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The Hippo pathway component WW domain-containing transcription regulator 1 (TAZ) is a transcriptional coactivator and an oncogene in breast and lung cancer. Transcriptional targets of TAZ that modulate immune cell function in the tumor microenvironment are poorly understood. Here, we perform a comprehensive screen for immune-related genes regulated by TAZ and its paralog YAP using NanoString gene expression profiling. We identify the immune checkpoint molecule PD-L1 as a target of Hippo signaling. The upstream kinases of the Hippo pathway, mammalian STE20-like kinase 1 and 2 (MST1/2), and large tumor suppressor 1 and 2 (LATS1/2), suppress PD-L1 expression while TAZ and YAP enhance PD-L1 levels in breast and lung cancer cell lines. PD-L1 expression in cancer cell lines is determined by TAZ activity and TAZ/YAP/TEAD increase PD-L1 promoter activity. Critically, TAZ-induced PD-L1 upregulation in human cancer cells is sufficient to inhibit T-cell function. The relationship between TAZ and PD-L1 is not conserved in multiple mouse cell lines, likely due to differences between the human and mouse PD-L1 promoters. To explore the extent of divergence in TAZ immune-related targets between human and mouse cells, we performed a second NanoString screen using mouse cell lines. We show that many targets of TAZ may be differentially regulated between these species. These findings highlight the role of Hippo signaling in modifying human/murine physiologic/pathologic immune responses and provide evidence implicating TAZ in human cancer immune evasion.Significance: Human-specific activation of PD-L1 by a novel Hippo signaling pathway in cancer immune evasion may have a significant impact on research in immunotherapy. Cancer Res; 78(6); 1457–70. ©2018 AACR.

This file contains supplementary figures summarizing control experiments that support conclusions from the main text. Figure S1: Candidates from our NanoString screen are not upregulated due to EMT or doxycycline treatment. Figure S2: CYR61 mRNA expression is regulated by modulation of the Hippo pathway. Figure S3: Clinical datasets support a relationship between TAZ, YAP and PD-L1. Figure S4: Pd-l1, Pd-l2, S1pr1 and Nlrp3 are differentially regulated by YAP/TAZ in human and mouse cell lines. Figure S5: Candidates from our mouse NanoString screen are not upregulated due to doxycycline treatment. This file also contains Supplementary Methods.

The identification of clinically significant genes recurrently mutated in myeloid malignancies necessitates expanding diagnostic testing with higher throughput, such as targeted next-generation sequencing. We present validation of the Thermo Fisher Oncomine Myeloid Next-Generation Sequencing Panel (OMP), targeting 40 genes and 29 fusion drivers recurrently mutated in myeloid malignancies. The study includes data from a sample exchange between two Canadian hospitals demonstrating high concordance for detection of DNA and RNA aberrations. Clinical validation demonstrates high accuracy, sensitivity, and specificity of the OMP, with a lower limit of detection of 5% for single-nucleotide variants and 10% for insertions/deletions. Prospective sequencing was performed for 187 samples from 168 unique patients presenting with suspected or confirmed myeloid malignancy and other hematological conditions to assess clinical impact of identifying variants. Of detected variants, 48% facilitated or clarified diagnoses, 29% affected prognoses, and 25% had the potential to influence clinical management. Of note, OMP was essential to identifying patients with premalignant clonal states likely contributing to cytopenias. We also found that the detection of even a single variant by the OMP assay, versus 0 variants, was predictive of overall survival, independent of age, sex, or diagnosis (P = 0.03). This study demonstrates that molecular profiling of myeloid malignancies with the OMP represents a promising strategy to advance molecular diagnostics.

Introduction: The ductus arteriosus (DA) connects the fetal pulmonary artery and aorta, diverting placentally oxygenated blood from the developing lungs to the systemic circulation. The DA constricts in response to increased oxygen (O 2 ) immediately post-partum, resulting in functional DA closure, with anatomic closure ensuing within the first days of life. Failure of DA closure results in patent ductus arteriosus (PDA), a common complication of extreme preterm birth. The DA response to O 2 , though modulated by the endothelium, is intrinsic to DA smooth muscle cells (DASMC). DA constriction is mediated by mitochondria-derived reactive oxygen species, which increase in proportion to PaO 2 . The molecular signature of the DASMC response to O 2 is not well understood. Methods & Results: DASMC were isolated from DA obtained from 10 infants at the time of congenital heart surgery. Cells were purified by flow cytometry, negatively sorted to eliminate fibroblasts (CD90+) or endothelial cells (CD31+). DASMC purity was confirmed by α-smooth muscle actin staining. Cells were grown for 96 hours in hypoxia (2.5% O 2 ) or normoxia (19% O 2 ) and confocal calcium imaging confirmed O 2 -responsiveness of all cell lines: O 2 exposure increased intracellular calcium 18.1%±4.4% and decreased cell length 27%±1.5% relative to hypoxic baseline. RNA sequencing (Seq) of the cells grown in hypoxia and normoxia revealed significant regulation of 1344 genes (corrected p2 , revealing enrichment of functional groups including mitochondria, cellular respiration and transcription. Multiple subunits of electron transport chain complex I, NDUF (NADH:ubiquinone oxidoreductase) family genes, are upregulated in hDASMC following normoxia. Upregulated NDUF genes were validated with qPCR (t-test p Conclusion: This first examination of the effects of O 2 on human DA transcriptomics supports a putative role for mitochondria as O 2 sensors, with mitochondrial subunits as potential future therapeutic targets for PDA.

The Ductus Arteriosus (DA) is a fetal vessel that connects the aorta to the pulmonary artery ensuring that placental oxygenated blood is diverted from the lungs to the systemic circulation. Following exposure to oxygen (O2), in the first few days of life, the DA responds with a functional closure that is followed by anatomical closure. Here, we study human DA smooth muscle cells (DASMC) taken from 10 term infants during congenital heart surgery. Purification of these cells using flow cytometry ensured a pure population of DASMCs, which we confirmed as responsive to O2. An oxygen-induced increase in intracellular calcium of 18.1%±4.4% and SMC constriction (-27%±1.5% shortening) occurred in all cell lines within five minutes. These cells were maintained in either hypoxia (2.5% O2), mimicking in utero conditions or in normoxia (19% O2) mimicking neonate conditions. We then used 3’ RNAsequencing to identify the transcriptome of DASMCs in each condition [1]. In this paper, we present the full differentially regulated gene list from this experiment.

The mechanisms by which patients with RUNX1 familial platelet disorder with propensity to myeloid malignancies (FPDMM) develop myeloid malignancies (MM) are not fully understood. We report the results of targeted next-generation sequencing on three patients with RUNX1 FPDMM who developed acute myeloid leukaemia or myelodysplastic syndromes (AML/MDS). DNA samples were collected from bone marrow, peripheral blood and buccal swabs at different time points. One patient had clonal haematopoiesis, represented by an SRSF2 p.P95R variant, prior to his AML diagnosis, when he developed an additional NRAS p.G12D variant. His sister presented to us with MDS, with a TET2 p.S471fs and identical NRAS p.G12D variant. The third patient, from another family, had an additional RUNX1 p.R204X and an NFE2 p.Q139fs variant at AML diagnosis. This constitutes the first report of NFE2 variants in AML without extramedullary disease and NRAS variants in AML/MDS in the setting of FPDMM. A systematic review of the literature including our findings distinguishes two genetic landscapes at AML transformation from FPDMM characterized by either the presence or absence of somatic abnormalities in RUNX1 with or without variants in genes usually associated with MM. Whether clonal haematopoiesis precedes transformation only in patients without somatic abnormalities in RUNX1 needs further confirmation.

Assessment of cardiac function is essential to conduct cardiovascular and pulmonary-vascular preclinical research. Pressure-volume loops (PV loops) generated by recording both pressure and volume during cardiac catheterization are vital when assessing both systolic and diastolic cardiac function. Left and right heart function are closely related, reflected in ventricular interdependence. Thus, recording biventricular function in the same animal is important to get a complete assessment of cardiac function. In this protocol, a closed chest approach to cardiac catheterization consistent with the way catheterization is performed in patients is adopted in mice. While challenging, the closed chest strategy is a more physiological approach, because opening the chest results in major changes in preload and afterload that create artifacts, most notably a fall in systemic blood pressure. While high-resolution echocardiography is used to assess rodents, cardiac catheterization is invaluable, particularly when assessing diastolic pressures in both ventricles. Described here is a procedure to perform invasive, closed chest, sequential left and right ventricular pressure-volume (PV) loops in the same animal. PV loops are acquired using admittance technology with a mouse pressure-volume catheter and pressure-volume system acquisition. The procedure is described, beginning with the neck dissection, which is required to access the right jugular vein and the right carotid artery, to the insertion and positioning of the catheter, and finally the data acquisition. Then, the criteria required to ensure the acquisition of high-quality PV loops are discussed. Finally, the analysis of the left and right ventricular PV loops and the different hemodynamic parameters available to quantify systolic and diastolic ventricular function are briefly described.

Background: Pulmonary arterial hypertension (PAH) is a lethal vasculopathy. Hereditary cases are associated with germline mutations in BMPR2 and 16 other genes; however, these mutations occur in TET2 ), a key enzyme in DNA demethylation, occur in cardiovascular disease and are associated with clonal hematopoiesis, inflammation, and adverse vascular remodeling. The role of TET2 in PAH is unknown. Methods: To test for a role of TET2 , we used a cohort of 2572 cases from the PAH Biobank. Within this cohort, gene-specific rare variant association tests were performed using 1832 unrelated European patients with PAH and 7509 non-Finnish European subjects from the Genome Aggregation Database (gnomAD) as control subjects. In an independent cohort of 140 patients, we quantified TET2 expression in peripheral blood mononuclear cells. To assess causality, we investigated hemodynamic and histological evidence of PAH in hematopoietic Tet2 -knockout mice. Results: We observed an increased burden of rare, predicted deleterious germline variants in TET2 in PAH patients of European ancestry (9/1832) compared with control subjects (6/7509; relative risk=6; P =0.00067). Assessing the whole cohort, 0.39% of patients (10/2572) had 12 TET2 mutations (75% predicted germline and 25% somatic). These patients had no mutations in other PAH-related genes. Patients with TET2 mutations were older (71±7 years versus 48±19 years; P P =0.02), and had increased inflammation (including elevation of interleukin-1β). Circulating TET2 expression did not correlate with age and was decreased in >86% of PAH patients. Tet2 -knockout mice spontaneously developed PAH, adverse pulmonary vascular remodeling, and inflammation, with elevated levels of cytokines, including interleukin-1β. Long-term therapy with an antibody targeting interleukin-1β blockade resulted in regression of PAH. Conclusions: PAH is the first human disease related to potential TET2 germline mutations. Inherited and acquired abnormalities of TET2 occur in 0.39% of PAH cases. Decreased TET2 expression is ubiquitous and has potential as a PAH biomarker.

Pulmonary arterial hypertension (PAH) is a lethal vasculopathy. Hereditary cases are associated with germline mutations inTo test for a role ofWe observed an increased burden of rare, predicted deleterious germline variants inPAH is the first human disease related to potential

The Hippo pathway component WW domain-containing transcription regulator 1 (TAZ) is a transcriptional coactivator and an oncogene in breast and lung cancer. Transcriptional targets of TAZ that modulate immune cell function in the tumor microenvironment are poorly understood. Here, we perform a comprehensive screen for immune-related genes regulated by TAZ and its paralog YAP using NanoString gene expression profiling. We identify the immune checkpoint molecule PD-L1 as a target of Hippo signaling. The upstream kinases of the Hippo pathway, mammalian STE20-like kinase 1 and 2 (MST1/2), and large tumor suppressor 1 and 2 (LATS1/2), suppress PD-L1 expression while TAZ and YAP enhance PD-L1 levels in breast and lung cancer cell lines. PD-L1 expression in cancer cell lines is determined by TAZ activity and TAZ/YAP/TEAD increase PD-L1 promoter activity. Critically, TAZ-induced PD-L1 upregulation in human cancer cells is sufficient to inhibit T-cell function. The relationship between TAZ and PD-L1 is not conserved in multiple mouse cell lines, likely due to differences between the human and mouse PD-L1 promoters. To explore the extent of divergence in TAZ immune-related targets between human and mouse cells, we performed a second NanoString screen using mouse cell lines. We show that many targets of TAZ may be differentially regulated between these species. These findings highlight the role of Hippo signaling in modifying human/murine physiologic/pathologic immune responses and provide evidence implicating TAZ in human cancer immune evasion.Significance: Human-specific activation of PD-L1 by a novel Hippo signaling pathway in cancer immune evasion may have a significant impact on research in immunotherapy. Cancer Res; 78(6); 1457–70. ©2018 AACR.

Immune dysregulation is a common feature of myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia (CMML), particularly in early stages. However, the genetic basis remains poorly understood. We recently reported that macrophages from mice deficient in tet methylcytosine dioxygenase 2 (Tet2), a model of MDS/CMML, are hyperinflammatory and have increased expression of arginase 1 (Arg1). In macrophages and myeloid derived suppressor cells (MDSCs) expression of Arg1 contributes to T-cell suppression and immune evasion by L-arginine depletion, in the setting of chronic inflammation and cancer. Since human MDS and CMML are driven by TET2 mutations and associated with chronic inflammation, we hypothesized that arginase enzymatic activity and ARG1 expression would be increased in human MDS/CMML bone marrow. Elevated arginase activity was observed in bone marrow mononuclear cells of MDS and CMML patients with lower-grade features. Immunohistochemical studies confirmed that myelomonocytic cells overexpress ARG1. Additionally, mutations in the epigenetic regulators TET2 and DNMT3A corresponded to high ARG1 expression and activity. These findings suggest ARG1 is a biomarker of immune dysregulation in early MDS and CMML. Recent murine findings have implicated Tet2 and Dnmt3a in regulation of innate immunity. Our study suggests similar changes may be driven by human TET2 and DNMT3A mutations.

Recent studies suggest that lung cancer stem cells (CSCs) may play major roles in lung cancer. Therefore, identification of lung CSC drivers may provide promising targets for lung cancer. TAZ is a transcriptional co-activator and key downstream effector of the Hippo pathway, which plays critical roles in various biological processes. TAZ has been shown to be overexpressed in lung cancer and involved in tumorigenicity of lung epithelial cells. However, whether TAZ is a driver for lung CSCs and tumor formation in vivo is unknown. In addition, the molecular mechanism underlying TAZ-induced lung tumorigenesis remains to be determined. In this study, we provided evidence that constitutively active TAZ (TAZ-S89A) is a driver for lung tumorigenesis in vivo in mice and formation of lung CSC. Further RNA-seq and qRT-PCR analysis identified Aldh1a1, a well-established CSC marker, as critical TAZ downstream target and showed that TAZ induces Aldh1a1 transcription by activating its promoter activity through interaction with the transcription factor TEAD. Most significantly, inhibition of ALDH1A1 with its inhibitor A37 or CRISPR gene knockout in lung cancer cells suppressed lung tumorigenic and CSC phenotypes in vitro, and tumor formation in mice in vivo. In conclusion, this study identified TAZ as a novel inducer of lung CSCs and the first transcriptional activator of the stem cell marker ALDH1A1. Most significantly, we identified ALDH1A1 as a critical meditator of TAZ-induced tumorigenic and CSC phenotypes in lung cancer. Our studies provided preclinical data for targeting of TAZ-TEAD-ALDH1A1 signaling to inhibit CSC-induced lung tumorigenesis in the future.

Aging hematopoietic stem cells acquire mutations that sometimes impart a selective advantage. Next-generation DNA sequencing (NGS) can be used to detect expanded peripheral blood progeny of a mutant clone, usually carrying just one cancer-driver mutation, most often in the epigenetic regulator genes, DNMT3A or TET2. This phenomenon is known as clonal hematopoiesis (CH), age-related CH (ARCH) when considering its association with age, and CH of indeterminate potential (CHIP) when the variant allele fraction (VAF) is at least 2% in peripheral leukocytes. CHIP is present in at least 10-15% of adults older than 65 years and is a risk factor for hematological neoplasms and diseases exacerbated by mutant, hyper-inflammatory, monocytes/macrophages, such as atherosclerotic cardiovascular disease. Therefore, the detection of CHIP has important clinical consequences. Herein, we present a protocol for the generation of targeted, amplicon-based, NGS libraries for ion semiconductor sequencing and CHIP detection, using Ion Torrent platforms.

Aging hematopoietic stem cells acquire mutations that sometimes impart a selective advantage. Next-generation DNA sequencing (NGS) can be used to detect expanded peripheral blood progeny of a mutant clone, usually carrying just one cancer-driver mutation, most often in the epigenetic regulator genes, DNMT3A or TET2. This phenomenon is known as clonal hematopoiesis (CH), age-related CH (ARCH) when considering its association with age, and CH of indeterminate potential (CHIP) when the variant allele fraction (VAF) is at least 2% in peripheral leukocytes. CHIP is present in at least 10-15% of adults older than 65 years and is a risk factor for hematological neoplasms and diseases exacerbated by mutant, hyper-inflammatory, monocytes/macrophages, such as atherosclerotic cardiovascular disease. Therefore, the detection of CHIP has important clinical consequences. Herein, we present a protocol for the generation of targeted, amplicon-based, NGS libraries for ion semiconductor sequencing and CHIP detection, using Ion Torrent platforms.

Tet methylcytosine dioxygenase 2 (TET2) is one of the earliest and most frequently mutated genes in clonal hematopoiesis of indeterminate potential (CHIP) and myeloid cancers, including myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia (CMML). TET2 catalyzes the oxidation of 5-methylcytosine to 5-hydroxymethylcytosine, leading to DNA demethylation, and also affects transcription by recruiting histone modifiers. Inactivating TET2 mutations cause epigenetic dysregulation, clonal hematopoietic stem cell (HSC) dominance, and monocytic lineage skewing. Here, we found that Tet2 was the most highly expressed Tet enzyme in murine macrophage (MΦ) differentiation. Tet2 transcription was further induced by lipopolysaccharide (LPS), but not interleukin (IL)-4, stimulation, potentially in a nuclear factor κβ-dependent manner. Tet2 loss did not affect early LPS gene responses in vitro, but increased Il-1b, Il-6, and Arginase 1 (Arg1) mRNA expression at later stages of stimulation in bone-marrow-derived MΦs (BMMΦs). Tet2-deficient peritoneal MΦs, however, demonstrated profound, constitutive expression of LPS-induced genes associated with an inflammatory state in vivo. In contrast, Tet2 deficiency did not affect alternative MΦ gene expression significantly in response to IL-4. These results suggested impaired resolution of inflammation in the absence of Tet2 both in vitro and in vivo. For the first time, we also detected TET2 mutations in BMMΦs from MDS and CMML patients and assayed their effects on LPS responses, including their potential influence on human IL-6 expression. Our results show that Tet2 restrains inflammation in murine MΦs and mice, raising the possibility that loss of TET2 function in MΦs may alter the immune environment in the large elderly population with TET2-mutant CHIP and in TET2-mutant myeloid cancer patients.

BACKGROUND: Clonal hematopoiesis of indeterminate potential (CHIP) involves the peripheral blood (PB) expansion of progeny of a hematopoietic stem or progenitor cell that is somatically mutated in a hematological cancer-associated gene (most often TET2 or DNMT3A). CHIP associates with comorbid diseases of aging such as cardiovascular disease. Murine knockout (Tet2 or Dnmt3a) and engraftment models of CHIP develop exacerbated cardiovascular disease and their mutated myeloid cells are more reactive to inflammatory stimuli. However, whether blood leukocytes in human CHIP are hyper-inflammatory remains speculative. We recently found people with CHIP have higher serum levels of certain pro-inflammatory cytokines and chemokines than controls (Cook et al, ASH 2017). Thus, we hypothesized that PB effector cells in people with CHIP will be enriched for pro-inflammatory gene expression and pathways. METHODS: The presence of CHIP (variant allele frequency, VAF>0.02) was determined in the whole PB of 30 hematologically healthy adults >65 years old at Baycrest and Sunnybrook Health Sciences Centers (Toronto, Canada) using Ion Proton DNA sequencing targeting 48 commonly mutated genes in myeloid neoplasms. RNA-Seq (HISeq 4000, Illumina, 75bp paired-end sequencing reads with a depth of >50 million/sample) was performed on corresponding ribo-depleted whole PB samples (PAXgene), reads were aligned with HISAT2, gene counts quantified with featureCount, and analyzed with DESeq2. FDR RESULTS: People with CHIP ("CHIP+", n: males=8, females=13; TET2=12, DNMT3A=8, SF3B1=1; VAF range=0.03-0.40) compared to those without CHIP ("CHIP-", n: males=3, females=6) had six significantly downregulated genes (e.g. GZMM) and 10 upregulated genes (e.g. DEFA4, LTF, MPO, see Figure 1A). Hierarchical clustering of these top genes yielded two groups, one consisting of most of the CHIP- cases (8/9 cases, in a cluster of 11, see Figure 1A). The three CHIP+ cases that clustered with CHIP- had VAFs lower than 0.15. Of the 16 differentially regulated genes between CHIP+ and CHIP-, nine were recognized by reactome, and most overlapped (≥6 genes) with pathways involving neutrophil degranulation and innate immunity (Figure 1B). DEFA4, LTF, CRISP3, BPI and MPO specifically encode components of neutrophil granules, with various anti-microbial and homeostatic functions. However, mean neutrophil counts (4.6±1.6 vs. 4.4±1.6 10^9/L for CHIP+ vs. CHIP-) and neutrophil to lymphocyte ratios (3.2±1.4 vs. 2.8±2.1 in CHIP+ vs. CHIP-) did not significantly differ between the groups. This suggests that mutations of CHIP may affect neutrophil/immune-related function or phenotype, potentially contributing to comorbid disease. For example, greater expression of alpha-defensins (i.e. DEFA4) in CHIP may involve dysregulated granulocyte maturation and inflammatory function as seen in myelodysplasia (Droin et al, 2010 Blood), suggesting a potential dysregulation of inflammation and immunity. Higher VAFs (>0.15) associated with higher ECOG scores (poorer overall daily functioning: odds ratio=44, 95% CI=4-500, p=0.002), suggesting that larger proportions of mutated cells may have greater effects on gene expression profiles. Accordingly, there were linear correlations between the VAFs of the mutated cell populations and the levels of differentially expressed genes (Figure 1C). CONCLUSIONS: The connection between mutant clones of CHIP and disease remains poorly elucidated. For the first time, to our knowledge, we studied gene expression in CHIP leukocytes. We report that the most prominent gene expression differences between people with CHIP and those without CHIP involve neutrophil degranulation and the innate immune system. Additionally, higher VAFs may have a greater influence on gene expression levels and health than lower VAFs. We plan to validate these candidate genes in a larger cohort. These novel data warrant further investigation of the cellular pathways perturbed by somatic mutations of CHIP. Disclosures Buckstein: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Introduction: The expanding diagnostic and clinical significance of genes associated with myeloid malignancies necessitates higher throughput testing. Targeted next-generation sequencing (NGS) of DNA and RNA may be an ideal approach to capture these variants and eventually streamline, or replace, conventional cytogenetic testing. We present our clinical validation of the OncomineTM Myeloid NGS assay and early experience regarding the clinical impact in patients with suspected or known myeloid malignancies. Methods: OncomineTM Myeloid (Thermo Fisher Scientific) panel (OMP) validation was conducted at Kingston Health Sciences Centre (KHSC) on anonymized and commercial samples according to IQMH and ACMG standards for NGS (PMID: 23887774). As of April 2018 the test has been formally validated and offered at KHSC. Prior to this, patients were consented at the time of blood (PB) or bone marrow (BM) collection for research-based testing. BM and PB samples were collected, gDNA and RNA extracted, and libraries prepared using the Ion ChefTM System. Sequencing was completed using the OMP on Ion PGM (research phase) and S5XL (clinical) sequencers. The OMP uses amplicon-based semiconductor sequencing technology to cover 40 DNA genes recurrently mutated in myeloid malignancies (23 hotspots and 17 full coding), 29 RNA fusion driver genes and 10 gene expression targets. Sequencing data were analyzed using Torrent Suite, Torrent Variant Caller, Ion Reporter (v.5.2-5.6) and Alamut software. The OMP includes a Torrent Suite FLT3-ITD "plug-in" and Ion Reporter "Oncomine filter". Orthogonal clinical assays were performed where necessary for additional confirmation. Variants were interpreted based on a 4-tier classification system developed by Li et al (PMID: 27993330). Variants in the top 3 tiers were reported after assessment by at least one clinician and one certified medical geneticist. Actionability was assessed in the context of electronic diagnostic and clinical information and molecular board review. Results: Validation involved 24 paired DNA-RNA samples previously run at KHSC on an early access OMP version and also at an external site (kind exchange by Dr. N. Carson, Saint John, NB), along with SeraCareTM DNA and RNA controls. Validation run metrics were as follows: DNA means (mapped reads 1306216, read length 226bp, uniformity 98%, depth 2518), RNA means (mapped reads 260350, read length 114bp). Complete concordance was found for RNA variants while the DNA filter chain was only modified to better capture a JAK2 exon 12 variant in a SeraCare control. The minimum depth of coverage was subsequently set to 400X for somatic DNA variants and sensitivity set at 5% variant allele fraction (VAF) for clinical reporting. To date, we have performed sequencing for 38 samples from 35 unique patients (avg age = 64.5y). Of these, 12/35 (34%) involved suspected or known myelodysplastic syndromes (MDS), 12/35 (34%) with acute myeloid leukemia (AML), 4/35 (12%) with query myeloproliferative neoplasms (MPNs), 2/35 (5%) with MDS/MPN, and 5/35 (14%) other (2 B-ALL, 1 aplastic, 1 hairy cell + ?MDS, 1 ?CHIP). OMP detected 56 variants (in 80% of cases), including 43% tier 1, 48% tier 2, and 9% tier 3 variants. The most frequently mutated genes included TET2 (21%), SRSF2 (14%), SF3B1 (9%), TP53 (5%), and RUNX1 (5%). The average VAF was 36% for all samples, with an average of 1.5 variants per sample. Of detected variants, 73% were potentially actionable. Specifically, 51% of actionable variants facilitated or clarified diagnosis (38% of total variants), 32% (23%) affected prognosis, and 17% (13%) had the potential to affect treatment decisions. Among 10 patients with suspected (but subthreshold for) MDS or MPN, clonality was detected in 5, while 5 had no variants, prompting further investigation of reversible cytopenia or cytosis. We are rapidly accruing patients and expect to have OMP profiles for at least 100 patients at the time of the ASH 2018 meeting. Conclusions: Oncomine Myeloid DNA and RNA molecular profiling has found greatest clinical use at our centre in the realm of MDS and AML. In the majority of patients, OMP permits the identification of actionable variants that may aid clinicians in achieving or clarifying diagnoses, inform follow-up and prognosis, and guide treatment decisions. The OMP represents a promising strategy to address the challenge of expanding, important molecular features of myeloid malignancies. Disclosures No relevant conflicts of interest to declare.

The Hippo signaling pathway has recently emerged as a cellular network that is dysregulated in cancer. In breast cancer cells, aberrant activation of the Hippo transducers (and transcriptional co-activators) TAZ and YAP leads to altered expression of their downstream gene targets and endows cells with multiple “hallmarks of cancer”. While several transcriptional targets of TAZ and YAP underlying their pro-tumorigenic activity have been described (e.g. CTGF, CYR61), gene targets of TAZ and YAP that modulate immune cell behavior in the tumor microenvironment are poorly understood. We have performed a comprehensive screen for immune-related transcriptional targets of TAZ and YAP in human breast cells. We have identified many candidate genes that are potentially regulated by TAZ and YAP including the immune checkpoint molecules PD-L1 and PD-L2, the lymphocyte regulator S1PR1 and the inflammasome component NLRP3. We have further validated PD-L1 as a bona fide transcriptional target of TAZ and YAP. These findings reveal new functions for the Hippo pathway in modifying immune responses and implicate TAZ and YAP in cancer immune evasion. Citation Format: Helena J. Janse van Rensburg, Taha Azad, Min Ling, Yawei Hao, Lori M. Minassian, Brooke Snetsinger, Prem Khanal, Michael J. Rauh, Charles H. Graham, Xiaolong Yang. Screen for immune-related transcriptional targets of the Hippo pathway in human breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5434.

Introduction: Immune dysregulation is a common feature of myeloid cancers including MDS and CMML. Precisely how cells such as macrophages (MΦs) contribute to or are impacted by aberrant innate immunity is not fully understood. Previously, our group demonstrated Arginase 1 (Arg1) overexpression in MDS/CMML patient samples as well as in a murine model of Tet2-deficiency (Cull et al., Blood Abstract 2013: 1578). Arg1, an L-arginine metabolizing enzyme, is highly expressed by alternatively activated (M2) macrophages and myeloid-derived suppressor cells. In the current study, our goals were to (1) establish whether the presence of certain mutations (i.e. in TET2) corresponds with high Arg1 immunohistochemical (IHC) staining, and (2) determine the environmental vs. cell-intrinsic effects of Tet2-deficiency on Arg1 expression in murine peritoneal macrophages (PMΦs). Methods: H&E staining and anti-human Arg1 IHC was performed on FFPE BM biopsies obtained from Sunnybrook and Kingston General Hospitals (8 age-matched controls and 30 MDS/CMML cases including 9 RCMD, 5 RAEB-1, 2 RAEB-2, 2 RARS, 1 RA, 1 del(5q) and 10 CMML-1). IHC staining was scored independently by 2 blinded Hematopathologists. Genomic DNA from each patient was subjected to mutation profiling using a 1,552-amplicon Ion Torrent Sequencing Panel. Primary PMΦ were obtained from peritoneal lavages performed on hematopoietic-specific Vav1-Cre-driven Tet2 knockout (Tet2-/-) mice generated from parental floxed (Tet2f/f) and Vav1-Cre mice (JAX) in accordance with Queen's Animal Care protocols. Results: Following an initial pilot study, we generated Arg1 IHC data and corresponding genome sequences from 8 age-matched control and 30 MDS/CMML patient FFPE BM biopsies. In keeping with our previous findings, we observed increased BM biopsy Arg1 IHC expression in CMML and low-grade MDS for both hospital cohorts. Taking all patient samples together, we found a significant correlation between high Arg1 IHC status and the presence of either TET2 or DNMT3A mutations (Fisher's exact test, p-value = 0.0173), both of which are highly prevalent in MDS and CMML patients. While this result - that mutations in a DNA methyltransferase and a DNA demethylating protein could lead to increased Arg1 protein expression and ultimately a similar disease phenotype - may appear counterintuitive, a recent publication provided evidence that Tet2 and Dnmt3a act cooperatively at specific sites within the genome (PMID 27428748). In addition, we noted upregulation of Ccr5, Tgfbi, Socs3, Bst2, Ccl6, Ifit2, Ccl2, Cxcl1, Arg1 and Ccl7 in Dnmt3a knockout vs. wild-type mouse PMΦs (PMID 27240213). Intriguingly, our results showed that all of these genes were also upregulated in Tet2-/- knockout PMΦs. Taken together, this suggests a biological basis linking high Arg1 IHC levels with mutations in TET2 and DNMT3A. In order to study environmental influences that may impact Arg1 expression, we isolated PMΦs from Tet2f/f and Tet2-/- mice and treated these cells with peritoneal lavage fluid derived from both control and Tet2-knockout animals. We observed a 10.2-fold increase in Arg1 mRNA expression in Tet2f/f cells treated with Tet2-/- peritoneal lavage fluid. Moreover, Tet2-/- lavage fluidtreatment of Tet2-/- PMΦs induced a 2.2-fold increase in Arg1 expression (from 5.8- to 12.8-fold compared to untreated Tet2f/f control cells). Given these findings, we hypothesized that Tet2-/- peritoneal lavage fluid contains a factor(s) which leads to induction of Arg1. In a pilot experiment, Tet2f/f and Tet2-/- lavage fluid (n=3/genotype) samples were sent for 31-plex cytokine/chemokine array (Eve Technologies). Levels of the pro-inflammatory molecules IL-12 (p70) and MIG (CXCL9) were elevated in knockout lavage fluid versus controls, both demonstrating trends towards significance. While Arg1 upregulation may be occurring in response to aberrant inflammation, we have also observed that PMΦs can be cultured ex vivo over a period of at least 72h all the while maintaining elevated Arg1 expression. This suggests a cell-intrinsic mechanism by which these cells maintain high levels of Arg1 transcript independent of the peritoneal microenvironment. Conclusion: Arg1 overexpression in both TET2/DNMT3A mutant human MDS/CMML and Tet2-deficient mouse models points to the potential utility of this enzyme as a marker of aberrant inflammation. Disclosures Buckstein: Celgene: Honoraria, Research Funding; Novartis: Honoraria.

Introduction: The epigenetic regulator, TET2, catalyzes the conversion of methylcytosine to 5-hydroxymethylcytosine. Inactivating TET2 mutations are common in myeloid cancers such as chronic myelomonocytic leukemia (CMML). Although TET2 has been characterized in hematopoietic stem and progenitor cells, little is known about its role in disease-relevant monocytes/macrophages (MΦ). Previously, we found increased expression of M2 MΦ-associated arginase 1 (Arg1) in TET2 -mutant CMML and Tet2 -deficient MΦ. Therefore, our goals were to (1) characterize Tet family expression during normal murine MΦ differentiation and polarization, (2) determine the effect of Tet2 -deficiency on broader M1-M2 MΦ spectrum gene signatures. Methods: Hematopoietic-specific Tet2+/- and Tet2-/- knockout mice were generated by breeding floxed Tet2(f/f) with Vav-Cre mice (JAX), in accordance with Queen's University's Animal Care protocols. MΦs obtained by peritoneal lavage (PMΦ) and bone marrow differentiation (BMMΦ) from 9-13 week old Tet2-/- and 20-40 week old Tet2+/- mice were treated with an M1 stimulus (100ng/mL LPS) or an M2 stimulus (10ng/mL Il-4). Comparative gene expression analysis was conducted using a 591 candidate gene Mouse Immunology Gene Expression CodeSet (NanoString). Blood plasma samples collected from Tet2f/f and Tet2-/- mice were sent for cytokine/chemokine array analysis (Eve Technologies). Results: A survey of Tet mRNA expression in wild-type C57BL/6 mouse whole BM showed that Tet1 was most abundantly expressed, with Tet2 and Tet3 having relative abundances of 0.56±0.05 and 0.09±0.01 respectively. In contrast, Tet2 expression peaked, while Tet1 expression diminished during BMMΦ differentiation. Suggesting a functional role, loss of murine Tet2 is associated with skewed myelomonocytic differentiation (i.e. CMML phenotype). In terminally-differentiated MΦ, Tet2 was the most abundantly expressed Tet gene, suggesting MΦ-specific functions. Consistent with this, following a 3-hour LPS stimulation, Tet2 mRNA levels increased 2- to 4-fold, whereas Il-4 failed to induce a similar increase in expression. Overall, our results suggested that Tet2 plays a role in M1 but not M2 macrophage polarization. Based on these findings, we hypothesized that loss of Tet2 would lead to M1 program dysregulation. PMΦs were obtained from Tet2f/f and Tet2-/- mice (n=2/ genotype) and RNA was harvested from untreated and LPS- or Il-4-treated cells. Pools of these RNA samples were then screened using Nanostring. Overall, M1-associated markers such as Stat1, Socs1, Nfkbiz, Il-6, Il-27, Il-12, Il-1 and Ccl2 were markedly increased by 2- to 50-fold in resting Tet2-/- PMΦs compared to matched Tet2f/f samples. These same M1 genes demonstrated a reduced ability to be induced by LPS treatment. We also found that while the expression of most M2 genes was similar in controls versus knockouts, Il-1rn and Arg1 were overexpressed, and Marco was decreased. This suggested that Tet2 -deficient MΦs possess a complex phenotype with a potential homeostatic response to M1 gene dysregulation. We have previously seen variable upregulation of Arg1 in mouse BMMΦs and PMΦs. Approximately 60% of Tet2-deficient mice (+/- and -/-) (n=20) tested for MΦ Arg1 mRNA expression demonstrated 2- to 90-fold increases in Arg1 compared to pooled Tet2f/f controls (n=5). We were interested in investigating the underlying mechanisms contributing to this dramatic increase in expression. Using Nanostring on pooled Tet2-deficient PMΦs with low (n=7) or high (n=8) Arg1 mRNA expression, we were able to identify genes whose expression significantly correlated with Arg1 overexpression: Cxcl3 (p=0.0329), Ppbp (p=0.0015), Cxcl1 (p=0.0104) and Ccl6 (p=0.0185). Of note, Ppbp was the most divergently expressed gene (46-fold difference) in Arg1 low vs Arg1 high macrophages, followed by Arg1 itself (14-fold difference). Suggesting a further environmental influence, blood plasma levels of TNF-alpha, Il-1b, Il-4, Il-10, Il-12 and Il-13 were significantly elevated in mice with high PMΦ Arg1 mRNA expression (n=5) compared to those with low expression (n=10). Conclusions: Tet2 is a novel regulator of murine MΦ, induced during MΦ differentiation and M1-polarization. Tet2 loss leads to complex disruption of the M1-M2 spectrum. We are currently exploring whether human TET2 mutations contribute to the abnormal immune environment of myeloid cancers. Disclosures No relevant conflicts of interest to declare.

Introduction: When investigations for myelodysplastic syndrome (MDS) reveal sub-threshold dysplasia and a normal karyotype, patients may be labeled with equivocal/suspicious/idiopathic cytopenia or dysplasia, discharged and lost to follow-up, or subjected to serial bone marrow investigations and diagnostic delays. Although not part of the current WHO classification, targeted somatic mutation profiling increases clonality detection in MDS and clonal hematopoiesis of indeterminate potential (CHIP). Hypothesis: The application of targeted DNA sequencing to suspicious MDS cases may improve diagnostic yield, inform the natural history of MDS-like CHIP, and shift the paradigm to earlier intervention. Methods: With IRB approval, frozen BM mononuclear cells or air-dried smears were obtained from Kingston General & Sunnybrook Hospitals. These included: a) 16 age-matched controls (8 negative lymphoma staging; 8 non-MDS cytopenia), b) 18 BM suspicious for MDS, c) 20 diagnosed MDS & d) 16 MDS/MPN. Genomic DNA was subjected to mutation profiling of 589 coding regions in 48 recurrently-mutated genes using a 1,662-amplicon Ion Torrent sequencing panel at Queen's University (QGLO). Variant calling was performed with Ion Reporter v4.6 (Life Technologies) & Integrative Genomics Viewer (Broad Institute). Selected variants were confirmed by PCR/Sanger-sequencing. Statistical analysis was conducted using Prism GraphPad software. Results: Clinical characteristics of suspicious MDS: Compared to age-matched controls (n=16), suspicious MDS cases (n=18) were associated with significantly reduced mean hemoglobin (119.6 ± 3.7 vs 96.0 ± 3.1 g/l; p Mutation profiles: We found suspected mutations in 14/18 (78%) of suspicious cases, 17/20 (85%) of MDS & 16/16 (100%) of MDS/MPN. While the mean number of mutations per suspicious patient (1.33 ± 0.3; most commonly in SF3B1, TET2, DNMT3A and ASXL1) was significantly lower than MDS (2.15 ± 0.3; p=0.03) & MDS/MPN (3.75 ± 0.4; p CHIP in control subjects and suspicious cases: CHIP was also detectable in 31% of control marrows (5/16) drawn from negative lymphoma staging & non-MDS cytopenia, albeit with significantly lower mutational and clonal burdens than suspicious MDS (0.31 ± 0.12 mutations/patient, p=0.002; 23 ± 0.1% avg. VAF, p=0.049). Among control & suspicious cases, CHIP (n=19) was associated with a 13.3 g/l reduction in mean hemoglobin (p=0.03). SRSF2, SETPB1, CBL & PTNPN11 mutations were statistically absent or under-represented in CHIP (as compared to MDS+MDS/MPN), with similar trends for EZH2, RUNX1, & TP53 mutations. CHIP versus low-grade MDS: Importantly, given the diagnostic uncertainty between CHIP & low blast count, normal karyotype MDS (i.e. hinges on the presence of 10% dysplasia), we next stratified our control/suspicious & diagnostic low-grade MDS cases by the presence or absence of detectable mutations. The control/suspicious group (n=34) included 19 patients with & 15 patients without mutations; low-grade MDS (n=12) included 9 mutant & 3 non-mutant. Ignoring whether 10% subjective dysplasia was present in this cohort, using Kaplan-Meier analysis we found the presence of a mutation was associated with reduced median survival (677 days, n=28) versus the absence of a mutation (median survival undefined, n=18) (p=0.045 by Gehan-Breslow-Wilcoxon test). Conclusions: CHIP is commonly detected in suspicious but non-MDS-diagnostic cases (78%). While the average clone size (42% VAF) is indistinguishable from MDS & MDS/MPN, CHIP-positive suspicious marrows have a reduced & more restricted mutation profile. CHIP is associated with increased anemia & worse outcome. These findings suggest clinical utility for mutation profiling in suspicious MDS cases & call for larger prospective studies of the natural history of MDS-like CHIP. Disclosures No relevant conflicts of interest to declare.

Introduction Among FVIII-treated hemophilia A (HA) patients, 25-30% develop FVIII antibodies that inhibit its pro-coagulant function. However, the etiology of FVIII inhibitor formation remains poorly understood, particularly the role played by myeloid innate immune cells. Myeloid-derived suppressor cells (MDSC – CD11b/Gr1 co-expressing, immunosuppressive myeloid cells found in peripheral blood, lymphoid tissue and bone marrow) are expanded in cancer and inhibit adaptive immune responses against tumors. MDSC also mediate organ transplant tolerance. Therefore, we investigated MDSC dynamics during the course of FVIII exposure in HA mice and the potential to harness MDSC as a novel means of mediating FVIII tolerance. Methods 6 to 12 week-old F8-knockout Balb/c hemophilia A mice (HA mice) were used in accordance with Queen's University Animal Care Committee protocols. Over 3 independent experiments, HA mice were either untreated (Week 0; n = 4-6) or tail vein infused weekly for 2 or 4 weeks with 2 IU rhFVIII (Kogenate-FS for 2 experiments or Advate for one; n = 6 and 7, at 2 and 4 weeks) or 200 μl HBSS vehicle alone (n = 4 and 5). Alternatively HA mice were infused weekly with 2 pdFVIII (Wilate) for 2 (n = 2) or 4 weeks (n = 2) or subjected to a 4-day G-CSF preconditioning regimen during Week minus-1 (10 μg/day, SC, in 200 μl HBSS; n = 4) or HBSS alone (n = 4), followed by 2 or 4 weekly rFVIII infusions. Red cell-lysed blood, spleen and bone marrow suspensions were subjected to MDSC flow cytometry using anti-CD11b(Mac1)-APC and anti-Gr1-PE (Miltenyi Biotec). Week 2 and 4 plasma was subjected to anti-FVIII antibody ELISA and Bethesda assays. Means were compared using student's t-test. Results HA mice contained CD11b+Gr1+ MDSC in expected proportions in the blood, bone marrow and spleen. Peripheral blood MDSC proportions declined significantly from baseline following 4 weeks of rFVIII infusions (baseline mean 12.6%, versus 5.9% at 4 weeks, p = 0.041) (Figure 1A). More striking, however, was the observation of diminishing CD11b expression within all three MDSC compartments during the course of rFVIII exposure (magnitude of reduction: 46-65%; at 4 weeks in blood, p = 0.0011; at 2 and 4 weeks in bone marrow, p = 0.0014 and 0.0002; spleen, p = 0.057 and Conclusions For the first time, to our knowledge, we assessed the dynamics of endogenous MDSC in FVIII-treated HA mice, revealing decreased circulating MDSC after 4 weeks of rFVIII exposure, and diminished CD11b expression in all examined MDSC compartments. We hypothesize this phenomenon leads to impaired MDSC function, necessary to mount an optimal adaptive immune response to FVIII, although this awaits further confirmation. Finally, our studies suggest it is feasible to expand MDSC ex vivo from na•ve HA bone marrow for future adoptive transfer experiments, and to expand endogenous HA MDSC using growth factors such as G-CSF, as novel investigative approaches to mediating FVIII tolerance. If warranted by murine studies, G-CSF may be an attractive novel immune tolerance strategy, given extensive clinical experience with human G-CSF administration. Disclosures: No relevant conflicts of interest to declare.

Introduction Our group and others have reported the murine SHIP1-deficient mouse model of MDS/CMML is characterized by expansion of immunosuppressive, arginase 1 (Arg1)-positive M2-macrophages and myeloid-derived suppressor cells (MDSC) (Rauh et al., Immunity, 2005). Moreover, translational studies confirmed increased Arg1 in bone marrow aspirate cells of subsets of MDS and CMML patients (Rauh et al., Blood Abstract 2010: 1855), although involving impractical Arg1 enzymatic assays and Western blots. Herein, our goals were to 1) confirm this Arg1 immune signature in an independent cohort of patients, using more clinically applicable immunohistochemistry (IHC), 2) determine the associated risk profile with recent prognostic scoring systems, and 3) begin to connect this Arg1 immune signature with recurring MDS/CMML-acquired mutations. Methods With ethics approval, 29 BM biopsies (decalcified, FFPE) and clinical parameters were retrieved from the archives of Kingston General Hospital: 6 controls (4 negative lymphoma staging BM and 2 mild anemia NYD; mean age +/- std = 57 +/- 13 y), 13 MDS (1 RA, 1 RARS, 1 del(5q), 5 RCMD, 3 RAEB-1, 2 RAEB-2; 76 +/- 14 y), and 10 CMML patients (7 CMML-1, 3 CMML-2; 72 +/- 10 y). H&E and anti-human Arg1 IHC (1/2500 dilution, clone HPA003595, Sigma) were conducted under optimized, automated conditions (Ventana). IHC scoring was recorded independently by 2 blinded Hematopathologists. IPSS, IPSS-R (Greenberg), WPSS (Malcovati), CMML PSS (Such) and Mayo CMML (Patnaik) scores were calculated. Floxed TET2 and Vav-Cre mice were obtained from JAX and used according to approved Queen's University Animal Care protocols. TET2 sequences were obtained from genomic DNA using custom AmpliSeq primer pools and the Ion Torrent PGM platform (LifeTech). Linear regression and student's t-tests were conducted with Prism software (GraphPad). Results 1) We demonstrated increased BM biopsy Arg1 IHC expression in CMML (22 +/- 17% Arg1-positive cells; n = 10; p = 0.0093) and low-grade MDS, particularly RCMD (14 +/- 11%; n = 5; p = 0.012) relative to control subjects (0.4 +/- 1%; n = 6) (Figure 1). Significantly increased mean Arg1 expression was not seen in other MDS subtypes (n = 8). These findings were consistent with our previously reported 40-subject (Toronto) BM aspirate Arg1 assay/Western blot cohort (Rauh et al. Blood Abstract 2010:1855), suggesting the reproducibility of these findings and the clinical utility of IHC-based assessment. Parallel Arg1 IHC is underway on the Toronto cohort, to determine correlations with enzymatic assays/Westerns. 2) We previously reported Arg1 over-expression in the Toronto cohort was significantly associated with the lowest IPSS and WPSS MDS clinical risk categories and now extend this to IPSS-R. In our Kingston cohort, only trends to lower MDS risk were observed. In contrast, increased Arg1 expression was not associated with clinical risk (CPSS and Mayo scores) in either CMML cohort. Thus, Arg1 expression associated with neutral risk in CMML and neutral to lower risk in MDS patients. Both the clinical risk profiles and proportions of Arg1 over-expressing MDS/CMML patients were reminiscent of reported TET2 mutation risk profiles/percentages. 3) TET2-deficient mice demonstrated increased monocytes, macrophages and CD11b+Gr1+ splenocytes (immuno-phenotypically consistent with MDSC), phenocopying SHIP1-deficient mice. We are currently determining whether TET2-/- mouse macrophages are similarly M2-skewed. In parallel, were are obtaining TET2 genomic sequences (along with other recurring mutated genes) for our Toronto and Kingston cohorts, in order to determine if the Arg1 immune signature associates with a particular mutation(s). These findings will be discussed. Conclusions 1) Using two independent patient cohorts, we demonstrated significant Arg1 over-expression in CMML and low-grade (RCMD) MDS. Arg1 IHC warrants further investigation as an ancillary diagnostic test. 2) Increased Arg1 expression had neutral prognostic significance in CMML and neutral to low-risk MDS associations. 3) Increased Arg1 expression in MDS/CMML may be driven by mutant TET2, impacting the epigenetics of MDSC and M2-macrophage expansion, controlled by SHIP1 and related signaling networks. Confirmatory studies are in progress. Disclosures: No relevant conflicts of interest to declare.