The SP/KLF family of transcription factors harbour three C-terminal C2H2 zinc fingers interspersed by two linkers which confers DNA-binding to a 9-10 bp motif. Mutations in KLF1, the founding member of the family, are common. Missense mutations in linker two result in a mild phenotype. However, when co-inherited with loss-of-function mutations, they result in severe non-spherocytic hemolytic anemia. We generate a mouse model of this disease by crossing Klf1+/- mice with Klf1H350R/+ mice that harbour a missense mutation in linker-2. Klf1H350R/- mice exhibit severe hemolysis without thalassemia. RNA-seq demonstrate loss of expression of genes encoding transmembrane and cytoskeletal proteins, but not globins. ChIP-seq show no change in DNA-binding specificity, but a global reduction in affinity, which is confirmed using recombinant proteins and in vitro binding assays. This study provides new insights into how linker mutations in zinc finger transcription factors result in different phenotypes to those caused by loss-of-function mutations.

Introduction: Erythropoietin (EPO) regulates expression of genes that drive proliferation, survival and differentiation of erythroid progenitor cells into mature erythrocytes. The EPO receptor signals via JAK2 and STAT, PI3K and MAPK pathways. Only a few direct target genes of these pathways have been identified to date. Aim: To determine the repertoire of EPO-induced target genes in human erythroid cells Methods: We employed a well characterized conditionally immortalized EPO-dependent human erythroid progenitor model (HUDEP-2 cells). The cells require stem cell factor and EPO for growth, but can be starved of both and then re-challenged with either growth factor. A multi-omics approach was employed to find direct target genes of EPO stimulation. We performed ChIP-seq for pSTAT5, ATAC-seq, and a new method of primary RNA-seq based on SLAM-seq but using full length cDNA libraries. Results: Three hours of starvation followed EPO (5U/ml) stimulation for 1 hour resulted in rapid phosphorylation of STAT5, but not STAT1 or STAT3. ChIP-seq or pSTAT5 identified 3128 EPO-induced binding sites. The majority of peaks contain a palindromic 'GAS' motif (TTCYXRGAA), and are located at intronic (50%), distal (29%) and intergenic (15%) enhancers; only 3% are located at promoters. De novo motif discovery identified significant enrichment of DNA-binding motifs for GATA and KLF transcription factors (TFs), suggesting co-operativity between EPO signalling and the essential basal erythroid TFs, GATA1 and KLF1. We show GATA1 and KLF1 bind most of these by ChIP-seq. We found 14,535 new ATAC-seq peaks after EPO stimulation, at both enhancers and promoters. Only ~8% overlap with pSTAT5 ChIP-seq peaks, suggesting EPO-mediated phosphorylation and DNA-binding of undiscovered TFs. By searching for enriched motifs within these regions, we identified binding sites for TFs of the NFY, EGR, and NRF1 families and others, suggesting a hidden complexity of transcription factors that mediate responses to EPO. ChIP-seq for EGR1 is in progress and will be reported. We developed a novel metabolic labelling technique and bio-informatic pipeline, called BodySLAM-seq, to determine the immediate transcriptional targets of EPO. We used this to find ~100 differentially transcribed genes (DTGs) as well as differentially expressed genes (DEGs) in immediate response to EPO (45 min labelling period). Some are direct pSTAT5 target genes such as BCL2L1, PIM1, and CISH; others are novel targets involved in transcription regulation, erythropoiesis and signalling. Some are known immediate early genes such as EGR1. The RNAs for these are rapidly degraded in the absence of EPO and rapidly induced. Other genes are not degraded in the absence of EPO but are nevertheless rapidly induced. These were only detectable by BodySLAM-seq. Conclusions: We have discovered new pSTAT5 direct target genes, and also new DNA motifs that mediate responses to EPO, in human erythropoiesis. We have discovered a set of immediate early genes (IEGs) that are rapidly degraded in the absence of an EPO stimulus and rapidly induced again by EPO. These are involved in negative regulation of JAK-STAT signalling, cell proliferation, cell survival, and transcription. They are not involved in erythroid cell differentiation specifically. This suggests the main function of EPO is to induce cell proliferation and survival rather than differentiation.

Acute myeloid leukemia (AML) is a malignancy of immature progenitor cells. AML differentiation therapies trigger leukemia maturation and can induce remission, but relapse is prevalent and its cellular origin is unclear. Here we describe high resolution analysis of differentiation therapy response and relapse in a mouse AML model. Triggering leukemia differentiation in this model invariably produces two phenotypically distinct mature myeloid lineages in vivo. Leukemia-derived neutrophils dominate the initial wave of leukemia differentiation but clear rapidly and do not contribute to residual disease. In contrast, a therapy-induced population of mature AML-derived eosinophil-like cells persists during remission, often in extramedullary organs. Using genetic approaches we show that restricting therapy-induced leukemia maturation to the short-lived neutrophil lineage markedly reduces relapse rates and can yield cure. These results indicate that relapse can originate from therapy-resistant mature AML cells, and suggest differentiation therapy combined with targeted eradication of mature leukemia-derived lineages may improve disease outcome.

Three driver mutations (JAK2, CALR and MPL) comprise one of the major diagnostic criteria for Philadelphia negative (Ph-) myeloproliferative neoplasms (MPN) in the current World Health Organisation (WHO) classification. Although driver mutations were previously thought to be mutually exclusive, recent reports have demonstrated their uncommon co-existence. The impact of single driver mutations on phenotype and prognosis has been established, though the clinical implication of coexisting driver mutations remains unclear. With more sensitive and cost-effective next generation sequencing (NGS) applications for patients with blood cancers, novel mutations are being discovered that have important clinical and prognostic significance. Herein, we report a case of three stages of molecular and clinical evolution of a MPN from polycythemia vera (PV) to post-PV myelofibrosis (MF), and then to accelerated phase MF. We found the acquisition of new mutations and copy number changes using an NGS panel that correlated with changes in clinical features; the late acquisition of a new MPL p.Y591D mutation in addition to baseline JAK2 p.V617F and ASXL1 p.R1415Ter mutations resulted in accelerated disease with severe leukocytosis. Our case is of a 73-year-old woman who was diagnosed with JAK2 p.V617F mutant PV in 2008. The initial diagnosis of PV was made based on WHO criteria, including a positive test for the JAK2 p.V617F mutation and a low serum erythropoietin (EPO) level (2 IU/mL); a bone marrow biopsy was not performed. Initial treatment included hydroxycarbamide, aspirin and intermittent venesections. Retrospective molecular assessment using a custom designed AmpliSeq panel run on an Illumina MiniSeq analyser showed heterozygosity for the JAK2 p.V617F mutation (variant allele frequency [VAF]=0.51) and a heterozygous single nucleotide variant (SNV) in the ASXL1 gene, leading to a nonsense amino acid change, p.R1415Ter (VAF=0.43) (Table 1). This variant has been reported as a pathogenic mutation in the Catalogue of Somatic Mutations in Cancer (COSMIC) database (v90). Six years after diagnosis, the patient developed new fatigue, night sweats and splenomegaly. Full blood examination (FBE) showed anemia (hemoglobin 9.1g/dL), leucocytosis (white cell count [WCC] 17x10/L) (Figure 1) and thrombocytosis (platelets 661x10/L) (Figure 2, blue arrow). There was also new monocytosis, basophilia and eosinophila in the blood (Figure 1, blue arrow). The blood smear showed leucoerythroblastosis with prominent teardrop poikilocytes. The lactate dehydrogenase was 1049 U/L (reference range: 125-255 U/L). A bone marrow biopsy demonstrated a markedly hypercellular and fibrotic marrow with increased granulocytic proliferation and abnormal megakaryocyte clustering, consistent with post-PV MF. Molecular analysis of DNA extracted from the trephine detected the same two sequence variants; i.e., (i) a JAK2 p.V617F missense mutation with a VAF of 0.82 suggesting uniparental disomy (UPD) in a fraction of the cells of the malignant clone, and (ii) the same ASXL1 mutation at the same VAF of 0.45 (Table 1) (Figure 3). No other variants were detected in the coding regions of 86 genes that includes most variants documented to be associated with progression of PV to MF. 8 Hydroxycarbamide was changed to ruxolitinib 15 mg twice daily (Figure 2) with improvement in constitutional symptoms and reduction in spleen size. The FBE remained stable with a mild anemia (hemoglobin 9.5-11 g/dL), moderate leucocytosis white cell count (WCC) 2535x10/L) and mild thrombocytopenia (platelets 75125x10/L). Four years after commencing ruxolinitib, there was sudden progression into an accelerated phase of disease with marked leucocytosis (WCC 115x10/L) (Figure 1, red arrow) and splenomegaly. The hemoglobin and platelet values (Figure 2) remained stable. A blood smear showed prominence of myelocytes and basophils. A repeat bone marrow trephine was fibrotic with increased abnormal megakaryocytes, but not markedly different from the bone marrow performed four years earlier. A BCR-ABL1 fusion transcript was not detected by quantitative real-time PCR. Molecular analysis on the peripheral blood using the same NGS panel now detected three sequence variants: (i) the same JAK2 p.V617F missense mutation with a VAF=0.97, consistent with a selective advantage for the clone with UPD, (ii) the same ASXL1 nonsense mutation with a VAF=0.48, and (iii) a new SNV in the MPL gene, leading to a p.Y591D amino acid change (Table 1 and Figure 3). This is not one of the common p.W515L/K/A gain of function mutations in MPL, but has been reported to be pathogenic. This new MPL variant was present at a VAF =0.96, so we performed concurrent peripheral blood cytogenetic testing, with no abnormalities found. In particular, monosomy 1, a common cytogenetic abnormality in MPL-driven MPN, was not present, so we conclude that there must be UPD

Objectives: The aim of this birth cohort study was to identify concurrent associations between early childhood caries and putative risk and protective factors. Methods: Data were collected in seven waves over five years. The study outcome measure, d(3-6) mfs, was modelled in a set of sequential negative binomial regressions that introduced the variables in steps starting from health determinants most distal to the child and ending with the more proximal ones. The goodness of fit of each model at each step was tested using the quasi-likelihood under independence model criterion (QIC). A final model included all significant factors identified in the sequential modelling. Bacterial composition of the child's saliva was determined by 16S RNA gene sequencing. Results: Overall, 467 children (48.6 % female) participated, of whom 419 (89.7 %) had at least one follow-up visit after baseline. Of the 419 children included in the analyses, 133 (31.7 %) had their saliva samples sequenced for microbiomic determination. Independent protectors of surface cavitation included water fluoridation, and older age of mothers. Risk for d(3-6) mfs was significantly higher among children whose mothers were current smokers (IRR 3.29, 95 % CI 1.09-9.88, p = 0.034), children who went to bed with a bottle (IRR 2.67, 95-6.88, p = 0.041) and whose saliva sample sequencing over time showed higher percentages of Streptococcus mutans (IRR 1.39, 95 % CI 1.11-1.74, p = 0.005). Model fit was mostly improved by child's proximal variables. Household and mother covariates did not substantially improve model fit. Conclusion: This analysis highlights the relevance and importance of child-proximal risk factors in childhood dental cavitation. Clinical significance: The study findings inform clinical decision making for the management of early childhood caries at both the individual and population level. At an individual and family level these risk factors should be incorporated into caries risk assessment tools for more precise identification of risk and evidence-informed interventions by health professionals.

Modulators of epithelial-to-mesenchymal transition (EMT) have recently emerged as novel players in the field of leukemia biology. The mechanisms by which EMT modulators contribute to leukemia pathogenesis, however, remain to be elucidated. Here we show that overexpression of SNAI1, a key modulator of EMT, is a pathologically relevant event in human acute myeloid leukemia (AML) that contributes to impaired differentiation, enhanced self-renewal, and proliferation of immature myeloid cells. We demonstrate that ectopic expression of Snai1 in hematopoietic cells predisposes mice to AML development. This effect is mediated by interaction with the histone demethylase KDM1A/LSD1. Our data shed new light on the role of SNAI1 in leukemia development and identify a novel mechanism of LSD1 corruption in cancer. This is particularly pertinent given the current interest surrounding the use of LSD1 inhibitors in the treatment of multiple different malignancies, including AML.

Krüppel-like factors (KLFs) are a family of transcription factors that play essential roles in the development and differentiation of the hematopoietic system. These transcription factors possess highly conserved C-terminal zinc-finger motifs, which enable their binding to GC-rich, or CACC-box, motifs in promoter and enhancer regions of target genes. The N-terminal domains of these proteins are more varied and mediate the recruitment of various co-factors, which can form a complex with either activator or repressor function. Acting primarily as a gene repressor through its recruitment of CtBPs and histone deacetylases (HDACs) [1], we have recently shown that KLF3 competes with KLF1 bound sites in the genome to repress gene expression during erythropoiesis [2]. However, the function of Klf3 in other lineages has been less well studied. This widely expressed transcription factor has reported roles in the differentiation of marginal zone B cells, eosinophil function and inflammation [3]. We utilised the Klf3-null mouse model [4] to more closely examine the role of Klf3 in innate inflammatory cells. These mice exhibit elevated white cell counts, including monocytes (Figure 1A), and inflammation of the skin. Conditional knockout of Klf4 in myeloid cells leads to a deficiency of inflammatory macrophages [5]. To test our hypothesis KLF3 normally represses inflammation, perhaps by antagonising the action of KLF4, bone-marrow derived macrophages (BMDM) were generated from wild-type or Klf3-null mice and stimulated with the bacterial toxin lipopolysaccharide (LPS). In wild type BMDM, LPS induces Klf3 gene expression and activation then delayed repression of target genes such as Lgals3 (galectin-3) over a 21 hour time course (Figure 1B). Quantitative real-time PCR and mRNA-seq of WT v Klf3-null macrophages identified ~100 differentially expressed genes involved in proliferation, macrophage activation and inflammation. We transduced the monocyte cell line, RAW264.7 (that expresses Klf4, Klf3 and Klf2), with a retroviral vector expressing a tamoxifen-inducible KLF3-ER fusion construct. KLF3 induced cell cycle arrest and macrophage differentiation. We will report on KLF3-induced gene expression changes (repression and activation), and ChIP-seq for KLF3, in RAW cells. The results shed light on the mechanism by which KLF3 normally represses monocyte/macrophage responses to infection. This study highlights the importance of key transcriptional regulators that tightly control gene expression during inflammation. Loss of Klf3 leads to alterations in this process, resulting in hyper-activation of inflammatory macrophages, increased white cell counts and inflammation of the skin. A greater knowledge of the inflammatory process and how it is regulated is important for our understanding of acute infection and inflammatory disease. Further studies are planned to investigate the role of the KLF3 transcription factor in response to inflammation in vivo. References: 1. Pearson, R., et al., Kruppel-like transcription factors: A functional family. Int J Biochem Cell Biol, 2007. W2. Ilsley, M.D., et al., Kruppel-like factors compete for promoters and enhancers to fine-tune transcription. Nucleic Acids Res, 2017. 45(11): p. 6572-6588. W3. Knights, A.J., et al., Kruppel-like factor 3 (KLF3) suppresses NF-kappaB-driven inflammation in mice. J Biol Chem, 2020. 295(18): p. 6080-6091. W4. Sue, N., et al., Targeted disruption of the basic Kruppel-like factor gene (Klf3) reveals a role in adipogenesis. Mol Cell Biol, 2008. 28(12): p. 3967-78. W5. Alder, J.K., et al., Kruppel-like factor 4 is essential for inflammatory monocyte differentiation in vivo. J Immunol, 2008. 180(8): p. 5645-52. Figure 1: Elevated WCC (A) and inflammatory markers (B) in BMDM after LPS stimulation. 1. Total WCC in adult mice (3-6 months old) of the indicated genotypes. There is a statistically significant increase in the WCC in Klf3-/- v wild type mice (P<0.001 by student's t test). B. Time course (hours) after LPS stimulation of confluent BMDM. Klf3 is induced 3-fold by LPS and KLF3-target genes such as Lgals3 are not fully repressed by 21 hours in knockout mice. Figure 1 Disclosures Perkins: Novartis Oncology: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Sickle cell disease (SCD) affects millions of people worldwide and represents the most common monogenic disease of mankind (1). It is due to a homozygous T to A transversion in the β-globin gene that results in an amino acid variant - G6V - and production of HbS, which polymerises in red blood cells (RBCs) under hypoxic conditions. This generates irreversibly sickled cells that fail to traverse the microcirculation, resulting in micro-infarcts, hypoxia and pain, or 'sickle cell crises'. During gestation RBCs utilise different sets of globin genes to produce embryonic and fetal hemoglobins (HbF), so it is not until after birth when adult hemoglobin (HbA) is first produced that the first signs of SCD become apparent. This process termed 'hemoglobin switching' has been the focus of research efforts for decades because it offers an opportunity to reactivate HbF in adult cells of patients with hemoglobinopathies. A number of transcription factors, including Krüppel-like factor 1 (KLF1), play critical roles in hemoglobin switching. KLF1 is an essential erythroid transcription factor that co-ordinates the expression of more than a thousand genes critical to the formation of adult RBCs. KLF1 directly binds the β-globin gene promoter to up regulate its expression, whilst regulating the expression of additional factors like BCL11A and LRF that directly repress γ-globin expression (HbF). Heterozygosity for loss of function mutations in KLF1 leads to a significant increase in HbF that is beneficial to patients with β-thalassemia. We propose this can be recreated by advanced gene editing techniques to provide an effective therapy for SCD. We have employed CRISPR-based gene editing to knockout the expression of KLF1 in human cells. We designed two separate sgRNAs with corresponding HDR templates to target the second exon of KLF1 and ablate its function. We optimised transfection protocols and tested the on-target specificity of our sgRNAs achieving >90% efficacy in all cell types assayed. Using HUDEP-2 cells (2), a conditionally immortalised erythroid cell line which harbors three copies of KLF1 (3), we have demonstrated that these cells require at least one copy (>1/3) for survival; heterozygous cells (+/-/- or +/+/-) proliferate at a reduced rate, but are able to differentiate normally. Using RNA-seq, we identified some genes, including ICAM-4 and BCAM, which are down-regulated accordingly in a KLF1 gene dosage-dependent manner. ICAM-4 and BCAM are cellular adhesion molecules implicated in triggering vaso-occlusive episodes (4; 5), so it is anticipated their reduced expression may provide additional benefit in treating SCD. Gamma-globin is upregulated 10-fold, BCL11A down-regulated 3-fold, and HbF+ RBCs generated at ~20% of total RBCs in KLF1 +/-/- HUDEP-2 cell lines. We also engineered the ablation of KLF1 in CD34+ cells harvested from the peripheral blood of SCD patients undergoing exchange transfusions. Following transfection of the two guides, we performed directed differentiation using an erythroid differentiation medium and analysed the levels of HbF. We observed HbF at levels of between 40-60% of total Hb by HPLC, and HbF+ cells of ~50% by FACS. There was no measurable block in erythroid differentiation by FACS. We documented the types of gene editing using a high throughout NGS assay (6). We compared efficiencies of CRISPR repair of the HbS mutation with CRIPSR damage of the KLF1 gene. Lastly, we transplanted gene-edited CD34 cells into NSGW41 mice (where human erythropoiesis is established) to determine the efficiency and safety of editing long term HSCs from SCD patients. We will report on the results of these xenotransplantation assays. Taken together these results reveal the potential utility in targeting KLF1 to cure SCD. References: Wastnedge, E. et al..J Glob Health 8, 021103 (2018). Kurita, R. et al.PLoS One 8, e59890 (2013). Vinjamur, D. S. & Bauer, D. E. Methods Mol Biol 1698, 275-284 (2018). Bartolucci, P. et al..Blood 116, 2152-9 (2010). Zhang, J., et al. PLoS One 14, e0216467 (2019). Bell, C. C., et al. BMC Genomics 15, 1002 (2014). Perkins, A. et al..Blood 127, 1856-62 (2016). Disclosures Kaplan: Celgene: Honoraria; Novartis: Honoraria. Perkins:Novartis Oncology: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Background: Acute myeloid leukaemia (AML) is an aggressive malignancy characterized by the accumulation of transformed immature myeloid blasts. Although standard cytotoxic chemotherapy routinely induces disease remission, most AML patients ultimately relapse with resistant disease. A notable exception is the AML subtype known as acute promyelocytic leukemia, where retinoic acid induces leukaemia maturation and transient remission as a single agent and is frequently curative in combination with arsenic trioxide. Recently agents including mutant IDH1/2 inhibitors and DHODH inhibitors have been shown to induce maturation and regression of other AML subtypes, sparking renewed interest in AML differentiation therapy. Aims: Our aims for this project included: Determining the kinetics and mechanisms of AML blast differentiation in vivo Identifying the source of relapse in our genetic model of differentation therapy Preventing relapse in our mouse model of AML Methods: To examine differentiation therapy dynamics in vivo we have generated a novel mouse AML model driven by reversible knockdown of PU.1, a myeloid transcription factor functionally compromised in >50% of AML cases. High resolution flow cytometry was used to determine the immunophenotype of AML derived cells as they undergo differentiation. IVIS imaging was also used to determine the location of disease during disease remission. Results: RNAi-mediated PU.1 knockdown results in disseminated AML in vivo, and subsequent restoration of endogenous PU.1 in established AML triggers synchronous differentiation of leukemic blasts followed by widespread disease clearance. Despite near-complete remission upon PU.1 restoration, mice reproducibly relapse with immature AML within several months. Notably, time course studies of in vivo AML treatment reveal that one week after differentiation stimulus leukemic blasts mature into two myeloid lineages with distinct immunophenotype and morphology. AML-derived SSClowLy6G+ cells resembling neutrophils initially predominate but are eradicated in vivo within two weeks, consistent with the rapid turnover of normal neutrophils. In contrast, high resolution flow cytometry and imaging indicate that mature AML-derived SSChighF4/80+SigF+ eosinophil-like cells persist at low numbers in specific organs during disease remission and seed relapse within the spleen. In mice transplanted with AML blasts lacking the essential eosinophil lineage transcription factor GATA1, in vivo PU.1 restoration triggers neutrophil but not eosinophil lineage differentiation resulting in the elimination of residual disease. Summary/Conclusion: These results demonstrate that AML differentiation therapy can produce long-lived sublineages of mature AML-derived cells from which relapse can originate. Understanding the multilineage potential of AML blasts in individual patients may inform strategies that preclude or eradicate mature AML-derived cells to improve differentiation therapy outcomes.

Dental caries is associated with plaque dysbiosis, leading to an increase in the proportions of acidogenic and aciduric bacteria at the expense of alkali-generating commensal species. Stannous fluoride (SnF2) slows the progression of caries by remineralization of early lesions but has also been suggested to inhibit glycolysis of aciduric bacteria. Casein phosphopeptide–amorphous calcium phosphate (CPP-ACP) promotes fluoride remineralization by acting as a salivary biomimetic that releases bioavailable calcium and phosphate ions, and the peptide complex has also been suggested to modify plaque composition. We developed a polymicrobial biofilm model of caries using 6 bacterial species representative of supragingival plaque that were cultured on sound human enamel and pulsed with sucrose 4 times a day to produce a high cariogenic challenge. We used this model to explore the mechanisms of action of SnF2 and CPP-ACP. Bacterial species in the biofilms were enumerated with 16S rRNA gene sequence analyses, and mineral loss and lesion formation were determined in the enamel directly under the polymicrobial biofilms via transverse microradiography. The model tested the twice-daily addition of SnF2, CPP-ACP, or both. SnF2 treatment reduced demineralization by 50% and had a slight effect on the composition of the polymicrobial biofilm. CPP-ACP treatment caused a similar inhibition of enamel demineralization (50%), a decrease in Actinomyces naeslundii and Lactobacillus casei abundance, and an increase in Streptococcus sanguinis and Fusobacterium nucleatum abundance in the polymicrobial biofilm. A combination of SnF2 and CPP-ACP resulted in a greater suppression of the acidogenic and aciduric bacteria and a significant 72% inhibition of enamel demineralization.

Objectives: To determine if chewing gum containing casein phosphopeptide stabilised amorphous calcium phosphate (CPP-ACP) promoted an increase in the abundance of Streptococcus sanguinis and other species associated with dental health in supragingival plaque in a clinical study. Materials and methods: Nineteen participants were recruited for a three-leg cross-over, randomised, controlled clinical trial. Participants chewed a sugar-free gum with or without CPP-ACP six times daily for 20 min over two weeks. The study also involved no gum chewing (no gum) for the same two week period. Participants were randomly assigned to one of the test gums or no gum for each intervention period. Participants abstained from oral hygiene and had washout periods of two weeks between intervention periods. After each intervention period, supragingival plaque was collected and analysed for bacterial composition by sequencing the V4 variable region of the 16S rRNA gene. Data were analysed using a linear mixed model. Results: The CPP-ACP gum intervention produced a significant (p < 0.01) increase in the proportions of S. sanguinis (112%), as well as the commensal species Rothia dentocariosa (127%), Corynebacterium thrum (80%) and Streptococcus mitis (55%) when compared with the no gum intervention. All the species that were promoted by the CPP-ACP gum are known to possess one or both of the alkali-producing enzymes arginine deiminase and nitrate reductase. Conclusion: This clinical study demonstrated that chewing a sugar-free gum containing CPP-ACP promoted prebiosis by significantly increasing the proportion of S. sanguinis and other health-associated bacterial species in supragingival plaque. Clinical significance: Regular chewing of CPP-ACP sugar-free gum increases the proportions of health-associated commensal species in supragingival plaque to promote prebiosis and oral homeostasis.

Acute myeloid leukaemia (AML) is characterized by the accumulation of transformed immature myeloid blasts. While most AML patients treated with standard therapy have poor outcomes, in the APL disease subtype retinoic acid induces leukaemia maturation and can be curative in combination with arsenic trioxide. Recently approved mutant IDH1/2 inhibitors also induce AML maturation, renewing interest in AML differentiation therapy. To examine differentiation therapy dynamics in vivo we have generated a novel mouse AML model driven by reversible RNAi-mediated knockdown of the myeloid transcription factor PU.1. Restoration of endogenous PU.1 in established AML in vivo triggers synchronous differentiation of leukemic blasts and disease clearance. However, despite near-complete remission, mice reproducibly relapse with immature AML. Notably, in vivo time course studies reveal that one week after PU.1 restoration leukemic blasts differentiate into two mature myeloid lineages with distinct immunophenotype and morphology. AML-derived SSClowLy6G+ cells resembling neutrophils initially predominate but are rapidly eradicated in vivo. In contrast, high resolution flow and imaging indicates that mature AML-derived SSChighF4/80+SigF+ eosinophil-like cells persist at low numbers in specific organs during disease remission and appear to seed relapse. In mice transplanted with AML blasts lacking the essential eosinophil lineage transcription factor GATA1, in vivo PU.1 restoration triggers neutrophil but not eosinophil lineage differentiation and thereby eliminates residual disease. These results demonstrate that AML differentiation therapy can produce long-lived sublineages of mature AML-derived cells from which relapse can originate. Understanding the multilineage potential of AML blasts in individual patients may inform new strategies to improve differentiation therapy outcomes.

Myeloproliferative neoplasms (MPNs) are characterised by excess production of mature blood cells accompanied by an increased risk of thrombosis and progression to marrow fibrosis and AML. Most are driven by a mutation (V617F) in the pseudo-kinase domain of JAK2, which leads to unrestrained cell proliferation. To find direct targets of JAK2-STAT signalling in MPNs, we undertook ChIP-Seq for pSTAT5 and pSTAT3 in cell lines, HEL and SET2. HEL cells have 13-16 copies of JAK2-V617F as determined by FISH and SNP arrays 1. They have high levels of pSTAT5 and pSTAT3 by phosphoflow and Western blotting. SET-2 cells were derived from a patient with JAK2-V617F+ ET; they also have high levels of pSTATs. We found ∼690 pSTAT5-occupied sites and >10,000 pSTAT3-occupied sites in HEL cells. The majority reside in distal enhancers. We found new enhancers in well-known target genes such as BCL2L1, which encodes the pro-survival protein, BCL-X. The human enhancer is in a similar relative position to the recently described mouse EPO-dependent enhancer 2. We found new direct target genes that encode for proteins with interesting predicted functions. SET-2 cells have similar pSTAT5 and pSTAT3-bound sites suggesting commonality of functions. We show pSTAT3 and pSTAT5 bind as dimers in vivo to typical GAS elements, but with slightly different site preferences. This has implications for differential target gene regulation. We undertook expression profiling using SLAM-seq following treatment with ruxolitinib and validated novel target genes by qRT-PCR. In short, we have discovered hundreds of direct JAK-STAT target genes involved in cell survival, proliferation, down regulation of cytokine signalling, and novel functions. The genes provide new insights into the biology of MPNs, and a source of potential new biomarkers and drug targets. We have validated some in primary MPN samples.

Human microbiomes are predicted to assemble in a reproducible and ordered manner yet there is limited knowledge on the development of the complex bacterial communities that constitute the oral microbiome. The oral microbiome plays major roles in many oral diseases including early childhood caries (ECC), which afflicts up to 70% of children in some countries. Saliva contains oral bacteria that are indicative of the whole oral microbiome and may have the ability to reflect the dysbiosis in supragingival plaque communities that initiates the clinical manifestations of ECC. The aim of this study was to determine the assembly of the oral microbiome during the first four years of life and compare it with the clinical development of ECC. The oral microbiomes of 134 children enrolled in a birth cohort study were determined at six ages between two months and four years-of-age and their mother's oral microbiome was determined at a single time point. We identified and quantified 356 operational taxonomic units (OTUs) of bacteria in saliva by sequencing the V4 region of the bacterial 16S RNA genes. Bacterial alpha diversity increased from a mean of 31 OTUs in the saliva of infants at 1.9 months-of-age to 84 OTUs at 39 months-of-age. The oral microbiome showed a distinct shift in composition as the children matured. The microbiome data were compared with the clinical development of ECC in the cohort at 39, 48, and 60 months-of-age as determined by ICDAS-II assessment. Streptococcus mutans was the most discriminatory oral bacterial species between health and current disease, with an increased abundance in disease. Overall our study demonstrates an ordered temporal development of the oral microbiome, describes a limited core oral microbiome and indicates that saliva testing of infants may help predict ECC risk.

Porphyromonas gingivalis is a keystone pathogen of chronic periodontitis. The virulence of P. gingivalis is reported to be strain related and there are currently a number of strain typing schemes based on variation in capsular polysaccharide, the major and minor fimbriae and adhesin domains of Lys-gingipain (Kgp), amongst other surface proteins. P. gingivalis can exchange chromosomal DNA between strains by natural competence and conjugation. The aim of this study was to determine the genetic variability of P. gingivalis strains sourced from international locations over a 25-year period and to determine if variability in surface virulence factors has a phylogenetic basis. Whole genome sequencing was performed on 13 strains and comparison made to 10 previously sequenced strains. A single nucleotide polymorphism-based phylogenetic analysis demonstrated a shallow tri-lobed phylogeny. There was a high level of reticulation in the phylogenetic network, demonstrating extensive horizontal gene transfer between the strains. Two highly conserved variants of the catalytic domain of the major virulence factor the Kgp proteinase (KgpcatI and KgpcatII) were found. There were three variants of the fourth Kgp C-terminal cleaved adhesin domain. Specific variants of the cell surface proteins FimA, FimCDE, MfaI, RagAB, Tpr, and PrtT were also identified. The occurrence of all these variants in the P. gingivalis strains formed a mosaic that was not related to the SNP-based phylogeny. In conclusion P. gingivalis uses domain rearrangements and genetic exchange to generate diversity in specific surface virulence factors.

Revised MS IAI01500-15R1 1 2 Porphyromonas gulae has similar virulence and immunological characteristics to the 3 human periodontal pathogen Porphyromonas gingivalis 4 5 Jason C. Lenzo, Neil M. O’Brien-Simpson, Rebecca K. Orth, Helen L. Mitchell, Stuart G. 6 Dashper and Eric C. Reynolds 7 8 Oral Health Cooperative Research Centre, Melbourne Dental School, Bio21 Institute, The 9 University of Melbourne, Melbourne, Victoria, Australia 10

ABSTRACT Periodontitis is a significant problem in companion animals, and yet little is known about the disease-associated microbiota. A major virulence factor for the human periodontal pathogen Porphyromonas gingivalis is the lysyl- and arginyl-specific proteolytic activity of the gingipains. We screened several Porphyromonas species isolated from companion animals— P. asaccharolytica , P. circumdentaria , P. endodontalis , P. levii , P. gulae , P. macacae , P. catoniae , and P. salivosa —for Lys- and Arg-specific proteolytic activity and compared the epithelial and macrophage responses and induction of alveolar bone resorption of the protease active species to that of Porphyromonas gingivalis . Only P. gulae exhibited Lys-and Arg-specific proteolytic activity. The genes encoding the gingipains (RgpA/B and Kgp) were identified in the P. gulae strain ATCC 51700 and all publicly available 12 draft genomes of P. gulae strains. P. gulae ATCC 51700 induced levels of alveolar bone resorption in an animal model of periodontitis similar to those in P. gingivalis W50 and exhibited a higher capacity for autoaggregation and binding to oral epithelial cells with induction of apoptosis. Macrophages (RAW 264.7) were found to phagocytose P. gulae ATCC 51700 and the fimbriated P. gingivalis ATCC 33277 at similar levels. In response to P. gulae ATCC 51700, macrophages secreted higher levels of cytokines than those induced by P. gingivalis ATCC 33277 but lower than those induced by P. gingivalis W50, except for the interleukin-6 response. Our results indicate that P. gulae exhibits virulence characteristics similar to those of the human periodontal pathogen P. gingivalis and therefore may play a key role in the development of periodontitis in companion animals.

Recent microbiome studies have shown that the human oral microbiome is composed of over 260 abundant bacterial species that predominantly live as polymicrobial biofilms accreted to the non-shedding hard surfaces of the teeth. In addition representatives of both Archaea and Fungi are found in the oral cavity and there is considerable colonisation of the soft tissues of the mouth. Most of these species are commensal and form complex biofilm communities that restrict the colonisation of the oral cavity by exogenous bacteria. Changes in the polymicrobial biofilm microenvironment such as those resulting from the effects of chronic inflammation for subgingival plaque, can lead to the emergence of opportunistic pathogens resulting in dysbiosis and the development of chronic diseases such as periodontitis in a susceptible host. The application of microbiomic studies to the analysis of these complex and dynamic communities in rigorously designed human clinical studies will provide valuable mechanistic insight into the bacterial succession and complex interactions involved in the development of dysbiosis and disease.