Alexander Thompson, PhD
Centre for Cancer Research and Cell Biology, Queen’s University Belfast
A study of differential gene expression in cytogenetically normal subtype of acute myeloid leukemia cells reveals a HOX/TALE signature that may have specific prognostic value. Direct gene targeting of HOXA/PBX3 sensitizes these cells to standard chemotherapy, laying a foundation for a novel therapeutic strategy.
HOX/TALE genes are master regulators of developmental processes including blood cell formation. They encode transcription factors, some of which have been shown to be proto-oncogenes and several of which have been shown to be associated with cancer progression. One of the main aims of the project was to identify a clinically relevant signature of HOX/TALE gene expression in acute myeloid leukaemia (AML). To achieve this we uploaded microarray data obtained from the global MILE study (NCBI Gene Expression Omnibus Accession number: GSE13204 ) into Partek Genomics Suite software as .CEL files and generated unsupervised hierarchical cluster and heatmaps depicting clustering of genes by subgroup-defined prognostic risk based on cytogenetics (n=420). It was clear from this approach that HOX and TALE gene expression correlated with cytogenetic status and furthermore was able to partition patient samples with no gross cytogenetic rearrangement, termed cytogenetically normal AML (CN-AML) that have intermediate risk. Statistical analysis identified probe-sets for 11 of the HOX and TALE genes with false discovery rate-corrected P-values < 0.0000005. These probe-sets were examined further to identify more defined subgroups of CN-AML with associated HOX/TALE expression.
To further evaluate these initial findings, two independent cohorts with more defined mutation and clinical status were also examined. De novo CN-AML samples (n=235) with known mutation status and a custom cDNA array (n=114 patients) were interrogated as before with Affymetrix CEL files and imported into Partek Genomics Suite software where ANOVA and Kaplan-Meier analysis of microarray experiments were completed. Unsupervised hierarchical clustering confirmed partitioning of a cohort of de novo CN-AML patients (n=235) by four genes HOXA6, HOXA9, PBX3 and MEIS1 expression. Probe-sets for the four genes were significantly differentially expressed against NPM-1 mut status with a false discovery rate-corrected P-value < 0.0000005. Unsupervised hierarchical clustering analysis of the custom array also supported the previous findings for HOXA9, PBX3 and MEIS1 probe-sets with well-defined mutation status. In addition, four distinct gene expression-based clusters associated more with the clinical status of these patients and not strictly dependent on cytogenetic status were identified. Significant correlation between the probe-set clusters and survival (P=0.015) was demonstrated by a Kaplan-Meier plot generated within Partek Genomics Suite.
Taken together, the data derived from Partek Genomics Suite software supported a key role for four HOX/TALE genes in leukaemia maintenance and patient survival. To address this, the four genes were specifically targeted by shRNA approaches and the functional consequences examined by conventional haematological readouts (cell growth, apoptosis, morphology and colony forming assays) alone or in combination with standard-of-care therapies. Targeting of expression of these genes sensitized the leukaemic cells to chemotherapy. Partek Genomics Suite software allowed us to identify and confirm a restricted yet clinically relevant signature of HOX/TALE genes (four genes) for therapeutic targeting.