http://hdl.handle.net/10366/132633 2026-05-04T00:24:00Z http://hdl.handle.net/10366/158982 [EN]BIRC3 is monoallelically deleted in up to 80% of chronic lymphocytic leukemia (CLL) cases harboring del(11q). In addition, truncating mutations in the remaining allele of this gene can lead to BIRC3 biallelic inactivation, which has been shown to be a marker for reduced survival in CLL. Nevertheless, the biological mechanisms by which these lesions could contribute to del(11q) CLL pathogenesis and progression are partially unexplored. We implemented the CRISPR/Cas9-editing system to generate isogenic CLL cell lines harboring del(11q) and/or BIRC3 mutations, modeling monoallelic and biallelic BIRC3 loss. Our results reveal that monoallelic BIRC3 deletion in del(11q) cells promotes non-canonical NF-κB signaling activation via RelB-p52 nuclear translocation, being these effects allelic dose-dependent and therefore further enhanced in del(11q) cells with biallelic BIRC3 loss. Moreover, we demonstrate ex vivo in primary cells that del(11q) cases including BIRC3 within their deleted region show evidence of non-canonical NF-κB activation which correlates with high BCL2 levels and enhanced sensitivity to venetoclax. Furthermore, our results show that BIRC3 mutations in del(11q) cells promote clonal advantage in vitro and accelerate leukemic progression in an in vivo xenograft model. Altogether, this work highlights the biological bases underlying disease progression of del(11q) CLL patients harboring BIRC3 deletion and mutation. 2021-01-01T00:00:00Z http://hdl.handle.net/10366/158976 2021-01-01T00:00:00Z http://hdl.handle.net/10366/158975 [EN]The deletion of 11q (del(11q)) invariably comprises ATM gene in chronic lymphocytic leukemia (CLL). Concomitant mutations in this gene in the remaining allele have been identified in 1/3 of CLL cases harboring del(11q), being the biallelic loss of ATM associated with adverse prognosis. Although the introduction of targeted BCR inhibition has significantly favored the outcomes of del(11q) patients, responses of patients harboring ATM functional loss through biallelic inactivation are unexplored, and the development of resistances to targeted therapies have been increasingly reported, urging the need to explore novel therapeutic approaches. Here, we generated isogenic CLL cell lines harboring del(11q) and ATM mutations through CRISPR/Cas9-based gene-editing. With these models, we uncovered a novel therapeutic vulnerability of del(11q)/ATM-mutated cells to dual BCR and PARP inhibition. Ex vivo studies in the presence of stromal stimulation on 38 CLL primary samples confirmed a synergistic action of the combination of olaparib and ibrutinib in del(11q)/ATM-mutated CLL patients. In addition, we showed that ibrutinib produced a homologous recombination repair impairment through RAD51 dysregulation, finding a synergistic link of both drugs in the DNA damage repair pathway. Our data provide a preclinical rationale for the use of this combination in CLL patients with this high-risk cytogenetic abnormality. 2020-01-01T00:00:00Z http://hdl.handle.net/10366/147481 [EN]The clonal basis of relapse in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is complex and not fully understood. Next-generation sequencing (NGS), array comparative genomic hybridization (aCGH), and multiplex ligation-dependent probe amplification (MLPA) were carried out in matched diagnosis–relapse samples from 13 BCP-ALL patients to identify patterns of genetic evolution that could account for the phenotypic changes associated with disease relapse. The integrative genomic analysis of aCGH, MLPA and NGS revealed that 100% of the BCP-ALL patients showed at least one genetic alteration at diagnosis and relapse. In addition, there was a significant increase in the frequency of chromosomal lesions at the time of relapse (p = 0.019). MLPA and aCGH techniques showed that IKZF1 was the most frequently deleted gene. TP53 was the most frequently mutated gene at relapse. Two TP53 mutations were detected only at relapse, whereas the three others showed an increase in their mutational burden at relapse. Clonal evolution patterns were heterogeneous, involving the acquisition, loss and maintenance of lesions at relapse. Therefore, this study provides additional evidence that BCP-ALL is a genetically dynamic disease with distinct genetic profiles at diagnosis and relapse. Integrative NGS, aCGH and MLPA analysis enables better molecular characterization of the genetic profile in BCP-ALL patients during the evolution from diagnosis to relapse 2020-01-01T00:00:00Z http://hdl.handle.net/10366/147480 [EN]Background: The t(12;21)(p13;q22), which fuses ETV6 and RUNX1 genes, is the most common genetic abnormality in children with B-cell precursor acute lymphoblastic leukaemia. The implication of the fusion protein in leukemogenesis seems to be clear. However, its role in the maintenance of the disease continues to be controversial. Methods: Generation of an in vitro ETV6/RUNX1 knock out model using the CRISPR/Cas9 gene editing system. Functional characterization by RNA sequencing, proliferation assays, apoptosis and pharmacologic studies, and generation of edited-cell xenograft model. Results: The expression of ETV6/RUNX1 fusion gene was completely eliminated, thus generating a powerful model on which to study the role of the fusion gene in leukemic cells. The loss of fusion gene expression led to the deregulation of biological processes affecting survival such as apoptosis resistance and cell proliferation capacity. Tumour cells showed higher levels of apoptosis, lower proliferation rate and a greater sensitivity to PI3K inhibitors in vitro along as a decrease in tumour growth in xenografts models after ETV6/RUNX1 fusion gene abrogation. Conclusions: ETV6/RUNX1 fusion protein seems to play an important role in the maintenance of the leukemic phenotype and could thus become a potential therapeutic target. 2020-01-01T00:00:00Z http://hdl.handle.net/10366/147479 [EN]Background: B-acute lymphoblastic leukemia (B-ALL) is a hematological neoplasm of the stem lymphoid cell of the B lineage, characterized by the presence of genetic alterations closely related to the course of the disease. The number of alterations identified in these patients grows as studies of the disease progress, but in clinical practice, the conventional techniques frequently used are only capable of detecting the most common alterations. However, techniques, such as next-generation sequencing (NGS), are being implemented to detect a wide spectrum of new alterations that also include point mutations. Methods: In this study, we designed and validated a comprehensive custom NGS panel to detect the main genetic alterations present in the disease in a single step. For this purpose, 75 B-ALL diagnosis samples from patients previously characterized by standard-of-care diagnostic techniques were sequenced. Results: The use of the custom NGS panel allowed the correct detection of the main genetic alterations present in B-ALL patients, including the presence of an aneuploid clone in 14 of the samples and some of the recurrent fusion genes in 35 of the samples. The panel was also able to successfully detect a number of secondary alterations, such as single nucleotide variants (SNVs) and copy number variations (CNVs) in 66 and 46 of the samples analyzed, respectively, allowing for further refinement of the stratification of patients. The custom NGS panel could also detect alterations with a high level of sensitivity and reproducibility when the findings obtained by NGS were compared with those obtained from other conventional techniques. Conclusions: The use of this custom NGS panel allows us to quickly and efficiently detect the main genetic alterations present in B-ALL patients in a single assay (SNVs and insertions/deletions (INDELs), recurrent fusion genes, CNVs, aneuploidies, and single nucleotide polymorphisms (SNPs) associated with pharmacogenetics). The application of this panel would thus allow us to speed up and simplify the molecular diagnosis of patients, helping patient stratification and management. 2020-01-01T00:00:00Z http://hdl.handle.net/10366/138196 2017-01-01T00:00:00Z http://hdl.handle.net/10366/138195 2018-01-01T00:00:00Z http://hdl.handle.net/10366/138194 2018-04-07T00:00:00Z http://hdl.handle.net/10366/138193 [EN]Background: In B-cell precursor acute lymphoblastic leukaemia (B-ALL), the identification of additional genetic alterations associated with poor prognosis is still of importance. We determined the frequency and prognostic impact of somatic mutations in children and adult cases with B-ALL treated with Spanish PETHEMA and SEHOP protocols. Methods: Mutational status of hotspot regions of TP53, JAK2, PAX5, LEF1, CRLF2 and IL7R genes was determined by next-generation deep sequencing in 340 B-ALL patients (211 children and 129 adults). The associations between mutation status and clinicopathological features at the time of diagnosis, treatment outcome and survival were assessed. Univariate and multivariate survival analyses were performed to identify independent prognostic factors associated with overall survival (OS), event-free survival (EFS) and relapse rate (RR). Results: A mutation rate of 12.4% was identified. The frequency of adult mutations was higher (20.2% vs 7.6%, P=0.001). TP53 was the most frequently mutated gene (4.1%), followed by JAK2 (3.8%), CRLF2 (2.9%), PAX5 (2.4%), LEF1 (0.6%) and IL7R (0.3%). All mutations were observed in B-ALL without ETV6-RUNX1 (P=0.047) or BCR-ABL1 fusions (P<0.0001). In children, TP53mut was associated with lower OS (5-year OS: 50% vs 86%, P=0.002) and EFS rates (5-year EFS: 50% vs 78.3%, P=0.009) and higher RR (5-year RR: 33.3% vs 18.6% P=0.037), and was independently associated with higher RR (hazard ratio (HR)=4.5; P=0.04). In adults, TP53mut was associated with a lower OS (5-year OS: 0% vs 43.3%, P=0.019) and a higher RR (5-year RR: 100% vs 61.4%, P=0.029), whereas JAK2mut was associated with a lower EFS (5-year EFS: 0% vs 30.6%, P=0.035) and a higher RR (5-year RR: 100% vs 60.4%, P=0.002). TP53mut was an independent risk factor for shorter OS (HR=2.3; P=0.035) and, together with JAK2mut, also were independent markers of poor prognosis for RR (TP53mut: HR=5.9; P=0.027 and JAK2mut: HR=5.6; P=0.036). Conclusions: TP53mut and JAK2mut are potential biomarkers associated with poor prognosis in B-ALL patients. 2017-05-01T00:00:00Z http://hdl.handle.net/10366/138183 [EN]CRISPR/Cas9 technology was used to abrogate p210 oncoprotein expression in the Boff-p210 cell line, a pro-B line derived from interlukin-3-dependent Baf/3, that shows IL-3-independence arising from the constitutive expression of BCR-ABL p210. Using this approach, pools of Boff-p210-edited cells and single edited cell-derived clones were obtained and functionally studied in vitro. The loss of p210 expression in Boff-p210 cells resulted in the loss of ability to grow in the absence of IL-3, as the Baf/3 parental line, showing significantly increased apoptosis levels. Notably, in a single edited cell-derived clone carrying a frame-shift mutation that prevents p210 oncoprotein expression, the effects were even more drastic, resulting in cell death. These edited cells were injected subcutaneously in immunosuppressed mice and tumor growth was followed for three weeks. BCR/ABL-edited cells developed smaller tumors than those originating from unedited Boff-p210 parental cells. Interestingly, the single edited cell-derived clone was unable to develop tumors, similar to what is observed with the parental Baf/3 cell line. CRISPR/Cas9 genomic editing technology allows the ablation of the BCR/ ABL fusion gene, causing an absence of oncoprotein expression, and blocking its tumorigenic effects in vitro and in the in vivo xenograft model of CML. The future application of this approach in in vivo models of CML will allow us to more accurately assess the value of CRISPR/Cas9 technology as a new therapeutic tool that overcomes resistance to the usual treatments for CML patients. 2017-04-08T00:00:00Z