Dnmt3a Mutation Promotes Expansion of a Quiescent Myeloid Progenitor Population in Npm1^c-Flt3^ITD Acute Myeloid Leukemia

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, DNMT3A^R882H, NPM1^c and FLT3^ITD, leads to inferior patient survival after intensive chemotherapy in comparison to AML containing NPM1^c and FLT3^ITD (5-year survival-rate DNMT3A^R882H-NPM1^c-FLT3^ITD vs. NPM1^c-FLT3^ITD=38% vs 55%, overall-survival p=0.004; Straube et al. 2018). Achieving a long-term cure for DNMT3A^R882H-NPM1^c-FLT3^ITD 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 Npm1^c-Flt3^ITD murine models, with and without a novel Dnmt3a^R878H knock-in allele (equivalent to human DNMT3A^R882H), we have developed isogenically matched Dnmt3a^R878H-Npm1^c-Flt3^ITD and Npm1^c-Flt3^ITD 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 Dnmt3a^R878H-Npm1^c-Flt3^ITD (LSC frequency in Dnmt3a^R878H-Npm1^c-Flt3^ITD vs Npm1^c-Flt3^ITD MPP3s= 1/49.6 vs 1/4341.7, p=0.0001, n=3 per group). This increased LSC frequency suggests that the Dnmt3a^R878H mutation confers additional characteristics that enhance leukemia persistence. Cell cycle analysis identifies increased quiescence (G0; ki-67 negative population) in MPP3s co-expressing Dnmt3a^R878H compared to Npm1^c-Flt3^ITD (Dnmt3a^R878H-Npm1^c-Flt3^ITD vs Npm1^c-Flt3^ITD; % 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<0.0001) and quiescent LSC signatures (NES=1.9, FDR=0.001) in Dnmt3a^R878H-Npm1^c-Flt3^ITD vs. Npm1^c-Flt3^ITD MPP3s. Consistent with the role of Dnmt3a in de novo DNA methylation, Dnmt3a^R787H mutation leads to epigenetic changes with predominant loss of methylation and enhanced gene expression. Specifically, Whole Genome Enzymatic Methyl-seq demonstrates that Dnmt3a^R878H-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<2.2e-16), where gain of promoter accessibility is associated with loss of methylation in Dnmt3a^R878H-Npm1^c-Flt3^ITD vs. Npm1^c-Flt3^ITD MPP3s (p<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 Dnmt3a^R878H-Npm1^c-Flt3^ITD AMLs compared to Npm1^c-Flt3^ITD when treated with a combination of doxorubicin and cytarabine (DA). Cultured Dnmt3a^R878H-Npm1^c-Flt3^ITD vs. Npm1^c-Flt3^ITD 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 Npm1^c-Flt3^ITD AML cells. In contrast, DA chemotherapy is unable to reduce Dnmt3a^R878H-Npm1^c-Flt3^ITDAML disease burden (49% reduction in Npm1^c-Flt3^ITD AML cells, p=0.004 vs. 17% reduction in Dnmt3a^R878H-Npm1^c-Flt3^ITD, p=0.3, n=15 per group). This difference in response is also evident in the LSC population, with Dnmt3a^R878H-Npm1^c-Flt3^ITD MPP3s exhibiting reduced chemosensitivity (60% reduction in absolute number of Npm1^c-Flt3^ITDMPP3s, p=0.03 vs 16% reduction in Dnmt3a^R878H-Npm1^c-Flt3^ITD 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 DNMT3A^R882H-NPM1^c-FLT3^ITDAML.

Tavakoli:Bristol Myers Squibb: Other: Drugs, Research Funding; Millennium Science: Other: reagents. Bywater:Bristol-Myers Squibb: Other: Drugs, Research Funding; PharmaEssentia: Other: Drugs; Megan Bywater: Patents & Royalties: PCT/GB2020/050350. Straube:PharmaEssentia: Other: Drugs; Bristol-Myers Squibb: Other: Drugs, Research Funding. Grove:AbbVie: Other: institutional consultancy payment; Otsuka Australia Pharmaceutical: Other: institutional consultancy payment; Astellas Pharma: Other: institutional consultancy payment; institutional payment for educational event. Lane:GSK: Consultancy; Abbvie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS: Other: Drugs, Research Funding.