AbstractBackgroundAcute Myeloid Leukemia Research Articles

Abstract 1764: Long read sequencing allows comprehensive molecular profiling of complex karyotype acute myeloid leukemia (CKAML) with 5q deletions

Abstract Background Acute myeloid leukemia with complex karyotype (CKAML) does frequently show del(5q), deletion of chromosome 5q. Previous work in MDS and AML showed that CKAML form clusters distinguishing del(5q) cases with commonly retained regions (CRRs) in the telomeric (≥5q34) and centromeric (≤5q14.2) ends from cases with loss of the telomeric and/or centromeric regions. Interestingly, del(5q) patients with CRRs were shown to have less genomic lesions and were associated with a better prognosis than other 5q-deleted CKAML cases. Aim: Combining the advantages of two different long read sequencing technologies, Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT), we aimed to more comprehensively characterize the molecular landscape of CKAML with del(5q). Our integrative workflow enabled an in-depth characterization of copy number variations (CNV), genomic rearrangements, transcriptomic and epigenomic changes. Methods: Low coverage whole genome sequencing (WGS) using ONT was performed on 209 patients with CKAML to identify del(5q) cases for further in-depth analysis. For 10 selected cases with known CRR status, genomic deep sequencing using PacBio Revio and ONT PromethION was performed, as well as PacBio Revio MAS-seq for fusion transcript detection. Results: A mean sequencing depth of 3-fold (range 0.4-17.3-fold) was obtained by low coverage WGS of the 209 CKAML cases. With a resolution of 0.1 Mbp n=134 del(5q) cases were identified in our CKAML cohort (64%) and commonly deleted regions on 5q could be resolved at 5q21.2 to 5q23.3 and 5q33.3 to 5q35 (Fisher test, FDR Benjamini-Hochberg, p<10−30). These regions separated del(5q) in n=52 cases showing CRR retention and n=82 showing partially no CRR, (either the centromeric or telomeric regions, n=74, or of both regions, n=8 involved). Subsequent co-occurrence analysis provided evidence of an association between the loss of telomeric 5q region with a 3p12-25 deletion. In-depth analysis (30-50 fold) of ten distinct del(5q) cases by PacBio and ONT revealed additional insights into the complex clonal composition of del(5q) cases. For example, one case showed two distinct subclones with 5q14.2-5q35.1 deletion and with translocation t(3;5)(p14;q14) respectively. Moreover, we could demonstrate that 5q-aberrations are far more complex than initially thought showing, e.g. inverted terminal duplications that resulted in fusion transcripts involving PDE4D and leading to its deregulated expression. Conclusion: Using our long read deep sequencing approach we could further delineate the structural complexity of del(5q) CKAML. While gene expression profiling, methylation analysis and correlation of findings with clinical outcome are ongoing and will be presented at the meeting, our results help to further pinpoint the biology underlying del(5q) and to further refine this heterogeneous CKAML subgroup. Citation Format: Felix Schick, Ekaterina Slonova, Eric Straeng, Sarah Huet, Maurizio Raso, Marlon Tilgner, Frederik Damm, Pierre Sujobert, Olga Blau, Lars Bullinger, Anna Dolnik. Long read sequencing allows comprehensive molecular profiling of complex karyotype acute myeloid leukemia (CKAML) with 5q deletions [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 1764.

How to adjust therapy for elderly AML patients in developing countries?

Abstract Background Acute myeloid leukemia (AML) with advancing age represents a therapeutic challenge. The tools used for comprehensive geriatric assessment (CGA) in elderly AML are not uniform. Methods This is a retrospective study that included 226 elderly AML patients (≥60 years), identified from the electronic database and followed till the end of 2021. Comorbidities were assessed using Charlson Comorbidity Index (CCI), Cumulative Illness Rating Scale-Geriatric (CIRS-G), Hematopoietic cell transplantation-specific comorbidity index (HCT-CI) scores, and Chia-Jen Liu and colleagues simplified proposed models. The primary outcome was 30 days mortality. The secondary outcomes were the achievement of complete remission. Results 226 patients were included, the median age was 67 years, and 86.2% of patients had ECOG greater than or equal to 2 performance status at diagnosis. 82 patients received intensive induction treatment, 85 patients received reduced-intensity treatment, and 59 patients were unfit for therapy. 30-Day mortality was reported in 49.1% of included patients. With multivariate analysis; ECOG-PS (3-4), high CIRS-G or HCT-CI scoring system (≥3), and intermediate-high Chia-Jen Liu score were considered as prognostic factors for early mortality in elderly AML patients. Complete remission (CR) was achieved in 38.3% and 4.7% of the patients who received intensive induction and low-intensity therapy, respectively. Conclusion Geriatric and comorbidity assessment tools can be used to predict early mortality in elderly AML. Higher scores and poor PS are predictors for early death rather than a prediction of response to treatment.

Identification of TIM-3 Expression in Acute Myeloid Leukemia: Clinical and Laboratory Correlation

Abstract Background Acute myeloid leukemia (AML) is an aggressive highly heterogenous hematological malignancy associated with poor prognosis and high mortality rate. Both cancer cells and immune cells participate in tumor initiation, growth and progression and might affect clinical outcomes. Indeed, leukemic cells exploit a variety of mechanisms to evade immune response, leading to disease progression and relapse. T-cell immunoglobulin and mucin domain 3 (TIM-3), an immune checkpoint molecule, is expressed not only on immune cells but also on leukemic stem cells (LSCs) in AML. Studies relating to TIM-3 function in AML are limited, and the role of TIM3 in maintaining leukemic stem cells and contributing to the suppression of the anti-tumor immune response is still poorly understood. Aim of the Work To investigate TIM-3 expression in patients with AML and explore the impact of its expression on the clinico-laboratory characteristics and prognostic behavior of denovo-AML patients. Methods A total of 45 patients newly diagnosed AML patients were tested for TIM-3 expression by flowcytometry at diagnosis. Results TIM-3 was found to be over expressed in patients with AML with significant association with the different FAB subtypes (p = 0.010). TIM-3 positivity was negatively correlated with CD34 expression (p = 0.019.). However, no significant association between TIM-3 expression and extramedullary disease (EMD) as well as cytogenetic risk groups. Conclusion TIM-3 is overexpressed in AML patients. The prognostic value of TIM3 in AML is not clear and remains to be further explored.

Expression Pattern and Prognostic Implication of SALL4 Gene in Acute Myeloid Leukemia

Abstract Background Acute myeloid leukemia (AML) is a clonal disorder arises from the differentiation arrest of myeloid precursor and malignant proliferation of a bone marrow derived, self-renewing stem or progenitor cells in the bone marrow (BM) and blood. Aim of the Work To characterize the expression pattern of SALL4 gene in patients with acute myeloid leukemia and to determine its prognostic impact. Patients and Methods This study was conducted on 20 Egyptian AML patients (recruited from hematology units of Internal Medicine department, Ain shams University Hospitals) and 10 patients as controls. All patients were subjected to full history taking, thorough clinical examination, complete blood count, BM aspiration cytochemical analysis, flow-cytometric immunophenotyping of BM blasts using acute leukemia panel and cytogenetic analysis using FISH technique and RT-PCR to measure SALL4 gene expression in AML cases and controls. Results Our results showed highly significant difference between expression of SALL 4 gene in AML patients than control group with RQ median in AML cases 15.46 and in control group 0.4 (p value 0.000). Non significant results about prognosios was reached Conclusion SALL4 gene expression is constitutive in De-novo AML cases.

EIF4A inhibition targets bioenergetic homeostasis in AML MOLM-14 cells in vitro and in vivo and synergizes with cytarabine and venetoclax.

Acute myeloid leukemia (AML) is an aggressive hematological cancer resulting from uncontrolled proliferation of differentiation-blocked myeloid cells. Seventy percent of AML patients are currently not cured with available treatments, highlighting the need of novel therapeutic strategies. A promising target in AML is the mammalian target of rapamycin complex 1 (mTORC1). Clinical inhibition of mTORC1 is limited by its reactivation through compensatory and regulatory feedback loops. Here, we explored a strategy to curtail these drawbacks through inhibition of an importanteffector of themTORC1signaling pathway, the eukaryotic initiation factor 4A (eIF4A). We tested the anti-leukemic effect of a potent and specific eIF4A inhibitor (eIF4Ai), CR-1-31-B, in combination with cytosine arabinoside (araC) or the BCL2 inhibitor venetoclax. We utilized the MOLM-14 human AML cell line to model chemoresistant disease both in vitro and in vivo. In eIF4Ai-treated cells, we assessed for changes in survival, apoptotic priming, de novo protein synthesis, targeted intracellular metabolite content, bioenergetic profile, mitochondrial reactive oxygen species (mtROS) and mitochondrial membrane potential (MMP). eIF4Ai exhibits anti-leukemia activity in vivo while sparing non-malignant myeloid cells. In vitro, eIF4Ai synergizes with two therapeutic agents in AML, araC and venetoclax. EIF4Ai reduces mitochondrial membrane potential (MMP) and the rate of ATP synthesis from mitochondrial respiration and glycolysis. Furthermore, eIF4i enhanced apoptotic priming while reducing the expression levels of the antiapoptotic factors BCL2, BCL-XL and MCL1. Concomitantly, eIF4Ai decreases intracellular levels of specific metabolic intermediates of the tricarboxylic acid cycle (TCA cycle) and glucose metabolism, while enhancing mtROS. In vitro redox stress contributes to eIF4Ai cytotoxicity, as treatment with a ROS scavenger partially rescued the viability of eIF4A inhibition. We discovered that chemoresistant MOLM-14 cells rely on eIF4A-dependent cap translation for survival in vitro and in vivo. EIF4A drives an intrinsic metabolic program sustaining bioenergetic and redox homeostasis and regulates the expression of anti-apoptotic proteins. Overall, our work suggests that eIF4A-dependent cap translation contributes to adaptive processes involved in resistance to relevant therapeutic agents in AML.

Incidence of Invasive Fungal Infections in Patients With Previously Untreated Acute Myeloid Leukemia Receiving Venetoclax and Azacitidine.

BackgroundAcute myeloid leukemia (AML) is associated with poor prognosis, particularly in elderly patients with comorbidities. Combining azacitidine (AZA) with BCL-2 inhibitor venetoclax (VEN) demonstrated significant improvement in outcomes for newly-diagnosed AML patients compared to AZA alone. However, this regimen is myelosuppressive, and the incidence of invasive fungal infections (IFIs) and impact of antifungal prophylaxis are not well defined.MethodsThis retrospective cohort study evaluated newly-diagnosed AML patients treated with VEN/AZA at the University of Colorado Hospital from January 2014 to August 2020. Patients with history of prior IFI were excluded. Primary outcome was IFI incidence during VEN/AZA therapy. χ2 and Fisher exact tests assessed the impact of patient demographics, AML-specific risk factors, and receipt of antifungal prophylaxis on IFI incidence.Results144 VEN/AZA-treated AML patients were included in the study. 25 (17%) patients developed IFI: 8% (n = 2) “proven,” 24% (n = 6) “probable,” and 68% (n = 17) “possible” per European Organization for Research and Treatment of Cancer and the Mycoses Study Group Education and Research Consortium criteria. There was no statistically significant association between IFI incidence with age, sex, or European LeukemiaNet classification. 10 patients received antifungal prophylaxis; none developed IFI. IFI incidence rate per 1000 patient-days was greatest 0–9 days after starting VEN/AZA, at 8.39.ConclusionsIncidence of “proven” and “probable” IFI in our VEN/AZA-treated AML cohort was 5.6%, in-line with incidence rates reported by recent similar studies. Furthermore, IFI incidence decreased as days from starting VEN/AZA therapy increased.

Super-enhancer profiling identifies novel critical and targetable cancer survival gene LYL1 in pediatric acute myeloid leukemia

BackgroundAcute myeloid leukemia (AML) is a myeloid neoplasm makes up 7.6% of hematopoietic malignancies. Super-enhancers (SEs) represent a special group of enhancers, which have been reported in multiple cell types. In this study, we explored super-enhancer profiling through ChIP-Seq analysis of AML samples and AML cell lines, followed by functional analysis.MethodsChIP-seq analysis for H3K27ac was performed in 11 AML samples, 7 T-ALL samples, 8 B-ALL samples, and in NB4 cell line. Genes and pathways affected by GNE-987 treatment were identified by gene expression analysis using RNA-seq. One of the genes associated with super-enhancer and affected by GNE-987 treatment was LYL1 basic helix-loop-helix family member (LYL1). shRNA mediated gene interference was used to down-regulate the expression of LYL1 in AML cell lines, and knockdown efficiency was detected by RT-qPCR and western blotting. The effect of knockdown on the growth of AML cell lines was evaluated by CCK-8. Western blotting was used to detect PARP cleavage, and flow cytometry were used to determine the effect of knockdown on apoptosis of AML cells.ResultsWe identified a total of 200 genes which were commonly associated with super-enhancers in ≧10 AML samples, and were found enriched in regulation of transcription. Using the BRD4 inhibitor GNE-987, we assessed the dependence of AML cells on transcriptional activation for growth and found GNE-987 treatment predominantly inhibits cell growth in AML cells. Moreover, 20 candidate genes were selected by super-enhancer profile and gene expression profile and among which LYL1 was observed to promote cell growth and survival in human AML cells.ConclusionsIn summary, we identified 200 common super-enhancer-associated genes in AML samples, and a series of those genes are cancer genes. We also found GNE-987 treatment downregulates the expression of super-enhancer-associated genes in AML cells, including the expression of LYL1. Further functional analysis indicated that LYL1 is required for AML cell growth and survival. These findings promote understanding of AML pathophysiology and elucidated an important role of LYL1 in AML progression.

SEMA4D/PlexinB1 promotes AML progression via activation of PI3K/Akt signaling

BackgroundAcute myeloid leukemia (AML) is the most common type of acute leukemia in adults. SEMA4D is a 150 kDa transmembrane protein that belongs to the IV class of the subfamily of semaphorin family. Previous studies have reported that SEMA4D is a multifunctional target in many solid tumors, involving multiple physiological systems, and there are emerging therapies to target these pathways. The role of SEMA4D in AML has not yet been explored.MethodsThe SEMA4D expression prolile, clinical data and potential prognostic analysis were acquired via the cBioPortal and GEPIA databases. SEMA4D expression was measured using real-time quantitative PCR and western blot. Cell counting kit-8 (CCK8) and flow cytometry were used to evaluate the malignant biological characteristics.ResultsWe observed that SEMA4D was increased in AML patients and correlated with risk stratification and prognosis. Moreover, SEMA4D promotes the proliferation and inhibits apoptosis of AML cells by binding to its receptor, PlexinB1, and reduces the sensitivity of AML cells to daunorubicin. In addition, SEMA4D/PlexinB1 promotes the proliferation and survival of AML cells by activating the PI3K/Akt signaling pathway. VX15/2503, an anti-SEMA4D antibody, can inhibit the proliferation of AML cells in xenograft mouse models, thereby inhibiting the development of AML.ConclusionSEMA4D will serve as a unique predictive biomarker and a possible therapeutic target in AML.

Up-Regulation of TRIM32 Associated With the Poor Prognosis of Acute Myeloid Leukemia by Integrated Bioinformatics Analysis With External Validation.

BackgroundAcute myeloid leukemia (AML) is a malignant and molecularly heterogeneous disease. It is essential to clarify the molecular mechanisms of AML and develop targeted treatment strategies to improve patient prognosis.MethodsAML mRNA expression data and survival status were extracted from TCGA and GEO databases (GSE37642, GSE76009, GSE16432, GSE12417, GSE71014). Weighted gene co-expression network analysis (WGCNA) and differential gene expression analysis were performed. Functional enrichment analysis and protein-protein interaction (PPI) network were used to screen out hub genes. In addition, we validated the expression levels of hub genes as well as the prognostic value and externally validated TRIM32 with clinical data from our center. AML cell lines transfected with TRIM32 shRNA were also established to detect the proliferation in vitro.ResultsA total of 2192 AML patients from TCGA and GEO datasets were included in this study and 20 differentially co-expressed genes were screened by WGCNA and differential gene expression analysis methods. These genes were mainly enriched in phospholipid metabolic processes (biological processes, BP), secretory granule membranes (cellular components, CC), and protein serine/threonine kinase activity (molecular functions, MF). In addition, the protein-protein interaction (PPI) network contains 15 nodes and 15 edges and 10 hub genes (TLE1, GLI2, HDAC9, MICALL2, DOCK1, PDPN, RAB27B, SIX3, TRIM32 and TBX1) were identified. The expression of 10 central genes, except TLE1, was associated with survival status in AML patients (p<0.05). High expression of TRIM32 was tightly associated with poor relapse-free survival (RFS) and overall survival (OS) in AML patients, which was verified in the bone marrow samples from our center. In vitro, knockdown of TRIM32 can inhibit the proliferation of AML cell lines.ConclusionTRIM32 was associated with the progression and prognosis of AML patients and could be a potential therapeutic target and biomarker for AML in the future.

MECOM gene overexpression in pediatric patients with acute myeloid leukemia

Background Acute myeloid leukemia (AML) is characterized by blocked or aberrant differentiation of hematopoietic stem cells. The MECOM gene overexpression in hematopoietic progenitors induces myeloid differentiation block, resulting in increased self-renewal and survival of these transformed progenitors. However, its exact role in AML remains unclear. We aimed to estimate the prevalence of MECOM overexpression among pediatric AML patients, and assess its impact on clinical outcome. Patients and Methods Real-time quantitative polymerase chain reaction and Livak method (2ΔΔCt) were used to determine relative MECOM expression level among 243 pediatric patients with AML. MECOM overexpression was considered if the cumulative relative expression was above 1 (2-ΔΔCt) and was designated as MECOMpos . Results Of 243 AML patients tested 57(23.5%) demonstrated MECOMpos . Patients with MECOMpos had significantly lower median age. The frequency of MECOMpos was significantly higher among AML patients with 11q23 abnormalities, complex karyotypes and among high- and intermediate-risk groups compared to low-risk group (p = .014). MECOMpos patients had significantly lower overall survival (OS) (38.7 vs. 78.9%, p < .001), event-free survival (EFS) (37.3% vs. 68.4%, p < .001), and had higher cumulative incidence of relapse (49.5% vs. 23.5%, p = .002) at 36 months compared to MECOMneg patients. Multivariate analysis revealed that MECOMpos was an adverse prognostic factor for OS (hazards ratio (HR) = 2.11, 95% confidence interval (CI) 1.24–3.60, p = .006) and EFS (HR= 1.71, 95% CI 1.07–2.75, p = .025). The logistic regression model showed that MECOMpos was an independent prognostic factor regardless of minimal residual disease status post first induction therapy in the intermediate-risk group (odds ratio 2.89; 95% CI 1.19–6.57, p = .018). Conclusion The aberrant MECOM gene expression is an adverse prognostic factor, especially in patients without previously known cytogenetic risk factors. Our results suggest the potential benefit from pretreatment screening for MECOM gene overexpression in newly diagnosed AML patients for better risk stratification and treatment adjustment.

986. Incidence of Invasive Fungal Infections in Previously Untreated Patients with Acute Myeloid Leukemia Receiving Venetoclax and Azacitadine

Abstract Background Acute myeloid leukemia (AML) is associated with poor prognosis, particularly in elderly patients with co-morbidities. Low-intensity therapies like azacitidine (aza) were the standard of care and were associated with low response rates and limited survival. Combining venetoclax (ven) with aza demonstrated significant improvements in responses and survival compared to aza alone, and represents the new standard of care for this population. However, as a myelosuppressive regimen, infectious complications, especially invasive fungal infections (IFI), are a potential concern. The incidence of IFI and the role for antifungal prophylaxis have not been well defined for newly-diagnosed AML patients receiving ven/aza. Methods We conducted a retrospective cohort review of AML patients treated with ven/aza at the University of Colorado Hospital from January 2014 to August 2020. Duration of therapy was defined as the time from initiation of treatment through one of the following endpoints (1) patient discontinuation, (2) progression of disease, (3) bone marrow transplantation, or (4) death. Four patients with a history of prior IFI were excluded. We assessed the impact of patient age, sex, duration of neutropenia, antifungal prophylaxis, and AML specific risk factors on the incidence of IFI as defined by the European Mycoses Study Group. Results One hundred forty-four AML patients were included in the study. Ten patients received antifungal prophylaxis and none developed IFI (p=0.21). Twenty-five (17%) patients developed IFI: 2 (8%) had proven IFI, 6 (24%) probable IFI, and 17 (68%) possible IFI. Invasive pulmonary aspergillosis represented all 25 cases of proven, probable, and possible IFI. There was a statistically significant association between prolonged neutropenia ( &amp;gt;60 days) and IFI (p=0.007), whereas age, sex, and SWOG classification were not significantly associated with IFI. Conclusion The incidence of IFI in our AML cohorts treated with ven/aza was 17%, lower than that reported at other institutions. Neutropenia &amp;gt; 60 days was significantly associated with IFI in our AML cohort treated with ven/aza. Although we were not powered to determine whether antifungal prophylaxis impacted IFI, there was no significant difference in IFI for patients who received prophylaxis. Disclosures All Authors: No reported disclosures

Bayesian variable selection for high-dimensional data with an ordinal response: identifying genes associated with prognostic risk group in acute myeloid leukemia

BackgroundAcute myeloid leukemia (AML) is a heterogeneous cancer of the blood, though specific recurring cytogenetic abnormalities in AML are strongly associated with attaining complete response after induction chemotherapy, remission duration, and survival. Therefore recurring cytogenetic abnormalities have been used to segregate patients into favorable, intermediate, and adverse prognostic risk groups. However, it is unclear how expression of genes is associated with these prognostic risk groups. We postulate that expression of genes monotonically associated with these prognostic risk groups may yield important insights into leukemogenesis. Therefore, in this paper we propose penalized Bayesian ordinal response models to predict prognostic risk group using gene expression data. We consider a double exponential prior, a spike-and-slab normal prior, a spike-and-slab double exponential prior, and a regression-based approach with variable inclusion indicators for modeling our high-dimensional ordinal response, prognostic risk group, and identify genes through hypothesis tests using Bayes factor.ResultsGene expression was ascertained using Affymetrix HG-U133Plus2.0 GeneChips for 97 favorable, 259 intermediate, and 97 adverse risk AML patients. When applying our penalized Bayesian ordinal response models, genes identified for model inclusion were consistent among the four different models. Additionally, the genes included in the models were biologically plausible, as most have been previously associated with either AML or other types of cancer.ConclusionThese findings demonstrate that our proposed penalized Bayesian ordinal response models are useful for performing variable selection for high-dimensional genomic data and have the potential to identify genes relevantly associated with an ordinal phenotype.

High expression levels and the C3435T SNP of the ABCB1 gene are associated with lower survival in adult patients with acute myeloblastic leukemia in Mexico City

BackgroundAcute myeloid leukemia (AML) is a heterogeneous hematologic malignancy characterized by different genetic alterations that cause changes in the normal mechanisms of differentiation, which are associated with chemoresistance. The ABCB1 gene is part of a family of ATP-binding cassette (ABC) transporter genes involved in the progression of various types of cancer. The following work aimed to evaluate the expression levels of the ABCB1 gene and the C3435T SNP with the response to first-line treatment and survival in patients with AML.MethodsIn total 135 samples were taken to isolate total RNA and DNA at the beginning of the treatment. Expression analysis by RT-qPCR and SNP C3435T assessment method were performed for real-time Polymerase chain reaction (qPCR).ResultsThe expression levels impact on the survival of patients with AML compared to low or absent levels; the CC genotype was found in 22.9%, the CT genotype was found in 47.4%, and the TT genotype was found in 29.6%, the presence of the C3435T SNP, the TT genotype also impacts with a lower survival compared to CT and CC genotypes. In addition, it was shown that the dominant model significantly impacts survival.ConclusionIn conclusion, we have found that the overexpression of the ABCB1 gene, as well as the presence of the TT genotype of the C3435T SNP, contributes to a worse prognosis in AML.

Mutant NPM1-regulated lncRNA HOTAIRM1 promotes leukemia cell autophagy and proliferation by targeting EGR1 and ULK3

BackgroundAcute myeloid leukemia (AML) with mutated nucleophosmin (NPM1), which displays a distinct long noncoding RNA (lncRNA) expression profile, has been defined as a unique subgroup in the new classification of myeloid neoplasms. However, the biological roles of key lncRNAs in the development of NPM1-mutated AML are currently unclear. Here, we aimed to investigate the functional and mechanistic roles of the lncRNA HOTAIRM1 in NPM1-mutated AML.MethodsThe expression of HOTAIRM1 was analyzed with a public database and further determined by qRT-PCR in NPM1-mutated AML samples and cell lines. The cause of upregulated HOTAIRM1 expression was investigated by luciferase reporter, chromatin immunoprecipitation and ubiquitination assays. The functional role of HOTAIRM1 in autophagy and proliferation was evaluated using western blot analysis, immunofluorescence staining, a Cell Counting Kit-8 (CCK-8) assay, a 5-ethynyl-2′-deoxyuridine (EdU) incorporation assay, flow cytometric analyses and animal studies. The action mechanism of HOTAIRM1 was explored through RNA fluorescence in situ hybridization, RNA pulldown and RNA immunoprecipitation assays.ResultsHOTAIRM1 was highly expressed in NPM1-mutated AML. High HOTAIRM1 expression was induced in part by mutant NPM1 via KLF5-dependent transcriptional regulation. Importantly, HOTAIRM1 promoted autophagy and proliferation both in vitro and in vivo. Mechanistic investigations demonstrated that nuclear HOTAIRM1 promoted EGR1 degradation by serving as a scaffold to facilitate MDM2-EGR1 complex formation, while cytoplasmic HOTAIRM1 acted as a sponge for miR-152-3p to increase ULK3 expression.ConclusionsTaken together, our findings identify two oncogenic regulatory axes in NPM1-mutated AML centered on HOTAIRM1: one involving EGR1 and MDM2 in the nucleus and the other involving the miR-152-3p/ULK3 axis in the cytoplasm. Our study indicates that HOTAIRM1 may be a promising therapeutic target for this distinct leukemia subtype.

Platelet transcriptome profiles provide potential therapeutic targets for elderly acute myelocytic leukemia patients

BackgroundAcute myeloid leukemia (AML) is the most common acute leukemia in adults, with a median age of 68 in clinical diagnosis. About 60% patients are over 60 years old. There are various treatment options for AML patients. But for elderly patients, the complete remission rates are disappointing due to genetic, molecular, and age-related factors. Development of next-generation sequencing technologies makes it possible to seek individual strategies for patients in different ages. This study analyzed transcriptome profiles in platelets of AML patients in different ages for the first time.MethodsPlatelet RNA sequencing in AML of ten elderly and seven young patients were performed with Illumina TruSeq Stranded mRNA library Prep Kit and Illumina HiSeq4000 sequencing instrument. With the FASTQ sequencing data obtained, statistical analyses between elderly with young AML patients were analyzed by R program. GO and KEGG enrichment analyses were performed via R package clusterProfiler. TOP 10 down-regulated/up-regulated genes in elderly patients compared to young patients were selected with the threshold of |L2FC| > 2 and padj ≤ 0.0001. The down-regulated gene ATF4 was chosen by GSEA analysis and ROC analysis with AUC > 0.95.ResultsWe found 3059 genes with differential transcript levels (GDTLs) in AML patients of different age. Among them, 2048 genes are down-regulated and 651 genes are up-regulated in elderly patients. We found that gene transcript profiles in elderly patients is obviously different from those in young patients, including a collection of down-regulated genes related to proteins processing in endoplasmic reticulum and immunity. We further identified that genes of pathway in cancer and mitogen activated protein kinase (MAPK) pathway, involved in natural immunity and metabolism, are significantly down-regulated in elderly patients. Among all screened genes with decreased transcript levels, we believe that activating transcription factor 4 (ATF4) is a biomarker indicating different chemotherapy strategies for elderly patients.ConclusionsIn summary, gene transcript profiles are different in platelets of elderly and young AML patients. And ATF4 can be a useful biomarker indicating different chemotherapy strategies for AML patients with different ages.

Impact of induction chemotherapy with intermediate-dosed cytarabine and subsequent allogeneic stem cell transplantation on the outcome of high-risk acute myeloid leukemia

BackgroundAcute myeloid leukemia (AML) with antecedent hematological disease (s-AML) and treatment-related AML (t-AML) predicts poor prognosis. Intensive treatment protocols of those high-risk patients should consider allogeneic stem cell transplantation (allo-HSCT) in first complete remission (CR). Despite allo-HSCT, relapse rate remains high. Induction chemotherapy with liposomal cytarabine and daunorubicin (CPX-351) has been approved for patients with AML with myeloid-related changes (AML-MRC) or t-AML based on improved survival and remission rates compared to standard 7 + 3 induction.Patients and methods110 patients with newly diagnosed s-AML or t-AML at a university hospital were analyzed retrospectively. Median age was 62 years (24–77 years). A total of 65 patients with s-AML after MDS (59%) and 23 patients (20.9%) with t-AML were included. Induction chemotherapy consisted of intermediate-dosed cytarabine (ID-AraC) in combination with idarubicin (patients up to 60 years) or mitoxantrone (patients over 60 years). In patients subsequently undergoing allo-HSCT, reduced conditioning regimens (RIC) were applied prior to transplantation in 47 of 62 patients (76%).ResultsInduction chemotherapy with ID-AraC resulted in an overall response rate of 83% including complete remission (CR/CRi) in 69 patients (63%) with a low rate of early death (2.7%). Most relevant non-hematologic toxicity consisted of infectious complications including sepsis with need of intensive care treatment in five patients (4.5%) and proven or probable invasive fungal disease in eight patients (7.2%). Relapse-free survival (RFS), event-free survival (EFS) and overall survival (OS) of the whole cohort were 19 months (0–167), 10 months (0–234) and 15 months (0–234), respectively (p < 0.0001). A significant improvement of OS was observed in patients who underwent allo-HSCT compared to those without subsequent allo-HSCT: 9 vs. 46 months, p < 0.0001. Rate of transplantation-related mortality (TRM) in the early phase post allo-HSCT was low (0.9% at day 30 and 1.8% at day 90, respectively). RIC conditioning results in OS rate of 60% after 60 months post allo-HSCT (median OS not reached).ConclusionS-AML and t-AML patients receiving induction chemotherapy with intermediate-dosed cytarabine showed satisfactory response rate and consolidation therapy with allo-HSCT after full or reduced-intensity conditioning further improved survival in these patients with similar outcome as reported for CPX-351.

Abstract 1018: SEL120, a CDK8/CDK19 inhibitor, possesses strong multilineage differentiation potential in AML

Abstract Background Acute myeloid leukemia (AML) is a heterogeneous disease characterized by rapid proliferation of leukemic blasts and high rate of acquired resistance to drugs. Differentiating agent ATRA has been established as a backbone of APL treatment, however its activity in other leukemias is limited. Knowing that Cyclin Dependent Kinase 8 (CDK8) can maintain tumor dedifferentiation and embryonic stem cell pluripotency, we investigated whether CDK8 inhibitor SEL120 could effectively target leukemia by induction of lineage commitment. SEL120 is a specific, selective inhibitor of CDK8 and its paralog CDK19. A first-in-human phase Ib clinical trial with SEL120 in AML or HR-MDS patients is currently ongoing. To better understand the mechanism of action of SEL120 in AML, we studied effects of SEL120 on differentiation as one of the important anti-leukemic activities of the compound. Methods Global transcriptional changes were analyzed by RNAseq at different time points to capture early and long-term effects of SEL120. Genome-wide profiling of DNA-binding was performed by CHIPseq. Cell cycle, proliferation and lineage specific markers were studied by flow cytometry. Differentiation potential of AML cells was studied in semi-solid methylcellulose-based media to asses colony formation of SEL120 treated blasts. Results SEL120 treatment leads to decreased CDK8 occupancy and increased RNA Pol II occupancy as well as changes in the peak distribution among promoter and enhancer regions. SEL120 could repress many “stemness” genes and induce the expression of genes involved in lineage commitment, including regulators of erythroid/megakaryocytic fate, such as RGS18, KLF1, FLI1 and GATA1/2. Moreover, for the first time, we showed that prolonged exposure of AML CD34+ cells to SEL120 could lead to colony formation in semi-solid media. Detailed analysis by flow cytometry at early and late time points reflected sequential changes in the expression of lineage-specific surface markers, characterizing differentiation and the presence of cells of myeloid or erythroid/megakaryocytic origin. Conclusion In addition to established role of CDK8 in regulation of tumor suppressor genes we present evidence for an essential role of CDK8 in lineage controlling-functions. Previously we reported specific cytotoxicity of SEL120 on cells positive for stemness markers such as CD34+. We further expanded on these studies, showing profound morphological changes in AML blasts during prolonged exposure to SEL120, correlating with increased expression of myeloid and erythroid/megakaryocytic markers. Based on these findings further studies are warranted to investigate the efficacy of SEL120 in anemia associated with bone marrow failures in AML and MDS. Combination of direct effects of SEL120 on viability of cells, with a strong differentiation potential, represents a promising profile for a drug in successful leukemia treatment. Citation Format: Urszula Pakulska, Elzbieta Adamczyk, Katarzyna Dziedzic, Katarzyna Wiklik, Michal Combik, Michal Mikula, Aniela Golas, Marta Obacz, Magdalena Masiejczyk, Przemyslaw Juszczynski, Magdalena Cybulska, Milena Mazan, Krzysztof Brzozka, Tomasz Rzymski. SEL120, a CDK8/CDK19 inhibitor, possesses strong multilineage differentiation potential in AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1018.

RUNX1 gene expression in Egyptian acute myeloid leukemia patients: may it have therapeutic implications?

BackgroundAcute myeloid leukemia represents the highest percentage of all adult acute leukemia variants. Runt-related transcription factor1 (RUNX1), a transcription factor with a known tumor suppressor function, was recently reported as a tumor promoter in acute myeloid leukemia (AML). We investigated the role of RUNX1 gene expression level in Egyptian AML patients and delineated its clinical significance.ResultsWe measured RUNX1 gene expression level using reverse transcription-quantitative polymerase chain reaction and found that the RUNX1 gene expression level was significantly higher than the control group (p < 0.001). Patients with FMS-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) mutations had a higher expression level of RUNX1 (p = 0.023). The male patients expressed a significantly higher level of RUNX1 (p = 0.046).ConclusionsThe RUNX1 gene is highly expressed in Egyptian AML patients. It has a relation to FLT3-ITD, which may give a clue that patients carrying this mutation may benefit from new treatments that target RUNX1 in the future. Further studies on a larger number of patients with different ethnic groups may give a clearer vision of the therapeutic implications of a new molecular target.

Cytogenetic and mutational analysis and outcome assessment of a cohort of 284 children with de novo acute myeloid leukemia reveal complex karyotype as an adverse risk factor for inferior survival

BackgroundAcute myeloid leukemia (AML) is rare in children. Although complex karyotype (CK) defined as ≥ 3 cytogenetic abnormalities is an adverse risk factor in adult AML, its prognostic impact on childhood AML remains to be determined.ResultsWe studied the prevalence, cytogenetic and mutational features, and outcome impact of CK in a cohort of 284 Chinese children with de novo AML. Thirty-four (12.0%) children met the criteria for CK-AML with atypical CK being more frequent than typical CK featured with -5/5q-, -7/7q-, and/or 17p aberration. Mutational prevalence was low and co-occurrence mutants were uncommon. Children with CK-AML showed shorter overall survival (OS) (5-year OS: 26.7 ± 10.6% vs. 37.5 ± 8.6%, p = 0.053) and event-free survival (EFS) (5-year EFS: 26.7 ± 10.6% vs. 38.8 ± 8.6%, p = 0.039) compared with those with intermediate-risk genetics. Typical CK tended to correlate with a decreased OS than atypical CK (5-year OS: 0 vs. 33 ± 12.7%.; p = 0.084), and CK with ≥ 5 cytogenetic aberrations was associated with an inferior survival compared with CK with ≤ 4 aberrations (5-year OS: 13.6 ± 11.7% vs. 50.0 ± 18.6%; p = 0.040; 5-year EFS: 13.6 ± 11.7% vs. 50.0 ± 18.6%; p = 0.048).ConclusionOur results demonstrate CK as an adverse risk factor for reduced survival in childhood AML. Our findings shed light on the cytogenetic and mutational profile of childhood CK-AML and would inform refinement of risk stratification in childhood AML to improve outcomes.

Double and single mixed-lineage leukemia-rearranged subclones in pediatric acute myeloid leukemia: a case report

BackgroundAcute myeloid leukemia (AML) is a disease with a significant amount of cytogenetic heterogeneity including mixed-lineage leukemia (MLL) gene rearrangements. Pediatric AML commonly has genetic rearrangements which involve chromosome 11q23 in 15–20% of cases, and these genetic abnormalities have been associated with a poorer prognosis (Grimwade et al. in Blood 92:2322–2333, 1998; Raimondi et al. in Blood 94:3707–3716, 1999; Lie et al. in Br J Haematol 122: 217–225). MLL rearrangements in AML have been shown to have multiple different fusion partners (Meyer et al. in Leukemia 23:1490–1499). Heterogeneity of these cytogenetic abnormalities makes it difficult to determine how to approach patients from a treatment standpoint. This difficulty is further complicated when patients have more than a single MLL rearrangement.Case presentationA 10-year-old Caucasian girl presented with easy bruising and was found to have acute myeloid leukemia. Her cytogenetics showed two different MLL rearrangements, t(9;11)(p22;q23) and t(11;19)(q23;p13.3). At initial presentation she had no other cytogenetic findings. She responded well to initial therapy and achieved remission following the first induction cycle and completed four rounds of chemotherapy. She subsequently had a relapse of her AML, and her cytogenetics were consistent with a single MLL rearrangement, t(9;11)(p22;q23), in addition to monosomy 7. She was treated with reduction therapy and a haplo-identical bone marrow transplant but ultimately succumbed to her disease.ConclusionMLL rearrangements are common in AML, but clinical significance continues to be elusive, and there is conflicting data on the prognostic significance. In the setting of multiple MLL rearrangements, there is concern for reduced survival, although treatment modifications are not currently done in this setting. This report details a case with multiple MLL rearrangements that initially responded to therapy but ultimately had disease progression with a selection of a leukemic clone containing a single MLL rearrangement.