scholarly journals Mutational Landscape, Clonal Evolution Patterns and Role of RAS Mutations in Relapsed Acute Lymphoblastic Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4068-4068
Author(s):  
Koichi Oshima ◽  
Hossein Khiabanian ◽  
Ana Carolina da Silva Almeida ◽  
Gannie Tzoneva ◽  
Francesco Abate ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Disease Progression ◽  
Lymphoblastic Leukemia ◽  
Clonal Evolution ◽  
Wild Type ◽  
Ras Mutations ◽  
Recurrent Mutations ◽  
Glucocorticoid Receptor Gene ◽  
Pediatric All

Abstract Acute Lymphoblastic Leukemia (ALL) is the most common malignancy in children. Altogether 90% of pediatric ALL patients achieve a complete hematologic remission with current high dose combination chemotherapy and 80% of them remain leukemia free. However, the outcome for patients showing refractory disease or those whose leukemia relapses after an initial transient response remains disappointingly poor with cure rates of less than 40%. To investigate genetic drivers of relapse and resistance and explore the specific roles of clonal evolution in disease progression and relapse here we performed whole-exome sequence analysis of matched diagnosis, germline (remission) and relapse DNA samples in a panel of 55 pediatric ALL patients including 33 T-cell ALLs and 22 B-cell precursor ALLs. These analyses identified an average of 9 mutations present in diagnostic samples and 17 mutations in relapsed leukemia DNAs. Phylogenetic tree analysis for each of the 48 cases with optimal variant call parameters analyzing their clonal evolution dynamics during disease progression, combined with whole genome sequencing of targeted samples with low exonic mutation input, showed that branched evolution in which relapse clones contain some, but not all genetic lesions present in the major clone at diagnosis as the primary mechanism driving tumor progression and relapse present in 45/48 (94%) cases. In addition, and consistent with previous reports we identified the presence of chemotherapy associated mutations in NT5C2 (10/55), TP53 (3/55), CREBBP (4/55) and the NR3C1 glucocorticoid receptor gene (2/55). However, and most strikingly, 23/27 (85%) recurrently mutated genes in this series with mutations preferentially selected or retained at the time of relapse (mutation never lost in the relapse clone) were not implicated in relapse ALL before (HTR3A, MED12, USP9X, CACNA1H, ODZ3, AACS, SAMD4A, ANO5, PAPPA, NAALADL2, HIST3H2A, FZD7, TBX15, NEB, GREB1L, PLXNA4, SGK223, TSC1, PTPRG, FGF10, SYCP2, TRPM3 and EYS). A branched pattern of genetic evolution and the presence of recurrent mutations selected at relapse support that chemotherapy imposes a strong Darwinian genetic selection in leukemic cell populations. In this context it is worth noting that RAS-MAPK pathway activating mutations in NRAS, KRAS and PTPN11 were present in 24/55 (44%) cases in our series. Interestingly, some leukemias showed retention or emergence of RAS mutant clones at relapse, while in others, RAS mutant clones present at diagnosis were replaced by RAS wild type populations, supporting a role for both positive and negative selection evolutionary pressures in clonal evolution of RAS-mutant leukemia. Most notably, and in agreement with this hypothesis, inducible expression of mutant KRAS in human ALL lines demonstrate that oncogenic KRAS G12D induces methotrexate resistance, but also improves leukemia response to vincristine; a phenotype perfectly recapitulated in a isogenic ALL leukemia model generated from a conditional inducible Kras G12D knockin mice. Mechanistically, KRAS G12 expression induces MAPK dependent abrogation of methotrexate induced apoptosis. Moreover, Kras mutant tumors show enhanced G2/M cell cycle arrest and apoptosis upon spindle poisoning with vincristine, a phenotype linked with increased PLK phosphorylation and transcriptional down-regulation of mitotic genes. Finally clonal competition assays demonstrate that the differential response to methotrexate and vincristine in isogenic Kras wild type and Kras mutant ALL cells results in clonal dominance of Kras G12D populations in cultures treated with methotrexate, while Kras wild type cells are selected the context of vincristine treatment. In all these results show novel insight on the genetics and mechanisms of clonal selection, disease progression and relapse in ALL and demonstrate a previously unrecognized dual role of RAS mutations in chemotherapy response. Disclosures Loh: Abbvie: Research Funding; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees.

scholarly journals Mutational landscape, clonal evolution patterns, and role of RAS mutations in relapsed acute lymphoblastic leukemia

2016 ◽  
Vol 113 (40) ◽  
pp. 11306-11311 ◽  
Author(s):  
Koichi Oshima ◽  
Hossein Khiabanian ◽  
Ana C. da Silva-Almeida ◽  
Gannie Tzoneva ◽  
Francesco Abate ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Mapk Pathway ◽  
Lymphoblastic Leukemia ◽  
Clonal Evolution ◽  
Wild Type ◽  
Ras Mutations ◽  
Viral Oncogene ◽  
Mutational Landscape ◽  
Viral Oncogene Homolog

Although multiagent combination chemotherapy is curative in a significant fraction of childhood acute lymphoblastic leukemia (ALL) patients, 20% of cases relapse and most die because of chemorefractory disease. Here we used whole-exome and whole-genome sequencing to analyze the mutational landscape at relapse in pediatric ALL cases. These analyses identified numerous relapse-associated mutated genes intertwined in chemotherapy resistance-related protein complexes. In this context, RAS-MAPK pathway-activating mutations in the neuroblastoma RAS viral oncogene homolog (NRAS), kirsten rat sarcoma viral oncogene homolog (KRAS), and protein tyrosine phosphatase, nonreceptor type 11 (PTPN11) genes were present in 24 of 55 (44%) cases in our series. Interestingly, some leukemias showed retention or emergence of RAS mutant clones at relapse, whereas in others RAS mutant clones present at diagnosis were replaced by RAS wild-type populations, supporting a role for both positive and negative selection evolutionary pressures in clonal evolution of RAS-mutant leukemia. Consistently, functional dissection of mouse and human wild-type and mutant RAS isogenic leukemia cells demonstrated induction of methotrexate resistance but also improved the response to vincristine in mutant RAS-expressing lymphoblasts. These results highlight the central role of chemotherapy-driven selection as a central mechanism of leukemia clonal evolution in relapsed ALL, and demonstrate a previously unrecognized dual role of RAS mutations as drivers of both sensitivity and resistance to chemotherapy.

Relapsed Childhood High Hyperdiploid Acute Lymphoblastic Leukemia: Genome-Wide Screening Reveals the Presence of Preleukemic Ancestral Clones and the Secondary Nature of Microdeletions and RTK-RAS Mutations.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2591-2591
Author(s):  
Josef Davidsson ◽  
Kajsa Paulsson ◽  
David Lindgren ◽  
Henrik Lilljebjörn ◽  
Tracy Chaplin ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Structural Changes ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Clonal Evolution ◽  
Snp Array ◽  
Prognostic Impact ◽  
Genetic Changes ◽  
Ras Mutations ◽  
Paired Samples

Abstract Abstract 2591 Poster Board II-567 Although childhood high hyperdiploid acute lymphoblastic leukemia is associated with a favorable outcome, 20% relapse. This makes it important to identify these patients already at diagnosis to ensure proper risk-stratification. To identify changes associated with relapse and ascertain the genetic evolution patterns, SNP array and mutation analyses of FLT3, KRAS, NRAS, and PTPN11 were performed on 11 paired diagnostic/relapse samples. The “triples trisomies” +4, +10, and +17 were detected in 64%, a frequency similar to the one generally observed at diagnosis, thus questioning their favorable prognostic impact. Structural changes, mainly cryptic hemizygous deletions, were significantly more common at relapse (P<0.05). No single aberration was linked to relapse, but four deletions, involving IKZF1, PAX5, CDKN2A/B or AK3, were recurrent. Based on the genetic relationship between the paired samples, three groups were delineated: 1) identical genetic changes at diagnosis and relapse (18%), 2) clonal evolution with all changes at diagnosis being present at relapse (18%), and 3) clonal evolution with some changes conserved, lost or gained (64%), suggesting the presence of a preleukemic clone. This ancestral clone was characterized by numerical changes only, with structural changes and RTK-RAS mutations being secondary to the high hyperdiploid pattern and perhaps necessary for overt leukemia. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Clinical Implications of Inflammation in Acute Myeloid Leukemia

2021 ◽  
Vol 11 ◽  
Author(s):  
Christian Récher
Keyword(s):  
Acute Myeloid Leukemia ◽  
Disease Progression ◽  
Myeloid Leukemia ◽  
Kinase Inhibitors ◽  
Lymphoblastic Leukemia ◽  
Clonal Evolution ◽  
Leukemic Cells ◽  
Prognostic Impact ◽  
Acute Myeloid

Recent advances in the description of the tumor microenvironment of acute myeloid leukemia, including the comprehensive analysis of the leukemic stem cell niche and clonal evolution, indicate that inflammation may play a major role in many aspects of acute myeloid leukemia (AML) such as disease progression, chemoresistance, and myelosuppression. Studies on the mechanisms of resistance to chemotherapy or tyrosine kinase inhibitors along with high-throughput drug screening have underpinned the potential role of glucocorticoids in this disease classically described as steroid-resistant in contrast to acute lymphoblastic leukemia. Moreover, some mutated oncogenes such as RUNX1, NPM1, or SRSF2 transcriptionally modulate cell state in a manner that primes leukemic cells for glucocorticoid sensitivity. In clinical practice, inflammatory markers such as serum ferritin or IL-6 have a strong prognostic impact and may directly affect disease progression, whereas interesting preliminary data suggested that dexamethasone may improve the outcome for AML patients with a high white blood cell count, which paves the way to develop prospective clinical trials that evaluate the role of glucocorticoids in AML.

scholarly journals IKZF1 deletions in pediatric acute lymphoblastic leukemia: still a poor prognostic marker?

Blood ◽  
2020 ◽  
Vol 135 (4) ◽  
pp. 252-260 ◽  
Author(s):  
Martin Stanulla ◽  
Hélène Cavé ◽  
Anthony V. Moorman
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Perceived Risk ◽  
Lymphoblastic Leukemia ◽  
Treatment Strategies ◽  
Response To Treatment ◽  
Primary Therapy ◽  
Disease Biology ◽  
Pediatric All ◽  
Shed Light

Abstract Improved personalized adjustment of primary therapy to the perceived risk of relapse by using new prognostic markers for treatment stratification may be beneficial to patients with acute lymphoblastic leukemia (ALL). Here, we review the advances that have shed light on the role of IKZF1 aberration as prognostic factor in pediatric ALL and summarize emerging concepts in this field. Continued research on the interplay of disease biology with exposure and response to treatment will be key to further improve treatment strategies.

Clonal Evolution Underlying the Progression from Myelodysplastic Syndromes to Ph-Positive Acute Lymphoblastic Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5514-5514
Author(s):  
Masataka Taguchi ◽  
Tomoko Kohno ◽  
Hiroyuki Mishima ◽  
Hiroaki Taniguchi ◽  
Takeharu Kato ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Acute Lymphoblastic Leukemia ◽  
Myelodysplastic Syndromes ◽  
Copy Number ◽  
Lymphoblastic Leukemia ◽  
Clonal Evolution ◽  
Intron 1

Abstract Introduction: Myelodysplastic syndromes (MDS) are considered as a "stem cell disorders", in which hematopoietic stem cells and lineage-committed progenitor cells acquire genetic and epigenetic alterations and provide aberrant, clonal hematopoiesis, sometimes resulted in the progression to acute myeloid leukemia (Elias HK et al, Oncogene 2014). We previously reported a rare case of which the patient developed Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ALL) 2.5 years after being diagnosed with MDS (Kohno T et al, Br J Haematol 1996). p190 BCR-ABL1 mRNA was detected in the Ph+ALL cells. Metaphase cytogenetics showed the karyotypes: 46, XY, 20q- in MDS phase and 46, XY, t(9;22)(q34;q11), 20q- in ALL phase, indicating that MDS and Ph+ALL in this patient were of the same clonal origin. To uncover the detail of the clonal evolution, we analyzed bone marrow samples of MDS and Ph+ALL in this patient by targeted massively parallel sequencing with a panel of 154 genes including known driver genes of hematologic malignancies. Methods: Genomic DNAs (gDNAs) were extracted from the bone marrow mononuclear cells of MDS and Ph+ALL in this patient. Targeted sequencing was performed on the Illumina HiSeq2500 platform. Single nucleotide variants (SNVs) and small insertions and deletions (INDELs) were called using HaplotypeCaller of Genome Analysis Toolkit (GATK) version 3.4-46. We also attempted to detect the breakpoint of BCR-ABL1 translocation from the targeted sequencing data using the computational method, BreaKmer (Abo RP et al, Nucleic Acids Research 2015). The candidates of the mutations and structural variations were validated by amplicon-based deep sequencing and Sanger sequencing. Copy number variations were analyzed using Affymetrix CytoScan HD Array. Results: The mutations in ASXL1 and U2AF1 genes were identified in the MDS sample with variant allele frequencies (VAFs) of about 45%. At the progression of Ph+ALL, the mutations in SETBP1, SMC1A, and SLC5A8 genes were newly acquired while the ASXL1 and U2AF1 mutations were also identified with the same level of VAFs (about 50%) as the other mutations. VAFs of all of the mutations were decreased to about 20% after the chemotherapy for Ph+ALL, and then increased to about 40% at the recurrence of the disease. Furthermore, we identified the breakpoint of BCR-ABL1 translocation at intron 1 of ABL1 genes and intron 1 of BCR genes, that is the well-known cluster region, m-bcr, only among the samples of Ph+ALL. Copy number analysis confirmed that both MDS and Ph+ALL samples harbored the deletion of chromosome 20q. And the deletion of IKZF1 gene, which is frequently identified in Ph+ALL cases (Mullighan CG et al, Nature 2008), was not identified during the progression from MDS to Ph+ALL. These results demonstrated that the MDS clone harboring 20q- and ASXL1 and U2AF1 mutations acquired the mutations in SETBP1, SMC1A, and SLC5A8 genes and the p190 BCR-ABL1, resulted in the development of Ph+ALL in this patient. Conclusion: The alterations of SETBP1, SMC1A, and SLC5A8 genes are usually reported in myeloid malignancies (Makishima H et al, Nat Genet 2013, Kon A et al, Nat Genet 2013, Whitman SP et al, Blood 2008). Previous study in transgenic mouse demonstrated the distinct role of p190 BCR-ABL1 in the development of an ALL (Voncken JW et al, Blood 1995). Recapitulating this scenario, p190 BCR-ABL1 may play a critical role in the development of Ph+ALL from the MDS stem cells in this patient. This study may provide a new insight into the stem cell origin of MDS and the role of p190 BCR-ABL1 in the development of Ph+ALL. Disclosures No relevant conflicts of interest to declare.

scholarly journals Clinical Significance of Ck2 (CSNK2) and C-Myc Expression in Childhood Acute Lymphoblastic Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5269-5269
Author(s):  
Paola Bonaccorso ◽  
Manuela La Rosa ◽  
Nellina Andriano ◽  
Valeria Iachelli ◽  
Emanuela Cannata ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Conflicts Of Interest ◽  
Residual Disease ◽  
Lymphoblastic Leukemia ◽  
Flow Analysis ◽  
State Level ◽  
Potential Candidate ◽  
Wild Type ◽  
Significant Difference ◽  
Pediatric All

Abstract Background. Acute lymphoblastic leukemia (ALL) is the most common malignancy of childhood and a major cause of childhood cancer-related mortality. Although the cure rate now approaches 90%, certain pediatric ALL subgroups present subsequent relapse. For this reason, analyses of cell signaling pathways will help to identify new markers and/or targets for tailored therapy. PI3K/AKT/mTOR activation is frequently found in both B-ALL and T-ALL. Protein kinase Ck2 (CSNK2) activity in pediatric ALL was increased and its inhibition restored PTEN phosphatase activity with subsequent inactivation of AKT. Moreover, Ck2 may serve the activity of oncogenes such as BCR-ABL and c-MYC, control the activation of other critical signaling cascades (JAK-STAT), and sustain multiple cellular stress-elicited pathway such as the proteotoxic stress, unfolded protein and DNA-damage responses. Ck2 has also been shown to have an essential role in tuning signals derived from the stromal tumor microenvironment (Piazza F et al, Oncogene2016). Material and Methods. We analyzed cDNA collected from 46 patients with B-ALL [19 High Risk (HR) for Minimal Residual Disease (MRD) and 27 NON-HR] and 25 with T-ALL (8 HR and 17 NON-HR), respectively, diagnosed in our Center from 2000 to 2012. The latter subgroup was screened fro PTEN-Exon7 mutations and TXL3 rearrangements. We evaluated the gene expression of Ck2 and c-Myc genes using RQ-PCR with Sybr-Green and a relative quantification method (ΔΔCt method), comparing gene's expression from patients with samples from 6 healty donors (HDs). In order to demonstrate the correlation between genetic alteration and signaling transduction, specifically in HR patients , we analyzed some phosphoproteins by Phospho-flow approach. We profiled 5 proteins (STAT3, STAT5, CREB, PTEN and pS6) in 4 T-ALL cases (3 with PTEN-Exon 7 mutation). Results. We observed a significant difference of Ck2 expression in T-ALL NON-HR patients vs HDs (Mean Ck2 Fold-Changes 3.494 vs 1.17, p=0.0315) and in T-ALL HR patients (6.384 vs 1.17, p=0.0219) vs HDs (Fig 1A and B). Comparing NON-HR vs HR cases, we found a statistically significant difference (p<0.0001) (Fig 1C). c-Myc mean expression was very similar between the two T-ALL subgroups. Moreover, among T-ALL cases, we identified 5 patients with PTEN-Exon7 mutations and 6 with TLX3 rearrangements. We observed that cases with PTEN-Exon7 mutation showed lower c-Myc expression than cases with PTEN-Exon7 wild-type (mean c-Myc 8.550 vs 1.920) whereas patients with TLX3 rearrangements showed higher c-Myc expression than TLX3 negative (mean c-Myc 18.260 vs 5.502) (p<0.005) (Fig 2A and B). We did not observe any correlation between these rearrangements and Ck2 expression. We also performed Ck2 and c-Myc expression analysis in B-ALL (NON-HR and HR) subgroups. We surprisingly observed a Ck2 overexpression in both NON-HR and HR B-ALLs compared to HDs. On the other side, we did not observed significant difference about c-Myc expression in cases with B-ALL vs HDs; whilst we observed an overexpression of c-Myc in HR vs NON-HR patients with B-ALL (mean 7.075 vs 2.095, respectively)(p<0.0004). Phospho-Flow analysis, in 3 cases with PTEN-Exon7 mutation (1 Ck2+/normal Myc, 1 normal Ck2/normal Myc, 1 Ck2-/normal Myc) showed PTEN null, very lower pS6 basal level and higher CREB basal level than in case with PTEN-Exon7 wild-type. Moreover, we observed that the latter patient, presented with a TLX3 rearrangements (Ck2+/Myc+) with higher c-Myc expression, showed higher STAT3 basal state level confirming that STAT3 induces the expression of c-Myc. Conclusions. Based on our preliminary findings, Ck2 could be considered as a marker and /or a potential candidate for targeted therapy, specifically in HR-ALL, as confirmed by the use of CK2 inhibitor (CX-4945) in ongoing clinical trials. c-Myc overexpression confirmed its association with HR features. The potential role as markers of both genes needs to be demonstrated in a larger population study. Combined application of genomic and phosphoproteomic strategies will lead us to better profile diagnostic samples of HR-ALL, addressing future tailored treatments. Disclosures No relevant conflicts of interest to declare.

Expression and Effect of CTCF in Pediatric Acute Lymphoblastic Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4298-4298
Author(s):  
Lin Zhu ◽  
Han Zhang ◽  
Xiao Liu ◽  
Chaohao Du ◽  
Shanshan Zhu ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Health System ◽  
Disease Progression ◽  
Cell Apoptosis ◽  
Research Funding ◽  
Lymphoblastic Leukemia ◽  
Paired Samples ◽  
Pediatric All ◽  
High Level ◽  
Lymphoblastic Cells

Abstract Abstract 4298 Acute lymphoblastic leukemia (ALL) is the most frequent-occurring malignant neoplasm in children, but the pathogenesis of the disease remains unclear. In a microarray assay using samples from 100 Chinese children with ALL, CTCF was found to be up-regulated. DNA-binding nuclear protein CTCF (encoded by CTCF gene) is a highly conserved zinc finger protein involved in multiple cellular processes including transcriptional activation/repression, insulation, imprinting and × chromosome inactivation. It has been shown to be associated with cell apoptosis and differentiation in tumors; however, the biological function of CTCF in pediatric ALL is presently unknown. To investigate the expression features of CTCF in pediatric ALL cells, matched newly diagnosis (ND), complete remission (CR) and relapse (RE) bone marrow samples from 24 patients were collected. Ten ND-CR paired samples (n=20) were selected to detect the mRNA levels of CTCF by Q-PCR. Besides, the protein levels of CTCF at different stages of disease progression were measured by western blot in all patients (20 ND-CR paired samples, n=40; 4 ND-CR-RE matched samples, n=12). To further explore the role of CTCF in the pathogenesis of leukemia, the potential effect of CTCF on the cell apoptosis in lymphoblastic cells was investigated by flow cytometry. We identified significant up-regulation of CTCF in the ND samples. Importantly, the expression of CTCF returned to normal level after CR, but rebounded in the RE samples. Knock-down of CTCF resulted in nearly 3–fold and 15–fold increases in early and late apoptosis of leukemic cells respectively, which indicated that CTCF is an anti-apoptotic factor and plays an anti-apoptotic role in lymphoblastic cells. Our results indicate that CTCF may represent a promising indicator of disease progression as well as reflecting the ongoing therapeutic effects of treatment. Furthermore, CTCF serves as an anti-apoptotic factor and potentially contributes to leukemogenesis in pediatric ALL patients. Disclosures: Zhang: Beijing Health System High-level Technical Personel Plan: Research Funding. Zheng:Beijing Health System High-level Technical Personel Plan: Research Funding.

scholarly journals Expression and Regulation of Bcl-2, Bcl-xl, and Bax Correlate With p53 Status and Sensitivity to Apoptosis in Childhood Acute Lymphoblastic Leukemia

Blood ◽  
1997 ◽  
Vol 89 (8) ◽  
pp. 2986-2993 ◽  
Author(s):  
Harry W. Findley ◽  
Lubing Gu ◽  
Andrew M. Yeager ◽  
Muxiang Zhou
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Lymphoblastic Leukemia ◽  
Mutant P53 ◽  
Wild Type ◽  
High Level Expression ◽  
Associated Proteins ◽  
Pediatric All ◽  
P53 Status ◽  
Induced Apoptosis ◽  
High Level

Abstract Bcl-2 and its homologue, Bcl-xl, encode membrane-associated proteins that protect neoplastic cells from DNA damage-induced apoptosis, whereas Bax is a Bcl-2 antagonist that promotes cell death. In the present study, we examined the expression and regulation of these genes at both the mRNA and protein level in 22 pediatric acute lymphoblastic leukemia (ALL) cell lines, as well as their sensitivity to apoptosis after exposure to ionizing radiation (IR). Eleven of 22 lines expressed wild-type (wt) p53, 4 expressed mutant p53, and 7 did not express p53 (p53-null). Nine of 22 (41%) lines expressed Bcl-2; of these, 8 were wt-p53+ and 1 expressed mutant p53. Bcl-2 was not expressed in any p53-null lines. In contrast, all 22 lines were positive for Bcl-xl and Bax, although expression level varied. Treatment with IR (10 Gy) induced both downregulation of Bcl-2 and upregulation of Bax at 2 to 5 hours post-IR in 5 of 8 (63%) wt-p53+ lines, leading to apoptosis. Conversely, lines that failed to both downregulate Bcl-2 and upregulate Bax after IR were resistant to apoptosis. Although levels of Bcl-xl expression varied among the 22 lines, high levels of Bcl-xl were observed in 5 of 7 (71%) p53− lines. There were no obvious changes in the expression of Bcl-xl in these lines after IR. However, among the p53-null lines, resistance to IR was observed only in those expressing high levels of Bcl-xl. These results suggest that expression of Bcl-2 but not Bcl-xl is p53-dependent and that IR-induced downregulation of Bcl-2 and upregulation of Bax occur in most wt-p53+ lines and are associated with radiosensitivity. Furthermore, high-level expression of Bcl-xl occurs predominantly in p53-null lines and is associated with resistance to IR-induced apoptosis in these lines, indicating differential expression and regulation of Bcl-2 and Bcl-xl in pediatric ALL.

scholarly journals Mechanisms of NT5C2 mutations Driving Thiopurine Resistance in Acute Lymphoblastic Leukemia

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 715-715
Author(s):  
Chelsea Dieck ◽  
Gannie Tzoneva ◽  
Farhad Forouhar ◽  
Zachary Carpenter ◽  
Alberto Ambesi-Impiombato ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Lymphoblastic Leukemia ◽  
Chemotherapy Resistance ◽  
Mechanisms Of Action ◽  
Terminal Segment ◽  
Full Length ◽  
Wild Type ◽  
Mutant Proteins ◽  
Solid Spheres

Abstract Relapse acute lymphoblastic leukemia (ALL) is associated with chemotherapy resistance and poor prognosis. Persistent leukemia initiating cells with increased self-renewal capacity, clonal heterogeneity and selection of resistance-driving genetic alterations have been proposed as drivers of leukemia relapse. Gain of function mutations in the 5'-Nucleotidase, Cytosolic II (NT5C2) gene are selectively present in relapsed ALL and confer resistance to chemotherapy with 6-mercaptopurine (6-MP). Yet, the specific mechanisms mediating constitutive activation of NT5C2 remain unknown. Here we performed detailed genetic, enzymatic, structural and functional analyses of relapsed leukemia-associated NT5C2 mutant alleles uncovering previously unrecognized functional heterogeneity in their mechanisms of action. Specifically, detailed nucleotidase assays in the presence of increased concentration of allosteric activators across 10 relapse-associated NT5C2 mutant proteins and analyses of the crystal structures of 7 distinct mutant proteins and wild type NT5C2 in the presence and absence of allosteric factors defined three classes of NT5C2 mutants with distinct mechanisms of action. Class I NT5C2 mutations force an active configuration of the NT5C2 catalytic center in the absence of allosteric activator. In contrast, class II NT5C2 mutations disrupt a novel switch-off mechanism mediated by the dynamic mobilization of the tip region of the arm segment responsible for returning the enzyme to its basal inactive configuration following allosteric activation. Finally, increased allosteric response in the class III p.Gln523* truncating mutation results from loss of the C-terminal acidic tail of NT5C2, which functions as a lock stabilizing the enzyme in its inactive basal configuration. The proposed role of the arm domain region and the C-terminal segment were verified in enzymatic assays incubating wild type NT5C2 with antibodies recognizing these regulatory elements, which phenocopied the increased enzymatic activity and response to allosteric effector of NT5C2 mutations involving these domains. In all, these results identify distinct molecular mechanisms mediating NT5C2 regulation targeted by activating NT5C2 mutant alleles and serve as a framework for the functional annotation of clinically-relevant NT5C2 mutations in relapsed ALL. Finally, the prominent role of the allosteric binding site as critical mediator of the mechanisms mediating thhe increased nucleotidase activity of NT5C2 mutant proteins point to this region as a preferred target for the design of NT5C2 inhibitors for the reversal of chemotherapy resistance in relapsed ALL. Figure 1 Relapse associated NT5C2 mutations and crystal structure of basal and activated wild type human NT5C2. (a) Graphical representation of relapse associated mutations in B-ALL (black) and T-ALL (red).(b-d) In vitro nucleotidase assays assessing the enzymatic activity of (b)class I, (c) class II, and (d)class IIINT5C2 mutations in the presence of increased concentrations of ATP. (e) A Ribbon diagram of the basal structure of wild type NT5C2-X537, displaying the HAD domain (cyan) and its three extensions: N-terminal segment (orange), arm (pink), and C-terminal segment (marine). The mutation sites are depicted as red solid spheres for Cα of each mutated amino acid. The phosphate (yellow for phosphorus) and Mg ions (dark green) are depicted as stick models and solid spheres respectively. (f) A Ribbon diagram of the activated structure of the full-length wild type NT5C2, in which the allosteric helix A (αA) is shown in dark purple. (g,h) Ribbon and surface (for subunit B) depictions of basal and activated dimers of (g) NT5C2-X537 and the (h) full-length, respectively. (i,j) Ribbon diagrams of the basal (i) NT5C2-X537 and activated (j) full-length wild type tetramers, respectively. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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