Ibrutinib directly reduces CD8+T cell exhaustion independent of BTK

Introduction: Cytotoxic CD8+ T cell (CTL) exhaustion is a dysfunctional state of T cells triggered by persistent antigen stimulation, with the characteristics of increased inhibitory receptors, impaired cytokine production and a distinct transcriptional profile. Evidence from immune checkpoint blockade therapy supports that reversing T cell exhaustion is a promising strategy in cancer treatment. Ibrutinib, is a potent inhibitor of BTK, which has been approved for the treatment of chronic lymphocytic leukemia. Previous studies have reported improved function of T cells in ibrutinib long-term treated patients but the mechanism remains unclear. We investigated whether ibrutinib directly acts on CD8+ T cells and reinvigorates exhausted CTLs. Methods: We used an established in vitro CTL exhaustion system to examine whether ibrutinib can directly ameliorate T cell exhaustion. Changes in inhibitory receptors, transcription factors, cytokine production and killing capacity of ibrutinib-treated exhausted CTLs were detected by flow cytometry. RNA-seq was performed to study transcriptional changes in these cells. Btk deficient mice were used to confirm that the effect of ibrutinib was independent of BTK expression. Results: We found that ibrutinib reduced exhaustion-related features of CTLs in an in vitro CTL exhaustion system. These changes included decreased inhibitory receptor expression, enhanced cytokine production, and downregulation of the transcription factor TOX with upregulation of TCF1. RNA-seq further confirmed that ibrutinib directly reduced the exhaustion-related transcriptional profile of these cells. Importantly, using btk deficient mice we showed the effect of ibrutinib was independent of BTK expression, and therefore mediated by one of its other targets. Discussion: Our study demonstrates that ibrutinib directly ameliorates CTL exhaustion, and provides evidence for its synergistic use with cancer immunotherapy.</p

Protein-carbohydrate and protein-protein interactions: using models to better understand and predict specific molecular recognition

Any molecular recognition event results in a change in the free energy of the system. The extent of this change is related to the association constant, such that the more negative the free energy change is, the tighter the interaction between receptor and ligand. Protein-carbohydrate interactions play a critical role in signal transduction, innate immunity, and metabolism. Modeling these interactions is somewhat complicated by the inherent flexibility of carbohydrates as well as their relatively large number of functional groups. An empirical scoring function for docking carbohydrates to proteins, specifically tailored to predict both the correct binding orientation and free energy of binding of the carbohydrate-ligand/protein-receptor complex, will be presented. This new scoring function can predict free energies of binding to within 1.1 kcal/mol residual standard error, a definite improvement over existing scoring functions that result in standard errors well over 2 kcal/mol. Application of automated docking methodology to determine carbohydrate recognition specificity of the C-type lectin, human surfactant protein D, will also be presented. In the second part of the thesis, the role of pi-stacking interactions (e.g. between Tyr side chains) in stabilizing protein folds will be discussed. A 17-residue peptide derived from the naturally occurring anti-microbial peptide tachyplesin I was investigated using NMR spectroscopy. NOE cross-peaks were observed, confirming the existence of this interaction in solution. In the final part of the thesis, a quantitative NMR investigation into the self-association behavior of the regulatory domains of several Tec family member kinases will be presented. Of particular interest, self-association within Bruton\u27s tyrosine kinase (Btk) regulatory domains occurs through the formation of an asymmetric homodimer. Together this work demonstrates the importance of rigorous biophysical characterization of biomolecular recognition events and the interdependence of computational modeling and experimentation

Overcoming TCF4-Driven BCR Signaling in Diffuse Large B-Cell Lymphoma

Diffuse Large B-cell Lymphoma (DLBCL) is the most common subtype of lymphoma. Despite a cure rate of 40% with standard R-CHOP therapy, patients that refract or relapse are subject to a dismal prognosis. Cases of DLBCL can be classified by their molecular expression phenotype, with the GCB-like subtype aligning with the profile of a germinal center B-cell and the ABC-like subtype aligning to that of an activated B-cell. Aggressive disease is often characterized by high levels of B-cell Receptor (BCR) signaling. This pathway engages downstream kinases responsible for stimulating proliferation and survival that play a key role under the normal circumstances of B-cell development. A comprehensive study aimed at delineating sources of inhibitor insensitivity within the BCR signaling pathway was conducted in order to identify novel drivers of disease and improve clinical outcome. A cohort of 39 aggressive lymphoma cell lines was assayed for sensitivity to Ibrutinib, a BTK inhibitor, and Umbralisib, a PI3Kδ inhibitor. Combined with intracellular phosphoflow measurements, these results revealed that higher levels of proximal BCR kinase (SYK, LYN, BTK, BLNK) and AKT (downstream of PI3K) signaling were highly linked and predictive of inhibitor insensitivity. Simultaneous inhibition of these pathways with Ibrutinib and Umbralisib consequently revealed a synergistic relationship. Following these results, a DNA copy number analysis of 673 DLBCL patient profiles was performed alongside 249 matching gene expression profiles to uncover the genomic drivers responsible for higher signaling. These results identified an enrichment of genes with transcription factor activity within regions of significant DNA copy number gain and matching transcript gain. The TCF4 transcription factor was identified within the most significant gain peak at chromosome 18q21 and led to increased transcript and protein translation. TCF4 gain was associated with the aggressive ABC-like phenotype, poor survival, and increased transcription of key BCR signaling component targets, such as BLNK, BTK, PIK3CA (PI3Kα), and the IgM heavy chain constant region. Collectively, these results identified sources of inhibitor insensitivity within DLBCL, and TCF4 was characterized as a driving force behind aggressive BCR signaling

A yeast-based assay for protein tyrosine kinase substrate specificity and inhibitor resistance

Phosphorylation of tyrosines by protein kinases is a fundamental mode of signal transduction in all eukaryotic cells, leading to a wide variety of cellular outcomes, including proliferation, differentiation, transcriptional activation, and programmed cell death. Perturbations to tyrosine kinase signaling networks by activation, overexpression, or mutation is the driving factor in many diseases, most notably cancers. The development of tyrosine kinase inhibitors, 37 of which are currently FDA-approved, has led to a revolution in cancer treatment. Imatinib, the first FDA-approved kinase inhibitor, has drastically improved prognosis for patients with Bcr-abl-positive leukemias. Despite this unprecedented success, however, up to one-third of patients lose response to imatinib due to mutations within the tyrosine kinase domain of Bcr-abl. Subsequent generations of Bcr-abl inhibitors, including dasatinib and ponatinib, have been developed to overcome these resistance mutations, but in each case, novel resistance mutations have arisen. We present a high-throughput yeast-based assay for the prediction of dasatinib- and ponatinib-resistant mutations in the ABL1 kinase domain. Our results not only recapitulate all known dasatinib-resistant mutations, but confirm recent patient data emphasizing the importance of compound mutations in ponatinib resistance. Furthermore, with hundreds of kinase inhibitors in development for the treatment of a wide range of diseases, understanding the cellular pathway of each kinase is critically important to the selection of ideal drug targets and avoiding potentially toxic side effects. Discovery of novel tyrosine kinase substrates is hindered by the presence of 90 human tyrosine kinases, which are often active in the same pathways. Phosphoproteomics, chemical genetics, and in vitro assays have been used to great success, yet only 30% of phosphorylated tyrosines in the human proteome have been assigned to a specific kinase. Recent advances in predicting tyrosine kinase substrates have been made by combining large data sets on kinase domain specificity, cellular localization, and protein-protein interactions in probabilistic algorithms. However, the high-quality data sets required for accurate predictions are often lacking. In chapter 2, we present a high-throughput yeastbased assay for screening millions of putative kinase substrates, which we then use to build a probabilistic model to accurately predict the in vitro phosphorylation of candidate substratesBiochemistr

Studies on the function of PRG2/PLPPR3 in neuron morphogenesis

Neuron development follows a multifaceted sequence of cell migration, polarisation, neurite elongation, branching, tiling, and pruning. The implementation of this sequence differs between neuronal cell types and even in individual neurons between sub-compartments such as dendrites and axons. Membrane proteins are at a prime position in neurons to couple extrinsic morphogenetic signals with their intrinsic responses to orchestrate this defined morphological progression. The Phospholipid phosphatase-related / Plasticity-related gene (PLPPR/PRG)-family comprises five neuron-enriched and developmentally regulated membrane proteins with functions in cellular morphogenesis. At the start of this project, no publication had characterised the function of PLPPR3/PRG2 during neuron development. The presented work describes PLPPR3 as an axon-enriched protein localising to the plasma membrane and internal membrane compartments of neurons. Mutagenesis studies in cell lines establish the plasma membrane localisation of PLPPR3 as a regulator of its function to increase filopodia density (Chapter 2). Furthermore, the generation of a Plppr3-/- mouse line using CRISPR/Cas9 genome editing techniques (Chapter 3) enabled characterising endogenous phenotypes of PLPPR3 in neurons. In primary neuronal cultures, PLPPR3 was found to specifically control branch formation in a pathway with the phosphatase PTEN, without altering the overall growth capacity of neurons (Chapter 4). Loss of PLPPR3 specifically reduced branches forming from filopodia without affecting the stability of branches. This precise characterisation of PLPPR3 function unravelled the existence of parallel, independent programs for branching morphogenesis that are utilised and implemented differentially in developing axons and dendrites (Chapter 5). Furthermore, this thesis establishes multiple tools to study PLPPR3, the membrane lipid phosphatidylinositol-trisphosphate, and neuron morphogenesis by providing molecular tools, protocols, and semi-automated and automated image analysis pipelines (Appendix Chapter 7) and discusses experiments to test, refine and extend models of PLPPR3 function (Chapter 6). In summary, this thesis generated and utilised several tools and a Plppr3-/- mouse model to characterise PLPPR3 as a specific regulator of neuron branching morphogenesis. This precise characterisation refined and expanded the understanding of axon-specific branching morphogenesis.Nervenzellen entwickeln ihre komplexe Morphologie durch das Zusammenwirken diverser molekularer Entwicklungs-Programme der Zellkörper-Migration, der Polarisierung und der Morphogenese durch Wachstum, Verzweigung, Stabilisierung und Koordinierung ihrer Neuriten. Dabei unterscheidet sich die exakte Implementierung zwischen Nervenzell-Typen und selbst innerhalb einzelner Zellen zwischen Axonen und Dendriten. Diese unterschiedliche Morphogenese wird dabei speziell durch Membranproteine stark beeinflusst, die durch ihre Präsenz an der Plasmamembran Zell-extrinsische Signale mit den Zell-intrinsischen Morphogeneseprogrammen verbinden und beeinflussen. Die Familie der Phospholipid phosphatase-related / Plasticity-related gene (PLPPR/PRG) Proteine umfasst fünf Nervenzell-spezifische Membranproteine mit Effekten auf die Morphologie von Zellen. Zu Beginn dieses Projektes hatte noch keine Studie die Funktion des Familienmitglieds PLPPR3/PRG2 in Nervenzellen untersucht. Diese Dissertation beschreibt die Lokalisation von PLPPR3 an der Plasmamembran und in Zell-internen Membranstrukturen von Nervenzellen. Experimente in Zellkultur zeigen eine erhöhte Filopodien-Dichte nach Überexpression von PLPPR3, Mutagenese-Studien deuten eine strikte Kontrolle der Plasmamembran-Lokalisation an (Kapitel 2). Die Generierung einer Plppr3 Knockout Mauslinie mittels CRISPR/Cas9 Genom-Modifizierung (Kapitel 3) erlaubte eine Charakterisierung der endogenen Funktion von PLPPR3 in Nervenzellen. In Primärzellkultur von Nervenzellen des murinen Hippocampus zeigte sich, dass PLPPR3 im Zusammenspiel mit der Phosphatase PTEN spezifisch die Verzweigung von Nervenzellen kontrolliert, ohne deren Wachstumspotential global zu verändern (Kapitel 4). Dadurch kann PLPPR3 als ein Schalter zwischen Verzweigung und Verlängerung eines Nervenzell-Fortsatzes agieren. Der Verlust von PLPPR3 verursachte reduzierte spezifisch die Anzahl an Verzweigungen, die aus Filopodien entstanden, ohne dabei die Stabilität dieser Verzweigungen zu beeinflussen. Die präzise Charakterisierung dieser Funktion von PLPPR3 deckte auf, dass Verzweigungen von Nervenzell-Fortsätzen durch voneinander unabhängige Entwicklungsprogramme ausgebildet und stabilisiert werden können (Kapitel 5). Diese Programme werden von Axonen und Dendriten in unterschiedlicher Weise eingesetzt. Zusätzlich etabliert diese Arbeit sowohl diverse molekulare Werkzeuge und Visualisierungs-Protokolle zur Analyse von PLPPR3 und dem Membranlipid Phosphatidylinositol-Trisphosphat, als auch automatisierte Quantifizierungssoftware zur Studie der Nervenzellmorphologie (Appendix-Kapitel 7). Abschließend entwickelt und verfeinert die Dissertation mögliche Modelle zur PLPPR3-Funktion und zeigt experimentelle Strategien auf, um diese Modelle besser charakterisieren zu können (Kapitel 6). Zusammenfassend wurden in dieser Promotionsarbeit diverse Experimental- und Analyse-Strategien und eine Plppr3-/- Mauslinie entwickelt und genutzt, um PLPPR3 als einen spezifischen Regulator der Nervenzell-Morphogenese zu etablieren. Diese präzise Charakterisierung des PLPPR3 Phänotyps erlaubte zusätzlich eine Verfeinerung und Erweiterung der Erkenntnisse zur Axon-spezifischen Entwicklung von Verzweigungen

Characterization of Chronic Lymphocytic Leukemia Progression to Richter Transformation: Patient-derived xenograft models as an evolutionary model

[eng] Chronic lymphocytic leukemia (CLL) is a prevalent form of leukemia among adults in western countries, marked by a monoclonal mature CD19+ CD5+ B cell expansion in the peripheral blood, secondary lymphoid tissues, and bone marrow. In 5-10% of cases, it transforms into Richter transformation (RT), a diffuse large B-cell lymphoma associated with a poor prognosis. However, the mechanisms responsible for driving RT are still not well understood. In this Thesis, we present novel insights into RT and a detailed development of two new patient-derived xenograft (PDX) models for RT. One of these models derived from a CLL sample, which accurately reflects the clinical transformation of CLL to RT observed in the patient's follow-up. In the first chapter, we analyzed 54 longitudinal samples using genomic, epigenomic and transcriptomic techniques that covered up to 19 years of disease progression. The genomic analysis revealed the early seeding of RT subclones that drive advanced stages of cancer evolution as early as 19 years before the final expansion. Furthermore, we identified new driver alterations and described a new transcriptional axis in RT with oxidative phosphorylation (OXPHOS)high – B-cell receptor (BCR)low signaling. The study demonstrated the effectiveness of inhibiting the proliferation of RT cells by targeting OXPHOS. In the second chapter, we established two new PDXs by injecting CLL (PDX12) and RT (PDX19) cells into immunocompromised NSG mice. Both PDXs exhibited similar morphological and phenotypic characteristics to those of RT. PDX progression analysis uncovered a genomic landscape similar to RT tumors from both patients and revealed unprecedented RT subclonal heterogeneity and clonal evolution during PDX generation. Transcriptomic analysis of PDXs demonstrated high OXPHOS and low BCR signaling that corresponded with RT levels in the patients. The OXPHOS inhibitor, IACS- 010759, decreased proliferation on these cells and circumvented resistance to venetoclax.[cat] La leucèmia limfocítica crònica (LLC) és una forma prevalent de leucèmia entre adults als països occidentals, caracteritzada per una expansió monoclonal de cèl·lules B madures CD19+ CD5+ en la sang perifèrica, teixits limfoides secundaris i medul·la òssia. En un 5-10 % dels casos, es transforma en la transformació de Richter (RT), un limfoma difús de cèl·lules B grans, associat amb un pronòstic desfavorable. No obstant això, els mecanismes responsables de la transformació en RT encara no s’entenen bé. En aquesta Tesi, presentem noves percepcions sobre la RT i detallem el desenvolupament de dos nous models de xenoempelt derivat de pacient (PDX) per a la RT. Un d’aquests models es va derivar d’una mostra de LLC, que reflecteix amb precisió la transformació clínica de la LLC a RT observada en el seguiment del pacient. En el primer capítol, vam analitzar 54 mostres longitudinals amb tècniques genòmiques, epigenòmiques i transcriptòmiques que abastaven fins a dinou anys de progressió de la malaltia. L'anàlisi genòmica va revelar la sembra primerenca de subclons de RT que impulsen estadis avançats de l'evolució del càncer fins a 19 anys abans de l'expansió final. A més, vam identificar noves alteracions i vam descriure un nou eix transcripcional en la RT amb senyalització elevada de la fosforilació oxidació (OXPHOS) i una disminució de l’activitat del receptor de cèl·lules B (BCR). L’estudi va demostrar l’eficàcia d’inhibir la proliferació de les cèl·lules RT mitjançant la inhibició d’OXPHOS. En el segon capítol, vam establir dos PDX injectant cèl·lules de LLC (PDX12) i RT (PDX19) en ratolins immunocompromesos NSG. Ambdós PDX van mostrar característiques morfològiques i fenotípiques similars a les de la RT. L’anàlisi de la progressió del PDX va descobrir un paisatge genòmic similar als tumors de RT dels pacients, ja que va revelar una gran heterogeneïtat subclonal de RT i una evolució clonal durant la generació del PDX. L’anàlisi transcriptòmica dels PDX va demostrar uns nivells d’OXPHOS elevats i una senyalització baixa de BCR que es corresponia amb el perfil d’expressió OXPHOSalt-BCRbaix en les mostres de pacients amb RT. L’inhibidor d’OXPHOS, IACS-010759, va disminuir la proliferació d’aquestes cèl·lules i va sensibilitzar aquestes cèl·lules a venetoclax

Peptide microarray of pediatric acute myeloid leukemia is related to relapse and reveals involvement of DNA damage response and repair

The majority of acute myeloid leukemia (AML) patients suffer from relapse and the exact etiology of AML remains unclear. The aim of this study was to gain comprehensive insights into the activity of signaling pathways in AML. In this study, using a high-throughput PepChip™ Kinomics microarray system, pediatric AML samples were analyzed to gain insights of active signal transduction pathway. Unsupervised hierarchical cluster analysis separated the AML blast profiles into two clusters. These two clusters were independent of patient characteristics, whereas the cumulative incidence of relapse (CIR) was significantly higher in the patients belonging to cluster-2. In addition, cluster-2 samples showed to be significantly less sensitive to various chemotherapeutic drugs. The activated peptides in cluster-1 and cluster-2 reflected the activity of cell cycle regulation, cell proliferation, cell differentiation, apoptosis, PI3K/AKT, MAPK, metabolism regulation, transcription factors and GPCRs signaling pathways. The difference between two clusters might be explained by the higher cell cycle arrest response in cluster-1 patients and higher DNA repair mechanism in cluster-2 patients. In conclusion, our study identifies different signaling profiles in pediatric AML in relation with CIR involving DNA damage response and repair