Bone marrow failure in patients with Fanconi anemia (FA) is often complicated by the clonal neoplasms myelodysplasia (MDS) and acute myelogenous leukemia (AML). FA stem cells (HSC) exhibit self-replicative defects and HSC and committed progenitor cells (HPC) from Fancc-deficient (Fancc −/−) mice and FA-C-deficient patients are hypersensitive to suppressive cytokines, particularly TNFα, a cytokine also known to be overproduced by Fancc−/− macrophages. In fact, in Fancc−/− mice overproduction of TNFα plays an important role in both marrow failure and evolution of TNF-resistant neoplastic clones. To test the notion that evolution of clones in patients with FA might follow a like adaptive pathway to TNF-resistance, we performed gene expression microarray analysis (Affymetrix GeneChip HG-U133A [22,283 probe sets] arrays) using RNA obtained from low-density marrow cells from; (a) normal volunteers (NV, n=11) (b) FA patients with clonal cytogenetic defects detected in at least 50% of metaphases (FA-CL, n=10) and (c) FA patients with no detectable cytogenetic defects (FA-APL, n=22). Data were normalized using the MAS 5.0 global scaling method with the target intensity of 325. The adapted nature of evolved clones was supported by unsupervised hierarchical clustering (GeneSifter) and principal component analyses (Partek) which revealed a high degree of relatedness between the NV and FA-CL samples while FA-APL samples were more distant from both NV and FA-CL in transcriptomal space. To identify genes whose differential expression might play a role in clonal evolution, two analytical approaches were taken. First, using the shrunken centroids method we identified a 30 gene signature that distinguished FA-CL and FA-APL samples. Secondly, we applied two filters to the data. The first revealed 2317 genes that were either over-expressed or under-expressed (>1.5 fold, p<0.05, adjusted for multiple comparisons using the Benjamini-Hochberg method) in a pairwise comparison of FA-APL to NV samples. The second filter utilized a pattern reading tool (GeneSifter) to identify those genes on the first list of 2317 whose expression did not differ in the comparison between NV and FA-CL samples. The majority (959 of 1063) of these genes were those whose expression was suppressed in FA-APL samples and either not-suppressed or overexpressed in the FA-CL samples. Three genes of interest were in the 30 gene signature identified using the shrunken centroids method and in those identified using the pattern reading tool. Specifically, HOXA9, a gene with known leukemogenic potential, and two genes known to enhance the expression of HOXA9 (MYB and KDM5B) were suppressed in FA-APL samples and over-expressed in the FA-CL samples. After confirming the RNA results using quantitative real time RT-PCR and subsequently determining that the differentially expressed genes in the FA-CL samples included most of the genes known to be regulated by HOXA9 expression in CD34+ marrow cells (Dorsam,S.T. et al, Blood 2004) (p<0.05, Fisher's Exact P.T.) we demonstrated the following: (a) retroviral expression of HOXA9 in normal human CD34+ marrow cells enhanced TNFα resistance in CFU-GM and BFU-E, (b) 3-day in vitro exposure of kit+/lin−/sca-1+ (KLS) cells from Fancc−/− mice to TNFα and multilineage growth factors (MGF; IL-11, Flt3L, SCF, IL-6) suppressed expression of HoxA9 and suppressed survival of KLS cells but enhanced expression of HoxA9 and enhanced survival of wild-type KLS cells, (c) ectopic expression of HOXA9 in Fancc−/− KLS cells enhanced KLS survival and blunted the inhibitory response to TNFα in 3-day suspension cultures with MGF. Taken together, these results indicate that clonal evolution in FA stem cell pools represents the ascendance of a fundamentally adapted clone and that over-expression of HOXA9 in the clonal progeny may represent at least one early step in the pathway of clonal adaptation.
Pasquini:Novartis: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria.
Asterisk with author names denotes non-ASH members.