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Significance: Iron is an essential element required for sustaining a normal healthy life. However, an excess amount of iron in the bloodstream and tissue generates toxic hydroxyl radicals through Fenton reactions. Henceforth, a balance in iron concentration is extremely important to maintain cellular homeostasis in both normal hematopoiesis and erythropoiesis. Iron deficiency or iron overload can impact hematopoiesis and is associated with many hematological diseases. Recent Advances: The mechanisms of action of key iron regulators such as erythroferrone and the discovery of new drugs, such as ACE-536/luspatercept, are of potential interest to treat hematological disorders, such as β-thalassemia. New therapies targeting inflammation-induced ineffective erythropoiesis are also in progress. Furthermore, emerging evidences support differential interactions between iron and its cellular antioxidant responses of hematopoietic and neighboring stromal cells. Both iron and its systemic regulator, such as hepcidin, play a significant role in regulating erythropoiesis. Critical Issues: Significant pre-clinical studies are on the way and new drugs targeting iron metabolism have been recently approved or are undergoing clinical trials to treat pathological conditions with impaired erythropoiesis such as myelodysplastic syndromes or β-thalassemia. Future Directions: Future studies should explore how iron regulates hematopoiesis in both benign and malignant conditions. Antioxid. Redox Signal. 35, 415–432.

Acute myeloid leukemia (AML) is a heterogeneous, aggressive malignancy with dismal prognosis and with limited availability of targeted therapies. Epigenetic deregulation contributes to AML pathogenesis. KDM6 proteins are histone-3-lysine-27-demethylases that play context-dependent roles in AML. We inform that KDM6-demethylase function critically regulates DNA-damage-repair-(DDR) gene expression in AML. Mechanistically, KDM6 expression is regulated by genotoxic stress, with deficiency of KDM6A-(UTX) and KDM6B-(JMJD3) impairing DDR transcriptional activation and compromising repair potential. Acquired KDM6A loss-of-function mutations are implicated in chemoresistance, although a significant percentage of relapsed-AML has upregulated KDM6A. Olaparib treatment reduced engraftment of KDM6A-mutant-AML-patient-derived-xenografts, highlighting synthetic lethality using Poly-(ADP-ribose)-polymerase-(PARP)-inhibition. Crucially, a higher KDM6A expression is correlated with venetoclax tolerance. Loss of KDM6A increased mitochondrial activity, BCL2 expression, and sensitized AML cells to venetoclax. Additionally, BCL2A1 associates with venetoclax resistance, and KDM6A loss was accompanied with a downregulated BCL2A1. Corroborating these results, dual targeting of PARP and BCL2 was superior to PARP or BCL2 inhibitor monotherapy in inducing AML apoptosis, and primary AML cells carrying KDM6A-domain-mutations were even more sensitive to the combination. Together, our study illustrates a mechanistic rationale in support for a novel combination therapy for AML based on subtype-heterogeneity, and establishes KDM6A as a molecular regulator for determining therapeutic efficacy.

Acute myeloid leukemia (AML) is a heterogeneous, aggressive malignancy with dismal prognosis and with limited availability of targeted therapies. AML exhibits epigenetic deregulation and transcriptional plasticity that contributes to pathogenesis. KDM6 proteins are histone-3 lysine-27 demethylases that play major context dependent roles in AML evolution and therapy resistance. Here, we demonstrate that KDM6 demethylase function critically regulates DNA damage repair (DDR) gene expression programs in AML. Mechanistically, KDM6 family protein expression is regulated by genotoxic stress, with deficiency of KDM6A (UTX) and KDM6B (JMJD3) impairing DDR transcriptional activation and compromising repair potential. Acquired KDM6A loss-of-function mutations have been implicated in chemoresistance, although a significant percentage of relapsed AML have upregulated KDM6A. Based on these mechanistic findings, olaparib treatment significantly reduced engraftment of patient-derived xenografts. Thus KDM6A-mutant human primary AML samples have increased susceptibility to Poly-(ADP-ribose)-polymerase (PARP) inhibition in vivo. Crucially, a higher KDM6A expression is correlated with venetoclax tolerance. Loss of KDM6A increased mitochondrial activity, BCL2 expression, and sensitized AML cells to venetoclax. Additionally, KDM6A loss was accompanied with a downregulated BCL2A1, which is commonly associated with venetoclax resistance. Corroborating these results, dual targeting of PARP and BCL2 was superior to PARP or BCL2 inhibitor monotherapy in inducing AML apoptosis, and primary AML cells carrying acquired KDM6A-domain mutations were even more sensitive to the combination. Together, our study illustrates a mechanistic rationale in support for a novel combination targeted therapy for human AML based on subtype heterogeneity, and establishes KDM6A as an important molecular regulator for determining therapeutic efficacy.

Somatic heterozygous mutations in genes encoding for RNA splicing factors (SF) SRSF2, U2AF1, and SF3B1 are frequently mutated in patients with hematologic malignancies, representing a unique genetic vulnerability for targeted therapy. In the current study, we performed a focused drug screen with inhibitors targeting different DNA damage response and DNA metabolic pathways to identify novel therapeutic vulnerabilities generated by SF mutations. We generated a murine leukemia model by overexpressing the MLL-AF9 fusion oncogene on an Srsf2P95H/+ background, a mutational combination that is found in ~10% of MLL-rearranged leukemias. We surprisingly found that MLL-AF9 Srsf2P95H/+ mutant leukemias are sensitive to inhibitors targeting ADP-ribosyltransferases (PARP). PARP inhibitor sensitivity was also observed in isogenic murine MLL-AF9 U2af1s34/+ cells compared to MLL-AF9 U2af1+/+ cells. Second, murine Srsf2P95H leukemias showed improved prolonged survival when treated with olaparib (PARPi) compared to vehicle treatment in vivo. Third, human primary AML patient samples that harbor SF mutations are sensitive to PARPi compared to non-SF mutant samples. These data highlight that both SRSF2P95H and U2AF1S34F mutations create a common vulnerability that is dependent on PARP activity for survival. To evaluate PARP activity, we used isogenic K562 leukemia cells expressing SRSF2P95H and U2AF1S34F mutations from their endogenous loci and monitored ADP-ribosylation (ADPr) levels, a marker of PARP activity. Both SRSF2P95H and U2AF1S34F cells exhibited elevated levels of ADPr compared to wildtype cells in a PARP1- dependent manner. PARPi preferentially induced DNA damage and cell death in SF mutant cells. Surprisingly, we found that SRSF2P95H and U2AF1S34F cells are not defective in homologous recombination repair. Instead, the increased PARP1-mediated ADPr in SF-mutant cells is caused by accumulated R loops, a group of transcription intermediates containing RNA:DNA hybrids and displaced single-stranded DNA. To determine whether PARPi sensitivity is due to R-loop accumulation, we overexpressed RNase H1, an enzyme that specifically cleaves the RNA moiety within RNA:DNA hybrids in U2AF1S34F cells. Overexpression of RNase H1 significantly reduced ADPr levels and suppressed the PARPi-induced U2AF1S34F cell growth inhibition. Collectively, these results suggest that spliceosome mutants induce R-loop accumulation and elicit an R-loop-associated PARP1 response to promote cell survival. In summary, our data establish a previously unknown link between R-loop-induced PARP1 response and RNA splicing perturbation and provide a mechanistic rationale to evaluate the clinical efficacy of PARP inhibitors in spliceosome-mutant malignancies. Furthermore, our study highlights a new therapeutic potential of targeting the R-loop tolerance pathways caused by different spliceosome gene mutations. Citation Format: Dang Hai Nguyen, Sayantani Sinha, Zhiyan Silvia Liu, Maxwell Henry Bannister, Erica Arriaga-Gomez, Axia Song, Dawei Zong, Martina Sarchi, Victor Corral, Wannasiri Chiraphapphaiboon, Jennifer Yoo, Matthew McMahon, Cassandra Leibson, Derek L. Stirewalt, H Joachim Deeg, Sumit Rai, Matthew Walter, Timothy A. Graubert, Sergei Doulatov, Stanley C. Lee. PARP inhibitors preferentially sensitize splicing factor mutant myeloid neoplasms. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6183.

The paternally imprinted neuronatin (NNAT) gene has been identified as a target of aberrant epigenetic silencing in diverse cancers, but no association with pediatric bone cancers has been reported to date. In screening childhood cancers, we identified aberrant CpG island hypermethylation in a majority of osteosarcoma (OS) samples and in 5 of 6 human OS cell lines studied but not in normal bone-derived tissue samples. CpG island hypermethylation was associated with transcriptional silencing in human OS cells, and silencing was reversible upon treatment with 5-aza-2’-deoxycytidine. Expression of NNAT was detectable in osteoblasts and chondrocytes of human bone, supporting a potential role in bone homeostasis. Enforced expression of NNAT in human OS cells lacking endogenous expression resulted in significant reduction in colony formation and in vitro migration compared to nonexpressor control cells. We next analyzed the effect of NNAT expression on intracellular calcium homeostasis and found that was associated with an attenuated decay of calcium levels to baseline following ATP-induced release of calcium from endoplasmic reticulum (ER) stores. Furthermore, NNAT expression was associated with increased cytotoxicity in OS cells from thapsigargin, an inhibitor of calcium reuptake into ER and an inducer of the ER stress response. These results suggest a possible tumor suppressor role for NNAT in human osteosarcoma. Additional study is needed ascertain sensitization to ER stress-associated apoptosis as a mechanism of NNAT-dependent cytotoxicity. In that case, epigenetic modification therapy to effect NNAT transcriptional derepression may represent a therapeutic strategy potentially of benefit to a majority of osteosarcoma patients.

There is a Blood Commentary on this article in this issue.

Heterotopic ossification (HO) is a common, potentially debilitating pathology that is instigated by inflammation caused by tissue damage or other insults, which is followed by chondrogenesis, osteogenesis, and extraskeletal bone accumulation. Current remedies are not very effective and have side effects, including the risk of triggering additional HO. The TGF-β family member activin A is produced by activated macrophages and other inflammatory cells and stimulates the intracellular effectors SMAD2 and SMAD3 (SMAD2/3). Because HO starts with inflammation and because SMAD2/3 activation is chondrogenic, we tested whether activin A stimulated HO development. Using mouse models of acquired intramuscular and subdermal HO, we found that blockage of endogenous activin A by a systemically administered neutralizing antibody reduced HO development and bone accumulation. Single-cell RNA-seq analysis and developmental trajectories showed that the antibody treatment reduced the recruitment of Sox9+ skeletal progenitors, many of which also expressed the gene encoding activin A (Inhba), to HO sites. Gain-of-function assays showed that activin A enhanced the chondrogenic differentiation of progenitor cells through SMAD2/3 signaling, and inclusion of activin A in HO-inducing implants enhanced HO development in vivo. Together, our data reveal that activin A is a critical upstream signaling stimulator of acquired HO in mice and could represent an effective therapeutic target against forms of this pathology in patients.

Summary We describe a protocol for a long-term co-culture assay to study the contribution of mesenchymal stromal cells (MSCs) in regulating hematopoietic stem/progenitor cell (HSPC) activity. In addition, we describe the use of a clonogenic assay to determine myelo-erythroid differentiation. This long-term culture-initiating cell assay can be used for qualitative analysis of MSCs capable of supporting hematopoiesis and may also be used as a proxy readout to study HSPC repopulation. For complete details on the use and execution of this protocol, please refer to Sinha et al. (2020)., Graphical Abstract, Highlights • We report long-term co-culture of mesenchymal stroma and hematopoietic stem/progenitors • End-point colony-forming analysis helps determine myelo-erythroid differentiation • This protocol analyzes mesenchymal stromal cell potential to support hematopoiesis • Long-term culture-initiating cell assay is a surrogate for hematopoietic engraftment, We describe a protocol for a long-term co-culture assay to study the contribution of mesenchymal stromal cells (MSCs) in regulating hematopoietic stem/progenitor cell (HSPC) activity. In addition, we describe the use of a clonogenic assay to determine myelo-erythroid differentiation. This long-term culture-initiating cell assay can be used for qualitative analysis of MSCs capable of supporting hematopoiesis and may also be used as a proxy readout to study HSPC repopulation.

We present a rare case of an aberrant right vertebral artery originating from the arch of aorta distal to the origin of the left subclavian artery. The incidence of this particular variant of aberrant origin of the right vertebral artery is extremely uncommon with only seventeen cases reported in literature to date. This case was incidentally detected on a staging positron emission tomography-computerized tomography (PET-CT) scan for lung cancer. We review the incidence, embryological mechanism, and clinical importance of this aberrant course of the right vertebral artery.

Somatic heterozygous mutations in spliceosome genes SRSF2, U2AF1, and SF3B1 commonly occur in patients with myeloid malignancies such as myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). Moreover, SRSF2 and U2AF1 mutations associate with poor survival and high risk of progression to AML, representing a unique genetic vulnerability for targeted therapy. We and others previously found that R-loops, a group of transcription intermediates containing RNA:DNA hybrids and displaced single-stranded DNA, are a source of genomic instability induced by different spliceosome mutants. We further showed that inhibition of ATR kinase activity preferentially kills spliceosome mutant cells in an R-loop-dependent manner. Inspired by ATR inhibition results, we performed a focused drug screen with inhibitors targeting additional DNA damage response pathways to identify novel therapeutic vulnerabilities generated by spliceosome mutations. We generated a murine leukemia model by overexpressing the MLL-AF9 fusion oncogene on an Srsf2 P95H/+background, a mutational combination that is found in ~10% of MLL-rearranged leukemias. Surprisingly, we found that Srsf2 P95H/+cells are more sensitive to five inhibitors targeting ADP-ribosyltransferases or PARP (olaparib, talazoparib, rucaparib, niraparib, veliparib) (Figs 1A-B). Olaparib (PARPi)-treated Srsf2 P95H/+cells exhibited increased apoptosis compared to Srsf2 +/+ cells as determined by AnnexinV (Fig 1C). PARPi sensitivity was also observed in isogenic murine MLL-AF9 U2af1 S34F/+cells compared to MLL-AF9 U2af1 +/+ cells (Fig 1D). These data highlight that both SRSF2 P95H and U2AF1 S34F mutations create a common vulnerability that is dependent on PARP activity for survival. To evaluate PARP activity in cells, we used isogenic K562 leukemia cells expressing SRSF2 P95H and U2AF1 S34F mutations from their endogenous loci and monitored PAR (poly(ADP-ribose)) chain levels, a marker of PARP activity. Both SRSF2 P95H and U2AF1 S34F cells exhibited elevated PAR levels compared to wildtype cells (Figs 1E-F). PARPi treatment significantly suppressed PAR signals in SRSF2 P95H and U2AF1 S34F cells. PARP inhibitors target both PARP1 and PARP2 enzymes, of which PARP1 plays a key role in DNA damage response. We used CRISPR-Cas9 to knockout PARP1 gene to determine the major PARP responsible for elevated PAR level in these leukemia cells. PARP1 deletion abrogated elevated PAR levels in U2AF1 S34F (Fig 1G) and SRSF2 P95H cells (data not shown). Altogether, we demonstrated that SRSF2 P95H and U2AF1 S34F cells trigger a PARP1 response critical for cell survival. To test whether increased PAR level arises from U2AF1 S34F-induced R-loops, we generated U2AF1 S34F cells that inducibly express RNaseH1, an enzyme that specifically cleaves the RNA moiety within RNA:DNA hybrids. Induction of RNaseH1 in U2AF1 S34F cells significantly reduced PAR levels, showing that U2AF1 S34F-induced PAR chains is R-loop-dependent (Fig 1H). Moreover, RNaseH1 overexpression suppressed the growth inhibition of PARPi-treated U2AF1 S34F cells (Fig 1I). Collectively, these results suggest that U2AF1 S34F mutants induce R-loop accumulation and elicit an R-loop-associated PARP1 signaling to promote cell survival. We next tested whether combining ATR inhibitor (ATRi) can further exacerbate PARPi sensitivity in spliceosome mutant cells. To examine ATR activity, we monitored phosphorylated RPA (Replication Protein A, or pRPA), a known ATR substrate. pRPA level was enhanced in PARPi-treated SRSF2 P95H cells compared to PARPi-treated SRSF2 WT cells but was suppressed when treated with ATRi (Fig 1J), suggesting that splicing factor mutant cells are more reliant on ATR function in the context of PARPi. Importantly, the combination of PARPi with ATRi, but not with ATMi, significantly promoted cell growth inhibition in SRSF2 P95H cells compared to SRSF2 WT cells or to SRSF2 P95H cells treated with individual compounds alone (Fig 1K). Collectively, these data provide a pre-clinical rationale that splicing factor mutant leukemias are preferentially sensitive to PARP1 modulation compared to their wildtype counterpart. Moreover, combining PARPi and ATRi may further sensitize spliceosome mutant cells and could represent a new therapeutic strategy in myeloid leukemia patients harboring these mutations (Fig 1L). Figure 1 Figure 1. Disclosures Graubert: Calico: Research Funding; Janssen: Research Funding; astrazeneca: Research Funding.

The hallmarks of β-thalassemia (BT) include ineffective erythropoiesis (IE), splenomegaly and iron overload (IO). Recent studies have pointed to iron restriction (IR) to improve both anemia and IO in BT (Rivella, Blood). The decreased iron-uptake by early erythroid cells reduces hemichrome toxicity and prevents premature RBC hemolysis. One such IR therapy targets the matriptase-2 (Tmprss6) gene using antisense oligonucleotides (T-ASO). Our group has previously shown that treatment of Hbb th3/+(th3/+) mice (a mouse model for BT-intermedia) with T-ASO improved anemia, lengthened red blood cell (RBC) lifespan, reduced levels of erythroferrone (ERFE), hemichromes and reactive oxygen species, and ameliorated splenomegaly (Casu et al. Blood). Another novel therapeutic approach to improve anemia targets the Transforming Growth Factor (TGF)-β pathway to increase erythroid maturation. Luspatercept, a TGF-β trap-ligand, gained FDA approval in 2019 to treat transfusion dependent BT patients (Cappellini and Taher, Blood Adv). In mouse models of BT, its murine analog (RAP-536) was found to promote EPO-independent maturation of late-stage erythroid cells, and resulted in increased RBC parameters in a dose-dependent manner (Surgani, et al. Nat Med). In this work we treated th3/+ mice with an agent analogous to murine Luspatercept (RAP-GRL) in combination with the iron restriction (IR) drug T-ASO, (RAP-GRL+T-ASO) with the goal of targeting distinct morbidities associated with BT. To test our RAP-GRL construct, primary fibroblasts were transduced with an adenovirus containing the RAP-GRL sequence (FB Ad5RAP-GRL) and used to deliver RAP-GRL to mice. As a second strategy, RAP-GRL was expressed in a mammalian cell line and purified. Wild-type (WT) or th3/+ mice were subcutaneously (s.c.) implanted with 1x10 6 FB Ad5RAP-GRL or injected s.c. with 10mg/kg of RAP-GRL and monitored by complete blood counts. Implantation of FB Ad5RAP-GRL ortreatment with purified RAP-GRL increased RBC parameters in both WT and th3/+ mice (n=3-9, 2-4-month-old females and males). In the first combination therapy experiment we implanted FB Ad5RAP-GRL s.c. and delivered T-ASO via intraperitoneal (i.p.) injection in th3/+ mice. RBC parameters were increased in all treatment groups except controls after 6 weeks. The RAP-GRL+ T-ASO group displayed the most pronounced increase in RBC parameters with a mean increase in RBC of 3.067±0.73 10 6 cells/µL, Hb of 3.02±0.77 g/dL, and Hct of 5.88±2.36 % (Table 1). Additionally, we also treated th3/+ mice with two different doses of protein purified RAP-GRL in combination with T-ASO (Table 1). The best results using the protein purified RAP-GRL were achieved in the RAP-GRL+T-ASO group that was treated with two weekly 10mg/kg s.c. injections of RAP-GRL and two weekly 5mg/kg i.p. injections of T-ASO (Group 2) for 6 weeks. Flow cytometry analysis using CD71, TER119, and CD44 antibodies showed improvements in the bone marrow (BM) and spleen (SPL) of all treatment groups compared to controls. Additionally, ROS levels and splenomegaly were also greatly reduced in all T-ASO and RAP-GRL+T-ASO treated groups compared to controls. Serum assessment of T-ASO and RAP-GRL+T-ASO treated animals showed decreased levels of iron and transferrin saturations with a simultaneous increase in hepcidin levels. ERFE levels were decreased in all T-ASO and RAP-GRL+T-ASO groups, however, erythropoietin (EPO) levels were increased only in the RAP-GRL and RAP-GRL+T-ASO cohorts of Group 2. Additionally, although EPO was elevated in all RAP-GRL treated animals of Group 2, only the RAP-GRL+T-ASO group had reduced ERFE. This result is in agreements with our findings of decreased early (ERFE-producing) erythroid progenitors in the BM and SPL of RAP-GRL+T-ASO treated mice. This finding also suggests that higher doses of RAP-GRL may result in elevated EPO. Luspatercept, through heightened iron consumption, may increases EPO synthesis in the kidney via activation of the transcription factor HIF2-α, which can be stabilized not only by hypoxia, but also by iron deficiency. In conclusion our results provide pre-clinical support for combining IR and TFG-β trap-ligands in the treatment of BT. Our data shows that IR, in conjunction with the enhancing erythroid maturation action of Luspatercept (and potential activation of EPO), may offer an additive and more effective therapeutic strategy for BT patients. Figure 1 Figure 1. Disclosures Guo: Ionis Pharmaceuticals, Inc.: Current Employment. Rivella: Ionis Pharmaceuticals: Consultancy; Meira GTx: Consultancy.

Anemia of inflammation (AI) is the second most common anemia after iron deficiency anemia. The predominant regulators of AI are the cytokine interleukin 6 (IL6) and the hormone hepcidin (HAMP). IL6 is an inflammatory cytokine which also limits iron absorption by inducing HAMP, which promotes the degradation of the iron exporter ferroportin. We hypothesized that knocking down both HAMP and IL6 simultaneously will help us to understand if their mode of action in AI is uniquely limited to iron absorption and erythroid iron intake or if they also have independent roles. Henceforth, we generated IL6/HampKO (DKO) mice and, unexpectedly, observed that IL6KO mice showed the best recovery in bone marrow (BM) erythropoiesis (using flow cytometry analysis and looking at the absolute number of erythroid progenitors) after BA administration when compared to wild type (WT), HampKO and DKO mice. The best differences were observed at 14 days post BA administration. In contrast, the extramedullary erythropoiesis in the spleen was more pronounced in HampKO and DKO mice compared to WT and IL6KO animals, indicating that the mechanism impairing erythropoiesis in the BM did not affect erythroid progenitors in the spleen. These observations suggest that HAMP and IL6 proteins contribute independently to AI, with IL6 having some effect on the erythropoiesis in the BM independent from the IL6-HAMP axis leading to iron restriction. Furthermore, these observations raised the question why both HampKO and DKO mice showed reduced BM erythropoiesis compared to IL6KO animals. We investigated inflammatory cytokines and altered iron parameters as potential mediators of impaired erythropoiesis. We compared several inflammatory cytokines, including IL6, TNFa and INFg following BA administration: cytokine levels were elevated 6 hrs, reduced 48hrs after BA administration and moderately increased again two weeks later. Interestingly, among all the cytokines the levels of IL1b were significantly attenuated in IL6KO mice at day 14 compared to WT and HampKO animals. Moreover, transferrin saturation and NTBI levels were higher in HampKO and DKO animals compared to IL6KO mice. These observations strongly suggested that BM erythropoiesis is more sensitive to inflammatory insult in presence of an excess of iron, while extramedullary erythropoiesis is mildly affected and can eventually thrive under supra-physiological transferrin saturation levels. To test if increased iron affects BM erythropoiesis in presence of inflammation, we treated both WT and IL6KO mice with combination of iron dextran and BA. Both WT and IL6KO mice were treated with a combination of BA and iron at day 0 followed by alternate day of iron injections showed the poorest erythropoiesis in the BM and became rapidly sick, although the effect was significantly more pronounced in WT animals, as suggested by their survival curve. Since mycobacterium infections lead to NLPR3 inflammasome activation and Caspase1 upregulation (Marim et al. Semin Immunopathology 2017), we investigated how erythroid progenitors were affected. By flow cytometry analyses, we observed a significantly higher upregulation of the Caspase1 protein in WT and DKO mice compared to IL6KO animals. This was also reproduced by culturing WT or IL6KO BM progenitor erythroid cells in presence of mouse serum derived from WT or IL6KO mice treated with BA. Most importantly, IL6KO mice treated with BA and iron showed the highest levels of Caspase1 compared to only BA treated IL6KO mice, indicating that excess of iron abrogates the beneficial effect of IL6 deficiency on erythropoiesis under conditions of AI. Furthermore, using flow cytometry, we observed in WT mice treated with BA or BA and iron a significant increase in mitochondrial mass, which is an indicator of mitochondrial stress. The mitochondrial mass was reduced in IL6KO mice treated with BA, but again increased in IL6KO mice treated with BA and iron. We have also observed an increase of mitochondrial superoxide by confocal microscopy in WT mice compared to IL6KO mice treated with BA. Altogether, these data support a model where inflammation in presence of an excess of iron impairs BM erythropoiesis through mechanisms at least in part mediated by Caspase1 and mitochondrial dysfunction, while iron excess itself is sufficient to boost extramedullary erythropoiesis to compensate and sustain RBC production. Disclosures Vinchi: PharmaNutra: Research Funding; Vifor Pharma: Research Funding; Silence Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Research Funding. Rivella: Ionis Pharmaceuticals: Consultancy; Meira GTx: Consultancy.

Acute myeloid leukemia (AML) is a heterogeneous, aggressive hematological malignancy with dismal prognosis where limited targeted therapies are currently available. Poly-(ADP-ribose)-polymerase (PARP) inhibition has emerged as an important therapeutic arsenal to target homologous recombination-deficient tumors. However, molecular understanding of PARP blockade in the context of epigenetic derangements and transcriptional plasticity in human elderly AML pathogenesis remains unexplored. KDM6 proteins are H3K27 demethylases that critically regulate chromatin architecture in multi-cellularity and tumorigenesis (Tran, Mol Cell Biol 2020). KDM6A escapes X-chr inactivation, and Utx-/- female mice spontaneously develop aging associated myeloid leukemia (Gozdecka, Nat Genet 2018; Sera, Blood 2021). In addition, KDM6A loss of function mutation is implicated in AML relapse (Stief, Leukemia 2020). In contrast, KDM6B primarily exerts an oncogenic function in heme-malignancies. Together, KDM6A and KDM6B play cell type-specific function in leukemia, and KDM6 proteins and associated signaling emerge as important focal point for developing molecular targeted therapy. We identify that KDM6 demethylase activity critically regulates DNA damage repair (DDR) gene expression program in AML. Transcriptome analysis indicated a significant downregulation of expression of DDR genesets in both KDM6A deficient human AML and Utx -/- pre-leukemic cells. Lentiviral shRNA screening performed in response to low-dose γ-irradiation in AML stem cells, revealed a radioprotective function of KDM6A. Expression of KDM6s is regulated by genotoxic stress in a time-dependent manner, and deficiency of JmjC catalytic function impaired DDR transcriptional activation and compromised repair potential. Mechanistically, quantitative ChIP experiments also revealed co-operation between KDM6A and SWI/SNF facilitating dynamic chromatin remodeling at TSS/promoter to induce DDR gene expression. To interrogate changes in chromatin accessibility we performed ATAC-seq analysis in KDM6 deficient AML. Motif enrichment highlighted that while KDM6A depletion led to reduced chromatin access to 140 transcription factors (TFs), only 56 TF binding sites showed increased accessibility. Overall, changes in chromatin accessibility, associated with a reduced binding of DDR regulatory TFs in KDM6 deficient AML, account for a compromised DDR function. In agreement with these findings an array of KDM6 deficient AML cells were more sensitive to PARP inhibition, and pre-clinical mice models xenotransplanted with KDM6A loss of function AML line showed an increased susceptibility to PARP blockade in vivo. FLT3-ITD positive AML with a lower KDM6A expression was more sensitive to olaparib. In addition, olaparib administration significantly reduced bone marrow engraftment of patient-derived xenografts of KDM6A-mutant primary AML. Interestingly, KDM6A expression is upregulated in venetoclax-resistant monocytic-AML compared to venet-sensitive primitive-AML. Using venet responsive isogenic lines we demonstrated that attenuation of KDM6 function increased mitochondrial activity, intracellular ROS levels, de-repressed BCL2 expression, and sensitized AML cells to venetoclax. Additionally, KDM6 loss resulted in transcriptional repression of BCL2A1, commonly associated with venet resistance (Zhang, Nat Cancer 2020). Corroborating these results, dual targeting of PARP with BCL2 was superior to PARP or BCL2 inhibitor monotherapy in inducing primary AML apoptosis, and KDM6A loss further enhanced this synergism. In sum, our study illustrates a molecular mechanistic rationale in support for a novel combination targeted therapy for AML, and posit KDM6A as a molecular determinant for therapeutic efficacy. Intriguingly, KDM6A functions as a gatekeeper of BCL2 and BCL2A1 expression. Similar to TET2 although bi-allelic Utx loss causes evolution to myeloid neoplasms, minimal KDM6 activity is important for survival of human AML cells. KDM6s have been implicated in solid tumors, and both PARP and BCL2 inhibitors are being tested in cancer patients, underscoring a wider scope of application. To conclude, KDM6A unfolds to be a central regulator for susceptibility of AML to both PARP and BCL2 inhibition, expanding the possibility to characterize effective combination targeted therapy for AML in clinical settings. Disclosures Minden: Astellas: Consultancy. Dick: Celgene, Trillium Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Summary: Bone morphogenic protein (BMP)/transforming growth factor β (TGF-β) signaling determines mesenchymal-stromal-cell (MSC) osteolineage commitment and tissue identity. However, molecular integration of developmental signaling with MSC-intrinsic chromatin regulation remains incompletely understood. SWI/SNF-(BAF) is an ATP-dependent chromatin remodeler implicated in multi-cellular development. We show that BMPs and long-term osteogenic signals in MSCs selectively induce expression of polybromo BAF (PBAF) components Pbrm1, Arid2, and Brd7. Loss of Pbrm1/Arid2/Brd7 profoundly impairs osteolineage gene expression and osteogenesis without compromising adipogenesis. Pbrm1 loss attenuates MSC in vivo ossification. Mechanistically, Pbrm1/PBAF deficiency impairs Smad1/5/8 activation through locus-specific epi-genomic remodeling, involving Pbrm1 bromodomains, along with transcriptional downregulation of Bmpr/TgfβrII affecting BMP-early-responsive gene expression. Gain of function of BmprIβ, TgfβrII in PBAF-deficient MSCs partly restores Smad1/5/8 activation and osteogenesis. Pbrm1 loss further affects hematopoietic stem and progenitor activity through non-cell-autonomous regulation of microenvironment and niche-factor expression. Together, these findings reveal a link illustrating epi-genomic feedforward control of BMP/TGF-β signaling to transcriptional and cellular plasticity in the mesenchymal microenvironment and account for stromal-SWI/SNF in hematopoiesis. : Sinha et al. examine the role of Pbrm1/polybromo-BAF in mammalian mesenchymal stromal cell osteolineage commitment and hematopoiesis. Osteogenic signals induce Pbrm1/PBAF expression, which in turn orchestrates chromatin remodeling at BmprIβ/TgfβrII loci. These findings reveal an epi-genomic connection to feedforward control of BMP/TGF-β signaling in the mesenchymal stromal microenvironment. Keywords: BMP/TGF-β signaling, mesenchymal stromal cell, hematopoietic microenvironment, hematopoiesis, osteolineage differentiation, SWI/SNF, chromatin remodeling, bromodomain, transcriptional regulation, feedforward control

Heterotopic ossification (HO) consists of ectopic cartilage and bone formation following severe trauma or invasive surgeries, and a genetic form of it characterizes patients with Fibrodysplasia Ossificans Progressiva (FOP). Recent mouse studies showed that HO was significantly inhibited by systemic treatment with a corticosteroid or the retinoic acid receptor γ agonist Palovarotene. Because these drugs act differently, the data raised intriguing questions including whether the drugs affected HO via similar means, whether a combination therapy would be more effective or whether the drugs may hamper each other's action. To tackle these questions, we used an effective HO mouse model involving subcutaneous implantation of Matrigel plus rhBMP2, and compared the effectiveness of prednisone, dexamathaosone, Palovarotene or combination of. Each corticosteroid and Palovarotene reduced bone formation at max doses, and a combination therapy elicited similar outcomes without obvious interference. While Palovarotene had effectively prevented the initial cartilaginous phase of HO, the steroids appeared to act more on the bony phase. In reporter assays, dexamethasone and Palovarotene induced transcriptional activity of their respective GRE or RARE constructs and did not interfere with each other's pathway. Interestingly, both drugs inhibited the activity of a reporter construct for the inflammatory mediator NF-κB, particularly in combination. In good agreement, immunohistochemical analyses showed that both drugs markedly reduced the number of mast cells and macrophages near and within the ectopic Matrigel mass and reduced also the number of progenitor cells. In sum, corticosteroids and Palovarotene appear to block HO via common and distinct mechanisms. Most importantly, they directly or indirectly inhibit the recruitment of immune and inflammatory cells present at the affected site, thus alleviating the effects of key HO instigators.

MicroRNAs (miRNAs) are 19–22 nt-long RNA molecules that mediate posttranscriptional gene silencing. Since their discovery in Caenorhabditis elegans , miRNAs gained attention after a plethora of reports suggested their contribution to cell fate commitment, cellular plasticity, and cancer. Tumorigenesis often involves tumor-cell autonomous progressive, even stochastic, accumulation of a series of genetic and epigenetic changes, somatic mutations, and genomic instability that eventually transform into an overt malignant phenotype. Hallmarks of cancer further highlight importance of tissue-resident cancer stem cells, tumor-initiating cells, and cross talk between tumor cells within the mesenchymal microenvironment, as an important prognostic index in cancer development. Fluctuation in miRNA levels causes derepression of gene expression and could lead to tumor cell dedifferentiation. In the following sections, we illustrate miRNA involvement across the crucial hallmarks of tumorigenesis. Given the current understanding of miRNA regulation in cancer, synthetic regulation with miRNA mimics and therapeutic intervention would play a key role in cancer prevention.

Acquired aplastic anemia (AA) is a bone marrow (BM) failure associated with autoimmune destruction of hematopoietic stem cells (HSCs). Although somatic mutations have been identified in AA patients, mutations alone do not explain AA pathophysiology. SWI/SNF is an evolutionarily conserved, multi-subunit, ATP-dependent chromatin-remodeling protein complex that plays an important role in mammalian hematopoiesis. Herein, gene expression analysis identified a significant loss of the SWI/SNF core component SMARCC1, along with ARID1B, ACTL6A, and SMARCD1, in human AA BM CD34+ HSCs and hematopoietic stem and progenitor cells (HSPCs) compared with normal HSPCs. However, expression of SMARCA4, SMARCB1, SMARCD3, and DPF2 remained intact in our AA cohort. PBRM1, BRD7, and SMARCA2 expression were significantly upregulated in both untreated and follow-up AA patients. Clonal hematopoiesis in AA is associated with evolution to late clonal disorders, including myelodysplastic syndromes (MDS). Apart from SMARCD1 loss, we did not observe significant alteration of SWI/SNF expression in MDS HSPCs, indicating SWI/SNF differential expression in AA and MDS. In addition, except for ACTL6A, SWI/SNF expression was unaltered in aged HSPCs. Importantly, our results provide evidence for loss of SWI/SNF in AA, and may implicate AA HSPC-autonomous defective SWI/SNF regulation as an integral component of BM failure, in addition to autoimmune destruction of AA HSCs. These findings illustrate for the first time SWI/SNF subunit expression heterogeneity in human AA HSPCs and require prognostic validation in a larger cohort.

Background Transient receptor potential (TRP) ion channels have emerged as key components contributing to vasoreactivity. Propofol, an anesthetic is associated with adverse side effects including hypotension and acute pain upon infusion. Our objective was to determine the extent to which TRPA1 and/or TRPV1 ion channels are involved in mediating propofol-induced vasorelaxation of mouse coronary arterioles in vitro and elucidate the potential cellular signal transduction pathway by which this occurs. Methods Hearts were excised from anesthetized mice and coronary arterioles were dissected from control C57Bl/6J, TRPA1-/-, TRPV1-/- and double-knockout mice (TRPAV-/-). Isolated microvessels were cannulated and secured in a temperature-controlled chamber and allowed to equilibrate for 1 hr. Vasoreactivity studies were performed in microvessels pre-constricted with U46619 to assess the dose-dependent relaxation effects of propofol on coronary microvascular tone. Results Propofol-induced relaxation was unaffected in vessels obtained from TRPV1-/- mice, markedly attenuated in pre-constricted vessels obtained from TRPA1-/- mice and abolished in vessels obtained from TRPAV-/- mice. Furthermore, NOS inhibition with L-NAME or endothelium denuding abolished the proporfol-induced depressor response in pre-constricted vessels obtained from all mice. In the absence of L-NAME, BKCa inhibition with penitrem A markedly attenuated propofol-mediated relaxation in vessels obtained from wild-type mice and to a lesser extent in vessels obtained from TRPV1-/-, mice with no effect in vessels obtained from TRPA1-/- or TRPAV-/- mice. Conclusions TRPA1 and TRPV1 appear to contribute to the propofol-mediated antagonism of U46619-induced constriction in murine coronary microvessels that involves activation of NOS and BKCa.

Anemia of inflammation, also known as anemia of chronic disease is the second most common anemia after iron deficiency anemia. The predominant regulators of AI are the cytokine-interleukin-6 (IL6) and the hormone hepcidin (Hamp). IL6 has been implicated in inducing expression of hepcidin. Published data from our lab have shown that lack of IL6 or hepcidin in knockout mouse models (IL6-KO and Hamp-KO) injected with the heat-killed pathogen Brucella abortus(BA) results in recovery from anemia but interestingly the pattern of the recovery was different in IL6-KO and Hamp-KO mice, suggesting that the two proteins contribute independently to AI. Here, we validated the independent role of IL6 and Hamp in AI by generating a double-knockout (DKO) mouse model lacking the expression of both. In the first few days following BA administration, we observed severe reduction in the total number of BM cells in each model followed by a slow recovery in erythroid and multilineage hematopoietic cells. The recovery, initially, was more sustained in the BA-treated-DKO model. In particular, in the first week, BA-treated-DKO mice showed an increased number of erythroblasts in the bone marrow (BM) and spleen as seen in comparison to IL6-KO and Hamp-KO. IL6-KO mice showed an intermediate recovery profile when compared to DKO and Hamp-KO, the last one showing the worst profile in the BM. Interestingly, when the reticulocyte count in the DKO mice was compared to that of IL6-KO and Hamp-KO mice, it showed a biphasic trend, with a significant increase in number during the 2nd week, followed by a significant reduction during the 3rd week. We hypothesized that the initial surge in reticulocyte count in DKO was due to lack of hepcidin, which increases iron availability to erythroid cells, and concurrent lack of IL6, which favors BM erythropoiesis in presence of inflammatory stimuli. However, we also speculated that the excess of iron (as NTBI), which accumulates during the first two weeks, leads to oxidative stress and erythroid cell death in presence of inflammatory cytokines, despite the absence of IL6. We also surmised that, during the second week, a second wave of inflammatory cytokines is triggered by the adaptive response in response to the BA that would explain the negative effect on erythropoiesis after the initial recovery. To assess this hypothesis, we utilized an inflammation panel to analyze the cytokine expression in WT animals treated with PBS or BA at 6 hours, 24 hours and then around ~2 weeks. The cytokine levels were normalized after 24 hours. However, around two weeks, we observed a novel surge of cytokines such as IFN-g and TNFa in the BA treated mice, indicating their role in innate (immediate effect; 6 hours) and adaptive immune response, which activated a second wave of inflammation (around 2 weeks, during the recovery of hematopoiesis in the BM). Interestingly, while we observed oxidative stress and defective erythropoiesis in the bone marrow, this was not seen in the spleen, where increased and extramedullary erythropoiesis sustained some level of RBC production. Since the BA-treated-IL6-KO did not show any major defect in the BM after two weeks, we challenged them with administration of iron dextran. Upon treatment, also the IL6-KO mice treated with both BA and iron dextran shown increased production of reactive oxygen species as well as a defect in bone marrow erythropoiesis, similarly as in DKO or Hamp-KO mice, thereby explaining the plausible reason of reduced erythropoiesis in the bone-marrow. Furthermore, to identify mechanisms leading to oxidative stress, we established an in-vitro culture system where primary murine bone marrow cells were cultured for 18-20 hours in presence of serum isolated after 6hrs from either PBS treated or BA treated C57BL/6 mice. With the help of confocal microscopy, we observed an increase in mitochondrial superoxide in the cells treated with BA serum; interestingly we have also seen a decrease in Ter 119 population in the cells cultured with BA treated serum implicating that the erythroid cells are dying. To further investigate the downstream players related to the death of erythroid progenitors we are currently investigating the role caspase 1 (a major regulator in pyroptosis) and Gata-1. In conclusion, this study is elucidating some of the mechanisms associated with the anemia triggered by inflammation with the potential to identify new targets and treatments. Disclosures Rivella: Disc medicine, Protagonist, LIPC, Meira GTx: Consultancy; Meira GTx, Ionis Pharmaceutical: Membership on an entity's Board of Directors or advisory committees.

Background: Transient receptor potential (TRP) ion channels of the A1 (TRPA1) and V1 (TRPV1) subtypes are key regulators of vasomotor tone. Propofol, an intravenous anesthetic known to cause vasore...

We previously demonstrated that the intravenous anesthetic, propofol, restores the sensitivity of transient receptor potential vanilloid channel subtype-1 (TRPV1) receptors via a protein kinase C epsilon (PKCε)-dependent and transient receptor potential ankyrin channel subtype-1 (TRPA1)-dependent pathway in sensory neurons. The extent to which the two pathways are directly linked or operating in parallel has not been determined. Using a molecular approach, our objectives of the current study were to confirm that TRPA1 activation directly results in PKCε activation and to elucidate the cellular mechanism by which this occurs. F-11 cells were transfected with complimentary DNA (cDNA) for TRPV1 only or both TRPV1 and TRPA1. Intracellular Ca(2+) concentration was measured in individual cells via fluorescence microscopy. An immunoblot analysis of the total and phosphorylated forms of PKCε, nitric oxide synthase (nNOS), and TRPV1 was also performed. In F-11 cells containing both channels, PKCε inhibition prevented the propofol- and allyl isothiocyanate (AITC)-induced restoration of TRPV1 sensitivity to agonist stimulation as well as increased phosphorylation of PKCε and TRPV1. In cells containing TRPV1 only, neither agonist induced PKCε or TRPV1 phosphorylation. Moreover, NOS inhibition blocked propofol-and AITC-induced restoration of TRPV1 sensitivity and PKCε phosphorylation, and PKCε inhibition prevented the nitric oxide donor, SNAP, from restoring TRPV1 sensitivity. Also, propofol-and AITC-induced phosphorylation of nNOS and nitric oxide (NO) production were blocked with the TRPA1-antagonist, HC-030031. These data indicate that the AITC- and propofol-induced restoration of TRPV1 sensitivity is mediated by a TRPA1-dependent, nitric oxide synthase-dependent activation of PKCε.

Hereditary Multiple Exostoses (HME) is a rare pediatric disorder caused by loss-of-function mutations in the genes encoding the heparan sulfate (HS)-synthesizing enzymes EXT1 or EXT2. HME is characterized by formation of cartilaginous outgrowths—called osteochondromas- next to the growth plates of many axial and appendicular skeletal elements. Surprisingly, it is not known whether such tumors also form in endochondral elements of the craniofacial skeleton. Here, we carried out a retrospective analysis of cervical spine MRI and CT scans from 50 consecutive HME patients that included cranial skeletal images. Interestingly, nearly half of the patients displayed moderate defects or osteochondroma-like outgrowths in the cranial base and specifically in the clivus. In good correlation, osteochondromas developed in the cranial base of mutant Ext1f/f;Col2-CreER or Ext1f/f;Aggrecan-CreER mouse models of HME along the synchondrosis growth plates. Osteochondroma formation was preceded by phenotypic alteration of cells at the chondro-perichondrial boundary and was accompanied by ectopic expression of major cartilage matrix genes -collagen 2 and collagen X- within the growing ectopic masses. Because chondrogenesis requires bone morphogenetic protein (BMP) signaling, we asked whether osteochondroma formation could be blocked by a BMP signaling antagonist. Systemic administration with LDN-193189 effectively inhibited osteochondroma growth in conditional Ext1-mutant mice. In vitro studies with mouse embryo chondrogenic cells clarified the mechanisms of LDN-193189 action that turned out to include decreases in canonical BMP signaling pSMAD1/5/8 effectors but interestingly, concurrent increases in such anti-chondrogenic mechanisms as pERK1/2 and Chordin, Fgf9 and Fgf18 expression. Our study is the first to reveal that the cranial base can be affected in patients with HME and that osteochondroma formation is amenable to therapeutic drug intervention., Author summary The rare musculoskeletal pediatric disorder Hereditary Multiple Exostoses (HME) is characterized by benign cartilaginous outgrowths called osteochondromas that cause limb and axial skeletal problems and can also transform into life threatening chondrosarcomas. Surprisingly, no report has yet identified osteochondromas in the craniofacial skeleton though it contains several endochondral structures, including the cranial base. We report here for the first time that the cranial base of many HME patients does in fact exhibit defects and osteochondroma-like outgrowths and that similar lesions form in the cranial base of mouse models of HME. Of similar major importance is our novel demonstration that osteochondroma formation in mutant mice can be effectively inhibited by systemic treatment with a drug that blocks BMP signaling, an essential pro-chondrogenic pathway. Our study thus represents a major step ahead in understanding the pathogenesis and complexities of HME and provides proof-of-principle evidence that osteochondroma formation is amenable to drug-based treatment.

Two prominent members of the transient receptor potential (TRP) family, the vanilloid subtype-1 (TRPV1) receptor and the ankyrin subtype-1 (TRPA1) receptor are extensively coexpressed in peripheral sensory neurons and function as sensory transducers of noxious stimuli.1–4 The relative sensitivity of these receptors to their respective agonists plays an important role in nociceptive signal transduction. The cellular mechanism(s) mediating sensitization, de-sensitization and resensitization of TRPV1 receptors is complex and involve calcium-dependent and independent mechanisms.5–9 Recent evidence has suggested that TRPV1 and TRPA1 receptors exhibit reciprocal regulation, indicating cross-talk between the two receptors.10,11 Specifically, these receptors have been shown to cross-sensitize or cross-desensitize each other in which activation of one enhances or inhibits the sensitivity of the other to agonist stimulation.10,12–16 Propofol is one of the most commonly used intravenous anesthetics for the induction and maintenance of general anesthesia and sedation. Apart from its anesthetic properties, propofol has several nonanesthetic effects, one of which is the modulation of TRP receptor function. A recent study indicated that propofol directly activates TRPA1 receptors in transfected human embryonic kidney 293 cells.17 In addition, our laboratory has recently shown that propofol restores sensitivity of TRPV1 receptors following agonist-induced de-sensitization and attenuates agonist-induced de-sensitization via a protein kinase C e-dependent signaling pathway in mouse dorsal root ganglion (DRG) sensory neurons.18 However, no study to date has investigated the role of TRPA1 receptor activation on restoration of TRPV1 sensitivity, or the role that TRPA1 may play in the propofol-induced restoration of TRPV1 sensitivity. In the current study, we tested the hypothesis that propofol restores TRPV1 receptor sensitivity to agonist stimulation via a TRPA1-dependent pathway. Moreover, we also tested the hypothesis that TRPA1 activation restores TRPV1 receptor sensitivity to agonist stimulation. The major findings are that in DRG neurons containing both TRPV1 and TRPA1, pretreatment with propofol or Allyl isothiocyanate (AITC), restores TRPV1 sensitivity to agonist stimulation in previously de-sensitized DRG neurons. In contrast, restoration of TRPV1 sensitivity to agonist stimulation by propofol or AITC was not observed in DRG neurons containing only TRPV1 receptors (lacking TRPA1). Moreover, the TRPA1 antagonist, HC-030031, prevents the propofol-induced restoration of TRPV1 sensitivity. In F-11 cells (DRG neuronal cell line) transfected with TRPV1 complementary DNA (cDNA) only (lacking TRPA1), neither propofol nor AITC restore TRPV1 sensitivity. However, in F-11 cells transfected with both TRPV1 and TRPA1, propofol and AITC restore TRPV1 sensitivity. Our current findings indicate that propofol can resensitize TRPV1 receptors thereby restoring TRPV1 function via a TRPA1-dependent signaling pathway. In addition, our findings also indicate that TRPA1 activation can restore TRPV1 receptor sensitivity to agonist stimulation.

BackgroundTransient receptor potential (TRP) ion channels of the A1 (TRPA1) and V1 (TRPV1) subtypes are key regulators of vasomotor tone. Propofol is an intravenous anesthetic known to cause vasorelaxation. Our objectives were to examine the extent to which TRPA1 and/or TRPV1 ion channels mediate propofol-induced depressor responses in vivo and to delineate the signaling pathway(s) involved.MethodsMice were subjected to surgery under 1.5-2.5% sevoflurane gas with supplemental oxygen. After a stable baseline in mean arterial pressure (MAP) was achieved propofol (2.5, 5.0, 10.0 mg/kg/min) was administered to assess the hemodynamic actions of the intravenous anesthetic. The effect of nitric oxide synthase (NOS) inhibition with L-NAME and/or calcium-gated K+ channel (BKCa) inhibition with Penetrim A (Pen A), alone and in combination, on propofol-induced decreases in mean arterial pressure were assessed in control C57Bl/6J, TRPA1-/-, TRPV1-/- and double-knockout mice (TRPAV-/-).ResultsPropofol decreased MAP in control mice and this effect was markedly attenuated in TRPA1-/- and TRPAV-/- mice but unaffected in TRPV1-/-mice. Moreover, pretreatment with L-NAME or Pen A attenuated the decrease in MAP in control and TRPV1-/- mice, and combined inhibition abolished the depressor response. In contrast, the markedly attenuated propofol-induced depressor response observed in TRPA1-/- and TRPAV-/- mice was unaffected by pre-treatment with Pen A or L-NAME when used either alone or in combination.ConclusionThese data demonstrate for the first time that propofol-induced depressor responses in vivo are predominantly mediated by TRPA1 ion channels with no involvement of TRPV1 ion channels and includes activation of both NOS and BKCa channels.