Biology subjects

Pichler, A.

Publications and source records attributed to Pichler, A..

7 recordsLinked to original sources

bioRxiv2025

A STAT5B-driven mouse model of hepatosplenic γδ T-cell lymphoma reveals therapeutic efficacy of JAK inhibition

Hepatosplenic T-cell lymphoma (HSTCL) is a rare and aggressive neoplasm associated with poor responses to standard chemotherapy regimens and low survival rates. No targeted therapies are available for HSTCL, and preclinical models to test new treatment options have not been established. The JAK-STAT signaling cascade is a key dysregulated pathway in HSTCL, and STAT5BN642His the most frequent somatic mutation in the disease. Here, we report on newly established clonal, murine {gamma}{delta} T-cell lymphoma cell lines initiated and driven by oncogenic STAT5BN642H, which recapitulate key immunophenotypic features, gene expression profiles and typically low cytolytic activity of patient-derived human HSTCL cells. CRISPR-Cas9 mediated knockout demonstrated growth dependence on STAT5BN642H. Murine C15 cells were allo-engrafted intravenously into both immunodeficient and immunocompetent mice to model an aggressive HSTCL-like disease at high penetrance, with recipient mice displaying hepatosplenomegaly and destructive {gamma}{delta} T cell organ infiltration, including bone marrow and blood involvement. We identified the potential of JAK inhibition as a targeted treatment strategy for HSTCL, and found the clinically approved JAK inhibitor upadacitinib to display selective anti-tumor efficacy against STAT5B-mutated HSTCL cell lines in vitro, in vivo, and in primary HSTCL patient samples. Overall, we describe the first robust STAT5B-driven preclinical model resembling features of HSTCL in an immune competent setting. This tool is expected to accelerate the study of HSTCL disease mechanisms and the testing of novel therapies. Our data further present the JAK inhibitor upadacitinib as a promising targeted treatment option for STAT5B-mutated HSTCL.

cancer biology↗

bioRxiv2022

STAT5 Gain-of-Function Variants Promote Precursor T-Cell Receptor Activation to Drive T-Cell Acute Lymphoblastic Leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive immature T-cell cancer. Hotspot mutations in JAK-STAT pathway members IL7R, JAK1 and JAK3 were analyzed in depth. However, the role of STAT5A or STAT5B mutations promoting their hyperactivation is poorly understood in the context of T-cell cancer initiation and acute leukemia progression. Importantly, the driver mutation STAT5BN642H encodes the most frequent activating STAT5 variant in T-ALL associated with poor prognosis. Here, we show that hyperactive STAT5 promotes early T-cell progenitor (ETP)-ALL-like cancer in mice and upregulated genes involved in T-cell receptor signaling (TCR), even in absence of surface TCR promoting. Importantly, these genes were also overexpressed in human T-ALL and other STAT5-dependent T-cell cancers. Moreover, human T-ALL cells were sensitive to pharmacologic inhibition by dual STAT3/5 degraders or ZAP70 tyrosine kinase blockers. Thus, we define STAT5 target genes in T-ALL that promote pre-TCR signaling mimicry. We propose therapeutic targeting using selective ZAP70 or STAT3/5 inhibitors in a subgroup of T-ALL patients with prominent IL-7R-JAK1/3-STAT5 activity. SignificanceWe provide detailed functional characterizations of hyperactive STAT5A or STAT5B in thymic T-cell development and transformation. We found that hyperactive STAT5 transcribes T-cell-specific kinases or pre-TCR signaling hubs to promote T-ALL. Biomolecular and next-generation-sequencing methods, transgenesis and pharmacologic interference revealed that hyperactive STAT5 is a key oncogenic driver that can be targeted in T-ALL using STAT3/5 or SYK family member tyrosine kinase inhibitors. Conflict of interestThe authors declare no potential conflicts of interest.

cancer biology↗

bioRxiv2022

SUMO Activated Target Traps (SATTs) enable the identification of a comprehensive E3-specific SUMO proteome.

Ubiquitin and ubiquitin-like conjugation cascades consist of dedicated E1, E2 and E3 enzymes with E3s providing substrate specificity. Mass spectrometry-based approaches have enabled the identification of more than 60,000 acceptor sites for ubiquitin and 40,000 acceptor sites for SUMO2/3. However, E3-to-target wiring is poorly understood. The limited number of SUMO E3s provides the unique opportunity to systematically study E3-substrate wiring. We developed SUMO Activated Target Traps (SATTs) and systematically identified substrates for eight different SUMO E3s, PIAS1, PIAS2, PIAS3, PIAS4, NSMCE2, ZNF451, LAZSUL(ZNF451-3) and ZMIZ2. SATTs enabled us to identify 590 SUMO1 and 1195 SUMO2/3 targets in an E3-specific manner. We found pronounced E3 substrate preference, even at the substrate isoform level. Quantitative proteomics enabled us to measure substrate specificity of E3s, quantified using the SATT index. Furthermore, we developed the Polar SATTs web-based tool (https://amsterdamstudygroup.shinyapps.io/PolaRVolcaNoseR/) to browse the dataset in an interactive manner, increasing the accessibility of this resource for the community. Overall, we uncover E3-to-target wiring of 1681 SUMO substrates, highlighting unique and overlapping sets of substrates for eight different SUMO E3 ligases.

molecular biology↗

bioRxiv2022

UBC9 and EME1 sumoylation foster ribosomal DNA damage repair in CPT response

UBC9 sumoylation (S*UBC9) in mammals contributes to SUMO target discrimination, biochemically1. Here, we present biological insights by characterizing a sumoylation mimetic UBC9-fusion (mCS~UBC9) in comparison to its wild-type (mCUBC9). We observe that sumoylation promotes UBC9s stability, nuclear localization and is beneficial for cell survival. We identified EME1, the regulatory subunit of the structure-specific endonuclease EME1-MUS81, as S*UBC9 substrate and demonstrate EME1 and UBC9 sumoylation being advantageous for survival upon Camptothecin (CPT) exposure. Moreover, mCS~UBC9 expression enhances double-strand breaks (DSBs) and replication upon CPT treatment, features assigned to the EME1-MUS81 complex2. EME1 and mCS~UBC9 co-localize in nucleolar repair-condensates, that enlarge and round when exposed to the drug. Together, these findings imply that S*UBC9-induced EME1 sumoylation improves ribosomal rDNA repair, which might prevent dangerous second template switches in highly repetitive ribosomal chromatin and allow the converging replication fork to complete DNA replication. Finally, we discuss implications of these findings for anti-cancer therapy.

molecular biology↗

bioRxiv2022

Multi-level monitoring of EME1-MUS81 in CPT induced nucleolar rDNA repair

We identified EME1, the regulatory subunit of the structure-specific endonuclease complex EME1-MUS81, as substrate for the sumoylated UBC9 and demonstrated synergistic functions in promoting Camptothecin (CPT)-induced nucleolar ribosomal DNA (rDNA) repair (Nagamalleswari et al, co-submitted). Sumoylation of EME1 appears complex involving mono- and poly-sumoylation. Hence, we addressed here whether these modifications differentially regulate EME1 functions by analyzing EME1-variant expressing cell lines including mono-sumo(1)ylation and poly-sumo(2)ylation mimetic fusions. We complemented our analysis with the regulated endogenous EME1 interactome and our observation that Trichostatin A (TSA) induced EME1 and UBC9 sumoylation. Our findings are substantiated by identifying several regulatory proteins and by detecting CPT-induced endogenous di- and poly-sumoylated EME1 in different cell fractions. Together, our data suggest that Histone H4 acetylation, two mono-sumoylation events, poly-sumoylation, ISG15 and ubiquitination sequentially and in mutual dependence tightly control the intracellular localization, recruitment to DNA lesions, enzymatic activity, withdrawal from DNA lesions and the stability of EME1.

molecular biology↗

bioRxiv2020

Core binding factor leukemia hijacks T-cell prone PU.1 antisense promoter

Summary paragraphThe blood system serves as a key model for cell differentiation and cancer. It is orchestrated by precise spatiotemporal expression of the hematopoietic master regulator PU.11-4. PU.1 gene expression is regulated through enhancer-promoter interactions within a topologically associated domain (TAD)5,6. PU.1 levels increase during myeloid differentiation while failure to do so results in myeloid leukemia7. In contrast, T-cell differentiation requires PU.1 to be completely switched off8-10. Little is known about the precise mechanisms of PU.1 repression, physiological as in T-cell differentiation, or pathological as in leukemia. Here we demonstrate that the down-regulation of PU.1 mRNA is a dynamic process involving an alternative promoter11 in intron 3 that is induced by RUNX transcription factors driving noncoding antisense transcription. Core binding factor (CBF) fusions, RUNX1-ETO and CBF{beta}-MYH11 in t(8;21) and inv(16) acute myeloid leukemia (AML)12, activate the PU.1 antisense promoter, thus shifting from sense towards antisense transcription and blocking myeloid differentiation. In patients with CBF-AML, we found that an elevated antisense/sense ratio represents a hallmark compared to normal karyotype AML or healthy CD34+ cells. Competitive interaction of the enhancer with the proximal or the antisense promoter are at the heart of differential PU.1 expression during myeloid and T-cell development. Leukemic CBF fusions thus utilize a physiologic mechanism employed by T-cells to decrease sense PU.1 transcription. Our results identify the first example of a sense/antisense promoter competition as a crucial functional switch for gene expression perturbation by oncogenes. This novel basic disease mechanism reveals a previously unknown Achilles heel for future precise therapeutic targeting of oncogene-induced chromatin remodeling.

cancer biology↗

bioRxiv2019

Physiological and pathophysiological homeostasis of astroglial channel proteins by Nedd4-2

Nedd4-2 is an E3 ubiquitin ligase, missense mutation of which is related to familial epilepsy, indicating its critical role in regulating neuronal network activity. However, Nedd4-2 substrates involved in neuronal network function have yet to be identified. Using mouse lines lacking Nedd4-1 and Nedd4-2, we identified astrocytic channel proteins inwardly rectifying K+ channel 4.1 (Kir4.1) and Connexin43 as Nedd4-2 substrates. We found that the expression of Kir4.1 and Connexin43 is increased upon conditional deletion of Nedd4-2 in astrocytes, leading to an elevation of astrocytic membrane ion permeability and gap junction activity, with a consequent reduction of {gamma}-oscillatory neuronal network activity. Interestingly, our biochemical data demonstrate that missense mutations found in familial epileptic patients produce gain-of-function of Nedd4-2 gene product. Our data reveal a process of coordinated astrocytic ion channel proteostasis that controls astrocyte function and astrocyte-dependent neuronal network activity, and elucidate a potential mechanism by which aberrant Nedd4-2 function leads to epilepsy.\n\nCONDENSED TITLEUbiquitination of Astrocytic Channels by Nedd4-2\n\nSUMMARYUbiquitination is a key regulatory machinery for protein expression. Our present work provides evidence that astrocytic ion channel proteostasis coordinated by an E3 ubiquitin ligase Nedd4-2 is of particular importance for the maintenance of neuronal network activity.

neuroscience↗