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Expression of NT5C2 mutations in ALL cells induces resistance to chemotherapy with 6-MP and 6-TG. (a) Viability assays in CCRF-CEM and CUTLL1 T-ALL cells expressing wild-type NT5C2, relapse-associated mutant NT5C2 alleles or a red fluorescent protein (RFP) control and treated with increasing concentrations of 6-MP. (b) 6-TG dose-response cell-viability curves. Data are shown as the means ± s.d.
Source publication
Acute lymphoblastic leukemia (ALL) is an aggressive hematological tumor resulting from the malignant transformation of lymphoid progenitors. Despite intensive chemotherapy, 20% of pediatric and over 50% of adult ALL patients fail to achieve a complete remission or relapse after intensified chemotherapy, making disease relapse and resistance to ther...
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Citations
... 14 After achieving remission, T-ALL has high incidence of relapse with an Overall Survival (OS) of 50-60% in adult patients. 5 With the application of risk-adapted therapy and increased supportive care, the survival rate of ALL among children has increased from 57 to 92%, however in 20% of children, relapses can still occur 23 , which have also been associated with poor outcomes. The incidence of high-risk leukemia and relapse is higher in adults (40-50%) 24 as compared to children. ...
Objectives
To evaluate the outcomes (relapse and mortality rate) and response of the bone marrow in early stages after combination chemotherapy in patients with T-cell Acute Lymphoblastic Leukemia (T-ALL)
Methods
A descriptive cross-sectional study was conducted at King Fahad Medical City, from January 2021 to December 2022, to evaluate bone marrow findings at the time of diagnosis and post-chemotherapy in 26 patients diagnosed with T-ALL. The study included all patients diagnosed with T-ALL of any age group during the study period. The patients’ bone marrows were examined at 0 days of treatment (diagnosis work-up), followed by examination at day 15 post induction therapy, and day 30 after treatment.
Results
In this study, 26 cases of T-lymphoblastic leukemia were analyzed. The mean age at diagnosis was 15.69±14.28 years, and eight cases had central nervous system involvement. The majority of cases (88.5%) were positive for Cytoplasmic-CD3 and CD7. Positive findings by fluorescence in situ hybridization (FISH) were: T cell receptor (TCR) α/δ in 6 (23.1%) of the patients, CDNK2A/CEP9 in five (19.2%), and TRCB in one (3.8%). Examination of the bone marrow on day 15 revealed a decrease in blasts to ≤1% in nine patients, and to ≤1% in 19 patients on day 30 post-therapy. Relapse was recorded in five (19.23%) patients. Three (11.53%) patients did not survive during treatment, of which two were <10 years old. The relapse rate for T-ALL was 19.23%, with an overall survival rate of about 64%. The overall mortality rate was 11.53%.
Conclusion
The relapse rate for T-ALL in our study was approximately 19%, but the mortality rate was 11.5%. A substantial decrease in blast percentages was observed, suggesting a favorable initial reaction of the bone marrow to the combined chemotherapy. This suggests that the use of aggressive and more effective chemotherapy has led to better outcomes.
... Genomic analyses of relapsed ALL patients have revealed several somatic mutations acquired during chemotherapy that could cause drug resistance of leukemia cells. These include mutations in NT5C2 5,6 , which increases cell resistance to purine analogs, PRPS1/PRPS2 7 , FPGS 8 , NR3C1/NR3C2 9 , and CREBBP 10 , among others. However, these genomic aberrations could only be detected in a subset of relapsed tumors, and the mechanisms of drug resistance and relapse remain unknown for nearly half of such patients. ...
For around half of the pediatric B-lineage acute lymphoblastic leukemia (B-ALL) patients, the molecular mechanism of relapse remains unclear. To fill this gap in knowledge, here we characterize the chromatin accessibility landscape in pediatric relapsed B-ALL. We observe rewired accessible chromatin regions (ACRs) associated with transcription dysregulation in leukemia cells as compared with normal B-cell progenitors. We show that over a quarter of the ACRs in B-ALL are in quiescent regions with high heterogeneity among B-ALLs. We identify subtype-specific and allele-imbalanced chromatin accessibility by integrating multi-omics data. By characterizing the differential ACRs between diagnosis and relapse in B-ALL, we identify alterations in chromatin accessibility during drug treatment. Further analysis of ACRs associated with relapse free survival leads to the identification of a subgroup of B-ALL which show early relapse. These data provide an advanced and integrative portrait of the importance of chromatin accessibility alterations in tumorigenesis and drug responses.
... Owing to the application of risk-adapted therapy and improved supportive care, the five-year survival rate for pediatrics with ALL has considerably increased from 57 to 92% (Della et al., 2019). However, relapses still develop in 20%of children with ALL and are associated with a poor outcome (Tzoneva et al., 2013). A regular evaluation of treatment response gives a clue as to how responsive leukemic cells are to chemotherapy and how effective the treatment is all around. ...
Introduction:
Recently, the identification of minimal residual disease (MRD) that persists after chemotherapy has emerged as the most powerful tool in determining the prognosis of patients with acute lymphoblastic leukemia (ALL). Multiple methods to detect MRD exist, each with its own benefits and drawback. Multiparameter flow cytometry and quantitative polymerase chain reaction are the most commonly used methods of MRD detection in clinical practice.
Objective:
to evaluate the impact of minimal residual disease detection by Next Generation Flow Cytometry on Outcome of Egyptian Patients with Acute Lymphoblastic Leukemia.
Patients &methods:
The study conducted on 93 patients with recently diagnosed acute lymphoblastic leukemia. MRD detection was evaluated during follow up of patient (at End of induction EOI and End of consolidation EOC by next generation flow cytometry.
Results:
Out of 93 patients, 28 (30%) had positive MRD at EOI. Age, BCR-ABL, risk assessment, and relapse had a substantial impact on MRD at EOI (P <0.005). Fourteen patients (17.9%) at EOC were MRD positive; age, hemoglobin, blast count at diagnosis, BCR-ABL, risk stratification, relapse and overall survival showed significant association.
Conclusion:
Positive MRD was a major risk factor for predicting poor survival and relapse at both EOI and EOC by cox regression analysis.
... So, unsurprisingly, they are called into play in a highly repetitive or convergent fashion in the face of potentially lethal therapeutic challenge [81,82]. In the case of highly targeted drugs, the nature of the resistance mechanism repetitively selected in different patients mirrors the target specificity of the drug applied [79,83,84], similarly in resistance to immunotherapy via immunoediting [85], demonstrating the adaptive logic of resistance selection. As in earlier bacterial models, multiple parallel experiments with drug exposure of barcoded cancer cells in vitro [86] reveals the deterministic, repetitive nature of resistance. ...
Cancer cell populations evolve by a stepwise process involving natural selection of the fittest variants within a tissue ecosystem context and as modified by therapy. Genomic scrutiny of patient samples reveals an extraordinary diversity of mutational profiles both between patients with similar cancers and within the cancer cell population of individual patients. Does this signify highly divergent evolutionary trajectories or are there repetitive and predictable patterns?
Major evolutionary innovations or adaptations in different species are frequently repeated, or convergent, reflecting both common selective pressures and constraints on optimal solutions. We argue this is true of evolving cancer cells, especially with respect to the TP53 gene. Functional loss variants in TP53 are the most common genetic change in cancer. We discuss the likely microenvironmental selective pressures involved and the profound impact this has on cell fitness, evolvability and probability of subsequent drug resistance.
... We then considered more carefully the nature of other AS events contributing to this cluster (Table 1). Notably, they were related to resistance to all commonly used B-ALL therapeutics: glucocorticoids (NR3C1 (13)), thiopurines (PRPS1 (14) & NT5C2 (15,16), and anti-folates (FPGS (17,18)). In additional, they affected relapse genes associated with more general, not exclusively cancer cellintrinsic mechanisms. ...
... We assessed 5′-nucleotidase activity of purified recombinant wild-type and mutant NT5C2 proteins using the 5′-NT enzymatic test kit (Diazyme) according to the manufacturer's instructions, as described previously (15). We calculated 5′-nucleotidase activity levels using a (which was not certified by peer review) is the author/funder. ...
Relapse-specific mutations do not account for all chemotherapy failures in patients with B-cell acute lymphoblastic leukemia (B-ALL). By mining RNA-seq datasets of paired diagnostic/relapse pediatric B-ALL samples, we discovered pervasive aberrant splicing (AS) patterns linked with relapse. They affected drivers of resistance to glucocorticoids, anti-folates, and thiopurines. Most splicing variations represent exon skipping, “poison” exon inclusion, and intron retention, phenocopying well-documented loss-of-function mutations. In addition, relapse-associated AS of NT5C2 mRNA yields an isoform with a cryptic 24-nt in-frame exon 6a. Inclusion of the extra 8 amino acids into this enzyme results in elevated nucleosidase activity, a known consequence of gain-of-function mutations in NT5C2 and a common determinant of 6-mercaptopurine resistance. Furthermore, in B-ALL cells NT5C2ex6a and the R238W hotspot variant confers comparable levels of resistance to 6-mercaptopurine in vitro and in vivo . These results support a role for alternative splicing as a prevalent mechanism driving chemotherapy resistance in relapsed B-ALL.
Statement of significance
Mutations in chemoresistance genes are found in relapsed/refractory acute lymphoblastic leukemia. However, in this low-mutational-burden disease, up to 30% of cases have no known relapse-specific genetic alterations. Our identification of aberrant splicing as an alternative mechanism of acquired drug resistance fills this gap and suggests new opportunities for therapeutic interventions.
... NT5C2, as a nucleotidase, dephosphorylates and inactivates HGPRT, effectively inhibiting conversion of 6-MP into its active form and reducing its therapeutic effectiveness. [29] Consequently, leukemia cells with NT5C2 mutations develop resistance to 6-MP. Overcoming this resistance can be achieved either through direct targeting of NT5C2 or by the inhibition of compensatory pathways, which are notably active in cells with NT5C2 mutations. ...
Nucleoside-based drugs, recognized as purine or pyrimidine analogs, have been potent therapeutic agents since their introduction in 1950, deployed widely in the treatment of diverse diseases such as cancers, myelodysplastic syndromes, multiple sclerosis, and viral infections. These antimetabolites establish complex interactions with cellular molecular constituents, primarily via activation of phosphorylation cascades leading to consequential interactions with nucleic acids. However, the therapeutic efficacy of these agents is frequently compromised by the development of drug resistance, a continually emerging challenge in their clinical application. This comprehensive review explores the mechanisms of resistance to nucleoside-based drugs, encompassing a wide spectrum of phenomena from alterations in membrane transporters and activating kinases to changes in drug elimination strategies and DNA damage repair mechanisms. The critical analysis in this review underlines complex interactions of drug and cell and also guides towards novel therapeutic strategies to counteract resistance. The development of targeted therapies, novel nucleoside analogs, and synergistic drug combinations are promising approaches to restore tumor sensitivity and improve patient outcomes.
... Multi-agent chemotherapy is known to induce variable stress on leukaemic clones, leading to clonal evolution and the development of drug-resistant mutations in the genes related to chemotherapy-dose response (CREBBP, WHSC1, NT5C2, PRPS1, PRPS2, MSH2, MSH6, PMS2, NR3C1, NR3C2, Tp53 and FPGS). Such therapy-acquired somatic mutations in chemotherapy-dose-response genes drive >20% of early and late relapses [17][18][19][20][21][22][23]. However, Diagnostics 2023, 13, 884 2 of 15 most early relapses occur when the 'persister clone' escapes treatment and acquires a second hit, in one of the genes mentioned above, under chemotherapy stress, giving it a proliferative advantage. ...
... The NT5C2 relapse-specific clonal mutations, R363L and R367Q, noted in two of our samples were already been shown by Li et al. to be functionally resistant to 6-mercaptopurine [17]. Loss-of-function mutations in the DNA mismatch repair gene, PMS2, have also been linked with thiopurine response in different studies [19][20][21]. All major mutant clones of PMS2 noted at relapse were either nonsense or frameshift mutations, with one clone showing a high VAF, of 79%, later noted as having a loss of heterozygosity at Chr. 7p. ...
Methods:
Forty pediatric (0-12 years) B-ALL DNA samples (20 paired Diagnosis-Relapse) and an additional six B-ALL DNA samples (without relapse at 3 years post treatment), as the non-relapse arm, were retrieved from the biobank for advanced genomic analysis. Deep sequencing (1050-5000X; mean 1600X) was performed using a custom NGS panel of 74 genes incorporating unique molecular barcodes.
Results:
A total 47 major clones (>25% VAF) and 188 minor clones were noted in 40 cases after bioinformatic data filtering. Of the forty-seven major clones, eight (17%) were diagnosis-specific, seventeen (36%) were relapse-specific and 11 (23%) were shared. In the control arm, no pathogenic major clone was noted in any of the six samples. The most common clonal evolution pattern observed was therapy-acquired (TA), with 9/20 (45%), followed by M-M, with 5/20 (25%), m-M, with 4/20 (20%) and unclassified (UNC) 2/20 (10%). The TA clonal pattern was predominant in early relapses 7/12 (58%), with 71% (5/7) having major clonal mutations in the NT5C2 or PMS2 gene related to thiopurine-dose response. In addition, 60% (3/5) of these cases were preceded by an initial hit in the epigenetic regulator, KMT2D. Mutations in common relapse-enriched genes comprised 33% of the very early relapses, 50% of the early and 40% of the late relapses. Overall, 14/46 (30%) of the samples showed the hypermutation phenotype, of which the majority (50%) had a TA pattern of relapse.
Conclusions:
Our study highlights the high frequency of early relapses driven by TA clones, demonstrating the need to identify their early rise during chemotherapy by digital PCR.
... Previous reports showed that patients with ribosomal mutations have a higher risk to develop cancer in their life, moreover, in particular cancer the risk even up to 200-fold higher [16][17][18], and a variety of tumors were discovered somatic mutations in RPs. Accordingly, the tumor cells have mutations in ribosome-associated genes frequently, for example, heterozygous loss of RPL5 occurs in 30% of multiple myeloma, melanoma, glioblastoma and breast cancers [19][20][21][22], and 2% of T-ALL patients [23,24]. Furthermore, the oncogenes and loss of suppressor genes will enhance the activities of ribosome biogenesis [25,26]. ...
Background: The diffuse large B-cell lymphoma (DLBCL) is a heterogeneous lymphoma with a dismal outcome, due to approximately 40% patients will be relapsed or refractory to the standard therapy of rituximab plus cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP). Therefore, we need urgently to explore the approach to classify the risk of DLBCL patients accurately and accurately targeting therapy. The ribosome is a vital cellular organelle that is mainly responsible for translation mRNA into protein, moreover, more and more reports revealed that ribosome was associated with cellular proliferation and tumorigenesis. Therefore, our study aimed to construct a prognostic model of DLBCL patients using ribosome-related genes (RibGs).
Method: We screened differentially expressed RibGs between healthy donors' B cells and DLBCL patients' malignant B cells in GSE56315 dataset. Next, we performed analyses of univariate Cox regression, the least absolute shrinkage and selection operator (LASSO) regression and multivariate Cox regression analyses to establish the prognostic model consisting of 15 RibGs in GSE10846 training set. Then, we validated the model by a range of analyses including Cox regression, Kaplan-Meier survival, ROC curve, and nomogram in training and validation cohorts.
Results: The RibGs model showed a reliably predictive capability. We found the upregulated pathways in high-risk group most associated with innate immune reaction such as interferon response, complement and inflammatory responses. In addition, a nomogram including age, gender, IPI score and risk score was constructed to help explain the prognostic model. We also discovered the high-risk patients were more sensitive to some certain drugs. Finally, knocking out the NLE1 could inhibit the proliferation of DLBCL cell lines.
Conclusion: As far as we know, it is the first time to predict the prognosis of DLBCL using the RibGs and give a new sight for DLBCL treatment. Importantly, the RibGs model could be acted as a supplementary to the IPI in classifying the risk of DLBCL patients.
... Li et al., 2015;Meyer et al., 2013;Somazu et al., 2021). NT5C2 plays a crucial role in the de novo purine synthesis pathway, which hydrolyzes inosine 5'monophosphate (IMP) and other purine nucleotides (Dieck & Ferrando, 2019;Dieck et al., 2018;Hoell et al., 2019;Li et al., 2020;Meyer et al., 2013;Tzoneva et al., 2018;Tzoneva et al., 2013). R238W/L/G/Q, R367Q, D407A/Y/E/H, and R39Q are the most common mutations of the NT5C2 gene (Dieck & Ferrando, 2019;Dieck et al., 2018). ...
... In the case of NT5C2 and PRPS1 mutations, thiopurine-resistant phenotypes were confirmed in ALL cell lines transduced with mutated NT5C2 or PRPS1 cDNAs using lentiviral vectors (B. Li et al., 2015;Tzoneva et al., 2013). ...
... Considering the off-target effect of the CRISPR/Cas9 system, we selected the NT5C2-R39Q and PRPS1-S103N mutations with the highest off-target scores among hotspot mutations as candidates. In the clinical setting, the NT5C2-R39Q mutation was collectively reported in 5 cases (3.9%) out of 129 relapsed ALL cases with NT5C2 gene mutations (Dieck & Ferrando, 2019;Dieck et al., 2018;Hoell et al., 2019;Li et al., 2020;Meyer et al., 2013;Tzoneva et al., 2018;Tzoneva et al., 2013), while This article has not been copyedited and formatted. The final version may differ from this version. ...
6-Mercaptopurine (6-MP) is a key component in maintenance therapy for childhood acute lymphoblastic leukemia (ALL). Recent next-generation sequencing analysis of childhood ALL clarified the emergence of the relapse-specific mutations of the NT5C2 and PRPS1 genes, which are involved in thiopurine metabolism. In this scenario, minor clones of leukemia cells could acquire the 6-MP-resistant phenotype as a result of the NT5C2 or PRPS1 mutation during chemotherapy (including 6-MP treatment) and confer disease relapse after selective expansion. Thus, to establish new therapeutic modalities overcoming 6-MP resistance in relapsed ALL, human leukemia models with NT5C2 and PRPS1 mutations in the intrinsic genes are urgently required. Here, mimicking the initiation process of the above clinical course, we sought to induce two relapse-specific hotspot mutations (R39Q mutation of the NT5C2 gene and S103N mutation of the PRPS1 gene) into a human lymphoid leukemia cell line by homologous recombination (HR) using the CRISPR/Cas9 system. After 6-MP selection of the cells transfected with Cas9 combined with single-guide RNA and donor DNA templates specific for either of those two mutations, we obtained the sublines with the intended NT5C2-R39Q and PRPS1-S103N mutation as a result of HR. Moreover, diverse in-frame small insertion/deletions were also confirmed in the 6-MP-resistant sublines at the target sites of the NT5C2 and PRPS1 genes as a result of nonhomologous end joining. These sublines are useful for molecular pharmacological evaluation of the NT5C2 and PRPS1 gene mutations in the 6-MP sensitivity and development of therapy overcoming the thiopurine resistance of leukemia cells. SIGNIFICANCE STATEMENT: Mimicking the initiation process of relapse-specific mutations of the NT5C2 and PRPS1 genes in childhood acute lymphoblastic leukemia treated with 6-mercaptopurine (6-MP), this study sought to introduce NT5C2-R39Q and PRPS1-S103N mutations into a human lymphoid leukemia cell line by homologous recombination using the CRISPR/Cas9 system. In the resultant 6-MP-resistant sublines, the intended mutations and diverse in-frame small insertions/deletions were confirmed, indicating that the obtained sublines are useful for molecular pharmacological evaluation of the NT5C2 and PRPS1 gene mutations.
... Despite the progressively improved 5-year survival rate [5][6][7][8][9][10], about 20% of children with ALL still relapse [11,12], and in adults, the risk of relapse affects up to 50% of patients [13][14][15]. ...
Simple Summary
Minimal/measurable residual disease (MRD) monitoring is a powerful and independent predictor of outcomes in both children and adult acute lymphoblastic leukemia (ALL). MRD monitoring enables patients’ stratification into different risk-adapted treatment arms; it guides treatment decisions in clinical practice, including stem cell transplantation, and represents an early marker of impending relapse. Real-time quantitative PCR is the most widely used molecular method for MRD assessment, but there are some limitations that new approaches may overcome. In this review, we discuss the most recent technological advances in MRD monitoring that are allowing to increase the number of evaluable patients and the levels of quantification and also have the potential to study different disease compartments.
Abstract
Minimal/measurable residual disease (MRD) evaluation has resulted in a fundamental instrument to guide patient management in acute lymphoblastic leukemia (ALL). From a methodological standpoint, MRD is defined as any approach aimed at detecting and possibly quantifying residual neoplastic cells beyond the sensitivity level of cytomorphology. The molecular methods to study MRD in ALL are polymerase chain reaction (PCR) amplification-based approaches and are the most standardized techniques. However, there are some limitations, and emerging technologies, such as digital droplet PCR (ddPCR) and next-generation sequencing (NGS), seem to have advantages that could improve MRD analysis in ALL patients. Furthermore, other blood components, namely cell-free DNA (cfDNA), appear promising and are also being investigated for their potential role in monitoring tumor burden and response to treatment in hematologic malignancies. Based on the review of the literature and on our own data, we hereby discuss how emerging molecular technologies are helping to refine the molecular monitoring of MRD in ALL and may help to overcome some of the limitations of standard approaches, providing a benefit for the care of patients.
