Matthias Stahl

Cancer heterogeneity at the proteome level may explain differences in therapy response and prognosis beyond the currently established genomic and transcriptomic-based diagnostics. The relevance of proteomics for disease classifications remains to be established in clinically heterogeneous cancer entities such as chronic lymphocytic leukemia (CLL). Here, we characterize the proteome and transcriptome alongside genetic and ex-vivo drug response profiling in a clinically annotated CLL discovery cohort (n = 68). Unsupervised clustering of the proteome data reveals six subgroups. Five of these proteomic groups are associated with genetic features, while one group is only detectable at the proteome level. This new group is characterized by accelerated disease progression, high spliceosomal protein abundances associated with aberrant splicing, and low B cell receptor signaling protein abundances (ASB-CLL). Classifiers developed to identify ASB-CLL based on its characteristic proteome or splicing signature in two independent cohorts (n = 165, n = 169) confirm that ASB-CLL comprises about 20% of CLL patients. The inferior overall survival in ASB-CLL is also independent of both TP53- and IGHV mutation status. Our multi-omics analysis refines the classification of CLL and highlights the potential of proteomics to improve cancer patient stratification beyond genetic and transcriptomic profiling.

The complexity of the functional proteome extends significantly beyond the protein coding genome resulting in millions of proteoforms. Investigation of proteoforms and their functional roles is important to understand cellular physiology and its deregulation in diseases, but challenging to perform systematically. Here, we apply thermal proteome profiling with deep peptide coverage to detect functional proteoforms in acute lymphoblastic leukemia cell lines with different cytogenetic aberrations. We detect 15,846 proteoforms, capturing differently spliced, post-translationally modified, and cleaved proteins expressed from 9,290 genes. We identify differential coaggregation of proteoform pairs and establish links to disease biology. Moreover, we systematically make use of measured biophysical proteoform states to find specific biomarkers of drug sensitivity. Our approach thus provides a powerful and unique tool for systematic detection and functional annotation of proteoforms.

Acute lymphoblastic leukemia (ALL) is the most common childhood cancer. Although standard-of-care chemotherapeutics are sufficient for most ALL cases, there are subsets of patients with poor response who relapse in disease. The biology underlying differences between subtypes and their response to therapy has only partially been explained by genetic and transcriptomic profiling. Here, we perform comprehensive multi-omic analyses of 49 readily available childhood ALL cell lines, using proteomics, transcriptomics, and pharmacoproteomic characterization. We connect the molecular phenotypes with drug responses to 528 oncology drugs, identifying drug correlations as well as lineage-dependent correlations. We also identify the diacylglycerol-analog bryostatin-1 as a therapeutic candidate in the MEF2D-HNRNPUL1 fusion high-risk subtype, for which this drug activates pro-apoptotic ERK signaling associated with molecular mediators of pre-B cell negative selection. Our data is the foundation for the interactive online Functional Omics Resource of ALL (FORALL) with navigable proteomics, transcriptomics, and drug sensitivity profiles at https://proteomics.se/forall .

Acute lymphoblastic leukemia (ALL) is the most common childhood cancer. Although standard-of-care chemotherapeutics are sufficient for most ALL cases, there are subsets of patients with poor response who relapse in disease. The biology underlying differences between subtypes and their response to therapy has only partially been explained by genetic and transcriptomic profiling. To characterize ALL subtypes and identify novel pharmacologic vulnerabilities, we performed comprehensive multi-omic analyses of 49 widely-used childhood ALL cell lines, using proteomics, transcriptomics, and pharmacoproteomic characterization. This enabled us to characterize the functional impact of genetic fusions and cellular differentiation states. The proteomics data revealed differences in spliceosome and p53 levels not evident in the transcriptomics data and with improved correlation to drug sensitivity. Focusing on BCP-ALL cell lines, we connected the genotype, molecular phenotype, and functional phenotype with drug response data on 528 oncology drugs. Here, we identified the DAG-analog Bryostatin-1 as a novel therapeutic candidate in the MEF2D-HNRNPUL1 fusion high-risk subtype, for which this drug activated pro-apoptotic ERK signaling associated with molecular mediators of pre-B cell negative selection. Our data also forms an interactive online resource with navigable proteomics, transcriptomics, and drug sensitivity profiles at https://lehtio-lab.se/forall/.

Legionella pneumophila infects eukaryotic cells by forming a replicative organelle – the Legionella containing vacuole. During this process, the bacterial protein DrrA/SidM is secreted and manipulates the activity and post-translational modification (PTM) states of the vesicular trafficking regulator Rab1. As a result, Rab1 is modified with an adenosine monophosphate (AMP), and this process is referred to as AMPylation. Here, we use a chemical approach to stabilise low-affinity Rab:DrrA complexes in a site-specific manner to gain insight into the molecular basis of the interaction between the Rab protein and the AMPylation domain of DrrA. The crystal structure of the Rab:DrrA complex reveals a previously unknown non-conventional Rab-binding site (NC-RBS). Biochemical characterisation demonstrates allosteric stimulation of the AMPylation activity of DrrA via Rab binding to the NC-RBS. We speculate that allosteric control of DrrA could in principle prevent random and potentially cytotoxic AMPylation in the host, thereby perhaps ensuring efficient infection by Legionella.

Die caseinolytische Protease P (ClpP) ist die proteolytische Komponente des Protein‐Abbau‐Komplexes ClpXP. Ihre genaue Funktion und Regulation sind größtenteils unerforscht. Hier stellen wir eine niedermolekulare Verbindung (D9) vor, die durch Nachahmung des natürlichen Chaperons ClpX als potenter und Spezies‐selektiver Aktivator der humanen ClpP (hClpP) wirkt. Struktur‐Aktivitäts‐Studien hoben die Wichtigkeit eines halogenierten Benzyl‐Motivs innerhalb von D9 hervor. Dieses interagiert mit einem einzigartigen aromatischen Aminosäure‐Netzwerk in hClpP. Mutations‐ und Strukturstudien legen nahe, dass dieses sogenannte YYW‐Motiv die hClpP‐Aktivität streng kontrolliert und den Substratumsatz durch Interaktion mit passenden Liganden reguliert. Ferner ist dieses Motiv besonders für ClpP höherer Organismen charakteristisch und existiert nicht in getesteten bakteriellen Homologen. Dies erlaubt eine Spezies‐selektive Analyse, womit D9 ein vielseitiges Werkzeug für die Untersuchung von mechanistischen Eigenschaften der hClpP werden kann.

Caseinolytic protease P (ClpP) is the proteolytic component of the ClpXP protein degradation complex. Eukaryotic ClpP was recently found to act within the mitochondria‐specific unfolded protein response (UPR^mt). However, its detailed function and dedicated regulation remain largely unexplored. A small molecule (D9) acts as a potent and species‐selective activator of human ClpP (hClpP) by mimicking the natural chaperone ClpX. Structure–activity relationship studies highlight the importance of a halogenated benzyl motif within D9 that interacts with a unique aromatic amino acid network in hClpP. Mutational and structural studies suggest that this YYW motif tightly controls hClpP activity and regulates substrate turnover by interaction with cognate ligands. This signature motif is unique to ClpP from higher organisms and does not exist in tested bacterial homologues, allowing a species‐selective analysis. Thus, D9 is a versatile tool to analyze mechanistic features of hClpP.

Caseinolytic proteases (ClpP) are important for recognition and controlled degradation of damaged proteins. While the majority of bacterial organisms utilize only a single ClpP, Listeria monocytogenes expresses two isoforms (LmClpP1 and LmClpP2). LmClpPs assemble into either a LmClpP2 homocomplex or a LmClpP1/2 heterooligomeric complex. The heterocomplex in association with the chaperone ClpX, exhibits a boost in proteolytic activity for unknown reasons. Here, we use a combined chemical and biochemical strategy to unravel two activation principles of LmClpPs. First, determination of apparent affinity constants revealed a 7-fold elevated binding affinity between the LmClpP1/2 heterocomplex and ClpX, compared to homooligomeric LmClpP2. This tighter interaction favors the formation of the proteolytically active complex between LmClpX and LmClpP1/2 and thereby accelerating the overall turnover. Second, screening a diverse library of fluorescent labeled peptides and proteins with various ClpP mutants allowed the individual analysis of substrate preferences for both isoforms within the heterocomplex. In addition to Leu and Met, LmClpP2 preferred a long aliphatic chain (2-Aoc) in the P1 position for cleavage. Strikingly, design and synthesis of a corresponding 2-Aoc chloromethyl ketone inhibitor resulted in stimulation of proteolysis by 160% when LmClpP2 was partially alkylated on 20% of the active sites. Determination of apparent affinity constants also revealed an elevated complex stability between partially modified LmClpP2 and the cognate chaperone LmClpX. Thus, the stimulation of proteolysis through enhanced binding to the chaperone seems to be a characteristic feature of LmClpPs.