DNA methyltransferase inhibitors in hematological malignancies and solid tumors.

Abbreviations

cytarabine and anthracycline (daunorubicin/idarubicin/mitoxantrone) based standard induction therapy

adverse events

allogeneic hematopoietic stem cell transplantation

acute myeloid leukemia

all‐trans retinoic acid

B‐cell lymphoma 2

best supportive care

Bruton's tyrosine kinase

Cancer and Leukemia Group B

chimeric antigen receptor

conventional care regimen

cluster of differentiation

chronic lymphocytic leukemia

chronic myelomonocytic leukemia

complete remission (incomplete)

cullin‐RING ligases

cytotoxic T‐lymphocyte associated protein 4

disease‐free survival

donor lymphocyte infusion

DNA methyltransferase (inhibitor)

event‐free survival

European Medicine Agency

European Organisation for Research and Treatment of Caner

U.S. Food and Drug Administration

Fms‐like tyrosine kinase 3

granulocyte‐colony stimulating factor

gastrointestinal

Gruppo Italiano Malattie EMatologiche dell 'Adulto

graft‐versus‐host disease

graft‐versus‐leukemia

histone deacetylase inhibitor

intensive chemotherapy

isocitrate dehydrogenase 1/2

juvenile myelomonocytic leukemia

leukemia stem cell

myeloid cell leukemia protein‐1

myelodysplastic syndrome

myeloproliferative neoplasm

measurable residual disease

National Cancer Institute

non‐hodgkin lymphoma

nucleophosmin 1

non‐small cell lung cancer

overall response rate

overall survival

programmed cell death 1 (ligand 1) protein

progression‐free survival

partial remission

quality of life

relapsed and refractory

red blood cell

relapse‐free survival

study alliance leukemia

signal regulatory protein α

(stromal) tumor‐infiltrating lymphocytes

treatment of choice

Tet‐methylcytosine‐dioxygenase 2

transfusion independency

T‐cell immunoglobulin domain and mucin domain‐3

triple‐negative breast cancer

tumor protein 53

INTRODUCTION

With the updated 2022 version of the “Hallmarks of Cancer”, epigenetics has been recognized as a fundamental cornerstone in the understanding of oncogenesis and tumor progression. 1 This recognition is the result of more than 50 years of extensive research spanning from the initial discovery of gene regulatory processes and culminating in the development of novel targeted therapies that modulate these pathways. 2 , 3 Probably the most extensively studied epigenetic modifications is DNA methylation, a biochemical reaction in which a methyl residue is transferred to the 5 position of a cytosine nucleotide, yielding 5‐methylcytosine. While the majority of the genome in adult eukaryotic cells is methylated, methylation at CpG islands—often found within promoter regions—shows the strongest association to transcriptional regulation, thereby determining whether genes are activated or silenced.

The regulation of DNA methylation is orchestrated by a specific set of enzymes known as DNA methyltransferases (DNMT), which include DNMT1, DNMT3A, and DNMT3B. DNMT1 serves a dual role, maintaining methylation patterns during DNA replication, thereby ensuring the fidelity of epigenetic information through cellular divisions, but also serves as a transcriptional corepressor integrated into lineage master transcription factor hubs to regulate gene expression. 4 , 5 , 6 By contrast, DNMT3A and DNMT3B establish “de novo” methylation patterns during development and in response to environmental stimuli. In cancer, dysregulation of DNA methylation is a frequent occurrence, with profound implications for cellular function. Aberrant methylation patterns can lead to the silencing of tumor suppressor genes, the activation of oncogenes, and the disruption of other regulatory genes essential for cell differentiation and genomic stability. A refined understanding of these mechanisms has paved the way for the development of therapies aimed at DNMT inhibition. Initially, this approach was primarily conceptualized as a way to reverse pathological hypermethylation and thereby reactivate silenced tumor suppressor genes; however, it has become increasingly clear that the clinical efficacy of DNMT inhibition does not appear to rely predominantly on such targeted gene reaction. Rather, current concepts suggest that cancer cells are highly dependent on the maintenance of a tightly controlled DNA methylation program and therefore particularly vulnerable to global perturbations in methylation. 7 , 8 The ultimate goal remains to exploit these vulnerabilities while minimizing the toxicities typically associated with standard, intensive chemotherapy (IC).

Azacitidine: preclinical development

Azacitidine (5‐azacytidine, 5‐azaCR) was first synthesized in Czechoslovakia about 60 years ago by František Šorm and his team when they were exploring a variety of antineoplastic agents. 9 , 10 Chemically, it differs from the naturally occurring pyrimidine nucleoside cytidine only by having the fifth carbon in cytidine replaced by nitrogen. It can be described as 4‐amino‐1‐b‐D‐ribofuranosyl 1–1,3,5 triazine‐2‐one or 1‐b‐D‐ribofuranosyl‐5‐azacytosine; C 8 H 12 N 4 O 5 and has a molecular weight of 244 g/mol. 11

Azacitidine, together with the structurally related agent decitabine, is a prodrug and belongs to the class of cytosine analogues. 12 Following their cellular uptake by nucleoside specific transporters, azacitidine (and decitabine) requires sequential phosphorylation by key pyrimidine metabolism enzymes to become metabolically active. 13 Azacitidine's main anticancer effects are assumed to be related to two distinct modes of action. After cellular intake and phosphorylation, azacitidine is incorporated mostly (80%–90%) into RNA, which leads to disruption of cellular and nuclear RNA metabolism and, consequently, disruption of protein synthesis. 14 , 15 , 16 The effect of azacitidine on RNA function and protein synthesis is likely responsible for the liver and kidney toxicities observed at high doses in early clinical studies. 17 , 18 However, apart from RNA incorporation, a minor fraction of azacitidine (10%–20%) is also incorporated into DNA (via ribonucleotide reductase). In 1980, azacitidine's distinct mode of action, that is, its DNA demethylating activity, was first described. 19 After DNA incorporation, azacitidine irreversibly binds to DNMT1, which results in intracellular depletion of the enzyme. Consequently, demethylation results in the reactivation and thus transcription of previously silenced genes, such as tumor suppressor genes. 20 , 21

By the late 1980s, it had become evident that aberrant DNA methylation constitutes a fundamental driver in the pathogenesis of most if not all neoplasias, including myeloid malignancies such as myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). 22 , 23 , 24 The pronounced efficacy of DNMTi in hematological malignancies is largely attributed to the comparatively severalfold higher expression of pyrimidine metabolism in myeloid cells. In contrast, other cell types such as lymphoid cells display enhanced expression of enzymes involved in the de novo pyrimidine synthesis, resulting in increased intracellular pyrimidine pools that compete with DNMTi incorporation and thereby attenuate their therapeutic activity. 25 At the clinically applied lower doses today, this demethylating effect is considered the main contributor to its activity rather than direct cytotoxicity. 26 , 27 , 28

Azacitidine and decitabine: clinical development for the treatment of MDS and AML

Azacitidine in MDS

It took almost another two decades from the landmark discovery of the DNA‐demethylating effects of DNMTi to the initiation of large clinical studies in MDS, with the first larger trials conducted by the Cancer and Leukemia Group B (CALGB). 29 Their seminal clinical successes have paved the way for monotherapy approaches in MDS and AML (Table 1 summarizes single‐agent therapy randomized clinical trials; Table 3 summarizes all FDA/EMA approved single/combination therapies), replacing the former standard of “sole best supportive care” for patients who are not candidates for intensive therapies in AML or MDS. 30 , 31

AML and MDS are clonal hematological diseases characterized by aberrant proliferation and disturbed differentiation of myeloid cells, often driven by critical molecular alterations such as hypermethylation events. 32 , 33 To counteract these observed changes, the two first‐generation DNA methyltransferase inhibitors (DNMTi) azacitidine and decitabine (5‐aza‐2′‐deoxycytidine, 5azaCdR) have been clinically developed. 13 Although not initially recognized as an epigenetic drug, early preclinical studies in cell culture and mouse models demonstrated their high potency, despite significant side effects. 10 In the 1970s, azacitidine was introduced as a treatment for childhood and adult leukemia in North America and Europe. 34 , 35 Despite encouraging results, its usage was limited by severe myelosuppression and gastrointestinal (GI) toxicity, which can be primarily attributed to the high doses used in these early studies.

Azacitidine gained approval by the U.S. Food and Drug Administration (FDA) for the treatment of MDS in 2004, following the results of the above‐mentioned National Cancer Institute (NCI)‐sponsored phase III clinical trial conducted by the CALGB, along with two supportive phase II CALGB studies. 36 , 37 , 38 The phase III trial, chaired by Dr. Lewis Silverman and published in 2002, 29 involved 191 patients with MDS, demonstrating a 60% disease control rate in the azacitidine arm compared to only 5% in the best supportive care (BSC) arm. This study also established the standard dose of subcutaneous azacitidine as 75 mg/m 2 for 7 days in a 28‐day cycle. In 2007, the FDA expanded its approval to include intravenous administration based on an uncontrolled phase II study, with pharmacokinetic bioavailability data. 39 By 2008, the European Medicines Agency (EMA) had approved azacitidine for higher‐risk MDS patients based on the results of the Aza‐001 trial. This multicenter, open‐label randomized phase III trial included 358 patients who received either a 7‐day treatment of 75 mg/m 2 subcutaneous azacitidine for at least six cycles or conventional care, which included BSC, low dose cytarabine (20 mg/m 2 ) or IC, consisting of cytarabine in combination with an anthracycline. 40 , 41 After a median follow‐up of 21.1 months, the study met its primary endpoint, with an overall survival (OS) of 24.5 months in the azacitidine arm, compared to 15.0 months for the conventional care arm (HR 0.58, 95% CI 0.43–0.77, p = 0.0001).

Azacitidine in AML

In 2015, the applicability of azacitidine was further evaluated in medically non‐fit AML patients with at least 30% blasts in an international, multicenter, open‐label phase III trial also comparing azacitidine to conventional care regimens (CCR). 42 This study, which included 488 patients assigned 1:1 to both interventions, demonstrated improved OS in the azacitidine arm of 10.4 months (95% CI 8.0–12.7), compared to the 6.5 months in the CCR arm (95% CI 5.0–8.6). Concurrently, efforts have been made to enhance treatment efficacy and durability by incorporating azacitidine into standard IC regimens in fit older AML patients. However, the phase II AML‐AZA trial of the Study Alliance Leukemia (SAL) German Cooperative Group, which enrolled 214 patients, failed to demonstrate an additional benefit of azacitidine and IC over IC alone in event‐free survival (EFS), while simultaneously leading to a marked increase in treatment‐related toxicities. 43 Most recently, azacitidine has also been evaluated as a maintenance therapy in AML patients who achieved complete remission (CR) after IC. The HOVON97 trial demonstrated the superiority of an azacitidine maintenance (50 mg/m 2 for 7 days in a 28‐day cycle) compared to observation at 12 months disease‐free survival (DFS, 64% vs. 42%, p = 0.04); OS, however, remained unaffected. 44

Oral azacitidine (CC‐486)

In recent years, an oral azacitidine formulation (also referred to as CC‐486) has been granted approval by the FDA and EMA based on the results of the QUAZAR AML‐001 trial, which investigated CC‐486 as a maintenance therapy for older AML patients in first remission after IC. 45 The trial randomized 472 patients to receive either 14 days of 300 mg oral azacitidine once daily or placebo, repeated every 28 days. The primary endpoint of median OS was met, with an OS of 24.7 months in the oral azacitidine cohort compared to 14.8 months in the placebo cohort ( p < 0.001). The secondary endpoint of relapse‐free survival (RFS) was also met. Side effects of CC‐486 were overall comparable to those of the subcutaneous and intravenous administration, mainly being grade I‐II (GI) side effects (more pronounced than with parenteral administration) and grade III‐IV hematological side effects.

In addition, the effects of CC‐486 on transfusion dependency in lower‐risk MDS patients have been evaluated. 46 The AZA‐MDS‐003 trial assigned a total of 216 patients to receive either CC‐486 or placebo. The primary endpoint of red blood cell (RBC) transfusion independence (TI) for at least 56 days was reached in 30.8% of patients receiving CC‐486 compared to 11.1% in the respective placebo cohort (OR 3.6, 95% CI 1.7–7.4, p = 0.0002). Likewise, 24.3% of patients receiving CC‐486 achieved improvement in platelet counts compared to 6.5% in the placebo arm.

Decitabine in MDS

In parallel with the development of azacitidine, the development of decitabine, the chemically very closely related deoxyribonucleoside and first generation DNMTi, has also been steadily pursued as a less‐intensive alternative to standard IC in patients with myeloid malignancies. Similar to azacitidine, decitabine was first synthesized in the 1960s, with initial phase I clinical data as a traditional chemotherapeutic being discouraging due to its high toxicities. 47 However, the repurposing of decitabine, an azanucleoside drug that is incorporated solely into DNA, with adjusted dosage regimens, has led to its resurgence. It was investigated both in the US and in Europe (particularly in Italy, the Netherlands and Germany), and in the early 1990s, the MD Anderson Cancer Center started incorporating decitabine in their clinical trials. In 2006, Kantarjian et al. first reported results of a randomized controlled phase III clinical trial in MDS patients. 48 In this trial, 170 MDS patients were randomized to receive either 15 mg/m 2 intravenous decitabine every 8 h for a total of 3 days, repeated every 6 weeks, or BSC. The trial met its primary endpoint of increased overall response rate (ORR), with 17% in the decitabine group vs. 0% in the BSC group ( p < 0.001) and a trend toward longer time to progression to AML or death. Based on these trials, the FDA approved decitabine in 2006 for the treatment of higher‐risk MDS.

These results were confirmed by a European EORTC (European Organisation for the Research and Treatment of Cancer) Leukemia Cooperative Group phase III clinical trial assessing decitabine versus BSC in older intermediate‐ or higher‐risk MDS patients. 49 Decitabine, while significantly prolonging progression‐free survival (PFS) from 3.3 to 6.0 months (HR 0.68; 95% CI, 0.52–0.88; p = 0.004), did not yield significantly prolonged OS (10.1 months in the decitabine cohort versus 8.5 months in the BSC arm).

Decitabine in AML: non‐fit patients

In 2012, the DACO‐016 trial, a multicenter, open‐label phase III trial followed, which investigated the effect of decitabine in newly diagnosed, medically non‐fit AML patients. 50 The study did not initially detect an increase in OS with decitabine (7.7 months, 95% CI 6.5–9.2) that reached statistical significance compared to treatment of choice (TC, 5.0 months, 95% CI 4.3–6.3), which was, however, reached in a second OS analysis. The CR(p) rates of patients treated with decitabine were significantly higher (17.8%) compared to the TC cohort (7.8%, OR 2.5, 95% CI 1.4–4.8, p = 0.001). This study also established the benchmark dosing regimen of 20 mg/m 2 intravenous decitabine daily for 5 days, repeated every 4 weeks.

A randomized study conducted at the MD Anderson Cancer Center addressed the question of the value of decitabine dose intensification in medically non‐fit elderly AML patients, comparing the 10‐day to the 5‐day treatment schedule. In a phase II clinical trial involving 71 patients with newly diagnosed AML, Short et al. reported similar clinical CCR rates (43% vs. 40%) as well as 1‐year OS outcomes (25% vs. 25%) between the 5‐day and 10‐day decitabine treatment groups. 51 However, the validity of these findings has been called into question due to the study's limited statistical power, leaving the equivalence between the two regimens unresolved, particularly in these elderly and non‐fit patients. 52

Decitabine in AML: fit patients

The EORTC Leukemia Cooperative Group, the Gruppo Italiano Malattie EMatologiche dell 'Adulto (GIMEMA) and German MDS Study Group subsequently conducted the Intergroup trial AML21, which compared standard “7 + 3” intensive induction combination chemotherapy with 10‐day decitabine monotherapy followed by allogeneic hematopoietic stem cell transplantation (allo‐HCT). 53 , 54 This first trial to investigating a de‐escalation approach versus intensive induction in a large, multinational phase III European trial randomized 606 patients in a 1:1 ratio and demonstrated equivalence in 4‐year OS (26% vs. 30%, HR 1.04, 95% CI 0.86–1.26, p = 0.68) between the DNMTi and IC treatments. Of note, grade ≥3 adverse events (AE) were significantly less frequent in the decitabine cohort, highlighting its potential for reduced treatment‐related toxicity. Importantly, health‐related quality of life (QoL) in the decitabine arm was less compromised than with IC, a result which, while very much intuitive, had not been demonstrated before in a randomized trial. This result had major implications for the use of DNMTi‐based “bridging” therapy of AML/MDS patients to the curative approach of allo‐HCT, as patients having received less toxic pre‐treatment (by the principle of “less is more”) may have a more favorable transplant outcome than those having received repetitive cycles of standard chemotherapy.

Decitabine in AML: maintenance treatment

The ECOG‐ACRIN E2906 trial led by Foran et al. confirmed the significance of a decitabine maintenance after IC in an older AML patient population. 55 In this trial, a total of 120 patients were randomized 1:1 to a 3‐day course of decitabine maintenance schedule (20 mg/m 2 , repeated every 4–6 weeks), first reported by Lübbert et al., 56 or observation only. Median OS was significantly improved in the decitabine cohort compared to observation only (HR 0.69, 80% CI 0.51–0.93, p = 0.06) with DFS indicating a trend toward favoring decitabine as well, although it is worth noting that the authors set a rather unusual significance level at 0.1, with an 80% CI.

Decitabine in chronic myelomonocytic leukemia (CMML)

Interestingly, decitabine's therapeutic potential has also been investigated in chronic myelomonocytic leukemia (CMML), a hematological disease that has often been included in traditional DNMTi trials due to its risk of progression to AML but rarely been studied on its own. In 2023, a phase III European Myelodysplastic Neoplasms Cooperation Group trial was reported that assigned 170 CMML patients to a 5‐day decitabine treatment versus BSC including hydroxyurea. 57 While the study failed to demonstrate superiority of decitabine as regards EFS and OS, the risk of CMML progression or transformation to AML was significantly reduced in the decitabine compared to the hydroxyurea cohort (HR 0.62, 95% CI 0.41–0.94, p = 0.005).

Azacitidine versus decitabine: prospective comparative studies

Until now, most clinical investigators have chosen to investigate either azacitidine or decitabine, with only very few directly comparing these two therapies. 53 Among the very few notable studies is a randomized phase II clinical trial that assigned 113 lower‐risk MDS patients to standard dose azacitidine and decitabine in order to compare transfusion dependency. 58 Out of 59 patients' transfusion‐dependency at baseline, a significantly higher proportion reached TI in the decitabine versus azacitidine cohort (41% vs. 15%, p = 0.0039), suggesting decitabine being superior in this head‐to‐head comparison. In another randomized trial, Jabbour et al. compared responses of a 3‐day decitabine treatment to a 3‐day azacitidine treatment in 113 lower‐risk MDS and MDS/MPN (myelodysplastic syndrome/myeloproliferative neoplasms) overlap patients. 59 While the ORR was significantly higher in the decitabine arm (70% vs. 49%), this was not reflected by median EFS nor transfusion independency.

Guadecitabine (SGI‐110)

Guadecitabine (SGI‐110), a second generation DNMTi, is a dinucleotide that acts as a prodrug of decitabine. Guadecitabine has a longer half‐life compared to decitabine, potentially enhancing its hypomethylating activity. 60 Initial data from phase II trials in both AML and high‐risk MDS demonstrated good feasibility of guadecitabine with similar ORR and CR rates between the two investigated doses of 60 mg/m 2 and 90 mg/m 2 . 61 , 62 However, in a large randomized phase III trial of guadecitabine vs. investigator's choice in newly diagnosed AML, the trial could not demonstrate superiority in OS (7.1 vs. 8.5 months) nor CR rate (19% vs. 17%) for a guadecitabine treatment regimen of 60 mg/m 2 over 5 days in comparison to TC. 63 However, the phase III ASTRAL‐2 trial (in relapsed/refractory [r/r] AML patients) which investigated a longer, 10‐day course of 60 mg/m 2 guadecitabine, reported higher CCR rates in the guadecitabine arm (27% vs. 14%, p < 0.01) than the TC, albeit without affecting OS. 64

Oral decitabine formulation (ASTX727)

Similar to CC‐486, an oral formulation of decitabine (ASTX727) has been developed to facilitate at‐home administration. This was with the goal of improving patients' QoL by limiting time spent in the hospital, therefore reducing the “time toxicity” that can burden non‐curative treatment conducted with parenteral drug administration. The most promising approach involves the oral formulation of the combination of decitabine with cedazuridine, a small‐molecule inhibitor of cytidine deaminase that limits the degradation of oral decitabine. This fixed‐dose combination, marketed as ASTX727, was compared to i.v. decitabine in the ASCERTAIN trial, a multicenter, randomized, open‐label, crossover pharmacokinetic study in adults with intermediate‐ or higher‐risk MDS and CMML. 65 , 66 The trial demonstrated pharmacological and pharmacodynamical equivalence to i.v. decitabine, leading to FDA approval for MDS, as this oral formulation could provide patients with a more convenient and equally effective treatment option. These results have also been validated in another crossover phase III study, conducted in Europe, comparing, in newly diagnosed AML patients, ASTX727 with i.v. decitabine and indicating similar systemic exposure and demethylation rates, 67 resulting in the EMA approval of ASTX727 in newly diagnosed, IC ineligible AML patients in 2023.

In addition to the currently approved hypomethylating agents, a promising alternative class of direct enzymatic DNMT1i are emerging. 68 , 69 GSK3685032 is a first‐in‐class, reversible DNMT1i that has shown preclinical efficacy by inducing loss of DNA methylation, transcriptional activation and cancer cell growth inhibition in various cancer cell lines, however clinical data is still lacking. 68 In r/r MDS/AML, the novel oral DNMT1i inhibitor NTX‐301 is actively tested in a phase I trial ( NCT04167917 ) to assess the safety and tolerability as well as the dose‐limiting toxicities. 69 This trial is expected to complete in 2027 and will include the secondary endpoint of ORR, PFS and OS. These novel developments underscore the translational potential of enzymatic DNMT1‐targeted therapy as a more selective and potentially less toxic alternative to traditional hypomethylating agents, marking an important future direction in epigenetic treatment modalities.

Demethylating agents and cellular therapies in MDS and AML

Decitabine conditioning for allogeneic stem cell transplantation

A Chinese phase III trial evaluated the impact of a modified conditioning regimen for allo‐HCT, incorporating decitabine as well as G‐CSF in addition to standard busulfan and cyclophosphamide in patients with relapsed MDS or secondary AML. 70 The study enrolled 202 patients across six Chinese sites and demonstrated a significant reduction in 2‐year cumulative incidence of relapse in the decitabine and G‐CSF augmented treatment arm compared to standard busulfan and cyclophosphamide conditioning (10.9% vs. 24.8%, HR 0.39, 95% CI 0.19–0.79, p = 0.011). Notably, the cumulative day 100 post allo‐HCT incidences of infections, GI side effects, and death were comparable between the two cohorts, thereby suggesting broader applicability of this modified conditioning.

Azacitidine maintenance after transplant

While both AML and MDS are potentially curable diseases after allo‐HCT, relapse remains one of the most significant causes of death in these patients. 71 As a result, DNMTi maintenance and relapse treatment strategies have been explored, with the rationale of both enhancing the Graft‐versus‐Leukemia (GvL) effect and instigating direct antitumor effects. Initial evidence supporting the use of low‐dose azacitidine as a maintenance or salvage therapy post allo‐HCT emerged around 2009. 72 In this early trial, Jabbour et al. reported an actuarial 1‐year EFS of 55% as well as an OS of 90% with low‐dose azacitidine. However, this study was limited by a small cohort of 17 patients and a median follow‐up of only 16 months after allo‐HCT and 11 months post‐azacitidine treatment. A subsequent, larger follow‐up trial of 45 patients who received low‐dose azacitidine as a maintenance therapy after allo‐HCT in heavily pretreated MDS/AML cases demonstrated an EFS of 58% at 18.2 months (95% CI 11.9–NA) and OS of 77% at a median of 30.8 months (95% CI 14.3–NA), indicating improved clinical outcomes. 73

CC‐486 maintenance after transplant

Additionally, the oral azacitidine formulation CC‐486 has been evaluated as a maintenance therapy after allo‐HCT in MDS/AML patients, demonstrating a feasible toxicity profile and low rates of relapse and Graft‐versus‐Host Disease (GvHD). 74 Mechanistic studies have also suggested that azacitidine can reinitiate the expression of silenced leukemia antigen genes and increase the frequencies of regulatory T cells, thereby further enhancing GvL and GvHD effects. 75 , 76 These findings prompted the initiation of a phase III clinical trial. However, an open‐label, randomized phase III clinical trial enrolling 187 higher‐risk MDS/AML patients to compare azacitidine maintenance versus observation after allo‐HCT failed to demonstrate differences in RFS (2.07 vs. 1.28 years) and OS (2.52 vs. 2.56 years). 77 In this context, another phase III clinical trial comparing CC‐486 maintenance versus placebo in MDS/AML patients after allo‐HCT is expected to finish recruiting in 2025 (AMADEUS, NCT04173533 ).

Decitabine maintenance after transplant

Maintenance therapy with decitabine following allo‐HCT in MDS/AML patients has also been exploited. A phase I, dose‐escalating trial, indicating manageable toxicities, 78 as well as a currently ongoing phase Ib clinical trial investigating the combination of oral decitabine and cedazuridine as maintenance after allo‐HCT for MDS, are currently ongoing, with results anticipated soon ( NCT04980404 ). However, larger, especially phase II/III clinical trials for decitabine maintenance after allo‐HCT are still lacking.

MRD‐driven relapse treatment after transplant: RELAZA trials

In AML and MDS patients who displayed features of imminent relapse after allo‐HCT, indicated by measurable residual disease (MRD) positivity, the phase II RELAZA‐1 trial demonstrated the feasibility of azacitidine with an ORR of restored or stable donor chimerism of 80% and a subsequently delayed relapse at a median of 231 days. 79 The follow‐up RELAZA‐2 trial confirmed these results, investigating 60 high‐risk MDS or AML patients who became MRD‐positive after IC or allo‐HCT and were subsequently eligible for an azacitidine salvage treatment. Patients who remained MRD positive had a significantly lower 12‐month RFS at 46% in comparison to the 88% seen in MRD‐negative converters (HR 6.6, 95% CI 3.7–11.8, p < 0.0001). 80

Combination of azacitidine or decitabine with donor‐lymphocyte infusions

For patients experiencing relapse of MDS/AML, combination treatment of donor lymphocyte infusions (DLI) and DNMTi has been investigated. A pilot study by Lübbert, Wäsch et al. demonstrated the feasibility of combining DLI with a 3‐day 100 mg (flat dose) subcutaneous azacitidine in relapsed AML patients, even within an adverse cytogenetic population. 81 A subsequent single‐arm phase II clinical trial investigating the effects of DLI and azacitidine in r/r MDS/AML followed shortly thereafter, demonstrating an ORR of 30%. 82 These results were later confirmed by an even larger retrospective analysis of 154 post‐allo‐HCT relapsed MDS/AML patients demonstrating an ORR of 33% (27% CR, 6% partial remission [PR]) and a 2‐year OS of 29%. 83 Similarly, a retrospective analysis of 36 patients receiving DLI and decitabine post allo‐HCT relapse showed a similar efficacy with an ORR of 25%. 84 However, the 2‐year OS was significantly lower at 11%, thereby suggesting DLI and decitabine may be more feasible as a second‐line treatment after azacitidine failure. Most recently, novel dual treatment combinations have been under investigation.

DNMTi and nivolumab in AML patients relapsing after transplant: the NIFAR trial

A prospective phase II clinical trial (EudraCT‐No. 2017–002194‐18) combining azacitidine and the anti‐PD1 inhibitor nivolumab in relapsed AML patients after allo‐HCT demonstrated an ORR of 25% (12.5% CR (i) , 12.5% PR) and a median survival of 15.6 months. 85 This response rate was attributed to higher frequencies of activated, non‐quiescent CD8 + T cells as well as a shift toward pro‐inflammatory expression profiles of T and myeloid cells.

DNMTi and CAR‐T cell therapies in AML

Besides the combination with immune checkpoint inhibitors for the treatment of AML, DNMTi were also combined with chimeric antigen receptor (CAR)‐T cells directed against CD123 to treat AML in mice. 86 These studies used anti‐CD123 CAR‐T cells with a CD28‐OX40‐CD3ζ intracellular signaling domain. Interestingly, azacitidine treatment enhanced CAR‐T cell‐mediated AML blast elimination by increasing CD123 expression on leukemia cells. In addition, leukemia‐bearing mice treated with azacitidine displayed lower frequencies of exhausted cytotoxic CTLA‐4 negative anti‐CD123 CAR‐T cells phenotype with higher TNFα production. 86 Building on these promising preclinical data, clinical trials assessing the synergistic effects of DNMTi and CAR‐T therapies in AML patients are still to be awaited.

DNMTi‐based combination studies in MDS and AML

DNMTi in combination with BCL‐2 inhibition

DNMTi have been extensively studied in various combination therapies, primarily as doublets, in frail patients or those for whom IC is not suitable (Table 2 summarizes combination therapy randomized clinical trials; Table 3 summarizes all FDA/EMA approved single/combination therapies). Combination therapies in both newly diagnosed and r/r AML/MDS patients have become an area of intensive study, especially with an increasing number of novel therapeutics becoming available and possible synergistic effects of dual treatment. 13 , 87 Among the most promising combination therapies are DNMTi in combination with inhibitors of the antiapoptotic protein BCL‐2, such as venetoclax. (Pre)‐clinical data suggest that these agents synergistically enhance each other's effect, offering a more potent therapeutic approach than either agent alone. 88 , 89 For instance, azacitidine was shown to reduce levels of antiapoptotic MCL‐1 in AML blasts and induce an integrated stress response via PAIMP1 induction, making blasts susceptible to venetoclax‐induced apoptosis. 90 Conversely, venetoclax inhibits dihydroorotate dehydrogenase, a key enzyme in conferring resistance to DNMTi. 91 Additional mechanisms, such as selective alteration of the citric acid cycle energy metabolism in leukemia stem cells (LSCs), inhibiting cellular energy supply, are discussed and may further contribute to the efficacy of this combination treatment. 92 Common side effects of this dual treatment include myelosuppression, infections, and GI symptoms. 93

In 2020, the combination treatment of azacitidine, decitabine, or low‐dose cytarabine in combination with venetoclax received full FDA approval for the treatment of newly diagnosed AML in adults who are 75 years or older, or in those with comorbidities that preclude IC based on the results of the VIALE‐A trial. 94 This pivotal study, which involved 431 patients older than 75 years or ineligible for IC, demonstrated significant improvements in response rates and OS with the dual treatment compared to azacitidine or decitabine alone. After a median follow‐up of 20.5 months, the combination therapy resulted in an increased OS of 14.7 months (95% CI 11.9–18.7) compared to the OS of 9.6 months (95% CI 7.4–12.7) in the control regimen (HR 0.66, 95% CI 0.52–0.85, p < 0.001). 94 The study also reported a higher rate of composite CR in the dual treatment arm (66.4%, 95% CI 60.6–71.9) compared to the control arm (28.3%, 95% CI 21.1–36.3, p < 0.001). Notably, the combination treatment demonstrated superiority in a subset analysis of patients with IDH1/2, NPM1 , and FLT3 mutations. Encouraging results have also been reported from a phase I/II study of azacitidine plus venetoclax in patients with high‐risk MDS or CMML. 95 The confirmatory randomized phase III VERONA trial ( NCT04401748 ) of venetoclax with azacitidine has been closed for accrual and is reported to be negative, though the publication is still pending (AbbVie press release 16th June 2025, NCT04401748 ).

DNMTi in combination with retinoic acid

In contrast, the combination of DNMTi with many other targeted agents has yet, with very few exceptions, to show improved outcomes in randomized trials. DNMTi have also been studied in combination with agents that stimulate cell maturation. A randomized phase II study (DECIDER) of valproate or retinoic acid in combination with decitabine in elderly unfit AML patients revealed that all‐trans retinoic acid (ATRA) plus decitabine induced significantly higher remission rates than decitabine alone (21.9% vs. 13.5%, OR 1.80; 95% CI 0.86–3.79, p = 0.06) as well as significantly improved OS (8.2 vs. 5.1 months, HR 0.65, 95% CI 0.44–0.89, p = 0.006). 96 Valproic acid had no effect in this trial. Based on this study, a randomized, placebo‐controlled, phase III trial (DECIDER‐2) investigating the effects of ATRA in combination with decitabine and venetoclax is ongoing (EUDRACT 2020‐005495‐36; DRKS 00023646, P001516).

Similar results have also been reported by a Chinese group investigating 223 MDS‐EB1/2 patients randomized 1:1 to decitabine and ATRA versus decitabine only, indicating significantly higher ORR (78% vs. 51%, OR 3.4, 95% CI 1.90–6.09, p < 0.001), higher CR rates (23% vs. 12%, OR 2.05, 95% CI 1.02–4.25, p = 0.042) and PFS (14.9 vs. 10.5 months, HR 0.70, 95% CI 0.51–0.97, p = 0.032). The SELECT‐MDS‐1 trial ( NCT04797780 ), evaluating tamibarotene, an oral selective retinoic acid receptor α agonist, in combination with DNMTi was closed for futility.

DNMTi in combination with ascorbic acid

In addition to clinical trials on vitamin A derivatives, investigating vitamin C, a cofactor of the epigenetic regulator TET2 , has also sparked interest recently. 97 , 98 A phase II trial investigating a 12‐month oral 1000 mg/day vitamin C supplementation in lower‐risk MDS and MDS/MPN overlap patient cohorts has shown early positive results in OS, however, more extensive research will be needed to draw final conclusions. 99

Other DNMTi‐based dual combination studies

Combining several epigenetic drugs, thereby potentially eliciting a synergistic effect by the reactivation of silenced genes, has also sparked significant interest. Candidates include the histone deacetylase inhibitors (HDACi) entinostat and pracinostat, and the IDH1 inhibitor ivosidenib. 100 In the phase III PRUMULA trial, 406 AML patients were assigned 1:1 to receive azacitidine in combination with either pracinostat or placebo. Unfortunately, the trial failed to demonstrate superiority in both OS (both 9.95 months) and CCR (36% vs. 31.5%). 101 In MDS, a phase II study reported similar outcomes with no significant differences in OS (16 vs. 19 months) and PFS (11 vs. 9 months). 102 Likewise, the combination of azacitidine with another oral HDACi, entinostat, did not demonstrate improvement in hematological normalization (32% vs. 27%) nor ORR (46% vs. 44%) in MDS/AML patients when compared to single‐agent treatment only. 103 Fortunately, the combination of other epigenetic drugs has been more successful. Ivosidenib, an oral IDH1 inhibitor, was shown to augment 12‐month EFS (37% vs. 12%; HR 0.33 95% CI 0.16–0.69, p = 0.002) and improve OS (24.0 vs. 7.9 months; HR 0.44, 95% CI 0.27–0.73, p = 0.001) in a randomized, placebo controlled phase III clinical trial investigating newly diagnosed IDH1 mut AML patients. 104 Unsurprisingly, the most common AEs ≥3 grade were hematological and included febrile neutropenia and bleeding.

Beyond these dual epigenetic drug combinations, combination of DNMTi with several other, already well‐established single agents has also been exploited. In the open‐label, multicenter phase III LACEWING trial, 123 FLT3 mut AML patients were randomized 2:1 to the FLT3 inhibitor gilteritinib in combination with azacitidine or simply azacitidine. 105 At an interim analysis, OS (9.82 vs. 8.87 months) and EFS (0.03 months in both) did not display a significant improvement, thereby leading to the termination of this trial (by set study criteria defined previously). Likewise, the first generation FLT3 inhibitor midostaurin failed to demonstrate superiority over single agent decitabine treatment in inducing CR/CRi and 1‐year OS in a cohort of 140 higher‐risk MDS/AML patients. 106 Ibrutinib, an oral Bruton's tyrosine kinase (BTK) inhibitor, has been investigated in a mixed MDS/AML patient cohort of 165 patients in combination with the DNMTi decitabine. 107 No significant differences in OS (10.0 vs. 11.5 months) nor ORR (41% vs. 50%) could be reported. Additionally, bortezomib and lenalidomide, two very well‐established drugs in multiple myeloma, have undergone investigation, potentially repurposing them in the dual combination with an DNMTi. In a phase II trial of 165 AML patients, the addition of bortezomib to decitabine, however, did not improve OS (8.9 vs. 9.3 months) nor ORR (38% vs. 39%). 108 Likewise, a combined phase II/III trial of the Northern American Study group investigated, in MDS and CMML, the effects of azacitidine with or without lenalidomide and vorinostat. ORR (38 vs. 49% vs. 27%) of the azacitidine only vs. azacitidine and lenalidomide vs. azacitidine and vorinostat cohort were similar. 109

In addition to these well‐known therapeutics, novel drugs with new targets and mechanisms are quickly entering the stage, with some of them being investigated in combination with DNMTi in AML/AMS. Pevonedistat (TAK924, MLN4924) is a first‐in‐class, small molecule inhibitor of NEDD8‐activating enzyme that selectively prevents activation of Cullin‐RING ligases (CRLs) and thereby diminishes proteasomal degradation of CRL substrates. NAE inhibition demonstrated preclinical efficacy in multiple cancer models, including myeloid malignancies. 110 Pevonedistat plus azacitidine was compared to azacitidine in higher‐risk MDS or low‐blast percentage AML. 111 However, the phase III PANTHER trial failed to demonstrate improvements in EFS, or median OS, with only a subgroup analysis suggesting potential benefit in high‐risk MDS patients receiving more than three (23.8 vs. 20.6 months, p = 0.021) cycles or more than six cycles (27.1 vs. 22.5 months, p = 0.008). 112 The most heavily investigated and targeted agent for patients with TP53 ‐mutant MDS and AML has been APR‐246 (eprenetapopt), a p53 reactivator, in combination with azacitidine, but also in triplets with venetoclax. 113 , 114 Despite positive results in phase II trials, a phase III trial did not confirm superior response or improved survival ( NCT03745716 ).

DNMTi based‐combination with immune checkpoint inhibition

The dual combination of an antibody‐based backbone in conjunction with DNMTi has also been investigated intensely in recent years. Magrolimab, a first‐in‐class investigational monoclonal antibody against CD47 and macrophage checkpoint inhibitor is designed to interfere with recognition of CD47 by the SIRPα receptor on macrophages, thus blocking the “don't eat me” signal used by cancer cells. Despite positive results, the ENHANCE‐3 trial comparing magrolimab and azacitidine to azacitidine and placebo in untreated patients with MDS was terminated because of futility and excessive deaths in February 2024 (Gilead press release, NCT05079230 ). Bexmarilimab (FP‐1305) is a novel humanized anti‐CLEVER‐1 IgG4‐antibody capable of inducing a phenotypic M2 to M1 immune switch of tumor‐associated macrophages. It is studied in an ongoing phase I/II study in combination with azacitidine with or without venetoclax ( NCT05428969 ) in MDS and AML.

Checkpoint inhibition in combination with DNMTi is the focus of several trials ongoing in MDS and AML. While early data appeared promising, confirmatory studies thus far have been largely disappointing. Sabatolimab, an experimental immunotherapy with immuno‐myeloid activity that targets TIM‐3 on immune cells and leukemic blasts, is being evaluated for the treatment of myeloid malignancies as part of the STIMULUS clinical trial program, in combination with azacitidine. However, the randomized phase III trial ( NCT04266301 ) failed to reach superiority in OS in intermediate‐, high‐ or very high‐risk MDS not eligible for IC or allo‐HCT. Although data from this trial have not yet been published, preliminary results from the STIMULUS‐MDS2 trial appear to be consistent with the negative findings of the STIMULUS‐MDS1 trial, which evaluated the combination of azacitidine with sabatolimab in previously untreated higher‐risk MDS patients. 115

Two randomized phase II trials investigating azacitidine with or without durvalumab (anti‐PD‐L1) as first‐line therapy for older, frail patients with higher‐risk MDS (<20% blasts) or AML (≥20% blasts) demonstrated that the addition of durvalumab did not improve ORR (61.9% vs. 47.6% for MDS, 31.3% vs. 35.4% for AML) or OS (11.6 vs. 16.7 months for MDS, 13.0 vs. 14.4 months for AML) compared to single agent azacitidine treatment. 116 , 117 Nivolumab (anti‐PD‐1) with or without ipilimumab (anti‐CTLA‐4) is being studied in a non‐randomized phase II study in patients with MDS. The study is closed for accrual, however results are pending ( NCT02530463 ). The currently ongoing LEAP trial ( NCT03092674 ) is a randomized controlled phase II/III clinical trial investigating various treatments for older patients with newly diagnosed AML or MDS. Per design of this study, azacitidine is either administered as single agent (arm A), combined with either nivolumab (arm B) or midostaurin (arm C), whereas in arm D, 5‐day decitabine is followed by cytarabine. The study is expected to finish recruiting by late 2025.

Pembrolizumab (anti‐PD‐1) is being studied in a phase Ib trial in combination with decitabine with/without venetoclax, for r/r MDS or AML. Interim results of six patients with AML demonstrated that four patients responded to this dual treatment ( NCT03969446 ), final results are awaited. Another phase II study of azacitidine with pembrolizumab in patients with intermediate‐1 or higher‐risk MDS, including patients who were deemed DNMTi refractory, displayed only moderate activity. 118 A study of atezolizumab (anti‐PD‐L1) in conjunction with azacitidine was terminated early by the sponsor due to high early death rate and lack of efficacy. 119

DNMTi in lymphoid malignancies

While azacitidine and decitabine are firmly established as a therapeutic backbone in MDS and AML patients who cannot tolerate IC, their use in lymphoid malignancies, specifically non‐Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia (CLL), is exploratory and thus off‐label. Clinical trials indicate potential benefits, particularly in combination therapies, but these applications require further validation through larger, randomized studies. Several studies have explored the combination of azacitidine (and, less frequently, decitabine) with lenalidomide in CLL and NHL, both sequentially and concurrently. 120 , 121 In a phase II study of decitabine in r/r aggressive NHL, the hypomethylating agent was well‐tolerated and exhibited modest clinical activity, with an ORR of 40% versus 33% for the standard of care, though this difference was not statistically significant. 120 In addition, others have tried to boost the efficacy of standard lymphoma therapy (R‐CHOP) by combining this treatment with a DNMTi. A phase I study of 59 patients in intermediate/high‐grade diffuse large B‐cell lymphoma showed potential clinical benefit by combining oral CC‐486 with standard R‐CHOP. ORR was 94.1% (88.1% in CR) with a 1‐year PFS of 84.1%. 122 However, larger follow‐up trials are needed to further validate these results.

DNMTi in solid tumors

DNMTi have been investigated for the treatment of solid tumors since the 1970s, with the most notable agents being decitabine and azacitidine. These compounds have shown some potential, particularly when used in combination with other therapeutic agents. However, clinical outcomes have been variable, and ongoing research is critical to better define their role and optimize their therapeutic efficacy.

Lung cancer

Preliminary pilot studies in the 1990s by Momparler et al. suggested that decitabine may demonstrate clinical efficacy in treating metastatic non‐small cell lung cancer, NSCLC. 123 However, a phase I/early‐phase II clinical trial combining cisplatin and decitabine in patients with metastatic and inoperable NSCLC did not demonstrate significant clinical benefit. 124 Conversely, a phase II trial using decitabine as a priming agent before chemotherapy in advanced NSCLC showed that the combination of decitabine and chemotherapy demonstrated some clinical benefit, with a disease control rate of about 60%. 125 Despite this, the OS benefit was modest.

Ovarian cancer

In recurrent, platinum‐resistant ovarian cancer, several small trials have been conducted. 126 , 127 , 128 , 129 A phase II trial of decitabine followed by carboplatin demonstrated that low‐dose decitabine could sensitize tumors to carboplatin, leading to an ORR of 35%. 127 The combination was well tolerated, exhibiting manageable toxicity. In another phase II clinical trial, which enrolled 100 patients, epigenetic priming was assessed by comparing guadecitabine in combination with carboplatin to standard TC chemotherapy in recurrent, platinum‐resistant ovarian cancer. 129 While this study showed improved PFS at 6 months, it did not significantly impact median PFS.

Breast cancer

In breast cancer, DNMTi have been studied preclinically since 2006, often in combination with other therapeutic modalities such as HDACi and tamoxifen, summarized in a review by Schröder et al. 130 Preclinical results have been promising, with one study suggesting re‐expression of estrogen receptor after application of a dual combination of HDACi and DNMTi in triple‐negative breast cancer (TNBC) cell lines, 131 while another study demonstrated reversibility of tamoxifen resistance in cell lines upon azacitidine treatment. 132 There are also promising preclinical data for guadecitabine plus HDACi in a TNBC model. 133 However, early clinical trials conducted in the 1970s failed to report an additional benefit of azacitidine in breast cancer. 134 , 135 Furthermore, despite preclinical evidence of synergy between DNMTi and HDACi, a clinical study published in 2017 found only one responder in a series of 40 patients with either TNBC or hormone refractory breast cancer treated with the combination of azacitidine and entinostat, an HDACi. 136 The combination was well tolerated, with biomarker studies indicating re‐expression of silenced genes; however, clinical efficacy was limited. A recent study reported on pre‐neoadjuvant treatment in advanced breast cancer with pembrolizumab and decitabine. 137 The study found higher levels of stromal tumor‐infiltrating lymphocytes (sTILs) after administration of the two agents prior to standard neoadjuvant therapy. Higher sTILs in breast cancer are associated with an increased likelihood of pCR. A modest, yet statistically significant increase in sTILs could be demonstrated in a subset of 46 patients evaluated.

Colorectal cancer

In previously treated metastatic colorectal cancer, guadecitabine was evaluated in combination with irinotecan in a phase I and randomized phase II clinical trial. 138 , 139 The phase II trial failed to demonstrate a benefit of the combination over standard of care in terms of response rates and PFS. 139

Melanoma

The combination of oral azacitidine with pembrolizumab in advanced melanoma demonstrated promising anti‐tumor activity. 140 In checkpoint inhibitor‐naive patients, the initial response rate was approximately 55%. However, an immune checkpoint‐pretreated cohort did not respond to this treatment, supporting the hypothesis that azacitidine can modulate the tumor microenvironment to enhance immune response in immune checkpoint treatment‐naïve patients.

CONCLUSIONS AND OUTLOOK

The clinical development of DNMTi for the treatment of patients with myeloid neoplasias has been moving steadily forward over the last four decades, with continuous improvements regarding low‐dose development, scheduling, modes of administration, extension of treatment duration and, importantly, the combination with cooperating agents, as well as the use of “bridging” to subsequent allo‐HCT.

During the early phases of the development of low‐cytotoxic doses and schedules of azacitidine and decitabine, clinical studies were primarily focused on MDS patients who were elderly and not medically fit to tolerate more intensive treatment. Administration of either drug in these early trials was complex, particularly for decitabine, where patients were hospitalized to receive six daily 2‐h infusions over a total of 72 h (hence with infusions administered also at midnight and at 2 AM). In several of these trials, the total number of treatment cycles to be given was limited, thereby not allowing for prolonged maintenance treatment to stabilize and thus prolong remissions in responding patients. In addition, DNMTi regimens historically followed traditional pulse‐cycled models used for other anti‐metabolic treatments; however, their subsequent recognition of low‐dose molecular targeted DNMT depletion guided the development of currently used low‐dose regimens. 100 Regarding their particular mechanism of action and distinct clinical activity, it is now well accepted that these agents induce in vivo gene de‐methylation and de‐repression of silenced genes; importantly, also the—at first surprising and counterintuitive—highly reproducible observation of recurrent responses in patients with complex‐monosomal karyotypes and TP53 mutations is now accepted, and DNMTi‐based drug combinations are aimed at improving the outcome of these patients despite the formidable challenges of this genotype.

At present, DNMTi have been FDA‐approved as monotherapy for high‐risk MDS, with doses and schedules that are feasible on an outpatient basis, with an FDA approval also for the oral formulation of decitabine in conjunction with cedazuridine. In AML, the combination of a DNMTi with venetoclax in patients ineligible for standard intensive induction chemotherapy was approved by both FDA and EMA. For AML and MDS patients ineligible for the curative approach of allo‐HCT, continued treatment with a DNMTi, alone or in combination with venetoclax, may stabilize complete remissions over years. A metronomic, very low‐dose treatment of decitabine (alone or with venetoclax) administered weekly has also resulted in very promising, remarkably well‐tolerated results. 141

As shown in the EORTC Intergroup study AML21, in patients aged >60 years and fit for standard induction chemotherapy, a de‐escalation approach investigating monotherapy with an extended 10‐day decitabine schedule resulted in similar overall survival, similar transplant rate (overall >50%), and survival from transplant as standard IC, albeit with a lower rate of toxicities, better quality of life, reduced numbers of hospitalizations, use of antibiotics, and antimycotics. This trial thus provides a blueprint for the ongoing ( NCT04801797 , NCT05904106 , NCT05177731 ) and future studies randomizing fit AML patients, also those below 60 years of age, for either IC or a hypomethylating agent in combination with venetoclax. Attempts to challenge the current standard of care IC with a de‐escalation approach in the frontline setting for younger, medically fit AML patients are currently ongoing. Lu et al. provided compelling evidence that a venetoclax‐decitabine dual treatment is non‐inferior to IC in regard to CCR rates with similar MRD‐negativity and OS, while simultaneously reducing treatment‐related AE such as infections and thrombocytopenia. 142 It is to be hoped that the de‐escalation strategy of this “less is more” approach will supersede traditional IC in different AML subgroups.

Relapse treatment after allografting is frequently approached by DLI, that is, cellular immunotherapy, with hypomethylating agents, achieving disease control in the majority of patients, and in a subgroup even has curative potential. DNMTi are also developed for the treatment of MPN, MDS/MPN overlap syndromes, and juvenile myelomonocytic leukemia (JMML): azacitidine is FDA approved as first‐line treatment in JMML (a myeloid cancer of childhood with a very poor prognosis unless treated with DNMTi followed by allografting).

The goal to improve the survival of elderly, medically non‐fit patients has also driven the implementation of QoL assessments before and during treatment. Quantification of patients' time not spent in the hospital and with good quality of life has also prompted QoL assessments in younger AML/MDS patients, raising awareness for the important objective of the reduction of “time toxicity” in hematology‐oncology (and medicine in general). In that regard, it is intriguing that de‐escalating treatment intensity, without sacrificing efficacy, that is, the starting point of the development of low‐dose DNMTi treatment for elderly non‐fit patients 40 years ago, may now convey benefit also for younger patients with myeloid malignancies. 142 It is to be hoped that this trend will also benefit patients with lymphoid neoplasias and solid tumors in the future.

AUTHOR CONTRIBUTIONS

Valentin Wenger: Writing – review and editing; writing – original draft; conceptualization; investigation. Guillermo Garcia‐Manero: Writing – review and editing. Robert Zeiser: Writing – review and editing; writing – original draft; funding acquisition. Michael Lübbert: Writing – review and editing; writing – original draft; funding acquisition; project administration; supervision; conceptualization; investigation.

CONFLICT OF INTEREST STATEMENT

Valentin Wenger has received honorarium from AbbVie. Robert Zeiser has received honoraria from Novartis, Incyte, VectorBio, Medae, Sanofi, and Therakos. Michael Lübbert is on the advisory boards of AbbVie, Astex, Janssen, and Otsuka, is receiving research support from Janssen, and is provided with study drug by Cheplapharm. Guillermo Garcia‐Manero reports no conflicts of interest.

ACKNOWLEDGMENTS

We wish to thank Johanna Thomas, M. Sc., for excellent clerical support during manuscript development. Robert Zeiser was supported by the DFG—SFB‐1479—Project ID: 441891347 (P01 to Robert Zeiser), DFG: RU5659 TARGET‐MPN: ZE 872/6‐1 (TP 7), Project‐ID 259373024—TRR 167 (to Robert Zeiser), CRC1160 (Project ID 256073931 to Robert Zeiser). Michael Lübbert has received funding from the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation)—CRC992—MEDEP (C04), Research Consortium FOR 2674 (A05, A09) of the Deutsches Konsortium für Translationale Krebsforschung (DKTK, German Cancer Research Center) and the DFG program “Clinical Trials.” Guillermo Garcia‐Manero and Valentin Wenger report no additional funding. Open Access funding enabled and organized by Projekt DEAL.

Wenger V, Garcia‐Manero G, Zeiser R, Lübbert M. DNA methyltransferase inhibitors in hematological malignancies and solid tumors. Int J Cancer. 2026;158(2):433‐461. doi: 10.1002/ijc.70197

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