Comprehensive analysis of ID genes reveals the clinical and prognostic value of ID3 expression in acute myeloid leukemia using bioinformatics identification and experimental validation.

BACKGROUND: Dysregulation of inhibitor of differentiation/DNA binding (ID) genes is linked to cancer growth, angiogenesis, invasiveness, metastasis and patient survival. Nevertheless, few investigations have systematically determined the expression and prognostic value of ID genes in acute myeloid leukemia (AML). METHODS: The expression and clinical prognostic value of ID genes in AML were first identified by public databases and further validated by our research cohort. RESULTS: Using public data, the expression of ID1/ID3 was markedly downregulated in AML, and the expression of ID2 was greatly upregulated in AML, whereas ID4 showed no significant difference. Among the ID genes, only ID3 expression may be the most valuable prognostic biomarker in both total AML and cytogenetically normal AML (CN-AML) and especially in CN-AML. Clinically, reduced ID3 expression was greatly associated with higher white blood cell counts, peripheral blood/bone marrow blasts, normal karyotypes and intermediate cytogenetic risk. In addition, low ID3 expression was markedly related to FLT3 and NPM1 mutations as well as wild-type TP53. Despite these associations, multivariate Cox regression analysis revealed that ID3 expression was an independent risk factor affecting overall survival (OS) and disease free survival (DFS) in CN-AML patients. Biologically, a total of 839 mRNAs/lncRNAs and 72 microRNAs were found to be associated with ID3 expression in AML. Importantly, the expression of ID3 with discriminative value in AML was further confirmed in our research cohort. CONCLUSION: The bioinformatics analysis and experimental verification demonstrate that low ID3 expression independently affects OS and DFS in patients with CN-AML, which might be seen as a potential prognostic indicator in CN-AML.

Pan-cancer analysis reveals distinct clinical, genomic, and immunological features of the LILRB immune checkpoint family in acute myeloid leukemia.

Leukocyte immunoglobulin (Ig)-like receptor Bs (LILRBs), a family of type I transmembrane glycoproteins, are known to inhibit immune activation. Here, we comprehensively evaluated the molecular, prognostic, and immunological characteristics of LILRB members in a broad spectrum of cancer types, focusing on their roles in acute myeloid leukemia (AML). We showed that LILRBs were significantly dysregulated in a number of cancers and were associated with immune-inhibitory phenotypes. Clinically, high expression of LILRB1-LILRB4 predicted poor survival in six independent AML cohorts. Genetically, LILRB1 was associated with more mutational events than other LILRB members, and multiple genes involved in immune activation were deleted in LILRB1 high patients. Epigenetically, LILRB4 was significantly hypomethylated and marked by MLL-associated histone modifications in AML. Immunologically, LILRBs were positively associated with monocytic cells, including M2 macrophages, but were negatively associated with tumor-suppressive CD8 T cells. Importantly, patients with higher LILRB expression generally showed a better response to immune checkpoint blockade (ICB) in five independent immunotherapy cohorts. Our findings reveal critical immunological and clinical implications of LILRBs in AML and indicate that LILRBs may represent promising targets for immunotherapy of AML.

DNA methylation-mediated differential expression of DLX4 isoforms has opposing roles in leukemogenesis.

BACKGROUND: Previously, we reported the expression of DLX4 isoforms (BP1 and DLX7) in myeloid leukemia, but the functional role of DLX4 isoforms remains poorly understood. In the work described herein, we further determined the underlying role of DLX4 isoforms in chronic myeloid leukemia (CML) leukemogenesis. METHODS: The expression and methylation of DLX4 isoforms were detected by real-time quantitative PCR (RT-qPCR) and real-time quantitative methylation-specific PCR (RT-qMSP) in patients with CML. The functional role of DLX4 isoforms was determined in vitro and in vivo. The molecular mechanism of DLX4 isoforms in leukemogenesis was identified based on chromatin immunoprecipitation with high-throughput sequencing (ChIP-Seq)/assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-Seq) and RNA sequencing (RNA-Seq). RESULTS: BP1 expression was increased in patients with CML with unmethylated promoter, but DLX7 expression was decreased with hypermethylated promoter. Functionally, overexpression of BP1 increased the proliferation rate of K562 cells with S/G2 promotion, whereas DLX7 overexpression reduced the proliferation rate of K562 cells with G1 arrest. Moreover, K562 cells with BP1 overexpression increased the tumorigenicity in NCG mice, whereas K562 cells with DLX7 overexpression decreased the tumorigenicity. Mechanistically, a total of 91 genes including 79 messenger RNAs (mRNAs) and 12 long noncoding RNAs (lncRNAs) were discovered by ChIP-Seq and RNA-Seq as direct downstream targets of BP1. Among the downstream genes, knockdown of RREB1 and SGMS1-AS1 partially revived the proliferation caused by BP1 overexpression in K562 cells. Similarly, using ATAC-Seq and RNA-Seq, a total of 282 genes including 151 mRNA and 131 lncRNAs were identified as direct downstream targets of DLX7. Knockdown of downstream genes PTPRB and NEAT1 partially revived the proliferation caused by DLX7 overexpression in K562 cells. Finally, we also identified and validated a SGMS1-AS1/miR-181d-5p/SRPK2 competing endogenous RNA (ceRNA) network caused by BP1 overexpression in K562 cells. CONCLUSIONS: The current findings reveal that DNA methylation-mediated differential expression of DLX4 isoforms BP1 and DLX7 plays opposite functions in leukemogenesis. BP1 plays an oncogenic role in leukemia development, whereas DLX7 acts as a tumor suppressor gene. These results suggest DLX4 as a therapeutic target for antileukemia therapy.

[Detection of NPM1 Mutation in Acute Myeloid Leukemia by Droplet Digital PCR and Its Clinical Application Value].

OBJECTIVE: To establish the droplet digital PCR (ddPCR) assay for the detection of NPM1 type A mutation in patients with acute myeloid leukemia (AML), and to evaluate its specificity, sensitivity and its value in clinical application. METHODS: NPM1 mutant and wildtype plasmids were used to verify the performance of ddPCR. Both ddPCR and Sanger sequencing were used to detect the bone marrow samples of 87 AML patients, which were confirmed by next generation sequencing (NGS). Moreover, NPM1 mutation burden was dynamically monitored in five patients by ddPCR. RESULTS: The limit of blank (LOB) of ddPCR established for NPM1 mutation detection was 1.1 copies/µl, and the limit of detection (LOD) was 2.43 copies/µl, which had good linearity. Among the 87 newly diagnosed AML patients, ddPCR identified seventeen cases positive for NPM1 mutation (19.5%)， which was consistent with Sanger sequencing. NGS confirmed 12 positive cases, including 8 of type A mutations, 2 of type D mutations, and 2 of rare type mutations. The results of dynamic monitoring of NPM1 mutation burden in 5 patients showed that the NPM1 mutation burden decreased obviously even close to 0, when patients achieve complete remission after chemotherapy. However, the mutation burden was increased again at the time of relapse. CONCLUSION: In this study, we established a ddPCR method for detection of NPM1 mutation with good sensitivity and repeatability, which can be used for screening NPM1 mutation in newly diagnosed AML patients and for minimal residual disease monitoring after remission in positive AML patients to guide treatment.

[Clinical Significance of Low Expression of LncRNA CASC15 in Acute Myeloid Leukemia with NPM1 Mutations].

AbstractObjective: To identify the expression and methylation patterns of lncRNA CASC15 in bone marrow (BM) samples of acute myeloid leukemia (AML) patients, and further explore its clinical significance. METHODS: Eighty-two de novo AML patients and 18 healthy donors were included in the study. Meanwhile, seven public datasets from Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) were included to confirm the expression and methylation data of CASC15. Receiver operating characteristic (ROC) curve analysis was applied to determine the discriminative capacity of CASC15 expression to identify AML. The patients were divided into CASC15high group and CASC15low group by X-tile method, and the prognostic value of CASC15 was identified by Kaplan-Meier method and univariate and multivariate Cox regression analysis. RESULTS: The expression level of CASC15 was significantly decreased in BM cells of AML patients compared with healthy donors (P<0.001). ROC curve analysis suggested that CASC15 expression might be a potential biomarker to discriminate AML from controls. The expression of CASC15 was high at the early stage of hematopoiesis, and reached a peak at the stage of multipotent progenitors differentiation, then decreased rapidly, and was at a range of low level fluctuations in the subsequent process. Among FAB subtypes, CASC15 expression in M0 was significantly higher than that in M1-M7. Clinically, CASC15low patients were more likely to have NPM1 mutations than CASC15high patients (P=0.048), while CASC15high patients had a significantly higher frequency of IDH1 and RUNX1 mutations (P=0.021 and 0.014, respectively). Moreover, CASC15low group had a shorter overall survival (OS) in patients with NPM1 mutations. Furthermore, multivariate analysis confirmed that CASC15 expression was a significant independent risk factor for OS in NPM1 mutated AML patients. In addition, CASC15 methylation level in BM samples of AML patients was significantly decreased compared with healthy donors. Patients with CASC15 high methylation had poor OS and disease-free survival. CONCLUSION: The expression of CASC15 is decreased in AML, and low CASC15 expression may predict adverse prognosis in AML patients with NPM1 mutations. Moreover, CASC15 methylation level in AML is significantly decreased, and high CASC15 methylation may predict poor prognosis in AML.

Abnormal expression and methylation of PRR34-AS1 are associated with adverse outcomes in acute myeloid leukemia.

It was previously reported that PRR34-AS1 was overexpressed in some solid tumors. PRR34-AS1 promoter was shown to have a differential methylation region (DMR), and was hypomethylated in acute myeloid leukemia (AML). Therefore, the present study used real-time quantitative PCR (RQ-PCR) to explore the expression characteristics of PRR34-AS1 in AML. In addition, the correlation between the expression of PRR34-AS1 and clinical prognosis of AML was determined. The findings of this study indicated that high PRR34-AS1 expression was bound up with shorter overall survival (OS) in AML patients (p = 0.002). Moreover, patients with high expression of PRR34-AS1 had significantly lower complete remission (CR) rate compared with those with low expression of PRR34-AS1 after induction chemotherapy. Furthermore, multivariate analysis confirmed that PRR34-AS1 expression was an independent factor affecting CR in whole-AML, non-APL-AML, and CN-AML patients (p = 0.032, 0.039, and 0.036, respectively). Methylation-specific PCR (MSP) and bisulfite sequencing PCR (BSP) were used to explore the methylation status of PRR34-AS1. PRR34-AS1 promoter showed a pattern of hypomethylation in AML patients compared with normal controls (p = 0.122). Notably, of whole-AML and non-APL-AML patients, PRR34-AS1 hypomethylated patients presented a significantly shorter OS than those with a hypermethylated PRR34-AS1 (p = 0.010 and 0.037, respectively). Multivariate analysis confirmed that the hypomethylation of PRR34-AS1 served as an independent prognostic indicator in both whole-cohort AML and non-APL-AML categories (p = 0.057 and 0.018, respectively). In summary, the findings of this study showed that abnormalities in PRR34-AS1 are associated with poor prognosis in AML. Therefore, monitoring this index may be important in the prognosis of AML and can provide information on effective chemotherapy against the disease.

Genome-wide methylation sequencing identifies progression-related epigenetic drivers in myelodysplastic syndromes.

The potential mechanism of myelodysplastic syndromes (MDS) progressing to acute myeloid leukemia (AML) remains poorly elucidated. It has been proved that epigenetic alterations play crucial roles in the pathogenesis of cancer progression including MDS. However, fewer studies explored the whole-genome methylation alterations during MDS progression. Reduced representation bisulfite sequencing was conducted in four paired MDS/secondary AML (MDS/sAML) patients and intended to explore the underlying methylation-associated epigenetic drivers in MDS progression. In four paired MDS/sAML patients, cases at sAML stage exhibited significantly increased methylation level as compared with the matched MDS stage. A total of 1090 differentially methylated fragments (DMFs) (441 hypermethylated and 649 hypomethylated) were identified involving in MDS pathogenesis, whereas 103 DMFs (96 hypermethylated and 7 hypomethylated) were involved in MDS progression. Targeted bisulfite sequencing further identified that aberrant GFRA1, IRX1, NPY, and ZNF300 methylation were frequent events in an additional group of de novo MDS and AML patients, of which only ZNF300 methylation was associated with ZNF300 expression. Subsequently, ZNF300 hypermethylation in larger cohorts of de novo MDS and AML patients was confirmed by real-time quantitative methylation-specific PCR. It was illustrated that ZNF300 methylation could act as a potential biomarker for the diagnosis and prognosis in MDS and AML patients. Functional experiments demonstrated the anti-proliferative and pro-apoptotic role of ZNF300 overexpression in MDS-derived AML cell-line SKM-1. Collectively, genome-wide DNA hypermethylation were frequent events during MDS progression. Among these changes, ZNF300 methylation, a regulator of ZNF300 expression, acted as an epigenetic driver in MDS progression. These findings provided a theoretical basis for the usage of demethylation drugs in MDS patients against disease progression.

Identification and validation of prognosis-related DLX5 methylation as an epigenetic driver in myeloid neoplasms.

The deregulated DLX gene family members DLX1/2/3/4/5/6 (DLXs) caused by DNA methylation has been demonstrated in various cancers with therapeutic target value. However, the potential role of DLXs methylation in myeloid neoplasms such as acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) remains to be elucidated. Clinical significance of DLXs methylation/expression was analyzed in patient with AML and MDS. The functional roles of DLXs were determined in vitro. In the identification stage, we found that lower DLX5 expression was correlated with prognosis in AML among all DLXs analyzed by The Cancer Genome Atlas datasets. In the validation stage, we revealed that reduced DLX5 expression was frequently occurred, and was also correlated with promoter hypermethylation in AML evaluated by targeted bisulfite sequencing. Epigenetic studies also showed that DLX5 promoter DNA methylation was associated with its expression. By quantitative polymerase chain reaction, we also validated that DLX5 hypermethylation was frequent event in both AML and MDS, and also correlated with MDS transformation to leukemia. Moreover, DLX5 hypermethylation was associated with lower rate of complete remission and shorter time of leukemia-free/overall survival, and was also confirmed by Logistic/Cox regression analysis. Functional studies revealed the antiproliferative and pro-apoptotic effects of DLX5 in MDS-derived AML cell-line SKM-1. Finally, bioinformatics analysis demonstrated that DLX5 functioned in leukemogenesis may be through the association with PI3K/Akt signaling pathway. Collectively, our findings demonstrated that DLX5 methylation, negatively correlated DLX5 expression, was a potential prognostic and predictive indicator in patients with AML and MDS, which could also act as an epigenetic driver in myeloid neoplasms.

SOX7 methylation is an independent prognostic factor in myelodysplastic syndromes.

OBJECTIVE: SOX7 downregulation caused by its promoter methylation was associated with poor survival in several types of human solid tumors. However, the pattern of SOX7 methylation and its clinical significance are less studied in hematological malignancies. Herein, we evaluated the methylation pattern of SOX7 in myelodysplastic syndrome (MDS) and determined its clinical implication in patients with MDS. METHODS: SOX7 methylation was determined by real-time quantitative methylation-specific PCR (RQ-MSP) in 99 MDS patients. Bisulfite sequencing PCR was applied to confirm the results of RQ-MSP. RESULTS: SOX7 methylation was detected in 55.6% of 99 patients but not in healthy donors. No correlation was found between SOX7 methylation and clinical parameters including patient age, gender, white blood cell count, hemoglobin, and platelet count. However, patients with SOX7 methylation harbored more U2AF1 mutation than patients without SOX7 methylation (P = 0.015). Kaplan-Meier curves indicated that the patients with SOX7 methylation presented reduced overall survival (OS) (P = 0.034). Furthermore, subgroup analysis indicated that SOX7 methylation was associated with poor OS in male patients (P = 0.034) and in patients older than 60 years (P = 0.019). According to the multivariate analysis, SOX7 methylation remained as an independent prognosis factor in MDS patients both as dichotomous (HR = 2.14, P = 0.041) and as continuous (HR = 1.55, P = 0.042) variable. Importantly, SOX7 methylation was significantly increased during progression from MDS to secondary acute myeloid leukemia (sAML). CONCLUSIONS: Our findings demonstrated that SOX7 methylation conferred adverse prognosis in MDS patients and was associated with leukemia progression.