Thioguanine and decitabine can overcome hypomethylating agent resistance in patients with advanced myeloid malignancies

Abstract BACKGROUND Despite active therapies such as hypomethylating agents (HMA) and venetoclax advanced myeloid malignancies, outcomes in relapsed/refractory (R/R) disease or secondary acute myeloid leukemia (AML) remain poor. We previously showed that combination thioguanine (6TG) and decitabine (DEC) restores therapeutic efficacy in vitro in R/R AML (O'Dwyer K et al. Blood 2010;114:2657). Building on these observations, we completed a Phase I dose-escalation study of 6TG with DEC in advanced myeloid malignancies followed by a retrospective analysis of 42 patients treated with this regimen at our institution (Cicero KI et al. Blood 2019;134:3899). In this cohort, which included patients on- and off-study, responses were seen in 33% of patients, without a significant difference between patients who had or had not received prior HMA. This suggested that combination therapy with 6TG and DEC could overcome HMA resistance. We now present updated clinical and mechanistic data from an expanded cohort, including insights into regimen-specific effects on malignant clones and immune constitution. METHODS A retrospective chart review was performed of all adult patients at our institution with advanced myeloid malignancies who received at least 1 cycle of 6TG and DEC between 2013-2025 with formal disease assessment evaluable by bone marrow biopsy or blood. The regimen utilized the maximum tolerated dose of 6TG identified in the phase I trial. Up to 2 cycles of induction with oral 6TG 80 mg/m2/day in 2 divided doses was given on Days 1-12. Decitabine 20 mg/m2 IV was given on Days 3-12. Following this, maintenance cycles consisted of 6TG on Days 1-7 and decitabine on Days 3-7 at the same doses. Treatment continued until the time of hematopoietic stem cell transplant (HSCT), disease progression, or toxicity. The primary objective of this retrospective analysis was to determine the overall response rate (ORR). Secondary objectives were to evaluate progression-free survival (PFS) and overall survival (OS) in this population. RESULTS Sixty-seven patients were identified, 53% of whom were female, with a median age of 68 years (range, 23-86 years). Thirty-four patients (51%) had secondary AML, 26 (39%) had de novo AML, 5 (7%) had higher-risk MDS, and 2 (3%) had chronic myelomonocytic leukemia (CMML). By ELN2022 risk stratification among AML patients, 40 (60%), 6 (9%), 6 (9%), and 8 (12%) had adverse, intermediate, favorable, and unknown risk disease, respectively. Patients received a median of 2 cycles of therapy (range, 1-10). Eight (12%) patients achieved complete remission (CR), 7 (10.5%) obtained a CR with incomplete count recovery (CRi), and 5 (7.5%) had morphologic leukemia-free state (MLFS), for an ORR of 30% (CR+CRi+MLFS). Nine of 20 responders (45%) had prior HMA exposure; 3 of these patients had exposure to prior HMA plus venetoclax. For the responders, the median PFS was 24 weeks (range, 7-NR), while the median OS was 50 weeks (range, 7-NR). For responders achieving composite CR (CRc: CR + CRi), median PFS was 35 weeks and median OS was 54 weeks. Two notable patients, one with CMML and another with relapsed AML, were bridged to allogeneic HSCT and are currently alive and in remission over 10 years after treatment. To elucidate response mechanisms, we performed methylome-aware whole-genome long-read sequencing and Pixelgen single-cell proteomic analysis. Pre-treatment samples from non-responders showed elevated CD44 co-localized with HLA and β2-microglobulin, while responders exhibited low CD44, uniform HLA/β2M expression, and expanded effector T-cell pools, suggesting immune activation as a key therapeutic axis. Post-treatment samples revealed persistent CD44 in non-responders, while responders expressed unexpectedly high CD36 expression in the context of re-emergence of normal stem cell marker expression. CONCLUSIONS: 6TG/DEC is a clinically active regimen in high-risk and R/R AML with a 30% ORR and favorable survival outcomes compared to historical controls. Single-cell proteomic data reveal immune-modulatory mechanisms that may underlie therapeutic response, offering a novel lens into resistance biology. Taken together, this combination remains a viable therapeutic option for elderly and unfit patients with high-risk and R/R AML who cannot tolerate intensive chemotherapy. A phase II study of 6TG and decitabine-cedazuridine as an entirely oral regimen for R/R AML irrespective of molecular phenotype is in preparation.