Factor Acute Myeloid Leukemia Research Articles

Abstract 4181: AXL-specific single domain antibodies show diagnostic potential and anti-tumor activity in acute myeloid leukemia

Abstract Rationale: AXL expression has been identified as a prognostic factor in acute myeloid leukemia (AML) and is detectable in approximately 50% of AML patients. In this study, we developed AXL-specific single domain antibodies (sdAbs), cross-reactive for both mouse and human AXL protein, to non-invasively image and treat AXL-expressing cancer cells. Methods: AXL-specific sdAbs were induced by immunizing an alpaca with mouse and human AXL proteins. SdAbs were characterized using ELISA, flow cytometry, surface plasmon resonance and the AlphaFold prediction software. A lead compound was selected and labeled with 99mTc for evaluation as a diagnostic tool in mouse models of human (THP-1 cells) or mouse (C1498 cells) AML using SPECT/CT imaging. For therapeutic purposes, the lead compound was fused to a mouse IgG2a-Fc tail and in vitro functionality tests were performed including viability, apoptosis and proliferation assays in human AML cell lines and primary patient samples. Using these in vitro models, its anti-tumor effect was evaluated as a single agent, and in combination with standard of care agents venetoclax or cytarabine. Results: Based on its cell binding potential, cross-reactivity, nanomolar affinity and GAS6/AXL blocking capacity, we selected sdAb20 for further evaluation. Using SPECT/CT imaging, we observed tumor uptake of 99mTc-sdAb20 in mice with AXL-positive THP-1 or C1498 tumors. In THP-1 xenografts, an optimized protocol using pre-injection of cold sdAb20-Fc was required to maximize the tumor-to-background signal. Besides its diagnostic value, we observed a significant reduction in tumor cell proliferation and viability using sdAb20-Fc in vitro. Moreover, combining sdAb20-Fc and cytarabine synergistically induced apoptosis in human AML cell lines, while these effects were less clear when combined with venetoclax. Conclusions: Because of their diagnostic potential, sdAbs could be used to screen patients eligible for AXL-targeted therapy and to follow-up AXL expression during treatment and disease progression. When fused to an Fc-domain, sdAbs acquire additional therapeutic properties that can lead to a multidrug approach for the treatment of AXL-positive cancer patients. Citation Format: Niels Vandewalle, Hatice Satilmis, Emma M. Verheye, Rong Fan, Yanmeng Wang, Timo W. De Groof, Tessa Kerre, Nathan De Beule, Ann De Becker, Elke De Bruyne, Eline Menu, Karin Vanderkerken, Karine Breckpot, Nick Devoogdt, Kim De Veirman. AXL-specific single domain antibodies show diagnostic potential and anti-tumor activity 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 4181.

Identification of mIDH1 R132C/S280F Inhibitors from Natural Products by Integrated Molecular Docking, Pharmacophore Modeling and Molecular Dynamics Simulations.

Mutant isocitrate dehydrogenase 1 (mIDH1) is a common driving factor in acute myeloid leukemia (AML), with the R132 mutation accounting for a high proportion. The U.S. Food and Drug Administration (FDA) approved Ivosidenib, a molecular entity that targets IDH1 with R132 mutations, as a promising therapeutic option for AML with mIDH1 in 2018. It was of concern that the occurrence of disease resistance or recurrence, attributed to the IDH1 R132C/S280F second site mutation, was observed in certain patients treated with Ivosidenib within the same year. Furthermore, it should be noted that most mIDH1 inhibitors demonstrated limited efficacy against mutations at this specific site. Therefore, there is an urgent need to investigate novel inhibitors targeting mIDH1 for combating resistance caused by IDH1 R132C/S280F mutations in AML. This study aimed to identify novel mIDH1 R132C/S280F inhibitors through an integrated strategy of combining virtual screening and dynamics simulations. First, 2000 hits were obtained through structure-based virtual screening of the COCONUT database, and hits with better scores than -10.67 kcal/mol were obtained through molecular docking. A total of 12 potential small molecule inhibitors were identified through pharmacophore modeling screening and Prime MM-GBSA. Dynamics simulations were used to study the binding modes between the positive drug and the first three hits and IDH1 carrying the R132C/S280F mutation. RMSD showed that the four dynamics simulation systems remained stable, and RMSF and Rg showed that the screened molecules have similar local flexibility and tightness to the positive drug. Finally, the lowest energy conformation, hydrogen bond analysis, and free energy decomposition results indicate that in the entire system the key residues LEU120, TRP124, TRP267, and VAL281 mainly contribute van der Waals forces to the interaction, while the key residues VAL276 and CYS379 mainly contribute electrostatic forces.

Identification of red blood cell distribution width as a prognostic factor in acute myeloid leukemia

Many prognostic factors have been identified in acute myeloid leukemia (AML). In this study, we investigated novel prognostic biomarkers using machine learning and Cox regression models in a prospective cohort of 591 patients with AML and tried to identify potential therapeutic targets based on transcriptomic data. We found that elevated red blood cell distribution width (RDW) at diagnosis was an adverse prognostic factor for AML, independent of the 2022 European LeukemiaNet (ELN2022) genetic risk. As a continuous variable, higher RDW was associated with shorter overall survival (OS) (hazard ratio [HR] 1.087, 95% confidence interval [CI] 1.036-1.139, p < 0.001) and event-free survival (EFS) (HR 1.078, 95% CI 1.033-1.124, p < 0.001). Elevated RDW returned to normal after consolidation therapy, which indicated that leukemia cells resulted in abnormal RDW. We further investigated the relationship between RDW and transcriptome in another cohort of 191 patients with AML and public datasets using gene set enrichment analysis (GSEA) and cell-type identification by estimating relative subsets of RNA transcripts (CIBERSORT). We found that patients in the high-RDW group were significantly enriched in the positive regulation of erythroid differentiation and inflammation-related pathways. Finally, we identified the inflammation-associated gene IL12RB2 and verified its prognostic relevance with patients with AML in public databases, suggesting it as a potential therapy target.

[Analysis of the Efficacy and Prognosis Factors of Acute Myeloid Leukemia with a Combination Therapy of Venetoclax].

To explore the efficacy and prognosis factors of acute myeloid leukemia with a combination therapy of venetoclax. A retrospective analysis was performed on the clinical data of AML patients treated with a combination therapy of venetoclax from March 2020 to April 2023 in the First Hospital of Lanzhou University. The efficacy, adverse reactions and survival were observed, and the influencing factors were analyzed. A total of 74 AML patients were included in this study, including 43 initially treated AML and 31 relapsed or refractory AML (R/R AML). The median age of 43 initially treated AML patients was 65 years old, the composite complete remission (cCR) rate was 67.4% (29/43), the objective response rate (ORR) was 72.1% (31/43), and the median overall survival (OS) was 17.3 months. The median age of 31 R/R AML patients was 51 years old, with a cCR rate of 38.7% (12/31), an ORR of 58.1% (18/31), and a median OS of 7.1 months. Sex, the blood cell count before VEN, gene mutation and prognosis stratification were related to whether to obtain cCR. Failure to obtain cCR was an independent risk factor for adverse outcomes. A combination therapy of venetoclax is safe and efficacious for AML. Its efficacy and survival are affected by molecular biology, cytogenetics and other factors.

Evaluation of Neddylation and Apoptosis-Related Gene Expression in Patients with Acute Myeloid Leukemia

Introduction Regulated cell death (RCD) is a process commonly dysregulated in patients (pts) with acute myeloid leukemia (AML), resulting in the survival of genetically unstable cells. Apoptosis, as the best-known form of RCD, interacts with neddylation, a process that contributes to tumorigenesis and apoptosis resistance. Aims This study was conducted to determine the expression of the selected genes involved in the neddylation pathway and to assess their clinical importance along with selected interacting proapoptotic genes. In addition, we correlated the expression levels of these genes with known prognostic factors in AML and determined their impact on the survival of AML pts. Methods The expression of selected proapoptotic genes BIK, BAX, BAK, BCL2L11, BBC3 , PMAIP1 CASP3, CASP7, and neddylation-related genes: CUL1, CUL2 , CUL4A, CUL5, CUL7, CUL9, NEDD8 was determined in duplicates in bone marrow samples of newly diagnosed AML pts. Real-Time PCR was carried out using the TaqMan chemistry and the QuantStudio7 thermal cycler (Applied Biosystems-Thermo Fisher Scientific). Gene expression was normalized using reference genes selected by the Normirazor tool. The normalization was performed by ∆Ct = Ct (reference) - Ct (mRNA of interest). This approach results in higher values for higher mRNA expression. Differential expression analysis was performed using a Welch t-test. Kaplan-Meier method with the log-rank test was used to assess overall survival (OS). Results In a prospective study, we included 40 newly diagnosed AML pts (mean age 62; range 26-87) with a median (m) follow-up of 6.7 months (95% CI: 4.5-9.1). High-risk AML was present in 52.5%, intermediate-risk in 32.5%, and low-risk in 15% of pts, according to European LeukemiaNet (ELN) 2022 risk stratification. NPM1 mutation was present in 23%, whereas FLT3 ITD or TKD mutation in 25% of pts. Intensive treatment was introduced in 43% of pts, while 25% were treated with azacitidine+venetoclax. Non-intensive treatment with azacitidine in monotherapy was administered to the remaining pts. BAK expression was detected in 70%, BIK in 98%, while expression of the other genes was found in all pts. In the high-risk group, we detected lower expression of both proapoptotic BAX (fold change - FC=0.7, p=0.01) and BIK gene (FC=0.4, p=0.01), as well as lower expression of CUL9 (FC=0.7, p=0.03). Accordingly, in pts with NPM1 mutation we revealed upregulation of BIK (FC=6.1, p=0.001), and CUL9 (FC=1.6, p=0.01). Whereas pts with FLT3 mutation had lower expression of proapoptotic BBC3 (FC=0.5, p=0.01), and neddylation-related CUL1 (FC=0.6, p=0.01). In cases of initial bone marrow blasts infiltration above median value (&amp;gt;49%) BCL2L11 CUL5 , NEDD8 and PMAIP1were downregulated ( p=0.001-0.02), while CUL7 ( p=0.00) and CASP3 ( p=0.04) were upregulated. Patients with initial white blood cell count &amp;gt;20 G/l had lower expression of BCL2L11 ( p=0.005), and CUL4A ( p=0.02), while higher expression of CASP7 ( p=0.04). The mOS in the study cohort was not reached. OS was longer in pts with higher (&amp;gt; -2.061, cutoff was determined with CutOff Finder) BAX expression (log-rank: p=0.03) [Figure 1]. The mOS was not reached in this group, whereas the mOS in pts with lower BAX expression was 1.3 months (95% CI: 0.6-1.3). In univariate Cox regression analysis, higher BAX expression (HR 0.31, 95%CI: 0.11-0.84, p=0.022), higher albumin level (HR 0.13, 95%CI: 0.04-0.41, p=0.0004), and intensive treatment (HR 0.18, 95%CI: 0.04-0.69, p=0.012) were factors influencing the outcome. In the multivariate model for OS, higher expression of BAX (HR 0.03, 95%CI: 0.003-0.33, p=0.004) retained its significant protective effect in the context of established prognostic factors [Table 1]. Conclusions To our knowledge, we were the first to evaluate such a complex spectrum of neddylation and apoptosis-related genes in AML. We revealed dysregulation of these genes in AML. Our data indicate that upregulation of BAX at the diagnosis is associated with longer OS. Moreover, we reported significantly lower BAX expression in ELN high-risk group. Thus, indicating BAX may be a potential prognostic factor in AML. Our preliminary results did not prove significant differences in the effect of neddylation gene expression levels on the prognosis of AML patients. As studies are emerging on the potential of neddylation-targeted therapies, we are conducting further research to verify the effect of neddylation on AML.

G Protein-Coupled Receptor 183 Plays a Prognostic Factor in Acute Myeloid Leukemia By Regulating Immune Regulation

CONCLUSION G protein-coupled receptor 183 (GPR183) is a critical oligoheptameric transmembrane receptor that regulates cellular immune responses. Its malfunction causes a variety of immunological and inflammatory disorders. GPR183 overexpression enhances B cell migration from the germinal center and plays a significant role in the pathogenesis of Chronic myeloid leukemia. However, its prognostic value and regulatory role in AML are not clear. This study aimed to reveal the prognostic significance of GPR183 in AML and the mechanism of its regulatory role in AML. The expression level of GPR183 in 300 deno AML patients was analyzed by RT-qPCR. Combined with the clinical characteristics and treatment response of patients, the expression characteristics and clinical prognostic significance of GPR183 in AML were analyzed. The regulatory function of GPR183 in AML was investigated using shRNA-targeted knockdown or over expression of GPR183 in AML, and the impact of knockdown of GPR183 on tumor burden and survival of AML mice was further confirmed in vivo using an AML mouse model. By using bioinformatics techniques, the potential regulatory mechanisms of GPR183's effects on AML cells were further examined and verified. The expression level of GPR183 in AML cells was higher than that in normal samples, and its expression was significantly higher in older AML patients than in younger ones. By univariate and multivariate analyses, it was found that the high expression of GPR183 was an independent poor prognostic factor in AML patients for overall survival(OS) and eventfree survival(EFS), and the knockdown of GPR183 in AML cells could inhibit the proliferation of AML cells, promote apoptosis, and inhibit the migration of AML cells, and the in vivo model of AML mice showed that the knockdown of GPR183 could reduce the tumor load and prolong the survival of AML mice. Overexpression of GPR183 in AML cells promoted the growth and increased the drug resistance of AML drug. Single-gene GESA revealed that AML patients with high GPR183 expression had HALLMARK_ALLOGRAFT_REJECTION, HALLMARK_TNFA_SIGNALING_VIA_NFKB, HALLMARK_INFLAMMATORY_RESPONSE, HALLMARK_COMPLEMENT, and HALLMARK_CLEARING_VIA_NFKB, and HALLMARK_INFLAMMATORY_RESPONSE. COMPLEMENT, HALLMARK_IL6_JAK_STAT3_SIGNALING, and HALLMARK_IL2_STAT5_SIGNALING pathways were activated, and all of these top 5 activated pathways were associated with immune regulation. GPR183 plays a prognostic factor in Acute Myeloid Leukemia by regulating immune regulation

Identification of ZFP91 As the Master Transcription Factor for EVI1 Gene Expression

Ecotropic viral integration site 1 (EVI1) is essential for hematopoiesis, particularly for the maintenance of hematopoietic stem cells. Aberrant expression of EVI1 is widely known to be an independent poor prognostic factor in acute myeloid leukemia (AML). Previous studies have shown that enhancer hijacking or gene fusion caused by chromosomal translocation or inversion involving 3q26 leads to EVI1 overexpression. Intriguingly, however, aberrant EVI1 expression is seen in many cases of AML without 3q26 rearrangement. The prognosis of these cases is similarly dismal, but the mechanism that causes aberrant EVI1 expression in the absence of 3q26 chromosomal abnormalities is largely unknown. To identify key regulatory factors essential for EVI1 expression in AML, we performed a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) knockout screen. We knocked in the green fluorescent protein (GFP) coding sequence at the EVI1 locus in the human myeloid leukemia cell line K562, which expresses high levels of EVI1 without a 3q26 chromosomal aberration. After transduction of a genome-wide CRISPR/Cas9 knockout library, transduced cells were separated into GFP High and GFP Low subpopulations by flow cytometry. By sequencing the uniquely attached barcodes, we estimated the effect of gene knockout for each gene. Genes counted more frequently in the GFP Low fraction were listed as candidates for activators of EVI1. The top 13 genes, including three apparent positive controls such as EVI1 and two ribosome-related genes, had a false discovery rate (FDR) of less than 1%. We validated these candidate genes in K562 and two other EVI1-overexpressing AML cell lines by transducing individual single guide RNAs targeting these genes. These analyses identified zinc finger protein 91 (ZFP91) as a potential regulatory factor that significantly upregulates EVI1 expression common to the three independent myeloid leukemia cell lines. ZFP91 is an atypical E3 ubiquitin ligase, a DNA-binding protein that functions as a transcription factor. ZFP91 promotes cell growth and inhibits apoptosis in AML and has been shown in previous studies to activate multiple oncogenic pathways, including nuclear factor-kappa B (NF-κB), mitogen-activated protein kinase (MAPK), and hypoxia-inducible factor-1α (HIF-1α). We demonstrated that knockout of ZFP91 in K562 substantially reduced EVI1 protein expression to undetectable levels by Western blotting (Figure 1). EVI1 mRNA expression also decreased dramatically after ZFP91 knockout. These results suggest that ZFP91 may play an indispensable role in EVI1 transcription. We performed chromatin immunoprecipitation quantitative polymerase chain reaction (ChIP-qPCR) targeting the EVI1 promoter in K562 and demonstrated that ZFP91 knockout clones showed approximately 10-fold weaker signals for active histone modifications (H3K4me3 and H3K27ac) at the EVI1 promoter (Figure 2). In addition, chromatin immunoprecipitation sequencing (ChIP-seq) for ZFP91 in K562 revealed the presence of a distinct peak as well as the ZFP91-specific motif sequence within the EVI1 promoter region. These data strongly suggest that ZFP91 functions as an essential transcription factor for EVI1 gene overexpression in some EVI1-associated myeloid leukemias. We confirmed that ZFP91 knockout in K562 inhibited cell proliferation, which is consistent with previous studies in other AML cell lines. Based on these results, we believe that ZFP91 has the potential to serve as a therapeutic target in EVI1-associated AML. To conclude, we have identified the transcription factor ZFP91 as a novel prominent activator of EVI1 expression in some EVI1-associated AML. Our study suggests that ZFP91 may be the key to unraveling the pathogenesis of EVI1-associated AML and an attractive therapeutic target in EVI1-associated AML.

Integrated Analysis of KIT Exon 17 Mutations and Flow-MRD Refines Risk Stratification in Pediatric Acute Myeloid Leukemia with RUNX1:: RUNX1T1

INTRODUCTION: Pediatric acute myeloid leukemia (AML) with RUNX1:: RUNX1T1 is considered a favorable risk group, with a high probability of event-free survival (EFS) in recent clinical trials. However, 20-30% of patients still experience relapse within three years and some patients have poor outcome. Several prognostic factors, including secondary genetic abnormalities and treatment response, have been previously reported. Flow cytometry-based measurable residual disease (flow-MRD) has been suggested as a poor prognostic factor in AML with RUNX1:: RUNX1T1. Additionally, KIT mutations, particularly mutations of exon 17, have been associated with poor EFS in both adult and pediatric patients. However, the combined impact of KIT exon 17 mutations and flow-MRD on prognosis has not been thoroughly investigated. This study aims to evaluate the clinical and genetic features of pediatric patients with RUNX1:: RUNX1T1-positive AML enrolled in the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG) AML-12 trial and determine if integrated analysis of flow-MRD and KIT exon 17 mutations can improve risk stratification. METHOD: A total of 101 pediatric AML patients with RUNX1:: RUNX1T1 were enrolled in the study. The patients were treated in the JPLSG AML-12 trial, and their clinical and genetic characteristics were analyzed. Targeted capture sequencing was performed on bone marrow samples obtained at diagnosis. The custom gene panel was designed for mutation profiling of pediatric AML, including 504 genes. Flow-MRD was centrally monitored at the end of induction 1 (EOI1) and the end of induction 2 but was not used to guide subsequent therapies. Leukemia-associated immunophenotypes were identified in diagnostic specimens and marker combinations that allowed detection of leukemia with a sensitivity of at least 0.1% were applied to subsequent samples. RESULTS: Patients with KIT exon 17 mutations had a higher white blood cell count at diagnosis and a significantly higher rate of MRD positivity (≥0.1%) at EOI1 compared to patients without KIT exon 17 mutations. Immunophenotypic analysis showed no significant differences in CD117 or CD33 expression between patients with and without KIT exon 17 mutations; whereas CD19 expression was observed less frequently and, although not significantly, CD56 expression was observed more frequently in the patients with KIT exon 17 mutations. The genetic landscape analysis revealed that KIT mutations were the most common and tended to coexist with ARID1B mutations, while NRAS mutations were mutually exclusive. The 5-year EFS and overall survival (OS) for the entire cohort were 67.3% and 82.7%, respectively. Negative MRD at EOI1 was associated with significantly superior 5-year EFS and OS (71.6% and 85.9%) compared to positive MRD (12.5% and 37.5%)(p&amp;lt;0.01). Patients with KIT exon 17 mutations had significantly inferior 5-year EFS and OS (46.9% and 62.2%) compared to those without KIT exon 17 mutations (76.8% and 92.4%) (p&amp;lt;0.01). Furthermore, we evaluated a prognostic impact of MRD within the KIT exon 17 mutated and non-mutated cases. As for the non-mutated cases, the 5-year OS in the patients with negative MRD was 92.2%. Among the KIT-mutated cases, the 5-year OS in the patients with negative and positive MRD was 68.0% and 33.3%, respectively. CONCLUSION: Patients with both non-mutated KIT exon 17 and negative MRD have the best prognosis, while positive MRD and KIT exon 17 mutations are associated with poorer outcomes. These findings indicated that integrated analysis of flow-MRD and KIT exon 17 status enables optimal risk assignment strategies in pediatric AML with RUNX1:: RUNX1T1.

Easyab Unravels Oncokinase Driven Regulation of Splicing Factors in AML

Acute myeloid leukemia (AML) is the most commonly diagnosed leukemia in adults. Gene mutations involved in various cellular functions are known to cause AML. A large proportion of AML is driven by aberrant activation of kinases and signal transducers such as FLT3, cKIT, PTPN11, JAK2, KRAS, NRAS, and to a lesser extent, JAK1, JAK3, and CSF3R. Treatment for AML has remained the same in the past five decades. Recent developments led to the availability of targeted therapies and enabled the practice of additional drugs like tyrosine kinase inhibitors. However, drug resistance and tolerance mechanism remain a challenge to finding a cure for the disease. Though we can identify and target kinase mutations individually, we have yet to learn how these mutations, like FLT3-ITD, change the cell's intrinsic signalling environment and cooperate with other oncogenes. Understanding these signalling behaviors is crucial for effective treatment outcomes and avoiding the development of drug resistance and tolerance mechanisms. Protein post-translational modifications (PTM) such as phosphorylation, ubiquitination, methylation, control the activity, fate, and spatial location of the proteins, thereby controlling processes such as signal transduction. Mass spectrometry-based proteomics offers unbiased profiling of PTMs. However, comprehensive analyses of PTMs require conventional antibody-based enrichment approaches and large starting materials, laborious, low throughput, cost prohibitive, and time-consuming, thus limiting the application to cell line-based studies. It also limits application in exploring unbiased-global cell signaling in patient samples. To overcome these limitations, we developed a loss-less sample preparation method named ‘EasyAb’. We show that our short, high throughput and low-cost approach enables capturing PTM peptides from very small inputs. As proof of principle, we enriched tyrosine-phosphorylated peptides from Molm13 and MV4-11 cell lines expressing FLT3-ITD kinase. Enrichment resulted in the ultra-deep identification of 4,416 tyrosine phosphosites, double the number of sites found in the conventional protocols, and took only one day of processing. About 40 % of the tyrosine phosphorylated proteins enriched are membrane proteins, including seven tyrosine phosphorylation sites on FLT3 kinase, of which FLT3 pY401 is a novel site discovered. To test the applicability of the EasyAb protocol in patient samples, we performed phosphotyrosine profiling of AML samples expressing FLT3 WT (n=9) and FLT3-ITD (n=5) mutation. EasyAb enrichment for phosphotyrosine peptides resulted in identifying 6,339 phosphotyrosine sites, of which 2300 sites were quantified. Comparing FLT3WT and ITD samples revealed more than 500 phosphosites significantly regulated. The significantly changing sites include several known signalling molecules such as STAT5, SYK, BTK, GAB2, GRB2, and PTPN11. The data also revealed differential tyrosine phosphorylation of proteins involved in various cellular functions such as autophagy, apoptosis, cell cycle, DNA damage repair proteins, phosphatases, and kinases. Interestingly, we also uncovered a class of proteins belonging to mRNA processing and splicing pathways distinctly tyrosine phosphorylated in FLT3-ITD samples. It is known that splicing factor mutations drive leukemia. Conversely, little is known about the regulatory mechanism of splicing factors by singling in cancer. To further investigate FLT3-ITD signalling regulation of splicing factors, we treated Molm13 and MV4-11 cells with Quizartinib and profiled tyrosine phosphorylation. Inhibition of FLT3-ITD lead to downregulation in tyrosine phosphorylation of several splicing factors. We have previously reported that splicing factor phosphorylation by JAK2 kinase plays a crucial role in Jak inhibitor persistence phenotype. Importantly, FLT3-ITD-mediated tyrosine phosphorylation-based regulation of splicing factors is unknown, and this process could be crucial for FLT3-ITD-mediated tumorigenesis. The functional significance of this novel regulatory mechanism is being studied. In conclusion, we have developed an effective tool for studying post-translational modifications that require antibody-based enrichment (tyrosine phosphorylation, ubiquitination, methylation, acetylation, and sumoylation) relatively quickly in primary patient samples.

Association between Cytogenetic Risk and Obesity in Patients with Acute Myeloid Leukemia (AML): A Meta-Analysis

Background: The global obesity epidemic has been well characterized over decades of research, linking increased body mass index (BMI) ≥ 25 to multiple medical conditions including increased cancer risk, morbidity, and mortality. The International Agency for Research on Cancer (IARC) group found a positive correlation between obesity to 13 cancers and multiple studies suggest leukemia should be added to this list. The causation behind this risk in leukemia however is not well understood and the prognostic factors have not been well characterized. Acute Myeloid Leukemia (AML) specifically defines risk based on cytogenetics between favorable, intermediate and unfavorable which significantly affects treatment decisions. The link between obesity and cytogenic risk factors in AML have not previously been thoroughly evaluated. To address this knowledge gap, we conducted a comprehensive meta-analysis aiming to evaluate the cytogenetic risk with obesity and prognostic significance in patients with AML. Methods: A systematic search was conducted across major databases until June 2023, using tailored search structures, subject headings, and keywords developed in collaboration with a medical research librarian. Two investigators independently screened the studies for inclusion. The inclusion criteria encompassed retrospective and prospective clinical studies that reported on the association between established cytogenetic classifications and standardized measures of excessive weight (BMI 25-30) and obesity (BMI ≥ 30). Meta-analysis was conducted using restricted maximum likelihood random-effects model. Results: From an initial pool of 1673 identified studies, eleven studies met the inclusion criteria, encompassing a total of 5887 patients. Among these patients, 1363 met the criteria for obesity (BMI ≥ 30) and 2176 met the criteria for excessive weight (BMI 25-30). The median patient's age was 54 (ranging from 15 to 89). The included studies originated from diverse geographic regions, including the USA, Japan, and France. Baseline patient characteristics are summarized in Table 1. Compared to overweight patients, our meta-analysis revealed a significant association between obesity and higher odds of unfavorable/intermediate cytogenetic risk (odds ratio [OR] 1.27, 95% CI [1.04; 1.54]), as seen in Figure 1. However, no meaningful statistical inference could be made regarding the association between obesity and specific gene mutations (such as FLT3, NPM1, CEBPa, TET2, IDH1, IDH2, RUNX1, and KRAS) due to the limited number of studies and small number of patients involved. Conclusion: Our meta-analysis demonstrates a significant association between obesity and unfavorable/ intermediate cytogenetic risk in patients with AML. Although the precise biological mechanisms remain elusive, several potential explanations have been proposed. These include the impact of obesity leading to chronic low-grade inflammation, altered adipokine secretion and insulin resistance, and metabolic changes on leukemic cell growth and survival. Despite the limitations of unknown cytogenetic risk in 590 patients and limited mutation data in some studies, our findings suggest that obesity may impact the prognosis of AML. Future research should address these limitations and provide a more comprehensive understanding of the intricate interplay between obesity, cytogenetics, and AML prognosis.

Acute Myeloid Leukemia: Selecting Induction Therapy Based on Biological Disease Factors

Greater insight into the role of biologic disease factors in acute myeloid leukemia (AML) has led to more effective and personalized treatment options. Data presented at the NCCN 2023 Annual Congress: Hematologic Malignancies reflected the rapidly evolving treatment landscape, showcasing the significance of combination therapies and precision medicine in managing different AML subtypes. For patients deemed fit for induction with favorable-risk cytogenetics, the standard of care has moved to combining the 7 + 3 regimen with gemtuzumab ozogamicin. Alternatively, for patients with FLT3-mutated disease, a more intensified approach involving the addition of midostaurin or newer therapies, such as quizartinib, to standard induction treatment has shown improved outcomes. For patients with AML who are unfit for standard induction, treatments combining hypomethylating agents with venetoclax, as well as therapies involving IDH1/2 and FLT3 inhibitors for specific genetic subtypes, offer encouraging alternatives in managing the disease.

Reversal of MYB-dependent suppression of MAFB expression overrides leukaemia phenotype in MLL-rearranged AML

The transcription factor MYB plays a pivotal role in haematopoietic homoeostasis and its aberrant expression is involved in the genesis and maintenance of acute myeloid leukaemia (AML). We have previously demonstrated that not all AML subtypes display the same dependency on MYB expression and that such variability is dictated by the nature of the driver mutation. However, whether this difference in MYB dependency is a general trend in AML remains to be further elucidated. Here, we investigate the role of MYB in human leukaemia by performing siRNA-mediated knock-down in cell line models of AML with different driver lesions. We show that the characteristic reduction in proliferation and the concomitant induction of myeloid differentiation that is observed in MLL-rearranged and t(8;21) leukaemias upon MYB suppression is not seen in AML cells with a complex karyotype. Transcriptome analyses revealed that MYB ablation produces consensual increase of MAFB expression in MYB-dependent cells and, interestingly, the ectopic expression of MAFB could phenocopy the effect of MYB suppression. Accordingly, in silico stratification analyses of molecular data from AML patients revealed a reciprocal relationship between MYB and MAFB expression, highlighting a novel biological interconnection between these two factors in AML and supporting new rationales of MAFB targeting in MLL-rearranged leukaemias.

The causal relationship between gut microbiota and leukemia: a two-sample Mendelian randomization study

BackgroundThe association between gut microbiota and leukemia has been established, but the causal relationship between the two remains unclear.MethodsA bidirectional two-sample Mendelian randomization (MR) was used to analyze the causal relationship between gut microbiota and leukemia. Microbiome data (n = 14,306) and leukemia (n = 1,145) data were both sourced from European populations. Single nucleotide polymorphisms (SNPs) were selected as instrumental variables based on several criteria. We employed various MR methods, such as the inverse variance weighted (IVW) method, to evaluate the causal effect between exposure and outcomes and conducted sensitivity analyses to validate the heterogeneity and pleiotropy of the instrumental variables.Results5,742 qualified instrumental variables were included. In the primary MR results, a total of 10 gut microbial taxa were associated with leukemia risk. Genus Blautia and genus Lactococcus are risk factors for acute lymphoblastic leukemia [genus Blautia odds ratio (OR): 1.643, 95% confidence interval (CI): 1.592 ~ 1.695, Adjusted p &amp;lt; 0.001; genus Lactococcus OR: 2.152, 95% CI: 1.447 ~ 3.199, Adjusted p = 0.011]. Genus Rikenellaceae RC9 gut group, genus Anaerostipes, genus Slackia, and genus Lachnospiraceae ND3007 group are risk factors for acute myeloid leukemia [genus Rikenellaceae RC9 gut group OR: 1.964, 95% CI: 1.573 ~ 2.453, Adjusted p &amp;lt; 0.001; genus Anaerostipes OR: 2.515, 95% CI: 1.503 ~ 4.209, Adjusted p = 0.017; genus Slackia OR: 2.553, 95% CI: 1.481 ~ 4.401, Adjusted p = 0.022; genus Lachnospiraceae ND3007 group OR: 3.417, 95% CI: 1.960 ~ 5.959, Adjusted p = 0.001]. Genus Ruminococcaceae UCG011 and genus Ruminococcaceae UCG014 were risk factors for chronic myeloid leukemia (genus Ruminococcaceae UCG011 OR: 2.010, 95% CI: 1.363 ~ 2.963, Adjusted p = 0.044; genus Ruminococcaceae UCG014 OR: 3.101, 95% CI: 1.626 ~ 5.915, Adjusted p = 0.044). Genus Slackia was a protective factor for acute lymphoblastic leukemia (genus Slackia OR: 0.166, 95% CI: 0.062 ~ 0.443, Adjusted p = 0.017). Family Acidaminococcaceae was a protective factor for acute myeloid leukemia (family Acidaminococcaceae OR: 0.208, 95% CI: 0.120 ~ 0.361, Adjusted p &amp;lt; 0.001). Genus Desulfovibrio was a protective factor for chronic lymphoblastic leukemia (genus Desulfovibrio OR: 0.581, 95% CI: 0.440 ~ 0.768, Adjusted p = 0.020). Sensitivity analysis revealed no heterogeneity or pleiotropy between SNPs.ConclusionThis study revealed the causal relationship between the gut microbiota and leukemia, and identified potential pathogenic bacteria and probiotic taxa associated with the onset of leukemia. This research may aid in the early detection of various types of leukemia and offer a new direction for the prevention and treatment of leukemia.

A small molecule BCL6 inhibitor as chemosensitizers in acute myeloid leukemia

BCL6 is a transcriptional repressor that regulates multiple genes involved in immune cell differentiation, DNA damage repair, cell cycle, and apoptosis, and is a carcinogenic factor in acute myeloid leukemia (AML). AML is one of the four major types of leukemia with the 5-year survival rate of patients is less than 20% and chemotherapy resistance remains the major obstacle to the treatment failure of AML. We identified WK499, a small molecule compound that can bind to BCL6BTB structure. Treatment with WK499 hinders the interactions between BCL6 with its corepressor proteins, resulting in a remarkable change of BCL6 downstream genes and anti-proliferative effects in AML cells, and inducing cell cycle arrest and apoptosis. We verified that AraC and DOXo could induce BCL6 expression in AML cells, and found that WK499 had a synergistic effect when combined with chemotherapeutic drugs. We further proved that WK499 and AraC could achieve a better result of inhibiting the growth of AML in vivo. These findings indicate that WK499, a small molecule inhibitor of BCL6, not only inhibits the proliferation of AML, but also provides an effective therapeutic strategy for increasing AML sensitivity to chemotherapy.

Fluorescence In Situ Hybridization (FISH) for Detection of c-MYC Amplifications in AML Patients: A Potential Marker for Risk Stratification

Abstract Background c-MYC is a transcription factor (located on 8q24) which regulates genes involved in proliferation, cell growth, differentiation, and apoptosis. The c-MYC amplification is related to a rapid proliferation of the affected cell clone in patients with myeloid neoplasms. Gene amplification (amp) is one of the basic mechanisms connected with over-expression of oncogenes. c-MYC is routinely analyzed in mature lymphoid neoplasms especially in Burkitt’s Lymphoma while not assessed for acute myeloid leukemia (AML).Despite the fact the c- MYC oncogene is commonly over-expressed in AML it has remained an elusive target. Routine assessment for c- MYC in AML may pave the way for targeting the c-MYC in treatment of AML patients. Several experiments are ongoing investigating both direct and indirect c-MYC targeting therapies and further establishing cMYC as an oncogene of central importance to AML pathogenesis and treatment. Aim of the Work In this study we aimed to define the prognostic value of c-MYC amplification in adult AML patients, and to relate it to other prognostic factors in AML. Patients and Methods The study was a randomized control study, conducted at the Clinical Pathology Department, Ain-Shams University Hospitals during the period from January 2019 till June 2020. It was carried on 50 adult AML patients (30 newly diagnosed and 20 on Therapy) and 10 healthy age-matched volunteers as control. All subjects underwent full history taking, thorough clinical examination, laboratory investigations including; CBC, Examination of Leishman’s stained peripheral blood films, Bone marrow aspiration and examination of Leishman’s stained bone marrow smears, Cytochemical studies using Myeloperoxidase stain, Immunophenotyping of bone marrow or peripheral blood samples and Fluorescence in situ hybridization analysis (FISH). Results we had 9 patients with c-MYC amplification (6 in the newly diagnosed group and 3 in the on therapy group). So the percentage of c-MYC amplification in our patients was 9/50 (18%). To further elucidate the effect of c-MYC amplification on our patients’ clinical, laboratory and prognostic factors, an analytical comparison was done between negative and positive patients for cMYC amplification as regard-studied parameters in each group of patients. Both group of patients showed a statistical significant association between c-MYC amplification and outcome where the majority of patients with positive c-MYC amplification achieved incomplete remission. In addition, c-MYC amplification showed a statically significant association with Trisomy 8 in both patients’ groups and with 17p deletion and hepatosplenomegaly in on therapy group. Univariate logistic regression analysis for the association between c-MYC amplification and outcome showed statistically significant association between c-MYC amplification and incomplete remission in newly diagnosed AML patients with P-value = 0.013 and odds ratio (95% CI) of 14.0. Conclusion Our study has shown that c-MYC amplification is associated with adverse prognosis, poor treatment outcome and it might be involved in the process of AML progression. Targeting cMYC may be of value in the development of new-targeted AML therapeutics.

Spi1 R235C point mutation confers hypersensitivity to radiation-induced acute myeloid leukemia in mice

Ionizing radiation (IR) is a risk factor for acute myeloid leukemia (rAML). Murine rAMLs feature both hemizygous chromosome 2 deletions (Del2) and point mutations (R235) within the hematopoietic regulatory gene Spi1. We generated a heterozygous CBA Spi1 R235 mouse (CBASpm/+) which develops de novo AML with 100% incidence by ∼12months old and shows a dose-dependent reduction in latencyfollowing X-irradiation. These effects are reduced on an AML-resistant C57Bl6 genetic background. CBASpm/Gfp reporter mice show increased Gfp expression, indicating compensation for Spm-induced Spi1 haploinsufficiency. Del2 is always detected in both de novo and rAMLs, indicating that biallelic Spi1 mutation is required for AML. CBASpm/+ mice show that a single Spm modification is sufficient for initiating AML development with complete penetrance, via the "two-hit" mechanism and this is accelerated by IR exposure. Similar SPI1/PU.1 polymorphisms in humans could potentially lead to enhanced susceptibility to IR following medical or environmental exposure.

Association of social deprivation with survival in younger adult patients with AML: an Alliance study.

Survival of patients with acute myeloid leukemia (AML) is influenced by genetic factors, age, and race. Social deprivation is increasingly recognized as an important contributor to disparities in cancer outcomes, but studies of adult AML are lacking. We analyzed associations between social deprivation index (SDI) and outcome in 1,893 patients with AML treated on Cancer and Leukemia Group B/Alliance for Clinical Trials in Oncology frontline protocols. Patients with low (first quartile, lowest deprivation) and high (quartiles 2-4) SDI were analyzed for associations with baseline clinical, cytogenetic and molecular features, and outcomes. Except for racial-ethnic identity, SDI was not associated with baseline clinical characteristics. Patients aged <60 years with high SDI harbored BCOR (3% vs 7%, P=.03), IDH1 (6% vs 11%, P=.03) and STAG2 (2% vs 5%, P=.02) mutations less often than patients with low SDI, suggesting possible differences in the underlying disease biology. High SDI patients had shorter disease-free (median: 1.5 vs 2.8 years, P=.02) and overall survival (OS; median: 1.9 vs 3.0 years, P=.005) than low SDI patients. In multivariable analysis, high SDI was associated with worse OS (HR: 1.28, 95% CI: 1.06-1.55, P=.001). Survival differences occurred mainly in the 2017 European LeukemiaNet favorable genetic-risk group. There were no significant associations between SDI and molecular features or survival among patients aged ≥60 years. We conclude that high SDI associated with worse survival, independent from other pretreatment prognostic factors, in younger AML patients. The role of socioeconomic factors in AML should be further investigated to overcome potentially avoidable survival disparities.

Platelet is an unfavorable prognostic biomarker and associated with leukemia stem cells and immunomodulatory factors in acute myeloid leukemia.

Many clinical features, besides cytogenetic and molecular abnormalities, can affect the prognosis of the patients with acute myeloid leukemia (AML). Within this context it remains unclear if and how platelet counts affect the outcome of AML patients. In the present study, we examined the platelet counts at diagnosis in 633 newly diagnosed adult patients with AML from January 2010 to April 2021, and divided the cases into the group with low level of platelet counts (≤30×109/L, n=316) and high level of platelet counts (>30×109/L, n=317) according to the median platelet counts. We then validated the prognostic significance and potential mechanism of platelet counts on the relevance of spectral features for diagnostic risk stratification, initial induction therapy response, treatment effect maintenance, long-term survival, leukemia stem cells (LSCs) proportion, immunomodulatory cytokines level and immune cell subsets proportion. The results suggested that AML patients with a high level of platelet counts at diagnosis were associated with a high-risk molecular cytogenetic stratification, low complete remission (CR) rate, poor leukemia free survival (LFS), high proportion of LSCs, high level of transforming growth factor-β (TGF-β) and interleukin-1β (IL-1β), high proportion of regulatory T cells (Tregs) and monocytic myeloid-derived suppressor cells (M-MDSCs). It was demonstrated that platelet might be an unfavorable prognostic biomarker and was associated with LSCs and immunomodulatory cytokines as well as immune cell subsets in AML.

The Molecular Context of Oxidant Stress Response in Cancer Establishes ALDH1A1 as a Critical Target: What This Means for Acute Myeloid Leukemia.

The protein family of aldehyde dehydrogenases (ALDH) encompasses nineteen members. The ALDH1 subfamily consists of enzymes with similar activity, having the capacity to neutralize lipid peroxidation products and to generate retinoic acid; however, only ALDH1A1 emerges as a significant risk factor in acute myeloid leukemia. Not only is the gene ALDH1A1 on average significantly overexpressed in the poor prognosis group at the RNA level, but its protein product, ALDH1A1 protects acute myeloid leukemia cells from lipid peroxidation byproducts. This capacity to protect cells can be ascribed to the stability of the enzyme under conditions of oxidant stress. The capacity to protect cells is evident both in vitro, as well as in mouse xenografts of those cells, shielding cells effectively from a number of potent antineoplastic agents. However, the role of ALDH1A1 in acute myeloid leukemia has been unclear in the past due to evidence that normal cells often have higher aldehyde dehydrogenase activity than leukemic cells. This being true, ALDH1A1 RNA expression is significantly associated with poor prognosis. It is hence imperative that ALDH1A1 is methodically targeted, particularly for the acute myeloid leukemia patients of the poor prognosis risk group that overexpress ALDH1A1 RNA.

ACSL5, a prognostic factor in acute myeloid leukemia, modulates the activity of Wnt/β-catenin signaling by palmitoylation modification.

Acyl-CoA synthetase long chain family member 5 (ACSL5), is a member of the acyl-CoA synthetases (ACSs) family that activates long chain fatty acids by catalyzing the synthesis of fatty acyl-CoAs. The dysregulation of ACSL5 has been reported in some cancers, such as glioma and colon cancers. However, little is known about the role of ACSL5 in acute myeloid leukemia (AML). We found that the expression of ACSL5 was higher in bone marrow cells from AML patients compared with that from healthy donors. ACSL5 level could serve as an independent prognostic predictor of the overall survival of AML patients. In AML cells, the ACSL5 knockdown inhibited cell growth both in vitro and in vivo. Mechanistically, the knockdown of ACSL5 suppressed the activation of the Wnt/β-catenin pathway by suppressing the palmitoylation modification of Wnt3a. Additionally, triacsin c, a pan-ACS family inhibitor, inhibited cell growth and robustly induced cell apoptosis when combined with ABT-199, the FDA approved BCL-2 inhibitor for AML therapy. Our results indicate that ACSL5 is a potential prognosis marker for AML and a promising pharmacological target for the treatment of molecularly stratified AML.