JAK-STAT signaling maintains homeostasis in T cells and macrophages.

Immune cells need to sustain a state of constant alertness over a lifetime. Yet, little is known about the regulatory processes that control the fluent and fragile balance that is called homeostasis. Here we demonstrate that JAK-STAT signaling, beyond its role in immune responses, is a major regulator of immune cell homeostasis. We investigated JAK-STAT-mediated transcription and chromatin accessibility across 12 mouse models, including knockouts of all STAT transcription factors and of the TYK2 kinase. Baseline JAK-STAT signaling was detected in CD8+ T cells and macrophages of unperturbed mice-but abrogated in the knockouts and in unstimulated immune cells deprived of their normal tissue context. We observed diverse gene-regulatory programs, including effects of STAT2 and IRF9 that were independent of STAT1. In summary, our large-scale dataset and integrative analysis of JAK-STAT mutant and wild-type mice uncovered a crucial role of JAK-STAT signaling in unstimulated immune cells, where it contributes to a poised epigenetic and transcriptional state and helps prepare these cells for rapid response to immune stimuli.

Hyperactive STAT5 hijacks T cell receptor signaling and drives immature T cell acute lymphoblastic leukemia.

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.

A lineage-specific STAT5BN642H mouse model to study NK-cell leukemia.

Patients with T- and NK-cell neoplasms frequently have somatic STAT5B gain-of-function mutations. The most frequent STAT5B mutation is STAT5BN642H, which is known to drive murine T-cell leukemia although its role in NK-cell malignancies is unclear. Introduction of the STAT5BN642H mutation into human NK-cell lines enhances their potential to induce leukemia in mice. We have generated a mouse model that enables tissue-specific expression of STAT5BN642H and have selectively expressed the mutated STAT5B in hematopoietic cells (N642Hvav/+) or exclusively in NK cells (N642HNK/NK). All N642Hvav/+ mice rapidly develop an aggressive T-/NK T-cell leukemia, whereas N642HNK/NK mice display an indolent NK-large granular lymphocytic leukemia (NK-LGLL) that progresses to an aggressive leukemia with age. Samples from NK-cell leukemia patients have a distinctive transcriptional signature driven by mutant STAT5B, which overlaps with that of murine leukemic N642HNK/NK NK cells. We have generated the first reliable STAT5BN642H-driven pre-clinical mouse model that displays an indolent NK-LGLL progressing to aggressive NK-cell leukemia. This novel in vivo tool will enable us to explore the transition from an indolent to an aggressive disease and will thus permit the study of prevention and treatment options for NK-cell malignancies.

Electrophilic MiniFrags Revealed Unprecedented Binding Sites for Covalent HDAC8 Inhibitors.

Screening of ultra-low-molecular weight ligands (MiniFrags) successfully identified viable chemical starting points for a variety of drug targets. Here we report the electrophilic analogues of MiniFrags that allow the mapping of potential binding sites for covalent inhibitors by biochemical screening and mass spectrometry. Small electrophilic heterocycles and their N-quaternized analogues were first characterized in the glutathione assay to analyze their electrophilic reactivity. Next, the library was used for systematic mapping of potential covalent binding sites available in human histone deacetylase 8 (HDAC8). The covalent labeling of HDAC8 cysteines has been proven by tandem mass spectrometry measurements, and the observations were explained by mutating HDAC8 cysteines. As a result, screening of electrophilic MiniFrags identified three potential binding sites suitable for the development of allosteric covalent HDAC8 inhibitors. One of the hit fragments was merged with a known HDAC8 inhibitor fragment using different linkers, and the linker length was optimized to result in a lead-like covalent inhibitor.

Inhibitor Library Screening of SH2 Domains Through Denaturation-Based Assays.

Screening of inhibitor libraries for candidate ligands is an important step in the drug discovery process. Thermal denaturation-based screening strategies are built on the premise that a protein-ligand complex has an altered stability profile compared to the protein alone. As such, these assays provide an accessible and rapid methodology for stratifying ligands that directly engage with the protein target of interest. Here, we describe three denaturation-based strategies for examining protein-inhibitor binding, in the context of SH2 domains. This includes conventional dye-based Thermal Shift Assays (TSA), nonconventional labeled ligand-based TSA, and Cellular Thermal Shift Assays (CETSA). Conventional dye-based TSA reports on the fluorescence of an external hydrophobic dye as it interacts with heat-exposed nonpolar protein surfaces as the temperature is incrementally increased. By contrast, nonconventional-labeled ligand TSA involves a fluorescence-tagged probe (phosphopeptide for SH2 domains) that is quenched as it dissociates from the protein during the denaturation process. CETSA involves monitoring the presence of the protein via Western blotting as the temperature is increased. In all three approaches, performing the assay in the presence of a candidate ligand can alter the melting profile of the protein. These assays offer primary screening tools to examine SH2 domain inhibitors libraries with varying chemical motifs, and a subset of the advantages and limitations of each approach is also discussed.

Medicinal chemistry advances in targeting class I histone deacetylases.

Histone deacetylases (HDACs) are a class of zinc (Zn)-dependent metalloenzymes that are responsible for epigenetic modifications. HDACs are largely associated with histone proteins that regulate gene expression at the DNA level. This tight regulation is controlled by acetylation [via histone acetyl transferases (HATs)] and deacetylation (via HDACs) of histone and non-histone proteins that alter the coiling state of DNA, thus impacting gene expression as a downstream effect. For the last two decades, HDACs have been studied extensively and indicated in a range of diseases where HDAC dysregulation has been strongly correlated with disease emergence and progression-most prominently, cancer, neurodegenerative diseases, HIV, and inflammatory diseases. The involvement of HDACs as regulators in these biochemical pathways established them as an attractive therapeutic target. This review summarizes the drug development efforts exerted to create HDAC inhibitors (HDACis), specifically class I HDACs, with a focus on the medicinal chemistry, structural design, and pharmacology aspects of these inhibitors.

ABCC1 and glutathione metabolism limit the efficacy of BCL-2 inhibitors in acute myeloid leukemia.

The BCL-2 inhibitor Venetoclax is a promising agent for the treatment of acute myeloid leukemia (AML). However, many patients are refractory to Venetoclax, and resistance develops quickly. ATP-binding cassette (ABC) transporters mediate chemotherapy resistance but their role in modulating the activity of targeted small-molecule inhibitors is unclear. Using CRISPR/Cas9 screening, we find that loss of ABCC1 strongly increases the sensitivity of AML cells to Venetoclax. Genetic and pharmacologic ABCC1 inactivation potentiates the anti-leukemic effects of BCL-2 inhibitors and efficiently re-sensitizes Venetoclax-resistant leukemia cells. Conversely, ABCC1 overexpression induces resistance to BCL-2 inhibitors by reducing intracellular drug levels, and high ABCC1 levels predicts poor response to Venetoclax therapy in patients. Consistent with ABCC1-specific export of glutathionylated substrates, inhibition of glutathione metabolism increases the potency of BCL-2 inhibitors. These results identify ABCC1 and glutathione metabolism as mechanisms limiting efficacy of BCL-2 inhibitors, which may pave the way to development of more effective therapies.

STAT3/LKB1 controls metastatic prostate cancer by regulating mTORC1/CREB pathway.

Prostate cancer (PCa) is a common and fatal type of cancer in men. Metastatic PCa (mPCa) is a major factor contributing to its lethality, although the mechanisms remain poorly understood. PTEN is one of the most frequently deleted genes in mPCa. Here we show a frequent genomic co-deletion of PTEN and STAT3 in liquid biopsies of patients with mPCa. Loss of Stat3 in a Pten-null mouse prostate model leads to a reduction of LKB1/pAMPK with simultaneous activation of mTOR/CREB, resulting in metastatic disease. However, constitutive activation of Stat3 led to high LKB1/pAMPK levels and suppressed mTORC1/CREB pathway, preventing mPCa development. Metformin, one of the most widely prescribed therapeutics against type 2 diabetes, inhibits mTORC1 in liver and requires LKB1 to mediate glucose homeostasis. We find that metformin treatment of STAT3/AR-expressing PCa xenografts resulted in significantly reduced tumor growth accompanied by diminished mTORC1/CREB, AR and PSA levels. PCa xenografts with deletion of STAT3/AR nearly completely abrogated mTORC1/CREB inhibition mediated by metformin. Moreover, metformin treatment of PCa patients with high Gleason grade and type 2 diabetes resulted in undetectable mTORC1 levels and upregulated STAT3 expression. Furthermore, PCa patients with high CREB expression have worse clinical outcomes and a significantly increased risk of PCa relapse and metastatic recurrence. In summary, we have shown that STAT3 controls mPCa via LKB1/pAMPK/mTORC1/CREB signaling, which we have identified as a promising novel downstream target for the treatment of lethal mPCa.

Oncogenic TYK2 P760L kinase is effectively targeted by combinatorial TYK2, mTOR and CDK4/6 kinase blockade.

Tyrosine kinase 2 (TYK2) is a member of the Janus kinase/signal transducer and activator of transcription pathway, which is central in cytokine signaling. Previously, germline TYK2 mutations have been described in two patients developing de novo T-cell acute lymphoblastic leukemias (T-ALL) or precursor B-ALL. The mutations (P760L and G761V) are located within the regulatory pseudokinase domain and lead to constitutive activation of TYK2. We demonstrate the transformation capacity of TYK2 P760L in hematopoietic cell systems including primary bone marrow cells. In vivo engraftment of TYK2 P760L-expressing cell lines led to development of leukemia. A kinase inhibitor screen uncovered that oncogenic TYK2 acts synergistically with the PI3K/AKT/mTOR and CDK4/6 pathways. Accordingly, the TYK2-specific inhibitor deucravacitinib (BMS986165) reduces cell viability of TYK2 P760L-transformed cell models and ex vivo cultured TYK2 P760L-mutated patient- derived xenograft cells most efficiently when combined with mTOR or CDK4/6 inhibitors. Our study thereby pioneers novel treatment options for patients suffering from TYK2-driven acute leukemia.

Proposed refined diagnostic criteria and classification of eosinophil disorders and related syndromes.

Eosinophilia and eosinophil activation are recurrent features in various reactive states and certain hematologic malignancies. In patients with hypereosinophilia (HE), HE-induced organ damage is often encountered and may lead to the diagnosis of a hypereosinophilic syndrome (HES). A number of known mechanisms and etiologies contribute to the development of HE and HES. Based on these etiologies and the origin of eosinophils, HE and HES are divided into primary forms where eosinophils are clonal cells, reactive forms where an underlying reactive or neoplastic condition is detected and eosinophils are considered to be "non-clonal" cells, and idiopathic HE and HES in which neither a clonal nor a reactive underlying pathology is detected. Since 2012, this classification and the related criteria have been widely accepted and regarded as standard. However, during the past few years, new developments in the field and an increasing number of markers and targets have created a need to update these criteria and the classification of HE and HES. To address this challenge, a Working Conference on eosinophil disorders was organized in 2021. In this conference, a panel of experts representing the relevant fields, including allergy, dermatology, hematology, immunology, laboratory medicine, and pathology, met and discussed new markers and concepts as well as refinements in definitions, criteria and classifications of HE and HES. The outcomes of this conference are presented in this article and should assist in the diagnosis and management of patients with HE and HES in daily practice and in the preparation and conduct of clinical trials.

Blocking STAT3/5 through direct or upstream kinase targeting in leukemic cutaneous T-cell lymphoma.

Leukemic cutaneous T-cell lymphomas (L-CTCL) are lymphoproliferative disorders of skin-homing mature T-cells causing severe symptoms and high mortality through chronic inflammation, tissue destruction, and serious infections. Despite numerous genomic sequencing efforts, recurrent driver mutations have not been identified, but chromosomal losses and gains are frequent and dominant. We integrated genomic landscape analyses with innovative pharmacologic interference studies to identify key vulnerable nodes in L-CTCL. We detected copy number gains of loci containing the STAT3/5 oncogenes in 74% (n = 17/23) of L-CTCL, which correlated with the increased clonal T-cell count in the blood. Dual inhibition of STAT3/5 using small-molecule degraders and multi-kinase blockers abolished L-CTCL cell growth in vitro and ex vivo, whereby PAK kinase inhibition was specifically selective for L-CTCL patient cells carrying STAT3/5 gains. Importantly, the PAK inhibitor FRAx597 demonstrated encouraging anti-leukemic activity in vivo by inhibiting tumor growth and disease dissemination in intradermally xenografted mice. We conclude that STAT3/5 and PAK kinase interaction represents a new therapeutic node to be further explored in L-CTCL.

High Efficacy and Drug Synergy of HDAC6-Selective Inhibitor NN-429 in Natural Killer (NK)/T-Cell Lymphoma.

NK/T-cell lymphoma (NKTCL) and γδ T-cell non-Hodgkin lymphomas (γδ T-NHL) are highly aggressive lymphomas that lack rationally designed therapies and rely on repurposed chemotherapeutics from other hematological cancers. Histone deacetylases (HDACs) have been targeted in a range of malignancies, including T-cell lymphomas. This study represents exploratory findings of HDAC6 inhibition in NKTCL and γδ T-NHL through a second-generation inhibitor NN-429. With nanomolar in vitro HDAC6 potency and high in vitro and in cellulo selectivity for HDAC6, NN-429 also exhibited long residence time and improved pharmacokinetic properties in contrast to older generation inhibitors. Following unique selective cytotoxicity towards γδ T-NHL and NKTCL, NN-429 demonstrated a synergistic relationship with the clinical agent etoposide and potential synergies with doxorubicin, cytarabine, and SNS-032 in these disease models, opening an avenue for combination treatment strategies.

JAK-STAT core cancer pathway: An integrative cancer interactome analysis.

Through a comprehensive review and in silico analysis of reported data on STAT-linked diseases, we analysed the communication pathways and interactome of the seven STATs in major cancer categories and proposed rational targeting approaches for therapeutic intervention to disrupt critical pathways and addictions to hyperactive JAK/STAT in neoplastic states. Although all STATs follow a similar molecular activation pathway, STAT1, STAT2, STAT4 and STAT6 exert specific biological profiles associated with a more restricted pattern of activation by cytokines. STAT3 and STAT5A as well as STAT5B have pleiotropic roles in the body and can act as critical oncogenes that promote many processes involved in cancer development. STAT1, STAT3 and STAT5 also possess tumour suppressive action in certain mutational and cancer type context. Here, we demonstrated member-specific STAT activity in major cancer types. Through systems biology approaches, we found surprising roles for EGFR family members, sex steroid hormone receptor ESR1 interplay with oncogenic STAT function and proposed new drug targeting approaches of oncogenic STAT pathway addiction.

Structural and mutational analysis of member-specific STAT functions.

BACKGROUND: The STAT family of transcription factors control gene expression in response to signals from various stimulus. They display functions in diseases ranging from autoimmunity and chronic inflammatory disease to cancer and infectious disease. SCOPE OF REVIEW: This work uses an approach informed by structural data to explore how domain-specific structural variations, post-translational modifications, and the cancer genome mutational landscape dictate STAT member-specific activities. MAJOR CONCLUSIONS: We illustrated the structure-function relationship of STAT proteins and highlighted their effect on member-specific activity. We correlated disease-linked STAT mutations to the structure and cancer genome mutational landscape and proposed rational drug targeting approaches of oncogenic STAT pathway addiction. GENERAL SIGNIFICANCE: Hyper-activated STATs and their variants are associated with multiple diseases and are considered high value oncology targets. A full understanding of the molecular basis of member-specific STAT-mediated signaling and the strategies to selectively target them requires examination of the difference in their structures and sequences.

Proteomic Analysis Identifies NDUFS1 and ATP5O as Novel Markers for Survival Outcome in Prostate Cancer.

We aimed to identify novel markers for aggressive prostate cancer in a STAT3-low proteomics-derived dataset of mitochondrial proteins by immunohistochemical analysis and correlation with transcriptomic data and biochemical recurrence in a STAT3 independent PCa cohort. Formalin-fixed paraffin-embedded tissue (FFPE) sample selection for proteomic analysis and tissue-microarray (TMA) generation was conducted from a cohort of PCa patients. Retrospective data analysis was performed with the same cohort. 153 proteins differentially expressed between STAT3-low and STAT3-high samples were identified. Out of these, 46 proteins were associated with mitochondrial processes including oxidative phosphorylation (OXPHOS), and 45 proteins were upregulated, including NDUFS1/ATP5O. In a STAT3 independent PCa cohort, high expression of NDUFS1/ATP5O was confirmed by immunocytochemistry (IHC) and was significantly associated with earlier biochemical recurrence (BCR). mRNA expression levels for these two genes were significantly higher in intra-epithelial neoplasia and in PCa compared to benign prostate glands. NDUFS1/ATP5O levels are increased both at the mRNA and protein level in aggressive PCa. Our results provide evidence that NDUFS1/ATP5O could be used to identify high-risk PCa patients.

Efficacy and Synergy of Small Molecule Inhibitors Targeting FLT3-ITD+ Acute Myeloid Leukemia.

Constitutive activation of FLT3 by ITD mutations is one of the most common genetic aberrations in AML, present in ~1/3 of cases. Patients harboring FLT3-ITD display worse clinical outcomes. The integration and advancement of FLT3 TKI in AML treatment provided significant therapeutic improvement. However, due to the emergence of resistance mechanisms, FLT3-ITD+ AML remains a clinical challenge. We performed an unbiased drug screen to identify 18 compounds as particularly efficacious against FLT3-ITD+ AML. Among these, we characterized two investigational compounds, WS6 and ispinesib, and two approved drugs, ponatinib and cabozantinib, in depth. We found that WS6, although not yet investigated in oncology, shows a similar mechanism and potency as ponatinib and cabozantinib. Interestingly, ispinesib and cabozantinib prevent activation of AXL, a key driver and mechanism of drug resistance in FLT3-ITD+ AML patients. We further investigated synergies between the selected compounds and found that combination treatment with ispinesib and cabozantinib or ponatinib shows high synergy in FLT3-ITD+ AML cell lines and patient samples. Together, we suggest WS6, ispinesib, ponatinib and cabozantinib as novel options for targeting FLT3-ITD+ AML. Whether combinatorial tyrosine kinase and kinesin spindle blockade is effective in eradicating neoplastic (stem) cells in FLT3-ITD+ AML remains to be determined in clinical trials.

The Diverse Roles of γδ T Cells in Cancer: From Rapid Immunity to Aggressive Lymphoma.

γδ T cells are unique players in shaping immune responses, lying at the intersection between innate and adaptive immunity. Unlike conventional αß T cells, γδ T cells largely populate non-lymphoid peripheral tissues, demonstrating tissue specificity, and they respond to ligands in an MHC-independent manner. γδ T cells display rapid activation and effector functions, with a capacity for cytotoxic anti-tumour responses and production of inflammatory cytokines such as IFN-γ or IL-17. Their rapid cytotoxic nature makes them attractive cells for use in anti-cancer immunotherapies. However, upon transformation, γδ T cells can give rise to highly aggressive lymphomas. These rare malignancies often display poor patient survival, and no curative therapies exist. In this review, we discuss the diverse roles of γδ T cells in immune surveillance and response, with a particular focus on cancer immunity. We summarise the intriguing dichotomy between pro- and anti-tumour functions of γδ T cells in solid and haematological cancers, highlighting the key subsets involved. Finally, we discuss potential drivers of γδ T-cell transformation, summarising the main γδ T-cell lymphoma/leukaemia entities, their clinical features, recent advances in mapping their molecular and genomic landscapes, current treatment strategies and potential future targeting options.

Proposed Diagnostic Criteria and Classification of Canine Mast Cell Neoplasms: A Consensus Proposal.

Mast cell neoplasms are one of the most frequently diagnosed malignancies in dogs. The clinical picture, course, and prognosis vary substantially among patients, depending on the anatomic site, grade and stage of the disease. The most frequently involved organ is the skin, followed by hematopoietic organs (lymph nodes, spleen, liver, and bone marrow) and mucosal sites of the oral cavity and the gastrointestinal tract. In cutaneous mast cell tumors, several grading and staging systems have been introduced. However, no comprehensive classification and no widely accepted diagnostic criteria have been proposed to date. To address these open issues and points we organized a Working Conference on canine mast cell neoplasms in Vienna in 2019. The outcomes of this meeting are summarized in this article. The proposed classification includes cutaneous mast cell tumors and their sub-variants defined by grading- and staging results, mucosal mast cell tumors, extracutaneous/extramucosal mast cell tumors without skin involvement, and mast cell leukemia (MCL). For each of these entities, diagnostic criteria are proposed. Moreover, we have refined grading and staging criteria for mast cell neoplasms in dogs based on consensus discussion. The criteria and classification proposed in this article should greatly facilitate diagnostic evaluation and prognostication in dogs with mast cell neoplasms and should thereby support management of these patients in daily practice and the conduct of clinical trials.