Acute myeloid leukemia fusion proteins deregulate genes involved in stem cell maintenance and DNA repair
Myriam Alcalay, Natalia Meani, Vania Gelmetti, Anna Fantozzi, Marta Fagioli, Annette Orleth, Daniela Riganelli, Carla Sebastiani, Enrico Cappelli, Cristina Casciari, Maria Teresa Sciurpi, Angela Rosa Mariano, Simone Paolo Minardi, Lucilla Luzi, Heiko Muller, Pier Paolo Di Fiore, Guido Frosina, Pier Giuseppe Pelicci
Myriam Alcalay, Natalia Meani, Vania Gelmetti, Anna Fantozzi, Marta Fagioli, Annette Orleth, Daniela Riganelli, Carla Sebastiani, Enrico Cappelli, Cristina Casciari, Maria Teresa Sciurpi, Angela Rosa Mariano, Simone Paolo Minardi, Lucilla Luzi, Heiko Muller, Pier Paolo Di Fiore, Guido Frosina, Pier Giuseppe Pelicci
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Article Hematology

Acute myeloid leukemia fusion proteins deregulate genes involved in stem cell maintenance and DNA repair

Abstract

Acute myelogenous leukemias (AMLs) are genetically heterogeneous and characterized by chromosomal rearrangements that produce fusion proteins with aberrant transcriptional regulatory activities. Expression of AML fusion proteins in transgenic mice increases the risk of myeloid leukemias, suggesting that they induce a preleukemic state. The underlying molecular and biological mechanisms are, however, unknown. To address this issue, we performed a systematic analysis of fusion protein transcriptional targets. We expressed AML1/ETO, PML/RAR, and PLZF/RAR in U937 hemopoietic precursor cells and measured global gene expression using oligonucleotide chips. We identified 1,555 genes regulated concordantly by at least two fusion proteins that were further validated in patient samples and finally classified according to available functional information. Strikingly, we found that AML fusion proteins induce genes involved in the maintenance of the stem cell phenotype and repress DNA repair genes, mainly of the base excision repair pathway. Functional studies confirmed that ectopic expression of fusion proteins constitutively activates pathways leading to increased stem cell renewal (e.g., the Jagged1/Notch pathway) and provokes accumulation of DNA damage. We propose that expansion of the stem cell compartment and induction of a mutator phenotype are relevant features underlying the leukemic potential of AML-associated fusion proteins.

Authors

Myriam Alcalay, Natalia Meani, Vania Gelmetti, Anna Fantozzi, Marta Fagioli, Annette Orleth, Daniela Riganelli, Carla Sebastiani, Enrico Cappelli, Cristina Casciari, Maria Teresa Sciurpi, Angela Rosa Mariano, Simone Paolo Minardi, Lucilla Luzi, Heiko Muller, Pier Paolo Di Fiore, Guido Frosina, Pier Giuseppe Pelicci

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(a) Semiquantitative PCR analysis of 14 predicted targets. For each gene...
(a) Semiquantitative PCR analysis of 14 predicted targets. For each gene, the number of cycles required to detect a signal in the linear range was previously determined (see “Supplementary Methods,” ref.12). (b) Semiquantitative PCR analysis of AML blasts compared to CD34+ normal precursors of 11 targets identified in the U937 system. Relative mRNA levels of induced target genes (c) and of repressed target genes (d) in U937 PML/RAR, U937 AML1/ETO or U937 PLZF/RAR cells assessed by real time RT-PCR. Mt was the control used in panels a, c, and d. (e) Expression levels of common target genes in U937 PML/RAR cells before and after 4 or 8 hours of treatment with 10–6 M RA. Values are calculated relatively to expression levels in Mt cells receiving the same treatment. (f) Expression levels of common target genes in blasts derived form two APL patients (APL no. 1 and APL no. 2) after 4 hours of in vitro treatment with 10–6 M RA. All values are shown as compared to the control (C); i.e., expression levels in blasts from the same individual, prior to RA treatment. All experiments shown in this figure were performed in triplicate. One representative experiment is shown for semiquantiative PCRs (a and b), whereas an average value of the three results was plotted for real time RT-PCR data (c–f). GAPDH gene expression was used to normalize all experiments.