Abstract Mature T cell leukemias/lymphomas (MaTCL) are a group of rare hematological malignancies of mostly incurable prospects due to limited efficient therapies and a lack of faithful pre-clinical models. STAT5 transcription factors are critical downstream effectors of cytokine and growth factor signaling through the JAK/STAT pathway, particularly in the hematopoietic compartment. The two gene products, STAT5A and STAT5B, play important roles in cell proliferation, survival and differentiation. It is increasingly evident that hyperactive JAK/STAT signaling plays a role in these MaTCL diseases. Notably, a hotspot gain-of-function (GOF) mutation in STAT5B, N642H, has been found in over 100 patients with T cell neoplasia, and is associated with more aggressive disease, therapy resistance and worse prognosis. To investigate the role of STAT5BN642H in MaTCL, we utilized our transgenic mouse model harboring moderate expression of human STAT5BN642H in the hematopoietic compartment. Notably, STAT5BN642H transgenic mice rapidly develop aggressive mature CD8+ T cell disease, suggesting that CD8+ T cells are particularly susceptible to transformation by this mutation. Further examination of these mice revealed prominent infiltration of neoplastic T cells into peripheral organs including lung, skin, brain and liver. Interestingly, in addition to CD8+ T cells, infiltration of CD4+ as well as γδ T cells was also observed in various organs of the STAT5BN642H mice, suggestive of STAT5BN642H-driven transformation of other T cell lineages. This would be more consistent with human patients carrying this mutation, who suffer predominantly from MaTCL of CD4+ or aggressive γδ T cell subtypes. Therefore, we isolated these T cell subsets from the transgenic mice and performed syngeneic transplant experiments to assess T cell transformation. Indeed, transplants of γδ or CD4+ T cells from STAT5BN642H transgenic mice into immunocompetent recipients resulted in the development of γδ or CD4+ T cell neoplasia, respectively, demonstrating the full transforming capacity of STAT5BN642H in multiple T cell lineages. From this, we have generated a novel γδ T cell line harboring the STAT5BN642H mutation, which we are utilizing together with the mouse models to interrogate oncogenic mechanisms of STAT5B (through immunophenotyping and sequencing efforts) as well as to screen and test new therapeutic options for these diseases. Overall, these data highlight the aggressive nature of the STAT5BN642H driver mutation in MaTCL. We report on novel pre-clinical models for aggressive γδ or CD4+ MaTCL that more closely recapitulate human disease. These models will be valuable for understanding STAT5BN642H-driven T cell disease and for testing new, urgently needed MaTCL treatment strategies. Citation Format: Heidi A. Neubauer, Tobias Suske, Susann Schönefeldt, Simone Tangermann, Auke Boersma, Thomas Rülicke, Vasileios Bekiaris, Lukas Kenner, Richard Moriggl. The gain-of-function STAT5BN642H mutation as a driver of mature T cell leukemia/lymphoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2752.
Abstract Background Anaplastic large cell lymphoma (ALCL) is an aggressive non-Hodgkin T cell lymphoma commonly driven by NPM-ALK. AP-1 transcription factors, cJUN and JUNb, act as downstream effectors of NPM-ALK and transcriptionally regulate PDGFRβ. Blocking PDGFRβ kinase activity with imatinib effectively reduces tumor burden and prolongs survival, although the downstream molecular mechanisms remain elusive. Methods and results In a transgenic mouse model that mimics PDGFRβ-driven human ALCL in vivo, we identify PDGFRβ as a driver of aggressive tumor growth. Mechanistically, PDGFRβ induces the pro-survival factor Bcl-x L and the growth-enhancing cytokine IL-10 via STAT5 activation. CRISPR/Cas9 deletion of both STAT5 gene products, STAT5A and STAT5B, results in the significant impairment of cell viability compared to deletion of STAT5A, STAT5B or STAT3 alone. Moreover, combined blockade of STAT3/5 activity with a selective SH2 domain inhibitor, AC-4-130, effectively obstructs tumor development in vivo. Conclusions We therefore propose PDGFRβ as a novel biomarker and introduce PDGFRβ-STAT3/5 signaling as an important axis in aggressive ALCL. Furthermore, we suggest that inhibition of PDGFRβ or STAT3/5 improve existing therapies for both previously untreated and relapsed/refractory ALK + ALCL patients.
Abstract Androgen deprivation therapy (ADT) remains a key approach in the treatment of prostate cancer (PCa). However, PCa inevitably relapses and becomes ADT resistant. Besides androgens, there is evidence that thyroid hormone thyroxine (T4) and its active form 3,5,3’-triiodo-L-thyronine (T3) are involved in the progression of PCa. Epidemiologic evidence indicates a higher incidence of PCa in men with elevated thyroid hormone levels. The thyroid hormone binding protein μ-Crystallin (CRYM) mediates intracellular thyroid hormone action by sequestering T3 and blocks its binding to cognate receptors (TRa/TRb) in target tissues. We show in this study that low CRYM expression levels in PCa patient samples are associated with early BCR and poor prognosis. Moreover, we found a disease stage-specific expression of CRYM in PCa. CRYM counteracted thyroid and androgen signaling and blocked intracellular choline uptake. CRYM inversely correlated with [18F]fluoromethylcholine (FMC) levels in PET/MRI imaging of PCa patients. Our data suggest CRYM as a novel antagonist of T3 and androgen-mediated signalling. The role of CRYM could therefore be an essential control mechanism for the prevention of aggressive PCa growth. Highlights Thyroid and androgen hormone driven pathways in prostate cancer (PCa) are antagonized by μ- Crystallin (CRYM). [18F]fluoromethylcholine uptake and prognostic values in PCa correlate with CRYM protein levels. Reduced CRYM expression predicts early biochemical recurrence (BCR) in PCa patients.
Src Homology 2 (SH2) domains arose within metazoan signaling pathways and are involved in protein regulation of multiple pleiotropic cascades. In signal transducer and activator of transcription (STAT) proteins, SH2 domain interactions are critical for molecular activation and nuclear accumulation of phosphorylated STAT dimers to drive transcription. Sequencing analysis of patient samples has revealed the SH2 domain as a hotspot in the mutational landscape of STAT proteins although the functional impact for the vast majority of these mutations remains poorly characterized. Despite several well resolved structures for SH2 domain-containing proteins, structural data regarding the distinctive STAT-type SH2 domain is limited. Here, we review the unique features of STAT-type SH2 domains in the context of all currently reported STAT3 and STAT5 SH2 domain clinical mutations. The genetic volatility of specific regions in the SH2 domain can result in either activating or deactivating mutations at the same site in the domain, underscoring the delicate evolutionary balance of wild type STAT structural motifs in maintaining precise levels of cellular activity. Understanding the molecular and biophysical impact of these disease-associated mutations can uncover convergent mechanisms of action for mutations localized within the STAT SH2 domain to facilitate the development of targeted therapeutic interventions.
Signal transducer and activator of transcription (STAT)3 and STAT5 are important transcription factors that are able to mediate or even drive cancer progression through hyperactivation or gain-of-function mutations. Mutated STAT3 is mainly associated with large granular lymphocytic T-cell leukemia, whereas mutated STAT5B is associated with T-cell prolymphocytic leukemia, T-cell acute lymphoblastic leukemia and γδ T-cell-derived lymphomas. Hyperactive STAT3 and STAT5 are also implicated in various hematopoietic and solid malignancies, such as chronic and acute myeloid leukemia, melanoma or prostate cancer. Classical understanding of STAT functions is linked to their phosphorylated parallel dimer conformation, in which they induce gene transcription. However, the functions of STAT proteins are not limited to their phosphorylated dimerization form. In this review, we discuss the functions and the roles of unphosphorylated STAT3/5 in the context of chromatin remodeling, as well as the impact of STAT5 oligomerization on differential gene expression in hematopoietic neoplasms. The central involvement of STAT3/5 in cancer has made these molecules attractive targets for small-molecule drug development, but currently there are no direct STAT3/5 inhibitors of clinical grade available. We summarize the development of inhibitors against the SH2 domains of STAT3/5 and discuss their applicability as cancer therapeutics.
The Signal Transducer and Activator of Transcription (STAT)3 and 5 are activated by many cytokine receptors to regulate specific gene expression and mitochondrial functions. Their role in cancer is largely context dependent as they can both act as oncogenes and tumor suppressors. We review here the role of STAT3/5 activation in solid cancers and summarize their association to survival in cancer patients. The molecular mechanisms that underpins the oncogenic activity of STAT3/5 signaling includes the regulation of genes that control cell cycle, cell death, inflammation and stemness. In addition, STAT3 mitochondrial functions are required for transformation. On the other hand, several tumor suppressor pathways act on or are activated by STAT3/5 signaling including the p19ARF/p53 pathway, tyrosine phosphatases, suppressor of cytokine signaling 1 and 3, the sumo ligase PIAS3, the E3 ubiquitin ligase TMF/ARA160 and the miRNAs miR-124 and miR-1181. Cancer mutations and epigenetic alterations may alter the balance between pro-oncogenic and tumor suppressor activities associated to STAT3/5 signaling explaining their context dependent association to tumor progression both in human cancers and animal models.
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.
Alimentary lymphomas arising from T cells are rare and aggressive malignancies in humans. In comparison, they represent the most common anatomical form of lymphoma in cats. Due to the low prevalence in humans, the underlying pathomechanism for these diseases is poorly characterised, limiting experimental analysis and therapeutic exploration. To date, activating mutations of the JAK/STAT core cancer pathway and particularly the STAT5B oncoprotein have been identified in human enteropathy-associated T cell lymphoma. Here, we describe a high homology of human and feline STAT3 and STAT5B proteins and strong conservation at the genomic level. Analysis of 42 samples of feline T cell alimentary lymphoma reveals broad activation of STAT3 and STAT5B. Screening for known activating mutations in STAT3 or STAT5B identifies the presence of the STAT5BN642H driver mutation in feline enteropathy-associated T cell lymphoma in 7 out of 42 (16.67%) samples in total. Regarding lymphoma subtypes, the majority of mutations with 5 out of 17 (29.41%) cases were found in feline enteropathy-associated lymphoma type II (EATL II). This identification of an oncogenic STAT5B driver mutation in felines recapitulates the genetic situation in the corresponding human disease, thereby establishing the cat as a potential new model for a rare and incurable human T cell disease.
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.
Abstract 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.
