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© 2019 Ferrata Storti Foundation., B-cell acute lymphoblastic leukemia is the commonest childhood cancer. In infants, B-cell acute lymphoblastic leukemia remains fatal, especially in patients with t(4;11), present in ~80% of cases. The pathogenesis of t(4;11)/KMT2A-AFF1+ (MLL-AF4+) infant B-cell acute lymphoblastic leukemia remains difficult to model, and the pathogenic contribution in cancer of the reciprocal fusions resulting from derivative translocated-chromosomes remains obscure. Here, “multi-layered” genome-wide analyses and validation were performed on a total of 124 de novo cases of infant B-cell acute lymphoblastic leukemia uniformly diagnosed and treated according to the Interfant 99/06 protocol. These patients showed the most silent mutational landscape reported so far for any sequenced pediatric cancer. Recurrent mutations were exclusively found in K-RAS and N-RAS, were subclonal and were frequently lost at relapse, despite a larger number of non-recurrent/non-silent mutations. Unlike non-MLL-rearranged B-cell acute lymphoblastic leukemias, B-cell receptor repertoire analysis revealed minor, non-expanded B-cell clones in t(4;11)+ infant B-cell acute lymphoblastic leukemia, and RNA-sequencing showed transcriptomic similarities between t(4;11)+ infant B-cell acute lymphoblastic leukemias and the most immature human fetal liver hematopoietic stem and progenitor cells, confirming a “pre-VDJ” fetal cellular origin for both t(4;11) and RASmut. The reciprocal fusion AF4-MLL was expressed in only 45% (19/43) of the t(4;11)+ patients, and HOXA cluster genes are exclusively expressed in AF4-MLL-expressing patients. Importantly, AF4-MLL/HOXA-expressing patients had a significantly better 4-year event-free survival (62.4% vs. 11.7%, P=0.001), and overall survival (73.7 vs. 25.2%, P=0.016). AF4-MLL expression retained its prognostic significance when analyzed in a Cox model adjusting for risk stratification according to the Interfant-06 protocol based on age at diagnosis, white blood cell count and response to prednisone. This study has clinical implications for disease outcome and diagnostic risk-stratification of t(4;11)+ infant B-cell acute lymphoblastic leukemia., This work was supported by the European Research Council (CoG-2014- 646903 to PM; and StG-2014-637904 to IV), the Spanish Ministry of Economy and Competitiveness (SAF-SAF2013- 43065 to PM and SAF2016-76758-R to IV), the Asociación Española Contra el Cáncer (AECC-CI-2015), FERO Foundation, and the ISCIII (PI14-01191) to CB. PM/IV also acknowledge financial support from The Obra Social La CaixaFundaciò Josep Carreras, The Inocente Inocente Foundation, Fundación Ramón Areces and The Generalitat de Catalunya (SGR330). IR was supported by a Programme Grant from Bloodwise (LLR 13001) and by the Oxford NIHR Biomedical Centre based at Oxford University Hospitals NHS Trust and University of Oxford. PM is an investigator of the Spanish Cell Therapy cooperative network (TERCEL). AR was supported by a Clinician Scientist Fellowship from Bloodwise (14041).

© 2019 The Authors Although health, development, and environment challenges are interconnected, evidence remains fractured across sectors due to methodological and conceptual differences in research and practice. Aligned methods are needed to support Sustainable Development Goal advances and similar agendas. The Bridge Collaborative, an emergent research-practice collaboration, presents principles and recommendations that help harmonize methods for evidence generation and use. Recommendations were generated in the context of designing and evaluating evidence of impact for interventions related to five global challenges (stabilizing the global climate, making food production sustainable, decreasing air pollution and respiratory disease, improving sanitation and water security, and solving hunger and malnutrition) and serve as a starting point for further iteration and testing in a broader set of contexts and disciplines. We adopted six principles and emphasize three methodological recommendations: (1) creation of compatible results chains, (2) consideration of all relevant types of evidence, and (3) evaluation of strength of evidence using a unified rubric. We provide detailed suggestions for how these recommendations can be applied in practice, streamlining efforts to apply multi-objective approaches and/or synthesize evidence in multidisciplinary or transdisciplinary teams. These recommendations advance the necessary process of reconciling existing evidence standards in health, development, and environment, and initiate a common basis for integrated evidence generation and use in research, practice, and policy design.

Research Note, VITIS - Journal of Grapevine Research, Vol. 40 No. 2 (2001): Vitis

This study was carried out to determine the involvement of the antibiotic kanamycin, commonly used as a selective agent in transformation protocols, in the phenomenon of somaclonal variation. Both genetic and epigenetic events were looked for. Two complementary approaches were used to evaluate global methylation changes in the genome of callus derived from the leaves of Arabidopsis thaliana L.; immunolabelling using monoclonal-antibodies raised against 5-methylcytosine in order to define the relative abundance of methylated cytosine; the analysis of methylation-sensitive polymorphism technique (MSAP) to assess methylation changes at CCGG sequences. In addition, the same samples were analysed using AFLPs in order to determine the extent of genomic change with respect to callus grown in the absence of kanamycin and plants grown from seed. The use of kanamycin as a selective agent caused extensive methylation changes in the genome with both hyper- and hypomethylation events seen. However, the net result was genome-wide hypomethylation: this effect was dosage dependent; the higher the dose, the greater the effect. At the same time, sequence mutation was detected

A 2 kb DNA fragment, upstream of the rice beta-tubulin isotype 16 (Ostub16) coding sequence, was isolated using inverse PCR and screening of a tubulin-enriched lambda library. An intron (863 bp) present in the 5' untranslated region (5' UTR) is spliced out to produce the most abundant mRNA species which corresponds to the previously cloned Ostub16 cDNA. Transient expression assays performed on rice embryogenic calluses with chimeric Ostub16::GUS constructs demonstrated that the entire 2 kb upstream sequence has a strong promoter activity, and that the 863 bp intron is required for high-level GUS expression. In addition, the intron sequence is capable per se of sustaining a weak but consistent GUS expression. Two rare Ostub16 transcripts, with a start site mapping within this intron sequence, were detected in rice coleoptile cells. The transcription start site mapped at position 290 with respect to the ATG codon, and the shorter molecule originated from splicing of the same precursor mRNA. Therefore transcriptional expression of rice beta-tubulin isotype 16 results in the synthesis of two premRNA molecules (I and II) encoding for three different mRNA species. We discuss these findings in terms of function and molecular evolution of the mechanisms that control plant -tubulin gene expression.

Recently, we defined “CML-like” subtype of BCR::ABL1-positive acute lymphoblastic leukemia (ALL), resembling lymphoid blast crisis of chronic myeloid leukemia (CML). Here we retrospectively analyzed prognostic relevance of minimal residual disease (MRD) and other features in 147 children with BCR::ABL1-positive ALL (diagnosed I/2000–IV/2021, treated according to EsPhALL (n = 133) or other (n = 14) protocols), using DNA-based monitoring of BCR::ABL1 genomic breakpoint and clonal immunoglobulin/T-cell receptor gene rearrangements. Although overall prognosis of CML-like (n = 48) and typical ALL (n = 99) was similar (5-year-EFS 60% and 49%, respectively; 5-year-OS 75% and 73%, respectively), typical ALL presented more relapses while CML-like patients more often died in the first remission. Prognostic role of MRD was significant in the typical ALL (p = 0.0005 in multivariate analysis for EFS). In contrast, in CML-like patients MRD was not significant (p values > 0.2) and inapplicable for therapy adjustment. Moreover, in the typical ALL, risk-prediction could be further improved by considering initial hyperleukocytosis. Early distinguishing typical BCR::ABL1-positive ALL and CML-like patients is essential to enable optimal treatment approach in upcoming protocols. For the typical ALL, tyrosine-kinase inhibitors and concurrent chemotherapy with risk-directed intensity should be recommended; in the CML-like disease, no relevant prognostic feature applicable for therapy tailoring was found so far.

The most frequent genetic aberration leading to infant ALL (iALL) is the chromosomal translocation t(4;11), generating the fusion oncogenes KMT2A:AFF1 and AFF1:KMT2A, respectively. KMT2A-r iALL displays a dismal prognosis through high relapse rates and relapse-associated mortality. Relapse occurs frequently despite ongoing chemotherapy and without the accumulation of secondary mutations. A rational explanation for the observed chemo-resistance and satisfactory treatment options remain to be elucidated. We found that elevated ICOSLG expression level at diagnosis was associated with inferior event free survival (EFS) in a cohort of 43 patients with t(4;-11) iALL and that a cohort of 18 patients with iALL at relapse displayed strongly increased ICOSLG expression. Furthermore, co-culturing t(4;11) ALL cells (ICOSLGhi) with primary T-cells resulted in the development of Tregs. This was impaired through treatment with a neutralizing ICOSLG antibody. These findings imply ICOSLG (1) as a relapse-predicting biomarker, and (2) as a therapeutic target involved in a potential immune evasion relapse-mechanism of infant t(4;11) ALL.

Background: Despite intensive risk-based treatment protocols, 15% of paediatric patients with B-Cell Precursor Acute Lymphoblastic Leukaemia (BCP-ALL) experience relapse. There is urgent need of novel strategies to target poor prognosis subgroups, like PAX5 translocated. Methods: We considered 289 childhood BCP-ALL cases consecutively enrolled in Italy in the AIEOP-BFM ALL2000/R2006 protocols and we performed extensive molecular profiling, integrating gene expression, copy number analyses and fusion genes discovery by target-capture NGS. We developed preclinical strategies to target PAX5 fusion genes. Findings: We identified 135 cases without recurrent genetic rearrangements. Among them, 59 patients (43·7%) had a Ph-like signature; the remaining cases were identified as ERG-related (26%), High-Hyperdiploid-like (17%), ETV6::RUNX1-like (8·9%), MEF2D-rearranged (2·2%) or KMT2A-like (1·5%). A poor prognosis was associated with the Ph-like signature, independently from other high-risk features. Interestingly, PAX5 was altered in 54·4% of Ph-like compared to 16·2% of non-Ph-like cases, with 7 patients carrying PAX5 fusions (PAX5t), involving either novel (ALDH18A1, IKZF1, CDH13) or known (FBRSL1, AUTS2, DACH2) partner genes. PAX5t cases have a specific driver activity signature, extending to multiple pathways including LCK hyperactivation. Among FDA-approved drugs and inhibitors, we selected Dasatinib, Bosutinib and Foretinib, in addition to Nintedanib, known to be LCK ligands. We demonstrated the efficacy of the LCK-inhibitor BIBF1120/Nintedanib, as single agent or in combination with conventional chemotherapy, both ex vivo and in patient-derived xenograft model, showing a synergistic effect with dexamethasone. Interpretation: This study provides new insights in high-risk Ph-like leukaemia and identifies a potential therapy for targeting PAX5-fusion poor risk group. Funding: Ricerca Finalizzata-Giovani Ricercatori (Italian Ministry of Health), AIRC, Transcall, Fondazione Cariparo.

B cell acute lymphoblastic leukemia (B-ALL) is the most common childhood cancer. As predicted by its prenatal origin, infant B-ALL (iB-ALL) shows an exceptionally silent DNA mutational landscape, suggesting that alternative epigenetic mechanisms may substantially contribute to its leukemogenesis. Here, we have integrated genome-wide DNA methylome and transcriptome data from 69 patients with de novo MLL-rearranged leukemia (MLLr) and non-MLLr iB-ALL leukemia uniformly treated according to the Interfant-99/06 protocol. iB-ALL methylome signatures display a plethora of common and specific alterations associated with chromatin states related to enhancer and transcriptional control in normal hematopoietic cells. DNA methylation, gene expression, and gene coexpression network analyses segregated MLLr away from non-MLLr iB-ALL and identified a coordinated and enriched expression of the AP-1 complex members FOS and JUN and RUNX factors in MLLr iB-ALL, consistent with the significant enrichment of hypomethylated CpGs in these genes. Integrative methylome-transcriptome analysis identified consistent cancer cell vulnerabilities, revealed a robust iB-ALL–specific gene expression–correlating dmCpG signature, and confirmed an epigenetic control of AP-1 and RUNX members in reshaping the molecular network of MLLr iB-ALL. Finally, pharmacological inhibition or functional ablation of AP-1 dramatically impaired MLLr-leukemic growth in vitro and in vivo using MLLr-iB-ALL patient–derived xenografts, providing rationale for new therapeutic avenues in MLLr-iB-ALL., We thank CERCA/Generalitat de Catalunya (SGR180) and Fundació Josep Carreras-Obra Social la Caixa for their institutional support. Financial support for this work was obtained from the European Research Council (CoG-2014-646903 and PoC-2018-811220 to PM), the Spanish Ministry of Economy and Competitiveness (SAF-2019-108160-R and SAF2016-76758-R to PM and IV, respectively), the Spanish Association against cancer (AECC-CI-2015 and PROYE18061FERN to CB and MFF), the Fundación Uno entre Cienmil (to PM), the Health Institute Carlos III (ISCIII/FEDER, PI17/01028, PI15/00892, PI18/01527 to CB and AFF/MFF, respectively). We also acknowledge the Plan de Ciencia, Tecnología e Innovación from the Asturias Government cofunding 2018–2022/FEDER (IDI/2018/146to MFF). MFF also acknowledges funding from Fundación General CSIC (0348_CIE_6_E). PM also acknowledges financial support from Fundación Leo Messi. JRT and MV are supported by Juan de la Cierva fellowships by the Spanish Ministry of Science and Innovation (FJCI-2015-26965, IJC2018-36825-I, IJCI-2017-3317) and IUOPA-ISPA-FINBA (The IUOPA is supported by the Obra Social Cajastur-Liberbank, Spain). RTR is supported by a fellowship from the AECC scientific foundation. RFP and PSO are supported by the Severo Ochoa program (BP17-114 and BP17-165, respectively).

B-cell precursor (BCP) acute lymphoblastic leukemia (BCP-ALL) in infants (ie, children age

MLL-rearranged acute lymphoblastic leukemia (ALL) occurring in infants is a rare but very aggressive leukemia, typically associated with a dismal prognosis. Despite the development of specific therapeutic protocols, infant patients with MLL-rearranged ALL still suffer from a low cure rate. At present, novel therapeutic approaches are urgently needed. Recently, the use of small molecule inhibitors targeting the epigenetic regulators of the MLL complex emerged as a promising strategy for the development of a targeted therapy. Herein, we have investigated the effects of bromodomain and extra-terminal (BET) function abrogation in a preclinical mouse model of MLL-AF4+ infant ALL using the BET inhibitor I-BET151. We reported that I-BET151 is able to arrest the growth of MLL-AF4+ leukemic cells in vitro, by blocking cell division and rapidly inducing apoptosis. Treatment with I-BET151 in vivo impairs the leukemic engraftment of patient-derived primary samples and lower the disease burden in mice. I-BET151 affects the transcriptional profile of MLL-rearranged ALL through the deregulation of BRD4, HOXA7/HOXA9, and RUNX1 gene networks. Moreover, I-BET151 treatment sensitizes glucocorticoid-resistant MLL-rearranged cells to prednisolone in vitro and is more efficient when used in combination with HDAC inhibitors, both in vitro and in vivo. Given the aggressiveness of the disease, the failure of the current therapies and the lack of an ultimate cure, this study paves the way for the use of BET inhibitors to treat MLL-rearranged infant ALL for future clinical applications. Mol Cancer Ther; 17(8); 1705–16. ©2018 AACR.

Leukemias bearing CRLF2 and JAK2 gene alterations are characterized by aberrant JAK/STAT signaling and poor prognosis. The HDAC inhibitor givinostat/ITF2357 has been shown to exert anti-neoplastic activity against both systemic juvenile idiopathic arthritis and myeloproliferative neoplasms through inhibition of the JAK/STAT pathway. These findings led us to hypothesize that givinostat might also act against CRLF2-rearranged BCP-ALL, which lack effective therapies. Here, we found that givinostat inhibited proliferation and induced apoptosis of BCP-ALL CRLF2-rearranged cell lines, positive for exon 16 JAK2 mutations. Likewise, givinostat killed primary cells, but not their normal hematopoietic counterparts, from patients carrying CRLF2 rearrangements. At low doses, givinostat downregulated the expression of genes belonging to the JAK/STAT pathway and inhibited STAT5 phosphorylation. In vivo, givinostat significantly reduced engraftment of human blasts in patient-derived xenograft models of CRLF2-positive BCP-ALL. Importantly, givinostat killed ruxolitinib-resistant cells and potentiated the effect of current chemotherapy. Thus, givinostat in combination with conventional chemotherapy may represent an effective therapeutic option for these difficult-to-treat subsets of ALL. Lastly, the selective killing of cancer cells by givinostat may allow the design of reduced intensity regimens in CRLF2-rearranged Down syndrome-associated BCP-ALL patients with an overall benefit in terms of both toxicity and related complications.

Metabolic reprogramming is a key hallmark of cancer, but less is known about metabolic plasticity of the same tumor at different sites. Here, we investigated the metabolic adaptation of leukemia in two different microenvironments, the bone marrow and the central nervous system (CNS). We identified a metabolic signature of fatty-acid synthesis in CNS leukemia, highlighting Stearoyl-CoA desaturase (SCD1) as a key player. In vivo SCD1 overexpression increases CNS disease, whilst genetic or pharmacological inhibition of SCD1 decreases CNS load. Overall, we demonstrated that leukemic cells dynamically rewire metabolic pathways to suit local conditions and that targeting these adaptations can be exploited therapeutically.

Infusion of patient-derived CD19-specific chimeric antigen receptor (CAR) T cells engineered by viral vectors achieved complete remission and durable response in relapsed and refractory (r/r) B-lineage neoplasms. Here, we expand on those findings by providing a preclinical evaluation of allogeneic non-viral cytokine-induced killer (CIK) cells transfected with the Sleeping Beauty (SB) transposon CD19CAR (CARCIK-CD19). Specifically, thanks to a large-scale 18-day manufacturing process, it was possible to achieve stable CD19CAR expression (62.425 ± 6.399%) and efficient T-cell expansion (23.36 ± 3.00-fold). Frozen/thawed CARCIK-CD19 remained fully functional both in vitro and in an established patient-derived xenograft (PDX) of MLL-ENL rearranged acute lymphoblastic leukemia (ALL). CARCIK-CD19 showed a dose-dependent antitumor response and prolonged persistence in a PDX, bearing the feature of a Philadelphia-like ALL with PAX5/AUTS2 translocation, and in a survival model of lymphoma, achieving complete eradication of disseminated tumors. Finally, the infusion of CARCIK-CD19 proved to be safe and well tolerated in a biodistribution and toxicity model. The infused cells persisted in the hematopoietic and post-injection perfused organs until the end of the study and consisted of CD8+, CD56+, and CAR+ T cells. Overall, these findings provide important implications for non-viral technology and the proof-of-concept that donor-derived CARCIK-CD19 are indeed effective against relapsed ALL, a possibility that will be tested in Phase I/II clinical trials after allogeneic hematopoietic stem-cell transplantation

Background: Essential hypertension arises from the combined effect of genetic and environmental factors. A pharmacogenomics approach could help to identify additional molecular mechanisms involved in its pathogenesis. Aim: The aim of SOPHIA study was to identify genetic polymorphisms regulating blood pressure response to the angiotensin II receptor blocker, losartan, with a whole-genome approach. Materials & methods: We performed a genome-wide association study on blood pressure response in 372 hypertensives treated with losartan and we looked for replication in two independent samples. Results: We identified a peak of association in CAMK1D gene (rs10752271, effect size -5.5 ± 0.94 mmHg, p = 1.2 × 10-8). CAMK1D encodes a protein that belongs to the regulatory pathway involved in aldosterone synthesis. We tested the specificity of rs10752271 for losartan in hypertensives treated with hydrochlorothiazide and we validated it in silico in the GENRES cohort. Conclusion: Using a genome-wide approach, we identified the CAMK1D gene as a novel locus associated with blood pressure response to losartan. CAMK1D gene characterization may represent a useful tool to personalize the treatment of essential hypertension. Original submitted 7 May 2014; Revision submitted 29 July 2014

Distinct from other forms of acute lymphoblastic leukemia (ALL), infant ALL with mixed lineage leukemia (MLL) gene rearrangement, the most common leukemia occurring within the first year of life, might arise without the need for cooperating genetic lesions. Through Ig/TCR rearrangement analysis of MLL-AF4+ infant ALL at diagnosis and xenograft leukemias from mice transplanted with the same diagnostic samples, we established that MLL-AF4+ infant ALL is composed of a branching subclonal architecture already at diagnosis, frequently driven by an Ig/TCR-rearranged founder clone. Some MLL-AF4+ clones appear to be largely quiescent at diagnosis but can reactivate and dominate when serially transplanted into immunodeficient mice, whereas other dominant clones at diagnosis can become more quiescent, suggesting a dynamic competition between actively proliferating and quiescent subclones. Investigation of paired diagnostic and relapse samples suggested that relapses often occur from subclones already present but more quiescent at diagnosis. Copy-number alterations identified at relapse might contribute to the activation and expansion of previously quiescent subclones. Finally, each of the identified subclones is able to contribute to the diverse phenotypic pool of MLL-AF4+ leukemia-propagating cells. Unraveling of the subclonal architecture and dynamics in MLL+ infant ALL may provide possible explanations for the therapy resistance and frequent relapses observed in this group of poor prognosis ALL.

Summary Induced pluripotent stem cells (iPSCs) are a powerful tool for disease modeling. They are routinely generated from healthy donors and patients from multiple cell types at different developmental stages. However, reprogramming leukemias is an extremely inefficient process. Few studies generated iPSCs from primary chronic myeloid leukemias, but iPSC generation from acute myeloid or lymphoid leukemias (ALL) has not been achieved. We attempted to generate iPSCs from different subtypes of B-ALL to address the developmental impact of leukemic fusion genes. OKSM(L)-expressing mono/polycistronic-, retroviral/lentiviral/episomal-, and Sendai virus vector-based reprogramming strategies failed to render iPSCs in vitro and in vivo. Addition of transcriptomic-epigenetic reprogramming “boosters” also failed to generate iPSCs from B cell blasts and B-ALL lines, and when iPSCs emerged they lacked leukemic fusion genes, demonstrating non-leukemic myeloid origin. Conversely, MLL-AF4-overexpressing hematopoietic stem cells/B progenitors were successfully reprogrammed, indicating that B cell origin and leukemic fusion gene were not reprogramming barriers. Global transcriptome/DNA methylome profiling suggested a developmental/differentiation refractoriness of MLL-rearranged B-ALL to reprogramming into pluripotency., Graphical Abstract, Highlights • Neither primary B-ALL blasts nor leukemic B cell lines can be reprogrammed to iPSCs • Global transcriptome and DNA methylome suggest a developmental refractoriness, Despite the interest in generating iPSCs from human primary acute leukemias for disease modeling, reprogramming leukemias is an extremely inefficient process. In this article, Menéndez, Bueno, and colleagues show that many reprogramming strategies reported to date are not sufficient to generate B-ALL-derived iPSCs. Global transcriptome/DNA methylome profiling suggested a developmental/differentiation refractoriness of B-ALL to reprogramming into pluripotency.

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. Leukemia (2010) 24, 169-176; doi:10.1038/leu.2009.203; published online 12 November 2009

Transformation of hematopoietic cells by the BCR/ABL oncogene is caused by perturbation of signal transduction pathways leading to altered patterns of gene expression and activity. By oligonucleotide microarray hybridization of polysomal RNA of untreated and STI571-treated 32D-BCR/ABL cells, we identified the beta-chemokine CCL9 as a gene regulated by BCR/ABL in a tyrosine kinase-dependent manner. BCR/ABL repressed CCL9 expression at the transcriptional level by mechanisms involving suppression of p38 MAP kinase, and modulation of the activity of CDP/cut and C/EBPalpha, two transcription regulators of myeloid differentiation. However, repression of C/EBP-dependent transcription did not prevent the induction of CCL9 expression by STI571, suggesting that C/EBPalpha is involved in maintaining rather than in inducing CCL9 expression. Restoration of CCL9 expression in 32D-BCR/ABL cells had no effect on the in vitro proliferation of these cells, but reduced their leukemogenic potential in vivo, possibly by recruitment of CD3-positive immune cells. Together, these findings suggest that downregulation of chemokine expression may be involved in BCR/ABL-dependent leukemogenesis by altering the relationship between transformed cells and the microenvironment.

The Opuntia genus includes over 181 species comprising, on the basis of morphological traits, a total of 29 series [The Cactaceae (1919)]. Starting from this classification, several authors have investigated the Opuntia genus taxonomy but the large morphological variation within different species, suggests that phenotypical characteristics will not serve to produce a stable classification. In this work chloroplastic simple sequence repeat (cpSSR) and amplified fragment length polymorphism (AFLP) were used to evaluate the usefulness of molecular markers in Opuntia species characterization and to study the relationships among different species. Results show that the combination of cpSSR and AFLP markers provide a quantitative estimation of genetic relationships among several Opuntia species. Both molecular analyses reveal a genetic similarity among species of series 20 and 21 [The Cactaceae (1919)] as suggested also by morphological traits. Particular attention was focused on the genetic relationship between Opuntia ficus-indica and Opuntia megacantha: individuals from different populations of the two species were analyzed with both molecular markers. A common genetic constitution of O. ficus-indica and O. megacantha was detected. On the basis of molecular data, morphological traits and biogeographical distribution, we suggest that O. ficus-indica should be considered as a domesticated form of O. megacantha. Our results suggest the importance of a revision of Opuntia genus classification using several tools: molecular, morphological and biogeographical analysis. © 2003 Elsevier Ireland Ltd. All rights reserved.

Bortezomib (BTZ) is an antineoplastic drug that reversibly binds to an active site threonine in the 26S proteasome. The inhibition of protein degradation in the cells leads to the accumulation of signalling molecules, blocking the tumour survival pathways. BTZ is mainly used for the treatment of multiple myeloma, but unfortunately its clinical use have evidenced the onset of a painful peripheral neuropathy (BIPN) that decreased patients’ quality of life. Despite the clinical relevance of BIPN, our knowledge of the molecular mechanism underlying the painful condition is still limited. In recent studies, inflammation has been described as a possible event in the onset of neuropathic pain induced by some chemotherapy drug. To investigate the putative role of immune response in the BTZ-induced painful peripheral neuropathy, we defined a new immune-suppressed murine model based on sub-lethal X-ray irradiation. Adult female BALB/c mice underwent X-ray irradiation, once a week for 4 weeks with a first dose of 350 RAD and 3 subsequent 100 RAD doses (X-ray group), to deplete the immune cells-mediated response. Part of these animals were treated with BTZ 0.8 mg/kg twice a week for 4 weeks (X-ray+BTZ group). To assess the BTZ-induced neurophysiological damage and the onset of neuropathic pain, nerve conduction velocity and mechanical nociceptive threshold were measured. Sciatic nerve, spinal cord and dorsal root ganglia were processed for light microscope observations. Moreover, flow cytometer analysis of CD45-positive cells were performed in tibiae bone marrow (BM) and peripheral blood (PB) to confirm the immune-suppression. After the first X ray irradiation piloerection and hypokinesia were observed, and significant body weight decreased was measured at the end of treatment in X-ray+BTZ-treated animals. The immune-suppression was persistent in the PB until the end of the study, while a partial recovery in BM was observed in the last time-point evaluation. In addition, BTZ induced the onset of the mechanical allodynia after the beginning of treatment and an impairment of nerve functions in both caudal and digital nerves. X ray irradiation per se was not able to induce any neurophysiological change. Further investigations are required to deeply understand the role of inflammation in the pathogenic mechanism of BTZ-induced painful peripheral neuropathy, and to identify novel therapeutic targets for an effective therapeutic approach. Supported in part by a research grant from the University of Milano-Bicocca F.A.R.

Over 90% of infants (

Implementation of array based whole-genome high-resolution technologies confirms the absence of secondary copy-number alterations in MLL-AF4 -positive infant ALL patients

Ogni giorno, circa mille miliardi di nuove cellule vengono prodotte dal midollo osseo e messe in circolazione nel sangue periferico per far fronte alle necessità del nostro organismo. Tutte le cellule che compongono il nostro sangue, derivano da una stessa cellula-madre, chiamata cellula staminale ematopoietica, un precursore primordiale e immaturo che ha la capacità di espandersi e la potenzilità di differenzare a eritrociti, linfociti, leucociti, piastrine. Il processo di differenziamento che va dalla cellula staminale alle cellule mature circolanti è detto emopoiesi. La leucemia è un tipo di tumore circolante che può avere origine da una cellula qualunque del sangue, a un qualsiasi stadio differenziativo, la quale inizia a proliferare all’impazzata, sfuggendo ad ogni controllo, e prende il sopravvento sulle altre cellule sane. Il risultato è l’espansione aberrante di un singolo tipo cellulare, a discapito degli altri compartimenti, che causa una disfunzione del sistema emopoietico. La leucemia non può essere considerata un’ unica malattia, poichè ne esistono molti diversi tipi, a seconda del tipo cellulare interessato, e del tipo di mutazione genica che la caratteizza. Nel nostro laboratorio, presso il Centro di Ricerca M. Tettamanti dell’Opedale S. Gerardo di Monza, ci occupiamo delle leucemie del bambino. In particolare, nel mio periodo di dottorato, mi sono occupata di studiare una particolare forma di leucemia linfoblastica acuta, detta infant LLA, ad insorgenza nel primo anno di vita, che presenta il riarrangiamento del gene MLL. Questa leucemia rappresenta la forma prevalente in età neonatale, ed è purtoppo una malattia molto aggressiva con prognosi infausta che in Italia colpisce circa 10-12 nuovi nati all’anno. In particolare, i casi che presentano la traslocazione t(4;11) con espressione del prodotto di fusione MLL-AF4 rappresentano il sottogruppo genetico a incidenza maggiore in età neonatale. Per moltissimi aspetti, sia dal punto di vista clinico che biologico, la LLA infant rappresenta una malattia peculiare, diversa dalle altre forme di leucemia dell’età pediatrica e adulta. In questi quattro anni di dottorato mi sono occupata nello specifico di due progetti. Nel primo, in collaborazione con il Prof. Jacobsen in Svezia, ci siamo proposti di identificare, isolare e caratterizzare dal punto di vista biologico e funzionale la ‘cellula staminale leucemica’ della LLA infant con riarrangiamento del gene MLL. Nel secondo studio, pubblicato su ‘Leukemia’ abbiamo voluto valutare se il solo riarrangiamento del gene MLL fosse di per se sufficiente per dare origine alla malattia, oppure se alterazioni genetiche ulteriori, secondarie al riarrangiamento di MLL che costituisce il primo evento, fossero necessarie per la manifestazione clinica della leucemia. La composizione cellulare di ogni singolo tumore umano è altamente eterogenea, e non tutte le cellule che lo compongono sono capaci di sostenerne la crescita. Per spiegare queste osservazioni è stata postulata l’esistenza di un sottogruppo ristretto di cellule, dette staminali tumorali (CSC), o staminali leucemiche (LSC) nel caso si tratti di leucemia, che hanno la potenzialità di auto-rigenerarsi e di riprodurre l’intera massa tumorale. Secondo il modello della ‘cellula staminale tumorale’, in tutti i tumori solo un ristretto gruppo di cellule è capace di generare l’intera massa tumorale e sostenerne la crescita all’infinito. Poter bersagliare in modo specifico questa cellula significa eradicare definitivamente la malattia, che altrimenti inevitabilmente si rigenererebbe. Questa ipotesi è in contrasto con il precedente modello stocastico secondo cui ogni cellula che compone il tumore è potenzialmente capace di sostenerne la crescita: tuttavia la eterogenicità tumorale, e la sua bassa tumorigenicità sono dovute alla scarsa probalilità per una cellula di iniziare il ciclo cellulare e proliferare. Tuttavia recenti studi mettono in discussione l’ipotesi stessa dell’esistenza di una CSC, ed attualmente l’opinione scientifica è divisa riguardo al fatto se il modello gerarchico della CSC, piuttosto che il classico modello stocastico, siano più adatti per spiagere il meccanismo biologico alla base dell’insorgenza dei tumori umani. Infatti, benchè il concetto di CSC sia intuitivamente immediato, la sua effettiva identificazione e caratterizzazione si è rivelata inaspettatamente complicata, e ad oggi, non esiste una definizione universale di CSC. Il termine ‘leucemia’ comprende in realtà tantissimi tipi differenti di tumore, e tale eterogeneità si riflette sulla LSC. La specifica alterazione genetica, la cellula di origine (cioè la cellula inizialmente colpita dal primo evento trasformante da cui il tumore ha avuto luogo), le diverse metodologie in vivo applicate, l’influenza del microambiente midollare e l’alta variabilità fenotipica tra diversi pazienti affetti dalla medesima forma di leucemia, hanno portato inevitabilmente alla generazione di risultati apparentementente contrastanti tra i vari gruppi di ricerca che indipendentemente si sono riproposti di identificare la LSC. Il modello sperimentale d’elezione per identificare la LSC è il trapianto in vivo di blasti leucemici in topi umanizzati immunocompromessi, capaci di accettare cellule umane senza rigettarle e permetterne l’attecchimento. Nel periodo all’estero trascorso nel laboratorio del Porf. Jacobsen a Lund, mi sono occupata di mettere a punto il modello di trapianto in vivo di cellule leucemiche da pazienti affetti da infant LLA in topi unamizzati. Previa irradiazione subletale del topo ricevente, che ha il duplice scopo di eliminare le cellule circolanti murine mature e creare spazio per le cellule umane trapiantate, i blasti leucemici vengono iniettati nel topo per via sistemica attraverso la vena della coda.. Se l’attecchimento è avvenuto, già dopo poche settimane, i topi mostreranno alti livelli di ricostituzione nel midollo e nei compartimenti extramidollari (come milza, fegato e sangue periferico), con conseguente manifestazione dei sintomi clinici della malattia (quali splenomegalia, pelo arruffato, letargia, anoressia, perdita di peso e decesso). L’attecchimento viene valutato eseguendo periodiche biopsie midollari (dopo 6, 9, 12 o più settimane dal trapianto) dalla sede femorale, in anestesia generale, e successivamente la presenza di cellule leucemiche umane è rilevata nell’aspirato midollare tramite analisi successive. Nel primo lavoro inviato a ‘Cancer Cell’ abbiamo osservato che nella LLA infant MLL-AF4 positiva la LSC risiede nella frazione CD19+, costituita da linfociti B a diversi stadi maturativi. Infatti, la frazione CD19- è composta sia da cellule staminali normali residue (non leucemiche) che danno luogo a una ricostituzione normale in vivo, che da cellule leucemiche (MLL riarrangiate), tuttavia prive di potenziale leucemogenico, poiché incapaci di ripopolare il midollo di un topo irradiato e dare origine alla leucemia. Inoltre abbiamo dimostrato che all’interno della frazione CD19+, esistono molteplici sottopolazioni LSC con diverso potenziale leucemogenico e diverse caratteristiche biologiche e funzionali in vivo (es. penetranza, latenza di insorgenza della malattia e fenotipo della leucemia risultante). In conclusione, i nostri risultati dimostrano che, a differenza del modello convenzionale, esistono molteplici sottopopolazioni LSC con caratteristiche biologiche e funzionali distinte, che possono essere distinte tra loro e dalla restante popolazione leucemica (non tumorigenica) sulla base dell’espressione di determinati antigeni di superficie. Nel secondo studio pubblicato su ‘Leukemia’, ci siamo chiesti se la sola presenza della traslocazione del gene MLL fosse di per se sufficiente per la manifestazione clinica della malattia, o se mutazioni accessorie cooperanti fossero necessarie. Per fare ciò abbiamo analizzato a livello genomico un gruppo consistente di pazienti affetti da LLA infant MLL-AF4 positiva, tramite tecniche sofisticate di screening su tutto il genoma ad alta risoluzione, e abbiamo osservato che, contrariamente ai pazienti affetti da altri tipi di LLA pediatrica ad insorgenza dopo l’anno, nella LLA infant MLL-AF4 positiva non si rilevano mutazioni aggiuntive. Questi risultati dimostrano che il solo riarrangiamento di MLL è necessario e sufficiente per la manifestazione, in tempi brevi, della malattia conclamata, a differenza di altre forme di leucemia del bambino e dell’adulto, in cui mutazioni genetiche aggiuntive sono indispensabili. Inoltre tali evidenze ribadiscono ulteriormente che la LLA infant con riarangiamento di MLL è una malattia nica dal punto di vista biologco, diversa dagli altri tipi di leucemia, e che che il meccanismo di leucemogenesi può essere distinto. Considerando l’alta mortalità, l’alto rischio di ricaduta, la prognosi infausta della LLA infant MLL positiva e l’urgente necessità di avere a disposizione nuove ed efficaci terapie antitumorali, questi studi sono di rilevante importanza, in quanto non solo aiutano a comprendere meglio i meccanismi di insorgenza della LLA infant e fare luce sulla biologia e l’aggressività di questa malattia, ma rappresentano anche il punto di partenza per identificare nuove strategie terapeutiche al fine bersagliare la LSC ed eradicare la malattia in modo definitivo.

Translational regulation by oncogenic proteins may be a rapid and efficient mechanism to modulate gene expression. We report here the identification of the CEBPB gene as a target of translational regulation in myeloid precursor cells transformed by the BCR/ABL oncogene. Expression of CEBPB was repressed in 32D-BCR/ABL cells and reinduced by imatinib (STI571) via a mechanism that appears to depend on expression of the CUG-repeat RNA-binding protein CUGBP1 and the integrity of the CUG-rich intercistronic region of c/ebpbeta mRNA. Constitutive expression or conditional activation of wild-type CEBPB induced differentiation and inhibited proliferation of 32D-BCR/ABL cells in vitro and in mice, but a DNA binding-deficient CEBPB mutant had no effect. The proliferation-inhibitory effect of CEBPB was, in part, mediated by the CEBPB-induced GADD45A gene. Because expression of CEBPB (and CEBPA) is low in the blast crisis (BC) stage of chronic myelogenous leukemia (CML) and is inversely correlated with BCR/ABL tyrosine kinase levels, these findings point to the therapeutic potential of restoring C/EBP activity in CML-BC and, perhaps, other types of acute leukemia.

C/EBP alpha and C/EBP beta, two transcription factors of the C/EBP family play important roles in the proliferation and differentiation of various cell types including myeloid progenitors. Expression of C/EBP alpha and C/EBP beta is repressed in myeloid blast crisis of Chronic Myelogenous Leukemia by mechanisms that involve translation repression which depends on the interaction of RNA-binding proteins with conserved binding sites in the 5'UTR of c/ebp alpha and c/ebp beta mRNA. Ectopic expression of C/EBP alpha and C/EBP beta in myeloid progenitors expressing the BCR/ABL oncogene inhibits proliferation, induces differentiation and suppresses leukemogenesis in mice, but C/EBP a is markedly more effective than C/EBP beta. The more potent effects of C/EBP a probably depends on protein-protein interaction with cell-cycle regulatory proteins, but the pattern of genes modulated by C/EBP alpha and C/EBP beta is not completely overlapping. This suggests that transcription-dependent and -independent effects are both involved and support the therapeutic potential of reactivating C/EBP alpha and C/EBP beta expression in leukemic cells

In viticulture, biotype identification problems have traditionally been solved using ampelography, ampelometry and chemical traits analysis. However, these tools have resulted in several false attributions, in particular when used at the clonal level. The availability of relatively cheap, reliable and reproducible tools to identify genetic differences at the clonal level would greatly facilitate the work of clonal patenting. In this work, 24 accessions of ‘Traminer’ cultivars were characterized using molecular markers. Three different approaches were applied: simple sequence repeats (SSR), amplified fragment length polymorphism (AFLP) and methyl-sensitive amplified length polymorphism (MSAP). Results showed that SSRs were not a powerful tool for clonal distinction. In contrast, the AFLP technique was able to distinguish 16 out of the 24 cultivars, even though the average similarity was high (97.1%). The MSAP technique was used to evaluate qualitative differences in the degree of DNA methylation among clones. Results suggest that morphological differences among clones are probably due to the synergetic effect of genetic and epigenetic modifications, and that clonal identification could be greatly improved using molecular tools such as AFLP and MSAP.

Bortezomib is the first proteasome inhibitor with significant antineoplastic activity for the treatment of relapsed/refractory multiple myeloma as well as other hematological and solid neoplasms. Peripheral neurological complications manifesting with paresthesias, burning sensations, dysesthesias, numbness, sensory loss, reduced proprioception and vibratory sensitivity are among the major limiting side effects associated with bortezomib therapy. Although bortezomib-induced painful peripheral neuropathy is clinically easy to diagnose and reliable models are available, its pathophysiology remains partly unclear. In this study we used well-characterized immune-competent and immune-compromised mouse models of bortezomib-induced painful peripheral neuropathy. To characterize the drug-induced pathological changes in the peripheral nervous system, we examined the involvement of spinal cord neuronal function in the development of neuropathic pain and investigated the relevance of the immune response in painful peripheral neuropathy induced by bortezomib. We found that bortezomib treatment induced morphological changes in the spinal cord, dorsal roots, dorsal root ganglia (DRG) and peripheral nerves. Neurophysiological abnormalities and specific functional alterations in Aδ and C fibers were also observed in peripheral nerve fibers. Mice developed mechanical allodynia and functional abnormalities of wide dynamic range neurons in the dorsal horn of spinal cord. Bortezomib induced increased expression of the neuronal stress marker activating transcription factor-3 in most DRG. Moreover, the immunodeficient animals treated with bortezomib developed a painful peripheral neuropathy with the same features observed in the immunocompetent mice. In conclusion, this study extends the knowledge of the sites of damage induced in the nervous system by bortezomib administration. Moreover, a selective functional vulnerability of peripheral nerve fiber subpopulations was found as well as a change in the electrical activity of wide dynamic range neurons of dorsal horn of spinal cord. Finally, the immune response is not a key factor in the development of morphological and functional damage induced by bortezomib in the peripheral nervous system.

Abstract 1432 Poster Board I-455 Whether a stochastic or hierarchical model best reflects the identity of leukemia initiating cell is still an open debate. In the case of acute lymphoblastic leukemia (ALL) little is known about the identity and the origin of leukemic stem cells (LSCs). In this study we focused on infant ALL carrying the t(4;11)/MLL-AF4, the most frequent and aggressive form of leukemia occurring within the first year of life. Using fluorescence-activated cell sorting (FACS) combined with fluorescence in situ hybridization (FISH) analysis, in vivo repopulating assay and clonal analysis, we investigated whether different BM subsets, sorted from infant ALL diagnostic samples on the bases of their CD34/CD19/NG2 phenotype, have similar or distinct LSC properties. Our studies demonstrate that in t(4;11)/MLL-AF4 infant ALL the 34+19- hematopoietic stem/progenitor cell pool is intact (MLL-AF4 negative by FISH), and that the LSC is not CD19-, as CD19- cells lacked repopulating ability (CD34-) or gave rise exclusively to normal multilineage reconstitution (CD34+). In contrast, CD19+ LSCs rapidly engraft and establish leukemia in NOD/SCID mice, with features recapitulating the human disease, and possess self-renewal potential in serial transplantation, regardless of CD34 and NG2 expression. However, although multiple leukemic subsets possess some “stemness”, we did observe differences in the kinetics and immunophenotypes of engraftment between phenotypically distinct LSC subpopulations, suggesting that in t(4;11)/MLL-AF4 infant ALL the LSCs are diverse but distinct. These findings might reconcile conflicting results with regard to previously proposed stochastic versus hierarchical models for LSCs. By designing patient-specific primers and probes on the junctional regions, we analyzed by RQ-PCR the clonal contribution to the leukemic grafts and found that, as in infant patients, ALL grafts in mice were oligoclonal, as several independent clones were able to sustain engraftment in recipient mice. However, among the engrafting clones, we observed different behaviours: either major or minor clones in the patient dominate in the graft; conversely some other clones which were predominant in the diagnostic sample failed to sustain leukemia in vivo. Specifically we observed that dominant clones in primary recipients reconstituted with leukemia can either persist over time or extinguish (or become dormant) in serial passages; while other clones, which were quiescent in primary recipents, never-the-less persisted, reactivated and became dominating in secondary recipients. In conclusion we provided evidences that LSC in infant ALL with t(4;11) is restricted to the CD19+ fraction and, among this, multiple phenotypically distinct BM subsets can initiate leukemia in NOD/SCID mice, with different kinetics and immunophenotypes of engraftment. Moreover, our clonal analysis results point out a further heterogeneity within purified LSC subsets and implicate the co-existence and competition between multiple clones. Disclosures No relevant conflicts of interest to declare.

// Valeria Cazzaniga 1 , Cristina Bugarin 1 , Michela Bardini 1 , Marco Giordan 2 , Geertruy te Kronnie 2 , Giuseppe Basso 2 , Andrea Biondi 1 , Grazia Fazio 1, * , Giovanni Cazzaniga 1, * 1 Centro Ricerca Tettamanti, Clinica Pediatrica, Universita di Milano-Bicocca, Ospedale San Gerardo/Fondazione MBBM, Monza 20900, Italy 2 Laboratory of Oncohematology, Department of Women’s and Children’s Health, University of Padova, Padova 35128, Italy * These authors contributed equally to this work Correspondence to: Giovanni Cazzaniga, e-mail: gianni.cazzaniga@hsgerardo.org Andrea Biondi, e-mail: abiondi.unimib@gmail.com Keywords: PAX5, ETV6, LCK, STAT5, BCP-ALL Abbreviations: BCP-ALL, B cell precursors acute lymphoblastic leukemia; wt, wild type; BCR, B cell receptor; IL7, interleukin 7; IL7R, IL7 receptor Received: August 14, 2014 Accepted: November 25, 2014 Published: January 08, 2015 ABSTRACT The PAX5 gene is altered in 30% of BCP-ALL patients and PAX5 chromosomal translocations account for 2–3% of cases. Although PAX5 fusion genes significantly affect the transcription of PAX5 target genes, their role in sustaining leukemia cell survival is poorly understood. In an in vitro model of PAX5/ETV6 leukemia, we demonstrated that Lck hyper-activation, and down-regulation of its negative regulator Csk, lead to STAT5 hyper-activation and consequently to the up-regulation of the downstream effectors, cMyc and Ccnd2 . More important, cells from PAX5 translocated patients show LCK up-regulation and over-activation, as well as STAT5 hyper-phosphorylation, compared to PAX5 wt and PAX5 deleted cases. As in BCR/ABL1 positive ALL, the hyper-activation of STAT5 pathway can represent a survival signal in PAX5 translocated cells, alternative to the pre-BCR, which is down-regulated. The LCK inhibitor BIBF1120 selectively reverts this phenomenon both in the murine model and in leukemic primary cells. LCK inhibitor could therefore represent a suitable candidate drug to target this subgroup of ALL patients.