scholarly journals Molecular Genetics of Pre-B Acute Lymphoblastic Leukemia Sister Cell Lines during Disease Progression

2021 ◽  
Vol 43 (3) ◽  
pp. 2147-2156
Author(s):  
Hilmar Quentmeier ◽  
Claudia Pommerenke ◽  
Hans G. Drexler
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Cell Lines ◽  
Tumor Suppressor Genes ◽  
Common Ancestor ◽  
Lymphoblastic Leukemia ◽  
Point Mutations ◽  
Genetic Alterations ◽  
Sister Cell ◽  
Dna And Rna ◽  
Clonal Development

For many years, immortalized tumor cell lines have been used as reliable tools to understand the function of oncogenes and tumor suppressor genes. Today, we know that tumors can comprise subclones with common and with subclone-specific genetic alterations. We sequenced DNA and RNA of sequential sister cell lines obtained from patients with pre-B acute lymphoblastic leukemia at different phases of the disease. All five pairs of cell lines carry alterations that are typical for this disease: loss of tumor suppressors (CDKN2A, CDKN2B), expression of fusion genes (ETV6-RUNX1, BCR-ABL1, MEF2D-BCL9) or of genes targeted by point mutations (KRAS A146T, NRAS G12C, PAX5 R38H). MEF2D-BCL9 and PAX R38H mutations in cell lines have hitherto been undescribed, suggesting that YCUB-4 (MEF2D-BCL9), PC-53 (PAX R38H) and their sister cell lines will be useful models to elucidate the function of these genes. All aberrations mentioned above occur in both sister cell lines, demonstrating that the sisters derive from a common ancestor. However, we also found mutations that are specific for one sister cell line only, pointing to individual subclones of the primary tumor as originating cells. Our data show that sequential sister cell lines can be used to study the clonal development of tumors and to elucidate the function of common and clone-specific mutations.

The Wee1 Inhibitor, MK-1775, Sensitizes Leukemic Cells to Different Antineoplastic Drugs Interfering with DNA Damage Response Pathway

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1276-1276 ◽  
Author(s):  
Andrea Ghelli Luserna Di Rora ◽  
Ilaria Iacobucci ◽  
Neil Beeharry ◽  
Simona Soverini ◽  
Cristina Papayannidis ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Acute Lymphoblastic Leukemia ◽  
Cell Viability ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Genetic Alterations ◽  
Additive Effect ◽  
Proliferation Index ◽  
Induction Of Apoptosis

Abstract Due to inadequate treatments, the survival rate of adult Acute Lymphoblastic Leukemia (ALL) patients with the exclusion of patients with particular genetic alterations, like the Philadelphia positive patients, is still very low. Moreover even the rate of patient that responds to specific treatment develops relapses during their life. Thus there is a need to improve the efficacy of conventional therapy and to discover novel specific targets. In eukaryotic cells Wee1, ATR/Chk1 and ATM/Chk2 are three pathways involved in cell cycle regulation, DNA damages response and DNA repair. Wee1 is a checkpoint kinase, involved mainly in the regulation of G2/M transition through the inhibitory phosphorylation of both Cyclin-dependent kinase 1 (CDK1) and 2 (CDK2) respectively. This study evaluates the effectiveness of MK-1775, a selective Wee1 inhibitor, as a monotherapy and as chemosensitizer agent for the treatment of B-/T-Acute Lymphoblastic Leukemia. Human B (BV-173, SUPB-15, NALM-6, NALM-19 and REH) and T (MOLT-4, RPMI-8402 and CEM) ALL cell lines were tested in this study. MK-1775 alone strongly reduced the cell viability in a dose and time-dependent manner in all the cell lines treated. The anti-proliferative activity of MK-1775 was accompanied by an increase in apoptotic cells (AnnexinV/Pi staining) and by DNA damage markers (gH2AX and Parp-1 cleavage). Moreover the inhibition of Wee1 disrupted the cell cycle profile by arresting the cells in late S and in G2/M phase. We hypothesized that targeting Chk1, a kinase upstream, of Wee1, would be more effective in reducing cell proliferation. Indeed, the concomitant inhibition of Chk1 and Wee1 kinases, using the PF-0477736 in combination with MK-1775, synergized in the reduction of the cell viability, inhibition of the proliferation index and induction of apoptosis. Moreover the immunofluorescence staining for the DNA damage marker gH2AX and the mitotic marker phosphor-Histone H3 showed that co-treatment with MK-1775 and PF-0477736 induced cell death by mitotic catastrophe. We undertook further studies to understand the immediate clinical potential of the compound, thus MK-1775 was combined with different drugs (Clofarabine, Bosutinib Authentic, and a particular isomer of this compound).The combination between MK-1775 and clofarabine showed an additive effect in terms of reduction of the cell viability and induction of apoptosis. Finally the Wee1 inhibitor was combined with the tyrosine kinase inhibitors Bosutinib and Bos-isomer (Bos-I). Both the isomers in combination with MK-1775 showed an additive effect in term of reduction of the cell viability. Interestedly the cytotoxic effect of Bos-I was stronger on the Philadelphia-negative cell lines in comparison to the positive counterpart. Western blot analysis highlighted that this compound, but not the Bosutinib authentic, interfered with the Chk1/Chk2 and Wee1 pathway. This supported our previous studies showing that Bosutinib and its isomer possess off-target effects against both Wee1 and Chk1 kinases and thus maybe used as a chemosensitizer (Beeharry et al. Cell Cycle 2014). The results of this study in our opinion identify the Wee1 kinase as a promising target for the treatment of ALL not only as a monotherapy but also as chemosensitizer agent to increase the cytotoxicity of different kind of drugs already used in clinical trials. Disclosures Soverini: Novartis, Briston-Myers Squibb, ARIAD: Consultancy. Martinelli:Novartis: Consultancy, Speakers Bureau; Ariad: Consultancy; AMGEN: Consultancy; ROCHE: Consultancy; BMS: Consultancy, Speakers Bureau; MSD: Consultancy; Pfizer: Consultancy.

scholarly journals Comprehensive Custom NGS Panel Validation for the Improvement of the Stratification of B-Acute Lymphoblastic Leukemia Patients

2020 ◽  
Vol 10 (3) ◽  
pp. 137
Author(s):  
Adrián Montaño ◽  
Jesús Hernández-Sánchez ◽  
Maribel Forero-Castro ◽  
María Matorra-Miguel ◽  
Eva Lumbreras ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Wide Spectrum ◽  
Lymphoblastic Leukemia ◽  
Point Mutations ◽  
Genetic Alterations ◽  
Copy Number Variations ◽  
Single Step ◽  
Fusion Genes ◽  
Single Nucleotide Variants ◽  
Single Nucleotide

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.

scholarly journals Predicting interactome network perturbations in human cancer: application to gene fusions in acute lymphoblastic leukemia

2014 ◽  
Vol 25 (24) ◽  
pp. 3973-3985 ◽  
Author(s):  
Leon Juvenal Hajingabo ◽  
Sarah Daakour ◽  
Maud Martin ◽  
Reinhard Grausenburger ◽  
Renate Panzer-Grümayer ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acute Lymphoblastic Leukemia ◽  
Human Cancer ◽  
Lymphoblastic Leukemia ◽  
Point Mutations ◽  
Genetic Alterations ◽  
Gene Fusions ◽  
Novel Approach ◽  
Interactome Network ◽  
Cell Acute Lymphoblastic Leukemia

Genomic variations such as point mutations and gene fusions are directly or indirectly associated with human diseases. They are recognized as diagnostic, prognostic markers and therapeutic targets. However, predicting the functional effect of these genetic alterations beyond affected genes and their products is challenging because diseased phenotypes are likely dependent of complex molecular interaction networks. Using as models three different chromosomal translocations—ETV6-RUNX1 (TEL-AML1), BCR-ABL1, and TCF3-PBX1 (E2A-PBX1)—frequently found in precursor-B-cell acute lymphoblastic leukemia (preB-ALL), we develop an approach to extract perturbed molecular interactions from gene expression changes. We show that the MYC and JunD transcriptional circuits are specifically deregulated after ETV6-RUNX1 and TCF3-PBX1 gene fusions, respectively. We also identified the bulk mRNA NXF1-dependent machinery as a direct target for the TCF3-PBX1 fusion protein. Through a novel approach combining gene expression and interactome data analysis, we provide new insight into TCF3-PBX1 and ETV6-RUNX1 acute lymphoblastic leukemia.

Refinement of IKZF1 Genomic Status in Pediatric Philadelphia Positive Acute Lymphoblastic Leukemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3785-3785
Author(s):  
Tobia Lana ◽  
Paola de Lorenzo ◽  
Silvia Bresolin ◽  
Ilaria Bronzini ◽  
Monique L. Den Boer ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Point Mutations ◽  
Genetic Alterations ◽  
Dominant Negative Effect ◽  
Prognostic Impact ◽  
Control Analysis ◽  
Frameshift Mutations ◽  
The Impact

Abstract Background: IKZF1 deletions are the most frequent secondary genetic alterations with prognostic impact in pediatric Philadelphia-positive B-cell precursor acute lymphoblastic leukemia (Ph+ BCP-ALL). Studies of IKZF1 status have mainly focused on deletions of the gene whereas very little is known about other genetic alterations in the IKZF1 locus. Aim: Using an amplicon deep sequencing approach to investigate IKZF1 mutations occurrence in pediatric Ph+ BCP-ALL reported as IKZF1wildtype (WT) for deletions by MLPA analysis. Methods: Six European centres participated in this study with a total of 98 patients.The 454 GS Junior system (Roche) was used to sequence the coding regions of IKZF1, Exon1 - Exon7 (Mullighan C. et al, 2009). An average depth of 250 reads (minimum of 102 reads, maximum of 853 reads) per amplicon was achieved. Quality control analysis excluded variants coverage of <50 reads in both forward and reverse directions. Variant analyses were performed using Amplicon Variant Analyzer (AVA) software (Roche). Variants at intronic regions or in homopolymeric stretches were excluded from further analysis, and a cut-off ≥10% mutated reads was applied to define mutations. Finally, variants described as SNPs or silent mutations were excluded. Results: In total, 882 amplicons were screened obtaining 337 variants. Of these, 14 variants corresponding to mutations with predicted deleterious impact on the function of IKZF1 passed the filters of our analysis pipeline. Twelve out of 98 (12%) IKZF1 non-deleted patients were mutated and 2 of them carried double mutations. Both cases showed different mutant allele frequencies of the 2 mutations, suggesting the presence of leukemic sub-populations with different IKZF1 status. Mutations can be subdivided in 2 categories: 5 miss-sense point mutations localized at the DNA binding domain with a dominant negative effect and 7 frameshift mutations leading to haploinsufficiency and impairing also the dimerization activity of IKZF1. The 5 point mutations were located in exon4 and 4 out of 5 were localized in the coding region of zinc finger 2 (ZF2), essential for DNA-protein interaction. Three patients carried the same c.698A>G substitution, that caused the change of Asparagine 159 into Serine. The 7 frameshift mutations (2 deletions, 4 insertions and 1 InDel) were identified all along the IKZF1locus. These nucleotide variations caused a shift in the reading frame and a consequent formation of premature stop codons depleting the C-terminal dimerization domain. Six out of 12 patients carrying IKZF1 mutations experienced an event (either relapse or death in CCR): 6 patients were treated in the pre-TKI (tyrosine kinase inhibitor) era and had 5 events while the remaining 6 patients received imatinib and showed a more favourable outcome with only one patient who died in CCR. This trend is in line with the findings recently reported by Van der Veer et al. (2014). In this study on IKZF1 deletions were identified in 126 of 191 patients, while 65 were defined IKZF1 WT on the basis of deletions analysis alone. Our study revealed that 6 of these 65 IKZF1 WT patients carried IKZF1 mutations (3 in the pre-TKI and 3 in the EsPhALL cohort). The outcome of mutated patients and deleted patients taken together (IKZF1 aberrant) is consistent with results reported in the Van der Veer study, both for the pre-TKI cohort [(4-year disease free survival (DFS) was 28.1% [SE 6.4] for IKZF1 aberrant vs 64.3% [SE 9.7] for IKZF1 WT, P=0.0036); Van der Veer study (30.0% [SE 6.8] for IKZF1 deleted vs 57.5% [SE 9.4] for IKZF1 WT, P=0.013)] and for the EsPhALL cohort [(4-year DSF was 55.7% [SE 6.8] for IKZF1 aberrant vs 59.8% [SE 11.6] for IKZF1 WT (P=0.348); Van der Veer study (4-year DFS was 53.6 [SE 7.0] for IKZF1 deleted vs 63.1 [SE 11.0] for IKZF1WT, P=0.1676]. Conclusion: This work revealed IKZF1 mutations in >10% of Ph+ BCP-ALL pediatric patients previously classified as IKZF1 WT on the basis of deletions analysis. Our results, together with data of IKZF1 deletions, highlight a 70-75% incidence of IKZF1 aberrations in pediatric Ph+ patients. Exon4 carried 9 of 14 aberrations: 7 point mutations and 2 deletions, suggesting a hotspot region for somatic mutations acquisition. The outcome of mutated patients is in line with the data previously published on the impact of IKZF1 deletions in pre and post TKI cohorts, suggesting a similar impact of mutations and deletions on clinical outcome of pediatric Ph+ patients. Disclosures No relevant conflicts of interest to declare.

Restraining the Proliferation of Acute Lymphoblastic Leukemia Cells by Genistein through Up-regulation of B-cell Translocation Gene-3 at Transcription Level

2019 ◽  
Vol 8 ◽  
Author(s):  
Masoumeh Abedi Nejad ◽  
Mohsen Nikbakht ◽  
Masoomeh Afsa ◽  
Kianoosh Malekzadeh
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
B Cell ◽  
Cancer Cells ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Suppressor Gene ◽  
Transcription Level ◽  
Hematopoietic Malignancy ◽  
Proliferative Effect ◽  
Cancer Agent

Background: Acute lymphoblastic leukemia (ALL) is a highly prevalent pediatric cancer accounting for approximately 78% of leukemia cases in patients younger than 15 years old. Different studies have demonstrated that B-cell translocation gene 3 (BTG3) plays a suppressive role in the progress of different cancers. Genistein is considered a natural and biocompatible compound and a new anti-cancer agent. In this study, we evaluate the effect of genistein on BTG3 expression and proliferation of ALL cancer cells. Materials and Methods: ALL cell lines (MOLT4, MOLT17, and JURKAT) were cultured in standard conditions. Cytotoxicity of genistein was detected using MTT assay. The cells were treated with different concentrations of genistein (10, 25, 40, and 55μM) for 24, 48, and 72 hours, and then cell viability and growth rate were measured. The quantitative real-time polymerase chain reaction was applied to investigate the effect of genistein on BTG3 expression. Results: The percentage of vital cells treated with genistein significantly decreased compared to the non-treated cells, showed an inverse relationship with an increasing genistein concentration. The present study suggests a dose of 40μM for genistein as a potent anticancer effect. Genistein could elevate BTG3 for 1.7 folds in MOLT4 and JURKAT and 2.7 folds in MOLT17 cell lines at transcription level conveged with 60 to 90% reduction in the proliferation rate of cancer cells. Conclusion: Up-regulation of BTG3 as a tumor suppressor gene can be induced by genistein. It seems that BTG3 reactivation can be introduced as another mechanism of anti-proliferative effect of genistein and could be considered as a retardant agent candidate against hematopoietic malignancy.[GMJ. 2019;inpress:e1229]

scholarly journals Mutational patterns and clonal evolution from diagnosis to relapse in pediatric acute lymphoblastic leukemia

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Shumaila Sayyab ◽  
Anders Lundmark ◽  
Malin Larsson ◽  
Markus Ringnér ◽  
Sara Nystedt ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Large Scale ◽  
Somatic Mutations ◽  
Lymphoblastic Leukemia ◽  
Clonal Evolution ◽  
Point Mutations ◽  
Driver Genes ◽  
Protein Coding ◽  
Pediatric Acute Lymphoblastic Leukemia ◽  
Evolutionary Trajectories

AbstractThe mechanisms driving clonal heterogeneity and evolution in relapsed pediatric acute lymphoblastic leukemia (ALL) are not fully understood. We performed whole genome sequencing of samples collected at diagnosis, relapse(s) and remission from 29 Nordic patients. Somatic point mutations and large-scale structural variants were called using individually matched remission samples as controls, and allelic expression of the mutations was assessed in ALL cells using RNA-sequencing. We observed an increased burden of somatic mutations at relapse, compared to diagnosis, and at second relapse compared to first relapse. In addition to 29 known ALL driver genes, of which nine genes carried recurrent protein-coding mutations in our sample set, we identified putative non-protein coding mutations in regulatory regions of seven additional genes that have not previously been described in ALL. Cluster analysis of hundreds of somatic mutations per sample revealed three distinct evolutionary trajectories during ALL progression from diagnosis to relapse. The evolutionary trajectories provide insight into the mutational mechanisms leading relapse in ALL and could offer biomarkers for improved risk prediction in individual patients.

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