Hypomethylating therapies using decitabine or azacitidine are actively investigated to treat acute myeloid leukemia, myelodysplastic syndromes, as maintenance therapy after allogenic stem cell transplant and hemoglobinopathies. The therapeutic mechanism is to de-repress genes that have been turned off through oncogenesis or development via methylation. The therapy can be non-cytotoxic at low dosage, sparing healthy stem cells and operating on committed precursors. Because the methods of determining maximum tolerated dose are not well suited to this paradigm, and because the mechanism of action, which is depletion of DNA methylase 1 (DNMT1), is complex and dependent on passing through a cell cycle, a pharmacodynamic assay that measures DNMT1 can inform clinical trials aimed at establishing and improving therapy. Herein, we provide an assay that measures DNMT1 relative levels in circulating T cells of peripheral blood.

The interaction of acute myeloid leukaemic (AML) blasts with the bone marrow (BM) microenvironment is a major determinant governing disease progression and resistance to treatment. The constitutive expression of E-selectin in the vascular compartment of BM, a key endothelial cell factor, directly mediates chemoresistance via E-selectin ligand/receptors. Despite the success of hypomethylating agent (HMA)-containing regimens to induce remissions in older AML patients, the development of primary or secondary resistance is common. We report that following treatment with 5-azacitidine, promoter regions regulating the biosynthesis of the E-selectin ligands, sialyl Lewis X, become further hypomethylated. The resultant upregulation of these gene products, in particular α(1,3)-fucosyltransferase VII (FUT7) and α(2,3)-sialyltransferase IV (ST3GAL4), likely causes functional E-selectin binding. When combined with the E-selectin antagonist uproleselan, the adhesion to E-selectin is reversed and the survival of mice transplanted with AML cells is prolonged. Finally, we present clinical evidence showing that BM myeloid cells from higher risk MDS and AML patients have the potential to bind E-selectin, and these cells are more abundant in 5-azacitidine-non-responsive patients. The collective data provide a strong rationale to evaluate 5-azacitidine in combination with the E-selectin antagonist, uproleselan, in this patient population.

The VEXAS syndrome, a genetically defined autoimmune disease, associated with various hematological neoplasms has been attracting growing attention since its initial description in 2020. While various therapeutic strategies have been explored in case studies, the optimal treatment strategy is still under investigation and allogeneic cell transplantation is considered the only curative treatment. Here, we describe 2 patients who achieved complete molecular remission of the underlying UBA1 mutant clone outside the context of allogeneic HCT. Both patients received treatment with the hypomethylating agent azacitidine, and deep molecular remission triggered treatment de-escalation and even cessation with sustained molecular remission in one of them. Prospective studies are necessary to clarify which VEXAS patients will benefit most from hypomethylating therapy and to understand the variability in the response to different treatment strategies.

DNA hypermethylation plays a critical role in the occurrence and progression of acute myeloid leukemia (AML). The mitochondrial serine transporter, SFXN3, is vital for onecarbon metabolism and DNA methylation. However, the impact of SFXN3 on the occurrence and progression of AML has not been reported yet.
In this study, we hypothesized that SFXN3 indicates a poor prognosis and suggested tailored treatment for AML patients.
We used GEPIA and TCGA repository data to analyze the expression of SFXN3 and its correlation with survival in AML patients. RT-qPCR was used to detect the SFXN3 level in our enrolled AML patients and volunteers. Additionally, Whole Genome Bisulfite Sequencing (WGBS) was used to detect the genomic methylation level in individuals.
Through the TCGA and GEPIA databases, we found that SFXN3 was enriched in AML patients, predicting shorter survival. Furthermore, we confirmed that SFXN3 was primarily overexpressed in AML patients, especially non-M3 patients, and that high SFXN3 in non-M3 AML patients was found to be associated with poor outcomes and frequent blast cells. Interestingly, non-M3 AML patients with high SFXN3 levels who received hypomethylating therapy showed a higher CR ratio. Finally, we found that SFXN3 could promote DNA methylation at transcription start sites (TSS) in non-M3 AML patients. These sites were found to be clustered in multiple vital cell functions and frequently accompanied by mutations in DNMT3A and NPM1.
In conclusion, SXFN3 plays an important role in the progression and hypermethylation in non-M3 AML patients and could be a potential biomarker for indicating a high CR rate for hypomethylating therapy.

Tet methylcytosine dioxygenase 2 (TET2) is one of the most frequently mutated genes in myelodysplastic syndrome (MDS). TET2 is known to involve a demethylation process, and the loss of TET2 is thought to cause DNA hypermethylation. Loss of TET2 function is known to be caused by genetic mutations and miRNA, such as miR-22. We analyzed 41 MDS patients receiving hypomethylating therapy (HMT) to assess whether TET2 mutation status and miR-22 expression status were associated with their clinical characteristics and treatment outcomes. Responsiveness to HMT was not affected by both TET2 mutation (odds ratio (OR) 0.900, p = 0.909) and high miR-22 expression (OR 1.548, p = 0.631). There was a tendency for TET2 mutation to be associated with lower-risk disease based on IPSS (Gamma = -0.674, p = 0.073), lower leukemic transformation (OR 0.170, p = 0.040) and longer survival (Hazard ratio 0.354, p = 0.059). Although high miR-22 expression also showed a similar tendency, this tendency was weaker than that of TET2 mutation. In summary, the loss of TET2 function, including both TET2 mutation and high miR-22 expression, was not a good biomarker for predicting the response to HMT but may be associated with lower-risk disease based on IPSS, lower leukemic transformation and longer survival.

BACKGROUND: Although the effects of U2 small nuclear RNA auxiliary factor 1 gene (U2AF1) mutations on the outcomes of patients with myelodysplastic syndromes (MDS) have previously been investigated, their prognostic significance remains controversial. We performed a meta-analysis to investigate the impact of U2AF1 mutations on MDS progression.
METHODS: Two reviewers independently extracted information such as hazard ratios (HRs) and 95% confidential intervals (CIs) for overall survival (OS) and leukemia-free survival (LFS) as well as the number of surviving patients each year after diagnosis from the included studies.
RESULTS: Thirteen studies with a total of 3038 patients were included. The summary odds ratio (OR) for U2AF1 mutations with an OS of 5 years was 0.37, the summary HR for U2AF1 mutations in OS was 1.60, and the summary OR for an OS of 5 years in patients with U2AF1S34 and U2AF1Q157 was 3.68. There were no significant differences in leukemia-free survival or hypomethylating therapy response between patients with and without U2AF1 mutations.
CONCLUSIONS: U2AF1 mutations were associated with poor survival in MDS patients, and patients with U2AF1Q157 had a worse OS than those with U2AF1S34. Our findings suggest that MDS patients with U2AF1 mutations could benefit more from hypomethylation therapy.

The prognosis of atypical chronic myeloid leukemia (aCML) patients is poor, but some patients with a suitable donor can be treated with allogeneic hematopoietic stem cell transplantation (HSCT). However, many of these patients cannot be treated with HSCT due to their age. The effectiveness of decitabine was recently indicated in case reports; however, the effectiveness of azacitidine (AZA) has not yet been reported. We report the case of a aCML patient successfully treated with AZA. A 66-year-old man with no remarkable medical history was admitted to our hospital because of leukocytosis. We diagnosed his disease as aCML and administered hydroxyurea (HU) and AZA. After four courses of AZA, his blood cell values improved, and he no longer needed transfusions and was able to stop HU. He continued receiving AZA without any severe complications. This is the first report that AZA is effective for the treatment of aCML.

We retrospectively analyzed the results of hypomethylating therapy in 586 patients (azacitidine in 423 and decitabine in 163) with International Prognostic Scoring System (IPSS) lower-risk myelodysplastic syndrome (MDS). The patients were reclassified with newer scoring systems (revised IPSS [R-IPSS], revised WHO classification-based Prognostic Scoring System [R-WPSS], and Lower Risk Prognostic Scoring System [LR-PSS]), and 21.8-38.4% of patients had high or very high risk features by the newer scoring systems. Median overall survival (OS) was 27.3 months and newer scoring systems well stratified the patients in terms of OS (R-IPSS, P=0.001; R-WPSS, P<0.001; LR-PSS, P<0.001). Hematologic improvement (HI) was observed in 279 patients (47.6%). OS differed by the achievement of HI (39.4% vs. 36.2%, P=0.067). The differences were significant only in patients of intermediate or high risk group by LR-PSS (P=0.034) or R-IPSS (P=0.018). In summary, IPSS lower-risk MDS included a broad range of prognosis, and hypomethylating therapy induced HI in approximately half of the patients. Achievement of HI was associated with longer survival, especially in patients with intermediate or high risk features by newer scoring systems. Hypomethylating therapy seems to have potential benefits in IPSS lower-risk MDS.

Although hypomethylating therapy (HMT) is the first line therapy in higher-risk myelodysplastic syndromes (MDS), predicting response to HMT remains an unresolved issue. We aimed to identify mutations associated with response to HMT and survival in MDS. A total of 107 Korean patients with MDS who underwent HMT (57 responders and 50 non-responders) were enrolled. Targeted deep sequencing (median depth of coverage 1,623X) was performed for 26 candidate MDS genes. In multivariate analysis, no mutation was significantly associated with response to HMT, but a lower hemoglobin level (<10g/dL, OR 3.56, 95% CI 1.22-10.33) and low platelet count (<50,000/μL, OR 2.49, 95% CI 1.05-5.93) were independent markers of poor response to HMT. In the subgroup analysis by type of HMT agents, U2AF1 mutation was significantly associated with non-response to azacitidine, which was consistent in multivariate analysis (OR 14.96, 95% CI 1.67-134.18). Regarding overall survival, mutations in DNMT1 (P=0.031), DNMT3A (P=0.006), RAS (P=0.043), and TP53 (P=0.008), and two clinical variables (male-gender, P=0.002; IPSS-R H/VH, P=0.026) were independent predicting factors of poor prognosis. For AML-free survival, mutations in DNMT3A (P<0.001), RAS (P=0.001), and TP53 (P=0.047), and two clinical variables (male-gender, P=0.024; IPSS-R H/VH, P=0.005) were independent predicting factors of poor prognosis. By combining these mutations and clinical predictors, we developed a quantitative scoring model for response to azacitidine, overall- and AML-free survival. Response to azacitidine and survival rates became worse significantly with increasing risk-scores. This scoring model can make prognosis prediction more reliable and clinically applicable.

Early therapeutic decision-making is crucial in patients with higher-risk MDS. We evaluated the impact of clinical parameters and mutational profiles in 134 consecutive patients treated with azacitidine using a combined cohort from Karolinska University Hospital (n=89) and from King\'s College Hospital, London (n=45). While neither clinical parameters nor mutations had a significant impact on response rate, both karyotype and mutational profile were strongly associated with survival from the start of treatment. IPSS high-risk cytogenetics negatively impacted overall survival (median 20 vs 10 months; p<0.001), whereas mutations in histone modulators (ASXL1, EZH2) were associated with prolonged survival (22 vs 12 months, p=0.01). This positive association was present in both cohorts and remained highly significant in the multivariate cox model. Importantly, patients with mutations in histone modulators lacking high-risk cytogenetics showed a survival of 29 months compared to only 10 months in patients with the opposite pattern. While TP53 was negatively associated with survival, neither RUNX1-mutations nor the number of mutations appeared to influence survival in this cohort. We propose a model combining histone modulator mutational screening with cytogenetics in the clinical decision-making process for higher-risk MDS patients eligible for treatment with azacitidine.