Poor Prognosis In Acute Myeloid Leukemia Research Articles

Epigenetic modifications in hematopoietic ecosystem: a key tuner from homeostasis to acute myeloid leukemia.

Hematopoietic stem cells (HSCs) maintain homeostasis in the hematopoietic ecosystem, which is tightly regulated at multiple layers. Acute myeloid leukemia (AML) is a severe hematologic malignancy driven by genetic and epigenetic changes that lead to the transformation of leukemia stem cells (LSCs). Since somatic mutations in DNA methylation-related genes frequently occur in AML, DNA methylation is widely altered and functions as a starting engine for initiating AML. Additionally, RNA modifications, especially N6-methyladenosine (m6A), also play an important role in the generation and maintenance of the hematopoietic ecosystem, and AML development requires reprogramming of m6A modifications to facilitate cells with hallmarks of cancer. Given the complex pathogenesis and poor prognosis of AML, it is important to fully understand its pathogenesis. Here, we mainly focus on DNA methylation and RNA m6A modification in hematopoiesis and AML and summarize recent advances in this field.

Blockade of the TIGIT-CD155/CD112 axis enhances functionality of NK-92 but not cytokine-induced memory-like NK cells toward CD155-expressing acute myeloid leukemia

TIGIT is an alternative checkpoint receptor (CR) whose inhibition promotes Graft-versus-Leukemia effects of NK cells. Given the significant immune-permissiveness of NK cells circulating in acute myeloid leukemia (AML) patients, we asked whether adoptive transfer of activated NK cells would benefit from additional TIGIT-blockade. Hence, we characterized cytokine-induced memory-like (CIML)-NK cells and NK cell lines for the expression of inhibitory CRs. In addition, we analyzed the transcription of CR ligands in AML patients (CCLE and Beat AML 2.0 cohort) in silico and evaluated the efficacy of CR blockade using in vitro cytotoxicity assays, CD69, CD107a and IFN-γ expression. Alternative but not classical CRs were abundantly expressed on healthy donor NK cells and even further upregulated on CIML-NK cells. In line with our finding that CD155, one important TIGIT-ligand, is reliably expressed on AMLs, we show improved killing of CD155+-AML blasts by NK-92 but interestingly not CIML-NK cells in the presence of TIGIT-blockade. Additionally, our in silico data (n = 671) show that poor prognosis AML patients rather displayed a CD86low CD112/CD155high phenotype, whereas patients with a better outcome rather exhibited a CD86high CD112/CD155low phenotype. Collectively, our data evidence that the complex CR ligand expression profile on AML blasts may be one explanation for the intrinsic NK cell exhaustion observed in AML patients which might be overcome with adoptive NK-92 transfer in combination with TIGIT-blockade.

The Study of SRSF1 Regulates Abnormal Alternative Splicing of BCL2L11 and the Role in Refractory Acute Myeloid Leukemia.

Abnormalities in splicing factors, such as mutations or deregulated expression, can lead to aberrant splicing of target genes, potentially contributing to the pathogenesis of acute myeloid leukemia (AML). Despite this, the precise mechanism underlying the abnormal alternative splicing (AS) induced by SRSF1, a splicing factor associated with poor AML prognosis, remains elusive. Using strict splicing criteria, we globally screened for AS events in NPMc-positive and NPMc-negative AML samples from TCGA. An AS network associated with AML prognosis was then established. Functional assays, including CCK-8, flow cytometry, and Western blot, were conducted on K562 and THP-1 cells overexpressing SRSF1. Cell viability following 72-h Omipalisib treatment was also assessed. To explore the mechanism of SRSF1-induced AS, we created a BCL2L11 miniGene with a site-specific mutation at its branch point. The AS patterns of both wild-type and mutant miniGenes were analyzed following SRSF1 overexpression in HEK-293T, along with the subcellular localization of different spliceosomes. SRSF1 was significantly associated with AML prognosis. Notably, its expression was markedly upregulated in refractory AML patients compared to those with a favorable chemotherapy response. Overexpression of SRSF1 promoted THP-1 cell proliferation, suppressed apoptosis, and reduced sensitivity to Omipalisib. Mechanistically, SRSF1 recognized an aberrant branch point within the BCL2L11 intron, promoting the inclusion of a cryptic exon 3, which in turn led to apoptosis arrest. Overexpression of SRSF1 and the resulting abnormal splicing of BCL2L11 are associated with drug resistance and poor prognosis in AML.

Preliminary safety, efficacy and molecular characterization of emavusertib (CA-4948) in patients with relapsed/refractory (R/R) acute myeloid leukemia (AML) with FLT3 mutation (FLT3m).

6539 Background: AML is a heterogenous disease and exhibits a dynamic mutational landscape as the disease progresses. Internal tandem duplication (ITD) of FLT3 is considered an acquired late-event mutation and is associated with a poor prognosis in AML. Emavusertib is a potent oral inhibitor of both IRAK4 and FLT3, conferring potential dual efficacy advantages compared to other IRAK4 or FLT3 inhibitors as single agents. IRAK4 is upregulated during anti-FLT3- or other cytotoxic therapies, which drives a resistance pathway of early relapse and progression. Methods: The safety, clinical activity, and potential biomarkers of emavusertib in R/R AML and higher-risk myelodysplastic syndrome are being investigated in the ongoing open-label, Phase 1/2a TakeAim Leukemia trial (NCT04278768). Next generation sequencing (NGS) of 68 genes was performed on genomic DNA from bone marrow or peripheral blood mononuclear cells at baseline and on treatment. Here, we present preliminary safety, efficacy data and molecular characterization in a subset of enrolled AML patients who carried FLT3m at baseline and were treated with emavusertib monotherapy. Results: In this study, we treated 11 R/R AML patients with FLT3m at dose levels of 200-400 mg BID. The median number of prior anti-cancer therapies was 2 (range 1-6). 8 of 11 patients had prior exposure to FLT3 inhibitors. Treatment-related adverse events (TRAEs) Grade ≥ 3 were reported in 2 of 11 (18%) patients. All responders were dosed at the Recommended Phase 2 Dose (RP2D) of 300 mg BID and with < 3 lines of prior therapy. In addition, responders demonstrated more than 90% bone marrow blast reduction compared to baseline. The most common co-mutations in this subset of patients included RUNX1, NRAS, and TET2. Emavusertib treatment significantly decreased the variant allele frequency of these mutations. FLT3-ITD levels were decreased or became undetectable in responders. Additional corelative analysis between bone marrow blast counts and mutation status indicates that AML patients with FLT3m demonstrated increased bone marrow blast reductions (P≤0.05) on emavusertib treatment, when compared to FLT3 WT AML patients. Conclusions: Emavusertib has a favorable safety and tolerability profile in pretreated AML patients with FLT3m and demonstrated monotherapy anti-cancer activity in patients with FLT3m, including patients who have progressed on prior FLT3 inhibitors. Mutational profiles are suggestive of disease-modifying activity of emavusertib. We will present updated safety and efficacy data, including data for patients who proceeded to stem cell transplant. Enrollment in this ongoing trial is continuing at RP2D in patients with < 3 lines of prior anti-cancer therapies. Clinical trial information: NCT04278768 .

A prospective study of all-trans retinoic acid plus venetoclax and azacitidine in newly diagnosed acute myeloid leukemia.

6545 Background: Over the decade, anthracycline and cytarabine-based intensive chemotherapy remains the standard of care for acute myeloid leukemia (AML) but is hampered by serious toxicities like myelosuppression, increased transfusion requirement and disappointing clinical outcomes among the so-called poor-prognosis AML subsets. BCL-2 inhibitor plus azacitidine (AZA) has become a preferred treatment for AML, but the remission rate and overall survival (OS) are suboptimal. Apart from hematologic toxicities, 70.0% of patients were RBC transfusion dependent and 58.6% were platelet transfusion dependent. At present, all-trans retinoic acid (ATRA) is used as a differentiation drug, and there is no combination regimen with BCL-2 inhibitor. This study aimed to assess the efficacy and safety of ATRA plus venetoclax and AZA for newly diagnosed AML. Methods: This study enrolled 35 patients (≥18 years) with newly diagnosed AML between 2022 and 2023. For induction, patients received AZA (75 mg/m²) from Day 1 to 7, venetoclax at a target dose of 400 mg/d from Day 2 to 10, and ATRA (45 mg/m²/d) from Day 12 to 28 of each 28-day cycle for up to 2 cycles or until disease progression. Patients who achieved complete remission (CR) after two cycles of induction therapy received bone marrow transplantation or consolidation at their own discretion. For consolidation, patients received ATRA (45 mg/m²/d) from Day 1 to 21 and AZA (75 mg/m²) from Day 1 to 7 of each 28-day cycle for up to 4 cycles or until disease progression. For maintenance, patients received ATRA (45 mg/m²/d) for 21 days per cycle plus AZA (75 mg/m²). Maintenance therapy was given every 3 months until disease progression and ATRA was provided during intermissions. The primary endpoint was the composite CR rate (CR and CR with incomplete hematologic recovery) at the end of cycle 1 of inductive therapy. Results: As of now, the composite CR rate reached 74% (26/35) and the objective response rate reached 91% (32/35). The composite CR was 73% (11/15) in the adverse risk group and 75% (15/20) in the favorable-intermediate risk group at the end of cycle 1. Thirty-three patients entered cycle 2. The cumulative composite CR rate reached 91%(30/33) after 2 cycles of induction therapy. The median event-free survival was 390 days, and the median OS was 573 days. Platelet transfusion independence occurred in 17% (6/35) patients in cycle 1 and 73% (24/33) in cycle 2. Furthermore, 20% (7/35) and 73% (24/33) patients were RBC transfusion independent in cycle 1 and 2, respectively. Any-grade infections occurred in 74% (26/35) patients, including 12 before admission. Conclusions: The treatment regimen achieved a high composite CR rate and was well tolerated, with reduced hematologic toxicities in cycle 2 of induction therapy. Both adverse risk and favorable-intermediate risk patients benefited from the regimen, which holds promise for treating AML in adult patients. Clinical trial information: NCT05654194 .

The Expression of METTL14 in Patients with Newly Diagnosed Acute Myeloid Leukemia and Its Clinical Significance

To detect the expression of RNA methyltransferase 14(METTL14) in bone marrow of patients with newly diagnosed acute myeloid leukemia (AML), and to investigate the clinical and prognostic significance of METTL14 expression in newly diagnosed AML. Bone marrow samples were collected from 100 patients with newly diagnosed AML as observation group and 60 patients with iron deficiency anemia AML as control group. And collected the clinical data of the AML patients. Real-time quantitative PCR (qRT-PCR) was used to detect the expression level of METTL14 in AML and IDA patients. The relationship between the expression level of METTL14 and clinicopathological features, prognosis was analyzed. Kaplan-Meier curves were used to analyze the effect of METTL14 on overall survival (OS) in AML patients. Cox risk regression model was used to analyze the prognostic factors affecting in patients with AML. Compared with the control group, the expression of METTL14 was significantly increased in AML patients (P < 0.05). Compared with the METTL14 low-expression group, patients in the METTL14 high-expression group had advanced age, high bone marrow cell number, poor efficacyand poor prognosis(P < 0.05). The overall survival time of patients with the METTL14 high-expression group was significantly shorter than that of the low-expression group (P < 0.05). The high expression of METTL14 was an independent risk factor for poor prognosis in AML. METTL14 is significantly overexpressed in AML patients, and its correlated with poor clinicopathological features and poor prognosis. It can be used as a prognostic marker and potential therapeutie target for AML patients.

Prognostic Value of IGF2BP3 Gene Expression Levels in Patients with Acute Myeloid Leukemia

To investigate the relationship between IGF2BP3 gene expression and prognosis in patients with acute myeloid leukemia (AML). High throughput transcriptome sequencing was performed on bone marrow primary leukemia cells from 27 patients with AML in our center, the relationship between IGF2BP3 expression levels and clinical characteristics were analyzed and verify the samples from patients with newly treated AML and refractory AML. The expression level of IGF2BP3 gene were analyzed in 20 healthy subjects and 26 patients with AML. The expression of IGF2BP3 in two anthracycline-resistant cell lines (HL60/ADR, K562/ADR) was detected by RT-qPCR and Western blot, and the expression difference of IGF2BP3 was compared with that in sensitive cells (HL60, K562). The relationship between the expression level of IGF2BP3 in patients with AML and prognostic were analyzed through data analysis of 746 patients with AML, and the prognostic value of IGF2BP3 in AML was analyzed by multivariate Cox regression analysis. In the bone marrow primary leukemia cells of 27 AML patients in our center, the expression level of IGF2BP3 in patients with refractory AML was significantly higher than that in chemotherapy sensitive patients (P =0.0343). The expression of IGF2BP3 in leukemia patients with extramedullary infiltration (EMI) was significantly higher than that in AML patients without extramedullary infiltration (P =0.0049). Compared with healthy subjects (n=20), IGF2BP3 expression in AML patients (n=26) was higher (P =0.0009). The expression of IGF2BP3 mRNA in the anthracycline resistant cell lines (HL60/ADR, K562/ADR) was significantly higher than that in the sensitive cell lines (K562/ADR vs K562,P =0.0430; HL60/ADR vs HL60, P =0.7369). Western blot results showed that the expression of IGF2BP3 protein in mycin resistant cells was significantly higher than that in sensitive cells (P < 0.001). qPCR results showed that the expression level of IGF2BP3 mRNA in refractory AML patients was significantly higher than that in patients with chemotherapy sensitive (P =0.002). High expression of IGF2BP3 was associated with poor prognosis in AML (P < 0.05) in 3 large sample cohorts of AML patients. Univariate and multivariate prognostic analyses demonstrated that high expression of IGF2BP3 was significantly associated with shorter event-free survival (EFS, HR=1.887, P =0.024) and overall survival (OS, HR=1.619, P =0.016). The high expression of IGF2BP3 gene may be an important factor in the poor prognosis of AML, suggesting that IGF2BP3 gene may be a new molecular marker for the clinical prognosis evaluation and treatment strategy of AML.

Aging and comprehensive molecular profiling in acute myeloid leukemia

Acute myeloid leukemia (AML) is an aging-related and heterogeneous hematopoietic malignancy. In this study, a total of 1,474 newly diagnosed AML patients with RNA sequencing data were enrolled, and targeted or whole exome sequencing data were obtained in 94% cases. The correlation of aging-related factors including age and clonal hematopoiesis (CH), gender, and genomic/transcriptomic profiles (gene fusions, genetic mutations, and gene expression networks or pathways) was systematically analyzed. Overall, AML patients aged 60 y and older showed an apparently dismal prognosis. Alongside age, the frequency of gene fusions defined in the World Health Organization classification decreased, while the positive rate of gene mutations, especially CH-related ones, increased. Additionally, the number of genetic mutations was higher in gene fusion-negative (GF-) patients than those with GF. Based on the status of CH- and myelodysplastic syndromes (MDS)-related mutations, three mutant subgroups were identified among the GF- AML cohort, namely, CH-AML, CH-MDS-AML, and other GF- AML. Notably, CH-MDS-AML demonstrated a predominance of elderly and male cases, cytopenia, and significantly adverse clinical outcomes. Besides, gene expression networks including HOXA/B, platelet factors, and inflammatory responses were most striking features associated with aging and poor prognosis in AML. Our work has thus unraveled the intricate regulatory circuitry of interactions among different age, gender, and molecular groups of AML.

MicroRNAs Associated with a Bad Prognosis in Acute Myeloid Leukemia and Their Impact on Macrophage Polarization.

MicroRNAs (miRNAs) are short, non-coding ribonucleic acids (RNAs) associated with gene expression regulation. Since the discovery of the first miRNA in 1993, thousands of miRNAs have been studied and they have been associated not only with physiological processes, but also with various diseases such as cancer and inflammatory conditions. MiRNAs have proven to be not only significant biomarkers but also an interesting therapeutic target in various diseases, including cancer. In acute myeloid leukemia (AML), miRNAs have been regarded as a welcome addition to the limited therapeutic armamentarium, and there is a vast amount of data on miRNAs and their dysregulation. Macrophages are innate immune cells, present in various tissues involved in both tissue repair and phagocytosis. Based on their polarization, macrophages can be classified into two groups: M1 macrophages with pro-inflammatory functions and M2 macrophages with an anti-inflammatory action. In cancer, M2 macrophages are associated with tumor evasion, metastasis, and a poor outcome. Several miRNAs have been associated with a poor prognosis in AML and with either the M1 or M2 macrophage phenotype. In the present paper, we review miRNAs with a reported negative prognostic significance in cancer with a focus on AML and analyze their potential impact on macrophage polarization.

Prognostic Relevance of Immunophenotyping in Adult Patients with Acute Leukemia in Turkey: A Single-Center Experience.

In acute leukemia, many associations have been identified between prognosis and some factors, such as individual antigen expression, cytogenetics, gender, age, high leukocyte level (WBC), platelet count (PLT), and lactate dehydrogenase (LDH), but few are consistent. In this study, we aimed to investigate the cell surface markers and other clinical pathological features for prognosis determination in acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) patients in our population. This cross-sectional study was performed from January 2017 to December 2023 in İstanbul Training and Research Hospital and included 113 patients (86 AML and 27 ALL) newly diagnosed with AML (non-APL) and ALL. The following tests were fulfilled for the included patients: complete blood count (CBC), LDH, and flow cytometric analysis using a blood sample or bone marrow aspirate. The effects of surface markers, gender, age, WBC, PLT, and LDH on 24-month survival were evaluated retrospectively. Among the investigated parameters, lack of CD13 expression and positive CD10, cTdT expressions were associated with poor prognosis in AML patients (p = 0.01, p = 0.04, and p = 0.04, respectively). We have found no association between the surface markers and other parameters with prognosis in ALL patients. Age > 65 years was associated with poor prognosis in both AML and ALL patients (p < 0.001). CD10, cTdT positivity, and CD13 negativity may predict poor prognosis for AML and can be considered as prognostic biomarkers for AML patients.

Variability in the perception of palliative care and end-of-life care among hematology professionals from the same reference center in Bahia, Brazil: A descriptive cross-sectional study.

There are several illness-specific cultural and system-based barriers to palliative care (PC) integration and end-of-life (EOL) care in the field of oncohematology. This study aimed to investigate the variability in the perceptions of PC and EOL care. A cross-sectional study was conducted in the Hematology Division of our University Hospital in Salvador, Bahia, Brazil. Twenty physicians responded to a sociodemographic questionnaire and an adaptation of clinical questionnaires used in previous studies from October to December 2022. The median age of the participants was 44 years, 80% of the participants identified as female, and 75% were hematologists. Participants faced a hypothetical scenario involving the treatment of a 65-year-old female with a poor prognosis acute myeloid leukemia refractory to first-line treatment. Sixty percent of the participants chose to follow other chemotherapy regimens, whereas 40% opted for PC. Next, participants considered case salvage for the patient who developed septic shock following chemotherapy and were prompted to choose their most probable conduct, and the conduct they thought would be better for the patient. Even though participants were from the same center, we found a divergence from the most probable conduct among 40% of the participants, which was due to personal convictions, legal aspects, and other physicians' reactions. We found considerable differences in the perception of PC and EOL care among professionals, despite following the same protocols. The study also demonstrated variations between healthcare professionals' beliefs and practices and persistent historical tendencies to prioritize aggressive interventions.

MiR-26b-5p Affects the Progression of Acute Myeloid Leukemia by Regulating the USP48-Mediated Wnt/β-Catenin Pathway.

Acute myeloid leukemia (AML) is a highly heterogeneous disease. Exploring the pathogenesis of AML is still an important topic in the treatment of AML. The expression levels of miR-26b-5p and USP48 were measured by qRT-PCR. The expression levels of related proteins were detected by Western blot. Cell proliferation and apoptosis were detected by CCK-8 and flow cytometry, respectively. Coimmunoprecipitation was used to examine the interaction between USP48 and Wnt5a. Bioinformatics analysis showed that high levels of miR-26b-5p and low levels of USP48 were associated with poor prognosis in AML. miR-26b-5p can negatively regulate the expression of USP48. Downregulation of miR-26b-5p inhibited EMT, cell viability and proliferation of AML cells and accelerated apoptosis. Furthermore, the influence of miR-26b-5p inhibition and USP48 knockdown on AML progression could be reversed by a Wnt/β-catenin signaling pathway inhibitor. This study revealed that miR-26b-5p regulates AML progression, possibly by targeting the USP48-mediated Wnt/β-catenin molecular axis to affect AML cell biological behavior.

Prognostic significance of ASXL1 mutations in acute myeloid leukemia: A systematic review and meta-analysis.

Although genetic mutations in additional sex-combs-like 1 (ASXL1) are prevalent in acute myeloid leukemia (AML), their exact impact on the AML prognosis remains uncertain. Hence, the present article was carried out to explore the prognostic importance of ASXL1 mutations in AML. We thoroughly searched electronic scientific databases to find eligible papers. Twenty-seven studies with an overall number of 8,953 participants were selected for the current systematic review. The hazard ratio (HR) and 95% confidence interval (CI) for overall survival (OS), event-free survival (EFS), and relapse-free survival (RFS) were extracted from all studies with multivariate or univariate analysis. Pooled HRs and p-values were also calculated as a part of our work. The pooled HR for OS in multivariable analysis indicated that ASXL1 significantly diminished survival in AML patients (pooled HR: 1.67; 95% CI: 1.342-2.091). ASXL1 mutations may confer a poor prognosis in AML. Hence, they may be regarded as potential prognostic factors. However, more detailed studies with different ASXL1 mutations are suggested to shed light on this issue.

Prognostic importance of NUP98-rearrangements in acute myeloid leukemia: A systematic review and meta-analysis.

NUP98 gene fusions in acute myeloid leukemia (AML) have recently attracted much interest. Despite substantial research illuminating the roles of NUP98 fusions in the course of AML, their impacts on the outcome of patients with AML should be explored in more detail. As a result, this meta-analysis was designed to provide further light on the prognostic implications of NUP98 fusions in AML. We completed an extensive search in PubMed, Scopus, and Web of Science to identify papers evaluating the prognostic effects of NUP98 rearrangements in patients with AML until August 22, 2022. In total, 15 publications with 6142 participants fulfilled the requirements for the current meta-analysis. All the qualified studies were examined for information regarding HRs and 95% confidence interval (95%CI) for overall survival (OS) and event-free survival (EFS). In addition, we utilized Comprehensive Meta-analysis software version 2 (CMA2) for calculating pooled HRs and 95% CI. Our Results : analyses for NUP98-NSD1 indicated that this fusion could significantly impact the outcome of patients with AML (pooled HR: 2.84; 95% CI: 2.49-3.24, P=0.000). Additionally, we observed a strong correlation between NUP98-KDM5A rearrangement and poor prognosis in AML (pooled HR: 2.65; 95% CI: 2.5-2.81; P=0.000). A subgroup analysis also showed that the NUP98-NSD1 and FLT3-ITD together confer a poor prognostic effect (pooled HR: 2.60, 95% CI: 1.61-4.18; P=0.000). NUP98 fusions could significantly impact the outcome of patients with AML. The use of these fusions as prognostic indicators in AML seems rational.

Epigenetic vulnerabilities of leukemia harboring inactivating EZH2 mutations

Epigenetic regulators, such as the polycomb repressive complex 2 (PRC2), play a critical role in both normal development and carcinogenesis. Mutations and functional dysregulation of PRC2 complex components, such as EZH2, are implicated in various forms of cancer and associated with poor prognosis. This study investigated the epigenetic vulnerabilities of acute myeloid leukemia (AML) and myelodysplastic/myeloproliferative disorders (MDS/MPN) by performing a chemical probe screen in patient cells. Paradoxically, we observed increased sensitivity to EZH2 and embryonic ectoderm development (EED) inhibitors in AML and MDS/MPN patient cells harboring EZH2 mutations. Expression analysis indicated that EZH2 inhibition elicited upregulation of pathways responsible for cell death and growth arrest, specifically in patient cells with mutant EZH2. The identified EZH2 mutations had drastically reduced catalytic activity, resulting in lower cellular H3K27me3 levels, and were associated with decreased EZH2 and PRC2 component EED protein levels. Overall, this study provides an important understanding of the role of EZH2 dysregulation in blood cancers and may indicate disease etiology for these poor prognosis AML and MDS/MPN cases.

An Overview on Lipid Droplets Accumulation as Novel Target for Acute Myeloid Leukemia Therapy.

Metabolic reprogramming is a key alteration in tumorigenesis. In cancer cells, changes in metabolic fluxes are required to cope with large demands on ATP, NADPH, and NADH, as well as carbon skeletons. In particular, dysregulation in lipid metabolism ensures a great energy source for the cells and sustains cell membrane biogenesis and signaling molecules, which are necessary for tumor progression. Increased lipid uptake and synthesis results in intracellular lipid accumulation as lipid droplets (LDs), which in recent years have been considered hallmarks of malignancies. Here, we review current evidence implicating the biogenesis, composition, and functions of lipid droplets in acute myeloid leukemia (AML). This is an aggressive hematological neoplasm originating from the abnormal expansion of myeloid progenitor cells in bone marrow and blood and can be fatal within a few months without treatment. LD accumulation positively correlates with a poor prognosis in AML since it involves the activation of oncogenic signaling pathways and cross-talk between the tumor microenvironment and leukemic cells. Targeting altered LD production could represent a potential therapeutic strategy in AML. From this perspective, we discuss the main inhibitors tested in in vitro AML cell models to block LD formation, which is often associated with leukemia aggressiveness and which may find clinical application in the future.

Phase Separation Mediates RUNX1- Mutation Leukemic Transformation

B ackground : The high degree of genetic heterogeneity in acute myeloid leukemia (AML) leads to refractory or relapse in some patients, so it is important to deepen the mechanistic studies and find specific drug targets.As a classical regulatory molecule associated with AML, the absence or down-regulation of RUNX1 can lead to the development of AML. Liquid-liquid phase separation is closely related to the occurrence and development of the disease, and it is not clear whether RUNX1 can participate in the pathogenesis of AML through phase separation. This study is the first to explore the involvement of RUNX1 in the pathogenesis of AML from the perspective of phase separation. Methods: The fluorescent expression of RUNX1 was detected by immunofluorescence. The extracellular phase separation of RUNX1 and the mutants were studied by prokaryotic expression of the target protein RUNX1. The intracellular phase separation of RUNX1 was studied by cellular transfection and live-cell imaging. The cellular differentiation ability after phase separation of AML cell lines was investigated by using Rachel Giemsa staining and flow cytometry. The specific mechanism of RUNX1 phase separation regulation of downstream target molecules was examined by Co-IP coupled with mass spectrometry and Western Blot. The effect of RUNX1 phase separation on the pathogenesis of AML was studied by using the AML mouse model. Results: In this study, we firstly confirmed that RUNX1 showed obvious spot aggregation during hematopoietic differentiation. We then confirmed that RUNX1 can perform liquid-liquid phase separation both in vitro and intracellularly. Furthermore, based on the structural domain of RUNX1 and the RUNX1 mutation sites in AML patients, it was confirmed that the IDR region of RUNX1 drove occurrence of phase separation, while the RHD region had an inhibitory effect on phase separation, and the RUNX1 point mutants associated with poor prognosis of AML had abnormal phase separation.The occurrence of phase separation of RUNX1 could regulate the differentiation function of AML cells, and the differentiation of AML cells in the phase separation-deficient type was inhibited. Transcriptome sequencing results showed that RUNX1 phase separation promoted the enrichment of signaling pathways such as cell differentiation, and it was also found that RUNX1 phase separation promoted downstream target molecules to participate in the regulation of AML cell function. In addition, we demonstrated that aberrant RUNX1 phase separation was involved in the pathogenesis of AML in the mouse model of AML. Conclusion: This study reveals for the first time that RUNX1 liquid-liquid phase separation is involved in the process of AML occurrence and development, and refines the functional regulation mechanism of RUNX1, providing new ideas for AML targeted therapy.

Novel Therapeutics Targeting Acute Myeloid Leukemia (AML) Stem Cells Identified through High-Throughput Screening

Acute myeloid leukemia (AML) is an aggressive form of blood cancer. Despite the use of cytotoxic standard-of-care drugs, patients often succumb to the disease partially due to the inability of medically unfit patients to withstand the cytotoxic treatments, regrowth from minimal residual disease and the chemo-resistant nature of leukemic stem cells (LSCs). Hence, novel therapies should focus on targeting the unique biology of LSCs to eliminate and avoid reoccurrence. To overcome challenges inherent of screening rare LSCs in vitro such as culturing and expanding, we optimized the conditions for a 4-week in vitro large-scale expansion (&amp;gt;600 million bulk) and enrichment of the CD34+ LSC-containing fraction (&amp;gt;90% purity) for a primary human AML sample (OCI-AML-8227), functionally validated to be enriched for LSCs in long-term xenotransplant assays (Eppert et al., 2011). Next, we performed a high-throughput screen of 11,140 chemical molecules in 3 stages. First, the viability of AML CD34+ cells and healthy cord blood (CB) CD34+ cells was read out at 1-2 doses using a CellTiter-Glo® Luminescent assay. 61 compounds had &amp;gt;70% inhibition of 8227 CD34+ cells and &amp;lt;30% inhibition on CB CD34+ cells. Next, we determined the dose response and refined the hits to 33 potent compounds with LC50 &amp;lt; 1 μM, including novel compounds and classes previously shown to target bulk and leukemic stem cells in AML. We are now presenting the follow up hits identified from the third stage of validation where we determined the LC 50 specifically in the CD34- (blast) and CD34+CD38- (LSC-enriched) OCI-AML-8227 populations using flow cytometry. We identified 25 novel anti-LSC compounds with high efficacy against CD34+CD38- AML cells (LC 50 &amp;lt; 500 nM). Venetoclax was among the top hits, a compound that revolutionized treatment for poor prognosis AML patients due to its anti-LSC activities, providing internal validation of the screen. We then tested these candidates in a second LSC-enriched model with poor prognosis, OCI-AML-20, to ensure the response of compounds on LSCs is not exclusive to OCI-AML-8227 and to investigate the influence of the microenvironment on drug efficiency. A total of 5 hits have significant responses (&amp;gt;50% reduction in LSC enriched populations) and nontoxic to stroma. Furthermore, we refined our top candidates to 7 compounds based on their efficiencies in the two LSC-enriched models (OCI-AML-20 and 8227) and low toxicity on stroma. To gain insights on their mechanisms of action in LSCs, the two models were treated with the 7 candidates and cytarabine as a control for 3 days and apoptosis was measured by flow cytometry. Out of 7 candidates, 5 induced apoptosis in the LSC-enriched fractions, suggesting elimination of LSCs through apoptosis. The remaining compounds may eliminate LSCs through different mechanisms. From these results, we focused on the three leading compounds and validated them for toxicity on CD34+ hematopoetic stem and progenitor cord blood cells (HSPCs) vs CD34+CD38- (LSC-enriched) OCI-AML-8227 by flow cytometry. Although slight toxicity was observed in HSPCs at higher doses, the LC 50 for the stem cell vs LSC-enriched populations differ by ~6-40-fold, indicating that a significantly lower concentration can be used to eradicate LSCs while sparing HSPCs (LC 50 for CD34+ population CB vs 8227; Compound A: 1558 nM vs 83 nM, Compound B: &amp;gt;2000 nM vs 305 nM and Compound C: 612 nM vs 16 nM, respectively). To functionally validate if HSPCs proliferation and differentiation was impaired and if leukemic progenitors were eradicated, a colony forming unit assay was performed for 12 days after 6 days of treatment. Candidates were found to eliminate leukemic progenitors by 50% or more, reducing leukemia initiating potential and having minimal or no impact on CB progenitor functionality. Overall, compound A is a potential candidate to move forward due to its ability to target and eliminate LSCs through apoptosis in two LSC-enriched models, its low toxicity on stroma and normal cord blood cells. This candidate is classified as an indole, a class shown by Pabst et al., 2014 to reduce CD34+CD15- AML cells in a drug screen for aryl hydrocarbon modulators, suggesting a potential mechanism for LSC elimination through apoptosis. We now aim to examine LSC eradication in a panel of genetically defined primary AMLs to be able to determine the broad applicability of this compound and translate the preliminary results for clinical use.

Development and validation of a disulfidptosis-related scoring system to predict clinical outcome and immunotherapy response in acute myeloid leukemia by integrated analysis of single-cell and bulk RNA-sequencing.

Background: Disulfidptosis is a metabolically relevant mode of cell death, and its relationship with acute myeloid leukemia (AML) has not been clarified. In this study, disulfidptosis scores were computed to examine the potential biological mechanisms. Methods: Consensus clustering was applied to detect disulfidptosis-related molecular subtypes. The least absolute shrinkage and selection operator (LASSO) regression analysis was used to construct a DRG prognostic model. Results: DRGs are upregulated in AML and associated with poor prognosis. The higher the disulfidptosis activity score, the worse the clinical outcome for patients, accompanied by increased immune checkpoint expression and tumor marker pathway activity. The two molecular subtypes exhibited distinct prognoses and tumor microenvironment (TME) profiles. A prognostic risk score model was established using six DRGs, and the AML cohort was divided into high- and low-risk score groups. Patients in the high-risk group experienced significantly worse prognosis, which was validated in seven AML cohorts. Receiver Operating Characteristic (ROC) curve analysis indicated that the area under the curve values for risk score prediction of 1-, 3-, and 5-year survival were 0.779, 0.714, and 0.778, respectively. The nomogram, in conjunction with clinicopathological factors, further improved the accuracy of prognosis prediction. The high-risk score group exhibited a higher somatic mutation frequency, increased immune-related signaling pathway activity, and greater immune checkpoint expression, suggesting a certain degree of immunosuppression. Patients with advanced age and higher cytogenetic risk also had elevated risk scores. According to drug prediction and AML anti-PD-1 therapy cohort analysis, the low-risk score group displayed greater sensitivity to chemotherapy drugs like cytarabine and midostaurin, while the high-risk score group was more responsive to anti-PD-1 therapy. Finally, clinical samples were collected for sequencing analysis, confirming that the progression of myeloid leukemia was associated with a higher risk score and a negative disulfidptosis score, suggesting that the poor prognosis of AML may be associated with disulfidptosis resistance. Conclusion: In conclusion, a systematic analysis of DRGs can help to identify potential disulfidptosis-related mechanisms and provide effective new biomarkers for prognosis prediction, TME assessment, and the establishment of personalized treatment plans in AML.

CD74 + Leukemia-Associated Macrophages Supporess T Cell Immune Surveillance and Promote Leukemic Cell Growth in Acute Myeloid Leukemia

The bone marrow microenvironment plays a pivotal role in the recurrence of acute myeloid leukemia (AML). However, the exact mechanisms are not fully understood. Single-cell technologies have emerged as powerful tools for unraveling the immune microenvironment in leukemia. In this study, utilizing scRNA-seq on a cohort of 13 paired AML patients, we have unveiled a distinctive subcluster of tumor-associated macrophages (TAMs) linked to relapse leukemia. To explore the heterogeneity of the bone marrow microenvironment during the progression of AML, we conducted a scRNA-seq analysis on bone marrow cells obtained from 13 paired AML patients at the time of diagnosis (DX) and relapse (RE) (Figure 1A). Employing UMAP for dimensionality reduction, we detected multiple subclusters and observed a notable rise in the Macrophage CD74 cluster in all paired RE patients, suggesting a pivotal role of CD74+ TAMs in AML relapse (Figure 1B-C). Furthermore, CD274, FOS, TGFβ1, and CD163, recognized as markers of M2 macrophages, were significantly enriched in the CD74+ TAMs population (Figure 1D). Intriguingly, a higher proportion of CD74+ TAMs correlated with unfavorable outcomes in TCGA database (Figure 1E). Meanwhile, we found that the ligand macrophage migration inhibitory factor (MIF) of CD74 was associated with poor prognosis of AML (Figure 1F). To investigate the association of CD74+ TAMs with AML cells, Gene Set Variation Analysis (GSVA) was employed and revealed restrained pathways related to T cell function (Figure 2A). Through in vitro experiments on patient samples, we found that CD74+TAM can inhibit the proliferation of T cells and the secretion of IFN-γ. Further monoclonal antibody block experiments unveiled that this inhibitory effect might be attributed to the heightened expression of PD-L1, TGFβ1, and IL-10 by the macrophages (Figure 2B-C). In conclusion, we have substantiated the inhibitory role of CD74+ TAMs on T cells, which hampers their ability to engage in effective tumor killing. To elucidate the interplay between CD74+ TAMs and AML cells, we examined the gene expression profile of cytokines, noting a significant increase in the expression level of IL-6 within the CD74+ TAMs subclusters of the RE group (Figure 3A). Furthermore, upon co-culture with CD74+ TAMs, THP-1 cells exhibited notable activation of IL-6 related pathways (Figure 3B). The injection of CD74+ TAMs resulted in heightened AML initiation and poorer survival in patient-derived xenograft (PDX) model, which was reversed by blocking the IL-6-IL-6R axis (Figure 3C-D). These findings collectively confirm that IL-6 secreted by CD74+ TAMs fosters the malignancy and drug resistance of AML cells. Subsequently, MLL-AF9-induced AML mouse model was employed to explore the spatial interplay between CD74+ TAMs, AML cells and T cells. Immunofluorescence (IF) analysis of mouse bone marrow tissue revealed that after chemotherapy, the proximity between AML cells and CD74+ TAMs decreased (Figure 4A-B). Conversely, the presence of T cells surrounding CD74+ TAMs increased (Figure 4C-D). These findings indicate that CD74+ TAMs can inhibit T cell function and directly influence AML cells within the tumor microenvironment. In summary, our study revealed a notable rise in the proportion of TAMs expressing CD74 in relapsed AML patients. These CD74+ TAMs were found to facilitate leukemia progression while concurrently inhibiting T cell immune surveillance in the context of AML. Thus, our findings indicate that CD74+ TAMs represent a distinct type of macrophages associated with AML relapse.