RNA-seq profiling of murine Tet-off MLL-AF9; NRAS acute myeloid leukemia at different timepoints upon doxycycline-induced MLL-AF9 down-regulation.

Using an acute myeloid leukemia (AML) mouse model driven by tet-regulated MLL-AF9 (fusion between the gene MLL1 (KMT2A/MLL) and MLLT3 (AF9)) co-expressed with oncogenic NRASG12D (Tet-off MLL-AF9), we investigated the effect of modulating the expression of the MLL-AF9 fusion oncogene on the transcriptome and proteome of established murine AML. Treatment in vitro or in vivo of these Tet-off MLL-AF9 AMLs with doxycycline (DOX) results in the efficient down-regulation of the expression of the driver oncogene MLL-AF9. RNA sequencing analysis was performed on primary Tet-Off MLL-AF9 AML cells obtained from the spleen of leukemic animals and cultured in vitro for either 2 or 4 days in the presence of doxycycline (1µg/ml) (DOX= down-regulation of MLL-AF9) or left untreated (UT). Overall design: Primary murine acute myeloid leukemias (Tet-off MLL-AF9 AML) driven by the doxycycline-reversible (Tet-OFF) expression of the oncogenic fusion protein MLL-AF9 linked to dsRED reporter, in association with oncogenic NRASG12D (Tet-off MLL-AF9) were generated by reconstituting lethally irradiated congenic mice with foetal liver cells co-transduced with a Tet-Off-MLL-AF9-dsRED retroviral vector and a second vector co-expressing NRASG12D together with the Tet-Off responsive transcriptional activator. Four primary Tet-Off MLL-AF9 leukemias obtained from the spleen of terminally sick animals were cultured in vitro in the presence of doxycycline (1µg/ml) or left untreated. The RNA was collected at day 2 and 4 after the start of the doxycycline treatment. Total RNA extraction was performed using Trizol reagent (Invitrogen) according to the manufacturer's instructions. RNA-seq libraries were prepared from 400 ng of total RNA using the Illumina Truseq V2 RNA-seq kit. Paired-end 50bp RNA-seq short reads were generated using HiSeq2500. 4 biological replicates (UT vs DOX) are provided for each of the 2 timepoints.