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From Stem Cells to Blastoids: Unraveling the Mechanisms of Early Embryonic Development and Gene Silencing
No full textUnderstanding the mechanisms of early embryonic development is essential for understanding the origins of aging and disease. Advancements in the culture of embryonic stem cells have opened new avenues for modeling critical timepoints of early development in vitro, such as blastocyst formation, implantation, and gastrulation. The work presented here shows the development of stem cell derived blastocyst like structures, termed blastoids, from pluripotent stem cell cultures in both humans and animals. Blastoids have increased our understanding of the signaling, genetic and epigenetic requirements for the formation of the early embryo. Finally, our work helps to uncover the critical epigenetic interplay that happens in the epiblast of the blastocyst before and after implantation, by using naïve and primed embryonic stem cells to respectively model these stages. We show that TASOR (transcription activator suppressor), part of the Human Silencing Hub (HUSH) complex, is a co-transcriptional platform for epigenetic and epitranscriptomic silencing. We show how the H3K9me3 deposition in naïve cells is essential for the proper establishment of long-term silencing via DNA methylation in primed or differentiated cells. Moreover, our work uncovers an innate immune checkpoint that is activated upon the exit of naïve pluripotency against endogenous retroviral LINE-1 elements and repeats. Overall, this dissertation provides significant insights into the genetic, epigenetic, immunological and signaling mechanisms of the early embryo, offering new perspectives on the intricate biological processes that govern the earliest stage of mammalian life
Structural Investigation of the Alkaline pH-Dependent Activation of Insulin Receptor-Related Receptor
No full textThe general metadata -- e.g., title, author, abstract, subject headings, etc. -- is publicly available, but access to the submitted files is restricted to UT Southwestern campus access and/or authorized UT Southwestern users.Insulin receptor family, a subset of receptor tyrosine kinases governing metabolic balance and growth. Among them, the insulin receptor-related receptor stands out for its activation mechanism, which responds to alkaline pH rather than ligand binding. However, the precise workings of this pH-induced IRR activation have remained elusive due to the lack of high-resolution structural data. Our breakthrough comes with the cryo-EM structures of human IRR in two key states: the inactive state at neutral pH and the active state at alkaline pH. Through mutation studies and cellular experiments, we've unveiled the mechanism: as pH rises, repulsion forces between IRR's pH-sensing motifs disrupt its auto-inhibition, initiating a scissor-like rotation between two units and forming a "T"-shaped active conformation. This activated IRR dimer relies on specific interactions to stabilize itself. Our findings offer an unprecedented insight into the pH-dependent activation of IRR, paving the way for a deeper understanding of this critical receptor's structure-function relationship
Therapeutic Targeting of Protocadherin 7 in Lung Adenocarcinoma
No full textThe general metadata -- e.g., title, author, abstract, subject headings, etc. -- is publicly available, but access to the submitted files is restricted to UT Southwestern campus access and/or authorized UT Southwestern users.Pages 38-92 are misnumbered as pages 41-95.Non-small cell lung cancer (NSCLC) is the most common lung cancer subtype and remains the leading cause of cancer associated deaths worldwide. Patients have few therapeutic options, and disease progression is inevitable, underscoring the critical need for the identification of new targets and therapeutic approaches to treat this disease. We identified a critical oncogenic role for PROTOCADHERIN 7 (PCDH7), a cell surface protein and member of the Cadherin superfamily in NSCLC. PCDH7 is frequently overexpressed in lung adenocarcinoma (LUAD) and associates with poor clinical outcome. Depletion of Pcdh7 reduces lung tumor burden and prolongs survival in mouse models of high-grade NSCLC, demonstrating that this cell surface protein is an actionable therapeutic target. Here we report the development and characterization of high affinity anti-PCDH7 monoclonal antibodies (mAbs) that inhibit downstream MAPK pathway activation and suppress tumor growth in multiple mouse models, including KRAS- and EGFR-mutant models. A lead mAb sensitized tumors to the FDA-approved MEK inhibitor Trametinib, the tyrosine kinase inhibitor Osimertinib, and the KRASG12C inhibitor Adagrasib. Moreover, a humanized mAb7 decreased tumor growth in an Fc-dependent manner and enhanced antibody dependent cell-mediated cytotoxicity and granzyme B production. These findings provide an important step towards the clinical development of PCDH7-targeting antibodies for the treatment of NSCLC and other tumor types with high PCDH7 expression
Mechanistic Basis of Skeletal Muscle Wasting Diseases
No full textSkeletal muscle is the largest tissue in the human body by biomass and it is essential for life. Impaired skeletal muscle function can negatively impact one's quality of life but in the case of diseases like cancer cachexia, it can directly contribute to death of patients. Skeletal muscle wasting is a key feature of many monogenic muscle diseases such as Duchenne muscular dystrophy (DMD) and Emery-Dreifuss muscular dystrophy (EDMD) but also more complex conditions such as cancer cachexia, starvation, and various neuromuscular diseases. Throughout this thesis, I focus on two muscle wasting diseases, cancer cachexia and EDMD. While these diseases have been studied for decades, with the disease-causing genes being identified for EDMD, the mechanistic basis has not been elucidated for either disease. Here we propose that downregulation of the nuclear envelope protein, Net39, contributes to EDMD pathogenesis. Adult deletion of Net39 in mice recapitulates many of the key features of EDMD, including nuclear envelope deformations, dysregulated gene expression, altered metabolism, and muscle wasting. Mechanistically, Net39 protects myonuclear envelopes from mechanical stretch and nuclear envelopes deficient of Net39 are structurally compromised, leading to DNA damage. Cancer cachexia on the other hand, is a highly prevalent and systemic wasting condition characterized by skeletal muscle wasting. We profiled the molecular changes at play in mouse and human cachexic muscle at single nuclear resolution, identifying the conserved activation of a denervation gene program. Mechanistically, we discovered that myogenin regulates myostatin during cancer cachexia and the inhibition of myostatin via AAV-Follistatin gene therapy rescues cancer cachexia and prolongs survival in preclinical models of cancer. Overall, our findings here highlight the importance of Net39 to EDMD pathogenesis and the myogenin-myostatin axis to cancer cachexia-induced muscle atrophy
Structures and Mechanisms of Lysosomal Transporters
No full textLysosomal membrane transporters are indispensable for maintaining lysosomal homeostasis and proper function. Indeed, mutations in these key proteins can lead to debilitating disorders known as lysosomal storage diseases. Cystinosin and Sialin are two such transporters. Both proteins utilize the low pH environment to transport their main substrates of interest from the lumen to the cytosol where they can be reused by the cell. Understanding how these proteins work at an atomic level can help us understand their overall function, the roles they play in lysosomal signaling pathways, and may enable the future development of therapeutics to treat their associated disorders.
Mutations in Cystinosin cause Cystinosis, a neurodegenerative disorder that occurs when Cystinosin's substrate, the dimeric form of cysteine called cystine, builds up in the lysosome. While there currently is a treatment for this disorder, how mutations disrupt this transport and how the protein utilizes the proton gradient remain a mystery. Similarly, Sialin causes a variety of free sialic acid storage diseases that result in developmental delays as well as neurodegeneration. Unlike Cystinosis, there is currently no approved treatment for these disorders. Like Cystinosin, the structure and mechanism of transport remain unknown.
The work presented herein reveals the cryo-EM structures of Cystinosin and Sialin at near atomic level resolution. These structures, captured in both cytosol- and lumen-open conformations as well as substrate bound states, reveal not only the mechanisms of conformational changes but also the residues involved in the substrate binding pocket(s). Along with the accompanying functional assays, I demonstrate that the majority of disease-causing mutations in both Cystinosin and Sialin center around their ability to bind their respective substrates. Additionally, I reveal the potential proton sensors of both proteins involved in their substrate symport. In the case of Sialin, I also hypothesize that its proton sensor could potentially double as a membrane potential sensor for its transport of neurotransmitters into synaptic vesicles. This work paves the way for understanding the greater PQ-loop family (Cystinosin) and SLC17 family (Sialin) of transporters. It also builds a foundation upon which future therapeutics can be designed to treat their associated disorders
An Ultra-pH-Sensitive Logic Decodes Cancer Imaging and Immunotherapy
No full textPages 186-213 are misnumbered as pages 219-246.Binary classification of cancer is a challenge in clinical diagnosis due to continuously varying cancer-associated signals in both time and space. To distinguish cancer from healthy tissue it's necessary to assess binary relationships between signals at the molecular scale. However, the naked eye cannot discriminate differences at that resolution. Further, contrast enhancement with responsive small molecules shows minute changes between cancer and healthy tissues. Therefore, our group previously developed an ultra-pH-sensitive (UPS) nanoparticle platform to discretely amplify differences of acidotic thresholds. Predicated on molecular cooperativity, a divergent phase transition behavior from nanoparticle to polymer enables an irreversible capture-and-integration in the tumor microenvironment for robust tumor margin visualization. Based on previous observations on applications of the UPS platform, here I employ a formal first-order logic to quantify ultra-pH-sensitivity in cancer diagnosis. Based on this framework, I demonstrate novel applications of UPS nanoparticles in lymph node metastasis imaging. I show that UPS nanoparticles accumulate in microscopic cancer foci inside of lymph nodes, enabling discrimination between metastatic and benign lymph nodes. Further, I determine contexts wherein near-neutral pKa UPS nanoparticles lose pH-reporting fidelity in vivo. Both UPS and PC7A nanoparticles (pKa 6.9) have a short circulation half-life and poor tumor biodistribution. To overcome this, I employ cybernetic control by co-formulating two UPS polymers into a single nanoparticle. Attenuation of micelle disassembly, through strengthened non-covalent interactions, stabilizes UPS nanoparticles for improved pharmacokinetics in blood circulation. I demonstrate two applications of these long-circulating, hybrid nanoparticles. First, I engineer HyUPS nanoparticles for improved lymph node delivery of UPS6.9 polymers through co-assembly with UPS5.3. Reporting dual pH-thresholds enables accumulation in metastatic lymph nodes. Second, I develop HySTING nanoparticles for improved accumulation of PC7A in tumors. HySTING retains the unique biological activity of PC7A, yet accumulates greater in tumors after intravenous injection. HySTING enables tumor growth inhibition in mouse models of cancer by synergizing with synthetic cyclic dinucleotide payloads. Overall, a formal logic reveals robust discrimination of cancer from normal tissue by UPS nanoparticles. However, this logic fails in certain contexts. Restoring high fidelity logic through engineering control enables ON-target cancer imaging and immunotherapy
Dynamic Regulation of Ribosome Biogenesis and Its Role in Early Brain Development
No full textChapter 1, including figures and a substantial amount of text, has been re-used, with or without modifications, from the following previous published works: [1] Ni, C., and Buszczak, M. (2023a). The homeostatic regulation of ribosome biogenesis. Semin Cell Dev Biol 136, 13-26. [2] Ni, C., and Buszczak, M. (2023b). Ribosome biogenesis and function in development and disease. Development 150.Chapter 2, including figures and a substantial amount of text, has been re-used, with or without modifications, from the following previous published work: Ni, C., Schmitz, D.A., Lee, J., Pawłowski, K., Wu, J., and Buszczak, M. (2022). Labeling of heterochronic ribosomes reveals C1ORF109 and SPATA5 control a late step in human ribosome assembly. Cell Reports 38, 110597.Chapter 3, including figures and a substantial amount of text, has been re-used, with or without modifications, from the following previous pre-print work: Chunyang Ni , Leqian Yu, Barbara Vona, Dayea Park, Yulei Wei, Daniel A Schmitz, Yudong Wei, Yi Ding, Masahiro Sakurai, Emily Ballard, Yan Liu, Ashwani Kumar, Chao Xing, Hyung-Goo Kim, Cumhur Ekmekci, Ehsan Ghayoor Karimiani, Shima Imannezhad, Fatemeh Eghbal, Reza Shervin Badv, Eva Maria Christina Schwaibold, Mohammadreza Dehghani, Mohammad Yahya Vahidi Mehrjardi, Zahra Metanat, Hosein Eslamiyeh, Ebtissal Khouj, Saleh Mohammed Nasser Alhajj, Aziza Chedrawi, César Augusto Pinheiro Ferreira Alves, Henry Houlden, Michael Kruer, Fowzan S. Alkuraya, Can Cenik, Reza Maroofian, Jun Wu, and Michael Buszczak (2023) Dynamic ribosome biogenesis shapes early brain development (Submitted).While features of ribosome assembly are shared between species, our understanding of the diversity, complexity, dynamics, and regulation of ribosome production in multicellular organisms remains incomplete. To gain insights into ribosome biogenesis in human cells, we performed a genome wide loss of function screen combined with differential labeling of pre-existing and newly assembled ribosomes. These efforts identified two functionally uncharacterized genes, C1ORF109 and SPATA5. We provide evidence that these factors, together with CINP and SPATA5L1, control a late step of human pre-60S maturation in the cytoplasm. Loss of either C1ORF109 or SPATA5 impairs global protein synthesis. These results raise the possibility that neurodevelopmental disorders associated with recessive SPATA5 mutations are caused by defects in ribosome assembly. Based on these findings, we propose the expanded repertoire of ribosome biogenesis factors likely enables multicellular organisms to regulate ribosome production in different ways in response to different developmental and environmental stimuli.
Lineage specific transcriptional programs orchestrate human brain development. Many neural developmental defects, however, are linked to mutations in general regulators of housekeeping genes, such as those encoding ribosome biogenesis and mRNA translation factors, suggesting additional layers of regulation. The molecular and cellular mechanisms by which minor disruptions in global protein synthesis capacity cause neurodevelopmental disorders and when these defects first arise remain unclear. Using cerebral organoids in combination with proteomic analysis, single-cell transcriptome analysis across multiple developmental stages, and single organoid translatome analysis, we discovered a previously unappreciated mechanism linking changes in ribosome levels and the timing of cell fate specification during early brain development. We find ribosome levels decrease during neuroepithelial differentiation, and differentiating cells are particularly vulnerable to defective ribosome biogenesis during this time. Reduced ribosome availability more profoundly impacts the translation of specific transcripts, disrupting both survival and cell fate commitment of transitioning neuroepithelia. Enhancing global protein synthesis ameliorates the growth and developmental defects associated with microcephaly linked variants. Together, these findings reveal that dynamic changes in ribosome levels regulate early development of central nervous system and provide insights into how disruptions in protein synthesis machinery can result in brain-specific malformations
Chemical Investigations in Complex Alkaloid Synthesis
No full textThe concise and efficient synthesis of complex biologically active natural products often requires the elaboration of innovative chemical design paired with the development of modern chemical methods to push the forefronts of chemical space to further biochemical investigations. Complex alkaloids have been of interest to synthetic chemists for decades where their intriguing chemical structures have led to the development of new reactions as well as provided biologically relevant compounds for potential therapeutic use. The growing Myrioneuron and Sarpagine- related alkaloid classes are only a small fraction of the larger realm of complex alkaloid space, however, chemical investigations into these families will hopefully provide insight to the synthetic community as a whole through new chemical methods, synthetic design, and expand further biochemical understandings.
Thus, Chapter 1 will serve to introduce the Myrioneuron alkaloids, beginning by detailing their breadth of biological activity, followed by their proposed biosynthetic origins. Lastly, the reported synthetic approaches to this family will be described, starting with early efforts towards simpler congeners, leading to recent, elegant approaches to the more elaborate family members.
Next, Chapter 2 will describe the evolution of our synthetic approach to the complex tetracyclic Myrioneuron alkaloid myrioneurinol, a strategy leveraging the hidden symmetry within the molecule to assemble the core scaffold. To begin this chapter, I will discuss our strategic plan towards the complex myrioneuron scaffold, then the early efforts to assemble the required symmetrical intermediate, an in-depth investigation into the key desymmetrizing double reductive amination, efficient elaboration of the scaffold, issues surrounding a challenging hydrogenation and our topologically controlled solution, and finally, the completion of myrioneurinol. Also described will be the development of a formal asymmetric synthesis, the first asymmetric entry towards this complex Myrioneuron alkaloid.
Chapter 3 will serve to introduce a similarly complex, yet structurally distinct, subset of indole alkaloids, the Sarpagine-related alkaloids. This chapter will cover this broad family's chemical diversity and wide range of biological activity, their biosynthetic origins, as well as discuss selected synthetic studies within the sarpagine-type alkaloids.
Finally, in Chapter 4, our efforts to develop a concise asymmetric approach to the unusual Sarpagine-type alkaloid rauvomine B will be described. First, I will introduce the strategic approach to the distinct sarpagine alkaloid, the challenges of achieving the preparation of the core methyl-containing scaffold will be discussed, including adjustments to the synthetic design. Lastly, the formation of the signature cyclopropane and associated quaternary carbon center via a key late- stage Rh-catalyzed cyclopropanation will be detailed, allowing for completion of this alkaloid target
Investigating the Role of Hippo/Warts Signaling in Sarcomatoid Renal Cell Carcinoma
No full textRenal cell carcinomas (RCCs) are the most common kidney malignancy and one of the most common cancers overall. Sarcomatoid RCC (sRCC) is a particularly aggressive subtype of RCC which displays a high rate of metastasis and poor prognosis, contributing to a disproportionately high clinical burden. Attempts to identify driver mutations of sarcomatoid transformation have revealed frequent mutations in components of the Hippo/Warts pathway, a highly conserved regulator of genes controlling proliferation and apoptosis which is frequently dysregulated in cancer.
Here, I describe the generation and characterization of a GEMM which gives rise to metastatic sRCC: deletion of Hippo pathway kinases Lats1/2 in adult kidney epithelia. Lats1/2 mutant tumors respond to immune checkpoint inhibition (ICI), as is seen in some human sRCCs. Bioinformatic analysis reveals an enrichment of serum response factor (Srf) targets amongst genes upregulated following Lats1/2 deletion. Ablating Srf in the Lats mutant background causes Lats;Srf mutant cells to undergo epithelial to mesenchymal transition but not progress to fulminant tumors in vivo. RNA-sequencing, CyTOF analysis, and implantation studies in immunocompromised mice suggest that Srf drives a pro-tumor microenvironment in Lats1/2 mutant sRCC. Additionally, transcriptional analysis identifies a subset of human RCCs that express an Srf transcriptional signature, indicating that Srf may contribute to the pathogenesis of RCC. This work has elucidated a novel role for Srf in immune modulation with potential implications for the treatment of human RCC
AXL-WRNIP1 Mediated Replication Stress Response Promotes Therapy Resistance and Metachronous Metastasis in HER2+ Breast Cancer
No full textTherapy resistance and metastatic progression are primary causes of cancer-related mortality. Disseminated tumor cells possess adaptive traits that enable them to reprogram their metabolism, maintain stemness, and resist cell death, facilitating their persistence to drive recurrence. The survival of disseminated tumor cells also depends on their ability to modulate replication stress in response to therapy while colonizing inhospitable microenvironments. In this study, we discovered that nuclear translocation of AXL, a TAM receptor tyrosine kinase, and its interaction with WRNIP1, a DNA replication stress response factor, promotes the survival of HER2 targeted therapy-resistant breast cancer cells in the brain. Using preclinical models, we demonstrated that knocking down or pharmacologically inhibiting AXL or WRNIP1 increased replication stress and attenuated metastatic latency and relapse. Our findings suggest that targeting the replication stress response, which is a shared adaptive mechanism in therapy-resistant and metastasis-initiating cells, could reduce metachronous metastasis and enhance the response to standard-of-care therapies