2A complexes. These studies have the potential to accelerate the drug discovery process and unlock PP2A activation as a new therapeutic modality for leukemia.

CCR4-NOT differentially controls host versus virus poly(a)-tail length and regulates HCMV infection.

Unlike most RNA and DNA viruses that broadly stimulate mRNA decay and interfere with host gene expression, human cytomegalovirus (HCMV) extensively remodels the host translatome without producing an mRNA decay enzyme. By performing a targeted loss-of-function screen in primary human fibroblasts, we here identify the host CCR4-NOT deadenylase complex members CNOT1 and CNOT3 as unexpected pro-viral host factors that selectively regulate HCMV reproduction. We find that the scaffold subunit CNOT1 is specifically required for late viral gene expression and genome-wide host responses in CCR4-NOT-disrupted cells. By profiling poly(A)-tail lengths of individual HCMV and host mRNAs using nanopore direct RNA sequencing, we reveal poly(A)-tails of viral messages to be markedly longer than those of cellular mRNAs and significantly less sensitive to CCR4-NOT disruption. Our data establish that mRNA deadenylation by host CCR4-NOT is critical for productive HCMV replication and define a new mechanism whereby herpesvirus infection subverts cellular mRNA metabolism to remodel the gene expression landscape of the infected cell. Moreover, we expose an unanticipated host factor with potential to become a therapeutic anti-HCMV target.

Distinct Clades of Protein Phosphatase 2A Regulatory B'/B56 Subunits Engage in Different Physiological Processes.

Protein phosphatase 2A (PP2A) is a strongly conserved and major protein phosphatase in all eukaryotes. The canonical PP2A complex consists of a catalytic (C), scaffolding (A), and regulatory (B) subunit. Plants have three groups of evolutionary distinct B subunits: B55, B' (B56), and B''. Here, the Arabidopsis B' group is reviewed and compared with other eukaryotes. Members of the B'α/B'β clade are especially important for chromatid cohesion, and dephosphorylation of transcription factors that mediate brassinosteroid (BR) signaling in the nucleus. Other B' subunits interact with proteins at the cell membrane to dampen BR signaling or harness immune responses. The transition from vegetative to reproductive phase is influenced differentially by distinct B' subunits; B'α and B'β being of little importance, whereas others (B'γ, B'ζ, B'η, B'θ, B'κ) promote transition to flowering. Interestingly, the latter B' subunits have three motifs in a conserved manner, i.e., two docking sites for protein phosphatase 1 (PP1), and a POLO consensus phosphorylation site between these motifs. This supports the view that a conserved PP1-PP2A dephosphorelay is important in a variety of signaling contexts throughout eukaryotes. A profound understanding of these regulators may help in designing future crops and understand environmental issues.

Disruption of the Mammalian Ccr4-Not Complex Contributes to Transcription-Mediated Genome Instability.

The mammalian Ccr4-Not complex, carbon catabolite repression 4 (Ccr4)-negative on TATA-less (Not), is a large, highly conserved, multifunctional assembly of proteins that acts at different cellular levels to regulate gene expression. It is involved in the control of the cell cycle, chromatin modification, activation and inhibition of transcription initiation, control of transcription elongation, RNA export, and nuclear RNA surveillance; the Ccr4-Not complex also plays a central role in the regulation of mRNA decay. Growing evidence suggests that gene transcription has a vital role in shaping the landscape of genome replication and is also a potent source of replication stress and genome instability. Here, we have examined the effects of the inactivation of the Ccr4-Not complex, via the depletion of the scaffold subunit CNOT1, on DNA replication and genome integrity in mammalian cells. In CNOT1-depleted cells, the elevated expression of the general transcription factor TATA-box binding protein (TBP) leads to increased RNA synthesis, which, together with R-loop accumulation, results in replication fork slowing, DNA damage, and senescence. Furthermore, we have shown that the stability of TBP mRNA increases in the absence of CNOT1, which may explain its elevated protein expression in CNOT1-depleted cells. Finally, we have shown the activation of mitogen-activated protein kinase signalling as evidenced by ERK1/2 phosphorylation in the absence of CNOT1, which may be responsible for the observed cell cycle arrest at the border of G1/S.

Protein-Protein Interactions and Quantitative Phosphoproteomic Analysis Reveal Potential Mitochondrial Substrates of Protein Phosphatase 2A-B'ζ Holoenzyme.

Protein phosphatase 2A (PP2A) is a heterotrimeric conserved serine/threonine phosphatase complex that includes catalytic, scaffolding, and regulatory subunits. The 3 A subunits, 17 B subunits, and 5 C subunits that are encoded by the Arabidopsis genome allow 255 possible PP2A holoenzyme combinations. The regulatory subunits are crucial for substrate specificity and PP2A complex localization and are classified into the B, B', and B" non-related families in land plants. In Arabidopsis, the close homologs B'η, B'θ, B'γ, and B'ζ are further classified into a subfamily of B' called B'η. Previous studies have suggested that mitochondrial targeted PP2A subunits (B'ζ) play a role in energy metabolism and plant innate immunity. Potentially, the PP2A-B'ζ holoenzyme is involved in the regulation of the mitochondrial succinate/fumarate translocator, and it may affect the enzymes involved in energy metabolism. To investigate this hypothesis, the interactions between PP2A-B'ζ and the enzymes involved in the mitochondrial energy flow were investigated using bimolecular fluorescence complementation in tobacco and onion cells. Interactions were confirmed between the B'ζ subunit and the Krebs cycle proteins succinate/fumarate translocator (mSFC1), malate dehydrogenase (mMDH2), and aconitase (ACO3). Additional putative interacting candidates were deduced by comparing the enriched phosphoproteomes of wild type and B'ζ mutants: the mitochondrial regulator Arabidopsis pentatricopeptide repeat 6 (PPR6) and the two metabolic enzymes phosphoenolpyruvate carboxylase (PPC3) and phosphoenolpyruvate carboxykinase (PCK1). Overall, this study identifies potential PP2A substrates and highlights the role of PP2A in regulating energy metabolism in mitochondria.

Cryo-EM structure of the Mon1–Ccz1–RMC1 complex reveals molecular basis of metazoan RAB7A activation

Understanding of the evolution of metazoans from their unicellular ancestors is a fundamental question in biology. In contrast to fungi which utilize the Mon1-Ccz1 dimeric complex to activate the small GTPase RAB7A, metazoans rely on the Mon1-Ccz1-RMC1 trimeric complex. Here, we report a near-atomic resolution cryogenic-electron microscopystructure of the Drosophila Mon1-Ccz1-RMC1 complex. RMC1 acts as a scaffolding subunit and binds to both Mon1 and Ccz1 on the surface opposite to the RAB7A-binding site, with many of the RMC1-contacting residues from Mon1 and Ccz1 unique to metazoans, explaining the binding specificity. Significantly, the assembly of RMC1 with Mon1-Ccz1 is required for cellular RAB7A activation, autophagic functions and organismal development in zebrafish. Our studies offer a molecular explanation for the different degree of subunit conservation across species, and provide an excellent example of how metazoan-specific proteins take over existing functions in unicellular organisms.

Venlafaxine antagonizes the noradrenaline-promoted colon cancer progression by inhibiting the norepinephrine transporter

Epidemiological studies have demonstrated that the use of antidepressants is associated with a decreased risk of colorectal cancer (CRC); however, the mechanisms behind this association are yet unknown. Adrenergic system contributes to the stress-related tumor progression, with norepinephrine (NE) mainly secreted from adrenergic nerve fibers. Norepinephrine serotonin reuptake inhibitors are successfully used antidepressants. This study demonstrates that a widely used antidepressant venlafaxine (VEN) antagonizes NE-promoted colon cancer in vivo and in vitro. Bioinformatic analysis suggested that NE transporter (NET, SLC6A2), a target of VEN, was closely associated with the prognosis of clinical patients with CRC. In addition, the knockdown of NET antagonized the effect of NE. The NET-protein phosphatase 2 scaffold subunit alpha/phosphorylated Akt/vascular endothelial growth factor pathway partially mediates the antagonizing effect of VEN on NE’s actions in colon cancer cells. These were also confirmed by in vivo experiments. Our findings revealed for the first time that, in addition to its primary function as a transporter, NET also promotes NE-enhanced colon cancer cell proliferation, tumor angiogenesis, and tumor growth. This provides direct experimental and mechanistic evidence for the use of antidepressant VEN in the treatment of CRC and a therapeutic potential for repurposing existing drugs as an anti-cancer approach to improve the prognosis of patients with CRC.

Abstract 6271: Discovery of BR1733 for the treatment of SWI/SNF deficient or highly vascularized solid tumor

Abstract Polycomb repressive complex 2 (PRC2) catalyze the writing of H3K27me3 epigenetic marker and suppresses the expression of genes including tumor suppressors. Activation mutation of EZH2, the catalytic subunit of the PRC2 complex, drives the progression of tumors such as DLBCL. The embryonic ectoderm development (EED) protein acts as the scaffolding and H3K27me3-binding subunit of PRC2 and is essential for its activity. We have developed potent, selective, and orally bioavailable EED inhibitor BR1733. BR1733 blocks the interaction between EED and H3K27me3, and inhibits catalytic activity of PRC2. BR1733 showed superior PK profile across species and exhibited tumor growth inhibition against several B cell lymphoma CDX models. Indication expansion studies demonstrated that BR1733 could inhibit solid tumors with SWI/SNF loss-of-function mutation only when both CDKN2A and TET1 are expressed. BR1733 also inhibited tumor angiogenesis and showed tumor growth inhibition in several CDX models of highly vascularized solid tumors such as HCC and RCC. BR1733 is currently in Phase I clinical trials. Citation Format: Xinghao Wang, Shichao Ma, Min (Bella) Xie, Lele Wang, Yajun Wang, Song Zhang, Sheng Chen, Lynette Zhang, Yongqi Zhou, Mengxi Zhao, Dandan Li, Guobin Li, Xiao Fan, Hailong Zhang. Discovery of BR1733 for the treatment of SWI/SNF deficient or highly vascularized solid tumor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6271.