Nucleoporin 98-rearranged (NUP98-r) acute myeloid leukemia (AML) is associated with poor outcomes and remains a major therapeutic challenge due to the absence of strategies that directly eliminate NUP98 fusion oncoproteins. Targeted degradation of cancer-driving oncofusions is an attractive approach, but the molecular mechanisms controlling NUP98 oncofusion stability are unknown. Using a CRISPR-Cas9 screen, we identify the E3 ligase Speckle-type POZ protein (SPOP) as a direct regulator of NUP98 fusion oncoprotein stability and a novel tumor suppressor in NUP98-r AML. Loss of SPOP increases NUP98 oncofusion levels and promotes leukemia cell proliferation. Exploiting this specificity, we demonstrate that induced proximity of SPOP and NUP98::lysine-specific demethylase 5A (KDM5A) through a biological proteolysis-targeting chimera (bioPROTAC) induces full clearance of the fusion oncoprotein, driving terminal differentiation and apoptosis of NUP98-r leukemia cells in vitro and in vivo. This study identifies SPOP as a direct regulator of NUP98 oncofusion stability and outlines a strategy to redirect the ubiquitin-proteasome system against oncogenic fusions.

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 STAT5BN642H is the most frequent somatic mutation in the disease. Here, we report on newly established clonal, murine γδ 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 γδ 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. ### Competing Interest Statement The authors have declared no competing interest. FWF Austrian Science Fund, https://ror.org/013tf3c58 Austrian Academy of Sciences, https://ror.org/03anc3s24

ABSTRACT:NUP98::NSD1 is one of the most recurring nucleoporin 98 (NUP98) fusions in acute myeloid leukemia (AML). NUP98::NSD1 positive AML is often associated with adverse outcomes and poor response to conventional treatments. However, limited studies have been done to identify new potential targets to develop better treatment approaches. The C-type lectin domain family 12 member A (CLEC12A) is a cell surface receptor that is differentially expressed in leukemic stem cells compared with healthy hematopoietic stem cells. We found a strong CLEC12A overexpression in both NUP98::NSD1 patients and murine AML cells transformed with the NUP98::NSD1 fusion oncogene. To understand the role of Clec12a in NUP98::NSD1 AML, we depleted Clec12a expression in NUP98::NSD1+NRASG12D-immortalized cells using the CRISPR/Cas9 approach. NUP98::NSD1+NRASG12D/Clec12a knockout cells had higher apoptosis levels and lower colony numbers in vitro compared with NUP98::NSD1+NRASG12D/Clec12a wild-type cells. Importantly, the deletion of Clec12a significantly reduced leukemic engraftment and prolonged survival of the NUP98::NSD1+NRASG12D murine model. Our data suggest to further explore CLEC12A as a potential target for the treatment of NUP98::NSD1 AML.

Cystoisospora suis, a member of the apicomplexan order Coccidia and causative agent of neonatal porcine coccidiosis, poses a challenge to pig production due to the emergence of reduced efficacy of toltrazuril, the only EU-approved treatment. To address the critical gaps in understanding toltrazuril resistance and possibilities of early diagnostics, our study investigated the genetic basis of resistance through whole-genome DNA sequencing and transcriptome analysis of two C. suis strains, the toltrazuril-susceptible Wien-I and the resistant Holland-I. Additionally, we studied the mitochondrial genome and analysed mitochondrial gene expression in both strains. Our results show that genes encoding proteins involved in host-cell invasion displayed variable expression patterns and genetic mutations, suggesting adaptive changes in invasion mechanisms. Moreover, substantial fluctuations in the expression of genes linked to retrotransposons, accompanied by genetic alterations, were observed, highlighting their potential involvement in genomic rearrangements. Finally, our mitochondrial genome analyses revealed important insights into its genetic organization and conservation. Notably, the marked downregulation of CoI, CoIII and Cytb mRNA levels in the resistant strain Holland-I upon toltrazuril exposure highlights the dynamic response of mitochondrial genes to toltrazuril. These mitochondrial adaptations appear to be closely linked to the parasite drug resistance mechanism, potentially facilitating its survival under pharmacological stress. These findings enhance our knowledge of drug resistance mechanisms in Coccidia and highlight the need for novel management strategies, leading to the development of targeted treatments and controls.

Nucleoporin 98 (NUP98) fusion oncoproteins are strong drivers of pediatric acute myeloid leukemia (AML) with poor prognosis. Here we show that NUP98 fusion-expressing AML harbors an epigenetic signature that is characterized by increased accessibility of hematopoietic stem cell genes and enrichment of activating histone marks. We employ an AML model for ligand-induced degradation of the NUP98::KDM5A fusion oncoprotein to identify epigenetic programs and transcriptional targets that are directly regulated by NUP98::KDM5A through CUT&Tag and nascent RNA-seq. Orthogonal genome-wide CRISPR/Cas9 screening identifies 12 direct NUP98::KDM5A target genes, which are essential for AML cell growth. Among these, we validate cyclin-dependent kinase 12 (CDK12) as a druggable vulnerability in NUP98::KDM5A-expressing AML. In line with its role in the transcription of DNA damage repair genes, small-molecule-mediated CDK12 inactivation causes increased DNA damage, leading to AML cell death. Altogether, we show that NUP98::KDM5A directly regulates a core set of essential target genes and reveal CDK12 as an actionable vulnerability in AML with oncogenic NUP98 fusions.

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive immature T cell cancer. Mutations in IL7R have been analyzed genetically, but downstream effector functions such as STAT5A and STAT5B hyperactivation are poorly understood. Here, we studied the most frequent and clinically challenging STAT5BN642H driver in T cell development and immature T cell cancer onset and compared it with STAT5A hyperactive variants in transgenic mice. Enhanced STAT5 activity caused disrupted T cell development and promoted an early T cell progenitor-ALL phenotype, with upregulation of genes involved in T cell receptor (TCR) signaling, even in absence of surface TCR. Importantly, TCR pathway genes were overexpressed in human T-ALL and mature T cell cancers and activation of TCR pathway kinases was STAT5 dependent. We confirmed STAT5 binding to these genes using ChIP-Seq analysis in human T-ALL cells, which were sensitive to pharmacologic inhibition by dual STAT3/5 degraders or ZAP70 tyrosine kinase blockers in vitro and in vivo. We provide genetic and biochemical proof that STAT5A and STAT5B hyperactivation can initiate T-ALL through TCR pathway hijacking and suggest similar mechanisms for other T cell cancers. Thus, STAT5 or TCR component blockade are targeted therapy options, particularly in patients with chemoresistant clones carrying STAT5BN642H.

Post-transcriptional mechanisms are fundamental safeguards of progenitor cell identity and are often dysregulated in cancer. Here, we identified regulators of P-bodies as crucial vulnerabilities in acute myeloid leukaemia (AML) through genome-wide CRISPR screens in normal and malignant haematopoietic progenitors. We found that leukaemia cells harbour aberrantly elevated numbers of P-bodies and show that P-body assembly is crucial for initiation and maintenance of AML. Notably, P-body loss had little effect upon homoeostatic haematopoiesis but impacted regenerative haematopoiesis. Molecular characterization of P-bodies purified from human AML cells unveiled their critical role in sequestering messenger RNAs encoding potent tumour suppressors from the translational machinery. P-body dissolution promoted translation of these mRNAs, which in turn rewired gene expression and chromatin architecture in leukaemia cells. Collectively, our findings highlight the contrasting and unique roles of RNA sequestration in P-bodies during tissue homoeostasis and oncogenesis. These insights open potential avenues for understanding myeloid leukaemia and future therapeutic interventions.

Introduction Clonal hematopoiesis with mono-allelic TP53 mutations confers a high-risk for progression to myeloid neoplasms, particularly in the context of exposure to cytotoxic stress. During leukemic transformation, bi-allelic TP53 aberrations frequently evolve - however, the exact molecular mechanisms driving transformation are not well understood. Here, we use genetically engineered human HSPCs to investigate the impact of mono- and bi-allelic TP53 aberrations on HSPC function and genomic integrity. Methods Cord blood derived HSPCs were engineered using a CRISPR-Cas9 and adeno-associated viral vector-mediated knock-in strategy to generate TP53R175H/WT, TP53R175H/KO, TP53R273H/WT, TP53R273H/KO, TP53KO/WT and TP53KO/KO aberrations, respectively. To control for gene editing, the AAVS1 safe harbor locus was targeted. To understand the functional impact of different TP53 allelic states, cell cycle analysis, colony formation and serial replating as well as bulk mRNA sequencing were performed. The emergence of structural chromosomal variants was investigated by single-cell template strand sequencing upon doxorubicin exposure. Results Successful editing of HSPCs at the TP53 locus was confirmed at the genomic DNA, RNA and protein levels, respectively. Bi-allelic TP53 aberrant HSPCs showed an increase of cells in S-phase and formed significantly more colonies with a high proportion of BFU-E as compared to control cells. Mono-allelic TP53 aberrant cells lost their replating capacity after three passages whereas bi-allelic ones were not exhausted after five replatings. Transcriptome analysis revealed the induction of an inflammatory signature in cells with both, mono-and bi-allelic TP53 aberrations, with a skewed expression rate of erythroid and myeloid genes. Single-cell template strand sequencing indicated that mono-allelic TP53 aberrations, especially in conjunction with doxorubicin treatment, already induced a substantial increase of structural genomic variants including chromothripsis. Conclusions Our data indicate that already mono-allelic TP53 aberrations induce an inflammatory signature and structural chromosomal alterations in human HSPCs. Alterations of the second TP53 allele increase the proliferative and self-renewing capacity of HSPCs further promoting their leukemic transformation and progression. These data contributes towards a better understanding of human, TP53 aberrant leukemogenesis.

The transcription factors STAT3, STAT5A, and STAT5B steer hematopoiesis and immunity, but their enhanced expression and activation promote acute myeloid leukemia (AML) or natural killer/T cell lymphoma (NKCL). Current therapeutic strategies focus on blocking upstream tyrosine kinases to inhibit STAT3/5, but these kinase blockers are not selective against STAT3/5 activation and frequent resistance causes relapse, emphasizing the need for targeted drugs. We evaluated the efficacy of JPX-0700 and JPX-0750 as dual STAT3/5 binding inhibitors promoting protein degradation. JPX-0700/-0750 decreased the mRNA and protein levels of STAT3/5 targets involved in cancer survival, metabolism, and cell cycle progression, exhibiting nanomolar to low micromolar efficacy. They induced cell death and growth arrest in both AML/NKCL cell lines and primary AML patient blasts. We found that both AML/NKCL cells hijack STAT3/5 signaling through either upstream activating mutations in kinases, activating mutations in STAT3, mutational loss of negative STAT regulators, or genetic gains in anti-apoptotic, pro-proliferative, or epigenetic-modifying STAT3/5 targets. This emphasizes a vicious cycle for proliferation and survival through STAT3/5. Both JPX-0700/-0750 treatment reduced leukemic cell growth in human AML or NKCL xenograft mouse models significantly, being well tolerated by mice. Synergistic cell death was induced upon combinatorial use with approved chemotherapeutics in AML/NKCL cells.

Signal transducer and activator of transcription 3 (STAT3) is frequently overexpressed in patients with acute myeloid leukemia (AML). STAT3 exists in two distinct alternatively spliced isoforms, the full-length isoform STAT3α and the C-terminally truncated isoform STAT3β. While STAT3α is predominantly described as an oncogenic driver, STAT3β has been suggested to act as a tumor suppressor. To elucidate the role of STAT3β in AML, we established a mouse model of STAT3β-deficient, MLL-AF9-driven AML. STAT3β deficiency significantly shortened survival of leukemic mice confirming its role as a tumor suppressor. Furthermore, RNA sequencing revealed enhanced STAT1 expression and interferon (IFN) signaling upon loss of STAT3β. Accordingly, STAT3β-deficient leukemia cells displayed enhanced sensitivity to blockade of IFN signaling through both an IFNAR1 blocking antibody and the JAK1/2 inhibitor Ruxolitinib. Analysis of human AML patient samples confirmed that elevated expression of IFN-inducible genes correlated with poor overall survival and low STAT3β expression. Together, our data corroborate the tumor suppressive role of STAT3β in a mouse model in vivo. Moreover, they provide evidence that its tumor suppressive function is linked to repression of the STAT1-mediated IFN response. These findings suggest that the STAT3β/α mRNA ratio is a significant prognostic marker in AML and holds crucial information for targeted treatment approaches. Patients displaying a low STAT3β/α mRNA ratio and unfavorable prognosis could benefit from therapeutic interventions directed at STAT1/IFN signaling.

ABSTRACT The myeloid transcription factor CEBPA is recurrently biallelically mutated (i.e., double mutated; CEBPA DM ) in acute myeloid leukemia (AML) with a combination of hypermorphic N-terminal mutations ( CEBPA NT ), promoting expression of the leukemia-associated p30 isoform, and amorphic C-terminal mutations. The most frequently co-mutated genes in CEBPA DM AML are GATA2 and TET2 , however the molecular mechanisms underlying this co-mutational spectrum are incomplete. By combining transcriptomic and epigenomic analyses of CEBPA - TET2 co-mutated patients with models thereof, we identify GATA2 as a conserved target of the CEBPA - TET2 mutational axis, providing a rationale for the mutational spectra in CEBPA DM AML. Elevated CEBPA levels, driven by CEBPA NT , mediate recruitment of TET2 to the Gata2 distal hematopoietic enhancer thereby increasing Gata2 expression. Concurrent loss of TET2 in CEBPA DM AML induces a competitive advantage by increasing Gata2 promoter methylation, thereby rebalancing GATA2 levels. Of clinical relevance, demethylating treatment of Cebpa-Tet2 co-mutated AML restores Gata2 levels and prolongs disease latency.

Gain-of-function mutations in the signal transducer and activator of transcription 3 (STAT3) gene are recurrently identified in patients with large granular lymphocytic leukemia (LGLL) and in some cases of natural killer (NK)/T-cell and adult T-cell leukemia/lymphoma. To understand the consequences and molecular mechanisms contributing to disease development and oncogenic transformation, we developed murine hematopoietic stem and progenitor cell models that express mutated STAT3Y640F. These cells show accelerated proliferation and enhanced self-renewal potential. We integrated gene expression analyses and chromatin occupancy profiling of STAT3Y640F-transformed cells with data from patients with T-LGLL. This approach uncovered a conserved set of direct transcriptional targets of STAT3Y640F. Among these, strawberry notch homolog 2 (SBNO2) represents an essential transcriptional target, which was identified by a comparative genome-wide CRISPR/Cas9-based loss-of-function screen. The STAT3-SBNO2 axis is also present in NK-cell leukemia, T-cell non-Hodgkin lymphoma, and NPM-ALK-rearranged T-cell anaplastic large cell lymphoma (T-ALCL), which are driven by STAT3-hyperactivation/mutation. In patients with NPM-ALK+ T-ALCL, high SBNO2 expression correlates with shorter relapse-free and overall survival. Our findings identify SBNO2 as a potential therapeutic intervention site for STAT3-driven hematopoietic malignancies.

The myeloid transcription factor CEBPA is recurrently biallelically mutated (i.e., double mutated; CEBPA DM ) in acute myeloid leukemia (AML) with a combination of hypermorphic N-terminal mutations ( CEBPA NT ), promoting expression of the leukemia-associated p30 isoform, and amorphic C-terminal mutations. The most frequently co-mutated genes in CEBPA DM AML are GATA2 and TET2 , however the molecular mechanisms underlying this co-mutational spectrum are incomplete. By combining transcriptomic and epigenomic analyses of CEBPA - TET2 co-mutated patients with models thereof, we identify GATA2 as a conserved target of the CEBPA - TET2 mutational axis, providing a rationale for the mutational spectra in CEBPA DM AML. Elevated CEBPA levels, driven by CEBPA NT , mediate recruitment of TET2 to the Gata2 distal hematopoietic enhancer thereby increasing Gata2 expression. Concurrent loss of TET2 in CEBPA DM AML induces a competitive advantage by increasing Gata2 promoter methylation, thereby rebalancing GATA2 levels. Of clinical relevance, demethylating treatment of Cebpa-Tet2 co-mutated AML restores Gata2 levels and prolongs disease latency.

Zusammenfassung Protocol Analysis (Ericsson und Simon 1993) ist eine Methode zum Gewinnen und Aufzeichnen von verbalen Protokollen. Versuchsleiter fertigen diese Protokolle aus den Verbalisierungen an, die die Versuchspersonen als so genanntes »Think aloud« zeitgleich zu Denkleistungen im Rahmen bestimmter Aufgaben äußern. Der Vorteil dieser Methode sei es, dass sie sich von introspektiven Berichten von Versuchspersonen unterscheide, die mit den bekannten Schwierigkeiten zu kämpfen haben: introspektive Berichte seien nicht realitätsgetreu (Konfabulationen) und durch die Selbstbeobachtung würden die zugrundeliegenden Denkprozesse verfälscht. Der vorliegende Beitrag befragt kritisch die theoretischen und methodischen Voraussetzungen von introspektiven Berichten und von Protokollen, die im Rahmen von »Protocol Analysis« erstellt werden. Der Beitrag endet mit einem Ausblick auf die Anwendbarkeit von Methoden der »Protocol Analysis« zur Beforschung von Rezeptionsprozessen beim Lesen und Verstehen von literarischen Texten.