Targeted dosing of anti-thymocyte globulin in adult unmanipulated haploidentical peripheral blood stem cell transplantation: A single-arm, phase 2 trial.

Anti-thymocyte globulin (ATG) is widely used in allogeneic hematopoietic stem cell transplantation to prevent severe graft-versus-host disease (GVHD) and graft failure. However, overexposure to ATG may increase cytomegalovirus (CMV), Epstein-Barr virus (EBV) reactivation, non-relapse mortality, and disease recurrence. To investigate the optimal dosing of ATG, we established a targeted dosing strategy based on ATG concentration monitoring for haploidentical peripheral blood stem cell transplantation (haplo-PBSCT). The aim of this phase 2 trial is to evaluate the safety and efficacy of the ATG-targeted dosing strategy in adult unmanipulated haplo-PBSCT. ATG was administered for 4 days (-5 days to -2 days) during conditioning. The ATG doses on -3 days and -2 days were adjusted by our dosing strategy to achieve the optimal ATG exposure. The primary endpoint was CMV reactivation on +180 days. Between December 2020 and January 2022, 66 haplo-PBSCT patients were enrolled and 63 of them were evaluable with a median follow-up of 632 days. The cumulative incidence of CMV reactivation was 36.7% and that of EBV was 58.7%. The 1-year disease-free survival was 82.5%, overall survival was 92.1%, and CD4+ T-cell reconstruction on +100 days was 76.8%. The most common severe regimen-associated toxicities (> grade 3) were infections (51.5%) and gastrointestinal toxicity (25.5%). A total of 102 haplo-PBSCT patients who received the conventional fixed ATG dose (cumulative 10 mg/kg) comprised historical control. The outcomes in historical control were inferior to those of phase 2 trial cohort (CMV reactivation: 70.8%, p < .001; EBV reactivation: 76.0%, p = .024; CD4 + T-cell reconstruction: 54.1%, p = .040). In conclusion, ATG-targeted dosing strategy reduced CMV/EBV reactivation and improved survival without increasing GVHD after haplo-PBSCT. These advantages may be associated with accelerated immune reconstitution.

Effect of Marital Status on the Survival of Patients With Adenocarcinoma of the Esophagogastric Junction: A Population-Based, Propensity-Matched Study.

BACKGROUND: Marital status has been reported as an independent prognostic factor in various types of malignancies. However, the association between marital status and outcomes of patients with adenocarcinoma of the esophagogastric junction (AEG) has not been fully explored. To this end, we aimed to investigate the effect of marital status on survival of AGE patients. METHODS: The Surveillance Epidemiology and End Results (SEER) database (2010-2015) was used to extract eligible patients with Siewert type II AEG. Meanwhile, propensity score matching was performed to match 1576 unmarried patients with 1576 married patients. Kaplan-Meier method with log-rank test was used to plot survival curves, univariate and multivariate Cox regression analyses were adopted to investigate the association of marital status with overall survival (OS) and cancer-specific survival (CSS) in AEG patients before and after matching. RESULTS: Multivariate analysis in the unmatched cohort revealed that marital status was an independent prognostic factor in patients with Siewert type II AEG. Unmarried patients had poorer OS (hazard ratio [HR]: 1.22, 95% confidence interval [CI]: 1.12-1.29, P < .001) and poorer CSS (HR: 1.19, 95% CI: 1.10-1.29, P < .001) than married patients before matching. Additionally, widowed patients had the poorest OS (HR: 1.26, 95% CI: 1.11-1.44, P < .001) and CSS (HR: 1.29, 95% CI: 1.12-1.48, P < .001) compared with married patients. Furthermore, unmarried status remained as an independent prognostic for both OS (HR: 1.20, 95% CI: 1.10-1.31, P < .001) and CSS (HR: 1.18, 95% CI: 1.08-1.30, P < .001) in 1:1 propensity score-matched analysis. Subgroup analysis further revealed that OS and CSS rates were significantly higher in married patients than unmarried ones in most subgroups stratified by different variables. CONCLUSIONS: This population-based study identified that marital status was an independent prognostic indicator for AEG patients. Married AEG patients had better prognosis than their unmarried counterparts.

[The Regulatory Effect of RNA m6 A Methylation Modification on KDM4B Gene Expression in t (8;21) AML Cells by MeRIP-qPCR].

OBJECTIVE: To confirm the direct regulatory effect of WTAP-mediated RNA m6A modification on the KDM4B gene in t (8;21) acute myeloid leukemia (AML) cells through MeRIP combined with reverse transcription real-time quantitative PCR (RT-qPCR) technology. METHODS: The lentivirus-mediated shRNA target WTAP or KDM4B gene was used to transfect the t (8;21) AML cell lines: Kasumi-1 and SKNO-1, and cells transfected with randomly shuffled shRNA as the control. Using the Ultrapure RNA Extraction Kit (DNase I) to extract RNA. The Magna MeRIPTM m6A Kit was used to enrich methylated modified fragments, and detect the m6A methylated RNA regions by RT-qPCR, and the protein and mRNA expression levels of WTAP and KDM4B in cells were detected by Western blot and reverse transcription real-time quantitative PCR (RT-qPCR). Colony formation assays were used to detect the colony ability of cells in vitro. RESULTS: Silencing the expression of WTAP in Kasumi-1 cells, the enrichment of m6A methylation modification was significantly decreased in the 3'UTR of KDM4B mRNA(P < 0.01), and the protein(P < 0.001) and mRNA (Kasumi-1:P < 0.001; SKNO-1: P < 0.01) expression levels of KDM4B were also significantly inhibited in Kasumi-1 and SKNO-1 cells upon WTAP knockdown (all P < 0.01), accompanied by a significant decrease in the colony-forming ability of both cell lines (both P < 0.01). CONCLUSION: In t(8;21) AML cell lines, WTAP could regulate the expression of KDM4B by regulating the m6A modification of the 3'UTR of KDM4B mRNA, and silencing the expression of KDM4B could inhibit the cellular proliferation in vitro.

HIF1α-mediated transactivation of WTAP promotes AML cell proliferation via m6A-dependent stabilization of KDM4B mRNA.

Hypoxia inducible factor 1α (HIF1α) is abnormally overexpressed in t(8;21) acute myeloid leukemia (AML) and functions as an oncogene through transactivating DNA methyltransferase 3 alpha leading to DNA hypermethylation. However, it remains unclear whether HIF1α influences RNA N6-methyladenosine (m6A) methyltransferases. Here, we show that HIF1α promotes the expression of Wilms tumor 1-associated protein (WTAP), a main component of the m6A methyltransferase complex, markedly alters the transcriptome-wide m6A distribution and enhances cell proliferation in t(8;21) AML. In agreement with this, WTAP is overexpressed and predicts poor prognosis in t(8;21) AML patients. Moreover, WTAP knockdown inhibits growth, and induces apoptosis and differentiation of leukemia cells. Mechanistically, HIF1α transactivates WTAP gene expression by directly binding to the hypoxia-response element of its promoter region. Pharmacological or genetic intervention in the HIF1α-WTAP axis results in the reduction of m6A level on lysine demethylase 4B (KDM4B) transcripts and increased its degradation, correlated with lower expression of KDM4B and higher trimethylation levels of histone H3 on lysine 9. KDM4B knockdown inhibits leukemia cell growth in vitro and in mice. Thus, HIF1α-mediated WTAP high expression enhances the malignant behavior of leukemia cells and drives a crosstalk between m6A RNA methylation and histone methylation through monitoring m6A-dependant KDM4B translation.

[The Relationship between HIF1α and WTAP Expression Level in t(8;21) Acute Myeloid Leukemia].

OBJECTIVE: To investigate the relationship between hypoxia inducible factor 1 (HIF1α) and Wilms' tumor 1associating protein (WTAP) expression level in t(8;21) acute myeloid leukemia cells. METHODS: The t(8;21) acute myeloid leukemia cell lines, including SKNO-1 and Kasumi-1 were treated by Echinomycin for 24 h, RT-qPCR and Western blot were used to detect the expression levels of WTAP mRNA and the protein. The CoCl 2 was used to induce the hypoxia of the cells for 24 h, the expression levels of HIF1α, WTAP protein were detected by Western blot. RESULTS: The expression level of WTAP mRNA and the protein in the echinomycin treated group was significantly lower than those in the control group (P<0.01). The expression level of WTAP protein in the CoCl2 treated group was significantly higher than those in the control group (P<0.05). CONCLUSION: The inhibition of HIF1-α could down-regulates the expression of WTAP, while the up-regulation of HIF1α could up-regulates the expression of WTAP, which shows that there is a positive correlation of HIF1α and WTAP expression. This result suggesting that HIF1α may be involves in the expression regulation of WTAP gene.

YTHDF2 is a potential target of AML1/ETO-HIF1α loop-mediated cell proliferation in t(8;21) AML.

The t(8;21) fusion product, AML1/ETO, and hypoxia-inducible factor 1α (HIF1α) form a feed-forward transcription loop that cooperatively transactivates the DNA methyltransferase 3a gene promoter that leads to DNA hypermethylation and drives leukemia cell growth. Suppression of the RNA N6-methyladenosine (m6A)-reader enzyme YTH N6-methyladenosine RNA binding protein 2 (YTHDF2) specifically compromises cancer stem cells in acute myeloid leukemia (AML) but promotes hematopoietic stem cell expansion without derailing normal hematopoiesis. However, the relevance of expression between AML1/ETO-HIF1α loop and YTHDF2, and its functional relationship with t(8;21) AML have not been documented. Here, we show that YTHDF2 is highly expressed in t(8;21) AML patients and associated with a higher risk of relapse and inferior relapse-free survival. Knockdown of YTHDF2 in leukemia cells causes an impaired cell proliferation rate in vitro and in mice. Mechanistically, HIF1α is able to bind to the hypoxia-response elements of the 5'-untranslated region of the YTHDF2 gene and promotes the transactivity of the YTHDF2 promoter. Knockdown and overexpression of either AML1/ETO or HIF1α resulted in decreased and increased YTHDF2 protein and mRNA expression in t(8;21) AML cells. In particular, knockdown of YTHDF2 resulted in increased global mRNA m6A levels in t(8;21) AML cells, accompanied by increased TNF receptor superfamily member 1b (TNFRSF1b) mRNA and protein expression levels. Last, we demonstrated that the m6A methylation and expression levels of the TNFRSF1b gene were both negatively correlated with HIF1α expression levels. In conclusion, YTHDF2 is a downstream target of the AML1/ETO-HIF1α loop and promotes cell proliferation probably by modulating the global m6A methylation in t(8;21) AML.