Processes In Acute Myeloid Leukemia Research Articles

Clinical Aspects and Significance of β-Chemokines, γ-Chemokines, and δ-Chemokines in Molecular Cancer Processes in Acute Myeloid Leukemia (AML) and Myelodysplastic Neoplasms (MDS).

Acute myeloid leukemia (AML) is a type of leukemia with a very poor prognosis. Consequently, this neoplasm is extensively researched to discover new therapeutic strategies. One area of investigation is the study of intracellular communication and the impact of the bone marrow microenvironment on AML cells, with chemokines being a key focus. The roles of β-chemokines, γ-chemokines, and δ-chemokines in AML processes have not yet been sufficiently characterized. This publication summarizes all available knowledge about these chemotactic cytokines in AML and myelodysplastic neoplasm (MDS) processes and presents potential therapeutic strategies to combat the disease. The significance of β-chemokines, γ-chemokines, and δ-chemokines is detailed, including CCL2 (MCP-1), CCL3 (MIP-1α), CCL5 (RANTES), CCL23, CCL28, and CX3CL1 (fractalkine). Additionally, the importance of atypical chemokine receptors in AML is discussed, specifically ACKR1, ACKR2, ACKR4, and CCRL2. The focus is on the effects of these chemokines on AML cells, particularly their influence on proliferation and resistance to anti-leukemic drugs. Intercellular interactions with non-AML cells, such as mesenchymal stem cells (MSC), macrophages, and regulatory T cells (Treg), are also characterized. The clinical aspects of chemokines are thoroughly explained, including their effect on overall survival and the relationship between their blood levels and AML characteristics.

Abstract 5588: Protein Arginine Methyltransferase 2 is involved in the control of inflammatory processes in acute myeloid leukemia

Abstract A direct link between chronic inflammation and development of Acute Myeloid Leukemia (AML) has been highlighted in the past few years, demonstrating an interconnection between marked inflammatory phenotype and aberrant myeloproliferation in AML patients. Treating AML patients exhibiting a higher inflammatory signature with anti-inflammatory molecules resulted in significant increase of overall survival. Protein Arginine Methyltransferases (PRMTs) are epigenetic factors known to regulate gene expression through methylation of histone tails. It has been previously reported that PRMT1, 4, and 5 inhibition exhibit anti-proliferative effects on AML models. In this study, we investigated the role of another PRMT, called PRMT2, in the development of AML through its regulatory roles in inflammatory pathways. We first determined from an AML cohort (The Leucegene project, IRIC, Montréal, QC, Canada) that patients with a low PRMT2 expression display an enrichment of proinflammatory pathways compared to patients with a high PRMT2 expression. Therefore, we hypothesized that PRMT2 could be a key regulator of inflammatory processes in AML. We thus used a PRMT2 knockout mouse model (Prmt2 KO) and a PRMT2 knockout human AML cell line to validate our hypothesis. Although we demonstrated no difference in the bone marrow progenitors or mature cell populations of Prmt2 KO mice compared to control, we observed that Prmt2 KO Bone-Marrow Derived Macrophages (BMDMs) are more sensitive to LPS stimulation and express higher levels of pro-inflammatory cytokines, supporting our previous findings for a role of PRMT2 in the negative regulation of inflammatory processes. PRMT2 depleted human AML cells displayed an increased pro-inflammatory signature due to overactivation of STAT3, which is caused by an enhanced activation of the NFkB signaling pathway, leading to an overproduction of IL6. Together, these findings demonstrate that PRMT2 is a key regulator of the control of inflammation in AML. Recognition of PRMT2 as a biomarker of inflammation in AML would help to adapt treatment possibly through the synergistic use of anti-inflammatory molecules with other cytotoxic drugs. Citation Format: Camille Sauter, Thomas Morin, Fabien Guidez, John Simonet, Cyril Fournier, Céline Row, Denis Masnikov, Baptiste Pernon, Anne Largeot, Aziza Aznague, Yann Herault, Guy Sauvageau, Marc Maynadie, Mary Callanan, Jean-Noël Bastie, Romain Aucagne, Laurent Levadny Delva. Protein Arginine Methyltransferase 2 is involved in the control of inflammatory processes in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5588.

AAT: does it have a place in acute leukemia?

Aim: In this study, the authors aimed to investigate the change of AAT, its effect on the response to induction and its effects on the treatment process in acute myeloid leukemia and acute lymphoblastic leukemia patients. Materials & methods: This study included 94 patients who were hospitalized and followed up inIstanbul Training and ResearchHospital, Hematology Clinic, between October 2019 and December 2021. Results: Patients with a complete response had higher serum AAT levels than those with a non-complete response (p<0.05). The mean serum AAT level was found to be significantly higher in patients without Gram-positive growth than in patients with Gram-positive growth. Conclusion: It can be thought that AAT can play a role during the course of acute leukemia management.

Construction of a prognostic model of acute myeloid leukemia associated with immunogenic cell death

ABSTRACT Background Immunogenic cell death (ICD)is a kind of regulatory cell death, which causes a series of antigen-specific adaptive immune responses by generating and emitting some danger signals or damage-associated molecular patterns (DAMPs). At present, little is known about the prognostic value of ICD and its related processes in acute myeloid leukemia (AML). The aim of the study was to explore the relationship between ICD and tumor immune microenvironment changes in AML. Research Design & Methods In the study, AML samples were divided into two groups by consensus clustering analysis, and then gene enrichment analysis and GSEA analysis were performed on the ICD high expression group. Furthermore, CIBERSORT was used to analyze the tumor microenvironment and immune characteristics of AML. Finally, a prognostic model related to ICD was constructed by using univariate and multivariate regression analysis. Results ICD was divided into two groups according to the level of ICD gene expression. The ICD high expression group was associated with good clinical results and high levels of immune cell infiltration. Conclusions The study constructed and verified the prognostic characteristics of AML related to ICD, which has important value in predicting the overall survival time of AML patients.

TET2 deficiency sensitizes tumor cells to statins by reducing HMGCS1 expression.

TET2 (ten-eleven-translocation) protein is a Fe(II)- and α-ketoglutarate-dependent dioxygenase that catalyzes DNA demethylation to regulate gene expression. While TET2 gene is frequently mutated in hematological cancer, its enzymatic activity is also compromised in various solid tumors. Whether TET2 deficiency creates vulnerability for cancer cells has not been studied. Here we reported that TET2 deficiency is associated with the change of lipid metabolism processes in acute myeloid leukemia (AML) patient. We demonstrate that statins, the inhibitors of β-Hydroxy β-methylglutaryl-CoA (HMG-CoA) reductase and commonly used cholesterol-lowering medicines, significantly sensitize TET2 deficient tumor cells to apoptosis. TET2 directly regulates the expression of HMG-CoA synthase (HMGCS1) by catalyzing demethylation on its promoter region, and conversely TET2 deficiency leads to significant down-regulation of HMGCS1 expression and the mevalonate pathway. Consistently, overexpression of HMGCS1 in TET2-deficient cells rescues statin-induced apoptosis. We further reveal that decrease of geranylgeranyl diphosphate (GGPP), an intermediate metabolite in the mevalonate pathway, is responsible for statin-induced apoptosis. GGPP shortage abolishes normal membrane localization and function of multiple small GTPases, leading to cell dysfunction. Collectively, our study reveals a vulnerability in TET2 deficient tumor and a potential therapeutic strategy using an already approved safe medicine.

P472: THE FLT3-ITD RECEPTOR CAN PROMOTE PROTEIN FOLDING IN THE ENDOPLASMIC RETICULUM THROUGH THE OXIDOREDUCTASE ERO1

Background: Acute myeloid leukemia (AML) is an aggressive hematologic malignancy and the most common myeloid tumour in adults. Internal tandem duplications (ITD) in FLT3 receptor appear in 30% of cases and is associated with poor disease outcome. This receptor is synthesized and processed in the endoplasmic reticulum (ER) before being transported to the plasma membrane, where it is activated by ligand binding. Upon activation, it regulates cell differentiation, proliferation, and survival. Mutations in FLT3, such as ITD mutations, not only constitutively activate this receptor, but also prevent its correct processing in the endoplasmic reticulum. Therefore, it is mostly retained in this organelle. Aims: We decided to study if the aberrant accumulation of FLT3-ITD affects the functioning of the ER. Methods: Experiments were performed on wild-type and FLT3-ITD AML cell lines, as well as on stably transfected FLT3 and FLT3-ITD Ba/F3 cell lines. Protein aggregation, reactive oxygen species (ROS) and glutathione (GSH) levels were determined by Thioflavin T, DCFH-DA and Thioltracker Violet fluorescence, respectively. Protein detection was performed by Western Blot. Cell viability and death was analysed by cell count and Trypan Blue exclusion. Results: Protein folding in the ER generates reactive oxygen species (ROS). Antioxidant molecules such as GSH are needed to maintain redox homeostasis. We found that FLT3-ITD cell lines have lower levels of protein aggregates, as well as ROS and GSH. This data could suggest that mutant cell lines have more efficient protein folding mechanisms. In fact, among the ER proteins that are involved in the protein folding process, we found the oxidoreductase ERO1 to be increased in FLT3-ITD cell lines. Supporting our findings, we saw that FLT3-ITD cell lines were less sensitive to protein folding blocking agents, DTT and 2-mercaptoethanol. This was reflected by a slight decrease in cell proliferation compared to wild-type cell lines. We confirmed that the decrease in cell viability in FLT3-ITD cell lines was not accompanied by cell death, as opposed to wild-type cell lines. Under ER stress conditions, such as an accumulation of misfolded proteins in the ER, the unfolded protein response (UPR) is activated. We decided to study if there were differences in the basal levels of UPR by western blot. We found FLT3-ITD cell lines to have lower levels of UPR, reflected by lower protein expression of the active forms of PERK and IRE (p-PERK and p-IRE respectively), together with the protein BiP, the major chaperone found in the ER and binds to misfolded proteins. Altogether, our data suggest that FLT3-ITD cell lines have less ER stress, reflected by lower levels of chaperones and UPR proteins, possibly because they rely on more efficient protein folding mechanisms. To confirm the importance of protein folding process in acute myeloid leukemia, we tested the effect of ERO1 inhibitor (EN460), and found it to have a greater impact in FLT3-ITD cell lines. In order to determine a possible relation between the ITD mutation in the FLT3 receptor and ERO1, we used stably transfected Ba/F3 cell lines carrying the normal or mutated FLT3 receptor. We found ERO1 to be overexpressed on FLT3-ITD Ba/F3 cell lines. Summary/Conclusion: Current AML treatments are mainly based on the administration of FLT3 inhibitors, with limited success. Our data suggest that FLT3-ITD cell lines, with greater resistance to treatments, have more efficient protein folding mechanisms. This process should be studied in more detail to determine its potential as a therapeutic target for AML treatment.

PF230 THE RNA HELICASE DDX17 PROTECTS ACUTE MYELOID LEUKEMIA CELLS AGAINST DNA DAMAGE

Background:Alternative splicing (AS) is essentially regulated by RNA‐binding proteins (RBPs) and is one of many deregulated cellular processes in Acute Myeloid Leukemia (AML). The alternative usage of exons substantially increases the coding potential of mRNAs and frequently leads to the expression of tumor‐promoting mRNA variants in cancer modulating apoptosis, proliferation, invasion and migration of cancer cells. This underlines the great plasticity of AS in cancer and justifies the need to elucidate the function of nuclear RBPs in specific cancer types. We found that the RNA helicase DDX17 is significantly more highly expressed in primary human AML specimens with high versus low Leukemia Stem Cell (LSC) frequency.Aims:In this study, we aimed to understand the functional role of DDX17 in normal hematopoietic stem and progenitor cells (HSPCs) and in AML cells. In particular, we sought to determine whether DDX17 exerts its function in the hematopoietic system primarily via AS or AS‐unrelated mechanisms.Methods:We performed knockdown (KD) experiments using small hairpin (sh) RNAs to determine whether DDX17 was essential for primary human cord blood CD34+ HSPCs and leukemia cells. Furthermore, mRFP tagged DDX17 was overexpressed through lentiviral transduction to track protein localization by immunofluorescence (IF) imaging. Lentivirally transduced HSPCs and leukemia cells were analyzed in vitro for proliferative output, viability, immunophenotype, and colony formation potential. RNA sequencing (RNA‐Seq) was used to determine how DDX17 KD affected gene expression and alternative exon usage. Pull down experiments combined with mass spectrometry revealed binding partners in presence and absence of chemotherapeutic agents. Finally, we performed homologous recombination (HR) and drug sensitivity assays to determine the role of DDX17 in DNA damage.Results:Knockdown of DDX17 induced DNA damage and severely impaired the function of healthy HSPCs and TP53 wild type AML cells. TP53 null cells maintained their proliferative capacity during short term in vitro expansion in absence of DDX17, but were significantly sensitized towards DNA damaging agents. In line, overexpression of DDX17 protected the cells against double strand break (DSB) inducing drugs. IF imaging revealed direct recruitment of DDX17 to DNA damage sites, while RNA‐Seq showed suppression of multiple DNA repair genes.Summary/Conclusion:RNA‐Seq, IF imaging, proteomics, and HR assays suggest an essential role of DDX17 in DNA repair via different mechanisms including direct recruitment to DNA damage sites, alternative splicing, and regulation of DNA repair genes on the transcriptional level.

CREB Controls C/Ebpd to Impair Myeloid Differentiation Process in Acute Myeloid Leukemia

Purpose. The cAMP response element binding protein (CREB) is a transcription factor documented to be crucial for normal and neoplastic hematopoiesis. Its overexpression has already been demonstrated to impair myelopoiesis and to aberrantly control cell proliferation, apoptosis and cell cycle progression, both in vitro and in vivo. Its protein overexpression has been found in patients with acute myeloid leukemia (AML) contributing to decrease survival. CREB guides the expression of more than 5000 different targets in a tissue/cell-type specific fashion, and to date its main network towards leukemogenesis remains unknown. Here we aim to identify through which genes CREB triggers AML by using a CREB overexpressing transgenic zebrafish which developed AML in adulthood.Patients and methods. We performed gene expression profile of RNA from the kidney marrow of 14-months old CREB-zebrafish (n=5) and control zebrafish (n=5). Principal component analysis was performed using Partek Genomic Suite software to integrate zebrafish AML signature with human signature found in pediatric AML to find common CREB target genes. AML cell lines were used to validate CREB targets in vitro.Results. By GEP analysis and integration data we found 20 differentially expressed genes in both the zebrafish and human leukemia. Several of them were involved in the myeloid differentiation process, such as JUN, FOS and C/EBPδ. We confirmed that C/EBPδ protein levels highy correlated with phosphoCREB levels in zebrafish tumor as well as in a cohort of 66 pediatric patients with AML (r=0.79). We silenced and overexpressed CREB in AML or healthy bone marrow primary cultures respectively, and found that C/EBPδ was controlled by CREB transcriptional activity. We took advantages of a previously created HL60(CREB-) cell line, where CREB protein levels was absent compared to the control cell line called HL60(CREB+), where CREB was overexpressed. HL60(CREB-) cell line expressed lowered C/EBPδ levels compared to HL60(CREB+). To investigate CREB's role on differentiation program, cell lines were treated with all-trans-retinoic acid (ATRA). The evaluation of CD11b expression revealed that HL60(CREB-) differentiated to a higher extent compared to HL60(CREB+).Enhanced myeloid differentiation was also confirmed by cell morphology examination. Then we silenced C/EBPδ gene in HL60(CREB+) cell line and treated with ATRA, confirming that C/EBPδ lowered expression increased CD11b expression, phenocopying the HL60(CREB-) behavior.Conclusion. These results indicated that C/EBPδ is a CREB target crucial for myeloid differentiation. The new role discovered for CREB proto-oncogene in blocking myeloid differentiation process opens for further opportunities with differentiation agents to cure AML. DisclosuresNo relevant conflicts of interest to declare.

RA-inducible gene-I induction augments STAT1 activation to inhibit leukemia cell proliferation

RA-inducible gene I (RIG-I/DDX58) has been shown to activate IFN-β promoter stimulator 1 (IPS-1) on recognizing cytoplasmic viral RNAs. It is unclear how RIG-I functions within the IFN and/or RA signaling process in acute myeloid leukemia (AML) cells, however, where obvious RIG-I induction is observed. Here, we show that the RIG-I induction functionally contributes to IFN-α plus RA-triggered growth inhibition of AML cells. Interestingly, although RIG-I induction itself is under the regulation of STAT1, a major IFN intracellular signal mediator, under circumstances in which it does not stimulate IPS-1, it conversely augments STAT1 activation to induce IFN-stimulatory gene expression and inhibit leukemia cell proliferation. Thus, our results unveil a previously undescribed RIG-I activity in regulating the cellular proliferation of leukemia cells via STAT1, which is independent of its classic role of sensing viral invasion to trigger type I IFN transcription.

Sequential Acquisition of Somatic Mutations In Progenitors with Differential Proliferative Potential In Human Acute Myeloid Leukemia

AbstractAbstract 755 Background:Evidence suggests that leukemic transformation is a multistep process in acute myeloid leukemia (AML). However, the cell of origin for human leukemogenesis remains ill defined. Experimental systems with murine hematopoetic cells indicate that not only immature multipotent hematopoietic stem cells but also more mature, committed myeloid precursors can be a target of murine AML transformation. These findings suggest that requisite mutations for leukemogenesis may be acquired sequentially in myeloid progenitors with differential proliferative potential. To investigate this possibility in human AML, we studied primary specimens from pediatric patients who had previously been identified as having 2 distinct, disease-relevant somatic mutations. Methods:Diagnostic bone marrow or peripheral blood specimens from pediatric AML patients with two distinct genomic alterations were obtained from Children's Oncology Group (COG) AML Reference Laboratory. Early myeloid CD34+/CD33- progenitors and more differentiated CD34+/CD33+ progenitors were isolated using fluorescence-activated cell sorting (FACS). Twenty-eight of 43 patient specimens had FACS-isolated CD34+/CD33- progenitor cells and molecular data available for both somatic alterations, and were included in this study. The genomic alterations included those with core-binding factor (CBF) translocations and FLT3/ITD (n=5), CBF translocations and KIT abnormalities (n=19), or nucleophosmin (NPM1) mutations and FLT3/ITD (n=4). Aliquots of sorted subpopulations were subjected to direct colony forming cell (CFC) assays. Freshly sorted cells and resulting colony forming units-granulocyte and/or monocyte (CFU-GM) were evaluated for the presence of the patient-specific genomic alterations. Results:In 20/28 specimens (71.4%), both mutations were detected in CD34+/CD33- cells. By comparison, in 6 (21.4%) patient specimens, the receptor tyrosine kinase (FLT3/ITD or KIT) alteration was not detected in CD34+/CD33- cells, whereas in 2 (7.1%) specimens, CD34+/CD33- cells did not harbor either of the two patient-specific alterations. In contrast to these less mature CD34+/CD33- progenitor cells, analysis of more differentiated CD34+/CD33+ progenitors revealed both genomic alterations in 26/28 samples. For the remaining 2 samples, only the CBF mutation was detected. FACS-isolated CD34+/CD33- cells from 22 specimens were subjected to CFC assay. There was sufficient colony growth and evaluable molecular data for the paired mutations for 7/22 samples. Within these specimens, the majority (43 of 60) of the evaluable CFU-GM demonstrated the CBF or NPM1 abnormality but lacked the RTK mutation; in the remaining colonies, both somatic alterations were detected (10 of 60), or neither mutation was detected (7 of 60). This finding suggests that the majority of CFU-GM analyzed were derived from an abnormal cell that had not yet acquired the requisite RTK mutation necessary for frank leukemic transformation. Conclusion:These findings suggest the potential for sequential acquisition of RTK mutations in some cases of pediatric CBF or NPM1+ AML. Specifically, the presumptive class I mutation confering a proliferative advantage (e.g. the receptor tyrosine kinase mutation) appears to arise, in some instances, in a more committed myeloid progenitor with limited proliferative potential. Disclosures:No relevant conflicts of interest to declare.

Increased angiogenesis and Fas-ligand expression are independent processes in acute myeloid leukemia

We evaluated the clinical significance of tumor angiogenesis and Fas-ligand (FasL) expression using parameters including the microvessel count (MVC), vascular endothelial growth factor (VEGF) level, and FasL expression in patients with acute myeloid leukemia (AML). Paraffin-embedded bone marrow (BM) sections from 43 AML patients at diagnosis, 20 patients after subsequent induction therapy, and 18 controls with non-invasive lymphoma were stained immunohistochemically for von Willebrand factor (vWF) and FasL. VEGF in BM mononuclear cells from 32 AML patients at diagnosis and 10 controls, including bone marrow transplantation donors, was assayed by an ELISA method. We found that the mean MVC, VEGF level, and FasL expression in AML patients at diagnosis were significantly higher than those of controls, with a significant correlation between the MVC and VEGF levels ( r=0.43). However, there were no correlations between FasL expression and MVC or VEGF level. The mean MVC and FasL expression after induction therapy were lower than those evaluated at diagnosis, but were higher than those of controls. There was a correlation between the MVC and percentage of BM blasts ( r=0.479), but no correlation between the MVC, VEGF level, or FasL expression and other hematologic or clinical variables. Our findings provide evidence of increased angiogenesis and tumor immune escape in AML, and both angiogenesis and tumor immune escape are independent processes in AML.