Acute Myeloid Leukemia Malignancies Research Articles

IRX-related homeobox gene MKX is a novel oncogene in acute myeloid leukemia.

Homeobox genes encode transcription factors which organize differentiation processes in all tissue types including the hematopoietic compartment. Recently, we have reported physiological expression of TALE-class homeobox gene IRX1 in early myelopoiesis restricted to the megakaryocyte-erythroid-progenitor stage and in early B-cell development to the pro-B-cell stage. In contrast, sister homeobox genes IRX2, IRX3 and IRX5 are aberrantly activated in the corresponding malignancies acute myeloid leukemia (AML) and B-cell progenitor acute lymphoid leukemia. Here, we examined the role of IRX-related homeobox gene MKX (also termed IRXL1 or mohawk) in normal and malignant hematopoiesis. Screening of public datasets revealed silent MKX in normal myelopoiesis and B-cell differentiation, and aberrant expression in subsets of AML and multiple myeloma (MM) cell lines and patients. To investigate its dysregulation and oncogenic function we used AML cell line OCI-AML3 as model which strongly expressed MKX at both RNA and protein levels. We found that IRX5, JUNB and NFkB activated MKX in this cell line, while downregulated GATA2 and STAT5 inhibited its expression. MKX downstream analysis was conducted by siRNA-mediated knockdown and RNA-sequencing in OCI-AML3, and by comparative expression profiling analysis of a public dataset from MM patients. Analysis of these data revealed activation of CCL2 which in turn promoted proliferation. Furthermore, MKX upregulated SESN3 and downregulated BCL2L11, which may together underlie decreased etoposide-induced apoptosis. Finally, myeloid differentiation genes CEBPD and GATA2 were respectively up- and downregulated by MKX. Taken together, our study identified MKX as novel aberrantly expressed homeobox gene in AML and MM, highlighting the function of IRX1 in normal myelopoiesis and B-cell development, and of IRX-related genes in corresponding malignancies. Our data merit further investigation of MKX and its deregulated target genes to serve as novel markers and/or potential therapeutic targets in AML patient subsets.

Germline Variants and Characteristic Features of Hereditary Hematological Malignancy Syndrome.

Due to the proliferation of genetic testing, pathogenic germline variants predisposing to hereditary hematological malignancy syndrome (HHMS) have been identified in an increasing number of genes. Consequently, the field of HHMS is gaining recognition among clinicians and scientists worldwide. Patients with germline genetic abnormalities often have poor outcomes and are candidates for allogeneic hematopoietic stem cell transplantation (HSCT). However, HSCT using blood from a related donor should be carefully considered because of the risk that the patient may inherit a pathogenic variant. At present, we now face the challenge of incorporating these advances into clinical practice for patients with myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML) and optimizing the management and surveillance of patients and asymptomatic carriers, with the limitation that evidence-based guidelines are often inadequate. The 2016 revision of the WHO classification added a new section on myeloid malignant neoplasms, including MDS and AML with germline predisposition. The main syndromes can be classified into three groups. Those without pre-existing disease or organ dysfunction; DDX41, TP53, CEBPA, those with pre-existing platelet disorders; ANKRD26, ETV6, RUNX1, and those with other organ dysfunctions; SAMD9/SAMD9L, GATA2, and inherited bone marrow failure syndromes. In this review, we will outline the role of the genes involved in HHMS in order to clarify our understanding of HHMS.

Deciphering Acute Myeloid Leukemia Associated Transcription Factors in Human Primary CD34+ Hematopoietic Stem/Progenitor Cells.

Hemato-oncological diseases account for nearly 10% of all malignancies and can be classified into leukemia, lymphoma, myeloproliferative diseases, and myelodysplastic syndromes. The causes and prognosis of these disease entities are highly variable. Most entities are not permanently controllable and ultimately lead to the patient's death. At the molecular level, recurrent mutations including chromosomal translocations initiate the transformation from normal stem-/progenitor cells into malignant blasts finally floating the patient's bone marrow and blood system. In acute myeloid leukemia (AML), the so-called master transcription factors such as RUNX1, KMT2A, and HOX are frequently disrupted by chromosomal translocations, resulting in neomorphic oncogenic fusion genes. Triggering ex vivo expansion of primary human CD34+ stem/progenitor cells represents a distinct characteristic of such chimeric AML transcription factors. Regarding oncogenic mechanisms of AML, most studies focus on murine models. However, due to biological differences between mice and humans, findings are only partly transferable. This review focuses on the genetic manipulation of human CD34+ primary hematopoietic stem/progenitor cells derived from healthy donors to model acute myeloid leukemia cell growth. Analysis of defined single- or multi-hit human cellular AML models will elucidate molecular mechanisms of the development, maintenance, and potential molecular intervention strategies to counteract malignant human AML blast cell growth.

Dissecting Lineage and Cell State-Specific Epigenetic Evolution in Relapsed Acute Myeloid Leukemia

Introduction: The prognosis for relapsed Acute Myeloid Leukemia (AML) following initial treatment and remission remains poor, largely due to a limited number of therapies with proven clinical efficacy. Epigenetic states and evolution are known to contribute to the initiation of AML, however, the role of epigenetic evolution in relapse remains understudied, and the effects of lineage-specific epigenetic changes at relapse are unknown. We hypothesized that epigenetic modifications confined to specific lineages or differentiation states may contribute to disease resistance and relapse. To test this, we generated single cell ATACseq data from four AML patients at initial diagnosis and at relapse post-induction chemotherapy. We utilized mapping of AML scATACseq to scATAC-derived healthy hematopoietic chromatin states, lineage trajectory analysis, and gene and transcription factor accessibility analysis to identify epigenetic changes occurring at discrete points in different AML differentiation trajectories. Methods: Malignant AML cells were purified from paired samples collected from four patients at diagnosis and relapse via FACS and were sequenced using the 10x chromium scATAC platform. ArchR, Monocle3, and custom R scripts were used for all analyses. AML cell scATAC data was mapped to a healthy hematopoietic scATAC reference using Latent Semantic Indexing (LSI) and projection onto a single cell ATAC-seq manifold derived from healthy hematopoietic cell types. Closest healthy cell types were identified for each AML cell, differentiation trajectories were generated using monocole3, and gene and transcription factor accessibility was quantified across the different AML differentiation states and lineages. Results: Our analysis showed varying patterns of lineage bias across AMLs. Some samples exhibited changes in differentiation state at relapse, becoming either less differentiated or less myeloid-like. Analysis of differences in TF activity between diagnosis and relapse within myeloid and lymphoid trajectories revealed lineage-specific and differentiation state-specific changes in TF activity. In patient SU360, overall differentiation states did not change, and AML cells at both diagnosis and relapse were primarily myeloid-like with a smaller lymphoid-like population. In this sample, GATA family TFs, which were active in progenitor-like cells at diagnosis, became significantly more active in terminally differentiated AML cells at relapse. No specific chromatin changes were observed in lymphoid-like AML cells at relapse. In patient SU484, where diagnosis cells were myeloid-differentiated and relapse cells were more progenitor-like, GATA and RUNX family TF activity, which was restricted to progenitor cells at diagnosis, showed broad activation in all cells at relapse, aligning with the overall de-differentiation observed in the AML. In SU142, a case with no significant change in overall chromatin or differentiation state at relapse, multiple SP family transcription factors showed significant activation specifically in progenitor-like AML cells at relapse. Conclusion: Our study of four distinct AMLs suggests the existence of lineage-specific epigenetic evolution in some relapsed AMLs. We found that epigenetic activation of specific transcription factors, such as GATA and RUNX, was restricted to certain steps of myeloid differentiation, suggesting that epigenetic changes contributing to resistance and relapse are relevant in specific AML lineages and differentiation states. Notably, we found that even in AMLs where overall epigenetic state remains stable, there is activation of specific TFs (e.g. SP1) in progenitor cells at relapse, consistent with literature demonstrating SP family TF relevance in Leukemic Stem Cell (LSC) drug resistance. These observations suggest that epigenetic modifications in less-differentiated AML cells may reflect changes occurring in LSCs, potentially contributing to resistance and relapse. Our study is limited due to a small sample size that precludes broad generalizations, but our findings provide a foundation for further characterization of AML relapse epigenetics. Ultimately, these findings suggest the existence of specific epigenetic changes at certain points in the AML differentiation hierarchy that contribute to relapse, warranting further exploration of cell state-specific epigenetic evolution.

A Rapid Test to Evaluate the Tumor Differentiation State of Acute Myeloid Leukemias

Introduction: The combination of the hypomethylating agent Azacitidine with Venetoclax, a BCL2 inhibitor (Aza+Ven), is the frontline therapy for patients with acute myeloid leukemia (AML) who are elderly or unfit for intensive chemotherapy. However, identifying responsiveness to Aza+Ven a priori remains a clinical challenge.Aza+Ven effectively targets the primitive leukemia stem cell population (Pollyea, 2018), whereas resistance is observed in differentiated tumors (Pei, 2020). Further insights into the hierarchical organization of hematopoiesis have led to a definition of malignant AML cell types that follow the progression from hematopoietic stem cell to myeloid differentiated cells based on gene expression signatures, and these have been used to classify AML tumors (van Galen, 2019). Classification schemes using gene expression to capture variations in leukemic composition represent key biomarkers of response to targeted agents (Zeng, 2022; Bottomly, 2022). However, the prompt availability of nuanced cell state signatures to guide clinical decision making is limited by the laborious nature of performing and analyzing RNA-sequencing studies. Rapid AML stratification based on these classifications permits the possibility of identifying patients most likely to benefit from a drug or combination. Methods: To classify cell states based on transcriptomic signatures, we developed two assay formats, involving custom Nanostring and TaqMan Array card panels, for evaluation of tumor cell states for AML patients. Both formats contained gene sets representative of the six hematopoietic lineage states (van Galen, 2019) and both provide outputs that are compatible with the timeline for clinical decision-making. Assays were run according to the manufacturers' specifications, and enrichment of each of the six gene signatures was assessed using the Singscore stable method (Foroutan, 2018). Banked AML patient samples with matched RNA-seq and ex vivo Aza+Ven drug sensitivity data were used to validate the assay formats. Data obtained from the rapid assays were validated against cell states determined by deconvolution of bulk RNA sequence and compared with respect to clinical immunophenotype data where available. Results : Both the Nanostring and quantitative PCR-based assay formats detected gene expression representative of the appropriate cell state as previously determined by bulk RNA sequencing (Fig. 1A). Correlation of Aza+Ven ex vivo sensitivity with tumor cell state signatures revealed patterns where primitive but not differentiated cell states associated with sensitivity. In contrast, RNA isolated from healthy donor samples showed expression of all six cell state signatures. Across a cohort of 50 primary AML patient specimens, enrichment scores for each gene set correlated well with those derived for the same patients using deconvolution of bulk RNA-seq (Pearson r values: 0.785 to 0.944). Cell state characterization by rapid gene expression assays also aligned well with clinical immunophenotype, a routinely available diagnostic characterization of tumor blasts. Surface expression of primitive (CD117) and mature (CD11b) markers associated with Aza+Ven ex vivo sensitivity and resistance overall, respectively (p=0.03 & 0.04); however, ex vivo sensitivity could be significantly further stratified among samples expressing these markers when considering the gene expression-based enrichment scores for progenitor- and monocyte-like gene signatures (low vs high p-values: 0.02-0.05) (Fig. 1B). Analysis of matched samples from individual patients before and after treatment also showed cell state signature changes that reflected their temporal disease progression and correlated with differences in ex vivo drug sensitivity and clinical immunophenotype. Conclusion:Incorporation of cell state into prospective evaluations of AML patients provides an early indication of responsiveness to Aza+Ven. With rapid turnaround, assays such as these may augment treatment stratification and improve patient outcomes. These assays offer additional versatility to detect changes in AML cells after forced differentiation or acquired drug resistance.

Seventeen-Gene Signature Identifies AML Cells in Single Cell RNA Sequencing Data and Resolves Treatment Resistant Subtypes

Background: Pediatric acute myeloid leukemia (AML) is a highly heterogeneous malignancy with disparate risk profiles between subtypes. While the advent of single-cell technologies enabled granular evaluation of disease biology, differentiation of malignant cells from healthy analogs remains challenging. Reliable identification of malignant cells has the potential to enhance transcriptomic characterization of high-risk AML subtypes. Methods: We applied Support Vector Machine (SVM) learning to four single-cell RNA sequencing (scRNAseq) datasets (101 bone marrow biopsies: 75 diagnoses, 15 post-treatment, 11 healthy controls) containing 204,836 cells to develop a geneset for discrimination of malignant and non-malignant cells. AML cells were identified based on canonical gene expression, patient occupancy scoring, clustering relative to healthy bone marrow, and inferred alterations (inferCNV) for known copy number variants. AML subtype clusters that persisted in end-of-induction (EOI) and relapse were correlated with clinical outcomes and underwent differential expression to identify high-risk transcriptomic profiles. High-risk subtypes were validated by the application of characteristic gene signatures to large bulk RNA sequencing datasets from the Therapeutically Applicable Research to Generate Effective Treatments initiative and National Center for Biotechnology Information Gene Expression Omnibus. Results: SVM classification using a 17-geneset discriminated malignant from non-malignant cells with areas under the curve from 0.84-0.95 (ARMH1, CLEC11A, NREP, AZU1, PRAME, IFITM2, CFD, DUSP6, SCN3A, CD163, SOX4, IFI30, HOXA9, CD44, SRGN, UBE2C, CXCL8). Malignant cells grouped into 1-5 clusters of monocyte-like, hematopoietic-stem-cell-like (HSC), granulocyte-monocyte-progenitor-like (GMP) AML cells. One HSC-like and one monocyte-like AML cluster persisted through treatment with both a > 1.5-fold proportional enrichment at EOI or relapse and at least a 50% clinical relapse rate. Differential expression (log 2FC>1, adjusted p<0.05) yielded characteristic gene-sets (HSC: 20 genes, Mono: 56 genes). The high-risk HSC subtype showed increased expression of genes associated with chemoresistance (CD69, Log 2FC: 1.46, adjusted p = 3.48x10 -50), angiogenesis (MDK: Log 2FC = 1.04, adjusted p = 9.25 x 10 -50), and poor survival (BCL3: Log 2FC = 1.18, adjusted p = 1.00 x 10 -47). Patients with similar transcriptomes in bulk RNAseq had worse overall survival with hazard ratios from 1.31-3.35 across datasets (Cox PH p-values 4.38x10 -2 to 7.20x10 -3). Conclusions: These findings illustrate that malignant AML cells can be differentiated from healthy analogs solely based on gene expression. Application of our 17-geneset revealed two high-risk transcriptomic profiles, with the HSC subtype having particularly poor outcomes. From this foundation, future work will identify malignant transcriptomic changes amenable to established chemotherapeutics.

Ceefourin-1, a MRP4/ABCC4 inhibitor, induces apoptosis in AML cells enhanced by histamine

BackgroundCeefourin-1 is a specific MRP4/ABCC4 inhibitor with potential antileukemic activity. In this study, we evaluate the ability of ceefourin-1 alone or in combination with histamine, an approved antileukemic agent, to induce cell differentiation or apoptosis in human acute myeloid leukemic cells. We also examine ceefourin-1 toxicity in mice. MethodsU937, HL-60, and KG1a cells were used as models for human acute myeloid leukemia. Cyclic AMP efflux was estimated by measuring intracellular and extracellular cAMP levels. Cell differentiation was assessed by levels of CD14 and CD11b by FACS, and CD88 by western blot, and by cell morphology. Apoptosis was evaluated by cleavage of caspase-3 and PARP by western blot, and by annexin V binding assay. Subacute toxicity study of ceefourin-1 was carried out in BALB/c mice. ResultsCeefourin-1 inhibits cAMP exclusion in AML cells and promotes intracellular signaling via CREB. Ceefourin-1 leads AML cells to apoptosis and histamine potentiates this effect, without evidence of cell differentiation. Intraperitoneal administration of ceefourin-1 shows no important alterations in mice blood parameters, hepatic, and renal functions, nor signs of histologic damage. ConclusionsThese results show that ceefourin-1 promotes apoptosis in AML cells that is enhanced by histamine.General significance:This work indicates that ceefourin-1 represents a promising molecule that could be used alone or in combination with histamine for in vivo evaluation in acute myeloid leukemia malignancies.

Single-Cell Profiling of Acute Myeloid Leukemia Identified ARMH1, a Novel Protein Associated with Proliferation, Migration, and Drug Resistance

Despite advancements in treatments, half of pediatric acute myeloid leukemia (AML) patients relapse (Chen, J. & Glasser, C.L., 2020), making it pertinent to identify novel biomarkers that can be used for better stratification of patients post end of induction (EOI) therapy as well as to develop targeted therapies that could lead to higher clinical remission (CR) rates. Single-cell RNA sequencing (scRNAseq) analysis has revolutionized cancer research by identifying novel cell types, genes, and pathways. Our group used scRNAseq analysis of pediatric AML bone marrow (BM) samples collected at diagnosis (Dx) and EOI to identify the upregulation of multiple genes including a novel gene, Armadillo like helical domain containing 1 (ARMH1)/C1orf228, in cancerous blast cells (Thomas, B.E. et al., 2020). ARMH1 has not been previously associated with AML. Also, ARMH1 expression was observed to be significantly higher at Dx in samples from patients with relapse compared to samples from patients with CR (P<2.2e-16). Independent validation for ARMH1 blast-association using single cell and bulk transcriptome profiling revealed that ARMH1 expression was significantly higher in scRNAseq data of AML Dx blast cells compared to EOI non-blast cells (fold change, FC=1.8, P<2.2e-31) as well as in bulk RNAseq data of Dx samples with high blast% (>90%) compared to EOI samples (FC=1.8, P=.003) (Ulukaya, G. et al., 2021). Further analysis on other leukemias like T cell-acute lymphocytic leukemia (T-ALL) and mixed phenotypic acute leukemia (MPAL) also revealed higher expression of ARMH1 in the blast cells as compared to non-blast cells. Survival analysis on bulk RNAseq data using the survival genie platform (Dwivedi, B. et al., 2022) revealed that higher expression of ARMH1 was significantly associated with poor overall survival (OS) in both AML (P=.003) (Ulukaya, G. et al., 2021) and T-ALL (P=.014) datasets. Comparative gene expression analysis using the the Pediatric Single Cell Cancer Atlas - PedSCAtlas - tool (Mumme, H.L. et al., 2021), revealed negligible expression of ARMH1 in normal lymphoid, myeloid, and erythroid cell lineages, further confirming the leukemic blasts-specific over-expression of ARMH1. ARMH1 encodes a cytosolic protein of yet unknown function. The malignant blast specificity of ARMH1 suggested a role in leukemic cells' survival/maintenance. To ascertain its functional involvement in cancer cells, ARMH1 was silenced using siRNAs in both normal and AML cell lines. Through in vitro assays, we demonstrate that ARMH1 silencing significantly reduced leukemic cells' capacity to proliferate (P=.0041) and migrate (P=.0001). We also observed slightly increased sensitivity to chemotherapy drug, Cytarabine in the ARMH1 silenced cancer cells compared to control cancer cells in which the gene is fully functional. However, suppression of ARMH1 expression had no effect on cell growth in the normal HEK293t cell line (P=0.913). On the other hand, overexpression of ARMH1 via transient transfection of ARMH1 gene into the AML cell line, made the cells more resistant to Cytarabine and significantly increased both migration (P=.041) and cell proliferation (P=.032). In conclusion, we demonstrate for the first time that ARMH1, a still poorly understood protein, contributes to the increased proliferation and migration of malignant AML cells. With specific overexpression in blast cells of several pediatric leukemias and the newly discovered roles in proliferation, migration, and chemotherapy resistance, ARMH1 may be a good candidate for targeted treatment of pediatric leukemias.

A canthin-6-one derivative induces cell death by apoptosis/necroptosis-like with DNA damage in acute myeloid cells.

Natural products have long been considered a relevant source of new antitumor agents. Despite advances in the treatment of younger patients with acute myeloid leukemia (AML), the prognosis of elderly patients remains poor, with a high frequency of relapse. The cytotoxicity of canthin-6-one alkaloids has been extensively studied in different cell types, including leukemic strains. Among the canthin-6-one analogs tested, 10-methoxycanthin-6-one (Mtx-C) showed the highest cytotoxicity in the malignant AML cells Kasumi-1 and KG-1. Thus, we evaluated the cytotoxicity and cell death mechanisms related to Mtx-C using the EC50 (80µM for Kasumi-1 and 36µM for KG-1) treatment for 24h. Our results identify reactive oxygen species production, mitochondrial depolarization, annexin V-FITC/7-AAD double staining, caspase cleave and upregulation of mitochondria-dependent apoptosis proteins (Bax, Bim, Bik, Puma and phosphorylation of p53) for both cell lineages. However, downregulation of Bcl-2 and the simultaneous execution of the apoptotic and necroptotic programs associated with the phosphorylation of the proteins receptor-interacting serine/threonine-protein kinase 3 and mixed lineage kinase domain-like pseudokinase occurred only in Kasumi-1 cells. About the lasted events, Kasumi-1 cell death was inhibited by pharmacological agents such as Zvad-FMK and necrostatin-1. The underlying molecular mechanisms of Mtx-C still include participation in the DNA damage and stress-signaling pathways involving p38 and c-Jun N-terminal mitogen-activated proteinkinases and interaction with DNA. Thus, Mtx-C represents a promising tool for the development of new antileukemic molecules.

Prevalence of Inherited Predisposition Syndromes in Young Patients with Acute Myeloid Leukemia and Aberrant Karyotype

Prevalence of Inherited Predisposition Syndromes in Young Patients with Acute Myeloid Leukemia and Aberrant Karyotype

The Impact of the Epigenetic Cancer Drug Azacitidine on Host Immunity: The Role of Myelosuppression, Iron Overload and tp53 Mutations in a Zebrafish Model.

The unsatisfactory real-world efficacy of the hypomethylating agent azacitidine in treating myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) has prompted us to investigate the hematological adverse events and host variables that may compromise the use of this epigenetic drug. Using the zebrafish, we found that azacitidine destroyed their myeloid precursors and impaired myeloid function by inhibiting antigen processing, allogeneic response and phagocytic activity, resulting in increased susceptibility to infection even by the normal flora E. coli. In addition, iron overload, a MDS-associated condition following repeated transfusions, exacerbated bacterial infection especially by V. vulnificus with known iron dependence. Furthermore, we show that the tp53M214K mutant zebrafish survived longer than the wild-type (WT) when challenged with bacteria following azacitidine treatment. This was attributed to the mutant’s hematopoietic cells rather than its general genetic background, since the WT animals reconstituted with the tp53M214K mutant kidney marrow became more resistant to bacterial infection following treatment with azacitidine. The clinical relevance of our findings was indicated by a MDS case with severe azacitidine-induced bone marrow suppression and by the association of hyperferritinemia with bacteremia in azacitidine-treated patients, while tp53M214K-mediated resistance to azacitidine-induced myelosuppression may explain the survival advantage of malignant MDS and AML clones over their normal counterparts under azacitidine treatment. Together, we propose that myelosuppression, iron overload and TP53 mutations may represent the host variables that compromise the azacitidine efficacy.

The Risk of Clonal Evolution of Granulocyte Colony-Stimulating Factor for Acquired Aplastic Anemia: A Systematic Review and Meta-Analysis

Objectives: This meta-analysis aimed to evaluate the risk of clonal evolution of granulocyte colony-stimulating factor (G-CSF) in acquired aplastic anemia (AA), and whether the use of G-CSF increases the occurrence of secondary malignant neoplasms, mainly myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) or paroxysmal nocturnal hemoglobinuria (PNH). Methods: Data were gathered from randomized controlled trials (RCTs) to evaluate the effect of G-CSF versus no G-CSF at the risk of developing the clonal complications of acquired AA. Electronic searches in PubMed, Embase, and the Cochrane Library were performed to identify studies up to 1 January 2017. Only RCTs performed on patients who were randomly assigned to receive G-CSF or not to receive G-CSF were included. Results: Four relevant trials that met the inclusion criteria were identified. In a pooled analysis, the G-CSF groups of AA patients were not associated with a statistically significant higher occurrence of secondary malignant neoplasm, mainly MDS and AML (relative risk [RR] 0.86; 95% confidence interval [CI] 0.34–2.19; 4 trials). No significant heterogeneity was found (p = 0.67, I² = 0%). There was no statistically significant higher occurrence of PNH in the G-CSF groups with AA (RR 1.17; 95% CI 0.51–2.71; 4 trials) and no significant heterogeneity was found (p = 0.42, I² = 0%). Conclusions: G-CSF for patients with AA is not associated with a higher occurrence of secondary malignant neoplasm, mainly MDS/AML, or PNH.

MiRNAs in acute myeloid leukemia.

MicroRNAs (miRNAs) are small, non-coding RNAs found throughout the eukaryotes that control the expression of a number of genes involved in commitment and differentiation of hematopoietic stem cells and tumorigenesis. Widespread dysregulation of miRNAs have been found in hematological malignancies, including human acute myeloid leukemia (AML). A comprehensive understanding of the role of miRNAs within the complex regulatory networks that are disrupted in malignant AML cells is a prerequisite for the development of therapeutic strategies employing miRNA modulators. Herein, we review the roles of emerging miRNAs and the miRNAs regulatory networks in AML pathogenesis, prognosis, and miRNA-directed therapies.

RNA Splicing Modulation Selectively Impairs Leukemia Stem Cell Maintenance in Secondary Human AML

Age-related human hematopoietic stem cell (HSC) exhaustion and myeloid-lineage skewing promote oncogenic transformation of hematopoietic progenitor cells into therapy-resistant leukemia stem cells (LSCs) in secondary acute myeloid leukemia (AML). While acquisition of clonal DNA mutations has been linked to increased rates of secondary AML for individuals older than 60 years, the contribution of RNA processing alterations to human hematopoietic stem and progenitor aging and LSC generation remains unclear. Comprehensive RNA sequencing and splice-isoform-specific PCR uncovered characteristic RNA splice isoform expression patterns that distinguished normal young and aged human stem and progenitor cells (HSPCs) from malignant myelodysplastic syndrome (MDS) and AML progenitors. Insplicing reporter assays and pre-clinical patient-derived AMLmodels, treatment with a pharmacologic splicing modulator, 17S-FD-895, reversed pro-survival splice isoform switching and significantly impaired LSC maintenance. Therapeutic splicing modulation, together with monitoring splice isoform biomarkers of healthy HSPC aging versus LSC generation, may be employed safely and effectively to prevent relapse, the leading cause of leukemia-related mortality.

Genetic Profile and Novel Thrombospondin-1(THBS1) Mutation of Familial AML Syndrome: A Genomic Analysis By Whole Genome Sequencing and Whole Transcriptome Sequencing

BackgroundFamilial form of acute myeloid leukemia (AML) is not well known except for extreme cases. BRCA1/2 and p53 germline mutation are well known genetic changes that is related to familial AML¡¯s. Because of diverse penetrance potential of germline mutations and complex pathogenesis for the development of AML, it is not easy to discern familial form of AML. In this study, we performed next generation sequencing (NGS) study of a family who has been suspected to have familial form of AML.Material and methodTwo relatively young patients have been referred to our institution for AML. One was a 41 years old male (son) and the other was 58 years old female (mother). To discover genetic factors involved in hematological familial cancer syndrome, we performed WGS/WTS using DNA of mother and son who are AML patients and WGS using DNA of healthy father. Tumor DNA and RNA of mother and son were extracted from bone marrow samples which were collected at the time of diagnosis and control DNA and RNA was extracted from saliva and bone marrow samples, respectively, at the time of CR. WGS were performed using HiSeq X ten (illumina, San Diego, USA). For WTS, we used HiSeq2000 platform (illumina, San Diego, USA). For the analysis of WGS, we used GATK unifiedGenotyper for caller in this study. The first filter was set an average depth >10 and conf_cut > 50. And next filter step was using several databases (dbSNP, clinvar, cosmnic70, nci60) and filtering tools (Meta SVM and Meta LR) to filter out of called loci. Meerkat was used to analyze structure variations (SVs). To analyze DEGs using WTS, we used HTSeq-count. To analyze DEGs, we used WTS and HTSeq-count.Results and discussionA total of 3,695,266 loci of normal DNA and 3,977,321 loci of tumor DNA were found in mom samples and 3,622,083 of these were commonly found in both normal and tumor samples. Similarly, a total of 3,513,806 loci of normal and 3,935,873 loci of tumor DNA were found in son samples, of which 3,476,405 were commonly found. Of these common loci, we identified 2,799,429 that are universally present in both mum and son. To determine genes that are found only in patients (Mon and son), we filtered out the loci of father¡¯s SNPs and excluded 2,191,882 loci We subsequently identified a total of 607,547 candidate loci which have association with AML malignancies. Using Support vector machines (SVMs) and DNM filter, we found 37 significant genes that are considered to be related with de-novo AML Among these, 12 were (AGL, COL12A1, IMPDH1, LIPN, MET, MYH13, PBX3, ROBO3, SLC34A3, SMO, THBS1 and TP63) already reported to have an association with hematopoietic disorder, while 25 were a novel mutation genes. The most interesting gene is THBS1. It is reported to affect hematopoietic differentiation function via CD36 and CD47 and a greater than 3 fold change was found in both mum and son in DEG analysis. In DEG analysis, total 1317 gene in mother and 473 genes in son were shown differently expressed above 3FC. The number of recurrent DEGs between mother and son is 144 and these genes were estimated to involve in Systemic lupus erythematosus, Chemokine signaling and bladder cancer pathway.We used Mutect to detect somatic mutation that acts as second hit. 29 nonsynonymous-SNVs were detected in son sample and 43 nonsynonymous-SNVs were detected in mother sample. OR11H1 gene was recurrently shown in both mother and son. We performed Structure variants analysis to identify second hit SVs using Meerkat in mother and son separately SVs of mother were detected in several regions and 69 genes were detected in exonic regions, 39 genes were somatic SVs and 12 of these were filtered out because the genes were also detected in the normal sample of son. 27 SVs genes are considered as a candidate of AML second hit in mother. On the other hand, we found 5 genes somatic SVs in son using the same method. Of note, FAM231A gene overlapped with mom tumor sample and son tumor sample.In conclusion, we found 37 significant genes may be related with de-novo AML. In addition, several genetic factors affect tumorigenesis through second hit. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Normal Hematopoietic Stem Cells within the AML Bone Marrow Have a Distinct and Higher ALDH Activity Level than Co-Existing Leukemic Stem Cells

Persistence of leukemic stem cells (LSC) after chemotherapy is thought to be responsible for relapse and prevents the curative treatment of acute myeloid leukemia (AML) patients. LSC and normal hematopoietic stem cells (HSC) share many characteristics and co-exist in the bone marrow of AML patients. For the development of successful LSC-targeted therapy, enabling eradication of LSC while sparing HSC, the identification of differences between LSC and HSC residing within the AML bone marrow is crucial. For identification of these LSC targets, as well as for AML LSC characterization, discrimination between LSC and HSC within the AML bone marrow is imperative. Here we show that normal CD34+CD38– HSC present in AML bone marrow, identified by their lack of aberrant immunophenotypic and molecular marker expression and low scatter properties, are a distinct sub-population of cells with high ALDH activity (ALDHbright). The ALDHbright compartment contains, besides normal HSC, more differentiated, normal CD34+CD38+ progenitors. Furthermore, we show that in CD34-negative AML, containing solely normal CD34+ cells, LSC are CD34– and ALDHlow. In CD34-positive AML, LSC are also ALDHlow but can be either CD34+ or CD34–. In conclusion, although malignant AML blasts have varying ALDH activity, a common feature of all AML cases is that LSC have lower ALDH activity than the CD34+CD38– HSC that co-exist with these LSC in the AML bone marrow. Our findings form the basis for combined functionally and immunophenotypically based identification and purification of LSC and HSC within the AML bone marrow, aiming at development of highly specific anti-LSC therapy.

A Novel Fc-Engineered Antibody to CD33 with Enhanced ADCC Activity for Treatment of AML

AbstractAbstract 1363 Introduction:Acute myeloid leukemia (AML), the most frequent form of adult acute leukemia, is a heterogeneous disease with respect to presentation and clinical outcome. Although there has been progress made in treatment for some subgroups of AML patients, there is still a high medical need to develop new anti-leukemic therapies. CD33 is a myeloid lineage-specific antigen which is expressed on the cell surface of normal myeloid cells and AML blasts. CD33 has been investigated as a target for antibody-based therapies for over a decade, leading to transient approval of Mylotarg®, an antibody-calicheamicin drug conjugate. Mylotarg®has been shown to substantially improve outcome of patients with AML in combination with chemotherapy, however is associated with additional toxicity. Here we describe a novel, unconjugated Fc-engineered antibody to CD33, mAb 33.1, which mediates potent antibody dependent cellular cytotoxicity (ADCC) against AML derived cell lines and malignant AML blasts. Methods:MAb 33.1 is a fully human, Fc-engineered, IgG1-type of antibody which has increased affinity to human Fc-gamma receptor IIIa and binds CD33 with low nanomolar affinity as determined by FACS scatchard analysis. ADCC of mAb 33.1 was determined on a panel of myeloid cell lines and primary AML cells. ADCC assays were performed using peripheral blood mononuclear cells (PBMC) from healthy donors as effector cells and cytotoxicity was determined by LDH release or FACS quantification of 7-AAD-positive cells. Internalization kinetics was assessed by FACS analysis using fluorochrome labelled antibody. Epitope mapping of mAb 33.1 was performed by direct competition experiments and hydrogen/deuterium exchange mass spectrometry (HXMS). Results:Antibody mAb 33.1 is able to mediate potent ADCC on a panel of AML derived cell lines and primary AML blasts in vitro. Direct comparison of mAb 33.1 to the humanized CD33 mAb lintuzumab (HuM195) demonstrates superior cytolytic activity on AML cell lines and primary AML cells. ADCC activity of mAb 33.1 is also evident on AML cell lines and primary AML blasts that do not respond to lintuzumab treatment in vitro. In contrast to lintuzumab, mAb 33.1 shows significantly slower internalization kinetics on HL60 cells, leading to prolonged exposure of antigen/antibody complexes on the cell surface. In direct competition experiments mAb 33.1 is competing with lintuzumab for antigen binding. HXMS experiments revealed that mAb 33.1 recognizes a novel epitope on CD33 different to that of lintuzumab. Conclusion:MAb 33.1 represents a novel, Fc-engineered antibody specific for CD33 which shows decelerated internalization compared to lintuzumab, and which recognizes a novel epitope on CD33 different to that of lintuzumab. MAb 33.1 displays strongly enhanced ADCC against leukemia cell lines and primary AML cells, resulting in significantly higher cytolysis than lintuzumab. MAb 33.1 is considered as a promising candidate for treatment of patients with CD33-positive myeloid malignancies and phase I clinical trials are currently under preparation. Disclosures:Heider:Boehringer Ingelheim: Employment. Konopitzky:Boehringer Ingelheim: Employment. Ostermann:Boehringer Ingelheim: Employment. Lamche:Boehringer Ingelheim: Employment. Kuepcue:Boehringer Ingelheim: Employment. Koessl:Boehringer Ingelheim: Employment. Adam:Boehringer Ingelheim: Employment. Borges:Boehringer Ingelheim: Employment.

Monocytic AML cells inactivate antileukemic lymphocytes: role of NADPH oxidase/gp91phox expression and the PARP-1/PAR pathway of apoptosis

AbstractDysfunction of T cells and natural killer (NK) cells has been proposed to determine the course of disease in acute myeloid leukemia (AML), but only limited information is available on the mechanisms of lymphocyte inhibition. We aimed to evaluate to what extent human malignant AML cells use NADPH oxidase-derived reactive oxygen species (ROS) as an immune evasion strategy. We report that a subset of malignant myelomonocytic and monocytic AML cells (French-American-British [FAB] classes M4 and M5, respectively), recovered from blood or BM of untreated AML patients at diagnosis, expressed the NADPH oxidase component gp91phox. Highly purified FAB M4/M5 AML cells produced large amounts of ROS on activation and triggered poly-[ADP-ribose] polymerase-1−dependent apoptosis in adjacent NK cells, CD4+ T cells, and CD8+ T cells. In contrast, immature (FAB class M1) and myeloblastic (FAB class M2) AML cells rarely expressed gp91phox, did not produce ROS, and did not trigger NK or T-cell apoptosis. Microarray data from 207 AML patients confirmed a greater expression of gp91phox mRNA by FAB-M4/M5 AML cells than FAB-M1 cells (P < 10−11) or FAB-M2 cells (P < 10−9). Our data are suggestive of a novel mechanism by which monocytic AML cells evade cell-mediated immunity.

Abstract 5414: Malignant monocytic AML cells trigger apoptosis in anti-leukemic lymphocytes: Role of NADPH oxidase gp91phox expression and PARP-1 activity

Abstract The dysfunction of natural killer (NK) cells and T cells in acute myeloid leukemia (AML) determines the course of disease, but only limited information is available on the mechanisms of lymphocyte inhibition. This study aimed to evaluate to what extent malignant AML cells utilize NADPH-oxidase derived reactive oxygen species (ROS) as an immune evasion strategy. We report that a subset of genetically defined malignant monocytic AML cells (FAB class M4/M5), recovered from blood or bone marrow of untreated AML patients at diagnosis, express gp91phox, a component of the NADPH oxidase. These AML cells produced large amounts of ROS upon activation and triggered PARP 1-dependent apoptosis in adjacent natural killer (NK) cells, CD4+ T cells, and cytotoxic T cells. In contrast, myeloblastic (FAB class M2) or immature (FAB class M1) AML cells did not express gp91phox, did not produce ROS, and did not trigger NK or T cell apoptosis. Our data are suggestive of a novel mechanism by which monocytic AML cells evade lymphocyte immunity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5414. doi:1538-7445.AM2012-5414

Novel agents inhibit human leukemic cells

Novel agents inhibit human leukemic cells