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.
Objective: Intravenous hydroxyethyl starch (HES) solutions are potentially nephrotoxic due to rapid renal tissue uptake, subsequent osmotic nephrosis, and long-lasting intracellular storage. This study aimed to investigate the severity of intracellular storage of HES in renal tissue samples from critically ill dogs receiving 6% HES 130/0.4. Materials and Methods: Fresh, post-mortem (<2 h after death) renal tissue samples were analyzed through histology, immunohistochemistry (HES 130/0.4-specific antibodies), and electron microscopy for the severity of renal tubular vacuolization (VAC), intravacuolar HES accumulation (ACC), and ultra-structure impairment. Moreover, we investigated the relationship between VAC or ACC grade and HES dose (mL/kg), duration of HES administration (h), and pre-HES plasma creatinine concentrations. Results: Histology revealed that 2/20 dogs (10%) had no, 11/20 dogs (55%) had mild, 5/20 dogs (25%) had moderate, and 2/20 dogs (10%) had severe VAC. Immunohistochemistry revealed that 5/20 dogs (25%) had no, 6/20 dogs (30%) had mild, 7/20 dogs (35%) had moderate, and 2/20 dogs (10%) had severe ACC. Both changes were predominantly found in the distal tubular epithelium of mild and moderate cases, and all tubular segments were affected in severe cases. Seven of 20 dogs (35%) had osmotic nephrosis (ON). On electron microscopy, large granules with an electron-dense content were repeatedly detected in individual cells, mainly in the distal tubules. No correlation was found between cumulative HES dose or duration of HES administration and VAC grade, ACC grade, or presence/absence of ON. Conclusion: A high percentage of dogs had renal tubular HES storage and one-third of dogs showed HES-induced ON. Short-term HES administration caused VAC and ACC, regardless of the dose or duration of administration. In contrast to previous studies, HES 130/0.4 deposits were mainly located in the renal distal tubule.
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.
The European Society of Toxicologic Pathology (ESTP) initiated a survey through its Pathology 2.0 workstream in partnership with sister professional societies in Europe and North America to generate a snapshot of artificial intelligence (AI) usage in the field of toxicologic pathology. In addition to demographic information, some general questions explored AI relative to (1) the current status of adoption across organizations; (2) technical and methodological aspects; (3) perceived business value and finally; and (4) roadblocks and perspectives. AI has become increasingly established in toxicologic pathology with most pathologists being supportive of its development despite some areas of uncertainty. A salient feature consisted of the variability of AI awareness and adoption among the responders, as the spectrum extended from pathologists having developed familiarity and technical skills in AI, to colleagues who had no interest in AI as a tool in toxicologic pathology. Despite a general enthusiasm for these techniques, the overall understanding and trust in AI algorithms as well as their added value in toxicologic pathology were generally low, suggesting room for the need for increased awareness and education. This survey will serve as a basis to evaluate the evolution of AI penetration and acceptance in this domain.
To the Editor, Nonobese diabetic severe-combined-immunodeficient γc−/− (NSG) mice engrafted with peripheral blood mononuclear cells (PBMC) from allergic donors develop respiratory allergy mainly mediated by allergen-specific CD4+ T cells.(1) Consequently, they represent an interesting in vivo model to assess therapeutic approaches that modulate the allergen-specific T-cell response causing IgE-mediated allergy. However, the frequency of allergen-specific CD4+ Th2-cells that produce high levels of IL-4, IL-5, and IL-13 but low levels of IFN-γ in the peripheral blood of allergic individuals is very low, only one of 103-106 CD4+ T cells is specific for pollen allergens.(2, 3) Our laboratory has long-standing expertise in the in vitro expansion of allergen-specific CD4+ T-cell lines (TCL) from allergic individuals which were successfully employed to characterize the specificity, cross-reactivity, and MHC-restriction of specific T cells and to evaluate different cell types for their ability to present allergen.(4, 5) Here, we assessed whether allergen-specific TCL are applicable in the NSG mouse model of respiratory allergy. Bet v 1-specific T cells were expanded from PBMC of seven birch pollen (BP)-allergic donors as described in the Supporting information 1. At day 21 of in vitro culture, TCL consisted mostly of CD3+CD4+ cells (Figure S1A) of which approximately 50% proliferated in response to recombinant (r) Bet v 1 (mean value: 56%, range 41%-61%; Figure S1B) and to BP extract containing natural Bet v 1 but not to the negative control Bos d 5 (Figure S1C). Intracellular cytokine staining demonstrated that the majority of rBet v 1-reactive CFSElow T cells produced IL-4, IL-5, and IL-13 (Figure S1D-F). In six tested TCL, on average less than 15% of IL-4+ T cells concomitantly synthesized IFN-γ and similar results were found for IL-5+ and IL-13+ T cells (Figure S1G). The majority of IL-4+ cells also produced IL-5 and IL-13 and more than 35% of T cells producing one of the Th2 cytokines concomitantly produced TNF-α. Accordingly, supernatants from allergen-stimulated TCL contained significantly more IL-5 than IFN-γ (P = .028, Wilcoxon signed-rank test) and supernatants from 5/6 tested TCL contained TNF-α (Table 1A). Epitope mapping confirmed that the cultures were oligoclonal as all but TCL4 and TCL5 responded to several peptides in the regions Bet v 14-24, Bet v 119-33, Bet v 128-42, Bet v 152-66, Bet v 164-78, Bet v 173-90, Bet v 185-99, Bet v 197-111, and Bet v 1112-129 (Figure S1H). All but TCL5 harbored cells reactive with the immunodominant T-cell–activating region Bet v 1139-159.(6) NSG mice intraperitoneally (i.p.) received allergen-specific TCL plus autologous CD3-depleted PBMC (CD3-PBMC) as source of antigen-presenting cells plus allergen (Figure 1A). At days 13-15, mice were intranasally (i.n.) challenged with allergen or PBS, and human cells detected in cell suspensions from lungs at day 17 contained a median of 70.3% vs 70.8% CD4+ T cells, 10.8% vs 10.4% CD8+ T cells, and 5.2% vs 5.9% B cells, whereas monocytes and NK cells were below 0.1% in either group (Figure 1B). Similar data were found in murine spleens (data not shown). Allergen-challenged mice showed higher percentages of basophils, eosinophils, and neutrophils in bronchoalveolar lavage fluids (BALF; Figure 1C) and significantly higher airway hyperreactivity (AHR) than PBS-challenged animals (Figure 1D). Histopathological analysis revealed a slightly increased peribronchial inflammation in allergen-challenged compared with PBS-challenged animals (Figure 1E) whereas goblet cell hyperplasia was not evident in lung sections of either group (data not shown). Together, NSG mice engrafted with oligoclonal allergen-specific Th2-cell cultures developed respiratory allergy with AHR as pre-eminent readout. These results confirm that allergen-specific CD4+ T cells are major players in this human/mouse allergy model. We observed perivascular inflammation in the lungs of PBMC-engrafted NSG mice which is considered as an early warning of graft-versus-host disease (GvHD, Figure 1F).(7) Furthermore, the numbers of CD8+ T cells in these animals increased resulting in significant lower ratios of CD4+/CD8+ T cells in lung suspensions than in PBMC before injection (Figure 1G). No perivascular inflammation or reduced ratios of CD4+/CD8+ T cells were evident in lungs of TCL-engrafted animals (Figure 1E,G). Moreover, mice did not show weight loss, skin rashes, ruffled fur, or increased mortality. Subclinical GvHD may cause perivascular and peribronchial inflammation and mucus production and thereby compromise allergen-induced respiratory responses. Accordingly, PBS-challenged PBMC-engrafted animals showed significantly higher AHR compared with untreated mice.(7) This enhanced background was not evident in PBS-exposed TCL-engrafted animals (Figure 1D) and a significantly lower AHR in PBS-exposed TCL-engrafted than PBMC-engrafted animals was found (Figure 1H). Allergen challenge triggered comparable AHR in both types of humanized mice. We speculate that the magnitude of allergen-induced AHR in TCL-engrafted mice is a consequence of an exclusive allergic response whereas it represents an overlap of allergen-specific and GvHD-induced inflammation in PBMC-engrafted mice. Consequently, NSG mice engrafted with allergen-specific TCL represent a more specific and possibly also more sensitive model of respiratory allergy. To monitor the preservation of the human Th2 phenotype in vivo, pooled sera from 5-7 mice i.n. challenged with allergen or PBS were analyzed for human IL-5, IFN-γ, and TNF-α (Table 1B). No cytokines were detectable in untreated mice (data not shown) and mice engrafted with TCL4. Solely TNF-α was detected in allergen-challenged mice engrafted with TCL3. Otherwise, IL-5 levels were higher in allergen-challenged mice (P = .043, Wilcoxon signed-rank test). Similarly, IFN-γ and TNF-α were predominantly found in allergen-challenged mice. Except for mice engrafted with TCL6, IFN-γ levels were lower than IL-5 levels. Overall, human cytokines detected in murine sera resembled those secreted from allergen-activated TCL in vitro (Table 1). IL-5 is a potent activator and attractor of eosinophils and together with eotaxin-2 induces AHR.(8) AHR, mucus production, and eosinophilia are also promoted by IL-13 (9) which was produced by most allergen-stimulated IL-5+ T cells (Figure S1G). Like IL-5 and IL-13, human and murine TNF-α demonstrate significant cross-species reactivity. We detected no differences in the murine cytokines IL-1β, IL-6, and IFN-γ in BALF and sera of allergen- and PBS-exposed animals excluding a relevant role in inflammation (data not shown). It is therefore tempting to speculate that human allergen-specific Th2 cells mediated murine airway inflammation after respiratory exposure to allergen. Together, NSG mice engrafted with allergen-specific TCL may represent a suitable preclinical model for therapeutic approaches that modulate allergen-specific Th2 cells. Dr Bohle reports grants from Austrian Science Funds during the conduct of the study and personal fees from Allergen Online Database, nonfinancial support from Paul Ehrlich Institute, personal fees from Christian Doppler Research Organisation, outside the submitted work. No potential conflicts of interest were disclosed by the other authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. 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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.
Abstract Candida albicans is the most common human fungal pathogen, causing diseases ranging from local to life-threating systemic infections. Tyrosine kinase 2 (TYK2), a crucial mediator in several cytokine signaling pathways, has been associated with protective functions in various microbial infections. However, its specific contribution in the immune response to fungal infections has remained elusive. In this study, we show that mice lacking TYK2 or its enzymatic activity exhibit enhanced resistance to C. albicans skin infections, limiting fungal spread and accelerating wound healing. Impaired TYK2-signaling prompted the formation of a distinctive layer of necrotic neutrophils around the fungal pathogens. Transcriptomic analysis revealed TYK2’s pivotal role in regulating interferon-inducible genes in neutrophils, thereby impacting their antifungal capacity during infection. Furthermore, we show that TYK2-dependent interferon-gamma (IFNγ) production contributes to fungal dissemination from the skin to the kidneys. Our study uncovers a hitherto unrecognized detrimental role of TYK2 in cutaneous C. albicans infections.
Introduction Prostate cancer (PCa) is the most frequently diagnosed malignancy in men and ranks as the third leading cause of cancer-related death in Western civilization. There is an urgent need to provide diagnostic tools able to reliably stratify aggressive from slow progressing tumours. As a result, over-diagnosis of PCa remains a prevalent issue and patients frequently suffer from severe side effects following therapeutic intervention. Recently, whole-exome sequencing has identified the MLL family member KMT2C amongst the most frequently mutated genes in human prostate cancer. However, the exact role of this histone lysine methyltransferase in tumorigenesis remains elusive. In this study we aim to investigate the contribution of the methyltransferase activity of Kmt2c to prostate tumorigenesis using a Pten-deficient mouse model of PCa. Material and methods We have developed a mouse model with a double deletion of the tumour suppressor Pten and the methyltransferase domain of Kmt2c in the prostate epithelium (PtenKmt2cPE-/-). Mice were sacrificed at 19 weeks of age and prostate tissue was analysed according to size, morphology and selected markers involved in tumorigenesis in comparison to Pten-deficient control mice (PtenPE-/-). To establish human relevance, we investigated the correlation of KMT2C expression to biochemical recurrence (BCR) and exploited publicly available databases to gain insight from the mutational frequency spectrum of KMT2C in PCa. Results and discussions Deletion of the Kmt2c methyltransferase activity (PtenKmt2cPE-/-) resulted in severely reduced life expectancy, increased tumour size and weight accompanied by a more aggressive histopathological morphology. PtenKmt2cPE-/- tumours showed increased proliferation rates and a deregulation of several histone marks. In a dataset of human PCa patients we found that low expression as well as truncating mutations of KMT2C are similarly associated with poor prognosis. Analysis of TMAs of human PCa patients showed decreased KMT2C expression in bad prognostic high Gleason grade PCa tumour samples. Conclusion Our findings indicate that loss of Kmt2c methyltransferase activity accelerates PCa tumorigenesis in the PtenKmt2cPE-/- mouse model as well as in human patient samples. We therefore propose Kmt2c as a tumour suppressor in prostate cancer.
Abstract Integrin-mediated interactions between hematopoietic cells and their microenvironment are important for the development and function of immune cells. Here, the role of the integrin adaptor Kindlin-3 in B cell homeostasis is studied. Comparing the individual steps of B cell development in B cell-specific Kindlin-3 or alpha4 integrin knockout mice, we found in both conditions a phenotype of reduced late immature, mature, and recirculating B cells in the bone marrow. In the spleen, constitutive B cell-specific Kindlin-3 knockout caused a loss of marginal zone B cells and an unexpected expansion of follicular B cells. Alpha4 integrin deficiency did not induce this phenotype. In Kindlin-3 knockout B cells VLA-4 as well as LFA-1-mediated adhesion was abrogated, and short-term homing of these cells in vivo was redirected to the spleen. Upon inducible Kindlin-3 knockout, marginal zone B cells were lost due to defective retention within 2 weeks, while follicular B cell numbers were unaltered. Kindlin-3 deficient follicular B cells displayed higher IgD, CD40, CD44, CXCR5, and EBI2 levels, and elevated PI3K signaling upon CXCR5 stimulation. They also showed transcriptional signatures of spontaneous follicular B cell activation. This activation manifested in scattered germinal centers in situ, early plasmablasts differentiation, and signs of IgG class switch.
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