BMP6 and VDR gene polymorphisms are associated with osteonecrosis in a sickle cell anaemia cohort.

The occurrence and severity of osteonecrosis in sickle cell anaemia (SCA) vary due to risk factors, including genetic modifiers. Bone morphogenetic proteins (BMPs), particularly BMP6, and the vitamin D receptor (VDR) play key roles in cartilage and bone metabolism, making them potential contributors to orthopaedic outcomes in SCA. Here, we evaluated the association of polymorphisms in BMP6 (rs3812163, rs270393 and rs449853) and VDR (FokI rs2228570 and Cdx2 rs11568820) genes with osteonecrosis risk in a Brazilian SCA cohort. A total of 177 unrelated SCA patients were selected. The AA genotype of BMP6 rs3812163 was independently associated with a lower osteonecrosis risk (p = 0.015; odds ratio (OR): 0.38; 95% confidence interval (CI): 0.18-0.83) and with the long-term cumulative incidence of osteonecrosis (p = 0.029; hazard ratio: 0.56, 95% CI: 0.34-0.94). The VDR rs2228570 TT genotype was independently associated with a lower osteonecrosis risk (p = 0.039; OR: 0.14; 95% CI: 0.02-0.90). In summary, our results provide evidence that BMP6 rs3812163 and the VDR rs2228570 might be implicated in osteonecrosis pathophysiology in SCA and might help identify individuals at high risk.

A-296G variant of THBS1 gene (rs1478605) is associated with a lower frequency of stroke in a Brazilian population with sickle cell anemia.

OBJECTIVES: Stroke is a devastating clinical outcome that significantly contributes to the morbidity and mortality of sickle cell anemia (SCA) patients. Despite its advantages in predicting stroke risk, transcranial Doppler screening has limitations that restrict its applicability, highlighting the need for emerging prognostic tools. Thrombospondin-1 plays a crucial role in endothelial injury, platelet adhesion, and nitric oxide metabolism and may be implicated in stroke pathophysiology. Here, we aimed to evaluate the association of THBS1 genetic variations with the occurrence of stroke in SCA patients MATERIALS AND METHODS: By real-time PCR, 512 SCA patients were fully genotyped for THBS1 A-296G (rs1478605) polymorphism RESULTS: THBS1 GG genotype was associated with a lower risk for stroke occurrence [odds ratio (OR): 0.30; 95% confidence interval (CI): 0.11-0.78; P = 0.011], although these findings were not consistent with multivariate logistic regression analysis (OR: 0.73, 95% CI: 0.12 - 4.37; P = 0.736). In agreement, the cumulative incidence of stroke for patients with AG/AA genotypes was higher when compared to the GG genotype (P = 0.018). However, the association was not maintained in the multivariate proportional hazards model (hazard ratio: 0.67, 95% CI: 0.12-3.61; P = 0.643) CONCLUSIONS: In summary, the present study shows that the THBS1 A-296G (rs1478605) polymorphism may be a potential modifier for stroke in SCA.

Prognostic implications of the ID1 expression in acute myeloid leukemia patients treated in a resource-constrained setting.

INTRODUCTION: The aberrant expression of the inhibitor of DNA binding (ID1) gene has been frequently associated with the leukemogenesis and prognostication acute myeloid leukemia (AML), although its clinical importance has never been investigated in patients treated outside well-controlled clinical trials. METHODS: Using quantitative real-time polymerase chain reaction, we investigated the role of the ID1 expression in the clinical outcomes of non-selected patients with acute myeloid leukemia treated in a real-life setting. RESULTS: Overall, 128 patients were enrolled. Patients with high ID1 expression had a lower 3-year overall survival (OS) rate of 9%, with the 95% confidence interval (95%CI) at 3 to 20%, compared to patients with a low ID1 expression (22%, 95%CI: 11 - 34%) (p = 0.037), although these findings did not retain significance after adjustment (hazard ratio (HR): 1.5, 95%CI: 0.98 - 2.28; p = 0.057). The ID1 expression had no impact on post-induction outcomes (disease-free survival, p = 0.648; cumulative incidence of relapse, p = 0.584). CONCLUSIONS: Although we are aware thar our data are confronted with many variables that cannot be fully controlled, including drug unavailability, risk-adapted treatment, comorbidities and the time from diagnosis to treatment initiation, we are firm believers that such an initiative can provide more realistic data on understudied populations, in particular those from low- and middle-income countries.

M2 macrophages drive leukemic transformation by imposing resistance to phagocytosis and improving mitochondrial metabolism.

It is increasingly becoming clear that cancers are a symbiosis of diverse cell types and tumor clones. Combined single-cell RNA sequencing, flow cytometry, and immunohistochemistry studies of the innate immune compartment in the bone marrow of patients with acute myeloid leukemia (AML) reveal a shift toward a tumor-supportive M2-polarized macrophage landscape with an altered transcriptional program, with enhanced fatty acid oxidation and NAD+ generation. Functionally, these AML-associated macrophages display decreased phagocytic activity and intra-bone marrow coinjection of M2 macrophages together with leukemic blasts strongly enhances in vivo transformation potential. A 2-day in vitro exposure to M2 macrophages results in the accumulation of CALRlow leukemic blast cells, which are now protected against phagocytosis. Moreover, M2-exposed "trained" leukemic blasts display increased mitochondrial metabolism, in part mediated via mitochondrial transfer. Our study provides insight into the mechanisms by which the immune landscape contributes to aggressive leukemia development and provides alternatives for targeting strategies aimed at the tumor microenvironment.

Clinical significance of mitochondrial DNA content in acute promyelocytic leukaemia.

Although a growing body of evidence demonstrates that altered mtDNA content (mtDNAc) has clinical implications in several types of solid tumours, its prognostic relevance in acute promyelocytic leukaemia (APL) patients remains largely unknown. Here, we show that patients with higher-than-normal mtDNAc had better outcomes regardless of tumour burden. These results were more evident in patients with low-risk of relapse. The multivariate Cox proportional hazard model demonstrated that high mtDNAc was independently associated with a decreased cumulative incidence of relapse. Altogether, our data highlights the possible role of mitochondrial metabolism in APL patients treated with ATRA.

Acute myeloid leukemia-derived bone marrow mesenchymal cells exhibit improved support for leukemic cell proliferation.

INTRODUCTION: The bone marrow (BM) microenvironment plays a significant role in acute myeloid leukemia (AML) genesis and there is evidence that BM mesenchymal stromal cells (BMMSCs) can support leukemia progenitor cell proliferation and survival and provide resistance to cytotoxic therapies. HYPOTHESIS AND METHOD: Nevertheless, currently unknown are the relevance of the spatial localization of AML cells relative to the BMMSCs and whether BMMSCs from patients with AML and healthy subjects have similar properties. To address these issues, we performed a differential gene expression analysis using RNA-sequencing data generated from healthy donors (HDs) and leukemic BMMSCs. RESULTS: The Gene Set Enrichment Analysis (GSEA) revealed that leukemic BMMSCs were associated with the terms "positive regulation of cell cycle", "angiogenesis" and "signaling by the estimated glomerular filtration rate (eGFR)", whereas healthy donor (HD)-derived BMMSCs were associated with "programmed cell death in response to the reactive oxygen species (ROS)", "negative regulation of the cytochrome C from the mitochondria" and "interferon signaling". Next, we evaluated the mitochondrial superoxide production in AML cells in a co-culture layered model. The superoxide production was reduced in leukemic cells in close contact (adhered to the surface or beneath the cell layer) with BMMSCs, indicating lower oxidative stress. CONCLUSION: Taken together, our results suggest that AML-derived BMMSCs are transcriptionally rewired and can reduce the metabolic stress of leukemic cells.

Inhibition of the succinyl dehydrogenase complex in acute myeloid leukemia leads to a lactate-fuelled respiratory metabolic vulnerability.

Metabolic programs can differ substantially across genetically distinct subtypes of acute myeloid leukemia (AML). These programs are not static entities but can change swiftly as a consequence of extracellular changes or in response to pathway-inhibiting drugs. Here, we uncover that AML patients with FLT3 internal tandem duplications (FLT3-ITD+) are characterized by a high expression of succinate-CoA ligases and high activity of mitochondrial electron transport chain (ETC) complex II, thereby driving high mitochondrial respiration activity linked to the Krebs cycle. While inhibition of ETC complex II enhances apoptosis in FLT3-ITD+ AML, cells also quickly adapt by importing lactate from the extracellular microenvironment. 13C3-labelled lactate metabolic flux analyses reveal that AML cells use lactate as a fuel for mitochondrial respiration. Inhibition of lactate transport by blocking Monocarboxylic Acid Transporter 1 (MCT1) strongly enhances sensitivity to ETC complex II inhibition in vitro as well as in vivo. Our study highlights a metabolic adaptability of cancer cells that can be exploited therapeutically.

The Glycolytic Gatekeeper PDK1 defines different metabolic states between genetically distinct subtypes of human acute myeloid leukemia.

Acute myeloid leukemia remains difficult to treat due to strong genetic heterogeneity between and within individual patients. Here, we show that Pyruvate dehydrogenase kinase 1 (PDK1) acts as a targetable determinant of different metabolic states in acute myeloid leukemia (AML). PDK1low AMLs are OXPHOS-driven, are enriched for leukemic granulocyte-monocyte progenitor (L-GMP) signatures, and are associated with FLT3-ITD and NPM1cyt mutations. PDK1high AMLs however are OXPHOSlow, wild type for FLT3 and NPM1, and are enriched for stemness signatures. Metabolic states can even differ between genetically distinct subclones within individual patients. Loss of PDK1 activity releases glycolytic cells into an OXPHOS state associated with increased ROS levels resulting in enhanced apoptosis in leukemic but not in healthy stem/progenitor cells. This coincides with an enhanced dependency on glutamine uptake and reduced proliferation in vitro and in vivo in humanized xenograft mouse models. We show that human leukemias display distinct metabolic states and adaptation mechanisms that can serve as targets for treatment.

High ME1 Expression Is a Molecular Predictor of Post-Transplant Survival of Patients with Acute Myeloid Leukemia.

Several laboratory and clinical variables have been reported to be associated with the outcome of intensive chemotherapy for acute myeloid leukemia (AML), but only a few have been tested in the context of hematopoietic stem cell transplant (HSCT). This study aimed to identify genes whose expression of AML at diagnosis were associated with survival after HSCT. For this purpose, three publicly available adult AML cohorts (TCGA, BeatAML, and HOVON), whose patients were treated with intensive chemotherapy and then subjected to allogeneic or autologous HSCT, were included in this study. After whole transcriptome analysis, we identified ME1 as the only gene whose high expression was associated with shorter survival in patients subjected to HSCT. In addition, the inclusion of ME1 expression was able to improve the European LeukemiaNet risk stratification. Pathways related to lipid biosynthesis, mainly fatty acids, and cholesterol were positively correlated with ME1 expression. Furthermore, ME1 expression was associated with an M2 macrophage-enriched microenvironment, mature AML blasts hierarchy, and oxidative phosphorylation metabolism. Therefore, ME1 expression can be used as biomarker of poor response to HSCT in AML.

STMN1 is highly expressed and contributes to clonogenicity in acute promyelocytic leukemia cells.

Stathmin 1 (STMN1) is a microtubule-destabilizing protein highly expressed in hematological malignancies and involved in proliferation and differentiation. Although a previous study found that the PML-RARα fusion protein, which contributes to the pathophysiology of acute promyelocytic leukemia (APL), positively regulates STMN1 at the transcription and protein activity levels, little is known about the role of STMN1 in APL. In this study, we aimed to investigate the STMN1 expression levels and their associations with laboratory, clinical, and genomic data in APL patients. We also assessed the dynamics of STMN1 expression during myeloid cell differentiation and cell cycle progression, and the cellular effects of STMN1 silencing and pharmacological effects of microtubule-stabilizing drugs on APL cells. We found that STMN1 transcripts were significantly increased in samples from APL patients compared with those of healthy donors (all p < 0.05). However, this had no effect on clinical outcomes. STMN1 expression was associated with proliferation- and metabolism-related gene signatures in APL. Our data confirmed that STMN1 was highly expressed in early hematopoietic progenitors and reduced during cell differentiation, including the ATRA-induced granulocytic differentiation model. STMN1 phosphorylation was predominant in a pool of mitosis-enriched APL cells. In NB4 and NB4-R2 cells, STMN1 knockdown decreased autonomous cell growth (all p < 0.05) but did not impact ATRA-induced apoptosis and differentiation. Finally, treatment with paclitaxel (as a single agent or combined with ATRA) induced microtubule stabilization, resulting in mitotic catastrophe with repercussions for cell viability, even in ATRA-resistant APL cells. This study provides new insights into the STMN1 functions and microtubule dynamics in APL.

The ratio of ATP11C/PLSCR1 mRNA transcripts has clinical significance in sickle cell anemia.

One of the physiologic mechanisms responsible to maintain asymmetric phospholipid distribution (in particular phosphatidylserine, PS) in human erythrocyte membranes is orchestrated by the balance between enzymes responsible for active transport of PS from the outer to the inner leaflet (ATP11C) and those whose counteracts these activities (PLSCR1). Using quantitative real-time polymerase chain reaction and standard flow cytometry procedures, we hypothesized that the aberrant expression of either or both ATP11C and PLSCR1 transcripts may disrupt the PS internalization/externalization process and become clinically relevant for patients with sickle cell anemia (SCA). Overall, neither ATP11C/PLSCR1 ratio or ATP11C and PLSCR1 (if analyzed separately) had impact on risk to present acute or chronic organ damage in 178 patients with SCA. By collecting a new set of samples from SCA patients during a vaso-occlusive crisis (VOC, crisis state, 13 patients) and comparing with new samples of patients in steady state (15 patients), we noticed that patients in steady state exhibited mean values of ATP11C/PLSCR1 ratio significantly higher (mean value: 18.2, range, 0.3-53) than those who were in crisis (mean value: 3.7, range, 0.5-9) (P = 0.013). Most importantly, there was a strong inverse correlation between PS exposure and ATP11C/PLSCR1 ratio in sickle erythrocytes (Pearson correlation coefficient, r: - 0.78). Based on these findings, it is conceivable that the ATP11C/PLSCR1 ratio may switch from high to low during a VOC, although the underlying reasons require further investigations.

The Combination of Gefitinib With ATRA and ATO Induces Myeloid Differentiation in Acute Promyelocytic Leukemia Resistant Cells.

In approximately 15% of patients with acute myeloid leukemia (AML), total and phosphorylated EGFR proteins have been reported to be increased compared to healthy CD34+ samples. However, it is unclear if this subset of patients would benefit from EGFR signaling pharmacological inhibition. Pre-clinical studies on AML cells provided evidence on the pro-differentiation benefits of EGFR inhibitors when combined with ATRA or ATO in vitro. Despite the success of ATRA and ATO in the treatment of patients with acute promyelocytic leukemia (APL), therapy-associated resistance is observed in 5-10% of the cases, pointing to a clear need for new therapeutic strategies for those patients. In this context, the functional role of EGFR tyrosine-kinase inhibitors has never been evaluated in APL. Here, we investigated the EGFR pathway in primary samples along with functional in vitro and in vivo studies using several APL models. We observed that total and phosphorylated EGFR (Tyr992) was expressed in 28% and 19% of blast cells from APL patients, respectively, but not in healthy CD34+ samples. Interestingly, the expression of the EGF was lower in APL plasma samples than in healthy controls. The EGFR ligand AREG was detected in 29% of APL patients at diagnosis, but not in control samples. In vitro, treatment with the EGFR inhibitor gefitinib (ZD1839) reduced cell proliferation and survival of NB4 (ATRA-sensitive) and NB4-R2 (ATRA-resistant) cells. Moreover, the combination of gefitinib with ATRA and ATO promoted myeloid cell differentiation in ATRA- and ATO-resistant APL cells. In vivo, the combination of gefitinib and ATRA prolonged survival compared to gefitinib- or vehicle-treated leukemic mice in a syngeneic transplantation model, while the gain in survival did not reach statistical difference compared to treatment with ATRA alone. Our results suggest that gefitinib is a potential adjuvant agent that can mitigate ATRA and ATO resistance in APL cells. Therefore, our data indicate that repurposing FDA-approved tyrosine-kinase inhibitors could provide new perspectives into combination therapy to overcome drug resistance in APL patients.

Association of KLOTHO polymorphisms with clinical complications of sickle cell anemia.

The clinical and phenotypic heterogeneity of patients with sickle cell anemia (SCA) is influenced by environmental and genetic factors. Several genetic modifiers, such as the KLOTHO (KL) gene, have been associated with SCA clinical outcomes. The KL gene and its encoded proteins are implicated in important biological pathways, which affect the disease's pathophysiology, such as expression of adhesion molecules VCAM-1 and ICAM-1, oxidative stress, and nitric oxide biology. Here, we evaluated the clinical relevance of two polymorphisms found on the KL gene (rs685417 and rs211239) in 588 unrelated patients with SCA. Genotyping analyses were performed using the TaqMan system. The KL rs211239 was associated with increased number of vaso-occlusive crisis (VOCs) per year (P = 0.001), while KL rs685417 was associated with increased frequency of stroke (P = 0.034), priapism (P = 0.011), number of complications (P = 0.019), and with a lower incidence of priapism (P = 0.036). Additionally, the associations with VOCs, stroke, and priapism remained consistent in multivariate analyses (P < 0.05). Our data highlight the clinical importance of KL in SCA.

MLL5 improves ATRA driven differentiation and promotes xenotransplant engraftment in acute promyelocytic leukemia model.

Although the mixed lineage leukemia 5 (MLL5) gene has prognostic implications in acute promyelocyte leukemia (APL), the underlying mechanism remains to be elucidated. Here, we demonstrate the critical role exerted by MLL5 in APL regarding cell proliferation and resistance to drug-induced apoptosis, through mtROS regulation. Additionally, MLL5 overexpression increased the responsiveness of APL leukemic cells to all-trans retinoic acid (ATRA)-induced differentiation, via regulation of the epigenetic modifiers SETD7 and LSD1. In silico analysis indicated that APL blasts with MLL5high transcript levels were associated with retinoic acid binding and downstream signaling, while MLL5low blasts displayed decreased expression of epigenetic modifiers (such as KMT2C, PHF8 and ARID4A). Finally, APL xenograft transplants demonstrated improved engraftment of MLL5-expressing cells and increased myeloid differentiation over time. Concordantly, evaluation of engrafted blasts revealed increased responsiveness of MLL5-expressing cells to ATRA-induced granulocytic differentiation. Together, we describe the epigenetic changes triggered by the interaction of MLL5 and ATRA resulting in enhanced granulocytic differentiation.

Influence of UGT1A1 promoter polymorphism, α-thalassemia and ßs haplotype in bilirubin levels and cholelithiasis in a large sickle cell anemia cohort.

Hyperbilirubinemia in patients with sickle cell anemia (SCA) as a result of enhanced erythrocyte destruction, lead to cholelithiasis development in a subset of patients. Evidence suggests that hyperbilirubinemia may be related to genetic variations, such as the UGT1A1 gene promoter polymorphism, which causes Gilbert syndrome (GS). Here, we aimed to determine the frequencies of UGT1A1 promoter alleles, alpha thalassemia, and ßS haplotypes and analyze their association with cholelithiasis and bilirubin levels. The UGT1A1 alleles, -3.7 kb alpha thalassemia deletion and ßS haplotypes were determined using DNA sequencing and PCR-based assays in 913 patients with SCA. The mean of total and unconjugated bilirubin and the frequency of cholelithiasis in GS patients were higher when compared to those without this condition, regardless of age (P < 0.05). Cumulative analysis demonstrated an early age-at-onset for cholelithiasis in GS genotypes (P < 0.05). Low fetal hemoglobin (HbF) levels and normal alpha thalassemia genotype were related to cholelithiasis development (P > 0.05). However, not cholelithiasis but total and unconjugated bilirubin levels were associated with ßS haplotype. These findings confirm in a large cohort that the UGT1A1 polymorphism influences cholelithiasis and hyperbilirubinemia in SCA. HbF and alpha thalassemia also appear as modulators for cholelithiasis risk.

Molecular-Based Score inspired on metabolic signature improves prognostic stratification for myelodysplastic syndrome.

Deregulated cellular energetics is formally incorporated as an emerging hallmark of cancer, however little is known about its processes in myelodysplastic syndromes (MDS). Using transcriptomic data of CD34+ cells from 159 MDS patients and 17 healthy donors, we selected 37 genes involved in cellular energetics and interrogated about its clinical and prognostic functions. Based on the low expression of ACLY, ANPEP, and PANK1, as well as high expression of PKM and SLC25A5, we constructed our Molecular-Based Score (MBS), that efficiently discriminated patients at three risks groups: favourable risk (n = 28; 3-year overall survival (OS): 100%); intermediate (n = 60; 76% [62-93%]) and adverse (n = 71; 35% [17-61%]). Adverse MBS risk was independently associated with inferior OS (HR = 10.1 [95% CI 1.26-81]; P = 0.029) in multivariable analysis using age, gender and the revised international prognostic score system as confounders. Transcriptional signature revealed that Favourable- and intermediate-risk patients presented enriched molecular programs related to mature myeloid progenitors, cell cycle progression, and oxidative phosphorylation, indicating that this cells differs in their origin, metabolic state, and cell cycle regulation, in comparison to the adverse-risk. Our study provides the first evidence that cellular energetics is transcriptionally deregulated in MDS CD34+ cells and establishes a new useful prognostic score based on the expression of five genes.

A multicenter comparative acute myeloid leukemia study: can we explain the differences in the outcomes in resource-constrained settings?

Outcomes in acute myeloid leukemia (AML) are dependent on patient- and disease-characteristics, treatment, and socioeconomic factors. AML outcomes between resource-constrained and developed countries have not been compared directly. We analyzed two cohorts: from São Paulo state, Brazil (USP, n = 312) and Oxford, United Kingdom (OUH, n = 158). USP cohort had inferior 5-year overall survival compared with OUH (29% vs. 49%, adjusted-p=.027). USP patients have higher early-mortality (23% vs. 6% p<.001) primarily due to multi-resistant Gram-negative bacterial and fungal infections. USP had higher 5-year cumulative incidence of relapse (60% vs. 50%, p=.0022), were less likely to undergo hematopoietic stem cell transplant (HSCT) (28% vs. 75%, p<.001) and waited longer for HSCT (median, 23.8 vs. 7.2 months, p<.001). Three-year survival in relapsed patients was worse in USP than OUH (10% vs. 39%, p<.001). Our study indicates that efforts to improve AML outcomes in Brazil should focus on infection prevention and control, and access to HSCT.