Knock-in Mutation Research Articles

Protein Kinase N Family Negatively Regulates Constitutive Androstane Receptor-Mediated Transcriptional Induction of Cytochrome P450 2b10 in the Livers of Mice.

In receptor-type transcription factors-mediated cytochrome P450 (P450) induction, few studies have attempted to clarify the roles of protein kinase N (PKN) in the transcriptional regulation of P450s. This study aimed to examine the involvement of PKN in the transcriptional regulation of P450s by receptor-type transcription factors, including the aryl hydrocarbon receptor, constitutive androstane receptor (CAR), and pregnane X receptor. The mRNA and protein levels and metabolic activity of P450s in the livers of wild-type (WT) and double-mutant (D) mice harboring both PKN1 kinase-negative knock-in and PKN3 knockout mutations [PKN1 T778A/T778A; PKN3 -/-] were determined after treatment with activators for receptor-type transcription factors. mRNA and protein levels and metabolic activity of CYP2B10 were significantly higher in D mice treated with the CAR activator phenobarbital (PB) but not with 1,4-bis((3,5-dichloropyridin-2-yl)oxy)benzene compared with WT mice. We examined the CAR-dependent pathway regulated by PKN after PB treatment because the extent of CYP2B10 induction in WT and D mice was notably different in response to treatment with different CAR activators. The mRNA levels of Cyp2b10 in primary hepatocytes from WT and D mice treated with PB alone or in combination with Src kinase inhibitor 1 (SKI-1) or U0126 (a mitogen-activated protein kinase inhibitor) were evaluated. Treatment of hepatocytes from D mice with the combination of PB with U0126 but not SKI-1 significantly increased the mRNA levels of Cyp2b10 compared with those from the corresponding WT mice. These findings suggest that PKN may have inhibitory effects on the Src-receptor for activated C kinase 1 (RACK1) pathway in the CAR-mediated induction of Cyp2b10 in mice livers. SIGNIFICANCE STATEMENT: This is the first report of involvement of PKN in the transcriptional regulation of P450s. The elucidation of mechanisms responsible for induction of P450s could help optimize the pharmacotherapy and improve drug development. We examined whether the mRNA and protein levels and activities of P450s were altered in double-mutant mice harboring both PKN1 kinase-negative knock-in and PKN3 knockout mutations. PKN1/3 negatively regulates CAR-mediated induction of Cyp2b10 through phosphorylation of a signaling molecule in the Src-RACK1 pathway.

Modeling a human CLP1 mutation in mouse identifies an accumulation of tyrosine pre-tRNA fragments causing pontocerebellar hypoplasia type 10

Cleavage factor polyribonucleotide kinase subunit 1 (CLP1), an RNA kinase, plays essential roles in protein complexes involved in the 3′-end formation and polyadenylation of mRNA and the tRNA splicing endonuclease complex, which is involved in precursor tRNA splicing. The mutation R140H in human CLP1 causes pontocerebellar hypoplasia type 10 (PCH10), which is characterized by microcephaly and axonal peripheral neuropathy. Previously, we reported that RNA fragments derived from isoleucine pre-tRNA introns (Ile-introns) accumulate in fibroblasts of patients with PCH10. Therefore, it has been suggested that this intronic RNA fragment accumulation may trigger PCH10 onset. However, the molecular mechanism underlying PCH10 pathogenesis remains elusive. Thus, we generated knock-in mutant mice that harbored a CLP1 mutation consistent with R140H. As expected, these mice showed progressive loss of the upper motor neurons, resulting in impaired locomotor activity, although the phenotype was milder than that of the human variant. Mechanistically, we found that the R140H mutation causes intracellular accumulation of Ile-introns derived from isoleucine pre-tRNAs and 5′ tRNA fragments derived from tyrosine pre-tRNAs, suggesting that these two types of RNA fragments were cooperatively or independently involved in the onset and progression of the disease. Taken together, the CLP1-R140H mouse model provided new insights into the pathogenesis of neurodegenerative diseases, such as PCH10, caused by genetic mutations in tRNA metabolism-related molecules.

Neuronal activity-induced BRG1 phosphorylation regulates enhancer activation.

SUMMARYNeuronal activity-induced enhancers drive gene activation. We demonstrate that BRG1, the core subunit of SWI/SNF-like BAF ATP-dependent chromatin remodeling complexes, regulates neuronal activity-induced enhancers. Upon stimulation, BRG1 is recruited to enhancers in an H3K27Ac-dependent manner. BRG1 regulates enhancer basal activities and inducibility by affecting cohesin binding, enhancer-promoter looping, RNA polymerase II recruitment, and enhancer RNA expression. We identify a serine phosphorylation site in BRG1 that is induced by neuronal stimulations and is sensitive to CaMKII inhibition. BRG1 phosphorylation affects its interaction with several transcription co-factors, including the NuRD repressor complex and cohesin, possibly modulating BRG1-mediated transcription outcomes. Using mice with knockin mutations, we show that non-phosphorylatable BRG1 fails to efficiently induce activity-dependent genes, whereas phosphomimic BRG1 increases enhancer activity and inducibility. These mutant mice display anxiety-like phenotypes and altered responses to stress. Therefore, we reveal a mechanism connecting neuronal signaling to enhancer activities through BRG1 phosphorylation.

Abstract 2296: Regulation of P-body dynamics and formation in tumors through EDC3 phosphorylation by PIM and AKT

Abstract Processing bodies (P-bodies) are cytoplasmic mRNA granules that form a hub to regulate mRNA translation via controlling decapping, degradation, and storage. P-bodies markedly increase as part of a stress response for example during nutrient deprivation and hypoxia. Thus, P-bodies could regulate cell fate by tuning protein levels during the stress, and thereby contribute to tumor initiation, growth, and metastasis. However, it is unclear how P-body formation is regulated by these stress and what biologic pathways P-body controls. Dysregulation of PIM and AKT kinases has been found in multiple cancers, including prostate and breast cancers. We find that the protein Enhancer of mRNA-decapping Protein 3 (EDC3), a P-body component, binds with the PIM protein kinase and is phosphorylated on serine 161 by the PIM and AKT kinases, suggesting these kinases could regulate P-body formation and function. Thus, we hypothesize that these oncogenic kinases regulate the EDC3 activity and P-body function via the phosphorylation of EDC3. Suppression of EDC3 phosphorylation by inhibiting PIM and AKT activities with drugs leads to an increase in P-body number. A knock-in mutation substituting alanine for serine 161 in PC3-LN4 prostate cancer cell line inhibited growth, migration and invasion in vitro. Prostate cancer tumor growth in these cells containing EDC3 mutation was reduced in mouse xenograft model, suggesting that this single phosphorylation is essential for regulating cell growth. These results suggest that the level of specific RNAs and its translation is being controlled by this mutation. Indeed, integrin family proteins, ITGB1 and ITGA6, are shown to be decreased in these cells consistent with the inability of these cells to attach and invade. High levels of phosphorylated EDC3 were detected in the breast cancer cell lines tested, whereas non-transformed immortalized breast epithelial cell lines showed much lower EDC3 phosphorylation. IHC staining of human breast cancer samples with phospho-specific antibodies demonstrated that EDC3 was highly phosphorylated in contrast to normal breast tissue which demonstrated low level of phosphorylation. These data indicate that in tumors, breast and prostate cancer, with activated PIM and AKT, high phosphorylation of EDC3 would increase the translation of target mRNAs via reducing their storage and destruction and affecting tumor cell growth and motility. Therefore, the phosphorylation of EDC3 by Pim and AKT kinases can be a driver of malignancy by controlling mRNA levels in breast and prostate cancers. Citation Format: Jeremiah Bearss, Sathish K. Padi, Neha Singh, Marina Cardo-Vila, Andrew S. Kraft, Koichi Okumura. Regulation of P-body dynamics and formation in tumors through EDC3 phosphorylation by PIM and AKT [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2296.

Abstract 2008: Targeting SKP2 by p27 Inhibits stemness and prolong the survival in osteosarcoma

Abstract Objective: Osteosarcoma is a highly aggressive malignancy for which treatment has remained essentially unchanged for years. Our previous studies found that the F-box protein SKP2 is overexpressed in osteosarcoma, acting as a proto-oncogene. p27Kip1 (p27) is an inhibitor of cyclin-dependent kinases and a downstream substrate of SKP2. The present study focused on the role of SCF-SKP2-mediated ubiquitination and degradation of p27 in osteosarcoma (OS) tumorigenesis. Methods: We generated a genetically engineered mouse model with double-knockout of Rb1 and Trp53 within cells of the osteoblastic lineage using the Osterix1-Cre (Osx1-Cre;Rb1lox/lox;Trp53lox/lox, DKO). To block the interaction between Skp2 and p27, we cross DKO mice into the p27T187A knock-in mutation (p27:187site Thr to Ala) background to create Osx1-Cre;Rb1lox/lox;Trp53lox/lox;p27T187A/T187A (DKOAA) animals. Mice of both genotypes were monitored for overall survival and tumor growth. Early passage osteosarcoma cells were harvested from mice tumors and used for in vitro analysis. Annexin V staining and TUNEL assay were used for apoptosis analysis. RNA-seq and RT-qPCR were used to compare transcriptional differences. Further, a small-molecule SKP2/CKS1 pocket inhibitor (C1) and a neddylation inhibitor, Pevenodistat, were tested both in vitro using adherent cells and organoid culture, and in vivo using xenograft. Results: All genotypes were born at the expected ratios, and mutant animals are viable, fertile, and developmentally normal. The p27T187A mutation significantly prolonged the overall survival of DKO osteosarcoma mice. Western and IHC staining showed an accumulation of p27 in DKOAA, accompanied by an increased TUNEL staining level and apoptotic subpopulation determined by Annexin V staining. Hence, DKOAA tumors proliferate slower than DKO both in vivo and in vitro organoid. RNA-seq based enrichment analysis further confirmed an increase of apoptosis and cell cycle arrest in DKOAA tumors than DKO. RNA-seq also revealed a significant downregulation of multiple cancer stemness markers in DKOAA tumors. Further GESA enrichment analysis, ALDH activity assay, and sphere-formation assay consistently demonstrated a decreased stemness status in DKOAA tumors. Finally, SKP2 inhibitors, both C1 and Pevenodistat, showed selective inhibition in DKO than DKOAA in adherent culture, osteosarcoma organoid, and xenograft model. Conclusion: Blocking p27 degradation by SKP2 significantly delayed osteosarcoma tumorigenesis and prolonged survival, promoted apoptosis, and reduced tumor-initiating properties in OS model. Given that RB1 and TP53 co-inactivation is common in osteosarcoma, our study suggests that inhibiting the SKP2-p27 axis may represent a desirable therapeutic strategy for this cancer. Citation Format: Jichuan Wang, Osama Aldahamsheh, Alexander Ferrena, Swapnil Singh, Amit Singla, Hasibagan Borjihan, Simon Yaguare, Valentina Viscarret, Janet Tingling, Xiaolin Zi, Yungtai Lo, Richard Gorlick, Deyou Zheng, Edward L. Schwartz, Hongling Zhao, Rui Yang, David S. Geller, Bang H. Hoang. Targeting SKP2 by p27 Inhibits stemness and prolong the survival in osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2008.

Abstract 2417: Mutant p53 and oncogenic KRAS converge on CREB1 to drive pancreatic cancer metastasis

Abstract Pancreatic ductal adenocarcinoma (PDAC) is almost uniformly fatal and characterized by early metastasis. Oncogenic KRAS mutations prevail in 95% of PDAC tumors and co-occur with genetic alterations in the TP53 tumor suppressor in nearly 70% of patients. Most p53 alterations are missense mutations that exhibit gain-of-function phenotypes that include increased invasiveness and metastasis. However, the extent of direct cooperation or synergism between KRAS effectors and mutant p53 remain undefined. To study how KRAS effectors and mutant p53 cooperate to drive PDAC development, we developed a novel somatic model that expresses mutant KRAS and p53 only in pancreas tumor cells, leaving the tumor microenvironment and immune system intact. PDAC genetically engineered mouse models (GEMMs) were generated by crossing a conditional oncogenic KRasG12D (K) allele with a conditional null p53 allele (Pfl) or a novel, conditional knock-in mutant p53R172H allele (Pwm) that selectively expresses mutant p53 in tumor cells while preserving wildtype p53 expression in all other animal cells. The incidence of metastatic lesions in the liver and lungs of KPwmC mice was over 2-fold greater (p=.03) relative to KPflC mice. Through orthogonal analyses of this model and human PDAC samples, we show that oncogenic KRAS effectors phosphorylate and activate cyclic AMP responsive element binding protein 1 (CREB1), allowing physical interactions with mutant p53 to transcriptionally upregulate the pro-metastatic transcription factor, FOXA1, which enhances β-catenin stabilization and activity. Targeting KRAS and mutant p53 cooperativity through pharmacologic inhibition of CREB1 dramatically dampens FOXA1 expression and PDAC metastasis. Our findings demonstrate a direct, mechanistic link between key oncogenic KRAS signaling elements and mutant p53 that result in a broad, multiplexed activation of cancer-associated transcriptional networks. Moreover, we identify CREB1 as a viable therapeutic strategy to undermine oncogenic KRAS and mutant p53 cooperation to mitigate PDAC metastasis. Citation Format: Michael Paul Kim, Xinqun Li, Jenying Deng, Yun Zhang, Bingbing Dai, Kendra Allton, Tara Hughes, Christian Siangco, Jithesh Augustine, Yaan Kang, Joy M. McDaniel, Shunbin Xiong, Eugene Koay, Florencia McAllister, Christopher Bristow, Timothy Heffernan, Anirban Maitra, Bin Liu, Michelle Barton, Amanda Wasylischen, Jason Fleming, Guillermina lozano. Mutant p53 and oncogenic KRAS converge on CREB1 to drive pancreatic cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2417.

CRISPR/Cas9-engineered Drosophila knock-in models to study VCP diseases.

ABSTRACTMutations in Valosin Containing Protein (VCP) are associated with several degenerative diseases, including multisystem proteinopathy (MSP-1) and amyotrophic lateral sclerosis. However, patients with VCP mutations vary widely in their pathology and clinical penetrance, making it difficult to devise effective treatment strategies. A deeper understanding of how each mutation affects VCP function could enhance the prediction of clinical outcomes and design of personalized treatment options. The power of a genetically tractable model organism coupled with well-established in vivo assays and a relatively short life cycle make Drosophila an attractive system to study VCP disease pathogenesis. Using CRISPR/Cas9, we have generated individual Drosophila knock-in mutants that include nine hereditary VCP disease mutations. Our models display many hallmarks of VCP-mediated degeneration, including progressive decline in mobility, protein aggregate accumulation and defects in lysosomal and mitochondrial function. We also made some novel and unexpected findings, including nuclear morphology defects and sex-specific phenotypic differences in several mutants. Taken together, the Drosophila VCP disease models generated in this study will be useful for studying the etiology of individual VCP patient mutations and testing potential genetic and/or pharmacological therapies.

Abstract 120: Compartmentalized functions of epidermal growth factor receptor (EGFR) in tumorigenesis and malignant phenotypes

Abstract Previous studies demonstrated that nuclear EGFR is associated with malignancy and resistance to tyrosine kinase inhibitor (TKI). To decipher the cellular compartmentalized functions and underlying mechanism of EGFR, we generated EGFR mutants at nuclear translocation signal (NLS) and nuclear export signal (NES). The results showed that EGFR NLS mutant with decreased nuclear translocation sensitized the cancer cells to TKI and impaired invasiveness and stemness of the cancer cells. In contrast, EGFR NES mutant with increased nuclear accumulation promoted aggressiveness of the cancer cells including TKI resistance. Next, we generated genetically engineered mouse model (GEMM) to validate the resulting phenotypes of the EGFR mutants. To this end, CRISPR-Cas9-mediated genome editing approach was used for generating EGFR mutant knock-in mouse, which harbors an internalization-deficient mutation (EGFRLL/AA), pro-nuclear (NES) mutation (EGFRL749P(S)) and the dual mutations. Upon EGF stimulation nuclear EGFR (nEGFR) accumulation in the hepatocytes from EGFRL749P heterozygous mice was increased. Conversely, a significant reduction of nEGFR was observed in the hepatocyte from EGFTRLL/AA homozygous mice. We found that germline expression of EGFRL749P(S) gave rise to deficiency in breeding. However, EGFRLL/AA mutant mice displayed no gross abnormal even in homozygous offspring, suggesting that depletion of nEGFR has no significant phenotype. We failed to generate homozygotes of EGFR NES mutant mice due to embryonic lethality. Of note, the female and male founders and F1 EGFRL798P(S) mice developed tumors with huge spleen. The IHC staining results indicated that the infiltrating lymphocytes in liver, stomach and kidney were B cells. The latency of the B cell lymphomagenesis was from 17 to 23 months, and tumor incidence of F1 offspring of EGFRL749P(S) knock-in mice was 10 of 15 (66.7%). These findings indicated that single allele mutation in EGFR NES domain is capable of driving lymphomagenesis, suggesting that the EGFR NES mutant possesses a strong oncogenic activity. Surprisingly, by searching database of EGFR mutation in human tumors (Cosmic database https://cancer.sanger.ac.uk/cosmic ), we noticed that among the somatic mutations in EGFR gene the gene amplification represents only small part of the gene alternations in human cancers, and EGFR L747P and L747S mutation occur in the lung cancer patients with intrinsic TKI resistance. In addition, the majority of human B lymphocyte originated tumors harboring EGFR NES mutation are also heterozygotes. Cosmic database shows that somatic mutations at the EGFR NES region occur in the patients with plasma cell lymphoma. Taken together, our findings suggest that nEGFR contributes to malignant potential of the tumors and EGFR with NES mutation is a tumor driver. Citation Format: Lei Nie, Junwei Hou, Yu-Yi Chu, Ying-Nai Wnag, Li-Chuan Chan, Longfei Huo, Paul Chiao, Shao-Chun Wang, Mien-Chie Hung, Michael Wang. Compartmentalized functions of epidermal growth factor receptor (EGFR) in tumorigenesis and malignant phenotypes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 120.

PI3Kinase Inactivation through Combined Loss of Catalytic Subunits p110α, p110β and p110δ Derails Hematopoietic Homeostasis

PI3Kinase is a lipid kinase that is involved in diverse cellular processes including cell growth, survival, differentiation and cell cycle. Deregulation of PI3K pathway has been associated with wide variety of cancers including several hematological disorders such as acute myeloid leukemia (AML). Hematopoietic growth factors such as Fms Related Tyrosine Kinase 3 (FLT3) ligand, stem cell factor (SCF), erythropoietin (EPO), and thrombopoietin (TPO) activate PI3K signaling pathway via tyrosine kinase receptors, growth factor receptors and G-protein coupled receptors. In mouse models of abrogation of PI3K signaling, involving AKT deletion, impairs the self-renewal potential of hematopoietic stem cells (HSCs). In contrast, mice models with constitutive activation of PI3K signaling, involving deletion of PTEN phosphatase, causes increased cycling of HSCs. PI3K regulatory subunits, P85α and P85β, are critical for fetal liver hematopoiesis. However, the role of PI3K in adult hematopoiesis and contribution of catalytic subunits of PI3K to hematopoietic homeostasis is not well studied. Given that HSCs express all three catalytic subunits of PI3K, we developed a mouse model for ablated PI3K pathway by depleting PI3K catalytic subunits p110α, p110β and p110δ. p110α and p110β subunits were conditionally deleted via MX-Cre system upon Poly-IC injection, and p110δ inactivation was achieved through p110δ inactivating knock-in mutation. Mouse tissues were harvested upon 5 weeks post poly-IC, at which time point the mice appeared moribund and were therefore analyzed for various hematopoietic lineages. These mice were anemic and peripheral blood analysis showed leukopenia, neutropenia and anemia with significantly reduced WBC, neutrophils, RBC, hemoglobin, hematocrits and platelet counts. Mice depleted of p110α, β and δ subunits-p110 triple knock out mice (p110-TKO) - demonstrated significantly reduced spleen size, reduced bone marrow cellularity with anemic bones relative to wild type mice. Hematopoietic stem cell compartment in p110-TKO mice showed increased frequency in Lineage-veSca1+veKit+ve (LSK) population as well as an increase in more primitive HSCs defined as SLAM cells (CD150+veCD48-ve). In contrast, there was reduction in total number of common lymphoid progenitors (CLP), common myeloid progenitors (CMP), granulocyte-macrophage progenitors (GMP) and megakaryocyte-erythroid progenitors (MEP) in the bone marrow. These mice also showed dramatically reduced total B-cells and significantly reduced T-cells. However, these mice show increased frequency of GR1-Mac1 positive granulocytes in all hematopoietic compartments. Erythroid compartment was analyzed for various stages of red blood cell development using Ter119 and CD71 markers. Hampered RBC development was observed in p110-TKO mice with significantly increased immature erythroid cells (Ter119lowCD71low) in the bone marrow, spleen and peripheral blood compartments, with significantly reduced terminally differentiated cells (Ter119+ve only). HSC self-renewal studies involving competitive repopulation assay showed that recipient mice have significantly reduced p110-TKO donor chimerism. Earlier studies from others have reported that p110α and p110δ subunits are dispensable for adult HSC function, however p110α is required for terminal erythroblast maturation and p110δ is required for leukocyte development. Our studies revealed that lack of all three catalytic subunits of PI3Kinase significantly impairs hematopoietic homeostasis with hampered stem cell development, erythroid cell development and a complete shut down in B-cell production. DisclosuresNo relevant conflicts of interest to declare.

Novel Mutations in RASGRP1 Are Associated with Immune Dysregulation and Predispose to EBV-Induced Lymphoma

& I. Somekh and B. Marquardt contributed equally§ C. Klein and R. Somech contributed equallyBackgroundRAS guanyl-releasing protein 1 (RASGRP1) deficiency has recently been shown to cause a primary immunodeficiency disorder in one patient (Salzer et al, Nat Immunol 2016). Clinically, the disease was characterized by recurrent episodes of pneumonia, bronchiectasis, severe failure to thrive, CD4 T-cell lymphopenia, elevated TCRγδ cell proportions as well as low-grade Epstein-Barr virus (EBV)-associated B cell lymphoma. Immunologically, RASGRP1 deficiency is associated with defective proliferation and activation of T and B cells, respectively. Here, we report 3 novel patients with RASGRP1 deficiency from 2 unrelated consanguineous kindred.MethodsOne patient of Turkish origin (P1) and 2 Palestinian 1st degree cousins (P2, P3) were evaluated. Genetic analysis using whole exome sequencing was conducted. Immunological and biochemical assays were performed on primary patient material and genetically engineered T-cell lines using CRISPR/Cas9.ResultsClinical findings for all three patients included failure to thrive, hepatosplenomegaly, lymphadenopathy, and recurrent pulmonary infections resulting in bronchiectasia. Two patients had overt auto-immunity (AIHA and autoimmune thrombocytopenia, respectively). One patient had increased anti-nuclear antibody titers. One patient (P1) developed EBV-associated diffuse large B cell lymphoma. He underwent autologous hematopoietic stem cell transplant due to relapse after combined rituximab and NHL-BFM-90 treatment protocol and is currently in remission at the age of 14 years. P2 had deceased at the age of 3 years due to disseminated intravascular coagulation. P3 developed diffuse large B cell lymphoma at the age of 3 years, treated with R-CHOP protocol and is currently in remission.Immune workup of P2 and P3 revealed low levels of T-cell receptor excision circles (TREC), an abnormal T-cell receptor (TCR) Vß repertoire and a markedly increased proportion of TCRγδ cells. Phytohemagglutinin (PHA)-induced lymphocyte proliferation and anti-CD3 mitogen proliferation responses were decreased. EBV-viral load in peripheral blood was elevated for both patients, as measured by PCR.Genetic analysis by whole exome sequencing identified an autosomal recessive homozygous mutations in RASGRP1. P1 had an inversion (c.649_650inv; p.Glu217Ser) and P2/P3 had a deleterious frameshift mutation (c.1111_1114del; p.Asp371Ilefs*7). The variants were confirmed by Sanger sequencing and segregated with the disease phenotype. To confirm the functional relevance of the RASGRP1 mutations, we engineered Jurkat cells via CRISPR/Cas9 editing. RASGRP1 -/- Jurkat cells showed decreased phosphorylation of ERK1/2 and decreased CD69 expression in comparison to wildtype cells. Jurkat cells with RASGRP1 knockin mutants are currently under analysis.ConclusionsRASGRP1 deficiency is a novel primary immunodeficiency disorder associated with immune dysregulation and susceptibility to EBV-induced B-cell malignancies. Early diagnosis of RASGRP1 deficiency is critical. Even though clinical remission of EBV-lymphoma may be reached using high dose chemotherapy regimens, long term cures may only be reached by allogeneic hematopoietic stem cell transplantation. DisclosuresNo relevant conflicts of interest to declare.

Severe Congenital Neutropenia-Associated Mutations Reveal Stage-Specific Gfi1-Dependent Checkpoints in Myeloid Development

Growth factor independent-1 (GFI1) is a zinc-finger transcription factor that is critically required for both murine and human granulopoiesis. Mutations in GFI1 zinc fingers (e.g. N382S in zinc finger 5) have previously been found in humans with severe congenital neutropenia (SCN). We noted that clinical resequencing of neutropenic patients identified a number of novel GFI1 sequence changes of unknown significance. To clarify their impact, we used viral vectors to force expression of 13 GFI1 polymorphisms/mutations in primary-murine progenitors followed by colony-forming-unit assays for differentiation. While these assays infer new potential clinical significance to some genetic alterations, we focused on the sixth zinc finger of GFI1. While Gfi1 zinc-finger 6 was previously found to be dispensable for DNA binding and repression of a high affinity GFI1 DNA-binding-site reporter, the biological impact of expressing GFI1 zinc finger 6 mutants suggests a functional role for this domain in granulopoiesis. Therefore, we generated mice with patient-derived SCN-associated mutations (zinc finger 5; N382S, zinc finger 6; K403R, and R412X) in the murine Gfi1 locus. The resulting defects in murine neutrophil production are proportional to the severity of SCN in patients with the same GFI1 mutation (ranging from mild to profound neutropenia); with neonates displaying more profound defects. Neonatal and adult Gfi1N382S/- and Gfi1R412X/- mice are neutropenic, but Gfi1K403R/- mice have normal steady-state neutrophil levels. While Gfi1-/- HSC are profoundly abnormal, SCN-associated mutant HSC are phenotypically normal; dissociating Gfi1 control of hematopoietic stem cell biology from granulopoiesis. Similar to Gfi1-/-, the Gfi1N382S/- and Gfi1R412X/- mice accumulate cells within the “granulocyte monocyte progenitor” (GMP) gate as well as those that express Sca1 (known as emergency GMP; eGMP); however, unlike Gfi1-/- mice, the serum levels of known cytokine inducers of eGMP are normal in SCN-associated mutants. Moreover, isolated progenitors from all three mutants form significantly more monocytic colonies at the expense of granulocytic colonies in methylcellulose assays. Together, these data suggest that the mutations induce a cell-intrinsic defect.We recently utilized single-cell RNA-Seq (scRNA-Seq) to identify a gene regulatory network (GRN) centered on the Gfi1 and Irf8 transcription factors that underlies the granulocytic versus monocytic lineage decision of myeloid progenitors at steady state. To pinpoint the molecular deregulation underlying Gfi1-mutant progenitors, we performed scRNA-Seq. Notably, Gfi1R412X/- induced deregulation of gene expression which increases in amplitude and complexity as myeloid differentiation proceeds; coincident with induction of Gfi1 expression. ChIP-Seq analysis reveals that most of the deregulated genes are normally bound by Gfi1. Using murine fluorescent-protein knock-in reporter alleles for Gfi1 and Irf8, we demonstrate the stage-specific regulation of the granulocyte-monocyte lineage decision, and how SCN mutations deregulate the Gfi1-Irf8 network. Finally, we developed an alternative flow-cytometric strategy that highlights individual Gfi1-dependent checkpoints in myeloid differentiation (identified by comparative analysis of Gfi1-/- versus knock-in mutants). We find that the accumulated Gfi1N382S/- GMP are unspecified (similar to Gfi1-/-); however, Gfi1R412X/- GMP exhibit only a modest reduction in the granulocytic specified population and instead display a profound block at granulocytic commitment. The latter effects can be partially explained by the protein-destabilizing effect of mutations in zinc finger 6; suggesting that there are dose-dependent roles for Gfi1 in granulopoietic specification and commitment. In summary, Gfi1 knock-in mice provide a mouse model of human SCN, and both flow-cytometric and scRNA-Seq dissection of hematopoiesis reveal myelopoietic-stage-specific deregulation to both block granulopoiesis and deregulate monocytic subsets. DisclosuresNo relevant conflicts of interest to declare.

MTOR Restricts Chromatin Access and Genomic Activity to Maintain Hematopoietic Stem Cell Quiescence and Engraftment

mTOR kinase is an anti-cancer drug target that integrates signals from nutrients, growth factors, and cellular energy status to control cell growth and proliferation, but its biological function in hematopoietic stem cells remains unclear. Numerous studies by using rapamycin or mTOR kinase inhibitors have indicated that mTOR inhibition suppresses chromatin access and gene expression and inhibits cell growth. Consistent with the notion that mTOR targeting suppresses gene activation, hyperactivation of mTOR by deletion of negative regulators of mTOR, TSC1/TSC2 or PTEN, causes a loss of quiescence and exhaustion of HSCs. To directly examine the function of mTOR and its kinase activity in HSC regulation, we used the conditional Mx-Cre;mTORflox/flox and the Mx-Cre;mTORflox/knockin mice that produce the mTOR-/- and the mTOR kinase-activity deficient D2338A mutant knockin genotype, respectively, upon poly I:C induced Cre recombination. We have determined the phenotypic, genomic and molecular effects of their HSCs. Our previous studies have found that depletion of mTOR drastically affected hematopoiesis, with a marked reduction in total BM cellularity and transient increase in the number of HSCs in bone marrow. Remarkably, mTOR-/- HSCs displayed a loss of quiescence, increased proliferation and a normal survival index (Guo et al., Haematologic a 98:1353). Our current study has found similar phenotypes of the HSCs in increased number and proliferation in the Mx-Cre;mTORflox/knockin mice where the mTOR kinase activity is abolished. Both mTOR-/- and mTORKI/- HSCs are defective in engraftment upon competitive transplant into recipient mice, with a clear molecular signature of drastically reduced downstream effectors p-S6K and p-4E-BP. ATAC-seq reveals that loss of mTOR or mTOR kinase activity results in selectively increased chromatin access and global gene activation, in contrast to the reported effects by mTOR inhibitors such as rapamycin or pp242/AZD2014. ChIP-seq of two active histone markers, H3K27Ac and H3K4Me3, yields consistent results that chromosome of the HSCs are activated upon the loss of mTOR or its kinase activity. In particular, increased H3K27Ac and H3K4Me3 ChIP-seq activities are observed in both the upstream and downstream region of c-myc locus, consistent with an activated c-myc expression. RNA-seq analysis reveals that upon mTOR KO or KI, thousands of genes exhibit differential expression. The up-regulated genes are particularly enriched in functions involved in ribosome biogenesis/protein translation, cell cycle progression, cell fate determination, and survival. Analysis using the STRING functional interaction network to search for the compensatory genes/pathways discovers a number of likely compensatorily upregulated genes, upon mTOR KO or KI, that share a significant number of interacting genes with mTOR, including c-myc, Fos, and Jun. These genes are enriched in pathways such as EGFR-MAPK pathway, PI3K-Akt pathway, Jak-Stat pathway, and NFkB pathway, which may compensate for mTOR loss. RT-PCR and Western blotting further verify that loss of mTOR or its kinase activity causes elevated mRNA and protein expression of selected compensatory genes, including c-myc, Fos, Jun, and Pik3cd. In addition, broad compensatory elevations in ribosome and translation machinery gene expression, activation of RNA pol-II, and activation of several interesting compensatory pathways are seen. The BET inhibitor, JQ1, that inhibits c-myc activity readily suppresses the hyper-proliferative phenotype of mTOR-/- and mTORKI/-HSCs. Thus, contrary to dogma established mostly by transient rapamycin or kinase inhibitor usages, our genetic and genomic studies demonstrate that mTOR, through its kinase activity, plays a restrictive role to maintain chromosomal access and genomic activity of HSCs. Loss of mTOR or its kinase activity unleashes a compensatory chromosomal and gene activation effects that involve master regulators of gene activities such as c-myc and Fos, protein translation and ribosomal proteins, and cell growth signaling pathways. These genomic changes may cause the HSC loss of quiescence and engraftment phenotypes seen upon mTOR loss, and need to be taken into consideration in the development of new generations of mTOR targeted therapy and combinatory therapy.Disclosures: No relevant conflicts of interest to declareASH Members: Yi Zheng DisclosuresNo relevant conflicts of interest to declare.

Constitutive protein kinase G activation exacerbates stress-induced cardiomyopathy.

Heart failure is associated with high morbidity and mortality, and new therapeutic targets are needed. Preclinical data suggest that pharmacological activation of protein kinase G (PKG) can reduce maladaptive ventricular remodelling and cardiac dysfunction in the stressed heart. However, clinical trial results have been mixed and the effects of long-term PKG activation in the heart are unknown. We characterized the cardiac phenotype of mice carrying a heterozygous knock-in mutation of PKG1 (Prkg1R177Q/+ ), which causes constitutive, cGMP-independent activation of the kinase. We examined isolated cardiac myocytes and intact mice, the latter after stress induced by surgical transaortic constriction or angiotensin II (Ang II) infusion. Cardiac myocytes from Prkg1R177Q/+ mice showed altered phosphorylation of sarcomeric proteins and reduced contractility in response to electrical stimulation, compared to cells from wild type mice. Under basal conditions, young PKG1R177Q/+ mice exhibited no obvious cardiac abnormalities, but aging animals developed mild increases in cardiac fibrosis. In response to angiotensin II infusion or fixed pressure overload induced by transaortic constriction, young PKGR177Q/+ mice exhibited excessive hypertrophic remodelling with increased fibrosis and myocyte apoptosis, leading to increased left ventricular dilation and dysfunction compared to wild type litter mates. Long-term PKG1 activation in mice may be harmful to the heart, especially in the presence of pressure overload and neurohumoral stress. This article is part of a themed issue on cGMP Signalling in Cell Growth and Survival. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.11/issuetoc.

MRNA translation is a therapeutic vulnerability necessary for bladder epithelial transformation.

Using genetically engineered mouse models, this work demonstrates that protein synthesis is essential for efficient urothelial cancer formation and growth but dispensable for bladder homeostasis. Through a candidate gene analysis for translation regulators implicated in this dependency, we discovered that phosphorylation of the translation initiation factor eIF4E at serine 209 is increased in both murine and human bladder cancer, and this phosphorylation corresponds with an increase in de novo protein synthesis. Employing an eIF4E serine 209 to alanine knock-in mutant mouse model, we show that this single posttranslational modification is critical for bladder cancer initiation and progression, despite having no impact on normal bladder tissue maintenance. Using murine and human models of advanced bladder cancer, we demonstrate that only tumors with high levels of eIF4E phosphorylation are therapeutically vulnerable to eFT508, the first clinical-grade inhibitor of MNK1 and MNK2, the upstream kinases of eIF4E. Our results show that phospho-eIF4E plays an important role in bladder cancer pathogenesis, and targeting its upstream kinases could be an effective therapeutic option for bladder cancer patients with high levels of eIF4E phosphorylation.

Phosphorylation-Dependent Interactome of Ryanodine Receptor Type 2 in the Heart.

: BackgroundHyperphosphorylation of the calcium release channel/ryanodine receptor type 2 (RyR2) at serine 2814 (S2814) is associated with multiple cardiac diseases including atrial fibrillation and heart failure. Despite recent advances, the molecular mechanisms driving pathological changes associated with RyR2 S2814 phosphorylation are still not well understood. Methods: Using affinity-purification coupled to mass spectrometry (AP-MS), we investigated the RyR2 interactome in ventricles from wild-type (WT) mice and two S2814 knock-in mutants: the unphosphorylated alanine mutant (S2814A) and hyperphosphorylated mimic aspartic acid mutant (S2814D). Western blots were used for validation. Results: In WT mouse ventricular lysates, we identified 22 proteins which were enriched with RyR2 pull-down relative to both IgG control and no antibody (beads-only) pull-downs. Parallel AP-MS using WT, S2814A, and S2814D mouse ventricles identified 72 proteins, with 20 being high confidence RyR2 interactors. Of these, 14 had an increase in their binding to RyR2 S2814A but a decrease in their binding to RyR2 S2814D. We independently validated three protein hits, Idh3b, Aifm1, and Cpt1b, as RyR2 interactors by western blots and showed that Aifm1 and Idh3b had significantly decreased binding to RyR2 S2814D compared to WT and S2814A, consistent with MS findings. Conclusion: By applying state-of-the-art proteomic approaches, we discovered a number of novel RyR2 interactors in the mouse heart. In addition, we found and defined specific alterations in the RyR2 interactome that were dependent on the phosphorylation status of RyR2 at S2814. These findings yield mechanistic insights into RyR2 regulation which may guide future drug designs.

Knock-in and precise nucleotide substitution using near-PAMless engineered Cas9 variants in Dictyostelium discoideum

The powerful genome editing tool Streptococcus pyogenes Cas9 (SpCas9) requires the trinucleotide NGG as a protospacer adjacent motif (PAM). The PAM requirement is limitation for precise genome editing such as single amino-acid substitutions and knock-ins at specific genomic loci since it occurs in narrow editing window. Recently, SpCas9 variants (i.e., xCas9 3.7, SpCas9-NG, and SpRY) were developed that recognise the NG dinucleotide or almost any other PAM sequences in human cell lines. In this study, we evaluated these variants in Dictyostelium discoideum. In the context of targeted mutagenesis at an NG PAM site, we found that SpCas9-NG and SpRY were more efficient than xCas9 3.7. In the context of NA, NT, NG, and NC PAM sites, the editing efficiency of SpRY was approximately 60% at NR (R = A and G) but less than 22% at NY (Y = T and C). We successfully used SpRY to generate knock-ins at specific gene loci using donor DNA flanked by 60 bp homology arms. In addition, we achieved point mutations with efficiencies as high as 97.7%. This work provides tools that will significantly expand the gene loci that can be targeted for knock-out, knock-in, and precise point mutation in D. discoideum.

Investigating the role of PKA type I in the heart

Norepinephrine and epinephrine provide the most powerful stimulation of cardiac function through activation of β-adrenergic receptors and the cAMP-dependent protein kinase (PKA). The PKA holoenzyme consists of a dimer of regulatory (R) subunits that binds and inhibits two catalytic subunits (C). Two types of PKA are expressed in the heart, type I and type II, differing by their R subunit, RIα and RIIα. Whereas PKA type II plays an important role in the regulation of cardiac contractility, the role of the PKA type I is little understood. To investigate the role of PKA type I in the adult heart. The cardiac phenotype of two mouse models was investigated. The first is a cardiac-specific and conditional knock-out of RIα (RIα-KO) which leads to a constitutive activation of PKA type I in the heart. The second is a knock-in mutation in the cAMP-binding site of RIα leading to a higher activation threshold for PKA type I (ADO-MUT). Echocardiography shows that young RIα-KO mice have an increased ejection fraction (EF) associated with greater phosphorylation of the key players of excitation-contraction coupling but no change in heart rate (HR). Besides, with age, these mice develop cardiac hypertrophy, dilatation and die from heart failure (HF). In contrast, young ADO-MUT mice have a slightly decreased EF and HR, and do not develop HF with age. In RIα-KO mice, gene and RIα protein deletion were not efficient in the sino-atrial node (SAN). Thus, a third model was developed in which RIα can be specifically and conditionally invalidated in the SAN and conduction system by the use of a HCN4 promoter to investigate the role of PKA type I in cardiac automatism. Thus far, our results indicate a previously unsuspected role of PKA type I in cardiac contraction and possibly also in automatism.

A phosphorylation of RIPK3 kinase initiates an intracellular apoptotic pathway that promotes prostaglandin2α-induced corpus luteum regression.

Receptor-interacting serine/threonine-protein kinase 3 (RIPK3) normally signals to necroptosis by phosphorylating MLKL. We report here that when the cellular RIPK3 chaperone Hsp90/CDC37 level is low, RIPK3 also signals to apoptosis. The apoptotic function of RIPK3 requires phosphorylation of the serine 165/threonine 166 sites on its kinase activation loop, resulting in inactivation of RIPK3 kinase activity while gaining the ability to recruit RIPK1, FADD, and caspase-8 to form a cytosolic caspase-activating complex, thereby triggering apoptosis. We found that PGF2α induces RIPK3 expression in luteal granulosa cells in the ovary to cause luteal regression through this RIPK3-mediated apoptosis pathway. Mice carrying homozygous phosphorylation-resistant RIPK3 S165A/T166A knockin mutations failed to respond to PGF2α but retained pro-necroptotic function, whereas mice with phospho-mimicking S165D/T166E homozygous knock-in mutation underwent spontaneous apoptosis in multiple RIPK3-expressing tissues and died shortly after birth. Thus, RIPK3 signals to either necroptosis or apoptosis depending on its serine 165/threonine 166 phosphorylation status.

Radiation inducible MafB gene is required for thymic regeneration

The thymus facilitates mature T cell production by providing a suitable stromal microenvironment. This microenvironment is impaired by radiation and aging which lead to immune system disturbances known as thymic involution. Young adult thymus shows thymic recovery after such involution. Although various genes have been reported for thymocytes and thymic epithelial cells in such processes, the roles of stromal transcription factors in these remain incompletely understood. MafB (v-maf musculoaponeurotic fibrosarcoma oncogene homolog B) is a transcription factor expressed in thymic stroma and its expression was induced a day after radiation exposure. Hence, the roles of mesenchymal MafB in the process of thymic regeneration offers an intriguing research topic also for radiation biology. The current study investigated whether MafB plays roles in the adult thymus. MafB/green fluorescent protein knock-in mutant (MafB+/GFP) mice showed impaired thymic regeneration after the sublethal irradiation, judged by reduced thymus size, total thymocyte number and medullary complexity. Furthermore, IL4 was induced after irradiation and such induction was reduced in mutant mice. The mutants also displayed signs of accelerated age-related thymic involution. Altogether, these results suggest possible functions of MafB in the processes of thymic recovery after irradiation, and maintenance during aging.

Generation of a chemical genetic model for JAK3

Janus Kinases (JAKs) have emerged as an important drug target for the treatment of a number of immune disorders due to the central role that they play in cytokine signalling. 4 isoforms of JAKs exist in mammalian cells and the ideal isoform profile of a JAK inhibitor has been the subject of much debate. JAK3 has been proposed as an ideal target due to its expression being largely restricted to the immune system and its requirement for signalling by cytokine receptors using the common γ-chain. Unlike other JAKs, JAK3 possesses a cysteine in its ATP binding pocket and this has allowed the design of isoform selective covalent JAK3 inhibitors targeting this residue. We report here that mutating this cysteine to serine does not prevent JAK3 catalytic activity but does greatly increase the IC50 for covalent JAK3 inhibitors. Mice with a Cys905Ser knockin mutation in the endogenous JAK3 gene are viable and show no apparent welfare issues. Cells from these mice show normal STAT phosphorylation in response to JAK3 dependent cytokines but are resistant to the effects of covalent JAK3 inhibitors. These mice therefore provide a chemical-genetic model to study JAK3 function.