Activation of Cgas-Sting Pathway By Hypomethylating Agent in TP53 Mutated MDS/AML
Authors: Ng Ka Lam Nelson , Ng Lam , Lam Stephen , Liu Xingliang , Chan Koon C. , Zheng Lichuan , Zhang Chunxiao , Tse Rachel L. S. , Lam Wing , Chan Natalie Nok-Man , Man Cheuk Him , Cheung Arthur K.L. , Ip Ho-Wan , Au Chun H. , Ma Edmond , Ng Chi T. , Ni Ying , Li Run S. , Ling Guang Sheng , Leung Suet Yi , Javed Asif , Leung Anskar Y. H. 
Published: December 2024

Myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) carrying TP53 mutation (TP53m) portend an extremely grave prognosis. Conventional treatments, including induction chemotherapy and allogeneic hematopoietic stem cell transplantation, have not been successful in curing these diseases. Recently, 10-day decitabine treatment has been shown to induce remission, albeit transient, in most patients. In this study, we examined the therapeutic mechanisms of decitabine in TP53m AML with the aim to formulate novel therapeutic strategies.

We examined the therapeutic mechanisms of decitabine with particular reference to TP53m AML based on cellular studies, transcriptomic analysis, nanopore sequencing and xenotransplantation model. Information pertaining to TP53 mutation was derived from AML cell lines and primary AML samples carrying mutant and wildtype TP53 and from isogenic system in which TP53 mutations were introduced into hitherto wildtype parental AML. We performed a Phase II clinical trial (ClinicalTrials.gov Identifier: NCT03080766) treating patients with complex or monosomy karyotype MDS/AML carrying wildtype or mutant TP53, with 10-day course of decitabine and performed nanopore sequencing and single-cell transcriptome analysis of serial samples.

To examine the relevance of TP53 mutation in the treatment response to decitabine, the effects of decitabine on TP53 mutant (K052, NOMO-1)and TP53 wildtype (MV4-11, OCI-AML3) AML cell lines were evaluated. Decitabine showed preferential effects on TP53 mutated AML with increased senescence, apoptosis, cytosolic double-stranded RNA and double-strand DNA breaks. Transcriptomic analysis showed increased expression of endogenous retrovirus and other transposable elements. Expression of cytokine/chemokine and genes pertinent to inflammation and immune activation were increased. These results were further validated in TP53 wildtype MV4-11 lines knocked-in with TP53 missense variants. cGAS-STING and RIG-1-MAVS pathways were activated as shown by the increase in key components of these pathways and the effects of gene knockdown on cytokine/chemokine expression. Macrophage activation could be demonstrated in co-culture studies and unleashing innate immunity checkpoint by anti-CD47 antibody enhanced the anti-leukemic effects of decitabine in vivo.

To validate results from the laboratory studies, 34 MDS/AML patients were treated with decitabine for 10 days in a Phase II clinical study. To ascertain if decitabine has induced DNA demethylation in patients receiving such treatment, nanopore sequencing was performed to evaluate their genomic cytosine methylation before and on Day 10 of drug exposure in 18 patients. The 5-methycytosine percentage at the promoter regions showed significant decrease in responders (N=13) but not non-responders (N=5) after decitabine treatment To examine the leukemic and immune cell responses to decitabine, single-cell transcriptome was performed in 47 samples (BM=39; PB=8) from 17 patients. Gene set enrichment analysis was performed to examine transcriptomic changes upon decitabine treatment Among the responders, TP53m leukemic populations showed positive enrichment in inflammatory response, antigen presentation and DNA methylation compared with TP53w counterparts. Immune populations of TP53m cases also showed positive enrichment of immune response gene sets such as TNF-a, IL2-STAT5, T cell receptor signaling, senescence-associated secretory phenotype and cytosolic DNA sensing gene sets At relapse, leukemic populations showed up-regulation of MYC signaling and heat shock response while T-cells showed exhaustion signature.

Our work highlighted the complex interplay between leukemic and immune populations in TP53m patients upon decitabine treatment that might account for clinical responses and subsequent relapses. Information arising from laboratory studies and single cell transcriptome analysis of bone marrow samples from patients receiving decitabine treatment have provided a new paradigm in which hypomethylating agents induced both intrinsic anti-leukemia effects and non-cell intrinsic effects via activation of innate and adaptive immunity through RIG-1-MAVS and cGAS-STING pathways in TP53m MDS/AML. This model might provide opportunities in the future design of clinical trials for this disease subtype.