Pro-inflammatory cytokines favor the emergence of ETV6-RUNX1-positive pre-leukemic cells in a model of mesenchymal niche.

ETV6-RUNX1 (E/R) fusion gene, arising in utero from translocation t(12;21)(p13:q22), is the most frequent alteration in childhood acute lymphoblastic leukemia (ALL). However, E/R is insufficient to cause overt leukemia since it generates a clinically silent pre-leukemic clone which persists in the bone marrow but fails to out-compete normal progenitors. Conversely, pre-leukemic cells show increased susceptibility to transformation following additional genetic insults. Infections/inflammation are the most accredited triggers for mutations accumulation and leukemic transformation in E/R+ pre-leukemic cells. However, precisely how E/R and inflammation interact in promoting leukemia is still poorly understood. Here we demonstrate that IL6/TNFα/ILß pro-inflammatory cytokines cooperate with BM-MSC in promoting the emergence of E/R+ Ba/F3 over their normal counterparts by differentially affecting their proliferation and survival. Moreover, IL6/TNFα/ILß-stimulated BM-MSC strongly attract E/R+ Ba/F3 in a CXCR2-dependent manner. Interestingly, E/R-expressing human CD34+ IL7R+ progenitors, a putative population for leukemia initiation during development, were preserved in the presence of BM-MSC and IL6/TNFα/ILß compared to their normal counterparts. Finally, the extent of DNA damage increases within the inflamed niche in both control and E/R-expressing Ba/F3, potentially leading to transformation in the apoptosis-resistant pre-leukemic clone. Overall, our data provide new mechanistic insights into childhood ALL pathogenesis.

Therapeutic targeting of mutant p53 in pediatric acute lymphoblastic leukemia.

Alterations of the tumor suppressor gene TP53 are found in different cancers, in particular in carcinomas of adults. In pediatric acute lymphoblastic leukemia (ALL), TP53 mutations are infrequent but enriched at relapse. As in most cancers, mainly DNA-binding domain missense mutations are found, resulting in accumulation of mutant p53, poor therapy response, and inferior outcome. Different strategies to target mutant p53 have been developed including reactivation of p53's wildtype function by the small molecule APR-246. We investigated TP53 mutations in cell lines and 62 B-cell precursor ALL samples and evaluated the activity of APR-246 in TP53-mutated or wildtype ALL. We identified cases with TP53 missense mutations, high (mutant) p53 expression and insensitivity to the DNA-damaging agent doxorubicin. In TP53-mutated ALL, APR-246 induced apoptosis showing strong anti-leukemia activity. APR-246 restored mutant p53 to its wildtype conformation, leading to pathway activation with induction of transcriptional targets and re-sensitization to genotoxic therapy in vitro and in vivo In addition, induction of oxidative stress contributed to APR-246-mediated cell death. In a preclinical model of patient-derived TP53-mutant ALL, APR-246 reduced leukemia burden and synergized strongly with the genotoxic agent doxorubicin, leading to superior leukemia-free survival in vivo Thus, targeting mutant p53 by APR-246, restoring its tumor suppressive function, seems to be an effective therapeutic strategy for this high-risk group of TP53-mutant ALL.

ActivinA: a new leukemia-promoting factor conferring migratory advantage to B-cell precursor-acute lymphoblastic leukemic cells.

B-cell precursor-acute lymphoblastic leukemia modulates the bone marrow (BM) niche to become leukemia-supporting and chemo-protective by reprogramming the stromal microenvironment. New therapies targeting the interplay between leukemia and stroma can help improve disease outcome. We identified ActivinA, a TGF-ß family member with a well-described role in promoting several solid malignancies, as a factor favoring leukemia that could represent a new potential target for therapy. ActivinA resulted over-expressed in the leukemic BM and its production was strongly induced in mesenchymal stromal cells after culture with leukemic cells. Moreover, MSCs isolated from BM of leukemic patients showed an intrinsic ability to secrete higher amounts of ActivinA compared to their normal counterparts. The pro-inflammatory leukemic BM microenvironment synergized with leukemic cells to induce stromal-derived ActivinA. Gene expression analysis of ActivinA-treated leukemic cells showed that this protein was able to significantly influence motility-associated pathways. Interestingly, ActivinA promoted random motility and CXCL12-driven migration of leukemic cells, even at suboptimal chemokine concentrations, characterizing the leukemic niche. Conversely, ActivinA severely impaired CXCL12-induced migration of healthy CD34+ cells. This opposite effect can be explained by the ability of ActivinA to increase intracellular calcium only in leukemic cells, boosting cytoskeleton dynamics through a higher rate of actin polymerization. Moreover, by stimulating the invasiveness of the leukemic cells, ActivinA was found to be a leukemia-promoting factor. Importantly, the ability of ActivinA to enhance BM engraftment and the metastatic potential of leukemic cells was confirmed in a xenograft mouse model of the disease. Overall, ActivinA was seen to be a key factor in conferring a migratory advantage to leukemic cells over healthy hematopoiesis within the leukemic niche.

What is the relevance of Ikaros gene deletions as a prognostic marker in pediatric Philadelphia-negative B-cell precursor acute lymphoblastic leukemia?

New prognostic markers are needed for upfront identification of patients with acute lymphocytic leukemia with a high risk of relapse or who are not likely to respond to the most aggressive chemotherapy. We focused our analysis on Ikaros (IKZF1) gene deletions in a homogeneous cohort of 410 pediatric patients with Philadelphia chromosome-negative, B-cell precursor acute lymphoblastic leukemia enrolled in Italy into the AIEOP-BFM ALL2000 study. We confirm their reported poor prognostic value, although the associated event-free survival was relatively high (approximately 70%). The difference in the cumulative incidence of relapse between patients positive or not for IKZF1 deletions was not marked: 24.2% (5.9) versus 13.1% (1.8) overall and 23.9% (6.6) versus 16.5% (2.5) in the intermediate-risk subgroup. In line with this, IKZF1 deletions were not an independent prognostic factor for the hazard of relapse. Most IKZF1-deleted cases stratified in the high-risk group relapsed, suggesting that once identified, patients with these deletions require an alternative treatment. In conclusion, the need of and benefit from introducing IKZF1 deletions as an additional stratification marker for patients with Philadelphia-negative B-cell precursor acute lymphoblastic leukemia remain questionable.

CirComPara: A Multi-Method Comparative Bioinformatics Pipeline to Detect and Study circRNAs from RNA-seq Data.

Circular RNAs (circRNAs) are generated by backsplicing of immature RNA forming covalently closed loops of intron/exon RNA molecules. Pervasiveness, evolutionary conservation, massive and regulated expression, and posttranscriptional regulatory roles of circRNAs in eukaryotes have been appreciated and described only recently. Moreover, being easily detectable disease markers, circRNAs undoubtedly represent a molecular class with high bearing on molecular pathobiology. CircRNAs can be detected from RNAseq data using appropriate computational methods to identify the sequence reads spanning backsplice junctions that do not colinearly map to the reference genome. To this end, several programs were developed and critical assessment of various strategies and tools suggested the combination of at least two methods as good practice to guarantee robust circRNA detection. Here,we present CirComPara (http://github.com/egaffo/CirComPara), an automated bioinformatics pipeline, to detect, quantify and annotate circRNAs from RNAseq data using in parallel four different methods for backsplice identification. CirComPara also provides quantification of linear RNAs and gene expression, ultimately comparing and correlating circRNA and gene/transcript expression level. We applied our method to RNAseqdata of monocyte and macrophage samples in relation to haploinsufficiency of the RNAbinding splicing factor Quaking (QKI). The biological relevance of the results, in terms of number, types and variations of circRNAs expressed, illustrates CirComPara potential to enlarge the knowledge of the transcriptome, adding details on the circRNAome, and facilitating further computational and experimental studies.

Deciphering KRAS and NRAS mutated clone dynamics in MLL-AF4 paediatric leukaemia by ultra deep sequencing analysis.

To induce and sustain the leukaemogenic process, MLL-AF4+ leukaemia seems to require very few genetic alterations in addition to the fusion gene itself. Studies of infant and paediatric patients with MLL-AF4+ B cell precursor acute lymphoblastic leukaemia (BCP-ALL) have reported mutations in KRAS and NRAS with incidences ranging from 25 to 50%. Whereas previous studies employed Sanger sequencing, here we used next generation amplicon deep sequencing for in depth evaluation of RAS mutations in 36 paediatric patients at diagnosis of MLL-AF4+ leukaemia. RAS mutations including those in small sub-clones were detected in 63.9% of patients. Furthermore, the mutational analysis of 17 paired samples at diagnosis and relapse revealed complex RAS clone dynamics and showed that the mutated clones present at relapse were almost all originated from clones that were already detectable at diagnosis and survived to the initial therapy. Finally, we showed that mutated patients were indeed characterized by a RAS related signature at both transcriptional and protein levels and that the targeting of the RAS pathway could be of beneficial for treatment of MLL-AF4+ BCP-ALL clones carrying somatic RAS mutations.

CRLF2 overexpression identifies an unfavourable subgroup of adult B-cell precursor acute lymphoblastic leukemia lacking recurrent genetic abnormalities.

BACKGROUND: A deregulated CRLF2 (d-CRLF2) expression was described in B-cell acute lymphoblastic leukemia without recurrent fusion genes (B-NEG ALL). While the role of d-CRLF2 in children has been extensively described, little is known about its role and impact in adult ALL. METHODS: Expression levels of CRLF2 were evaluated by quantitative real-time PCR in 102 newly-diagnosed adult B-NEG ALL and correlated with the clinico-biological characteristics and outcome. Incidence and clinical impact of the P2RY8/CRLF2 transcript was also assessed. RESULTS: High CRLF2 levels, as continuous variable, were significantly associated with hyperleucocytosis (p=0.0002) and thrombocytopenia (p=0.005); when a cut-point at ΔCt≤8 was applied, 35 cases (34.3%), mostly males (80%), proved positive for CRLF2 expression. High CRLF2 levels, as continuous or categorical variable, were associated with a worse disease-free (p=0.003 and p=0.015) and overall survival (p=0.017 and 0.0038). Furthermore, when CRLF2 was analyzed as a categorical variable, a high statistical association was found with IKZF1 deletion and mutations in the JAK/STAT pathway (p=0.001 and p<0.0001, respectively). Finally, the P2RY8/CRLF2 transcript, identified in 8/102 patients (7.8%), was associated with a poor outcome. CONCLUSIONS: In adult B-NEG ALL, high CRLF2 expression is associated with distinct clinico-biological features and an unfavourable prognosis in both univariate and multivariate analysis; similarly, P2RY8/CRLF2 positivity correlates with a poor outcome. The quantification of CRLF2 is an important prognostic marker in adult B-lineage ALL without known genetic lesions.

Epigenetic silencing of TFPI-2 in canine diffuse large B-cell lymphoma.

Epigenetic modifications are important early events during carcinogenesis. In particular, hypermethylation of CpG islands in the promoter region of tumor suppressor genes is a well-known mechanism of gene silencing that contributes to cancer development and progression. Tissue factor pathway inhibitor 2 (TFPI-2) is a tumor suppressor involved in invasiveness inhibition. Although TFPI-2 transcriptional silencing, through promoter hypermethylation, has been widely reported in several human malignancies, it has never been explored in lymphoma. In the present study TFPI-2 methylation and gene expression have been investigated in canine Diffuse Large B-cell lymphomas (cDLBCL). The methylation level of 23 CpGs located within the TFPI-2 promoter was investigated by bisulfite-specific PCR and next generation amplicon deep sequencing (GS Junior 454, Roche) in 22 cDLBCLs and 9 controls. For the same specimens, TFPI-2 gene expression was assessed by means of Real-time RT-PCR. Sequence analysis clearly demonstrated that TFPI2 is frequently hypermethylated in cDLBCL. Hypermethylation of the TFPI-2 promoter was found in 77% of DLBCLs (17 out of 22) and in one normal lymph node. Globally, dogs with DLBCL showed a mean methylation level significantly increased compared to controls (p<0.01) and analysis of hypermethylation by site identified 19 loci out of 23 (82%) with mean methylation levels from 2- to 120-fold higher in cDLBCL. Gene expression analysis confirmed a significant down-regulation of TFPI-2 (p<0.05) in DLBCLs compared with normal lymph nodes, suggesting that TFPI-2 hypermethylation negatively regulates its transcription. In addition, a significant positive correlation (p<0.01) was found between TFPI-2 methylation levels and age providing the first indication of age-associated epigenetic modifications in canine DLBCL. To conclude, our findings demonstrated that epigenetic dysregulation of TFPI-2, leading to its reduced expression, is frequently detected in canine DLBCL. In the next future, the aberrant TFPI-2 promoter hypermethylation may be considered in association with prognosis and therapy.

Clinical utility of microarray-based gene expression profiling in the diagnosis and subclassification of leukemia: report from the International Microarray Innovations in Leukemia Study Group.

PURPOSE: The Microarray Innovations in Leukemia study assessed the clinical utility of gene expression profiling as a single test to subtype leukemias into conventional categories of myeloid and lymphoid malignancies. METHODS: The investigation was performed in 11 laboratories across three continents and included 3,334 patients. An exploratory retrospective stage I study was designed for biomarker discovery and generated whole-genome expression profiles from 2,143 patients with leukemias and myelodysplastic syndromes. The gene expression profiling-based diagnostic accuracy was further validated in a prospective second study stage of an independent cohort of 1,191 patients. RESULTS: On the basis of 2,096 samples, the stage I study achieved 92.2% classification accuracy for all 18 distinct classes investigated (median specificity of 99.7%). In a second cohort of 1,152 prospectively collected patients, a classification scheme reached 95.6% median sensitivity and 99.8% median specificity for 14 standard subtypes of acute leukemia (eight acute lymphoblastic leukemia and six acute myeloid leukemia classes, n = 693). In 29 (57%) of 51 discrepant cases, the microarray results had outperformed routine diagnostic methods. CONCLUSION: Gene expression profiling is a robust technology for the diagnosis of hematologic malignancies with high accuracy. It may complement current diagnostic algorithms and could offer a reliable platform for patients who lack access to today's state-of-the-art diagnostic work-up. Our comprehensive gene expression data set will be submitted to the public domain to foster research focusing on the molecular understanding of leukemias.

Hepatocyte growth factor receptor c-MET is associated with FAS and when activated enhances drug-induced apoptosis in pediatric B acute lymphoblastic leukemia with TEL-AML1 translocation.

Expression of c-MET, the HGF (hepatocyte growth factor) tyrosine kinase receptor, was investigated in pediatric B-acute lymphoblastic leukemia (ALL) patients. c-MET was found to be expressed in normal B cells and in B-ALL patients with the t(12;21) TEL-AML1 translocation, but it is not expressed in the most part of B-ALL without the t(12;21). We also found that c-MET, related to proliferation and protection from apoptosis, is associated with the pro-apoptotic protein FAS in TEL-AML1 B-ALL cells and in normal B lymphocytes. The possible role of this protein complex in drug-induced apoptosis was thus investigated in REH TEL-AML1 B-ALL cell line. REH cells prestimulated with HGF and treated with doxorubicin had shown a higher apoptotic rate than non-HGF-prestimulated ones (p = 0.03). REH cells stimulated with IL-3 and treated with doxorubicin did not undergo apoptosis more than nonstimulated cells, demonstrating that increased proliferation in itself is not directly related to the higher apoptotic sensitivity observed with HGF stimulation. These results indicate that c-MET activation enhances specifically FAS-mediated apoptosis in TEL-AML1 ALL cells and, considering that the c-MET/FAS complex is present only in normal B lymphocytes and in TEL-AML1 leukemias, this implies that it may have an important contribution in cellular homeostasis and in high sensitivity of TEL-AML1 ALL to chemotherapeutic regimens.