ObjectiveAtrial fibrillation (AF) remains a highly prevalent arrhythmia with significant burden on morbidity and mortality. The impact of AF in the revascularised population remains incompletely ...described. Given the high prevalence of AF in the revascularised population, we sought to evaluate the incidence and prognosis in patients with pre-existing and new-onset AF following revascularisation.MethodsWe used the University of Ottawa Heart Institute Revascularisation Registry to identify patients who underwent revascularisation between August 2015 and March 2020, who were prospectively followed for an average of one year. We conducted a retrospective cohort study analysing the association between AF and clinical outcomes. The primary outcome of interest was 1-year major adverse cardiac events (MACE) defined as a composite of death, myocardial infarction, unplanned revascularisation and cerebrovascular accidents. Moreover, secondary outcomes include the individual components of MACE and bleeding.ResultsA total of 6704 patients underwent revascularisation and completed 1-year clinical follow-up. Median time to follow-up was 12.8 (IQR 11.2–15.9) months. One-year MACE occurred in 166 (21.8%) and 683 (11.5%) patients in AF and non-AF groups, respectively (adjusted HR, 1.61; 95% CI 1.29 to 2.01; p<0.0001). AF was independently predictive of 1-year mortality, myocardial infarction, unplanned revascularisation, cerebrovascular accident and bleeding. Within 1 year, 299 (4.5%) episodes of new-onset AF was observed. New-onset AF following revascularisation was also associated with 1-year MACE, mortality, myocardial infarction, cerebrovascular accident and unplanned revascularisation.ConclusionsPreprocedural and new-onset AF following revascularisation remains highly predictive 1-year MACE. AF should be considered in addition to traditional risk factors for adverse outcomes following revascularisation.
Leukemic stem cells (LSCs) rely on oxidative metabolism and are differentially sensitive to targeting mitochondrial pathways, which spares normal hematopoietic cells. A subset of mitochondrial ...proteins is folded in the intermembrane space via the mitochondrial intermembrane assembly (MIA) pathway. We found increased mRNA expression of MIA pathway substrates in acute myeloid leukemia (AML) stem cells. Therefore, we evaluated the effects of inhibiting this pathway in AML. Genetic and chemical inhibition of ALR reduces AML growth and viability, disrupts LSC self-renewal, and induces their differentiation. ALR inhibition preferentially decreases its substrate COX17, a mitochondrial copper chaperone, and knockdown of COX17 phenocopies ALR loss. Inhibiting ALR and COX17 increases mitochondrial copper levels which in turn inhibit S-adenosylhomocysteine hydrolase (SAHH) and lower levels of S-adenosylmethionine (SAM), DNA methylation, and chromatin accessibility to lower LSC viability. These results provide insight into mechanisms through which mitochondrial copper controls epigenetic status and viability of LSCs.
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•The sulfhydryl oxidase ALR is necessary for the viability of AML cells and LSCs•Loss of MIA/ALR substrate COX17 redistributes mitochondrial copper•Redistribution of mitochondrial copper reduces DNA methylation and accessibility•Copper-regulated epigenetic changes result in LSC differentiation
Singh et al. demonstrate that modulating the mitochondrial intermembrane assembly (MIA) pathway leads to decreased viability of AML cells. Targeting the sulfhydryl oxidase ALR results in loss of copper chaperone COX17 and redistribution of mitochondrial copper, causing decreased DNA methylation, chromatin accessibility, and self-renewal of leukemic stem cells (LSCs).
Abstract
Introduction
Golimumab is approved as a therapy for ulcerative colitis (UC) patients. Recent data also demonstrate efficacy in Crohn’s disease (CD); however, little is known about target ...drug levels to achieve endoscopic remission.
Methods
We performed a retrospective analysis of IBD patients on maintenance golimumab. Median trough levels were compared using Kruskal-Wallis test, and logistic regression was used to construct a probabilistic model to determine sensitivity and specificity of levels predicting mucosal healing.
Results
Fifty-eight patients on maintenance golimumab were included (n = 39 CD, n = 19 UC/IBD-unclassified IBDU). Forty percent (n = 23) were cotreated with an immunomodulator, 95% (n = 55) of patients were anti-TNF experienced, and 15.5% (n = 9) had 3 or more prior biologic therapies. Forty-four percent of patients achieved mucosal healing with endoscopic response in a further 26% of patients. Clinical remission was recorded in 41% of patients, and 82% had clinical response. Patients were treated with doses generally higher than the approved maintenance dose. In CD patients, median golimumab trough levels were higher in patients with mucosal healing (8.8 μg/mL vs 5.08 μg/mL, P = 0.03). After calculation of a receiver operating characteristic (ROC) curve for mucosal healing vs nonresponse, a trough level >8 μg/mL was associated with mucosal healing, with 67% sensitivity, 88% specificity, and a likelihood ratio of 3:4.
Conclusion
Treatment with golimumab was associated with mucosal healing in 44% of all IBD patients. Higher golimumab levels were associated with mucosal healing in CD. These findings support the need for prospective studies to determine target golimumab levels in IBD, which may impact current clinical practices in relation to selection of maintenance dosing.
Recent data have outlined the potential of golimumab to achieve mucosal healing in Crohn’s disease and ulcerative colitis, but drug monitoring targets are lacking. We have identified golimumab trough levels associated with mucosal healing in a largely anti-TNF experienced patient population.
A subset of AML and stem cells have increased mitochondrial stress and increased expression of mitochondrial proteases that degrade misfolded mitochondrial proteins. Given the recent findings of the ...interplay between mitochondrial homeostasis and mitochondrial protein import, we hypothesized that AML cells have an increased reliance on mitochondrial protein import as a compensatory mechanism for increased mitochondrial stress. To test this hypothesis, we measured expression levels of key mitochondrial protein import genes in publicly available datasets (GSE30377, GSE42414 and GSE 24759) and demonstrated their up regulation in a subset of AML cells over normal hematopoietic cells. Increased expression occurred across the spectrum of molecular mutations and cytogenetic abnormalities. Moreover, expression levels of mitochondrial protein import genes were enriched in functionally defined AML stem cells over bulk cells.
To assess the impact of inhibiting mitochondrial protein import in AML, we knocked down the outer mitochondrial membrane import channel TOM40, the inner membrane import channel Tim23 and the oxidase ALR, that folds proteins through a disulfide relay system in the mitochondrial inner membrane space and is a rate limiting step for the import of a subset of mitochondrial proteins. Knockdown of these targets in OCI-AML2, TEX and U937 leukemia cells with shRNA reduced growth and viability of AML cells. Knockdown of ALR targeted the leukemia initiating cells as it abrogated engraftment of TEX leukemia cells into immune deficient mice (shRNA ALR = 5.481 +/- 0.9 % engraftment vs shRNA control= 29.44 +/- 5.4 % engraftment; p =0.0004) . Mechanistically, knockdown of mitochondrial import genes reduced levels of nuclear (ATP5A, SDHB and NDUFB8), but not mitochondrial (CoxII) encoded proteins of the OXPHOS chain. This in turn led to decreased basal oxygen consumption in leukemic cells. As a chemical approach to investigate the impact of inhibiting mitochondrial protein import in AML and normal cells, we tested the effects of MitoBloCK-6 and related analogues that selectively inhibit ALR in zebrafish, hESCs and yeast models. MitoBloCK-6 and related analogues killed leukemia cell lines (OCI-AML2, TEX, Jurkat and NB4) with an IC50of 5-10 µM. At these concentrations, MitoBloCK-6 decreased levels of nuclear (ATP5A, SDHB and NDUFB8), but not mitochondrial encoded (CoxII) proteins of the OXPHOS chain. Demonstrating the functional importance of changes in mitochondrial metabolism by these compounds, rho zero 143B rhabdomyosarcoma cells that lack mitochondrial DNA and rely solely on glycolysis were resistant to MitoBloCK-6. Finally, we tested the effects of MitoBloCK-6 on primary AML and normal cells. Treatment with MitoBloCK-6 (2 µM) inhibited clonogenic growth of (4 of 5) primary AML > 64% but produced <3% loss of clonogenic growth in normal hematopoietic cells. Finally, in an OCI-AML2 xenograft model, systemic administration of MitoBloCK-6 reduced tumor growth > 50% of control without toxicity.
Thus, we have demonstrated that AML cells have a unique reliance on mitochondrial protein import and inhibition of this pathway may be a new therapeutic strategy to selectively target a subset of AML and AML stem cells.
Schimmer:Novartis: Honoraria.
The majority of mitochondrial proteins are encoded in the nucleus, translated in the cytoplasm and imported into the mitochondria. A subset of cysteine-rich proteins destined for the mitochondrial ...intermembrane space are oxidized and folded by the Mitochondrial IMS Assembly (MIA) pathway. We found that genes encoding substrates of the MIA pathway are over-expressed in leukemic stem cells compared to bulk AML cells. Therefore, we assessed the effects of inhibiting the MIA pathway in AML by targeting the FAD linked sulfhydryl oxidase ALR, an integral part of the MIA machinery.
Knockdown of ALR with shRNA reduced the growth and viability of OCI-AML2, TEX and NB4 leukemia cells. In addition, ALR knockdown reduced engraftment of TEX cells into mouse marrow, demonstrating an effect on the leukemia initiating cells. The small molecule selective ALR inhibitor, MitoBloCK-6 killed AML cells (IC50 of 5-10 mM) and preferentially reduced the clonogenic growth of primary AML cells over normal hematopoietic cells. MitoBloCK-6 treatment (80 mg/kg i.p. 5 of 7 days x 2 weeks) of mice engrafted with primary AML cells strongly reduced the leukemic burden without changing mouse body weight, serum chemistries, or organ histology. In contrast, MitoBloCK-6 did not change engraftment of normal cord blood in similar experiments. As evidenced by secondary transplants, MitoBloCK-6 also targeted leukemic stem cells.
As expression levels of ALR substrates are increased in AML stem cells we assessed the effects of ALR inhibition on differentiation in AML. Genetic or chemical inhibition of ALR induced the differentiation of AML cells as evidenced by changes in gene expression, increased differentiation associated CD surface marker expression and increased non-specific esterase.
Interrogation of the effects of ALR inhibition on its substrates identified the mitochondrial copper chaperone, Cox17 as the primary downstream target in leukemic cells. Genetic or chemical inhibition of ALR selectively reduced levels of Cox17 protein. Validating the functional importance of these findings, knockdown of Cox17 phenocopied ALR inhibition and reduced AML proliferation and induced AML differentiation.
Cox17 is a copper metallochaperone that promotes respiratory chain complex IV assembly by loading copper into the respiratory complex. COX17 knockdown slightly reduced the complex IV enzymatic activity, but did not change basal oxygen consumption or ROS production. However, COX17 knockdown increased intracellular levels of free copper 16-fold as measured by atomic mass spectrometry. Copper is a known inhibitor of adenosylhomocysteinase, a key enzyme involved in the preservation of S-adenosylmethionine (SAM):S-adenosylhomocysteine (SAH) ratio in cells. SAM is a global methyl donor and is critical for DNA methylation. Knockdown of COX17 or ALR inhibition with MitoBloCK-6 decreased levels of SAM and reduced DNA methylation in AML cells. Likewise, the enzymatic activity of adenosylhomocysteinase was reduced in OCI-AML2 cells after MitoBloCK-6 treatment. Importantly, co-treatment with the copper chelator, penicillamine, rescued reductions in SAM, DNA methylation, and cell viability after COX17 knockdown or MitoBloCK-6 treatment.
Thus, we have discovered a novel copper-dependent mechanism by which mitochondrial pathways regulate epigenetics and stemness in AML. Moreover, inhibitors of ALR or COX17 may be a novel therapeutic strategy to promote the differentiation of AML cells and stem cells.
Schimmer:Otsuka Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Medivir AB: Research Funding; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.
Background
Sarcopenia indicates poor prognosis in various malignancies. We evaluated the association of sarcopenia with overall (OS) and progression-free survival (PFS) in metastatic esophageal ...cancer (MEC) patients, a population often presenting with poor nutritional status.
Methods
In newly diagnosed MEC patients managed at the Princess Margaret (PM) Cancer Centre (diagnosed 2006–2015), total muscle area, visceral adiposity (VA), and subcutaneous adiposity (SA) were quantified on abdominal computed tomography at L3. Sarcopenia was determined using published cutoffs, based on sex and height.
Results
Of 202 MEC patients, most were male (166/82%), < 65 years (116/57%), and had adenocarcinoma histology (141/70%); 110/54% had recurrent MEC after initial curative-intent treatment; 92/46% presented with de novo MEC. At stage IV diagnosis, 20/10% were underweight, 97/48% were normal-weight and 84/42% were overweight/obese; 103/51% were sarcopenic. Sarcopenia was associated with worse median OS (4.6 vs
.
7.9 months; log-rank
p
= 0.03) and 1-year survival, even after adjusting for other body composition variables (e.g., BMI, VA, and SA): adjusted-HR 1.51 95% CI 1.1–2.2, p = 0.02. In post hoc analysis, sarcopenia was highly prognostic in adenocarcinomas (
p
= 0.003), but not squamous cell carcinomas (SCC). In patients receiving palliative systemic treatment (104/51%), sarcopenia was associated with shorter PFS (
p
= 0.004) in adenocarcinoma patients (75/72%).
Conclusions
In metastatic esophageal adenocarcinomas, sarcopenia is associated with worse PFS and OS. In metastatic esophageal SCC, there was a non-significant trend for worse PFS but no association with OS. In order to offset the poor prognosis associated with sarcopenia particularly in metastatic esophageal adenocarcinoma patients, future research should focus on possible countermeasures.
The vast majority of mitochondrial proteins are encoded in the nucleus, translated in the cytoplasm and then imported into the mitochondria. A subset of these imported proteins are folded into their ...mature and functional forms in the mitochondrial inter-membrane space (IMS) by the Mitochondrial IMS Assembly (MIA) pathway. We found that genes encoding substrates of the MIA pathway are over-expressed in leukemic stem cells compared to bulk AML cells. Therefore, we assessed the effects of inhibiting the MIA pathway in AML. We knocked down the mitochondrial sulfhydryl oxidase ALR, a key regulator of the MIA pathway. Knockdown of ALR with shRNA reduced the growth and viability of OCI-AML2, TEX and NB4 leukemia cells. In addition, knockdown of ALR reduced the engraftment of TEX cells into mouse marrow, demonstrating an effect on the leukemia initiating cells. The small molecule selective ALR inhibitor, MitoBloCK-6, mimicked the effects of ALR knockdown and killed AML cells with an IC50 of 5-10 μM. MitoBloCK-6 preferentially reduced the clonogenic growth of primary AML cells (n=4/5) over normal hematopoietic cells (n=4). However, only 3/10 bulk AML cells were sensitive to MitoBloCK-6 induced cell death by Annexin V/PI staining.
Next, we evaluated the efficacy and toxicity of ALR inhibition in vivo . We injected primary AML cells or normal cord blood into the femurs of mice and then treated mice with MitoBloCK-6 (80 mg/kg i.p. 5 of 7 days x 2 weeks). MitoBloCK-6 strongly reduced the engraftment of primary AML samples but did not affect engraftment of cord blood. In secondary transplants, MitoBloCK-6 also targeted leukemic stem cells. No change in mouse body weight, serum chemistries, or organ histology was seen.
As expression levels of ALR substrates are increased in AML stem cells, we assessed the effects of ALR inhibition on differentiation in AML. Genetic or chemical inhibition of ALR induced the differentiation of AML cells as evidenced by increased CD surface marker expression and increased non-specific esterase. In addition, ALR inhibition was preferentially cytotoxic towards undifferentiated cells and stem cells over differentiated bulk AML cells.
Interrogation of the effects of ALR inhibition on its substrates identified the mitochondrial copper chaperone, Cox17 as the primary downstream target in leukemic cells. Inhibition of ALR selectively reduced levels of Cox17 protein and altered mitochondrial cristae structure. Validating the functional importance of these findings, knockdown of Cox17 phenocopied ALR inhibition and reduced AML proliferation, induced differentiation of AML cells, and altered mitochondrial cristae structure, without changing respiratory chain activity or oxygen consumption. Of note, cristae remodelling independent of respiratory chain function has been recently implicated in cellular differentiation and in yeast, Cox17 regulates the cristae organizing machinery.
Thus, we have identified novel mechanisms by which mitochondrial pathways regulate the fate and differentiation of AML cells and stem cells Moreover, inhibition of ALR may be a novel therapeutic strategy to promote the differentiation of AML cells and stem cells.
Schimmer:Takeda Pharmaceuticals: Research Funding; Medivir: Research Funding; Novartis Pharmaceuticals: Honoraria.
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