We investigated the engraftment properties and impact on patient outcome of 50 pediatric acute lymphoblastic leukemia (ALL) samples transplanted into NOD/SCID mice. Time to leukemia (TTL) was determined for each patient sample engrafted as weeks from transplant to overt leukemia. Short TTL was strongly associated with high risk for early relapse, identifying an independent prognostic factor. This high-risk phenotype is reflected by a gene signature that upon validation in an independent patient cohort (n = 197) identified a high-risk cluster of patients with early relapse. Furthermore, the signature points to independent pathways, including mTOR, involved in cell growth and apoptosis. The pathways identified can directly be targeted, thereby offering additional treatment approaches for these high-risk patients.

A yet homogeneous leukemia entity was further subdivided, based on distinct genetic properties. This approach provided a simplified way to obtain robust and disease-specific gene signatures even in smaller cohorts.

Acute lymphoblastic leukemia (ALL) is a malignant disease of the white blood cells. The etiology of ALL is believed to be multifactorial and likely to involve an interplay of environmental and genetic variables. We performed a genome-wide association study of 355 750 single-nucleotide polymorphisms (SNPs) in 474 controls and 419 childhood ALL cases characterized by a t(12;21)(p13;q22) — the most common chromosomal translocation observed in childhood ALL — which leads to an ETV6–RUNX1 gene fusion. The eight most strongly associated SNPs were followed-up in 951 ETV6-RUNX1-positive cases and 3061 controls from Germany/Austria and Italy, respectively. We identified a novel, genome-wide significant risk locus at 3q28 (TP63, rs17505102, PCMH=8.94 × 10−9, OR=0.65). The separate analysis of the combined German/Austrian sample only, revealed additional genome-wide significant associations at 11q11 (OR8U8, rs1945213, P=9.14 × 10−11, OR=0.69) and 8p21.3 (near INTS10, rs920590, P=6.12 × 10−9, OR=1.36). These associations and another association at 11p11.2 (PTPRJ, rs3942852, P=4.95 × 10−7, OR=0.72) remained significant in the German/Austrian replication panel after correction for multiple testing. Our findings demonstrate that germline genetic variation can specifically contribute to the risk of ETV6–RUNX1-positive childhood ALL. The identification of TP63 and PTPRJ as susceptibility genes emphasize the role of the TP53 gene family and the importance of proteins regulating cellular processes in connection with tumorigenesis.

The MLLT10 gene, located at 10p13, is a known partner of MLL and PICALM in specific leukemic fusions generated from recurrent 11q23 and 11q14 chromosome translocations. Deep sequencing recently identified NAP1L1/12q21 as another MLLT10 partner in T-cell acute lymphoblastic leukemia (T-ALL). In pediatric T-ALL, we have identified 2 RNA processing genes, that is, HNRNPH1/5q35 and DDX3X/Xp11.3 as new MLLT10 fusion partners. Gene expression profile signatures of the HNRNPH1- and DDX3X-MLLT10 fusions placed them in the HOXA subgroup. Remarkably, they were highly similar only to PICALM-MLLT10-positive cases. The present study showed MLLT10 promiscuity in pediatric T-ALL and identified a specific MLLT10 signature within the HOXA subgroup.

Acute myeloid leukemia (AML) primary cells express high levels of phosphorylated Akt, a master regulator of cellular functions regarded as a promising drug target. By means of reverse phase protein arrays, we examined the response of 80 samples of primary cells from AML patients to selective inhibitors of the phosphatidylinositol 3 kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) axis. We confirm that >60% of the samples analyzed are characterized by high pathway phosphorylation. Unexpectedly, however, we show here that targeting Akt and mTOR with the specific inhibitors Akti 1/2 and Torin1, alone or in combination, result in paradoxical Akt phosphorylation and activation of downstream signaling in 70% of the samples. Indeed, we demonstrate that cropping Akt or mTOR activity can stabilize the Akt/mTOR downstream effectors Forkhead box O and insulin receptor substrate-1, which in turn potentiate signaling through upregulation of the expression/phosphorylation of selected growth factor receptor tyrosine kinases (RTKs). Activation of RTKs in turn reactivates PI3K and downstream signaling, thus overruling the action of the drugs. We finally demonstrate that dual inhibition of Akt and RTKs displays strong synergistic cytotoxic effects in AML cells and downmodulates Akt signaling to a much greater extent than either drug alone, and should therefore be explored in AML clinical setting.

Moreover, for eight patients with mutations in IKZF1 (n ¼ 3), RUNX1 (n ¼ 3), ASXL1 (n ¼ 1), WT1 (n ¼ 2) and IDH1 (n ¼ 2), matched DNA samples from initial diagnosis at chronic phase were available. In none of the chronic phase CML samples were the respective IKZF1 deletions or RUNX1 and ASXL1 mutations detectable, indicating that mutations in IKZF1 and RUNX1 were acquired at the time of transformation to BC-CML, and thus act as driver mutations in these cases. In contrast, WT1 and IDH1 mutations were detected at diagnosis in chronic phase in one case each.With respect to clinical data, associations with survival for RUNX1, ASXL1, IKZF1 and WT1 alterations were investigated. No molecular parameter was significantly associated with outcome, which may be due to the short median survival in BC-CML (n ¼ 34 patients with survival data available; median overall survival: 9.3 months).In conclusion, the aberrant BCR-ABL kinase causes genomic instability of the CML clone by inefficient DNA repair, resulting in chromosomal alterations and molecular aberrations of transcription factors. This study on 12 genes demonstrated for the first time that in 76.9% of the BC-CML patients, molecular mutations are detectable. The high mutation rate of RUNX1 (33.3%), ASXL1 (20.5%) and IKZF1 (17.9%) represented important molecular abnormalities in the progression of CML. In particular, IKZF1 and RUNX1 alterations, both involved in cell differentiation, were identified as important markers of disease progression from chronic phase to BC. Although this is a comprehensive study, further investigations are required to identify additional pathogenetic alterations, as in four cases (10.2%) of our cohort no chromosomal or molecular genetic alterations were observed in addition to t(9;22)(q34;q11). Conflict of interestCH, S Schnittger, WK, and TH have equity ownership of MLL Munich Leukemia Laboratory. VG, AK, MZ, CE, S Schindela, and SW are employed by MLL Munich Leukemia Laboratory. MCM and AH declare no conflict of interest.

The pathogenesis of infant acute lymphoblastic leukemia (ALL) is still not well defined. Short latency to leukemia and very high concordance rate for ALL in Mixed-Lineage Leukemia (MLL)-positive infant twins suggest that the MLL rearrangement itself could be sufficient for overt leukemia. Attempts to generate a suitable mouse model for MLL-AF4-positive ALL did not thoroughly resolve the issue of whether cooperating mutations are required to reduce latency and to generate overt leukemia in vivo. In this study, we applied single-nucleotide polymorphism array technology to perform genomic profiling of 28 infant ALL cases carrying t(4;11) to detect MLL-cooperating aberrations hidden to conventional techniques and to gain new insights into infant ALL pathogenesis. In contrast to pediatric, adolescent and adult ALL cases, the MLL rearrangement in infant ALL is associated with an exceptionally low frequency of copy-number abnormalities, thus confirming the unique nature of this disease. By contrast, additional genetic aberrations are acquired at disease relapse. Small-segmental uniparental disomy traits were frequently detected, mostly constitutional, and widely distributed throughout the genome. It can be argued that the MLL rearrangement as a first hit, rather than inducing the acquisition of additional genetic lesions, has a major role to drive and hasten the onset of leukemia.

BackgroundIn spite of leukemia therapy improvements obtained over the last decades, therapy is not yet effective in all cases. Current approaches in Acute Lymphoblastic Leukemia (ALL) research focus on identifying new molecular targets to improve outcome for patients with a dismal prognosis. In this light phosphoproteomics seems to hold great promise for the identification of proteins suitable for targeted therapy.Methodology/Principal FindingsWe employed Reverse Phase Protein Microarrays to identify aberrantly activated proteins in 118 pediatric B-cell precursor (BCP)-ALL patients. Signal transduction pathways were assayed for activation/expression status of 92 key signalling proteins. We observed an increased activation/expression of several pathways involved in cell proliferation in poor clinical prognosis patients. MLL-rearranged tumours revealed BCL-2 hyperphosphorylation through AMPK activation, which indicates that AMPK could provide a functional role in inhibiting apoptosis in MLL-rearranged patients, and could be considered as a new potential therapeutic target. Second, in patients with poor clinical response to prednisone we observed the up-modulation of LCK activity with respect to patients with good response. This tyrosine-kinase can be down-modulated with clinically used inhibitors, thus modulating LCK activity could be considered for further studies as a new additional therapy for prednisone-resistant patients. Further we also found an association between high levels of CYCLIN E and relapse incidence. Moreover, CYCLIN E is more expressed in early relapsed patients, who usually show an unfavourable prognosis.Conclusions/SignificanceWe conclude that functional protein pathway activation mapping revealed specific deranged signalling networks in BCP-ALL that could be potentially modulated to produce a better clinical outcome for patients resistant to standard-of-care therapies.