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BRCA1 functions as a tumor suppressor in DNA repair and centrosome regulation. Previously, Obg-like ATPase 1 (OLA1) was shown to interact with BARD1, a heterodimer partner of BRCA1. OLA1 binds to BRCA1, BARD1, and γ-tubulin and functions in centrosome regulation. This study determined that overexpression of wild-type OLA1 (OLA1-WT) caused centrosome amplification due to centriole overduplication in mammary tissue–derived cells. Centrosome amplification induced by overexpression of the cancer-derived OLA1 mutant, which is deficient at regulating centrosome number, occurred in significantly fewer cells than in that induced by overexpression of OLA1-WT. Thus, it was hypothesized that overexpression of OLA1 with normal function efficiently induces centrosome amplification, but not that of OLA1 mutants, which are deficient at regulating centrosome number. We analyzed whether overexpression of OLA1 missense mutants of nine candidate phosphorylation residues, three residues modified with acetylation, and two ATP-binding residues caused centrosome amplification and identified five missense mutants that are deficient in the regulation of centrosome number. Three of them did not bind to BARD1. Two phosphomimetic mutations restored the binding to BARD1 and the efficient centrosome amplification by their overexpression. Knockdown and overexpression of BARD1 also caused centrosome amplification. BARD1 mutant reported in cancer failed to bind to OLA1 and rescue the BARD1 knockdown-induced centrosome amplification and reduced its centrosomal localization. Combined, these data reveal that the OLA1–BARD1 interaction is important for the regulation of centrosome number.Implications: Regulation of centrosome number by BRCA1/BARD1 together with OLA1 is important for the genome integrity to prevent tumor development. Mol Cancer Res; 16(10); 1499–511. ©2018 AACR.

Supplementary Figure 4. BARD1 expression in Hs578T cells transfected with BARD1 siRNA and expression vector.

OLA1 residues responsible for the biological functions.

Summary of biological functions of OLA1 mutants.

Metabolic reprogramming of leukemia cells is important for survival, proliferation, and drug resistance under conditions of metabolic stress in the bone marrow. Deregulation of cellular metabolism, leading to development of leukemia, occurs through abnormally high expression of transcription factors such as MYC and Ecotropic Virus Integration site 1 protein homolog (EVI1). Overexpression of EVI1 in adults and children with mixed lineage leukemia-rearrangement acute myeloid leukemia (MLL-r AML) has a very poor prognosis. To identify a metabolic inhibitor for EVI1-induced metabolic reprogramming in MLL-r AML, we used an XFp extracellular flux analyzer to examine metabolic changes during leukemia development in mouse models of AML expressing MLL-AF9 and Evi1 (Evi1/MF9). Oxidative phosphorylation (OXPHOS) in Evi1/MF9 AML cells accelerated prior to activation of glycolysis, with a higher dependency on glutamine as an energy source. Furthermore, EVI1 played a role in glycolysis as well as driving production of metabolites in the tricarboxylic acid cycle. L-asparaginase (L-asp) exacerbated growth inhibition induced by glutamine starvation and suppressed OXPHOS and proliferation of Evi1/MF9 both in vitro and in vivo; high sensitivity to L-asp was caused by low expression of asparagine synthetase (ASNS) and L-asp-induced suppression of glutamine metabolism. In addition, samples from patients with EVI1+MF9 showed low ASNS expression, suggesting that it is a sensitive marker of L-asp treatment. Clarification of metabolic reprogramming in EVI1+ leukemia cells may aid development of treatments for EVI1+MF9 refractory leukemia.

Ecotropic viral integration site-1 (EVI1) has a critical role in normal and malignant hematopoiesis. Since we previously identified high expression of calcitonin receptor like receptor (CRLR) in acute myeloid leukemia (AML) with high EVI1 expression, we here characterized the function of CRLR in hematopoiesis. Since higher expression of CRLR and receptor activity modifying protein 1 (RAMP1) was identified in immature hematopoietic bone marrow (BM) cells, we focused on calcitonin gene-related peptide (CGRP), a specific ligand for the CRLR/RAMP1 complex. To elucidate the role of CGRP in hematopoiesis, Ramp1-deficient (Ramp1−/−) mice were used. The steady-state hematopoiesis was almost maintained in Ramp1−/− mice; however, the BM repopulation capacity of Ramp1−/− mice was significantly decreased, and the transplanted Ramp1−/− BM mononuclear cells had low proliferation capacity with enhanced reactive oxygen species (ROS) production and cell apoptosis. Thus, CGRP is important for maintaining hematopoiesis during temporal exposures with proliferative stress. Moreover, continuous CGRP exposure to mice for two weeks induced a reduction in the number of BM immature hematopoietic cells along with differentiated myeloid cells. Since CGRP is known to be increased under inflammatory conditions to regulate immune responses, hematopoietic exhaustion by continuous CGRP secretion under chronic inflammatory conditions is probably one of the important mechanisms of anti-inflammatory responses.

Here, we address the function of protein phosphatase 6 (PP6) loss on K-ras-initiated tumorigenesis in keratinocytes. To do so, we developed tamoxifen-inducible double mutant (K-rasG12D -expressing and Ppp6c-deficient) mice in which K-rasG12D expression is driven by the cytokeratin 14 (K14) promoter. Doubly-mutant mice showed early onset tumor formation in lips, nipples, external genitalia, anus and palms, and had to be killed by 3 weeks after induction by tamoxifen, while comparably-treated K-rasG12D -expressing mice did not. H&E-staining of lip tumors before euthanasia revealed that all were papillomas, some containing focal squamous cell carcinomas. Immunohistochemical analysis of lips of doubly-mutant vs K-rasG12D mice revealed that cell proliferation and cell size increased approximately 2-fold relative to K-rasG12D -expressing mutants, and epidermal thickness of lip tissue greatly increased relative to that seen in K-rasG12D -only mice. Moreover, AKT phosphorylation increased in K-rasG12D -expressing/Ppp6c-deficient cells, as did phosphorylation of the downstream effectors 4EBP1, S6 and GSK3, suggesting that protein synthesis and survival signals are enhanced in lip tissues of doubly-mutant mice. Finally, increased numbers of K14-positive cells were present in the suprabasal layer of doubly-mutant mice, indicating abnormal keratinocyte differentiation, and γH2AX-positive cells accumulated, indicating perturbed DNA repair. Taken together, Ppp6c deficiency enhances K-rasG12D -dependent tumor promotion.

Ppp6c, which encodes the catalytic subunit of phosphoprotein phosphatase 6 (PP6), is conserved among eukaryotes from yeast to humans. In mammalian cells, PP6 targets IκBε for degradation, activates DNA-dependent protein kinase to trigger DNA repair, and is reportedly required for normal mitosis. Recently, Ppp6c mutations were identified as candidate drivers of melanoma and skin cancer. Nonetheless, little is known about the physiological role of Ppp6c. To investigate this function in vivo, we established mice lacking the Ppp6c phosphatase domain by crossing heterozygous mutants. No viable homozygous pups were born, indicative of a lethal mutation. Ppp6c homozygous mutant embryos were identified among blastocysts, which exhibited a normal appearance, but embryos degenerated by E7.5 and showed clear developmental defects at E8.5, suggesting that mutant embryos die after implantation. Accordingly, homozygous blastocysts showed significant growth failure of the inner cell mass (ICM) in in vitro blastocyst culture, and primary Ppp6c exon4-deficient MEFs showed greatly reduced proliferation. These results establish for the first time that the Ppp6c phosphatase domain is indispensable for mouse embryogenesis after implantation.

G protein-coupled receptor 56 (GPR56) is highly expressed in acute myeloid leukemia (AML) cells with high EVI1 expression (EVI1high AML). Because GPR56 is a transcriptional target of EVI1 and silencing of GPR56 expression induces apoptosis, we developed a novel drug to suppress GPR56 expression in EVI1high AML cells. For this purpose, we generated pyrrole-imidazole (PI) polyamides specific to GPR56 (PIP/56-1 or PIP/56-2) as nuclease-resistant novel compounds that interfere with the binding of EVI1 to the GPR56 promoter in a sequence-specific manner. Treatment of EVI1high AML cell lines (UCSD/AML1 and Kasumi-3) with PIP/56-1 or PIP/56-2 effectively suppressed GPR56 expression by inhibiting binding of EVI1 to its promoter, leading to suppression of cell growth with increased rates of apoptosis. Moreover, intravenous administration of PIP/56-1 into immunodeficient Balb/c-RJ mice subcutaneously transplanted with UCSD/AML1 cells significantly inhibited tumor growth and extended survival. Furthermore, organ infiltration by leukemia cells in immunodeficient Balb/c-RJ mice, which were intravenously transplanted using UCSD/AML1 cells, was successfully inhibited by PIP/56-1 treatment with no apparent effects on murine hematopoietic cells. In addition, PIP treatment did not inhibit colony formation of human CD34+ progenitor cells. Thus, PI polyamide targeting of GPR56 using our compound is promising, useful, and safe for the treatment of EVI1high AML.

BRCA1 functions as a tumor suppressor in DNA repair and centrosome regulation. Previously, Obg-like ATPase 1 (OLA1) was shown to interact with BARD1, a heterodimer partner of BRCA1. OLA1 binds to BRCA1, BARD1, and γ-tubulin and functions in centrosome regulation. This study determined that overexpression of wild-type OLA1 (OLA1-WT) caused centrosome amplification due to centriole overduplication in mammary tissue–derived cells. Centrosome amplification induced by overexpression of the cancer-derived OLA1 mutant, which is deficient at regulating centrosome number, occurred in significantly fewer cells than in that induced by overexpression of OLA1-WT. Thus, it was hypothesized that overexpression of OLA1 with normal function efficiently induces centrosome amplification, but not that of OLA1 mutants, which are deficient at regulating centrosome number. We analyzed whether overexpression of OLA1 missense mutants of nine candidate phosphorylation residues, three residues modified with acetylation, and two ATP-binding residues caused centrosome amplification and identified five missense mutants that are deficient in the regulation of centrosome number. Three of them did not bind to BARD1. Two phosphomimetic mutations restored the binding to BARD1 and the efficient centrosome amplification by their overexpression. Knockdown and overexpression of BARD1 also caused centrosome amplification. BARD1 mutant reported in cancer failed to bind to OLA1 and rescue the BARD1 knockdown-induced centrosome amplification and reduced its centrosomal localization. Combined, these data reveal that the OLA1–BARD1 interaction is important for the regulation of centrosome number. Implications: Regulation of centrosome number by BRCA1/BARD1 together with OLA1 is important for the genome integrity to prevent tumor development. Mol Cancer Res; 16(10); 1499–511. ©2018 AACR.

Here, we address the function of protein phosphatase 6 (PP6) loss on K‐ras‐initiated tumorigenesis in keratinocytes. To do so, we developed tamoxifen‐inducible double mutant (K‐ras G12D‐expressing and Ppp6c‐deficient) mice in which K‐ras G12D expression is driven by the cytokeratin 14 (K14) promoter. Doubly‐mutant mice showed early onset tumor formation in lips, nipples, external genitalia, anus and palms, and had to be killed by 3 weeks after induction by tamoxifen, while comparably‐treated K‐ras G12D‐expressing mice did not. H&E‐staining of lip tumors before euthanasia revealed that all were papillomas, some containing focal squamous cell carcinomas. Immunohistochemical analysis of lips of doubly‐mutant vs K‐ras G12D mice revealed that cell proliferation and cell size increased approximately 2‐fold relative to K‐ras G12D‐expressing mutants, and epidermal thickness of lip tissue greatly increased relative to that seen in K‐ras G12D‐only mice. Moreover, AKT phosphorylation increased in K‐ras G12D‐expressing/Ppp6c‐deficient cells, as did phosphorylation of the downstream effectors 4EBP1, S6 and GSK3, suggesting that protein synthesis and survival signals are enhanced in lip tissues of doubly‐mutant mice. Finally, increased numbers of K14‐positive cells were present in the suprabasal layer of doubly‐mutant mice, indicating abnormal keratinocyte differentiation, and γH2AX‐positive cells accumulated, indicating perturbed DNA repair. Taken together, Ppp6c deficiency enhances K‐ras G12D‐dependent tumor promotion.

ADP-ribosylation factor (Arf) 1 is thought to affect the morphologies of organelles, such as the Golgi apparatus, and regulate protein trafficking pathways. Mice have six Arf isoforms. In knockdown experiments with HeLa cells, no single Arf isoform among Arf1-5 is required for organelle morphologies or any membrane trafficking step. This suggests that the cooperation of two or more Arfs is a general feature. Although many cell biological and biochemical analyses have proven the importance of Arf1, the physiological roles of Arf1 in mice remain unknown. To investigate the activity of Arf1 in vivo, we established Arf1-deficient mice. Arf(-/-) blastocysts were identified at the expected Mendelian ratio. The appearance of these blastocysts was indistinguishable from that of wild-type and Arf(+/-) blastocysts, and they grew normally in an in vitro culture system. However, Arf(-/-) embryos were degenerated at E5.5, and none survived to E12.5, suggesting that they died soon after implantation. These data establish for the first time that the Arf1 gene is indispensable for mouse embryonic development after implantation.

Epigenetic regulation is required to ensure the precise spatial and temporal pattern of gene expression that is necessary for embryonic development. Although the roles of some epigenetic modifications in embryonic development have been investigated in depth, the role of methylation at lysine 79 (H3K79me) is poorly understood. Dot1L, a unique methyltransferase for H3K79, forms complexes with distinct sets of co-factors. To further understand the role of H3K79me in embryogenesis, we generated a mouse knockout of Mllt10, the gene encoding Af10, one Dot1L complex co-factor. We find homozygous Mllt10 knockout mutants (Mllt10-KO) exhibit midline facial cleft. The midfacial defects of Mllt10-KO embryos correspond to hyperterolism and are associated with reduced proliferation of mesenchyme in developing nasal processes and adjacent tissue. We demonstrate that H3K79me level is significantly decreased in nasal processes of Mllt10-KO embryos. Importantly, we find that expression of AP2α, a gene critical for midfacial development, is directly regulated by Af10-dependent H3K79me, and expression AP2α is reduced specifically in nasal processes of Mllt10-KO embryos. Suppression of H3K79me completely mimicked the Mllt10-KO phenotype. Together these data are the first to demonstrate that Af10-dependent H3K79me is essential for development of nasal processes and adjacent tissues, and consequent midfacial formation.

In mammals, the small Arf GTPase-activating protein (SMAP) subfamily of Arf GTPase-activating proteins consists of closely related members, SMAP1 and SMAP2. These factors reportedly exert distinct functions in membrane trafficking, as manifested by different phenotypes seen in single knockout mice. The present study investigated whether SMAP proteins interact genetically. We report for the first time that simultaneous loss of SMAP1 and SMAP2 promotes apoptosis in the distal region of E7.5 mouse embryos, likely resulting in embryonic lethality. Thus, at least one SMAP gene, either SMAP1 or SMAP2, is required for proper embryogenesis.

We previously reported that deficiency in the gene encoding the catalytic subunit of protein phosphatase 6 (Ppp6c) predisposes mouse skin tissue to papilloma formation initiated by DMBA. Here, we demonstrate that Ppp6c loss acts as a tumor promoter in UVB-induced squamous cell carcinogenesis. Following UVB irradiation, mice with Ppp6c-deficient keratinocytes showed a higher incidence of skin squamous cell carcinoma than did control mice. Time course experiments showed that following UVB irradiation, Ppp6c-deficient keratinocytes upregulated expression of p53, PUMA, BAX, and cleaved caspase-3 proteins. UVB-induced tumors in Ppp6c-deficient keratinocytes exhibited a high frequency of both p53- and γH2AX-positive cells, suggestive of DNA damage. Epidemiological and molecular data strongly suggest that UVB from sunlight induces p53 gene mutations in keratinocytes and is the primary causative agent of human skin cancers. Our analysis suggests that PP6 deficiency underlies molecular events that drive outgrowth of initiated keratinocytes harboring UVB-induced mutated p53. Understanding PP6 function in preventing UV-induced tumorigenesis could suggest strategies to prevent and treat this condition.

Somatic mutations in the gene encoding the catalytic subunit of protein phosphatase 6 (Ppp6c) have been identified in malignant melanoma and are thought to function as a driver in B-raf- or N-ras-driven tumorigenesis. To assess the role of Ppp6c in carcinogenesis, we generated skin keratinocyte-specific Ppp6c conditional knockout mice and performed two-stage skin carcinogenesis analysis. Ppp6c deficiency induced papilloma formation with 7,12-dimethylbenz (a) anthracene (DMBA) only, and development of those papillomas was significantly accelerated compared with that seen following DMBA/TPA (12-O-tetradecanoylphorbol 13-acetate) treatment of wild-type mice. NF-κB activation either by tumor necrosis factor (TNF)-α or interleukin (IL)-1β was enhanced in Ppp6c-deficient keratinocytes. Overall, we conclude that Ppp6c deficiency predisposes mice to skin carcinogenesis initiated by DMBA. This is the first report showing that such deficiency promotes tumor formation in mice.

CALM is implicated in the formation of clathrin-coated vesicles, which mediate endocytosis and intracellular trafficking of growth factor receptors and nutrients. We previously found that CALM-deficient mice suffer from severe anemia due to the impaired clathrin-mediated endocytosis of transferrin receptor in immature erythroblast. However, CALM has been supposed to regulate the growth and survival of hematopoietic stem/progenitor cells. So, in this study, we focused on the function of CALM in these cells. We here show that the number of Linage−Sca-1+KIT+ (LSK) cells decreased in the fetal liver of CALM −/− mice. Also, colony forming activity was impaired in CALM−/− LSK cells. In addition, SCF, FLT3, and TPO-dependent growth was severely impaired in CALM−/− LSK cells, while they can normally proliferate in response to IL-3 and IL-6. We also examined the intracellular trafficking of KIT using CALM −/− murine embryonic fibroblasts (MEFs) engineered to express KIT. At first, we confirmed that endocytosis of SCF-bound KIT was not impaired in CALM −/− MEFs by the internalization assay. However, SCF-induced KIT trafficking from early to late endosome was severely impaired in CALM −/− MEFs. As a result, although intracellular KIT disappeared 30 min after SCF stimulation in wild-type (WT) MEFs, it was retained in CALM −/− MEFs. Furthermore, SCF-induced phosphorylation of cytosolic KIT was enhanced and prolonged in CALM −/− MEFs compared with that in WT MEFs, leading to the excessive activation of Akt. Similar hyperactivation of Akt was observed in CALM −/− KIT+ cells. These results indicate that CALM is essential for the intracellular trafficking of KIT and its normal functions. Also, our data demonstrate that KIT located in the early endosome can activate downstream molecules as a signaling endosome. Because KIT activation is involved in the pathogenesis of some malignancies, the manipulation of CALM function would be an attractive therapeutic strategy.

Protein phosphatase-6 (PP6) is a member of type-2A protein-Ser/Thr phosphatases, and reportedly regulates several cellular functions including DNA damage responses, NF-kB activation, and mitotic spindle formation. Additionally, some recent papers reported somatic mutations of PPP6c gene, encoding the catalytic subunit of PP6, in about 10 % of melanoma patients, suggesting its potential role as either driver or suppressor of melanoma development. In this study, we generated mice, in which the PPP6C gene is deleted in keratinocyte-specific manner (PP6-KKO). PP6-KKO mice were predisposed to formation of skin tumor upon DMBA-treatment. Our results demonstrate PPP6c has a tumor-suppressive function at least in skin tumorigenesis. Citation Format: Katsuhisa Hayashi, Honami Ogoh, Toshio Watanabe, Nobuhiro Tanuma, Hiroshi Shima. Promotion of skin tumorigenesis by deletion of Ppp6c gene. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5282. doi:10.1158/1538-7445.AM2014-5282