PAX7 Balances the Cell Cycle Progression via Regulating Expression of Dnmt3b and Apobec2 in Differentiating PSCs

PAX7 transcription factor plays a crucial role in embryonic myogenesis and in adult muscles in which it secures proper function of satellite cells, including regulation of their self renewal. PAX7 downregulation is necessary for the myogenic differentiation of satellite cells induced after muscle damage, what is prerequisite step for regeneration. Using differentiating pluripotent stem cells we documented that the absence of functional PAX7 facilitates proliferation. Such action is executed by the modulation of the expression of two proteins involved in the DNA methylation, i.e., Dnmt3b and Apobec2. Increase in Dnmt3b expression led to the downregulation of the CDK inhibitors and facilitated cell cycle progression. Changes in Apobec2 expression, on the other hand, differently impacted proliferation/differentiation balance, depending on the experimental model used.

FOXC1 promotes HCC proliferation and metastasis by Upregulating DNMT3B to induce DNA Hypermethylation of CTH promoter

Background Forkhead box C1 (FOXC1), as a member of the FOX family, is important for promote HCC invasion and metastasis. FOX family protein lays a pivotal role in metabolism. ROS is involved in tumor progression and is associated with the expression of lots of transcription factors. We next explored the mechanism underlying FOXC1 modulating the metabolism and ROS hemostasis in HCC. Methods We used amino acids arrays to verify which metabolism is involved in FOXC1-induced HCC. The kits were used to detect the ROS levels in HCC cells with over-expression or down-expression of FOXC1. After identified the downstream target genes and candidate pathway which regulated by FOXC1 during HCC progression in vitro and in vivo, we used western blot, immunohistochemistry, bisulfite genomic sequencing, methylation-specific PCR, chromatin immunoprecipitation analysis and luciferase reporter assays to explore the relationship of FOXC1 and downstream genes. Moreover, the correlation between FOXC1 and target genes and the correlation between target genes and the recurrence and overall survival were analyzed in two independent human HCC cohorts. Results Here, we reported that FOXC1 could inhibit the cysteine metabolism and increase reactive oxygen species (ROS) levels by regulating cysteine metabolism-related genes, cystathionine γ-lyase (CTH). Overexpression of CTH significantly suppressed FOXC1-induced HCC proliferation, invasion and metastasis, while the reduction in cell proliferation, invasion and metastasis caused by the inhibition of FOXC1 could be reversed by knockdown of CTH. Meanwhile, FOXC1 upregulated de novo DNA methylase 3B (DNMT3B) expression to induce DNA hypermethylation of CTH promoter, which resulted in low expression of CTH in HCC cells. Moreover, low levels of ROS induced by N-acetylcysteine (NAC) which is an antioxidant inhibited the cell proliferation, migration, and invasion abilities mediated by FOXC1 overexpression, whereas high levels of ROS induced by L-Buthionine-sulfoximine (BSO) rescued the suppression results mediated by FOXC1 knockdown. Our study demonstrated that the overexpression of FOXC1 that was induced by the ROS dependent on the extracellular regulated protein kinases 1 and 2 (ERK1/2)- phospho-ETS Transcription Factor 1 (p-ELK1) pathway. In human HCC tissues, FOXC1 expression was positively correlated with oxidative damage marker 8-hydroxy-2′-deoxyguanosine (8-OHdG), p-ELK1 and DNMT3B expression, but negatively correlated with CTH expression. HCC patients with positive co-expression of 8-OHdG/FOXC1 or p-ELK1/FOXC1 or FOXC1/DNMT3B had the worst prognosis, whereas HCC patients who had positive FOXC1 and negative CTH expression exhibited the worst prognosis. Conclusion In a word, we clarify that the positive feedback loop of ROS-FOXC1-cysteine metabolism-ROS is important for promoting liver cancer proliferation and metastasis, and this pathway may provide a prospective clinical treatment approach for HCC.

FOXC1 Promotes HCC Proliferation and Metastasis by Upregulating DNMT3B to Induce DNA Hypermethylation of CTH Promoter

Background: Forkhead box C1 (FOXC1), as a member of the FOX family, is important for promote HCC invasion and metastasis. FOX family protein lays a pivotal role in metabolism. ROS is involved in tumor progression and is associated with the expression of lots of transcription factors. We next explored the mechanism underlying FOXC1 modulating the metabolism and ROS hemostasis in HCC.Methods: We used amino acids arrays to verify which metabolism is involved in FOXC1-induced HCC. The kits were used to detect the ROS levels in HCC cells with over-expression or down-expression of FOXC1. After identified the downstream target genes and candidate pathway which regulated by FOXC1 during HCC progression in vitro and in vivo, we used western blot, immunohistochemistry, bisulfite genomic sequencing, methylation-specific PCR, chromatin immunoprecipitation analysis and luciferase reporter assays to explore the relationship of FOXC1 and downstream genes. Moreover, the correlation between FOXC1 and target genes and the correlation between target genes and the recurrence and overall survival were analyzed in two independent human HCC cohorts.Results: Here, we reported that FOXC1 could inhibit the cysteine metabolism and increase reactive oxygen species (ROS) levels by regulating cysteine metabolism-related genes, cystathionine γ-lyase (CTH). Overexpression of CTH significantly suppressed FOXC1-induced HCC proliferation, invasion and metastasis, while the reduction in cell proliferation, invasion and metastasis caused by the inhibition of FOXC1 could be reversed by knockdown of CTH. Meanwhile, FOXC1 upregulated de novo DNA methylase 3B (DNMT3B) expression to induce DNA hypermethylation of CTH promoter, which resulted in low expression of CTH in HCC cells. Moreover, low levels of ROS induced by N-acetylcysteine (NAC) which is an antioxidant inhibited the cell proliferation, migration, and invasion abilities mediated by FOXC1 overexpression, whereas high levels of ROS induced by L-Buthionine-sulfoximine (BSO) rescued the suppression results mediated by FOXC1 knockdown. Our study demonstrated that the overexpression of FOXC1 that was induced by the ROS dependent on the extracellular regulated protein kinases 1 and 2 (ERK1/2)- phospho-ETS Transcription Factor 1 (p-ELK1) pathway. In human HCC tissues, FOXC1 expression was positively correlated with oxidative damage marker 8-hydroxy-2'-deoxyguanosine (8-OHdG), p-ELK1 and DNMT3B expression, but negatively correlated with CTH expression. HCC patients with positive co-expression of 8-OHdG/FOXC1 or p-ELK1/FOXC1 or FOXC1/DNMT3B had the worst prognosis, whereas HCC patients who had positive FOXC1 and negative CTH expression exhibited the worst prognosis. Conclusion: In a word, we clarify that the positive feedback loop of ROS-FOXC1-cysteine metabolism-ROS is important for promoting liver cancer proliferation and metastasis, and this pathway may provide a prospective clinical treatment approach for HCC.

Pulsation Activates Mechanosensitive Piezo1 to Form Long-Term Hematopoietic Stem Cells

The temporal and spatial origin and development of long-term, self-renewing hematopoietic stem cells (LT-HSC) remain a mystery. The first set of definitive HSCs is born from the hemogenic endothelial cells residing in the ventral wall of the dorsal aorta (DA) of the aorta-gonad-mesonephros region during embryonic development. Blood flow- and shear-stress-mediated nitric oxide-induced vasodilation are responsible for the endothelial-to-HSC transition (EHT). However, it remains unknown why the ventral wall, and not the dorsal wall, of the DA is the restricted site of the EHT when blood flows through the entire DA and exerts shear stress on both the ventral and dorsal sides of the DA. Using single-particle tracking and fast Fourier Transform analyses of pulsating blood vessels, we demonstrate that the circumferential strain in the ventral wall, and not dorsal wall, is concurrent with and responsible for the magnitude, the site, and timing of the HSC formation. We extended our findings by developing a bioreactor to establish the functional link between pulsation in the blood vessels and HSC formation. Using serial transplant, limiting dilution, and serial replating assays, we found that pulsation mediated circumferential stretching of hemogenic endothelial cells or Piezo1 activation (Yoda1) yields 3-times higher amounts of Long Term (LT)-HSC formation; which reconstitute to normal multi-lineage adult blood. Using delayed-type hypersensitivity assay, adult globin expression, MPO enzyme activity, immunoglobulins, and T-cell receptor rearrangement analyses, we found that circumferential stretching or Piezo1 activation-derived HSCs reconstitute to functional T and B cells, adult erythrocytes, and myeloid cells. Our Piezo1fl/flxScl-Cre conditional knockout, gene-silencing, & confocal imaging further demonstrate that circumferential stretching of blood vessels activates Piezo1; which enhances epigenetic regulator Dnmt3b expression to stimulate the EHT. Our CUT&RUN CHIP-Sequencing & MASSArray methylation analyses demonstrate that Dnmt3b suppresses endothelial genes during EHT. To analyze the conserved role of PIEZO1-mediated mechanosensitive mechanisms in human hematopoiesis, we employed directed differentiation of constitutive RUNX1-mCherry human induced pluripotent stem cells (iPSCs) to hemogenic endothelial cells. We found that Yoda1-mediated PIEZO1 activation stimulated human endothelial-to-hematopoietic transition. In conclusion, pulsation-mediated circumferential strain activates Piezo1 to stimulate the endothelial-to-HSC transition via the induction of Dnmt3b expression. This leads to the formation of long-term self-renewing HSCs, which can engraft and reconstitute to multi-lineage, adult blood upon serial transplantations. Our identification of a novel biomechanical cue unravels the physiological mystery in HSC formation in the ventral wall of the DA. We also establish its cross-talk with mechanosensitive and epigenetic mechanisms to produce functional, long-term HSCs that reconstitute to form normal adult blood. This yields the therapeutic promise of developing transgene-free LT-HSC-based cellular therapies for the treatment of human blood disorders. Disclosures No relevant conflicts of interest to declare.

Molecular Mechanisms of Primary Resistance to Azacitidine in MDS/AML Patients - Data of the Hellenic MDS Study Group

Introduction: Azacitidine (AZA) is a hypomethylating agent that at low doses acts by inhibiting DNA methyltranferase activity. AZA is approved and widely used for the treatment of MDS patients and patients with AML not candidate for intensive chemotherapy. Unfortunately, even after an initial response, almost all patients relapse and so far -with the exception of a few clinical parameters and genetic mutations weakly correlated with favorable AZA response- the exact mechanisms underlying primary AZA resistance remain largely unknown. On the other hand, over the last years accumulated data suggest that hypoxia, an important regulatory factor of both, physiological and malignant, hematopoiesis, is also involved in MDS pathogenesis (Hayashi et al., 2018), while high Hif-1α levels in MDS have been previously correlated with poor overall survival and disease progression (Tong et al., 2012). Moreover, our group recently investigated the association between Hif-1α and response to AZA therapy and found that AZA-responders present with higher Hif-1α mRNA expression compared to non-responders/stable disease patients, while logistic regression analysis showed that Hif-1α mRNA expression is an independent predictor of response to AZA therapy (unpublished data). Aims: The current study focused on investigating the mechanisms underlying the observed association of Hif-1α over-expression with response to AZA-therapy, by examining the methyltransferase activity and mitochondrial dysfunction due to inactivation of complex II, which is reported to lead to increased Hif-1α expression. Methods: A total of 54 patients with a median age of 76 (52-89) years, and 10, age matched, healthy donors participated in the study. According to WHO 2016, 41 patients were classified as MDS (10 as MDS-EB-1, 24 as MDS-EB-2 and 7 as MDS-MLD) and 13 as AML. All patients received AZA treatment at the dose of 75mg/m2 x7 days SC. BM-derived mononuclear cells were isolated before treatment using the Ficoll-paque method, followed by RNA extraction using TRIzol reagent, and cDNA preparation using Superscript II reverse transcriptase. Hypoxia-inducible factor 1-alpha (Hif-1α), succinate dehydrogenase complex subunit D (DSHd) and DNA methyltrasferase beta (DNMT3b) expression were estimated by real time PCR TaqMan gene expression assays, using the appropriate primers and probes. Relative gene expression was calculated by comparative threshold cycle (2-ΔΔCt) method and normalized based on β-actin expression. Non-parametric tests were used for the statistical analysis of the results. Results: Out of the 54 examined patients, 28 responded to azacitidine treatment (R), (including CR, PR and HI), 9 failed to respond (NR), and 17 achieved stable disease status 9 (SD). NR and SD patients were considered as one group (NR/SD) in all analyses. Using Rt-PCR we found that the 2-ΔΔCt ratio of Hif-1α/β-Actin median expression for control samples was 1.18 (95% CI: 0.617-1.687), for AZA-responders 1.59 (95% CI: 1.029-3.18), while for NR/SD patients 0.754 (95% CI: 0.640-0.840), with a statistical significance between R and NR/SD patients (Mann-Whitney test, p=0.003). Moreover, the 2-ΔΔCt ratio of SDHd/β-Actin median expression for control samples was 1.2 (95% CI: 0.360-1.954), for R patients 0.81 (95% CI: 0.294-1.401), and for NR/SD patients 0.73 (95% CI: 0.542-0.793). Finally, for DNMT3b, the 2-ΔΔCt median expression ratio in control samples was 0.75 (95% CI: 0.637-1.526), for R patients 2.188 (95% CI: 1.547-3.630), while for NR/SD patients 1.338 (95% CI: 0.824-2.250). Conclusions: Our data suggest that both AZA-R and NR/SD patients present with low levels of SDHd mRNA, compared to control, in line with previous reports in MDS. For AZA-responders, this could be related to the observed Hif-1α mRNA over-expression, since the SDH inactivation (decreased Complex II activity) is known to cause HIF stabilization (Frezza et al., 2011; Selak et al., 2005). Nevertheless, NR/SD patients also appear with decreased SDHd activity, despite the observed low Hif-1α expression. Therefore, in those patients, Hif-1α- related AZA-therapy response seems to be independent from mitochondrial dysfunction and possibly relies on other hypoxia regulatory mechanisms. Moreover, our data suggest that AZA-responders appear with an increased DNMT3b expression compared to both control and NR/SD patients, which could also explain their better response to therapy. Disclosures Symeonidis: Pfizer: Research Funding; Sanofi: Research Funding; Tekeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; MSD: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Pappa:Novartis: Honoraria, Research Funding, Speakers Bureau; Celgene / GenesisPharma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Research Funding; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Honoraria, Research Funding; Amgen: Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Piezo1-Sensitive Biomechanical Pulsation Stimulates Long-Term Hematopoietic Stem Cell Formation

The birth and development of hemat\opoietic stem cells (HSCs) remain a mystery. During fetal development, a subset of endothelial cells transitions to become HSCs in the aorta-gonad-mesonephros (AGM) region. Blood flow-mediated shear stress and activation of nitric oxide synthase (NOS) were demonstrated to stimulate the endothelial-to-HSC transition in the AGM. However, we showed that malbec (mlbbw306), a zebrafish mutant for cadherin 5, produces HSCs despite circulation arrest and the inhibition of NOS, suggesting that other biomechanical forces, mechanosensation pathways, or epigenetic mechanisms might regulate HSC formation and could have utility in developing HSCs. Using zebrafish, murine, and human models, we show that Piezo1-sensitive biomechanical stretching of hemogenic endothelial cells enhances Dnmt3b expression for long-term (LT)-HSC formation. Our microangiography and time-lapse confocal imaging established that cdh5-MO embryos have a heartbeat and pulsation in blood vessels despite the absence of blood flow. We also employed light sheet and time-lapse confocal microscopy followed by Fourier transform analyses to establish that although pulsation is independent of blood flow in the AGM, it is concurrent with the endothelial-to-hematopoietic transition. To establish the functional link between pulsation and HSC formation, we developed a bioreactor simulating the pulsating blood vessel conditions. We found that the biomechanical stretching of hemogenic endothelial cells or the pharmacological activation of Piezo1 yields three times higher amounts of LT-HSC formation; which reconstitute to normal multi-lineage adult blood even upon serial transplantation. Our gene-silencing, time-lapse imaging, explant culture, and computational analyses further demonstrated that biomechanical stretching activates Piezo1; which enhances epigenetic regulator Dnmt3b expression to stimulate the endothelial-to-HSC transition. Our results demonstrate how pulsation-mediated biomechanical forces stimulate cell-fate transitions and stem cell formation by activating mechanosensitive channels as well as epigenetic machinery. We present a model that addresses major challenges in HSC transplantation and cellular therapies for treating blood and bone marrow diseases. In addition, we report a scalable bioreactor with potential widespread use and a pharmacological target to develop and expand LT-HSCs. Disclosures No relevant conflicts of interest to declare.