Super Enhancer Regulatory Gene FYB1 Promotes the Progression of T Cell Acute Lymphoblastic Leukemia by Activating IGLL1.

Background: Arising from T progenitor cells, T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignant tumor, accounting for 15% of childhood ALL and 25% of adult ALL. Composing of putative enhancers in close genomic proximity, super enhancer (SE) is critical for cell identity and the pathogenesis of multiple cancers. Belonging to the cytosolute linker protein group, FYB1 is essential for TCR signaling and extensively studied in terms of tumor pathogenesis and metastasis. Dissecting the role of FYN binding protein 1 (FYB1) in T-ALL holds the potential to improve the treatment outcome and prognosis of T-ALL. Methods: In this study, SEs were explored using public H3K27ac ChIP-seq data derived from T-ALL cell lines, AML cell lines and hematopoietic stem and progenitor cells (HSPCs). Downstream target of FYB1 gene was identified by RNA-seq. Effects of shRNA-mediated downregulation of FYB1 and immunoglobulin lambda-like polypeptide 1 (IGLL1) on self-renewal of T-ALL cells were evaluated in vitro and/or in vivo. Results: As an SE-driven gene, overexpression of FYB1 was observed in T-ALL, according to the Cancer Cell Line Encyclopedia database. In vitro, knocking down FYB1 led to comprised growth and enhanced apoptosis of T-ALL cells. In vivo, downregulation of FYB1 significantly decreased the disease burden by suppressing tumor growth and improved survival rate. Knocking down FYB1 resulted in significantly decreased expression of IGLL1 that was also an SE-driven gene in T-ALL. As a downstream target of FYB1, IGLL1 exerted similar role as FYB1 in inhibiting growth of T-ALL cells. Conclusion: Our results suggested that FYB1 gene played important role in regulating self-renewal of T-ALL cells by activating IGLL1, representing a promising therapeutic target for T-ALL patients.

[Expression of IGLL1 Gene and Its Clinical Significance in Pediatric T-ALL].

OBJECTIVE: To detect the relative expression of IGLL1 (immunoglobulin lambda-like polypeptide 1) mRNA in bone marrow of children with T-cell acute lymphoblastic leukemia (T-ALL), and analyze its correlation with the clinical characteristics and prognosis of the patients, so as to clarify the clinical significance of IGLL1 in pediatric T-ALL patients. METHODS: A total of 56 pediatric T-ALL patients hospitalized in Children's Hospital of Soochow University from June 2012 to December 2017 and treated with CCLG-ALL 2008 regimen were selected. Transcriptome sequencing technology was used to detect the transcription level of IGLL1 gene in children with T-ALL. According to 25% of the IGLL1 transcription level (cutoff value:448), the enrolled children were divided into IGLL1 low expression group (17 cases) and IGLL1 high expression group (39 cases). Combined with clinical data, the correlation between the expression level of IGLL1 and prognosis of the patients was analyzed. RESULTS: The comparative analysis showed that the transcription level of IGLL1 was not correlated with the clinical characteristics of the patients, such as sex, age, bone marrow blast, white blood cell (WBC) count at initial diagnosis. The 5-year OS rate of patients with high IGLL1 expression was significantly higher than that of patients with low IGLL1 expression (76.9%±6.7% vs 47.1%±12.1%, P =0.018). Further comparison of relapse-free survival (RFS) rate between the two groups showed that the 5-year RFS rate of patients with high IGLL1 expression was higher than that of patients with low IGLL1 expression, but the difference between the two groups was not statistically significant (P =0.095). Multivariate COX analysis was conducted on common clinical prognostic factors (age, sex, WBC count at diagnosis, prednisone response on the 7th day, bone marrow response on the 15th day after treatment) and IGLL1 expression level, and the results showed that IGLL1 expression (P =0.012) and prednisone response (P =0.017) were independent risk factors for overall survival in pediatric T-ALL patients. CONCLUSION: In pediatric T-ALL, the OS rate of children with high expression of IGLL1 gene was significantly higher than that of children with low expression of IGLL1 gene, and the expression level of IGLL1 gene was an independent factor affecting the survival of children with T-ALL, which suggests that IGLL1 is a marker of good clinical prognosis of children with T-ALL.

BRD4 PROTAC degrader MZ1 exhibits anti-B-cell acute lymphoblastic leukemia effects via targeting CCND3.

INTRODUCTION: B-cell acute lymphoblastic leukemia (B-ALL) is the most prevalent malignant tumor affecting children. While the majority of B-ALL patients (90%) experience successful recovery, early relapse cases of B-ALL continue to exhibit high mortality rates. MZ1, a novel inhibitor of Bromodomains and extra-terminal (BET) proteins, has demonstrated potent antitumor activity against hematological malignancies. The objective of this study was to examine the role and therapeutic potential of MZ1 in the treatment of B-ALL. METHODS: In order to ascertain the fundamental mechanism of MZ1, a sequence of in vitro assays was conducted on B-ALL cell lines, encompassing Cell Counting Kit 8 (CCK8) assay, Propidium iodide (PI) staining, and Annexin V/PI staining. Western blotting and quantitative real-time polymerase chain reaction (qRT-PCR) were employed to examine protein and mRNA expression levels. Transcriptomic RNA sequencing (RNA-seq) was utilized to screen the target genes of MZ1, and lentiviral transfection was employed to establish stably-expressing/knockdown cell lines. RESULTS: MZ1 has been observed to induce the degradation of Bromodomain Containing 4 (BRD4), Bromodomain Containing 3 (BRD3), and Bromodomain Containing 2 (BRD2) in B-ALL cell strains, leading to inhibited cell growth and induction of cell apoptosis and cycle arrest in vitro. These findings suggest that MZ1 exhibits cytotoxic effects on two distinct molecular subtypes of B-ALL, namely 697 (TCF3/PBX1) and RS4;11 (MLL-AF4) B-ALL cell lines. Additionally, RNA-sequencing analysis revealed that MZ1 significantly downregulated the expression of Cyclin D3 (CCND3) gene in B-ALL cell lines, which in turn promoted cell apoptosis, blocked cell cycle, and caused cell proliferation inhibition. CONCLUSION: Our results suggest that MZ1 has potential anti-B-ALL effects and might be a novel therapeutic target.

LMO2 promotes the development of AML through interaction with transcription co-regulator LDB1.

One of the characteristics of leukemia is that it contains multiple rearrangements of signal transduction genes and overexpression of non-mutant genes, such as transcription factors. As an important regulator of hematopoietic stem cell development and erythropoiesis, LMO2 is considered an effective carcinogenic driver in T cell lines and a marker of poor prognosis in patients with AML with normal karyotype. LDB1 is a key factor in the transformation of thymocytes into T-ALL induced by LMO2, and enhances the stability of carcinogenic related proteins in leukemia. However, the function and mechanism of LMO2 and LDB1 in AML remains unclear. Herein, the LMO2 gene was knocked down to observe its effects on proliferation, survival, and colony formation of NB4, Kasumi-1 and K562 cell lines. Using mass spectrometry and IP experiments, our results showed the presence of LMO2/LDB1 protein complex in AML cell lines, which is consistent with previous studies. Furthermore, in vitro and in vivo experiments revealed that LDB1 is essential for the proliferation and survival of AML cell lines. Analysis of RNA-seq and ChIP-Seq results showed that LDB1 could regulate apoptosis-related genes, including LMO2. In LDB1-deficient AML cell lines, the overexpression of LMO2 partially compensates for the proliferation inhibition. In summary, our findings revealed that LDB1 played an important role in AML as an oncogene, and emphasize the potential importance of the LMO2/LDB1 complex in clinical treatment of patients with AML.

Transcriptome sequencing identifies novel EVX fusions involved in transcriptional activation of HOX family genes in pediatric immature T-cell acute lymphoblastic leukemia: two cases reports and a literature review.

Driver genomic alterations in pediatric immature T-cell acute lymphoblastic leukemia are not fully known. We report two cases of novel EVX fusions involved in the transcriptional activation of HOX family genes, ETV6::EVX2 and MSI2::EVX1/HOXA13, which activate HOXD and HOXA cluster genes transcription through enhancer hijacking. HOXA and HOXD were the only key transcription factors activated in these cases, which indicates their important roles in leukemogenesis. Our findings elucidate potential drivers for development of T-cell lymphoblastic leukemia, and are valuable for diagnosis and risk stratification of pediatric T-ALL in the era of precision medicine.

Expression pattern of miR-16-2-3p and its prognostic values on pediatric acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) is a debilitating illness that easily occurs in adolescents. microRNAs (miRNAs) are potential biomarkers for multiple diseases. This paper was to elaborate on the expression of miR-16-2-3p in childhood ALL and its clinical values on ALL diagnosis and prognosis. First, serum miR-16-2-3p expression in ALL children and healthy volunteers was measured using RT-qPCR. Next, diagnostic potential and prognostic values of miR-16-2-3p on ALL were analyzed through receiver operating characteristic (ROC) curve, Kaplan-Meier survival curve, and multivariate Cox regression analysis, respectively. No significant difference was observed in the clinical baseline data between ALL patients and healthy children. ALL patients showed downregulated serum miR-16-2-3p (0.65 ± 0.27) (p < .01), whose area under the ROC curve was 0.837 with a cut-off value of 0.745 (67.92% sensitivity, 96.94% specificity). ALL patients with higher miR-16-2-3p expression had higher survival rates than those with lower miR-16-2-3p expression. Low miR-16-2-3p expression predicted poor prognosis of ALL patients. After adjusting LDH and lymphomyelocyte proportion (p = 0.003, HR = 0.003, 95%CI = 0.000-0.145), miR-16-2-3p was recognized as an independent prognostic factor for ALL patient survival. Briefly, low serum miR-16-2-3p expression in ALL children could aid ALL diagnosis and predict poor prognosis.

Association between CD34+ and CD3+ T-cells in allogeneic grafts and acute graft-versus-host disease in children undergoing allogeneic hematopoietic stem cell transplantation: A single-center study.

BACKGROUND: Acute graft-versus-host disease (aGVHD) is a major complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT). We examined the association between the composition of the cell subsets present in allogeneic grafts (allografts) and the occurrence and severity of aGVHD in pediatric patients. METHODS: We retrospectively analyzed 80 consecutive pediatric patients undergoing allo-HSCT at our center. RESULTS: Both univariate and multivariate analyses showed that the number of CD34+ and CD3+ T-cells in allografts were the two highest risk factors associated with II-IV aGVHD. Using receiver operating characteristic analysis, the cutoff levels of the allo-HSCT cell doses were used to divide the recipients into low-dose and high-dose groups. The 100-day cumulative incidence of II-IV aGVHD in the high-dose CD34+ and CD3+ T-cells group was significantly higher than that of the low-dose group (CD34+: 57% vs. 29%, p = 0.009; CD3+: 63% vs. 18%, p < 0.001). No other clinical factors or cell subsets correlated with aGVHD incidence. CONCLUSIONS: Our analysis indicates that the CD34+ and CD3+ T-cell numbers in the allografts could be the risk factors for the development of severe aGVHD (level II-IV). Further studies should aim to optimize the critical number of CD34+ and CD3+ T-cells to reduce the risk of severe aGVHD occurrence in pediatric patients.

BRD4 PROTAC degrader MZ1 exerts anticancer effects in acute myeloid leukemia by targeting c-Myc and ANP32B genes.

Acute myeloid leukemia (AML) is a highly cancerous and aggressive hematologic disease with elevated levels of drug resistance and relapse resulting in high mortality. Recently, bromodomains and extra-terminal (BET) protein inhibitors have been extensively researched in hematological tumors as potential anticancer agents. MZ1 is a novel BET inhibitor that mediates selective proteins degradation and suppression of tumor growth through proteolysis-targeting chimeras (PROTAC) technology. Accordingly, this study aimed to investigate the role and therapeutic potential of MZ1 in AML. In this study, we first identified that AML patients with high BRD4 expression had poor overall survival than those with low expression group. MZ1 inhibited AML cell growth and induced apoptosis and cycle arrest in vitro. MZ1 induced degradation of BRD4, BRD3 and BRD2 in AML cell strains. Additionally, MZ1 also initiated the cleavage of poly-ADP-ribose polymerase (PARP), which showed cytotoxic effects on NB4 (PML-RARa), K562 (BCR-ABL), Kasumi-1 (AML1-ETO), and MV4-11 (MLL-AF4) cell lines representing different molecular subtypes of AML. In AML mouse leukemia model, MZ1 significantly decreased leukemia cell growth and increased the mouse survival time. According to the RNA-sequencing analysis, MZ1 led to c-Myc and ANP32B genes significant downregulation in AML cell lines. Knockdown of ANP32B promoted AML cell apoptosis and inhibited cell growth. Overall, our data indicated that MZ1 had broad anti-cancer effects on AML cell lines with different molecular lesions, which might be exploited as a novel therapeutic strategy for AML patients.

BRD4 Inhibitor GNE-987 Exerts Anticancer Effects by Targeting Super-Enhancer-Related Gene LYL1 in Acute Myeloid Leukemia.

Background: AML (acute myeloid leukemia) is a common hematological malignancy in children with poor treatment effects and poor prognosis. Recent studies have shown that as a novel BRD4 (bromodomain containing 4) PROTACs (proteolysis targeting chimeras) degrader, GNE-987 can slow down the growth of various tumors and increase apoptosis, with promising clinical prospects. However, the function and molecular mechanism of GNE-987 in AML remain unclear. This study is aimed at investigating the therapeutic effect of GNE-987 on AML and its underlying mechanism. Methods: The association between BRD4 and AML was assessed by studying public databases. After GNE-987 was added to AML cells, cell proliferation slowed down, the cycle was disturbed, and apoptosis increased. Western blotting was used to detect BRD2 (bromodomain containing 2), BRD3 (bromodomain containing 3), BRD4, and PARP (poly ADP-ribose polymerase) proteins. The effect of GNE-987 on AML cells was analyzed in vivo. RNA-seq (RNA sequencing) and ChIP-seq (chromatin immunoprecipitation sequencing) validated the function and molecular pathways of GNE-987 in processing AML. Results: BRD4 expression was significantly elevated in pediatric AML samples compared with healthy donors. GNE-987 inhibited AML cell proliferation by inhibiting the cell cycle and inducing apoptosis. BRD2, BRD3, and BRD4 were consistent with decreased VHL (Von Hippel Lindau) expression in AML cells. In an AML xenograft model, GNE-987 significantly reduced the hepatosplenic infiltration of leukemia cells and increased the mouse survival time. Based on analysis of RNA-seq and ChIP-seq analyses, GNE-987 could target multiple SE- (super-enhancer-) related genes, including LYL1 (lymphoblastic leukemia 1), to inhibit AML. Conclusions: GNE-987 had strong antitumor activity in AML. GNE-987 could effectively inhibit the expression of SE-related oncogenes including LYL1 in AML. Our results suggested that GNE-987 had broad prospects in the treatment of AML.

A Novel BRD Family PROTAC Inhibitor dBET1 Exerts Great Anti-Cancer Effects by Targeting c-MYC in Acute Myeloid Leukemia Cells.

Acute myeloid leukemia (AML) represents an aggressive hematopoietic malignancy with a prognosis inferior to that of other leukemias. Recent targeted therapies offer new opportunities to achieve better treatment outcomes. However, due to the complex heterogeneity of AML, its prognosis remains dismal. In this study, we first identified the correlation between high expression of BRD4 and overall survival of patients with AML. Targeted degradation of BRD2, BRD3, and BRD4 proteins by dBET1, a proteolysis-targeting chimera (PROTAC) against the bromodomain and extra-terminal domain (BET) family members, showed cytotoxic effects on Kasumi (AML1-ETO), NB4 (PML-RARa), THP-1 (MLL-AF9), and MV4-11 (MLL-AF4) AML cell lines representing different molecular subtypes of AML. Furthermore, we determined that dBET1 treatment arrested cell cycling and enhanced apoptosis and c-MYC was identified as the downstream target. Collectively, our results indicated that dBET1 had broad anti-cancer effects on AML cell lines with different molecular lesions and provided more benefits to patients with AML.

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

BACKGROUND: Acute 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. METHODS: ChIP-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. RESULTS: We 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. CONCLUSIONS: In 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.

Effect of GATA3 rs3824662 gene polymorphism in Han Chinese children with pre-B-cell acute lymphoblastic leukemia with 10 years follow-up.

Purpose: To evaluate the influence of GATA3 rs3824662 on pre-B-cell acute lymphoblastic leukemia (pre-B-cell ALL) susceptibility and long-term prognosis in Han Chinese children with pre-B-cell ALL treated with the CCLG-2008 protocol at the Children's Hospital of Soochow University. Methods: A total of 256 patients with childhood pre-B-cell ALL under the CCLG-2008 protocol were enrolled in this study, and 174 healthy children were used as case controls. GATA3 rs3824662 genotyping was performed using a polymerase chain reaction, followed by Sanger sequencing. The association of genotype with clinical characteristics, treatment response, adverse events, and outcomes were analyzed. Results: The A allele frequency of GATA3 rs3824662 in patients with pre-B cell ALL was significantly higher than that in healthy children (OR = 1.41, 95% CI = 1.042-1.908; P = 0.026). Among patients with pre-B-cell ALL, the GATA3 rs3824662 AA genotype was associated with poor prednisolone response and high blast cell burden on day 15 of the induction therapy (P = 0.011 and 0.007, respectively). Patients with the rs3824662 AA variant suffered more episodes of sepsis than those with the CC or CA variants (P = 0.021). The GATA3 rs3824662 AA genotype was significantly associated with sepsis [hazard ratio (HR) = 3.375; P = 0.01]. No significant differences were found in the cumulative incidence of relapse, overall survival, and event-free survival among all genotypes. Conclusion: GATA3 rs3824662 was associated with susceptibility in Han Chinese children with pre-B-cell ALL and could be a possible risk factor for poor early treatment response and treatment-related sepsis.

CEBPG promotes acute myeloid leukemia progression by enhancing EIF4EBP1.

BACKGROUND: Acute myeloid leukemia (AML) is a myeloid neoplasm accounts for 7.6% of hematopoietic malignancies. AML is a complex disease, and understanding its pathophysiology is contributing to the improvement in the treatment and prognosis of AML. In this study, we assessed the expression profile and molecular functions of CCAAT enhancer binding protein gamma (CEBPG), a gene implicated in myeloid differentiation and AML progression. METHODS: shRNA mediated gene interference was used to down-regulate the expression of CEBPG 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. Genes and pathways affected by knockdown of CEBPG were identified by gene expression analysis using RNA-seq. One of the genes affected by knockdown of CEBPG was Eukaryotic translation initiation factor 4E binding protein 1 (EIF4EBP1), a known repressor of translation. Knockdown of EIF4EBP1 was used to assess its potential role in AML progression downstream of CEBPG. RESULTS: We explored the ChIP-Seq data of AML cell lines and non-AML hematopoietic cells, and found CEBPG was activated through its distal enhancer in AML cell lines. Using the public transcriptomic dataset, the Cancer Cell Line Encyclopedia (CCLE) and western blotting, we also found CEBPG was overexpressed in AML. Moreover, we observed that CEBPG promotes AML cell proliferation by activating EIF4EBP1, thus contributing to the progression of AML. These findings indicate that CEBPG could act as a potential therapeutic target for AML patients. CONCLUSION: In summary, we systematically explored the molecular characteristics of CEBPG in AML and identified CEBPG as a potential therapeutic target for AML patients. Our findings provide novel insights into the pathophysiology of AML and indicate a key role for CEBPG in promoting AML progression.

MI-773, a breaker of the MDM2/p53 axis, exhibits anticancer effects in neuroblastoma via downregulation of INSM1.

Neuroblastoma (NB) is a common pediatric malignancy associated with poor outcomes. Recent studies have shown that murine double minute2 homolog (MDM2) protein inhibitors are promising anticancer agents. MI-773 is a novel and specific antagonist of MDM2, however, the molecular mechanism of its anti-NB activity remains unclear. NB cell viability was measured by Cell Counting Kit-8 assay following MI-773 treatment. Cell cycle progression was analyzed using PI staining and apoptosis was assessed using Annexin V/PI staining. The molecular mechanisms by which MI-773 exerted its effects were investigated using a microarray. The results showed that disturbance of the MDM2/p53 axis by MI-773 resulted in potent suppression of proliferation, induction of apoptosis and cell cycle arrest in NB cells. In addition, microarray analysis showed that MI-773 led to significant downregulation of genes involved in the G2/M phase checkpoint and upregulation of hallmark gene associated with the p53 pathway. Meanwhile, knockdown of insulinoma-associated 1 decreased proliferation and increased apoptosis of NB cells. In conclusion, the present study demonstrated that MI-773 exhibited high selectivity and blockade affinity for the interaction between MDM2 and TP53 and may serve as a novel strategy for the treatment of NB.

The clinical outcomes and genomic landscapes of acute lymphoblastic leukemia patients with E2A-PBX1: A 10-year retrospective study.

The clinical outcomes and genomic features of E2A-PBX1 (TCF3-PBX1)-positive B-cell acute lymphoblastic leukemia (B-ALL) patients remain unclear. A total of 137 patients carrying E2A-PBX1 among 3164 B-ALL patients between 2009 and 2019 were retrospectively analyzed. The 5-year overall survival (OS) and disease-free survival (DFS) rates of the whole cohort were 68.6% and 61.0%, respectively. Age [DFS, p = 0.037; cumulative incidence of relapse (CIR), p = 0.005] and the level of minimal residual disease (MRD) after induction chemotherapy (OS, p = 0.020; DFS, p = 0.002; CIR, p = 0.006) were independent risk factors. In adolescents/adults, allogeneic hematopoietic stem cell transplantation (allo-HSCT) at first complete remission (CR1) significantly improved the 5-year prognosis (OS, p < 0.001; DFS, p < 0.001; CIR, p < 0.001). Haploidentical HSCT decreased the CIR compared with human leukocyte antigen-matched HSCT in adolescents/adults (p = 0.017). Mutations in PBX1, PAX5, CTCF and SETD2, amplification of AKT3, and deletion of CDKN2A/B were common in the total cohort, while transcriptome differences were found in the cell cycle, nerve growth factor (NGF) signaling pathway and transcriptional regulation by TP53 between adolescents/adults and children. Patients with multiple subclones at diagnosis tended to have unfavorable 3-year prognoses (DFS, p = 0.010; CIR, p = 0.021). Leukemia clones with DNA repair gene mutations showed aggressive and treatment-refractory phenotypes in this subtype of ALL. Our study indicated that age, the level of MRD and DNA repair gene mutations were associated with E2A-PBX1-positive B-ALL outcomes. Allo-HSCT, especially haploidentical HSCT, could improve the prognosis of adolescent/adult patients.

High Expression of Interleukin-3 Receptor Alpha Chain (CD123) Predicts Favorable Outcome in Pediatric B-Cell Acute Lymphoblastic Leukemia Lacking Prognosis-Defining Genomic Aberrations.

BACKGROUND: Aberrant expression of CD123 (IL-3Rα) was observed in various hematological malignancies including acute lymphoblastic leukemia (ALL), which is the most common malignancy in childhood. Although widely used for minimal residual disease (MRD) monitoring, the prognostic value of CD123 has not been fully characterized in pediatric B-ALL. This retrospective study aims to evaluate the association between the CD123 expression of leukemic blasts and the outcomes of the pediatric B-ALL patients. METHODS: A total of 976 pediatric B-ALL, including 328 treated with CCLG-ALL-2008 protocol and 648 treated with CCCG-ALL-2015 protocol, were recruited in this retrospective study. CD123 expression was evaluated by flow cytometry. Patients with >50, 20-50, or <20% of CD123 expressing blasts were grouped into CD123high, CD123low, and CD123neg, respectively. The correlation between CD123 expression and the patients' clinical characteristics, overall survival (OS), event-free survival (EFS), and relapse-free survival (RFS) were studied statistically. RESULTS: Of 976 pediatric B-ALL, 53.4% from the CCLG-ALL-2008 cohort and 49.2% from the CCCG-ALL-2015 cohort were CD123high. In the CCLG-ALL-2008 cohort, CD123high was significantly associated with chromosome hyperdiploidy (p < 0.0001), risk stratification (p = 0.004), and high survival rate (p = 0.005). By comparing clinical outcomes, patients with CD123high displayed favorable prognosis, with a significantly better OS (p = 0.005), EFS (p = 0.017), and RFS (p = 0.045), as compared to patients with CD123low and CD123neg. The prognostic value of CD123 expression was subsequently confirmed in the CCCG-ALL-2015 cohort. Univariate and multivariate cox regression model analysis showed that high CD123 expression was independently associated with favorable EFS (OR: 0.528; 95% CI: 0.327 to 0.853; p = 0.009) in this cohort. In patients without prognosis-defining genomic abnormalities, high CD123 expression strongly indicated superior survival rates and was identified as an independent prognosis factor for EFS and RFS in both cohorts. CONCLUSIONS: A group of B-ALL lacks prognosis-defining genomic aberrations, which proposes a challenge in risk stratification. Our findings revealed that high CD123 expression of leukemic blasts was associated with favorable clinical outcomes in pediatric B-ALL and CD123 could serve as a promising prognosis predictor, especially in patients without prognosis-defining genetic aberrations.

BRD4 PROTAC degrader ARV-825 inhibits T-cell acute lymphoblastic leukemia by targeting 'Undruggable' Myc-pathway genes.

BACKGROUND: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive disease with a high risk of induction failure and poor outcomes, with relapse due to drug resistance. Recent studies show that bromodomains and extra-terminal (BET) protein inhibitors are promising anti-cancer agents. ARV-825, comprising a BET inhibitor conjugated with cereblon ligand, was recently developed to attenuate the growth of multiple tumors in vitro and in vivo. However, the functional and molecular mechanisms of ARV-825 in T-ALL remain unclear. This study aimed to investigate the therapeutic efficacy and potential mechanism of ARV-825 in T-ALL. METHODS: Expression of the BRD4 were determined in pediatric T-ALL samples and differential gene expression after ARV-825 treatment was explored by RNA-seq and quantitative reverse transcription-polymerase chain reaction. T-ALL cell viability was measured by CCK8 assay after ARV-825 administration. Cell cycle was analyzed by propidium iodide (PI) staining and apoptosis was assessed by Annexin V/PI staining. BRD4, BRD3 and BRD2 proteins were detected by western blot in cells treated with ARV-825. The effect of ARV-825 on T-ALL cells was analyzed in vivo. The functional and molecular pathways involved in ARV-825 treatment of T-ALL were verified by western blot and chromatin immunoprecipitation (ChIP). RESULTS: BRD4 expression was higher in pediatric T-ALL samples compared with T-cells from healthy donors. High BRD4 expression indicated a poor outcome. ARV-825 suppressed cell proliferation in vitro by arresting the cell cycle and inducing apoptosis, with elevated poly-ADP ribose polymerase and cleaved caspase 3. BRD4, BRD3, and BRD2 were degraded in line with reduced cereblon expression in T-ALL cells. ARV-825 had a lower IC50 in T-ALL cells compared with JQ1, dBET1 and OTX015. ARV-825 perturbed the H3K27Ac-Myc pathway and reduced c-Myc protein levels in T-ALL cells according to RNA-seq and ChIP. In the T-ALL xenograft model, ARV-825 significantly reduced tumor growth and led to the dysregulation of Ki67 and cleaved caspase 3. Moreover, ARV-825 inhibited cell proliferation by depleting BET and c-Myc proteins in vitro and in vivo. CONCLUSIONS: BRD4 indicates a poor prognosis in T-ALL. The BRD4 degrader ARV-825 can effectively suppress the proliferation and promote apoptosis of T-ALL cells via BET protein depletion and c-Myc inhibition, thus providing a new strategy for the treatment of T-ALL.

Identification of novel CEBPA double mutations capable of promoting familial AML via the suppression of myeloid differentiation.

CCAAT-enhancer-binding protein α (CEBPA) gene carrying two mutations (CEBPA double mutations) is known to promote familial acute myeloid leukemia (AML). However, the underlying mechanism by which CEBPA double mutations promote AML remains poorly understood. Here we report that a family with three generations suffering from familial AML carries novel double mutations of CEBPA. Seven bases of GCGCGGG were inserted into the N-terminal c.113-114 of CEBPA as germline mutations and three bases of AAG were inserted into the C-terminal c.939-940 as a somatic mutation. To test the functional impact of this double mutation, we constructed plasmid encoding the double mutants of CEBPA and transfected it into the myeloid precursor 32Dcl3 cells. Lentiviral induced overexpression of CEBPA with these double mutations inhibited myeloid differentiation of these 32Dcl3 cells, and led to approximately 4-fold fewer frequency of CD11b expression. Our results confirm that the double mutations of CEBPA at both N- and C-terminals are potentially to induce leukemogenesis of AML.

Proteomic profiling and bioinformatics analysis identify key regulators during the process from fanconi anemia to acute myeloid leukemia.

Fanconi anemia (FA) is a congenital aplastic anemia, characterized as congenital bone marrow failure, developmental malformation, and the malignant tendency, which may develop into acute myeloid leukemia (AML). However, few studies have been conducted on the progression from FA to AML. In this study, we used proteomic profiling, together with bioinformatics analyses, to explore the molecular mechanisms by which FA progresses to AML. Quantitative proteomic analyses of bone marrow samples identified 168 differentially expressed proteins (DEPs), including 7 upregulated proteins and 161 downregulated proteins in the bone marrow of the FA patient compared with the healthy people. The upregulated proteins were enriched in response to stress, oxygen transport, and hydrogen peroxide catabolic process. The downregulated proteins were enriched in myeloid leukocyte mediated immunity, response to interleukin-12, platelet degranulation and regulation of ATPase activity. Based on these results, we discovered 155 DEPs (142 upregulated and 13 downregulated) in the bone marrow samples between FA and AML patients, of which HIST1H1D, HIST1H3A, PSME1 and THRAP3 may play important roles in the progression of FA to AML and may be used as markers for AML early diagnosis. Finally, cell-line based experiments confirmed that PSME1 had an important effect on the proliferation of leukemia cells.