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 4-gene prognostic index for enhancing acute myeloid leukaemia survival prediction.

Despite advancements in utilizing genetic markers to enhance acute myeloid leukaemia (AML) outcome prediction, significant disease heterogeneity persists, hindering clinical management. To refine survival predictions, we assessed the transcriptome of non-acute promyelocytic leukaemia chemotherapy-treated AML patients from five cohorts (n = 975). This led to the identification of a 4-gene prognostic index (4-PI) comprising CYP2E1, DHCR7, IL2RA and SQLE. The 4-PI effectively stratified patients into risk categories, with the high 4-PI group exhibiting TP53 mutations and cholesterol biosynthesis signatures. Single-cell RNA sequencing revealed enrichment for leukaemia stem cell signatures in high 4-PI cells. Validation across three cohorts (n = 671), including one with childhood AML, demonstrated the reproducibility and clinical utility of the 4-PI, even using cost-effective techniques like real-time quantitative polymerase chain reaction. Comparative analysis with 56 established prognostic indexes revealed the superior performance of the 4-PI, highlighting its potential to enhance AML risk stratification. Finally, the 4-PI demonstrated to be potential marker to reclassified patients from the intermediate ELN2017 category to the adverse category. In conclusion, the 4-PI emerges as a robust and straightforward prognostic tool to improve survival prediction in AML patients.

Dietary methionine starvation impairs acute myeloid leukemia progression.

Targeting altered tumor cell metabolism might provide an attractive opportunity for patients with acute myeloid leukemia (AML). An amino acid dropout screen on primary leukemic stem cells and progenitor populations revealed a number of amino acid dependencies, of which methionine was one of the strongest. By using various metabolite rescue experiments, nuclear magnetic resonance-based metabolite quantifications and 13C-tracing, polysomal profiling, and chromatin immunoprecipitation sequencing, we identified that methionine is used predominantly for protein translation and to provide methyl groups to histones via S-adenosylmethionine for epigenetic marking. H3K36me3 was consistently the most heavily impacted mark following loss of methionine. Methionine depletion also reduced total RNA levels, enhanced apoptosis, and induced a cell cycle block. Reactive oxygen species levels were not increased following methionine depletion, and replacement of methionine with glutathione or N-acetylcysteine could not rescue phenotypes, excluding a role for methionine in controlling redox balance control in AML. Although considered to be an essential amino acid, methionine can be recycled from homocysteine. We uncovered that this is primarily performed by the enzyme methionine synthase and only when methionine availability becomes limiting. In vivo, dietary methionine starvation was not only tolerated by mice, but also significantly delayed both cell line and patient-derived AML progression. Finally, we show that inhibition of the H3K36-specific methyltransferase SETD2 phenocopies much of the cytotoxic effects of methionine depletion, providing a more targeted therapeutic approach. In conclusion, we show that methionine depletion is a vulnerability in AML that can be exploited therapeutically, and we provide mechanistic insight into how cells metabolize and recycle methionine.

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.

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.

The Expression of NTAL and Its Protein Interactors Is Associated With Clinical Outcomes in Acute Myeloid Leukemia.

Non-T cell activation linker (NTAL) membrane protein depletion from lipid rafts by alkylphospholipids or downregulation by shRNA knockdown decreases cell viability through regulation of the Akt/PI3K pathway in mantle cell lymphoma and acute promyelocytic leukemia cells. Here, we confirmed that the knockdown of NTAL in acute myeloid leukemia (AML) cell lines was associated with decreased cell proliferation and survival. Similarly, a xenograft model using AML cells transduced with NTAL-shRNA and transplanted into immunodeficient mice led to a 1.8-fold decrease in tumor burden. Using immunoprecipitation, LC-MS/MS analysis, and label-free protein quantification, we identified interactors of NTAL in two AML cell lines. By evaluating the gene expression signatures of the NTAL protein interactors using the PREdiction of Clinical Outcomes from Genomic Profiles database, we found that 12 NTAL interactors could predict overall survival in AML, in at least two independent cohorts. In addition, patients with AML exhibiting a high expression of NTAL and its interactors were associated with a leukemic granulocyte-macrophage progenitor-like state. Taken together, our data provide evidence that NTAL and its protein interactors are relevant to AML cell proliferation and survival and represent potential therapeutic targets for granulocyte-macrophage progenitor-like leukemias.

Pharmacological Inhibition of PIP4K2 Potentiates Venetoclax-Induced Apoptosis in Acute Myeloid Leukemia.

Phosphatidylinositol-5-phosphate 4-kinase type 2 (PIP4K2) protein family members (PIP4K2A, PIP4K2B, and PIP4K2C) participate in the generation of PIP4,5P2, which acts as a secondary messenger in signal transduction, a substrate for metabolic processes, and has structural functions. In patients with acute myeloid leukemia (AML), high PIP4K2A and PIP4K2C levels are independent markers of a worse prognosis. Recently, our research group reported that THZ-P1-2 (PIP4K2 pan-inhibitor) exhibits anti-leukemic activity by disrupting mitochondrial homeostasis and autophagy in AML models. In the present study, we characterized the expression of PIP4K2 in the myeloid compartment of hematopoietic cells, as well as in AML cell lines and clinical samples with different genetic abnormalities. In ex vivo assays, PIP4K2 expression levels were related to sensitivity and resistance to several antileukemia drugs and highlighted the association between high PIP4K2A levels and resistance to venetoclax. The combination of THZ-P1-2 and venetoclax showed potentiating effects in reducing viability and inducing apoptosis in AML cells. A combined treatment differentially modulated multiple genes, including TAp73, BCL2, MCL1, and BCL2A1. In summary, our study identified the correlation between the expression of PIP4K2 and the response to antineoplastic agents in ex vivo assays in AML and exposed vulnerabilities that may be exploited in combined therapies, which could result in better therapeutic responses.

Phenformin increases early hematopoietic progenitors in the Jak2V617F murine model.

BACKGROUND: Myeloproliferative neoplasms (MPN) are disorders characterized by an alteration at the hematopoietic stem cell (HSC) level, where the JAK2 mutation is the most common genetic alteration found in classic MPN (polycythemia vera, essential thrombocythemia, and primary myelofibrosis). We and others previously demonstrated that metformin reduced splenomegaly and platelets counts in peripheral blood in JAK2V617F pre-clinical MPN models, which highlighted the antineoplastic potential of biguanides for MPN treatment. Phenformin is a biguanide that has been used to treat diabetes, but was withdrawn due to its potential to cause lactic acidosis in patients. AIMS: We herein aimed to investigate the effects of phenformin in MPN disease burden and stem cell function in Jak2V617F-knockin MPN mice. RESULTS: In vitro phenformin treatment reduced cell viability and increased apoptosis in SET2 JAK2V67F cells. Long-term treatment with 40 mg/kg phenformin in Jak2V617F knockin mice increased the frequency of LSK, myeloid progenitors (MP), and multipotent progenitors (MPP) in the bone marrow. Phenformin treatment did not affect peripheral blood counts, spleen weight, megakaryocyte count, erythroid precursors frequency, or ex vivo clonogenic capacity. Ex vivo treatment of bone marrow cells from Jak2V617F knockin mice with phenformin did not affect hematologic parameters or engraftment in recipient mice. CONCLUSIONS: Phenformin increased the percentages of LSK, MP, and MPP populations, but did not reduce disease burden in Jak2V617F-knockin mice. Additional studies are necessary to further understand the effects of phenformin on early hematopoietic progenitors.

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.

Combining gene mutation with gene expression analysis improves outcome prediction in acute promyelocytic leukemia.

By combining the analysis of mutations with aberrant expression of genes previously related to poorer prognosis in both acute promyelocytic leukemia (APL) and acute myeloid leukemia, we arrived at an integrative score in APL (ISAPL) and demonstrated its relationship with clinical outcomes of patients treated with all-trans retinoic acid (ATRA) in combination with anthracycline-based chemotherapy. Based on fms-like tyrosine kinase-3-internal tandem duplication mutational status; the ΔNp73/TAp73 expression ratio; and ID1, BAALC, ERG, and KMT2E gene expression levels, we modeled ISAPL in 159 patients (median ISAPL score, 3; range, 0-10). ISAPL modeling identified 2 distinct groups of patients, with significant differences in early mortality (P < .001), remission (P = .004), overall survival (P < .001), cumulative incidence of relapse (P = .028), disease-free survival (P = .03), and event-free survival (P < .001). These data were internally validated by using a bootstrap resampling procedure. At least for patients treated with ATRA and anthracycline-based chemotherapy, ISAPL modeling may identify those who need to be treated differently to maximize their chances for a cure.

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.

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.

Alpha thalassemia, but not ßS-globin haplotypes, influence sickle cell anemia clinical outcome in a large, single-center Brazilian cohort.

Alpha thalassemia and beta-globin haplotype are considered classical genetic disease modifiers in sickle cell anemia (SCA) causing clinical heterogeneity. Nevertheless, their functional impact on SCA disease emergence and progression remains elusive. To better understand the role of alpha thalassemia and beta-globin haplotype in SCA, we performed a retrospective study evaluating the clinical manifestations of 614 patients. The univariate analysis showed that the presence of alpha-thalassemia -3.7-kb mutation (αα/-α and -α/-α) decreased the risk of stroke development (p = 0.046), priapism (p = 0.033), and cholelithiasis (p = 0.021). Furthermore, the cumulative incidence of stroke (p = 0.023) and cholelithiasis (p = 0.006) was also significantly lower for patients carrying the alpha thalassemia -3.7-kb mutation. No clinical effects were associated with the beta-globin haplotype analysis, which could be explained by the relatively homogeneous haplotype composition in our cohort. Our results reinforce that alpha thalassemia can provide protective functions against hemolysis-related symptoms in SCA. Although, several genetic modifiers can impact the inflammatory state of SCA patients, the alpha thalassemia mutation remains one of the most recurrent genetic aberration and should therefore always be considered first.

Interleukin-8 is not a predictive biomarker for the development of the acute promyelocytic leukemia differentiation syndrome.

BACKGROUND: Differentiation syndrome (DS) is the main life-threatening adverse event that occurs in acute promyelocytic leukemia (APL) patients treated with all-trans retinoic acid (ATRA). Cytokine imbalances have been reported to play role during the developing of acute promyelocytic leukemia differentiation syndrome (APL-DS). However, the relationship between the plasma cytokine levels and their prognostic value for the prediction of DS developing in patients with APL during the treatment with ATRA and anthracyclines has not been previously reported. METHODS: In this study, we followed an APL cohort (n = 17) over 7 days of ATRA therapy in DS (n = 6) and non-DS groups (n = 11). Interleukin (IL)-1ß, IL-6, IL-8, IL-10, IL-12p70 and TNF-α were measured in the peripheral blood plasma from 17 patients with APL and 11 healthy adult controls by using the cytometric bead array method. RESULTS: In non-DS patients, IL-8 plasma levels were significantly reduced in the seventh day of ATRA treatment (34.16; 6.99 to 147.11 pg mL- 1 in D0 vs. 10.9; 0 to 26.81 pg mL- 1 in D7; p = 0.02) whereas their levels did not discriminate between DS and non-DS development during the entire induction period (all p > 0.05 in D0, D3, and D7). No significant differences were found in IL-6 levels between groups (p > 0.05 in D0-D7). Other cytokines tested were all undetectable in patients with APL or healthy controls. CONCLUSIONS: We demonstrated that the modulation of IL-8 following ATRA treatment may occur regardless of the development of DS and, therefore, does not appear to be a predictive biomarker to monitor the APL-DS.

High levels of proinflammatory cytokines IL-6 and IL-8 are associated with a poor clinical outcome in sickle cell anemia.

Sickle cell anemia (SCA) pathophysiology is characterized by the activation of sickle red blood cells, reticulocytes, leukocytes, platelets, and endothelial cells, and with the expression of several inflammatory molecules. Therefore, it is conceivable that variations in levels of proinflammatory cytokines may act as a signaling of differential clinical course in SCA. Here, we evaluated the clinical impact of proinflammatory cytokines interleukin 1-ß (IL-1ß), interleukin 6 (IL-6), and interleukin 8 (IL-8) in 79 patients with SCA, followed in a single reference center from northeastern Brazil. The main clinical/laboratory data were obtained from patient interview and medical records. The proinflammatory markers IL-1ß, IL-6, and IL-8 were evaluated by using commercially available enzyme-linked immunosorbent assay kits. According to levels of the proinflammatory markers, we observed that patients who had a higher frequency of VOC per year (P = 0.0236), acute chest syndrome (P = 0.01), leg ulcers (P = 0.0001), osteonecrosis (P = 0.0006), stroke (P = 0.0486), and priapism (P = 0.0347) had higher IL-6 levels compared with patients without these clinical complications. Furthermore, increased levels of IL-8 were found in patients who presented leg ulcers (P = 0.0184). No significant difference was found for IL-1ß levels (P > 0.05). In summary, the present study emphasizes the role of inflammation in SCA pathophysiology, reveals an association of IL-8 levels and leg ulcer occurrence, and indicates that IL-6 levels can be used as a useful predictor for poor outcomes in SCA.

Global gene expression reveals an increase of HMGB1 and APEX1 proteins and their involvement in oxidative stress, apoptosis and inflammation pathways among beta-thalassaemia intermedia and major phenotypes.

Beta-thalassaemia (BT) is classified according to blood transfusion requirement as minor (BTMi), intermedia (BTI) and major (BTM). BTM is the most severe form, requiring regular transfusions while transfusion need is only occasional in BTI. Differential gene expression between patients has not been assessed so far. Here, we evaluated the global gene expression profiles during differentiation of human erythroid cells of two patients carrying the same mutation [CD39, (C → T)], though displaying different phenotypes (BTI and BTM). Considering the role of reactive oxygen species (ROS) in the pathophysiology of thalassaemia, we focused on differentially expressed genes involved in metabolic pathways triggered by ROS, such as inflammation and apoptosis, and, from these, we selected the Apurinic/Apyrimidinic Endodeoxyribonuclease 1 (APEX1) and High Mobility Group Box1 (HMGB1) genes, whose role in BT is not well established. An in-depth expression analysis of transcriptional and protein levels in patients carrying a range of mutations associated with BT phenotypes indicated that APEX1 was increased in both BTI and BTM. Furthermore, higher amounts of HMGB1 was found in the plasma of BTI patients. Our findings suggest that these proteins have important roles in BT and could represent new targets for further studies aiming to improve the management of the disease.