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The transcription factor RUNX1 is mutated in familial platelet disorder with associated myeloid malignancies (FPDMM) and in sporadic myelodysplastic syndrome and leukemia. RUNX1 regulates inflammation in multiple cell types. Here we show that RUNX1 is required in granulocyte-monocyte progenitors (GMPs) to restrict the inflammatory response of neutrophils to toll-like receptor 4 (TLR4) signaling. Loss of RUNX1 in GMPs increased the TLR4 coreceptor CD14 on neutrophils, which contributed to neutrophils’ increased inflammatory cytokine production in response to the TLR4 ligand lipopolysaccharide. RUNX1 loss increased the chromatin accessibility of retrotransposons in GMPs and neutrophils and induced a type I interferon signature characterized by enriched footprints for signal transducer and activator of transcription (STAT1::STAT2) and interferon regulatory factors (IRF) in opened chromatin, and increased expression of interferon-stimulated genes. The overproduction of inflammatory cytokines by neutrophils was reversed by inhibitors of type I IFN signaling. We conclude that RUNX1 restrains the chromatin accessibility of retrotransposons in GMPs and neutrophils, and that loss of RUNX1 increases proinflammatory cytokine production by elevating tonic type I interferon signaling.

Syzygium filiforme var. filiforme is a plant variety from dicotyledonous plant family (Myrtaceae). Phytochemical studies on S. filiforme var. filiforme stem bark have successfully isolated and characterized arjunolic acid (1), alphitolic acid (2), betulinic acid (3), ursolic acid (4), ursolic acid 3-methyl ester (5), β-sitosterol (6) and stigmasterol (7). The inhibitory activities against free radical, starch, and bacteria for major compounds were tested by using DPPH, α-glucosidase and minimum inhibitory and bacterial concentration assays, respectively. No promising antioxidant activity was shown on tested samples except methanolic crude extract. For antidiabetic activity, methanolic and dichloromethane crude extracts displayed potent activity compared to 1-deoxynojirimycin. Minimum inhibitory concentration (MIC) and minimum bacterial concentration (MBC) assays for antibacterial activity were evaluated on Escherichia coli, Staphylococcus aureus, and Bacillus subtilis. All crude extracts and major compounds displayed weak and no promising activities for MIC method, respectively. Meanwhile, for MBC method, hexane crude extract and compound 1 showed inhibition against B. subtilis.

The outstanding evidence of phthalimide pharmacophore in securing enhanced biological activities had encouraged further research and development into phthalimide-based derivatives as potential new drugs. In this study, phthalimide core was hybridized with aldehydes giving integrated imines displaying different types of functionalities and at alternating positions. The resulting compounds, therefore, provide an innovative window to explore possible differential biological effects as antioxidants and anticancer agents. A total of sixteen compounds were synthesized, and each was verified by FT-IR, H NMR, C NMR, and MS characterization. Herein, a facile single-step synthesis method was employed substituting the conventional two-step chemical production routes. Among the sixteen tested compounds, the H7 compound with hydroxyl phenolic group has shown an eminent antioxidant activity with a 19.52% decrease to the IC50 value compared to that of the control standard BHT antioxidant. On the other hand, the halogenated H6 Schiff base structure was successful in securing effective cancer inhibition to both colon and breast cancer cell lines, while maintaining selective action toward normal tissues. Results have collectively indicated the importance and impactful effects of functional groups position and types within similar basic structures, in directing different biological outcomes.

New multipotent antioxidants (MPAOs), namely semicarbazides and thiosemicarbazides bearing thiolated butylated hydroxytoluene (BHT), were synthesized. The antioxidant activity of the synthesized compounds was evaluated using in vitro DPPH assay. Compounds containing thiosemicarbazides (5a′–h′) were found more active in free radical scavenger than semicarbazides (5a–h). Among the other compounds, compound 5f′ (IC50 of 25.47 ± 0.42 μM) showed the best antioxidant activity against DPPH radicals compared to standard antioxidant butylated hydroxytoluene (BHT). Based on DPPH results, compound 5f′ and its corresponding semicarbazide 5f were blended into trimethylolpropane trioleate (TMPTO) and isothermal differential scanning calorimetry (DSC) was carried out for the investigation of oxidative stability. At 125 °C isothermal DSC, TMPTO with 0.25 wt% of 5f′ showed 1.5 times higher oxidation stability than its corresponding semicarbazide 5f and was 2 times better than BHT. It was anticipated that due to the strong auto-synergistic effect, compound 5f′ showed promising oxidative stability to TMPTO by protecting from pre-mature oxidative degradation.

Patients with Familial Platelet disorder (FPD) have a germline RUNX1 mutation and are at high risk to developing hematologic malignancies (HM), primarily myelodysplastic syndrome and acute myeloid leukemia (lifetime risk~40%). To understand how germline RUNX1 mutations predispose to HM in vivo, we developed a Runx1 R188Q/+ mouse strain , mimicking the FPD-associated R201Q missense mutation. Analysis of the bone marrow cells in Runx1 R188Q/+ mice revealed a significant increase in the total number of bone marrow cells. Immunophenotypic analysis using Sca-1 and Cd86 markers revealed a significant increase in Sca-1 expression in hematopoietic stem and multi-potential progenitor cells, indicating a systemic inflammation in the bone marrow. In addition, the frequency of common-myeloid, granulocytic-monocytic and granulocytic progenitor cells were found significantly increased in the Runx1 R188Q/+ bone marrow. Accordingly, their colony-forming unit capacity was increased when compared to wildtype controls (wt/Runx1 R188Q/+ CFU average = 45/85), indicating a myeloid bias. The number and size of platelets were not altered in Runx1 R188Q/+ mice. However, platelet function was significantly reduced. The activation of the Cd41/Cd61 fibrinogen receptor complex in membrane after thrombin treatment was reduced in Runx1 R188Q/+ platelets. Similarly, the translocation of P-selectin by alpha granules and the secretion of serotonin by the dense granules were also reduced. Hematopoietic progenitor cells isolated from Runx1 R188Q/+ mice revealed a significant reduction in DNA-damage repair response in vitro. Quantitative analysis of nuclei with 53bp1-positive foci in response to ionizing radiation showed a marked increase in 53bp1-positive foci in Runx1 R188Q/+ nuclei, suggesting that Runx1 R188Q/+ cells have a defective repair of double strand DNA breaks. Furthermore, expression of DNA-damage repair pathway-associated Pmaip1 (Noxa) was significantly reduced in irradiated Runx1 R188Q/+ hematopoietic progenitor cells. To understand underlying mechanism responsible for the observed myeloid bias in Runx1 R188Q/+ cells, transcription profiling analysis was performed in myeloid progenitors from wildtype and Runx1 R188Q/+ mice, utilizing RNA-sequencing. A total of 39 genes were significantly deregulated (> 1.5 FC; FDR To study the FPD-associated pre-leukemic process in vivo, wildtype and Runx1 R188Q/+ mice were monitored for 20 months. Although Runx1 R188Q/+ mice remained healthy for 18 months, somatic mutations in their leukocytes were evident at 12 months. Targeted sequencing of 578 cancer genes (mIMPACT panel) in leukocyte DNA of two Runx1 R188Q/+ mice identified somatic mutations in Kdm6a, Setd1b, Amer1, and Esco1 (variant allele frequencies between 0.5% and 2.8%). These mutations were confirmed at stable frequency for eight following months. Since loss of the second Runx1 allele is a frequent somatic event in progression to FPD/HM, we evaluated the predisposition to HM in Mx1Cre-Runx1 R188Q/fl mice over time. Unlike Runx1 R188Q/+ mice, Runx1 R188Q/Δ mice succumbed to myeloid leukemia with a median latency of 37.5 weeks and full penetrance. In addition, the expression of oncogenic Nras-G12D, in Runx1 R188Q/Δ mice reduced the median latency to 14.7 weeks. These studies demonstrate that FPD-associated Runx1 germline mutations induce inflammation in hematopoietic stem cells, induce myeloid expansion with defective DNA-damage response and predispose to HM over time. These studies suggest that anti-inflammatory therapies in pre-symptomatic FPD patients may reduce clonal expansion and predisposition to HM. Disclosures Ebert: Exo Therapeutics: Membership on an entity's Board of Directors or advisory committees; Skyhawk Therapeutics: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Deerfield: Research Funding; GRAIL: Consultancy. Levine: Isoplexis: Membership on an entity's Board of Directors or advisory committees; Auron: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees; Zentalis: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; QIAGEN: Membership on an entity's Board of Directors or advisory committees; Ajax: Membership on an entity's Board of Directors or advisory committees; Imago: Membership on an entity's Board of Directors or advisory committees; Mission Bio: Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria; Prelude: Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy; Lilly: Honoraria; Morphosys: Consultancy; Roche: Honoraria, Research Funding; Incyte: Consultancy; Astellas: Consultancy; Amgen: Honoraria.

Signal transduction pathways regulate the proliferation and viability of acute myeloid leukemia (AML) blasts. The regulation in the expression of cytokine receptors in AML is not well understood. In this study, we investigated how the CBFβ-SMMHC fusion protein regulates expression of cytokine receptors in inv(16) AML, with focus on the co-receptor Neuropilin-1 (NRP1). Knock-down of CBFβ-SMMHC expression, utilizing shRNA transduction, induced G1 phase of cell cycle arrest and reduced the viability of inv(16) ME-1 cells in culture. Expression profile analysis of CBFβ-SMMHC knock-down cells revealed a significant repression of genes associated with transmembrane receptor protein kinase pathways, including NRP1 (-5 fold), FGFR1 (-4.2 fold) , FLT3 (-2 fold) and TGFBR2 (-1.2 fold). The expression of NRP1 was significantly upregulated in inv(16) AML cases when compared to other AML sub-types and to hematopoietic stem and progenitor cells. Functionally, NRP1 knock-down reduced the viability of ME-1 cells with a similar dynamics as when using CBFβ-SMMHC shRNAs. In addition, the proliferation of inv(16) AML cells was reduced 4.1-fold when treated with a function-blocking antibody for the FV/VIII extracellular NRP1 domain, while having no effect in non-inv(16) AML cells or when using blocking antibody for the CUB extracellular domain. Furthermore, deletion of Nrp1 by gene editing reduced the colony-forming unit capacity of primary mouse Cbfb +/MYH11 leukemic cells and extended the median leukemia latency in vivo. To identify the genes regulated by NRP1 in inv(16) AML, we analysed the transcription profile of NRP1 knock-down in ME-1 cells. Gene Set Enrichment and Pathway Analysis revealed a repression in STAT5 pathway, and in signalling receptor activity, including FLT3 (-1.8 fold) and TGFBR2 (-1.8 fold) expression, indicating that NRP1 mediates transcriptional regulation of FLT3 and TGFBR2 expression in inv(16) AML. Furthermore, the regulation of FLT3 and TGFB2 expression by CBFβ-SMMHC and by NRP1 was validated by gene editing in inv(16) AML blasts. Accordingly, NRP1 knock-down in AML cells reduced SMAD2/3 phosphorylation. The repression of RUNX1/CBFβ function, using small molecule inhibitors, in inv(16) AML cells with CBFβ-SMMHC knockdown restored NRP1 expression, suggesting that RUNX1 may repress NRP1 expression in AML cells. To evaluate if RUNX1 directly regulates NRP1 expression, we tested RUNX1 binding in the NRP1 locus of AML cells with CBFβ-SMMHC knockdown. RUNX1 binding at one of six sites with RUNX1 occupancy identified by chromatin immunoprecipitation followed by sequencing (RE5, regulatory element 5) was increased in the CBFβ-SMMHC knock-down cells. The RE5 is located 178kb upstream of the NRP1 transcription start site and it is evolutionarily conserved in vertebrates. The deletion of RE5 by gene editing (~50% editing efficiency) increased NRP1 expression 1.8-fold, suggesting that RUNX1 may repress NRP1 expression at by binding to the RE5 enhancer. Taken together, these studies demonstrate that CBFβ-SMMHC regulates expression of cytokine receptors in inv(16) AML. Specifically, it directly regulates expression of the co-receptor NRP1, which is essential for AML survival, acting (at least in part) by regulating FLT3 and TGFB pathways. Disclosures Guzman: SeqRx: Consultancy; BridgeMedicines: Consultancy; Cellectis: Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees.

In the present study, a series of multipotent antioxidants (MPAOs), namely sulfur-containing BHT (S-BHT), derivatives were rationally designed and synthesized, and their inhibitory activities against free radicals and human cancer cell lines, HT29 (colon cancer) and MCF7 (breast cancer) were further evaluated. The experimental results showed that the Six out-of-eight S-BHT compounds had excellent antioxidant activity against DPPH radical with major enhancement compared to BHT. Among them, compounds 2b, 2a and 3b attained over 45% lower IC50 values than BHT. In vitro cytotoxicity, MTT assay was carried out using two human cancer cell lines, HT29 (colon cancer) and MCF7 (breast cancer) in addition to their non-tumorigenic counterparts to explore selectivity. In line with antioxidant activity, compounds 2a and 2b displayed the highest cytotoxicity effect on both cancer types. Interestingly, 2b not only exhibited superior cancer inhibition but also scored high selectivity index (SI = 5.2, 12.5) in colon and breast tissues, respectively, exceeding that of the standard chemotherapeutic drugs used 5-Fluorouracil (5-FU) and Tamoxifen (Tmx), with lower IC50 values. The results indicated that the symmetric S-BHT derivatives were significantly enhanced by the antioxidant potency and their ability as useful and promising selective anticancer agents. A series of sulfur-containing phenols (S-BHT) as multipotent antioxidants structure have been rationally designed and synthesized by the combination of the antiradical activity of the most well-known phenol moiety, butylated hydroxytoluene (BHT, primary antioxidant) with the antiperoxide (secondary antioxidant) and anti-proliferative fragments, sulfur-containing groups into one structure aiming to enhance scavenging ability and antiproliferative activity of the radicals. Accordingly, the bioactivity of S-BHT was analysed by antioxidant and anticancer in vitro studies and demonstrated remarkable cancer inhibition rates along with significant cytotoxicity and selectivity against HT29 and MCF7 cancer cell lines. This work efficiently sets the ground for eminent sulfur-containing phenols developed based on a well-known standard antioxidant BHT, with features superior to common drawbacks faced in cancer therapeutic approaches, in addition to the great potential holding as selective and biocompatible agent for cancer applications.