Candidate Genes for Expansion and Transformation of Hematopoietic Stem Cells by NUP98-HOX Fusion Genes

BACKGROUND: Hox genes are implicated in hematopoietic stem cell (HSC) regulation as well as in leukemia development through translocation with the nucleoporin gene NUP98. Interestingly, an engineered NUP98-HOXA10 (NA10) fusion can induce a several hundred-fold expansion of HSCs in vitro and NA10 and the AML-associated fusion gene NUP98-HOXD13 (ND13) have a virtually indistinguishable ability to transform myeloid progenitor cells in vitro and to induce leukemia in collaboration with MEIS1 in vivo. METHODOLOGY/PRINCIPAL FINDINGS: These findings provided a potentially powerful approach to identify key pathways mediating Hox-induced expansion and transformation of HSCs by identifying gene expression changes commonly induced by ND13 and NA10 but not by a NUP98-Hox fusion with a non-DNA binding homedomain mutation (N51S). The gene expression repertoire of purified murine bone marrow Sca-1+Lin- cells transduced with retroviral vectors encoding for these genes was established using the Affymetrix GeneChip MOE430A. Approximately seventy genes were differentially expressed in ND13 and NA10 cells that were significantly changed by both compared to the ND13(N51S) mutant. Intriguingly, several of these potential Hox target genes have been implicated in HSC expansion and self-renewal, including the tyrosine kinase receptor Flt3, the prion protein, Prnp, hepatic leukemia factor, Hlf and Jagged-2, Jag2. Consistent with these results, FLT3, HLF and JAG2 expression correlated with HOX A cluster gene expression in human leukemia samples. CONCLUSIONS: In conclusion this study has identified several novel Hox downstream target genes and provides important new leads to key regulators of the expansion and transformation of hematopoietic stem cells by Hox

Correlation between HOXA7 and HOXA9 (A) and JAG2 and HLF (B) gene expression in human AML analyzed with TaqMan Low Density Array (TLDA).

<p>Spearman rank correlation analysis was done on the log ratio values obtained from the TLDA assay and calculated with the 2^−ΔΔCT method (n = 34).</p

Venn diagram of genes significantly activated (A) or repressed (B) in Sca1+, Lin- BM cells expressing the ND13, NA10 or ND13(N51S) mutant fusion genes compared to GFP control.

<p>Venn diagram of genes significantly activated (A) or repressed (B) in Sca1+, Lin- BM cells expressing the ND13, NA10 or ND13(N51S) mutant fusion genes compared to GFP control.</p

Gene expression levels in human AML relative to normal bone marrow.

<p>Gene expression levels in human AML relative to normal bone marrow.</p

Validation of microarray results with quantitative RT-PCR on unamplified RNA.

<p>Fold changes are calculated against an empty MIG control and <i>Gapdh</i> as endogenous control gene.</p

Genes changed by NA10 and ND13 but not by the ND13(N51S) mutant compared to the GFP control.

<p>Genes changed by NA10 and ND13 but not by the ND13(N51S) mutant compared to the GFP control.</p

Applicability of IG/TCR gene rearrangements as targets for minimal residual disease assessment in a population-based cohort of Swedish childhood acute lymphoblastic leukaemia diagnosed 2002-2006

Minimal residual disease (MRD) detection during the early treatment phase has become an important stratification parameter in many childhood acute lymphoblastic leukaemia (ALL) treatment protocols. Here, we aimed to address the applicability of rearranged antigen-receptor genes as potential MRD markers using real-time quantitative polymerase chain reaction (RQ-PCR) in a Swedish population-based cohort. From 334 childhood ALL cases diagnosed during 2002-2006, we analysed 279 diagnostic samples (84%) by screening for rearranged immunoglobulin (IG) and T-cell receptor (TCR) genes. Allele-specific oligonucleotides were designed, and the sensitivity and quantitative level was determined for each target. Overall, clonal IG/TCR rearrangements were detected in 97% (236/244) of B-cell precursor ALL (BCP ALL) and 94% (33/35) of T-ALL. A sensitive RQ-PCR analysis (= 10-3, which is applied in the current Nordic treatment protocol (NOPHO-ALL 2008) for the identification of high-risk patients, 93% of BCP ALL and 86% of T-ALL reached this quantitative range by at least one target gene. Taken together, this national retrospective study demonstrates that an IG/TCR target for MRD monitoring can be identified in the majority of childhood ALL cases, whereas identification of a second sensitive target gene needs to be improved