RAB27B controls palmitoylation-dependent NRAS trafficking and signaling in myeloid leukemia.

RAS mutations are among the most prevalent oncogenic drivers in cancers. RAS proteins propagate signals only when associated with cellular membranes as a consequence of lipid modifications that impact their trafficking. Here, we discovered that RAB27B, a RAB family small GTPase, controlled NRAS palmitoylation and trafficking to the plasma membrane, a localization required for activation. Our proteomic studies revealed RAB27B upregulation in CBL- or JAK2-mutated myeloid malignancies, and its expression correlated with poor prognosis in acute myeloid leukemias (AMLs). RAB27B depletion inhibited the growth of CBL-deficient or NRAS-mutant cell lines. Strikingly, Rab27b deficiency in mice abrogated mutant but not WT NRAS-mediated progenitor cell growth, ERK signaling, and NRAS palmitoylation. Further, Rab27b deficiency significantly reduced myelomonocytic leukemia development in vivo. Mechanistically, RAB27B interacted with ZDHHC9, a palmitoyl acyltransferase that modifies NRAS. By regulating palmitoylation, RAB27B controlled c-RAF/MEK/ERK signaling and affected leukemia development. Importantly, RAB27B depletion in primary human AMLs inhibited oncogenic NRAS signaling and leukemic growth. We further revealed a significant correlation between RAB27B expression and sensitivity to MEK inhibitors in AMLs. Thus, our studies presented a link between RAB proteins and fundamental aspects of RAS posttranslational modification and trafficking, highlighting future therapeutic strategies for RAS-driven cancers.

A swallowable X-ray dosimeter for the real-time monitoring of radiotherapy.

Monitoring X-ray radiation in the gastrointestinal tract can enhance the precision of radiotherapy in patients with gastrointestinal cancer. Here we report the design and performance, in the gastrointestinal tract of rabbits, of a swallowable X-ray dosimeter for the simultaneous real-time monitoring of absolute absorbed radiation dose and of changes in pH and temperature. The dosimeter consists of a biocompatible optoelectronic capsule containing an optical fibre, lanthanide-doped persistent nanoscintillators, a pH-sensitive polyaniline film and a miniaturized system for the wireless readout of luminescence. The persistent luminescence of the nanoscintillators after irradiation can be used to continuously monitor pH without the need for external excitation. By using a neural-network-based regression model, we estimated the radiation dose from radioluminescence and afterglow intensity and temperature, and show that the dosimeter was approximately five times more accurate than standard methods for dose determination. Swallowable dosimeters may help to improve radiotherapy and to understand how radiotherapy affects tumour pH and temperature.

Potent suppression of neuroendocrine tumors and gastrointestinal cancers by CDH17CAR T cells without toxicity to normal tissues.

Gastrointestinal cancers (GICs) and neuroendocrine tumors (NETs) are often refractory to therapy after metastasis. Adoptive cell therapy using chimeric antigen receptor (CAR) T cells, though remarkably efficacious for treating leukemia, is yet to be developed for solid tumors such as GICs and NETs. Here we isolated a llama-derived nanobody, VHH1, and found that it bound cell surface adhesion protein CDH17 upregulated in GICs and NETs. VHH1-CAR T cells (CDH17CARTs) killed both human and mouse tumor cells in a CDH17-dependent manner. CDH17CARTs eradicated CDH17-expressing NETs and gastric, pancreatic and colorectal cancers in either tumor xenograft or autochthonous mouse models. Notably, CDH17CARTs do not attack normal intestinal epithelial cells, which also express CDH17, to cause toxicity, likely because CDH17 is localized only at the tight junction between normal intestinal epithelial cells. Thus, CDH17 represents a class of previously unappreciated tumor-associated antigens that is 'masked' in healthy tissues from attack by CAR T cells for developing safer cancer immunotherapy.

Prolactin-regulated Pbk is involved in pregnancy-induced ß-cell proliferation in mice.

Gestational diabetes mellitus (GDM) is a condition of diabetes with onset or first recognition in pregnancy. Its incidence is increasing, and GDM deleteriously affects both mother and the fetus during and even after pregnancy. Previous studies in mice have shown that during pregnancy, ß-cell proliferation increases in the middle and late stages of pregnancy and returns to normal levels after delivery. Hormones, such as prolactin, estradiol, and progesterone as well as protein kinases, play important roles in regulating gestation-mediated ß-cell proliferation; however, the regulatory relationship between them is uncertain. We previously found that protein kinase Pbk was crucial for basal proliferation of mouse islet cells. Herein we show that Pbk is upregulated during pregnancy in mice and Pbk kinase activity is required for enhanced ß- cell proliferation during pregnancy. Notably, knock-in (KI) of a kinase-inactivating Pbk mutation leads to impaired glucose tolerance and reduction of ß-cell proliferation and islet mass in mice during pregnancy. Prolactin upregulates the expression of Pbk, but the upregulation is diminished by knockdown of the prolactin receptor and by the inhibitors of JAK and STAT5, which mediate prolactin receptor signaling, in ß-cells. Treatment of ß-cells with prolactin increases STAT5 binding to the Pbk locus, as well as the recruitment of RNA polymerase II, resulting in increased Pbk transcription. These results demonstrate that Pbk is upregulated during pregnancy, at least partly by prolactin-induced and STAT5-mediated enhancement of gene transcription, and Pbk is essential for pregnancy-induced ß-cell proliferation, increase in islet mass, and maintenance of normal blood glucose during pregnancy in preclinical models. These findings provide new insights into the interplay between hormones and protein kinases that ultimately prevent the development of GDM.

Menin-regulated Pbk controls high fat diet-induced compensatory beta cell proliferation.

Pancreatic beta cells undergo compensatory proliferation in the early phase of type 2 diabetes. While pathways such as FoxM1 are involved in regulating compensatory beta cell proliferation, given the lack of therapeutics effectively targeting beta cell proliferation, other targetable pathways need to be identified. Herein, we show that Pbk, a serine/threonine protein kinase, is essential for high fat diet (HFD)-induced beta cell proliferation in vivo using a Pbk kinase deficiency knock-in mouse model. Mechanistically, JunD recruits menin and HDAC3 complex to the Pbk promoter to reduce histone H3 acetylation, leading to epigenetic repression of Pbk expression. Moreover, menin inhibitor (MI) disrupts the menin-JunD interaction and augments Pbk transcription. Importantly, MI administration increases beta cell proliferation, ameliorating hyperglycemia, and impaired glucose tolerance (IGT) in HFD-induced diabetic mice. Notably, Pbk is required for the MI-induced beta cell proliferation and improvement of IGT. Together, these results demonstrate the repressive role of the menin/JunD/Pbk axis in regulating HFD-induced compensatory beta cell proliferation and pharmacologically regulating this axis may serve as a novel strategy for type 2 diabetes therapy.

Wild-type menin is rapidly degraded via the ubiquitin-proteasome pathway in a rat insulinoma cell line.

Menin is encoded by multiple endocrine neoplasia type 1 (MEN1) gene, the germ line mutations of which are the main cause of pancreatic neuroendocrine tumors (PNETs). To date, a large number of frameshift, nonsense and missense mutations of MEN1 have been identified to be responsible for part of MEN1-defficient PNETs patients due to truncation or rapid degradation of menin protein. However, the stability of the wild-type (WT) menin in PNETs is totally unknown. In the present study, we observed ubiquitination of WT menin in 293T cells by transfection of ectopic WT menin and HA-ubiquitin. As expected, either endogenous or ectopic WT menin is stable in 293T cells, whereas in INS-1 cells, a rat insulinoma cell line derived from PNETs, either endogenous or ectopic WT menin is rapidly degraded through ubiquitin-proteasome pathway. Furthermore, the degradation of WT menin is more rapid in the presence of serum. Our findings suggest that in part of PNETs patients with WT MEN1, a ubiquitin-proteasome system targeting menin is untimely activated.

GLP-1 signaling suppresses menin's transcriptional block by phosphorylation in ß cells.

Both menin and glucagon-like peptide 1 (GLP-1) pathways play central yet opposing role in regulating ß cell function, with menin suppressing, and GLP-1 promoting, ß cell function. However, little is known as to whether or how GLP-1 pathway represses menin function. Here, we show that GLP-1 signaling-activated protein kinase A (PKA) directly phosphorylates menin at the serine 487 residue, relieving menin-mediated suppression of insulin expression and cell proliferation. Mechanistically, Ser487-phosphorylated menin gains increased binding affinity to nuclear actin/myosin IIa proteins and gets sequestrated from the Ins1 promoter. This event leads to reduced binding of repressive epigenetic histone modifiers suppressor variegation 3-9 homologue protein 1 (SUV39H1) and histone deacetylases 1 (HDAC1) at the locus and subsequently increased Ins1 gene transcription. Ser487 phosphorylation of menin also increases expression of proproliferative cyclin D2 and ß cell proliferation. Our results have uncovered a previously unappreciated physiological link in which GLP-1 signaling suppresses menin function through phosphorylation-triggered and actin/myosin cytoskeletal protein-mediated derepression of gene transcription.

Menin Upregulates FOXO1 Protein Stability by Repressing Skp2-Mediated Degradation in ß Cells.

OBJECTIVES: Menin, a chromatin binding protein, interacts with various epigenetic regulators to regulate gene transcription, whereas forkhead box protein O1 (FOXO1) is a transcription factor that can be regulated by multiple signaling pathways. Both menin and FOXO1 are crucial regulators of ß-cell function and metabolism; however, whether or how they interplay to regulate ß cells is not clear. METHODS: To examine whether menin affects expression of FOXO1, we ectopically expressed menin complementary DNA and small hairpin RNA targeting menin via a retroviral vector in INS-1 cells. Western blotting was used to analyze protein levels. RESULTS: Our current work shows that menin increases the expression of FOXO1. Menin stabilizes FOXO1 protein level in INS-1 cells, as shown by increased half-life of FOXO1 by menin expression. Moreover, menin represses ubiquitination of FOXO1 protein and AKT phosphorylation, We found that menin stabilizes FOXO1 by repressing FOXO1 degradation mediated by S-phase kinase-associated protein 2 (Skp2), an E3 ubiquitin ligase, promoting caspase 3 activation and apoptosis. CONCLUSIONS: Because FOXO1 upregulates the menin gene transcription, our findings unravel a crucial menin and FOXO1 interplay, with menin and FOXO1 upregulating their expression reciprocally, forming a positive feedback loop to sustain menin and FOXO1 expression.

Human Proislet Peptide Promotes Pancreatic Progenitor Cells to Ameliorate Diabetes Through FOXO1/Menin-Mediated Epigenetic Regulation.

We investigated how human proislet peptide (HIP) regulates differentiation of human fetus-derived pancreatic progenitor cells (HFPPCs) and explored the potential link between HIP signaling and the menin pathway, which is key to regulating pancreatic islet differentiation. The data show that HIP promoted expression of proislet transcription factors (TFs), including PDX-1, MAFA, and NKX6.1, as well as other maturation markers of ß-cells, such as insulin, GLUT2, KIR6.2, SUR1, and VDCC. Moreover, HIP increased insulin content and promoted the ability of HFPPCs to normalize blood glucose in diabetic mice. HIP inhibited the TF FOXO1 by increasing AKT-mediated phosphorylation. HIP-induced repression of FOXO1 suppressed menin expression, leading to reducing menin binding to the promoter of the three key proislet TFs, decreasing recruitment of H3K9 methyltransferase SUV39H1, and thus reducing repressive H3K9me3 at the promoter. These coordinated actions lead to increased expression of the proislet TFs, resulting in induction of HFPPC differentiation. Consistently, constitutive activation of FOXO1 blocks HIP-induced transcription of these TFs. Together, these studies unravel the crucial role of the HIP/AKT/FOXO/menin axis in epigenetically controlling expression of proislet TFs, regulating the differentiation of HFPPCs, and normalizing blood glucose in diabetic mice.

G-Protein Gα13 Functions with Abl Kinase to Regulate Actin Cytoskeletal Reorganization.

Heterotrimeric G-proteins are essential cellular signal transducers. One of the G-proteins, Gα13, is critical for actin cytoskeletal reorganization, cell migration, cell proliferation, and apoptosis. Previously, we have shown that Gα13 is essential for both G-protein-coupled receptor and receptor tyrosine kinase-induced actin cytoskeletal reorganization such as dynamic dorsal ruffle turnover and cell migration. However, the mechanism by which Gα13 signals to actin cytoskeletal reorganization is not completely understood. Here we show that Gα13 directly interacts with Abl tyrosine kinase, which is a critical regulator of actin cytoskeleton. This interaction is critical for Gα13-induced dorsal ruffle turnover, endothelial cell remodeling, and cell migration. Our data uncover a new molecular signaling pathway by which Gα13 controls actin cytoskeletal reorganization.

Menin and PRMT5 suppress GLP1 receptor transcript and PKA-mediated phosphorylation of FOXO1 and CREB.

Menin is a scaffold protein that interacts with several epigenetic mediators to regulate gene transcription, and suppresses pancreatic ß-cell proliferation. Tamoxifen-inducible deletion of multiple endocrine neoplasia type 1 (MEN1) gene, which encodes the protein menin, increases ß-cell mass in multiple murine models of diabetes and ameliorates diabetes. Glucagon-like-peptide-1 (GLP1) is another key physiological modulator of ß-cell mass and glucose homeostasis. However, it is not clearly understood whether menin crosstalks with GLP1 signaling. Here, we show that menin and protein arginine methyltransferase 5 (PRMT5) suppress GLP1 receptor (GLP1R) transcript levels. Notably, a GLP1R agonist induces phosphorylation of forkhead box protein O1 (FOXO1) at S253, and the phosphorylation is mediated by PKA. Interestingly, menin suppresses GLP1-induced and PKA-mediated phosphorylation of both FOXO1 and cAMP response element binding protein (CREB), likely through a protein arginine methyltransferase. Menin-mediated suppression of FOXO1 and CREB phosphorylation increases FOXO1 levels and suppresses CREB target genes, respectively. A small-molecule menin inhibitor reverses menin-mediated suppression of both FOXO1 and CREB phosphorylation. In addition, ex vivo treatment of both mouse and human pancreatic islets with a menin inhibitor increases levels of proliferation marker Ki67. In conclusion, our results suggest that menin and PRMT5 suppress GLP1R transcript levels and PKA-mediated phosphorylation of FOXO1 and CREB, and a menin inhibitor may reverse this suppression to induce ß-cell proliferation.

Menin and Daxx Interact to Suppress Neuroendocrine Tumors through Epigenetic Control of the Membrane Metallo-Endopeptidase.

Neuroendocrine tumors (NET) often harbor loss-of-function mutations in the MEN1 and DAXX tumor suppressor genes. Here, we report that the products of these genes, menin and Daxx, interact directly with each other to suppress the proliferation of NET cells, to a large degree by inhibiting expression of the membrane metallo-endopeptidase (MME). Menin and Daxx were required to enhance histone H3 lysine9 trimethylation (H3K9me3) at the MME promoter, as mediated partly by the histone H3 methyltransferase SUV39H1. Notably, the menin T429K mutation associated with a NET syndrome reduced Daxx binding, MME repression, and proliferation of NET cells. Conversely, inhibition of MME in NET cells repressed proliferation and tumor growth in vivo Our findings reveal a previously unappreciated cross-talk between two crucial tumor suppressor genes thought to work by independent pathways, focusing on MME as a common target of menin/Daxx to treat NET. Cancer Res; 77(2); 401-11. ©2016 AACR.

Improving fascin inhibitors to block tumor cell migration and metastasis.

Tumor metastasis is the major cause of mortality of cancer patients, being responsible for ∼90% of all cancer deaths. One of the key steps during tumor metastasis is tumor cell migration which requires actin cytoskeletal reorganization. Among the critical actin cytoskeletal protrusion structures are antenna-like filopodia. Fascin protein is the main actin-bundling protein in filopodia. Here we report the development of fascin-specific small-molecules that inhibit the interaction between fascin and actin. These inhibitors block the in vitro actin-binding and actin-bundling activities of fascin, tumor cell migration and tumor metastasis in mouse models. Mechanistically, these inhibitors likely occupy one of the actin-binding sites, reduce the binding of actin filaments, and thus lead to the inhibition of the bundling activity of fascin. At the cellular level, these inhibitors impair actin cytoskeletal reorganization. Our data indicate that target-specific anti-fascin agents will have great potential for treating metastatic tumors.

Targeted inhibition of fascin function blocks tumour invasion and metastatic colonization.

One of the key steps during tumour metastasis is tumour cell migration and invasion, which require actin cytoskeletal reorganization. Among the critical actin cytoskeletal protrusion structures are the filopodia, which act like cell sensory organs to communicate with the extracellular microenvironment and participate in fundamental cell functions such as cell adhesion, spreading and migration in the three-dimensional environment. Fascin is the main actin-bundling protein in filopodia. Using high-throughput screening, here we identify and characterize small molecules that inhibit the actin-bundling activity of fascin. Focusing on one such inhibitor, we demonstrate that it specifically blocks filopodial formation, tumour cell migration and invasion in vitro, and metastasis in vivo. Hence, target-specific anti-fascin agents have a therapeutic potential for cancer treatment.

C-terminal Src kinase (Csk)-mediated phosphorylation of eukaryotic elongation factor 2 (eEF2) promotes proteolytic cleavage and nuclear translocation of eEF2.

Protein-tyrosine kinase C-terminal Src kinase (Csk) was originally purified as a kinase for phosphorylating Src and other Src family kinases. The phosphorylation of a C-terminal tyrosine residue of Src family kinases suppresses their kinase activity. Therefore, most physiological studies regarding Csk function have been focused on Csk as a negative regulator of Src family tyrosine kinases and as a potential tumor suppressor. Paradoxically, the protein levels of Csk were elevated in some human carcinomas. In this report, we show that eukaryotic elongation factor 2 (eEF2) is a new protein substrate of Csk and could locate in the nucleus. We demonstrate that Csk-mediated phosphorylation of eEF2 has no effect on its cytoplasmic function in regulating protein translation. However, phosphorylation of eEF2 enhances its proteolytic cleavage and the nuclear translocation of the cleaved eEF2 through a SUMOylation-regulated process. Furthermore, we show that cleaved fragments of eEF2 can induce nuclear morphological changes and aneuploidy similar to those in cancer cells, suggesting that there is an additional mechanism for Csk in tumorigenesis through regulation of eEF2 subcellular localization.