Potency and efficacy of pharmacological PIP4K2 inhibitors in acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL), a complex malignancy, displays varying expression profiles of PIP4K2-related genes in adult patients. While PIP4K2A expression is elevated in ALL bone marrow cells compared to healthy bone marrow cells, PIP4K2B is downregulated, and PIP4K2C remains relatively unchanged. Despite the correlation between increased PIP4K2A expression and increased percentage of peripheral blood blasts, clinical outcomes do not strongly correlate with the expression of these genes. Here we investigated the therapeutic potential of three PIP4K2 inhibitors (THZ-P1-2, a131, and CC260) in ALL cell models. THZ-P1-2 emerges as the most effective inhibitor, inducing cell death and mitochondrial damage while reducing cell viability and metabolism significantly. Comparative analyses highlight the superior efficacy of THZ-P1-2 over a131 and CC260. Notably, THZ-P1-2 uniquely disrupts autophagic flux and inhibits the PI3K/AKT/mTOR pathway, indicating a distinct molecular mechanism. In summary, our findings elucidate the differential expression of PIP4K2-related genes in ALL and underscore the potential role of PIP4K2A in disease pathogenesis. The therapeutic promise of THZ-P1-2 in ALL treatment, along with its distinct effects on cell death mechanisms and signaling pathways, enriches our understanding of PIP4K2's involvement in ALL development and offers targeted therapy prospects.

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.

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.

The nonessential amino acid cysteine is required to prevent ferroptosis in acute myeloid leukemia.

ABSTRACT: Cysteine is a nonessential amino acid required for protein synthesis, the generation of the antioxidant glutathione, and for synthesizing the nonproteinogenic amino acid taurine. Here, we highlight the broad sensitivity of leukemic stem and progenitor cells to cysteine depletion. By CRISPR/CRISPR-associated protein 9-mediated knockout of cystathionine-γ-lyase, the cystathionine-to-cysteine converting enzyme, and by metabolite supplementation studies upstream of cysteine, we functionally prove that cysteine is not synthesized from methionine in acute myeloid leukemia (AML) cells. Therefore, although perhaps nutritionally nonessential, cysteine must be imported for survival of these specific cell types. Depletion of cyst(e)ine increased reactive oxygen species (ROS) levels, and cell death was induced predominantly as a consequence of glutathione deprivation. nicotinamide adenine dinucleotide phosphate hydrogen oxidase inhibition strongly rescued viability after cysteine depletion, highlighting this as an important source of ROS in AML. ROS-induced cell death was mediated via ferroptosis, and inhibition of glutathione peroxidase 4 (GPX4), which functions in reducing lipid peroxides, was also highly toxic. We therefore propose that GPX4 is likely key in mediating the antioxidant activity of glutathione. In line, inhibition of the ROS scavenger thioredoxin reductase with auranofin also impaired cell viability, whereby we find that oxidative phosphorylation-driven AML subtypes, in particular, are highly dependent on thioredoxin-mediated protection against ferroptosis. Although inhibition of the cystine-glutamine antiporter by sulfasalazine was ineffective as a monotherapy, its combination with L-buthionine-sulfoximine (BSO) further improved AML ferroptosis induction. We propose the combination of either sulfasalazine or antioxidant machinery inhibitors along with ROS inducers such as BSO or chemotherapy for further preclinical testing.

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.

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.

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.

Oxidative Phosphorylation Fueled by Fatty Acid Oxidation Sensitizes Leukemic Stem Cells to Cold.

Dependency on mitochondrial oxidative phosphorylation (OxPhos) is a potential weakness for leukemic stem cells (LSC) that can be exploited for therapeutic purposes. Fatty acid oxidation (FAO) is a crucial OxPhos-fueling catabolic pathway for some acute myeloid leukemia (AML) cells, particularly chemotherapy-resistant AML cells. Here, we identified cold sensitivity at 4°C (cold killing challenge; CKC4), commonly used for sample storage, as a novel vulnerability that selectively kills AML LSCs with active FAO-supported OxPhos while sparing normal hematopoietic stem cells. Cell death of OxPhos-positive leukemic cells was induced by membrane permeabilization at 4°C; by sharp contrast, leukemic cells relying on glycolysis were resistant. Forcing glycolytic cells to activate OxPhos metabolism sensitized them to CKC4. Lipidomic and proteomic analyses showed that OxPhos shapes the composition of the plasma membrane and introduces variation of 22 lipid subfamilies between cold-sensitive and cold-resistant cells. Together, these findings indicate that steady-state energy metabolism at body temperature predetermines the sensitivity of AML LSCs to cold temperature, suggesting that cold sensitivity could be a potential OxPhos biomarker. These results could have important implications for designing experiments for AML research to avoid cell storage at 4°C. SIGNIFICANCE: Mitochondrial metabolism fueled by FAO alters the membrane composition and introduces membrane fragility upon cold exposure in OxPhos-driven AML and in LSCs. See related commentary by Jones, p. 2441.

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.

The PIP4K2 inhibitor THZ-P1-2 exhibits antileukemia activity by disruption of mitochondrial homeostasis and autophagy.

The treatment of acute leukemia is challenging because of the genetic heterogeneity between and within patients. Leukemic stem cells (LSCs) are relatively drug-resistant and frequently relapse. Their plasticity and capacity to adapt to extracellular stress, in which mitochondrial metabolism and autophagy play important roles, further complicates treatment. Genetic models of phosphatidylinositol-5-phosphate 4-kinase type 2 protein (PIP4K2s) inhibition have demonstrated the relevance of these enzymes in mitochondrial homeostasis and autophagic flux. Here, we uncovered the cellular and molecular effects of THZ-P1-2, a pan-inhibitor of PIP4K2s, in acute leukemia cells. THZ-P1-2 reduced cell viability and induced DNA damage, apoptosis, loss of mitochondrial membrane potential, and the accumulation of acidic vesicular organelles. Protein expression analysis revealed that THZ-P1-2 impaired autophagic flux. In addition, THZ-P1-2 induced cell differentiation and showed synergistic effects with venetoclax. In primary leukemia cells, LC-MS/MS-based proteome analysis revealed that sensitivity to THZ-P1-2 is associated with mitochondrial metabolism, cell cycle, cell-of-origin (hematopoietic stem cell and myeloid progenitor), and the TP53 pathway. The minimal effects of THZ-P1-2 observed in healthy CD34+ cells suggest a favorable therapeutic window. Our study provides insights into the pharmacological inhibition of PIP4K2s targeting mitochondrial homeostasis and autophagy, shedding light on a new class of drugs for acute leukemia.

Altered distribution and function of NK-cell subsets lead to impaired tumor surveillance in JAK2V617F myeloproliferative neoplasms.

In cancer, tumor cells and their neoplastic microenvironment can sculpt the immunogenic phenotype of a developing tumor. In this context, natural killer (NK) cells are subtypes of lymphocytes of the innate immune system recognized for their potential to eliminate neoplastic cells, not only through direct cytolytic activity but also by favoring the development of an adaptive antitumor immune response. Even though the protective effect against leukemia due to NK-cell alloreactivity mediated by the absence of the KIR-ligand has already been shown, and some data on the role of NK cells in myeloproliferative neoplasms (MPN) has been explored, their mechanisms of immune escape have not been fully investigated. It is still unclear whether NK cells can affect the biology of BCR-ABL1-negative MPN and which mechanisms are involved in the control of leukemic stem cell expansion. Aiming to investigate the potential contribution of NK cells to the pathogenesis of MPN, we characterized the frequency, receptor expression, maturation profile, and function of NK cells from a conditional Jak2V617F murine transgenic model, which faithfully resembles the main clinical and laboratory characteristics of human polycythemia vera, and MPN patients. Immunophenotypic analysis was performed to characterize NK frequency, their subtypes, and receptor expression in both mutated and wild-type samples. We observed a higher frequency of total NK cells in JAK2V617F mutated MPN and a maturation arrest that resulted in low-numbered mature CD11b+ NK cells and increased immature secretory CD27+ cells in both human and murine mutated samples. In agreement, inhibitory receptors were more expressed in MPN. NK cells from Jak2V617F mice presented a lower potential for proliferation and activation than wild-type NK cells. Colonies generated by murine hematopoietic stem cells (HSC) after mutated or wild-type NK co-culture exposure demonstrated that NK cells from Jak2V617F mice were deficient in regulating differentiation and clonogenic capacity. In conclusion, our findings suggest that NK cells have an immature profile with deficient cytotoxicity that may lead to impaired tumor surveillance in MPN. These data provide a new perspective on the behavior of NK cells in the context of myeloid malignancies and can contribute to the development of new therapeutic strategies, targeting onco-inflammatory pathways that can potentially control transformed HSCs.

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.

Probe ultrasonification of egg yolk plasma forms low-density lipoprotein nanoparticles that efficiently protect canine semen during cryofreezing.

Around the world, many couples have turned to in vitro fertilization as a viable solution to fertility issues. Low-density lipoprotein (LDL) is a protein best known for transporting fat molecules throughout the body, but it has also been shown to protect sperm cells during cryopreservation due to its micellar structure. In the present study, we aimed to evaluate different protocols for the preparation of nanoparticles from egg yolk plasma (EYP) containing LDL to improve the viability of cryopreserved canine semen. EYP was subjected to three distinct treatments: ultrasonification in an ultrasound bath at 40 kHz for 30 min (LDL-B); ultrasonification via an ultrasound probe at 50% amplitude for 30 min (LDL-P); and high-pressure homogenization at 10,000 PSI for six cycles (LDL-H). Sperm quality was assessed after thawing using computer-assisted sperm analysis and flow cytometry. The results revealed that compared to the EYP control, the LDL-P formulation presented significantly higher efficiency (p < 0.05) in maintaining total and progressive sperm motility, sperm membrane integrity and fluidity, and levels of intracellular reactive oxygen species. The LDL-P nanoparticles had an average size of approximately 250 nm, a PDI value of 0.3, and -1.15 mV of zeta potential, which are very important because it is an indicator of the stability of a colloidal dispersion. Therefore, we conclude that ultrasonication of EYP using a probe is an efficient method for the preparation of LDL nanoparticles that would enhance the cryoprotection of semen during freezing.

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.