Aly Karsan

We examined the AML-PMP, TCGA, TARGET pediatric, and Beat AML public datasets to identify genetic drivers of AML. We identified frequent somatic mutations in ubiquitin proteasome system genes, with recurrent inactivating mutations in FBXO11. FBXO11 encodes the substrate-recognizing component of the SKP1-CUL1-F-BOX (SCF) ubiquitin E3-ligase complex. In a mouse marrow transplant model generated by previous members of the Karsan lab, Fbxo11 knockdown cooperated with AML1-ETO expression to initiate AML. These leukemias were serially transplantable, consistent with the presence of leukemic stem cells (LSC). We hypothesized that FBXO11 depletion in hematopoietic stem and progenitor cells (HSPC) promotes self-renewal and drives leukemogenesis through dysregulated expression of its ubiquitination targets.First, we developed a serially transplantable human xenotransplant myeloid leukemia model based on FBXO11 depletion with cooperating mutations, and observed increased engraftment and expansion of primitive CD34⁺ cells with FBXO11 knockdown. Next, to identify FBXO11 targets that are dysregulated with FBXO11 depletion, we performed mass spectrometry analysis of FBXO11 co-immunoprecipitating proteins in the myeloid K562 cell line. We identified LONP1 as a top target, but LONP1 protein expression did not change with FBXO11 over-expression or loss, suggesting that LONP1 is not targeted for degradation. Knockdown of LONP1 in human CD34⁺ HSPCs resulted in similar phenotypes as FBXO11 knockdown: enriched hematopoietic stem cell, LSC, and mitochondrial defect transcriptomic signatures, increased myeloid cells, and reduced erythroid and megakaryocytic populations. In parallel, FBXO11 knockdown reduced mitochondrial membrane potential. These data suggest that FBXO11 normally positively regulates LONP1. We found that K63-linked polyubiquitination—a modification known to regulate protein activation and trafficking— of LONP1 is increased with FBXO11 overexpression, and reduced with FBXO11 loss. LONP1 overexpression alone had negligible effect on the transcriptome, and LONP1 knockdown blocked or reversed the effects of FBXO11 overexpression. This supports a model of FBXO11 regulating LONP1 mitochondrial function, presumably through K63-linked ubiquitination. These findings suggest that FBXO11 positively regulates LONP. We propose FBXO11 depletion in AML patients results in reduced LONP1 mitochondrial function, which promotes a primitive cell state. We have identified novel candidate targets of FBXO11, and demonstrated a unique link between the ubiquitin-proteosome system and the mitochondria.

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Embryonic development is a highly coordinated process during which cells undergo regulated division, differentiation, and migration to ultimately shape specialized tissues that perform particular functions. Throughout this course, cell fate is driven by intrinsic cell decisions in response to microenvironmental cues from their surroundings and understanding what governs these processes is at the core of developmental biology. Aberrant expression of essential developmental genes typically elicits apparent embryonic defects due to disruption of normal developmental processes. Thus, investigation of the resulting phenotype in animal models provides valuable insights into their role and overall biological pathways driving organogenesis. The work presented in this dissertation investigates the role of such developmental genes and uncovers significant signaling interactions involving endothelial cells to promote cell specification and maturation of non-vascular cells. First, this study explores the contribution of Sash1 during organogenesis. Examination of Sash1-knockout mouse embryos revealed cell non-autonomous functions in regulating both prenatal lung development and embryonic hematopoiesis. On one hand, endothelial Sash1 signaling in the lung promoted nitric oxide production which plays a critical role in the maturation of alveolar epithelial cells prior to birth. On the other hand, Sash1 expression in non-endothelial cells contributed to the emergence of hematopoietic stem and progenitor cells during the endothelial-to-hematopoietic transition (EHT) in the early embryo. Our findings suggest that Sash1 in the microenvironment impacts the progression of specified hemogenic endothelial cells into hematopoietic clusters and the work presented in chapter 4 identifies multiple candidate cell types as the origin of Sash1 signaling. In addition, early specification of a hemogenic endothelium distinct from its arterial counterpart is a critical event at the origin of EHT. Therefore, the last part of the study explored factors influencing endothelial cell fate and provided evidences to support a role for the developmental gene Meis1 in driving early hematopoietic commitment of the endothelium while showing that loss of its expression significantly impairs EHT. Altogether, the data presented in this dissertation highlights critical functions for Sash1 and Meis1 in embryogenesis and showcases examples of communication from, to, and within endothelial cells to influence cell fate in organogenesis.

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Acute myeloid leukemia (AML) is a blood-based cancer with dismal outcomes and limited therapeutic options. Clinical applications of next-generation sequencing (NGS) methods offer a potential avenue for improved diagnosis, stratification, and therapy selection for AML. In particular, RNA sequencing has the potential to provide a wealth of genome-wide information of potential clinical relevance. However, the analytic validity of RNA-Seq as a clinical assay for AML has not been established. In this thesis, I first compare RNA-Seq to whole-exome sequencing (WES) and whole-genome sequencing (WGS) approaches, and demonstrate that RNA-Seq offers the most broad benefits for clinical assessment of AML at diagnosis. To determine whether RNA-Seq information could be used to improve upon the current clinical stratification model for AMLs, I develop a novel gene expression signature. Together with demonstrating improved stratification performance, I find that a subset of high-risk AML cases are characterized by dysregulated integrin signaling, and are potentially amenable to treatment with existing inhibitors. To further characterize the dysregulated transciptome in AML, I analyze miRNA sequence data from the same patient cohorts, observing that the dysregulation of key miRNAs may be involved in up-regulation of integrin signaling. Taken together, the results presented in this thesis offer a path towards improved clinical diagnostics in AML, together with a deeper understanding of transcriptomic dysregulation at both the mRNA and miRNA levels.

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MicroRNAs are known to be upregulated or downregulated in various types of cancer, leading to changes in the expression of genes involved in cellular proliferation, anti-apoptosis, migration, and invasion. To study the effects of microRNA loss or gain in different neoplasms, numerous models have been described to decrease or increase expression of microRNAs, but the off-target effects of different methods have not been well investigated. I investigated the possibility of off-target effects in a model of miR-143 knockdown in myeloid leukemia cell lines that implemented a microRNA sponge, or decoy, as a method to reduce microRNA expression. The high expression of a sponge with repetitive sequence elements and low expression of the intended microRNA for knockdown, miR-143, created conditions with increased potential for non-specific microRNAs to bind to the sponge. Therefore, I investigated the potential binding sites present in the sponge and whether any novel microRNAs could bind to these sites. I found a number of potential candidates and eliminated them based on their likelihood of regulating protein targets and their resemblance to a microRNA in structure, leaving one potential candidate. I found genomic evidence of the existence of this novel microRNA, evolutionary conservation of function, and performed assays that confirmed the biological activity. Next, the original sponge was redesigned to inhibit the binding of the potential non-specific microRNA; miR-X, or the miR-143 binding sites were mutated to inhibit the binding of miR-143 and capture miR-X instead. This demonstrated that binding of non-specific microRNA could be abrogated and differential protein abundance specific to the knockdown of each microRNA separately was verified. I conclude that non-specific binding to the sponge is a distinct possibility in experiments using this method of microRNA knockdown, which needs to be taken into account when designing sponges in the future. This work also demonstrates that there remain novel microRNAs awaiting discovery.

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Myelodysplastic syndromes (MDS) are a collection of hematopoietic malignancies in which genomic abnormalities within the hematopoietic stem cell (HSC) compartment lead to pancytopenia and eventual bone marrow failure or progression to leukemia. The most common karyotypic abnormality in MDS is the interstitial deletion of chromosome 5q where a commonly deleted region (CDR) has been identified. Two microRNAs (miRNA) located within the CDR have been shown to be expressed at lower levels in del(5q) MDS compared to diploid MDS. Here I describe investigations of the functional consequence of loss of these two miRNAs, miR-143 and miR-145, on hematopoietic stem and progenitor cells. Loss of miR-143/145 in mice resulted in a decrease in hematopoietic stem cells and myeloid progenitors. I also identified a direct link between the loss of miR-143/145 and subsequent activation of both the canonical and non-canonical transforming growth factor beta (TGFβ) signaling pathways, mediated in part via the adaptor protein, Disabled-2 (DAB2). Analysis of del(5q) MDS patient bone marrow revealed an enriched TGFβ-signature compared to healthy controls and we show that TGFβ signaling is activated upon loss of miR-145 or enforced expression of its target, DAB2. Subsequent studies focused on the function of DAB2 within the hematopoietic system. Overexpression of DAB2 in mouse bone marrow resulted in a significant decrease in hematopoietic stem cell frequency, self-renewal, and colony forming activity. In competitive transplants, vector-transduced bone marrow cells were able to outcompete DAB2-overexpressing marrow in both primary transplants as well as in secondary limiting dilution assays. However, a subset of mice with enforced DAB2 expression alone developed a transplantable acute myeloid leukemia. In conclusion, our data suggest a role for miR-145 haploinsufficiency in the inappropriate activation of TGFβ signaling through derepression of DAB2 which leads to abnormal HSC function in del(5q) MDS.

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Hematopoiesis is a process essential for the maintenance of cells that mediate many vital functions such as the production of red blood cells necessary for transportation of oxygen and the removal of carbon dioxide from the body. It is also required for the production of platelets which are necessary for clotting, and the various types of white blood cells that make up the innate and adaptive immune systems that protect against viral, microbial, and parasitic infections. The cell responsible for the generation of all the downstream effector cells, the hematopoietic stem cell (HSC), is generated during embryogenesis. Through a series of symmetric and asymmetric cell divisions, the HSC is capable of maintaining all the cells of the hematopoietic hierarchy throughout the lifespan of an organism. A variety of genetic and epigenetic cues are necessary to maintain homeostasis, and perturbations in these cues lead to the development of hematopoietic malignancies and myelodysplastic syndromes. In recent years the innate immune pathway has emerged as an important player in hematopoietic homeostasis. This dissertation examines the role of dysregulation of innate immune signaling in the development of the myelodysplastic syndromes, one of the most common hematological malignancies. Using a murine bone marrow transplantation assay, I show that overexpression of the innate immune signaling adaptor, TIRAP, results in perturbations in normal hematopoiesis. Overexpression of TIRAP in the hematopoietic compartment results in an inability to produce mature hematopoietic cells, leading to pancytopenia and bone marrow failure (BMF). TIRAP-induced BMF is a result of both autonomous and non-autonomous effects mediated by the cytokine IFNγ. Interestingly, in an environment depleted of IFNγ, TIRAP-transplanted mice develop a myeloproliferative neoplasm suggesting that TIRAP activates both myelosuppressive pathways (through IFNγ) as well as myeloproliferative pathways. IFNγ acts in a paracrine manner inhibiting osteoclast proliferation and maturation in the bone marrow microenvironment, thus disrupting the HSC niche. In summary, this thesis shows the importance of immune regulation in hematopoietic homeostasis. Furthermore, it shows how defects in the hematopoietic stem/progenitor compartment can translate into a defect in the stem cell niche, contributing further to marrow failure and disease progression.

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Notch signaling is evolutionarily conserved and regulates various developmental and pathological processes. We are interested in the role of Notch signaling in cardiac valve formation and postnatal vascular remodeling. The heart is the first organ to form in a developing embryo and a common site of congenital defects. Valvuloseptal defects are the most common of the cardiac anomalies seen in the newborn with a prevalence of 1-2% and are often a result of deregulated atrioventricular canal (AVC) formation. The process of endothelial-to-mesenchymal-transition (EndMT) is required for the growth and maturation of the AVC and Notch signaling has been shown to induce this process. In chapter 3, we identify a novel link between Notch and Nitric Oxide (NO) signaling during EndMT. We show that Notch-activation directly induces GUCY1A3 and GUCY1B3, the soluble guanylyl cyclase that forms the heterodimeric NO receptor, during EndMT. We also show that Notch-induced expression and secretion of the TGFβ family member, Activin A, results in increased NO production via a PI3-kinase/Akt signaling mechanism. Paracrine activation of NO signaling by Activin A contributes to early onset of EndMT in the developing AVC. Functional arteries are essential for restoring blood flow and tissue regeneration in response to hypoxia, ischemia, or wound healing. Arterial obstruction can cause distal tissue ischemia and requires rapid reperfusion to limit tissue necrosis. Ischemia recovery requires two processes: angiogenesis (capillary sprouting) and arteriogenesis (expansion of existing vessels secondary to mechanical stress or chemical stimuli). Arteriogenesis involves two phases: a rapid vasodilatory phase followed by vascular expansion and remodeling. In chapter 4, we examine the relationship of endothelial Notch signaling and arteriogenesis using a hindlimb ischemia model. As the link between Notch and NO signaling unfolded in the heart study, we turned our attention to the NO pathway in the initial phase of arteriogenesis – vasodilation. The data presented in this dissertation defines a novel link between Notch signaling and NO signaling in the cardiovascular system and may help to explain Notch-induced EndMT in other pathologies.

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The Notch signaling pathway, which converges on RBPJ, is deregulated in a number of malignancies. Following pathway activation, RBPJ, the DNA-binding component of the pathway, associates with Notch to activate transcription of target genes. In the absence of Notch activity, RBPJ acts as a transcriptional repressor by recruiting a co-repressor complex that must be displaced to reinitiate the cycle of activation. As RBPJ is a key regulator of Notch signaling andis constitutively expressed in normal cells, we set out to evaluate the effect of RBPJ loss in the context of human cancer. Frequent RBPJ loss was detected in human breast and lung tumors.Moreover, depletion of RBPJ in a human breast cancer cell line accelerated xenograft tumorgrowth, whereas over-expression of a mutated version of RBPJ (which allows retained function as a transcriptional repressor but prevents activation via Notch) reduced tumor growth in amouse model. These findings were confirmed in a lymphoma knock-out cell line, where a complete loss of RBPJ strikingly increased tumor growth in mice. RBPJ-deficient tumor xenografts showed up-regulated expression of HEY family genes, which represent direct canonical RBPJ targets. Blockade of Notch activation had no effect on the magnitude of HEYgene derepression in the absence of RBPJ, indicating that Notch does not participate inderegulated signal activation resulting from loss of transcriptional repression. To identify other aberrantly induced genes that contribute to the oncogenic phenotype with RBPJ loss, we performed a global analysis. RBPJ removal led to enrichment of acetylated histone H4 at induced gene promoters. We therefore used this epigenetic mark as an indirect measure ofpromoter activity to identify processes that were differentially active in the RBPJ-depleted breastcancer cells compared to RBPJ-containing controls. RBPJ loss enriched for a Notch-like signal and increased acetyl marks at genes associated with cell survival. Indeed, resistance to cell death was observed in RBPJ-deficient breast cancer and lymphoma tumors both in vitro and in vivo. This work defines a new role for RBPJ as a tumor suppressor, the loss of which represents an alternate mechanism for deregulating Notch signaling in cancer.

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The endothelium plays a critical role in coordinating the innate immune response through the regulation of vascular tone, leukocyte recruitment and transmigration, and hemostasis. These functions are mediated, in part, by the signaling cascades initiated upon recognition of bacterial and viral products by a family of transmembrane receptors known as Toll-like receptors (TLRs). In endothelial cells, exposure to lipopolysaccharide (LPS), a major cell wall constituent of Gram negative bacteria, results in endothelial activation through TLR4. Recruitment of the adapter protein, MyD88, to the receptor facilitates association of serine threonine kinases of the IL-1 receptor associated kinase (IRAK) family. The IRAKs initiate a phosphorylation cascade through TNFR-associated factor 6 (TRAF6) culminating in activation of proinflammatory signaling pathways including NF-κB and c-Jun NH2-terminal kinase (JNK) pathways. This thesis investigates signaling molecules and pathways downstream of TLR4 in endothelial cells. Specifically, contained herein is a description of the role of heterotrimeric G proteins in endothelial TLR signaling. This thesis identifies for the first time the function of these proteins in multiple TLR signaling pathways. In addition, the work presented here describes the identification and characterization of a novel TLR4 signaling molecule, SAM and SH3 domain containing protein 1 (SASH1). SASH1 promotes LPS-induced NF-κB and JNK, by functioning as a scaffold molecule to bind TRAF6, transforming growth factor-β-activated kinase (TAK1) and IκB-kinase (IKK), thereby increasing proinflammatory cytokine production. The distinct functions of the endothelium in innate immunity highlight the need for an understanding of the signaling cascades initiated by LPS in endothelial cells and will be crucial to our understanding of the pathophysiology of sepsis in the clinic.

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The vasculature is essential for the delivery of oxygen and nutrients and the removal of metabolic wastes from tissues of the body. The embryonic vasculature is developed through the processes of vasculogenesis, angiogenesis, and arteriogenesis. Once the vasculature is fully developed and stabilized, the adult vasculature shows very little proliferation or cell death. Nevertheless, the endothelium, which lines the lumen of the blood vessels, is actively involved in the control of vascular tone, permeability, blood flow, coagulation, inflammation and tissue repair. An injury to the endothelium is important for progression of diseases such as atherosclerosis and the sepsis syndrome. The Notch signaling pathway has emerged in the recent decade as an important player in multiple vascular processes and endothelial behaviors. This thesis examines the role of the Notch signaling pathway in embryonic arteriogenesis and endothelial survival signaling. The first part of this thesis investigates the developmental source of vascular smooth muscle cells. This study presents the first in situ observation of an immediate smooth muscle precursor cell present in all embryonic arteries. This Tie1⁺/CD31⁺/VE-cadherin⁻ precursor requires Notch signaling to differentiate into vascular smooth muscle cells and to ensure vascular stability of newly formed arteries. However, Notch activation is not required in the precursor cells to maintain the medial layer of the arteries once the vessel is invested with vascular smooth muscle cells. In the second part of this thesis, the mechanism of Notch-induced endothelial survival signaling is examined. In endothelial cells, Notch signaling activates phosphotidylinositol-3 kinase (PI3K) through up-regulation of a secreted factor. Activity of PI3K is required to offset the parallel apoptotic signaling induced by Notch activation and to maintain endothelial survival through the up-regulation of Slug, a direct Notch target with anti-apoptotic activity. Upon treatment with apoptotic stimuli, Notch activation shows context-dependent effects on endothelial survival. Inhibition of PI3K activity and Slug expression by a stimulus abolishes Notch-induced endothelial survival and increases apoptotic death.The work presented in this thesis shows that the Notch signaling pathway is essential for the stability of the vasculature through regulation of vascular smooth muscle cell differentiation and endothelial cell survival.

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