Gene Expression Profile in Responsive and Non-Responsive Chronic Myeloid Leukemia Patients Treated with Dasatinib.

CML treatment with tyrosine kinase inhibitors induces high and durable rates of complete cytogenetic response. Despite treatment efficacy, a significant proportion of patients develop resistance to these drugs. We measured gene expression profiles in an attempt to identify gene pathways that may be associated with dasatinib resistance.

Mononuclear cells were separated from peripheral blood samples from seven CML patients resistant to imatinib, collected prior and after dasatinib treatment. Three patients who achieved partial cytogenetic response (Ph-positive cells: 1% - 35%) within twelve months were considered responders (R), whereas four patients who failed to achieve PCyR within 12 months of treatment were classified as non-responders. RNA samples prepared from peripheral mononuclear cells were hybridized to Agilent Technologies 4×44K Whole Human Genome Microarrays (WHGM) and 4×44K intronic-exonic custom oligoarrays. The latter was developed by Verjovski-Almeida's group (Nakaya et al, Genome Biology 2007, 8:R43) and contains sense and antisense probes that map to intronic regions in the human genome representing totally (TIN) and partially (PIN) intronic non-coding RNAs (ncRNAs), in addition to probes for the corresponding protein-coding genes of the same loci. Raw microarray data were normalized by the Affy package in statistical R language implemented in the Bioconductor platform. Each sample was labeled in replicate with Cy3 or Cy5 and the two were considered technical replicates. Two independent statistical approaches SAM (Significance Analysis of Microarrays) and Golub's discrimination score (SNR, Signal to Noise Ratio, with permutations) were performed to identify differentially expressed transcripts between responder and non-responder patients. For the intronic-exonic platform, the analysis parameters were FDR 10%, SNR>1.5 and p<0.01, and for WHGM platform parameters were FDR 5%, SNR>1.5 and p<0.001. For this latter platform, we also performed a patient leave-one-out analysis. Functions of transcripts differentially expressed were annotated and compared using GO Biological Process categories (www.genetools.microarray.ntu.no/egon).

We identified 34 ncRNAs with altered expression (26 over and 8 underexpressed in responders) in pre-treatment samples and 33 ncRNAs (20 over and 13 underexpressed in responders) in post-treatment samples. Functions associated with protein-coding genes from the same genomic loci as those of the intronic differentially expressed ncRNAs were: regulation of transcription (PRMT5, SOD2, SSBP3, BCL7A, MLL), signal transduction (PRKCB1, RASGRP2, NF1, PXN) and apoptosis (BCL2, PCSK6, TNFAIP8, EIF4G2). WHGM platform data analysis showed 63 and 250 protein-coding genes differentially expressed in pre and post-treatment samples, respectively. We observed a higher number of protein-coding genes with altered expression after treatment in the following functions: cell communication, immune response and metabolic process (p<0.02).

Overall, these findings indicate that protein-coding genes and intronic ncRNAs may be related to dasatinib resistance and response to treatment. In particular, altered expression of ncRNAs transcribed from the introns of ‘regulation of transcription' genes could be part of an important alternative mechanism of gene expression control during emergence of resistance.Support: FAPESP (2005/60266-8)

No relevant conflicts of interest to declare.