Loss of cardiac mitochondrial complex I persulfidation impairs NAD+ homeostasis in aging.

Protein persulfidation is a significant post-translational modification that involves addition of a sulfur atom to the cysteine thiol group and is facilitated by sulfide species. Persulfidation targets reactive cysteine residues within proteins, influencing their structure and/or function across various biological systems. This modification is evolutionarily conserved and plays a crucial role in preventing irreversible cysteine overoxidation, a process that becomes prominent with aging. While, persulfidation decreases with age, its levels in the aged heart and the functional implications of such a reduction in cardiac metabolism remain unknown. Here we interrogated the cardiac persulfydome in wild-type adult mice and age-matched mice lacking the two sulfide generating enzymes, namely cystathionine gamma lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3MST). Our findings revealed that cardiac persulfidated proteins in wild type hearts are less abundant compared to those in other organs, with a primary involvement in mitochondrial metabolic processes. We further focused on one specific target, NDUFB7, which undergoes persulfidation by both CSE and 3MST derived sulfide species. In particular, persulfidation of cysteines C80 and C90 in NDUFB7 protects the protein from overoxidation and maintains the complex I activity in cardiomyocytes. As the heart ages, the levels of CSE and 3MST in cardiomyocytes decline, leading to reduced NDUFB7 persulfidation and increased cardiac NADH/NAD+ ratio. Collectively, our data provide compelling evidence for a direct link between cardiac persulfidation and mitochondrial complex I activity, which is compromised in aging.

Disrupted Binding of Cystathionine γ-Lyase to p53 Promotes Endothelial Senescence.

BACKGROUND: Advanced age is unequivocally linked to the development of cardiovascular disease; however, the mechanisms resulting in reduced endothelial cell regeneration remain poorly understood. Here, we investigated novel mechanisms involved in endothelial cell senescence that impact endothelial cell transcription and vascular repair after injury. METHODS: Native endothelial cells were isolated from young (20±3.4 years) and aged (80±2.3 years) individuals and subjected to molecular analyses to assess global transcriptional and metabolic changes. In vitro studies were conducted using primary human and murine endothelial cells. A murine aortic re-endothelialization model was used to examine endothelial cell regenerative capacity in vivo. RESULTS: RNA sequencing of native endothelial cells revealed that aging resulted in p53-mediated reprogramming to express senescence-associated genes and suppress glycolysis. Reduced glucose uptake and ATP contributed to attenuated assembly of the telomerase complex, which was required for endothelial cell proliferation. Enhanced p53 activity in aging was linked to its acetylation on K120 due to enhanced activity of the acetyltransferase MOZ (monocytic leukemic zinc finger). Mechanistically, p53 acetylation and translocation were, at least partially, attributed to the loss of the vasoprotective enzyme, CSE (cystathionine γ-lyase). CSE physically anchored p53 in the cytosol to prevent its nuclear translocation and CSE absence inhibited AKT (Protein kinase B)-mediated MOZ phosphorylation, which in turn increased MOZ activity and subsequently p53 acetylation. In mice, the endothelial cell-specific deletion of CSE activated p53, induced premature endothelial senescence, and arrested vascular repair after injury. In contrast, the adeno-associated virus 9-mediated re-expression of an active CSE mutant retained p53 in the cytosol, maintained endothelial glucose metabolism and proliferation, and prevented endothelial cell senescence. Adenoviral overexpression of CSE in native endothelial cells from aged individuals maintained low p53 activity and reactivated telomerase to revert endothelial cell senescence. CONCLUSIONS: Aging-associated impairment of vascular repair is partly determined by the vasoprotective enzyme CSE.

Impact of mesenchymal stromal cell-derived vesicular cargo on B-cell acute lymphoblastic leukemia progression.

Leukemia cells reciprocally interact with their surrounding bone marrow microenvironment (BMM), rendering it hospitable to leukemia cell survival, for instance through the release of small extracellular vesicles (sEVs). In contrast, we show here that BMM deficiency of pleckstrin homology domain family M member 1 (PLEKHM1), which serves as a hub between fusion and secretion of intracellular vesicles and is important for vesicular secretion in osteoclasts, accelerates murine BCR-ABL1+ B-cell acute lymphoblastic leukemia (B-ALL) via regulation of the cargo of sEVs released by BMM-derived mesenchymal stromal cells (MSCs). PLEKHM1-deficient MSCs and their sEVs carry increased amounts of syntenin and syndecan-1, resulting in a more immature B-cell phenotype and an increased number/function of leukemia-initiating cells (LICs) via focal adhesion kinase and AKT signaling in B-ALL cells. Ex vivo pretreatment of LICs with sEVs derived from PLEKHM1-deficient MSCs led to a strong trend toward acceleration of murine and human BCR-ABL1+ B-ALL. In turn, inflammatory mediators such as recombinant or B-ALL cell-derived tumor necrosis factor α or interleukin-1ß condition murine and human MSCs in vitro, decreasing PLEKHM1, while increasing syntenin and syndecan-1 in MSCs, thereby perpetuating the sEV-associated circuit. Consistently, human trephine biopsies of patients with B-ALL showed a reduced percentage of PLEKHM1+ MSCs. In summary, our data reveal an important role of BMM-derived sEVs for driving specifically BCR-ABL1+ B-ALL, possibly contributing to its worse prognosis compared with BCR-ABL1- B-ALL, and suggest that secretion of inflammatory cytokines by cancer cells in general may similarly modulate the tumor microenvironment.

Extracellular Vesicles in Myeloid Neoplasms.

Myeloid neoplasms arise from malignant primitive cells, which exhibit growth advantage within the bone marrow microenvironment (BMM). The interaction between these malignant cells and BMM cells is critical for the progression of these diseases. Extracellular vesicles (EVs) are lipid bound vesicles secreted into the extracellular space and involved in intercellular communication. Recent studies have described RNA and protein alterations in EVs isolated from myeloid neoplasm patients compared to healthy controls. The altered expression of various micro-RNAs is the best-described feature of EVs of these patients. Some of these micro-RNAs induce growth-related pathways such as AKT/mTOR and promote the acquisition of stem cell-like features by malignant cells. Another well-described characteristic of EVs in myeloid neoplasms is their ability to suppress healthy hematopoiesis either via direct effect on healthy CD34+ cells or via alteration of the differentiation of BMM cells. These results support a role of EVs in the pathogenesis of myeloid neoplasms. mainly through mediating the interaction between malignant and BMM cells, and warrant further study to better understand their biology. In this review, we describe the reported alterations of EV composition in myeloid neoplasms and the recent discoveries supporting their involvement in the development and progression of these diseases.

The differential role of the lipid raft-associated protein flotillin 2 for progression of myeloid leukemia.

Lipid raft-associated proteins play a vital role in membrane-mediated processes. The lipid microdomain-associated protein flotillin 2 (FLOT2), which has a scaffolding function, is involved in polarization, as well as in actin cytoskeletal organization of primitive and mature hematopoietic cells and has been associated with different malignancies. However, its involvement in myeloid leukemias is not well studied. Using murine transplantation models, we show here that the absence of FLOT2 from leukemia-initiating cells (LICs) altered the disease course of BCR-ABL1+ chronic myeloid leukemia (CML), but not of MLL-AF9-driven acute myeloid leukemia (AML). While FLOT2 was required for expression of the adhesion molecule CD44 on both CML- and AML-LIC, a defect in the cytoskeleton, cell polarity, and impaired homing ability of LIC was only observed in FLOT2-deficient BCR-ABL1+ compared with MLL-AF9+ cells. Downstream of CD44, BCR-ABL1 kinase-independent discrepancies were observed regarding expression, localization, and activity of cell division control protein 42 homolog (CDC42) between wild-type (WT) and FLOT2-deficient human CML and AML cells. Inhibition of CDC42 by ML141 impaired the homing of CML LIC and, thereby, CML progression. This suggested that alteration of both CD44 and CDC42 may be causative of impaired CML progression in the absence of FLOT2. In summary, our data suggest a FLOT2-CD44-CDC42 axis, which differentially regulates CML vs AML progression, with deficiency of FLOT2 impairing the development of CML.

The age of the bone marrow microenvironment influences B-cell acute lymphoblastic leukemia progression via CXCR5-CXCL13.

B-cell acute lymphoblastic leukemia (B-ALL) occurs most commonly in children, whereas chronic myeloid leukemia is more frequent in adults. The myeloid bias of hematopoiesis in elderly individuals has been considered causative, but the age of the bone marrow microenvironment (BMM) may be contributory. Using various murine models of B-ALL in young vs old mice, we recapitulated B-ALL preponderance in children vs adults. We showed differential effects of young vs old BM macrophages on B-ALL cell function. Molecular profiling using RNA- and ATAC-sequencing revealed pronounced differences in young vs old BMM-derived macrophages and enrichment for gene sets associated with inflammation. In concordance with the role of C-X-C motif chemokine (CXCL) 13 for disease-associated B-cell chemoattraction, we found CXCL13 to be highly expressed in young macrophages on a translational compared with a transcriptional level. Inhibition of CXCL13 in BM macrophages impaired leukemia cell migration and decreased the proliferation of cocultured B-ALL cells, whereas recombinant CXCL13 increased pAKT and B-ALL cell expansion. Pretreatment of B-ALL-initiating cells with CXCL13 accelerated B-ALL progression. Deficiency of Cxcr5, the receptor for CXCL13, on B-ALL-initiating cells prolonged murine survival, whereas high expression of CXCR5 in pediatric B-ALL may predict central nervous system relapse. CXCL13 staining was increased in bone sections from pediatric compared with adult patients with B-ALL. Taken together, our study shows that the age of the BMM and, in particular, BM macrophages influence the leukemia phenotype. The CXCR5-CXCL13 axis may act as prognostic marker and an attractive novel target for the treatment of B-ALL.

The endolysosomal adaptor PLEKHM1 is a direct target for both mTOR and MAPK pathways.

The lysosome is a cellular signalling hub at the point of convergence of endocytic and autophagic pathways, where the contents are degraded and recycled. Pleckstrin homology domain-containing family member 1 (PLEKHM1) acts as an adaptor to facilitate the fusion of endocytic and autophagic vesicles with the lysosome. However, it is unclear how PLEKHM1 function at the lysosome is controlled. Herein, we show that PLEKHM1 coprecipitates with, and is directly phosphorylated by, mTOR. Using a phosphospecific antibody against Ser432/S435 of PLEKHM1, we show that the same motif is a direct target for ERK2-mediated phosphorylation in a growth factor-dependent manner. This dual regulation of PLEKHM1 at a highly conserved region points to a convergence of both growth factor- and amino acid-sensing pathways, placing PLEKHM1 at a critical juncture of cellular metabolism.

Vitamin K antagonism impairs the bone marrow microenvironment and hematopoiesis.

Vitamin K antagonists (VKAs) have been used in 1% of the world's population for prophylaxis or treatment of thromboembolic events for 64 years. Impairment of osteoblast function and osteoporosis has been described in patients receiving VKAs. Given the involvement of cells of the bone marrow microenvironment (BMM), such as mesenchymal stem cells (MSCs) and macrophages, as well as other factors such as the extracellular matrix for the maintenance of normal hematopoietic stem cells (HSCs), we investigated a possible effect of VKAs on hematopoiesis via the BMM. Using various transplantation and in vitro assays, we show here that VKAs alter parameters of bone physiology and reduce functional HSCs 8-fold. We implicate impairment of the functional, secreted, vitamin K-dependent, γ-carboxylated form of periostin by macrophages and, to a lesser extent, MSCs of the BMM and integrin ß3-AKT signaling in HSCs as at least partly causative of this effect, with VKAs not being directly toxic to HSCs. In patients, VKA use associates with modestly reduced leukocyte and monocyte counts, albeit within the normal reference range. VKAs decrease human HSC engraftment in immunosuppressed mice. Following published examples that alteration of the BMM can lead to hematological malignancies in mice, we describe, without providing a causal link, that the odds of VKA use are higher in patients with vs without a diagnosis of myelodysplastic syndrome (MDS). These results demonstrate that VKA treatment impairs HSC function via impairment of the BMM and the periostin/integrin ß3 axis, possibly associating with increased MDS risk.

Targeting the bone marrow microenvironment in acute leukemia.

Despite individual differences between certain leukemias, the overall survival rate in acute leukemia remains low at approximately 40%. Novel therapeutics, including targeted therapies like tyrosine kinase inhibitors, have been incorporated into treatment regimens, but most have failed at eradicating leukemic stem cells (LSCs). The causes of disease relapse, progression, and resistance to chemotherapy are as yet not entirely clear but thought to be linked to protection in the bone marrow microenvironment (BMM). In this review, we summarize current knowledge on the BMM in acute leukemias and examine the ongoing efforts to target the BMM, which include treatment strategies targeting (a) leukemia-BMM interactions, (b) leukemia-cell intrinsic pathways influenced by the BMM, and (c) direct BMM targeting strategies. It is likely that the future ploy against leukemia will involve these and other innovative strategies designed to eradicate the last remaining warrior - the LSC.

Association of the clock genes polymorphisms with colorectal cancer susceptibility.

BACKGROUND AND OBJECTIVES: The circadian rhythm regulates the cell cycle progression and DNA damage response. The aim of our study was to investigate the association between polymorphisms in the CLOCK1, PER2, and PER3 genes with the colorectal cancer (CRC) susceptibility and clinicopathological variables. METHODS: Four hundred two CRC patients and 480 healthy controls were included in a case-control study. Genotype and allelic frequencies of 311T>C (rs1801260) in CLOCK1 gene, G3853A (rs934945) in PER2 gene and 4/5 repeats polymorphisms in PER3 gene were evaluated by the polymerase chain reaction (PCR) restriction fragment length polymorphism method in the DNA extracted from the peripheral blood of patients and controls. RESULTS: The frequencies of the 311T>C CLOCK1 gene, CC genotype and C allele were significantly higher among CRC patients compared to controls (P < 0.0001) elevating the CRC risk by 2.78- and 1.78-fold respectively. No correlation was found between G3853A and 4/5 repeats polymorphisms and CRC risk. The C/G/5 and C/G/4 repeats haplotypes were higher in CRC patients (P = 0.0009 and P = 0.038) elevating the CRC risk by 60% and 89% respectively. No correlation was found between any polymorphism and clinicopathological characteristics of CRC patients. CONCLUSION: The 311T>C polymorphism in the CLOCK1 gene significantly increases the risk for CRC development while it does not affect the outcome of CRC patients.

Expression of clock genes in patients with colorectal cancer.

Recent studies have demonstrated the influence of clock genes in cell cycle regulation, cell proliferation, apoptosis and DNA damage recognition and repair. There is evidence suggesting the implication of clock genes in colorectal cancer (CRC) development and progression. The aim of this study is to evaluate the expression levels of clock genes in CRC and correlate them with patients' prognosis. Forty-two CRC samples (from 24 males and 18 females), their paired noncancerous tissues and 8 biopsies from healthy individuals were included. Quantitative real-time PCR was used to examine the expression levels of CLOCK1, BMAL1, PER1, PER2 and PER3 genes in all the samples. In the cancerous tissues CLOCK1 (p<0.0001) and BMAL1 (p<0.0001) expression levels were higher, while PER1 (p<0.0024) and PER3 (p<0.0001) expression levels were lower compared to matched healthy tissues. No difference was observed in the expression levels of PER2 (p=0.99). No correlation was found between clock gene expression and patients' clinicopathological characteristics or prognosis. The results suggest abnormal expression of CLOCK1, BMAL1, PER1 and PER3 genes in CRC but no correlation with patients' prognosis.