PU.1 is required to restrain myelopoiesis during chronic inflammatory stress.

Chronic inflammation is a common feature of aging and numerous diseases such as diabetes, obesity, and autoimmune syndromes and has been linked to the development of hematological malignancy. Blood-forming hematopoietic stem cells (HSC) can contribute to these diseases via the production of tissue-damaging myeloid cells and/or the acquisition of mutations in epigenetic and transcriptional regulators that initiate evolution toward leukemogenesis. We previously showed that the myeloid "master regulator" transcription factor PU.1 is robustly induced in HSC by pro-inflammatory cytokines such as interleukin (IL)-1ß and limits their proliferative activity. Here, we used a PU.1-deficient mouse model to investigate the broader role of PU.1 in regulating hematopoietic activity in response to chronic inflammatory challenges. We found that PU.1 is critical in restraining inflammatory myelopoiesis via suppression of cell cycle and self-renewal gene programs in myeloid-biased multipotent progenitor (MPP) cells. Our data show that while PU.1 functions as a key driver of myeloid differentiation, it plays an equally critical role in tailoring hematopoietic responses to inflammatory stimuli while limiting expansion and self-renewal gene expression in MPPs. These data identify PU.1 as a key regulator of "emergency" myelopoiesis relevant to inflammatory disease and leukemogenesis.

Dexamethasone and OLT1177 Cooperate in the Reduction of Melanoma Growth by Inhibiting STAT3 Functions.

The NLRP3 inflammasome is a multimolecular complex that processes inactive IL-1ß and IL-18 into proinflammatory cytokines. OLT1177 is an orally active small compound that specifically inhibits NLRP3. Here, B16F10 melanoma were implanted in mice and treated with OLT1177 as well as combined with the glucocorticoid dexamethasone. At sacrifice, OLT1177 treated mice had significantly smaller tumors compared to tumor-bearing mice treated with vehicle. However, the combined treatment of OLT1177 plus dexamethasone revealed a greater suppression of tumor growth. This reduction was accompanied by a downregulation of nuclear and mitochondrial STAT3-dependent gene transcription and by a significant reduction of STAT3 Y705 and S727 phosphorylations in the tumors. In vitro, the human melanoma cell line 1205Lu, stimulated with IL-1α, exhibited significantly lower levels of STAT3 Y705 phosphorylation by the combination treatment, thus affecting the nuclear functions of STAT3. In the same cells, STAT3 serine 727 phosphorylation was also lower, affecting the mitochondrial functions of STAT3. In addition, metabolic analyses revealed a marked reduction of ATP production rate and glycolytic reserve in cells treated with the combination of OLT1177 plus dexamethasone. These findings demonstrate that the combination of OLT1177 and dexamethasone reduces tumor growth by targeting nuclear as well as mitochondrial functions of STAT3.

PU.1 Expression Defines Distinct Functional Activities in the Phenotypic HSC Compartment of a Murine Inflammatory Stress Model.

The transcription factor PU.1 is a critical regulator of lineage fate in blood-forming hematopoietic stem cells (HSC). In response to pro-inflammatory signals, such as the cytokine IL-1ß, PU.1 expression is increased in HSC and is associated with myeloid lineage expansion. To address potential functional heterogeneities arising in the phenotypic HSC compartment due to changes in PU.1 expression, here, we fractionated phenotypic HSC in mice using the SLAM surface marker code in conjunction with PU.1 expression levels, using the PU.1-EYFP reporter mouse strain. While PU.1lo SLAM cells contain extensive long-term repopulating activity and a molecular signature corresponding to HSC activity at steady state, following IL-1ß treatment, HSCLT induce PU.1 expression and are replaced in the PU.1lo SLAM fraction by CD41+ HSC-like megakaryocytic progenitors (SL-MkP) with limited long-term engraftment capacity. On the other hand, the PU.1hi SLAM fraction exhibits extensive myeloid lineage priming and clonogenic activity and expands rapidly in response to IL-1ß. Furthermore, we show that EPCR expression, but not CD150 expression, can distinguish HSCLT and SL-MkP under inflammatory conditions. Altogether, our data provide insights into the dynamic regulation of PU.1 and identify how PU.1 levels are linked to HSC fate in steady state and inflammatory stress conditions.

PU.1 enforces quiescence and limits hematopoietic stem cell expansion during inflammatory stress.

Hematopoietic stem cells (HSCs) are capable of entering the cell cycle to replenish the blood system in response to inflammatory cues; however, excessive proliferation in response to chronic inflammation can lead to either HSC attrition or expansion. The mechanism(s) that limit HSC proliferation and expansion triggered by inflammatory signals are poorly defined. Here, we show that long-term HSCs (HSCLT) rapidly repress protein synthesis and cell cycle genes following treatment with the proinflammatory cytokine interleukin (IL)-1. This gene program is associated with activation of the transcription factor PU.1 and direct PU.1 binding at repressed target genes. Notably, PU.1 is required to repress cell cycle and protein synthesis genes, and IL-1 exposure triggers aberrant protein synthesis and cell cycle activity in PU.1-deficient HSCs. These features are associated with expansion of phenotypic PU.1-deficient HSCs. Thus, we identify a PU.1-dependent mechanism triggered by innate immune stimulation that limits HSC proliferation and pool size. These findings provide insight into how HSCs maintain homeostasis during inflammatory stress.

The anti-inflammatory cytokine interleukin-37 is an inhibitor of trained immunity.

Trained immunity (TI) is a de facto innate immune memory program induced in monocytes/macrophages by exposure to pathogens or vaccines, which evolved as protection against infections. TI is characterized by immunometabolic changes and histone post-translational modifications, which enhance production of pro-inflammatory cytokines. As aberrant activation of TI is implicated in inflammatory diseases, tight regulation is critical; however, the mechanisms responsible for this modulation remain elusive. Interleukin-37 (IL-37) is an anti-inflammatory cytokine that curbs inflammation and modulates metabolic pathways. In this study, we show that administration of recombinant IL-37 abrogates the protective effects of TI in vivo, as revealed by reduced host pro-inflammatory responses and survival to disseminated candidiasis. Mechanistically, IL-37 reverses the immunometabolic changes and histone post-translational modifications characteristic of TI in monocytes, thus suppressing cytokine production in response to infection. IL-37 thereby emerges as an inhibitor of TI and as a potential therapeutic target in immune-mediated pathologies.

Targeting tumor-derived NLRP3 reduces melanoma progression by limiting MDSCs expansion.

Interleukin-1ß (IL-1ß)-mediated inflammation suppresses antitumor immunity, leading to the generation of a tumor-permissive environment, tumor growth, and progression. Here, we demonstrate that nucleotide-binding domain, leucine-rich containing family, pyrin domain-containing-3 (NLRP3) inflammasome activation in melanoma is linked to IL-1ß production, inflammation, and immunosuppression. Analysis of cancer genome datasets (TCGA and GTEx) revealed greater NLRP3 and IL-1ß expression in cutaneous melanoma samples (n = 469) compared to normal skin (n = 324), with a highly significant correlation between NLRP3 and IL-1ß (P < 0.0001). We show the formation of the NLRP3 inflammasome in biopsies of metastatic melanoma using fluorescent resonance energy transfer analysis for NLRP3 and apoptosis-associated speck-like protein containing a CARD. In vivo, tumor-associated NLRP3/IL-1 signaling induced expansion of myeloid-derived suppressor cells (MDSCs), leading to reduced natural killer and CD8+ T cell activity concomitant with an increased presence of regulatory T (Treg) cells in the primary tumors. Either genetic or pharmacological inhibition of tumor-derived NLRP3 by dapansutrile (OLT1177) was sufficient to reduce MDSCs expansion and to enhance antitumor immunity, resulting in reduced tumor growth. Additionally, we observed that the combination of NLRP3 inhibition and anti-PD-1 treatment significantly increased the antitumor efficacy of the monotherapy by limiting MDSC-mediated T cell suppression and tumor progression. These data show that NLRP3 activation in melanoma cells is a protumor mechanism, which induces MDSCs expansion and immune evasion. We conclude that inhibition of NLRP3 can augment the efficacy of anti-PD-1 therapy.

Pro-inflammatory cytokine blockade attenuates myeloid expansion in a murine model of rheumatoid arthritis.

Rheumatoid arthritis (RA) is a debilitating autoimmune disease characterized by chronic inflammation and progressive destruction of joint tissue. It is also characterized by aberrant blood phenotypes including anemia and suppressed lymphopoiesis that contribute to morbidity in RA patients. However, the impact of RA on hematopoietic stem cells (HSC) has not been fully elucidated. Using a collagen-induced mouse model of human RA, we identified systemic inflammation and myeloid overproduction associated with activation of a myeloid differentiation gene program in HSC. Surprisingly, despite ongoing inflammation, HSC from arthritic mice remain in a quiescent state associated with activation of a proliferation arrest gene program. Strikingly, we found that inflammatory cytokine blockade using the interleukin-1 receptor antagonist anakinra led to an attenuation of inflammatory arthritis and myeloid expansion in the bone marrow of arthritic mice. In addition, anakinra reduced expression of inflammation-driven myeloid lineage and proliferation arrest gene programs in HSC of arthritic mice. Altogether, our findings show that inflammatory cytokine blockade can contribute to normalization of hematopoiesis in the context of chronic autoimmune arthritis.

CD34 and EPCR coordinately enrich functional murine hematopoietic stem cells under normal and inflammatory conditions.

Hematopoiesis is dynamically regulated to maintain blood system function under nonhomeostatic conditions such as inflammation and injury. However, common surface marker and hematopoietic stem cell (HSC) reporter systems used for prospective enrichment of HSCs have been less rigorously tested in these contexts. Here, we use two surface markers, EPCR/CD201 and CD34, to re-analyze dynamic changes in the HSC-enriched phenotypic SLAM compartment in a mouse model of chronic interleukin (IL)-1 exposure. EPCR and CD34 coordinately identify four functionally and molecularly distinct compartments within the SLAM fraction, including an EPCR+/CD34- fraction whose long-term serial repopulating activity is only modestly impacted by chronic IL-1 exposure, relative to unfractionated SLAM cells. Notably, the other three fractions expand in frequency following IL-1 treatment and represent actively proliferating, lineage-primed cell states with limited long-term repopulating potential. Importantly, we find that the Fgd5-ZSGreen HSC reporter mouse enriches for molecularly and functionally intact HSCs regardless of IL-1 exposure. Together, our findings provide further evidence of dynamic heterogeneity within a commonly used HSC-enriched phenotypic compartment under stress conditions. Importantly, they also indicate that stringency of prospective isolation approaches can enhance interpretation of findings related to HSC function when studying models of hematopoietic stress.

PI3 kinase alpha and delta promote hematopoietic stem cell activation.

Many cytokines and chemokines that are important for hematopoiesis activate the PI3K signaling pathway. Because this pathway is frequently mutated and activated in cancer, PI3K inhibitors have been developed for the treatment of several malignancies, and are now being tested in the clinic in combination with chemotherapy. However, the role of PI3K in adult hematopoietic stem cells (HSCs), particularly during hematopoietic stress, is still unclear. We previously showed that the individual PI3K catalytic isoforms P110α or P110ß have dispensable roles in HSC function, suggesting redundancy between PI3K isoforms in HSCs. We now demonstrate that simultaneous deletion of P110α and P110δ in double knockout (DKO) HSCs uncovers their redundant requirement in HSC cycling after 5-fluorouracil (5-FU) chemotherapy administration. In contrast, DKO HSCs are still able to exit quiescence in response to other stress stimuli, such as LPS. We found that DKO HSCs and progenitors have impaired sensing of inflammatory signals ex vivo, and that levels of IL1-ß and MIG are higher in the bone marrow after LPS than after 5-FU administration. Furthermore, exogenous in vivo administration of IL1-ß can induce cell cycle entry of DKO HSCs. Our findings have important clinical implications for the use of PI3K inhibitors in combination with chemotherapy.

Combinatorial regulation of a Blimp1 (Prdm1) enhancer in the mouse retina.

The mouse retina comprises seven major cell types that exist in differing proportions. They are generated from multipotent progenitors in a stochastic manner, such that the relative frequency of any given type generated changes over time. The mechanisms determining the proportions of each cell type are only partially understood. Photoreceptors and bipolar interneurons are derived from cells that express Otx2. Within this population, Blimp1 (Prdm1) helps set the balance between photoreceptors and bipolar cells by suppressing bipolar identity in most of the cells. How only a subset of these Otx2+ cells decides to upregulate Blimp1 and adopt photoreceptor fate is unknown. To understand this, we investigated how Blimp1 transcription is regulated. We identified several potential Blimp1 retinal enhancer elements using DNase hypersensitivity sequencing. Only one of the elements recapitulated Blimp1 spatial and temporal expression in cultured explant assays and within the retinas of transgenic mice. Mutagenesis of this retinal Blimp1 enhancer element revealed four discrete sequences that were each required for its activity. These included highly conserved Otx2 and ROR (retinoic acid receptor related orphan receptor) binding sites. The other required sequences do not appear to be controlled by Otx2 or ROR factors, increasing the complexity of the Blimp1 gene regulatory network. Our results show that the intersection of three or more transcription factors is required to correctly regulate the spatial and temporal features of Blimp1 enhancer expression. This explains how Blimp1 expression can diverge from Otx2 and set the balance between photoreceptor and bipolar fates.

Elucidating the Role of the Desmosome Protein p53 Apoptosis Effector Related to PMP-22 in Growth Hormone Tumors.

Densely granulated and sparsely granulated (SG) growth hormone (GH) pituitary adenomas differ in biological behavior, which may be correlated with their known differences in cytoplasmic keratin distribution and E-cadherin expression. We wanted to explore candidate genes that might further explain this behavior. Exon expression microarray was performed on 21 GH tumors (10 SG and 11 densely granulated) and 20 normal control pituitaries from autopsy. Bioinformatic analyses confirmed a differential molecular signature between normal pituitary and GH tumors as well as between the GH tumor subtypes. There was a consistent downregulation of transcripts involved in the structure and function of the desmosome, including desmoplakin (eightfold), desmoglein 2 (sixfold), plakophilin 2 (sevenfold), and p53 apoptosis effector related to PMP-22 (PERP; sixfold) in SG tumors compared with normal pituitary. PERP is lost in more aggressive SG human GH pituitary tumors. PERP re-expression in GH3 rat GH tumor cells resulted in decreased colony formation compared with vector transfectants, confirming the role of PERP as a tumor suppressor with no effects on proliferation. Increased PERP expression was associated with loss of a survival advantage in a hypoxic environment, as assessed by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (P < 0.05) and cleaved caspase-3 (P < 0.05). Downregulation of desmosomal formation transcripts including PERP may contribute to the aggressive phenotype seen in SG GH pituitary tumors and their behavior in response to surgery and medical therapy.

Structure-Based Screen Identification of a Mammalian Ste20-like Kinase 4 (MST4) Inhibitor with Therapeutic Potential for Pituitary Tumors.

Pituitary tumors of the gonadotrope lineage are often large and invasive, resulting in hypopituitarism. No medical treatments are currently available. Using a combined genetic and genomic screen of individual human gonadotrope pituitary tumor samples, we recently identified the mammalian sterile-20 like kinase 4 (MST4) as a protumorigenic effector, driving increased pituitary cell proliferation and survival in response to a hypoxic microenvironment. To identify novel inhibitors of the MST4 kinase for potential future clinical use, computational-based virtual library screening was used to dock the SelleckChem kinase inhibitor library into the ATP-binding site of the MST4 crystal structure. Several inhibitor candidates were identified with the potential to bind with high affinity. Using a TR-FRET in vitro recombinant kinase assay, hesperadin, initially described as an Aurora kinase inhibitor, exhibited potent inhibition of the MST4 kinase at nanomolar concentrations. The LßT2 gonadotrope pituitary cell hypoxic model was used to test the ability of this inhibitor to antagonize MST4 actions. Under short-term severe hypoxia (1% O2), MST4 protection from hypoxia-induced apoptosis was abrogated in the presence of hesperadin. Similarly, under chronic hypoxia (5%), hesperadin blocked the proliferative and colony-forming actions of MST4 as well as the ability to activate specific downstream signaling and hypoxia-inducible factor-1 effectors. Together, these data identify hesperadin as the first potent, selective inhibitor of the MST4 kinase with the capacity to block pituitary tumor cell growth in a hypoxic microenvironment.

Differential somatostatin receptor (SSTR) 1-5 expression and downstream effectors in histologic subtypes of growth hormone pituitary tumors.

PURPOSE: The aim of this study was to examine whether differential expression of somatostatin receptors (SSTR) 1-5 and downstream effectors are different in densely (DG) and sparsely (SG) granulated histological growth hormone (GH) pituitary tumor subtypes. METHODS: The study included 33 acromegalic patients with 23 DG and 10 SG tumors. SSTR1-5 were measured by qPCR and immunoblotting. Signaling candidates downstream of SSTR2 were also assessed. RESULTS: SSTR2 mRNA and protein levels were significantly higher in DG compared to SG tumors. Downstream of SSTR2, p27(kip1) was decreased (2.6-fold) in SG compared to DG tumors, suggesting a potential mechanism of SSA resistance in SG tumors with intact SSTR2 expression. Re-expression of E-cadherin in GH pituitary cell increased p27(kip1) levels. CONCLUSIONS: Histological subtyping correlated with SSTR2, E cadherin and p27(kip) protein levels and these may serve as useful biomarkers in GH tumors to predict behavior and response to therapy with SSA.