Innate immune system activation in zebrafish and cellular models of Diamond Blackfan Anemia.

Deficiency of ribosomal proteins (RPs) leads to Diamond Blackfan Anemia (DBA) associated with anemia, congenital defects, and cancer. While p53 activation is responsible for many features of DBA, the role of immune system is less defined. The Innate immune system can be activated by endogenous nucleic acids from non-processed pre-rRNAs, DNA damage, and apoptosis that occurs in DBA. Recognition by toll like receptors (TLRs) and Mda5-like sensors induces interferons (IFNs) and inflammation. Dying cells can also activate complement system. Therefore we analyzed the status of these pathways in RP-deficient zebrafish and found upregulation of interferon, inflammatory cytokines and mediators, and complement. We also found upregulation of receptors signaling to IFNs including Mda5, Tlr3, and Tlr9. TGFb family member activin was also upregulated in RP-deficient zebrafish and in RPS19-deficient human cells, which include a lymphoid cell line from a DBA patient, and fetal liver cells and K562 cells transduced with RPS19 shRNA. Treatment of RP-deficient zebrafish with a TLR3 inhibitor decreased IFNs activation, acute phase response, and apoptosis and improved their hematopoiesis and morphology. Inhibitors of complement and activin also had beneficial effects. Our studies suggest that innate immune system contributes to the phenotype of RPS19-deficient zebrafish and human cells

Modeling Diamond-Blackfan Anemia in the Mouse: Disease Pathogenesis and Evaluation of Novel Therapies

Diamond-Blackfan anemia (DBA) is a congenital erythroid hypoplasia that presents early in infancy. The main hematopoietic symptoms include macrocytic anemia with reticulocytopenia and selective absence of erythroid precursors in a normocellular bone marrow. In addition to the hematopoietic symptoms, DBA is characterized by the presence of physical abnormalities and cancer predisposition. Mutations in genes encoding ribosomal proteins have been identified in approximately 60-70% of DBA patients. Among these genes, ribosomal protein S19 (RPS19) is the most common disease gene (25% of the cases). All reported patients are heterozygous for the mutations and in most cases the mutations are predicted to result in haploinsufficiency of the respective ribosomal protein. Knowledge about DBA pathophysiology has been limited due to lack of appropriate animal models. The aim of this thesis was to generate mouse models for RPS19-deficient DBA and to use these models to study DBA pathophysiology and to evaluate novel therapies. In article I we generated and characterized novel mouse models for RPS19-deficient DBA. These models contain a doxycycline-regulatable Rps19-targeting shRNA that allows an inducible and graded downregulation of Rps19. We demonstrate that Rps19-deficient mice recapitulate many of the phenotypic and molecular features seen in patients, and are therefore well suited for the evaluation of novel therapies. In article II we used these mouse models to assess the therapeutic potential of the amino acid L-leucine in the treatment of DBA. We show that L-leucine treatment improves the anemia and alleviates the stress hematopoiesis in Rps19-deficient mice. In article III we evaluated the therapeutic efficacy of gene therapy using our mouse models for RPS19-deficient DBA. Using lentiviral vectors we demonstrate that enforced expression of RPS19 cures the lethal bone marrow failure in recipient mice transplanted with Rps19-deficient bone marrow cells. Taking together the generated mouse models provide novel tools to study DBA pathophysiology and to evaluate novel therapies. Our studies strengthen the rationale for clinical trials of L-leucine and provide a proof of principle for the development of clinical gene therapy in the treatment of RPS19-deficient DBA

Molecular bases of inherited bone marrow failures : Shwachman-Diamond syndrome and Diamond Blackfan anemia.

Inherited Bone Marrow Failure syndromes (IBMFS) are a heterogeneous class of diseases that converge on very few subjects. They are unified by a block in maturation of one or multiple blood lineages, are genetically inherited, and have an increased incidence of cancers and myelodisplastic syndromes. Several IBMFSs have been linked to defects in ribosome biogenesis. Diamond Blackfan anemia (DBA) which presents with a macrocytic anemia has 10 known affected genes all of which encode structural proteins of the large or small ribosomal subunit. In contrast, there is only a single gene known to be involved in the pathogenesis of Shwachman Diamond Syndrome (SDS) which usually presents as exocrine pancreatic insufficiency along with neutropenia. Investigation of DBA pathogenesis lead to the knowledge that deficiency of certain ribosomal proteins leads to a specific defect in ribosomal pre-RNA processing. In this work, we have identified a new DBA gene, RPL31, by isolating mononuclear cells from the patient’s blood and subsequently harvesting total RNA used for Northern blotting to identify delays in pre-rRNA processing in both this patient as well as those found in a patient with other large subunit DBA mutations. Additionally, we have created a human cell model of SDS that has allowed us to explore changes in respiration originally shown in yeast models. To this end, we have identified increases in reactive oxygen species, changes in oxygen consumption, mitochondrial membrane potential, and cell cycle delay, all linked to depletion of SBDS protein

Ribosome maturation in yeast models of Diamond-Blackfan anemia and Shwachman-Diamond syndrome.

The inherited bone marrow failure syndromes (IBMFS) encompass a heterogeneous collection of rare disorders characterized by hematological abnormalities, generalized growth delays, and an increased incidence of malignant transformation. These disorders include: Diamond-Blackfan anemia (DBA), Shwachman-Diamond syndrome (SDS), cartilage-hair hypoplasia (CHH), and dyskeratosis congenita (DC). Despite sharing overarching similarities, each of these disorders manifests distinct clinical phenotypes. Similar to their clinical features, the molecular underpinnings of the IBMFS have characteristics that are both shared and distinctive. Aberrations in ribosome synthesis have been associated with each of the IBMFS providing a common molecular target for pathogenic mutations in disease related genes. In some cases, the ribosome appears to be the major target of pathogenic lesions, whereas in others, effects on ribosome synthesis are secondary and appear to have a modifying influence on disease presentation. For example, the primary target of pathogenic lesions in dyskeratosis congenita is telomerase which distinguishes it from other IBMFS. The X-linked form of dyskeratosis congenita, however, affects both telomerase function and ribosome synthesis and is considerably more severe than the somatic forms of the disease that only affect telomerase. Thus, differences in primary targets of pathogenic lesions can account for the distinct clinical presentations of certain IBMFS. In other cases, where ribosome synthesis appears to be the major target of disease causing mutations, the basis for diverse clinical manifestations remains unknown. The body of work presented in this dissertation is focused on Shwachman-Diamond syndrome and Diamond-Blackfan anemia, two IBMFS where defects in ribosome synthesis appear to underlie disease pathophysiology. The approach was to use yeast models of both diseases to explore mechanisms by which ribosome synthesis was affected using the 60S ribosomal subunit as a common molecular target. My studies revealed that 60S subunit biogenesis was affected by distinct mechanisms in the two disease models and that these differences may provide the molecular underpinnings for the distinct clinical presentations observed in DBA and SDS patients. Further studies on the mechanism by which 60S subunit biogenesis was affected in the SDS model have clear implications for the treatment of this disorder

Study of the Ribosomal Stress Pathway in Pluripotency, Cancer and Disease

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología MolecularLa vía de estrés ribosomal se describió hace más de una década como una nueva vía activadora de p53. Dicha vía monitoriza la homeostasis de la biogénesis ribosomal. Perturbaciones en cualquiera de las etapas de la biosíntesis del ribosoma, transcripción del DNA ribosomal, procesamiento del RNA ribosomal, ensamblaje o transporte nuclear, conllevan un exceso de proteínas ribosomales no unidas al ribosoma. En este contexto, el complejo pre-ribosomal RPL11/RPL5/5S rRNA inhibe MDM2, activando así al supresor tumoral p53. La activación de p53 puede resultar en diferentes respuestas celulares que impiden que las células dañadas se conviertan en tumorales. Una de las preguntas que hemos abordado en este trabajo es si las células madre pluripotentes de ratón, caracterizadas por altas tasas de división celular, presentan mecanismos que monitoricen la homeostasis de la biogénesis ribosomal para salvaguardar la integridad de la progenie. Aquí demostramos que dichas células tienen funcional la vía de estrés ribosomal y que esta vía activa p53 en respuesta al estrés ribosomal y elimina aquellas células dañadas mediante un proceso de apoptosis. Las células tumorales requieren a una mayor producción de ribosomas para mantener las altas tasas de división celular que las caracterizan. En este trabajo hemos llevado a cabo un rastreo de compuestos que tengan como diana el nucléolo, la fábrica de ribosomas de la célula, con la finalidad de perturbar la producción de ribosomas y eliminar así las células cancerígenas. Hemos testado dos colecciones de compuestos químicos y hemos identificado un grupo de derivados de acridina que inhiben la transcripción del ADN ribosomal y, por tanto, generan la pérdida de la integridad del nucleolo. Esto resulta en la activación de p53 en ausencia de daño a través de la vía de estrés ribosomal. Finalmente, estos compuestos ralentizan el crecimiento celular y activan un proceso de apoptosis en distintas líneas de células tumorales. RPL11, proteína clave en la vía de estrés ribosomal, se encuentra mutada en heterozigosis en pacientes de anemia de Diamond-Blackfan. Hemos generado un alelo nulo condicional para Rpl11 y hemos demostrado que la pérdida de un alelo de Rpl11 en ratones adultos recapitula las principales características de la enfermedad, incluyendo un procesamiento inadecuado del ARN ribosomal, anemia macrocítica debida a una maduración eritroide defectuosa y una mayor predisposición a cáncer. Los ratones haploinsuficientes para Rpl11 muestran una linfomagénesis acelerada, lo que puede deberse a dos mecanismos no excluyentes: una vía de estrés ribosomal defectuosa y mayores niveles basales de la proto-oncoproteína c-MYC. En resumen, nuestro trabajo pone de manifiesto la importancia de la vía de estrés ribosomal y, en particular de RPL11, en la fisiología, incluyendo células madre embrionarias y el organismo adulto, así como para el desarrollo del cancer y su terapia.The ribosomal stress (RbS) pathway was described more than a decade ago as a new p53- activating pathway. This pathway monitors the homeostasis of the ribosome biogenesis. Perturbations in any of the steps comprising ribosome biosynthesis, rDNA transcription, rRNA processing and ribosome assembly and export, lead to the accumulation of ribosome-free ribosomal proteins. In this situation, RPL11/RPL5/5S rRNA pre-ribosomal complexes bind and inhibit MDM2, thus activating p53. Activation of p53 can result in different cellular outcomes that prevent damaged cells from becoming malignant. We wanted to explore whether mouse pluripotent stem cells, characterized by a rapid growth rate, present mechanisms to monitor the homeostasis of ribosome biogenesis as a way to ensure an optimal quality of their progeny. We have demonstrated that mouse pluripotent stem cells rely on an operative ribosomal stress pathway to eliminate damaged cells upon RbS. Importantly, p53 plays a key role in this process by eliciting apoptsis in embryonic stem cells following RbS. Cancer cells require high rates of ribosome biogenesis to sustain their rapid growth. To target this Achilles’ heel of cancer cells we have designed a cellular screen that monitors the integrity of the nucleolus, the ribosome factory, to identify small molecule compounds that disrupt ribosome biogenesis. By performing chemical library screens, we have identified a group of acridine derivatives that inhibits rDNA transcription, and thus cause nucleolar disruption. This results in p53 activation through the RbS pathway, and in the absence of detectable DNA damage. Remarkably, these compounds hamper proliferation and trigger apoptosis of different cancer cell lines, providing new therapeutical opportunities against cancer. Heterozygous mutations of RPL11, a key player in the RbS pathway, have been found in Diamond-Blackfan Anaemia (DBA) patients. To that end, we generated a conditional knockout mouse model for Rpl11. Here we demonstrate that partial loss of Rpl11 recapitulates the main pathologies of DBA, including impaired rRNA processing, macrocytic anaemia and cancer predisposition. Rpl11 haploinsufficient mice have reduced number of erythroid progenitors and delayed erythroid differentiation. These animals show accelerated lymphomagenesis, probably due to two non-exclusive mechanisms: defective activation of p53 through the RbS pathway and increased basal levels of c- MYC. In summary, our work provide insights into the biological relevance of the RbS pathway and, in particular of RPL11, in the physiology, including mouse pluripotent stem cells and the adult organism, as well as in cancer development and possible treatment

Ribosomal proteins in zebrafish haematopoiesis and human disease

PhDSeveral congenital disorders of human haematopoiesis including Diamond- Blackfan anaemia result from heterozygous loss of genes involved in ribosome biogenesis. Further, hemizygosity for ribosomal protein gene RPS14 has been implicated in the pathogenesis of myelodysplastic syndrome with loss of 5q, suggesting that genes involved in ribosome biogenesis may act as both haploinsufficient tumour suppressors and regulators of normal haematopoiesis. Ribosome biogenesis is highly conserved through evolution and readily studied in simple organisms such as yeasts. However the zebrafish provides a wellestablished genetic model system which is ideally suited to rapid assessment of vertebrate haematopoiesis. I have therefore used the zebrafish to study genes involved in ribosome biogenesis and their effects on developmental haematopoiesis relevant to human disease. Presented in this work is investigation of the effect of disruption of 4 genes known to be involved in ribosome biogenesis on zebrafish haematopoiesis. Firstly, I describe a gene, Dead-box 18 (ddx18), identified in a forward genetic screen, whose disruption results in defective haematopoiesis and embryonic lethality. Secondly, I have studied the effects of loss of zebrafish orthologues of the human nucleophosmin gene (NPM1), the most frequently mutated gene in human acute myeloid leukaemia. Loss of Npm1 resulted in aberrant numbers of myeloid cells. Heterologous overexpression of mutated NPM1(NPMc+) resulted in increased production of haematopoietic stem cells suggesting a role for NPMc+ in pathogenesis of AML. Finally, I have shown that loss of Rps14 and Rps19 result in anaemia in developing zebrafish and have investigated p53-independent mechanisms for this effect. The findings described herein demonstrate that disruption of normal ribosome biogenesis frequently results in abnormal developmental haematopoiesis. Further genetic assessment of these tissue-specific pathways deregulated by loss of normal ribosome function may represent an important common mechanism underlying the pathogenesis of congenital and acquired disorders of haematopoiesis, and may provide novel pathways for therapeutic targeting

SRP54 mutations induce Congenital Neutropenia via dominant-negative effects on XBP1 splicing

Heterozygous de novo missense variants of SRP54 were recently identified in patients presenting with Congenital Neutropenia (CN) or its syndromic form Shwachman-Diamond Syndrome (SDS). Ever since its discovery as a driver of CN and SDS, SRP54 has been increasingly studied in the context of disease and is nowadays considered the second most common cause of CN. Despite its hitherto unknown prevalence, the molecular mechanisms leading to the development of the disease are still largely unknown and patient treatments are far from specific. In this thesis, I aimed to investigate the underlying mechanisms and processes contributing to the pathophysiology of SRP54 deficiencies. To follow this aim, I characterized and established a transgenic srp54 KO zebrafish as the first in vivo model of srp54-driven disease. Interestingly, srp54-/- zebrafish show early embryonic mortality and suffer from severe neutropenia and developmental defects affecting multiple organs. srp54+/- zebrafish on the other hand are viable and only display mild neutropenia and no overt other defects. However, when injecting srp54+/- fish with human mRNA of three mutated SRP54 variants (T115A, T117Δ and G226E) identified in patients, the neutropenia intensified, and pancreatic defects developed – a phenotype accurately mimicking the characteristics of SDS patients. Of note, the induced phenotypes showed mutation-specific differences, indicating that different SRP54 lesions exert unique dominant-negative effects on the functionality of the residual wildtype SRP54 protein. Consistent with these findings, overexpression of SRP54 missense variants in human promyelocytic HL60 cells as well as in healthy CD34+ cord blood cells impaired granulocytic maturation. Mechanistically, we found that SRP54 defects significantly reduce the efficiency of the unconventional splicing of the transcription factor X-box binding protein 1 (XBP1), which is one of the major regulators of the unfolded protein response (UPR). Vice-versa, xbp1 morphant zebrafish recapitulate phenotypes observed in srp54 mutant fish, and the injection of spliced xbp1 but not unspliced xbp1 rescues the neutropenia in srp54+/- embryos. In order to identify additional mechanisms contributing to the pathophysiology of SRP54 deficient patients, we performed single cell RNA sequencing of srp54-mutated zebrafish. Sequencing analysis revealed several differentially expressed genes with most of them converging on the major signaling branches of the UPR, indicating the cell’s efforts to circumvent the impaired XBP1 activity aiming to alleviate unresolved ER-stress

DNA Damage, Oxidative Stress and Related Metabolic By-Products in Cancer and Environmental Studies

Computational fluid dynamics (CFD), which uses numerical analysis to predict and model complex flow behaviors and transport processes, has become a mainstream tool in engineering process research and development. Complex chemical processes often involve coupling between dynamics at vastly different length and time scales, as well as coupling of different physical models. The multiscale and multiphysics nature of those problems calls for delicate modeling approaches. This book showcases recent contributions in this field, from the development of modeling methodology to its application in supporting the design, development, and optimization of engineering processes

Gene-centric functional dissection of human genetic variation uncovers regulators of hematopoiesis

Genome-wide association studies (GWAS) have identified thousands of variants associated with human diseases and traits. However, the majority of GWAS-implicated variants are in non-coding regions of the genome and require in depth follow-up to identify target genes and decipher biological mechanisms. Here, rather than focusing on causal variants, we have undertaken a pooled loss-of-function screen in primary hematopoietic cells to interrogate 389 candidate genes contained in 75 loci associated with red blood cell traits. Using this approach, we identify 77 genes at 38 GWAS loci, with most loci harboring 1-2 candidate genes. Importantly, the hit set was strongly enriched for genes validated through orthogonal genetic approaches. Genes identified by this approach are enriched in specific and relevant biological pathways, allowing regulators of human erythropoiesis and modifiers of blood diseases to be defined. More generally, this functional screen provides a paradigm for gene-centric follow up of GWAS for a variety of human diseases and traits