Dnmt3a Mutation Promotes Expansion of a Quiescent Myeloid Progenitor Population in Npm1c-Flt3ITD Acute Myeloid Leukemia
Authors: Tavakoli Paniz , Bywater Megan , Straube Jasmin , Ling Victoria , Cooper Emily , Haldar Rohit , Janardhanan Yashaswini , Cooper Leanne , Bruedigam Claudia , Grove Carolyn S. , Lane Steven W 
Published: December 2024

The genetic profile of Acute Myeloid Leukemia (AML) is a key predictor of treatment response and survival. The combined presence of the three common oncogenic gene mutations, DNMT3AR882H, NPM1c and FLT3ITD, leads to inferior patient survival after intensive chemotherapy in comparison to AML containing NPM1c and FLT3ITD (5-year survival-rate DNMT3AR882H-NPM1c-FLT3ITD vs. NPM1c-FLT3ITD=38% vs 55%, overall-survival p=0.004; Straube et al. 2018). Achieving a long-term cure for DNMT3AR882H-NPM1c-FLT3ITD AML requires a precise understanding of how mutant DNMT3A leads to chemotherapy resistance and the development of novel approaches to effectively eradicate these cells.

Using genetically engineered Npm1c-Flt3ITD murine models, with and without a novel Dnmt3aR878H knock-in allele (equivalent to human DNMT3AR882H), we have developed isogenically matched Dnmt3aR878H-Npm1c-Flt3ITD and Npm1c-Flt3ITD AML. These AMLs are characterised by an expanded multipotent progenitor population (MPP3: Lineage neg/c-Kit+/Sca1+/CD48+/CD150-). Transplantation of MPP3s isolated from leukemic mice rapidly generates AML in secondary recipients, confirming this population contains Leukemia-driving stem cells (LSCs).

Limiting dilution assays demonstrate a higher LSC frequency in Dnmt3aR878H-Npm1c-Flt3ITD (LSC frequency in Dnmt3aR878H-Npm1c-Flt3ITD vs Npm1c-Flt3ITD MPP3s= 1/49.6 vs 1/4341.7, p=0.0001, n=3 per group). This increased LSC frequency suggests that the Dnmt3aR878H mutation confers additional characteristics that enhance leukemia persistence. Cell cycle analysis identifies increased quiescence (G0; ki-67 negative population) in MPP3s co-expressing Dnmt3aR878H compared to Npm1c-Flt3ITD (Dnmt3aR878H-Npm1c-Flt3ITD vs Npm1c-Flt3ITD; % of quiescent MPP3s=18.8 vs 7.01, p= 0.0009, n=8 per group).

Furthermore, gene expression analyses reveal an enrichment of HSC-like (NES=2.4, FDR&lt;0.0001) and quiescent LSC signatures (NES=1.9, FDR=0.001) in Dnmt3aR878H-Npm1c-Flt3ITD vs. Npm1c-Flt3ITD MPP3s. Consistent with the role of Dnmt3a in de novo DNA methylation, Dnmt3aR787H mutation leads to epigenetic changes with predominant loss of methylation and enhanced gene expression. Specifically, Whole Genome Enzymatic Methyl-seq demonstrates that Dnmt3aR878H-mutated MPP3s exhibit focal loss of methylation particularly at promoters and CpG rich regions (p=8.5e-07 and p=1.1e-07, respectively). ATAC-seq on purified MPP3s confirms a negative correlation between chromatin accessibility and DNA methylation (R=-0.33, p&lt;2.2e-16), where gain of promoter accessibility is associated with loss of methylation in Dnmt3aR878H-Npm1c-Flt3ITD vs. Npm1c-Flt3ITD MPP3s (p&lt;0.0001). This loss of methylation and gain of chromatin accessibility occurs at promoters of upregulated genes, such as Hoxa5 and Hlf regulating stemness and quiescence (p=0.0008).

Patients with mutated DNMT3A, FLT3 and NPM1 have inferior long-term survival, due to increased relapse. Consistent with this, we observe chemotherapy resistance in Dnmt3aR878H-Npm1c-Flt3ITD AMLs compared to Npm1c-Flt3ITD when treated with a combination of doxorubicin and cytarabine (DA). Cultured Dnmt3aR878H-Npm1c-Flt3ITD vs. Npm1c-Flt3ITD AML has 2-fold higher IC50 (DNF IC50=18.6/186 nM vs NF IC50=9.1/91 nM DA, p=0.016) after 48 hours of treatment. In vivo treatment with doxorubicin (1.5 mg/kg) + cytarabine (50 mg/kg) is able to efficiently eradicate Npm1c-Flt3ITD AML cells. In contrast, DA chemotherapy is unable to reduce Dnmt3aR878H-Npm1c-Flt3ITDAML disease burden (49% reduction in Npm1c-Flt3ITD AML cells, p=0.004 vs. 17% reduction in Dnmt3aR878H-Npm1c-Flt3ITD, p=0.3, n=15 per group). This difference in response is also evident in the LSC population, with Dnmt3aR878H-Npm1c-Flt3ITD MPP3s exhibiting reduced chemosensitivity (60% reduction in absolute number of Npm1c-Flt3ITDMPP3s, p=0.03 vs 16% reduction in Dnmt3aR878H-Npm1c-Flt3ITD MPP3s, p=0.3, n=15 per group)

Altogether, these data suggest that DNMT3A mutations, through changes in DNA methylation at gene promoters, enhance LSC characteristics of stemness and quiescence, mediating intrinsic resistance to combined chemotherapy. These mechanistic insights will help to inform alternative therapeutic strategies in DNMT3AR882H-NPM1c-FLT3ITDAML.