Heterozygous mutations in FLT3ITD, TET2 and DNMT3A are associated with human neoplasms. Whole exome sequencing of patients demonstrate co-occurrence of several of these mutations in myeloid malignancies; however it is unclear if and how these mutations cooperate and how does this cooperation manifest in disease. We assessed the consequence of co-existence of TET2, DNMT3A and FLT3ITD/WT mutations on AML development and overall survival. Mice were bred to obtain eight distinct genotypes WT, Tet2+/-, Flt3ITD/WT, Dnmt3aFlox/- MxCre, double het Tet2+/-; Dnmt3aFlox/- MxCre (TD), double het Tet2+/-; Flt3ITD/WT (TF), double het Flt3ITD/WT: Dnmt3aFlox/- MxCre (FD) and triple het Tet2+/-; Flt3ITD/WT; Dnmt3aFlox/+ MxCre (TFD) mice. These mice were treated with poly:IC and monitored for survival. Of the 8 genotypes examined, only TFD mice succumbed by 150 days. Peripheral blood (PB) counts, BM cellularity and splenomegaly were significantly higher in TFD mice compared to FD and TF mice. The absolute number of LSK cells in the BM was highest in TFD mice, although TF and FD mice also showed an increase relative to controls. A similar increase in the frequency of GMPs was noted in both double and triple heterozygous mice relative to controls as well as single heterozygous groups. An increase in Gr-1/Mac-1 cells was observed in TF and FD mice as well as in TFD mice relative to controls. To assess if the observed AML phenotype in TFD mice is transplantable, we transplanted BM cells from the 8 genotypes into lethally irradiated hosts. Recipients with TFD BM succumbed to rapid AML development and died within 45 days of transplantation. In contrast, none of the other recipients died during the entire monitoring period. A significant increase in PB neutrophil and monocyte counts with a significant reduction in red blood cells, and platelets counts was noted in TFD mice compared to other groups. Furthermore, a significant increase in BM cellularity, Lin- Kit+ progenitors, frequency and absolute number of LSK cells was also noted in mice with TF and FD BM but most significantly in recipients that received TFD BM. Given that the disease manifestation was qualitatively similar between FD, TF and TFD mice, although the severity greatest in TFD mice, we asked if these differences were due to quantitative or qualitative differences in gene expression between the various groups. RNA-seq analysis revealed distinct differences between WT and FD, TF and TFD BM cells. We observed 2328, 2168 and 1787 up-regulated genes and 1861, 1770 and 1430 downregulated genes in FD, TF and TFD vs. WT, respectively. The variations among FD, TF and TFD groups was smaller compared to the differences between WT and FD, TF and TFD groups, in which TF and TFD were more similar to each other compared to DF. Most of the cytokines regulating the differentiation of myeloid cells were downregulated in FD, TF and TFD, suggesting that all the mutant groups lost their differentiation ability. Of note, cytokines specific to stem cells and those involved in the differentiation of GMPs were all upregulated. Given the RNA-seq profile, we assessed the impact of using a combination of drugs that target Flt3ITD, inflammation as well as methylation status of cells in TFD mice. A cohort of mice was treated with a combination of AC220 (10 mg/kg, orally; Flt3 inhibitor), E3330 (20 mg/kg, i.p.; APE1 inhibitor; regulates the redox function of multiple transcription factors including NFkB and Stat3) and 2-dexoy-aza-cytidine (0.5 mg/kg, i.p). A significant correction in all PB, spleen and BM parameters was observed including in the frequency of Lin- Kit+ and LSK cells, HSCs, HPC1 and peripheral Gr1/Mac1 double positive cells in TFD mice. Collectively, these results demonstrate that a combination of three drugs targeting three different aspects of disease manifestation in TFD mice can significantly impact the leukemia progression both at the level of very primitive stem and progenitor cells as well as more mature myeloid and lymphoid cells. Disclosures No relevant conflicts of interest to declare.