Bulk RNA sequencing analysis of Lin- leukemia BCR-ABL and BCR-ABL/MSI2-HOXA9 cells (post-transplantation)

To understand how the MSI2-HOXA9 translocation triggers blast crisis CML (bcCML), we compared gene expression patterns in BCR-ABL and BCR-ABL/MSI2-HOXA9-driven disease. RNA-seq analysis was carried out on lineage negative cells from leukemia established with BCR-ABL alone or with a combination of  BCR-ABL and MSI2-HOXA9. Network mapping of all differentially expressed genes (q-value <0.05) using non-redundant functional grouping revealed an enrichment of metabolic processes with oncogenic pathways and developmental programs. Programs that were dominantly upregulated by MSI2/HOXA9 were those involved in development, including Aldh1a1, Erbb3, and Kit, consistent with BCR-ABL/MSI2-HOXA9 driving a more undifferentiated disease, known oncogenes, including Frat1, Map7, and Fzd3, and components of the mitochondria including mt-Co2, mt-Atp8, and mt-Nd5. Among the genes most enriched in BCR-ABL/MSI2-HOXA9-driven bcCML were key components of the mitochondrial respiratory chain, including Complex I, Complex III, Complex IV, and the F0 subunit of ATP synthase (mt-Atp6 and mt-Atp8).

Methods
Lin- leukemia cells were sorted from mice transplanted with BCR-ABL/Control or BCR-ABL/MSI2-HOXA9 transduced KLS cells. Total RNA was isolated using the RNAeasy Micro Plus kit (QIAGEN). RNA libraries were generated from 150ng of RNA using Illumina's TruSeq Stranded mRNA Sample PrepKit (Illumina). Libraries were pooled ands single end sequenced (1x75) on the Illumina NextSeq 500 using the High output V2 kit (Illumina).

Resultant fastq files were pseudoaligned into transcript-level summaries using Kallisto. Transcript level summaries were processed into gene-level summaries using Sleuth and differential expression analysis was performed using the Wald test.