Approximately 25% of childhood acute lymphoblastic leukemias carry the ETV6/RUNX1 fusion gene. Despite their excellent initial treatment response, up to 20% of patients relapse. To gain insight into the relapse mechanisms, we analyzed single nucleotide polymorphism arrays for DNA copy number aberrations (CNAs) in 18 matched diagnosis and relapse leukemias. CNAs were more abundant at relapse than at diagnosis (mean 12.5 vs 7.5 per case; P ‫؍‬ .01) with 5.3 shared on average. Their patterns revealed a direct clonal relationship with exclusively new aberrations at relapse in only 21.4%, whereas 78.6% shared a common ancestor and subsequently acquired distinct CNA. Moreover, we identified recurrent, mainly nonoverlapping deletions associated with glucocorticoid-mediated apoptosis targeting the Bcl2 modifying factor (BMF) (n ‫؍‬ 3), glucocorticoid receptor NR3C1 (n ‫؍‬ 4), and components of the mismatch repair pathways (n ‫؍‬ 3). Fluorescence in situ hybridization screening of additional 24 relapsed and 72 nonrelapsed ETV6/ RUNX1-positive cases demonstrated that BMF deletions were significantly more common in relapse cases (16.6% vs 2.8%; P ‫؍‬ .02). Unlike BMF deletions, which were always already present at diagnosis, NR3C1 and mismatch repair aberrations prevailed at relapse. They were all associated with leukemias, which poorly responded to treatment. These findings implicate glucocorticoid-associated drug resistance in ETV6/RUNX1-positive relapse pathogenesis and therefore might help to guide future therapies. (Blood. 2011;117(9):2658-2667)

Despite their apparently good prognosis B15% of high hyperdiploid (HD) childhood acute lymphoblastic leukemia (ALL) cases relapse. To search for responsible risk factors we determined copy number aberrations as well as copy neutral loss of heterozygosity (LOH) in 13 matched diagnosis and relapse samples and added the data of the only three available cases from the literature. Deletions and copy neutral LOH in 3 and 2 of the 16 cases directed us to the histone-modifying CREB-binding protein (CREBBP) gene, whose functional impairment is implicated in drug resistance. We therefore screened all samples for mutations in this gene and discovered 9 acquired sequence mutations in 7/16 cases, leading to an overall frequency of somatic CREBBP aberrations in HD ALL relapse cases of 63% that is considerably higher than that of the reported, mainly non-HD ALL (18.3%). Moreover, mutations in HD cases occur almost exclusively in the HAT domain (8/9; 89%). Hot spot mutations are present at diagnosis in 18.8% of relapsing HD ALL cases but in none of 40 respective cases remaining in long-term remission. Thus, the particular high incidence of CREBBP mutations in relapse-prone HD ALL cases could eventually be exploited for refined risk stratification and customized treatment in this genetic subgroup.

The ETV6/RUNX1 (E/R) gene fusion is generated by the t(12;21) and found in approximately 25% of childhood B-cell precursor acute lymphoblastic leukemia. In contrast to the overwhelming evidence that E/R is critical for the initiation of leukemia, its relevance for the maintenance of overt disease is less clear. To investigate this issue, we suppressed the endogenous E/R fusion protein with lentivirally transduced short hairpin RNA in the leukemia cell lines REH and AT-2, and found a distinct reduction of proliferation and cell survival. In line with the observed concurrent inactivation of the phosphoinositide 3-kinase (PI3K)/AKT/ mammalian target of rapamycin (mTOR) pathway, pharmacological inhibition diminished the phosphorylation of AKT and ribosomal protein S6, and significantly increased the apoptosis rate in E/R-positive leukemias. Moreover, PI3K/mTOR inhibitors sensitized glucocorticoid-resistant REH cells to prednisolone, an observation of potential relevance for improving treatment of drug-resistant relapses. Of note, knockdown of the E/R fusion gene also severely impaired the repopulation capacity of REH cells in non-obese deficient/severe combined immunodeficient mice. Collectively, these data demonstrate that the E/R fusion protein activates the PI3K/AKT/mTOR pathway and is indispensible for disease maintenance. Importantly, these results provide a first rationale and justification for targeting the fusion gene and the PI3K/AKT/mTOR pathway therapeutically.

The TEL/AML1 fusion gene results from the most frequent t(12;21)(p13;q22) translocation in childhood acute lymphoblastic leukemia (ALL). Its contribution to transformation is largely unknown, in particular with respect to survival and apoptosis. We therefore silenced TEL/AML1 expression in leukemic REH cells by RNA inhibition, which eventually led to programmed cell death. Microarray and 2D gel electrophoresis data demonstrated a differential regulation of heat-shock proteins (HSPs), among them HSP90, as well as of its client, survivin. Consistent with these findings, ectopic expression of TEL/ AML1 in Ba/F3 cells increased protein levels of HSP90 and survivin and conferred resistance to apoptotic stimuli. Our data suggest that TEL/AML1 not only contributes to leukemogenesis by affecting an antiapoptotic network but also seems to be indispensable for maintaining the malignant phenotype. The functional relationship between TEL/AML1, HSP90, and survivin provides the rational for targeted therapy, be it the fusion gene or the latter 2 proteins. (Blood. 2007;109: 2607-2610) IntroductionThe TEL/AML1 fusion gene is generated by t(12;21)(p13;q22), the most frequent chromosomal translocation in childhood acute lymphoblastic leukemia (ALL). 1 There is convincing evidence that the gene fusion already takes place in utero in the majority of children with TEL/AML1-positive ALL, and this suggests, together with studies in twins, that it represents an early, or even initiating, event in leukemia development. 2,3 This translocation, however, is not sufficient for the manifestation of a clinical disease, but seems rather to induce a protracted preleukemic state in which additional postnatal genetic events may accumulate, eventually leading to malignant transformation. 4,5 The role of TEL/AML1 expression in the affected cell was recently addressed in several studies and indicates an interference with differentiation in the B lineage as well as enhanced selfrenewal of B cells. [6][7][8] Of importance, such mutations that impair hematopoietic differentiation are assumed to interfere also with apoptosis. 9 The mechanisms by which the fusion protein breaches this crucial anticancer shield have, so far, not been identified.The function of genes, in particular of fusion genes, has been studied in recent years by gene silencing using RNA interference (RNAi), a powerful strategy to specifically target the gene of interest. 10 In this study, we investigated the functional contribution of TEL/AML1 to the malignant phenotype focusing on the evasion of programmed cell death by silencing the fusion gene in the t(12;21)-positive cell line REH. Materials and methods Cell cultureThe B-cell precursor (BCP) leukemic cell line REH (having the TEL/AML1 fusion gene, the second TEL allele deleted and AML1 retained) and the mouse IL3-dependent BCP Ba/F3 cells were grown in 24-well plates (Nunc, Roskilde, Denmark) at 1 ϫ 10 6 cells/mL. Short-interfering RNA (siRNA) design and transfection of cellssiRNAs targeting the fusion region of TEL/AML1 were desi...

Purpose We explored the mechanisms leading to the distinct overexpression of EPOR as well as the effects of EPO signaling on ETV6/RUNX1-positive acute lymphoblastic leukemias. Experimental Design ETV6/RUNX1-expressing model cell lines and leukemic cells were used for real-time PCR of EPOR expression. Proliferation, viability, and apoptosis were analyzed on cells exposed to EPO, prednisone, or inhibitors of EPOR pathways by [3H]thymidine incorporation, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and Annexin V/propidium iodide staining. Western blot analysis was done to detect activation of signaling proteins. Serum EPO levels and sequences of the EPOR (n = 53) as well as hemoglobin levels were taken from children with acute lymphoblastic leukemia enrolled in Austrian protocols. Results We show here that ectopic expression of ETV6/RUNX1 induced EPOR up-regulation. Anemia, however, did not appear to influence EPOR expression on leukemic cells, although children with ETV6/RUNX1-positive leukemias had a lower median hemoglobin than controls. Exposure to EPO increased proliferation and survival of ETV6/RUNX1-positive leukemias in vitro, whereas blocking its binding site did not alter cell survival. The latter was not caused by activating mutations in the EPOR but might be triggered by constitutive activation of phosphatidylinositol 3-kinase/Akt, the major signaling pathway of EPOR in these cells. Moreover, prednisone-induced apoptosis was attenuated in the presence of EPO in this genetic subgroup. Conclusions Our data suggest that ETV6/RUNX1 leads to EPOR up-regulation and that activation by EPO might be of relevance to the biology of this leukemia subtype. Further studies are, however, needed to assess the clinical implications of its apoptosis-modulating properties.