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MSH6 Haploinsufficiency at Relapse Contributes to the Development of Thiopurine Resistance in Pediatric B-Lymphoblastic Leukemia

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MSH6 Haploinsufficiency at Relapse Contributes to the Development of Thiopurine Resistance in Pediatric B-Lymphoblastic Leukemia View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Copenhagen University Research Information System MSH6 haploinsufficiency at relapse contributes to the development of thiopurine resistance in pediatric B-lymphoblastic leukemia Evensen, Nikki A.; Madhusoodhan, P. Pallavi; Meyer, Julia; Saliba, Jason; Chowdhury, Ashfiyah; Araten, David J.; Nersting, Jacob; Bhatla, Teena; Vincent, Tiffaney L.; Teachey, David; Hunger, Stephen P.; Yang, Jun; Schmiegelow, Kjeld; Carroll, William L. Published in: Haematologica DOI: 10.3324/haematol.2017.176362 Publication date: 2018 Document version Publisher's PDF, also known as Version of record Document license: CC BY-NC Citation for published version (APA): Evensen, N. A., Madhusoodhan, P. P., Meyer, J., Saliba, J., Chowdhury, A., Araten, D. J., ... Carroll, W. L. (2018). MSH6 haploinsufficiency at relapse contributes to the development of thiopurine resistance in pediatric B-lymphoblastic leukemia. Haematologica, 103(5), 830-839. https://doi.org/10.3324/haematol.2017.176362 Download date: 09. Apr. 2020 ARTICLE Acute Lymphoblastic Leukemia MSH6 haploinsufficiency at relapse Ferrata Storti contributes to the development of thiopurine Foundation resistance in pediatric B-lymphoblastic leukemia Nikki A. Evensen,1 P. Pallavi Madhusoodhan,1 Julia Meyer,2 Jason Saliba,1 Ashfiyah Chowdhury,1 David J. Araten,3 Jacob Nersting,4 Teena Bhatla,1 Tiffaney L. Vincent,5 David Teachey,5 Stephen P. Hunger,5 Jun Yang,6 Haematologica 2018 Kjeld Schmiegelow4 and William L. Carroll1 Volume 103(5):830-839 1Departments of Pediatrics and Pathology, Perlmutter Cancer Center, NYU-Langone Medical Center, New York, NY, USA; 2Huntsman Cancer Institute, University of Utah Medical Center, Salt Lake City, USA; 3Department of Medicine, Perlmutter Cancer Center, NYU-Langone Medical Center, New York NY, USA; 4Department of Pediatrics and Adolescent Medicine, The University Hospital Rigshospitalet, Copenhagen, Denmark; 5Department of Pediatrics and the Center for Childhood Cancer Research, Children’s Hospital of Philadelphia and The Perelman School of Medicine at The University of Pennsylvania, Philadelphia, PA, USA and 6St. Jude Children’s Research Hospital, Memphis, TN, USA ABSTRACT urvival of children with relapsed acute lymphoblastic leukemia is poor, and understanding mechanisms underlying resistance is Sessential to developing new therapy. Relapse-specific heterozygous deletions in MSH6, a crucial part of DNA mismatch repair, are frequent- ly detected. Our aim was to determine whether MSH6 deletion results in a hypermutator phenotype associated with generation of secondary mutations involved in drug resistance, or if it leads to a failure to initiate apoptosis directly in response to chemotherapeutic agents. We knocked down MSH6 in mismatch repair proficient cell lines (697 and UOCB1) Correspondence: and showed significant increases in IC50s to 6-thioguanine and 6-mer- [email protected] captopurine (697: 26- and 9-fold; UOCB1: 5- and 8-fold) in vitro, as well as increased resistance to 6-mercaptopurine treatmentin vivo. No shift in IC50 was observed in deficient cells (Reh and RS4;11). 697 MSH6 knock- down resulted in increased DNA thioguanine nucleotide levels com- Received: July 13, 2017. pared to non-targeted cells (3070vs. 1722 fmol/μg DNA) with no differ- Accepted: February 7, 2018. ence observed in mismatch repair deficient cells. Loss of MSH6 did not Pre-published: February 15, 2018. give rise to microsatellite instability in cell lines or clinical samples, nor did it significantly increase mutation rate, but rather resulted in a defect in cell cycle arrest upon thiopurine exposure. MSH6 knockdown cells doi:10.3324/haematol.2017.176362 showed minimal activation of checkpoint regulator CHK1, γH2AX (DNA damage marker) and p53 levels upon treatment with thiopurines, Check the online version for the most updated consistent with intrinsic chemoresistance due to failure to recognize information on this article, online supplements, thioguanine nucleotide mismatching and initiate mismatch repair. and information on authorship & disclosures: Aberrant MSH6 adds to the list of alterations/mutations associated with www.haematologica.org/content/103/5/830 acquired resistance to purine analogs emphasizing the importance of thiopurine therapy. ©2018 Ferrata Storti Foundation Material published in Haematologica is covered by copyright. All rights are reserved to the Ferrata Storti Foundation. Use of Introduction published material is allowed under the following terms and conditions: https://creativecommons.org/licenses/by-nc/4.0/legalcode. Relapsed B-precursor acute lymphoblastic leukemia (B-ALL) is a leading cause of Copies of published material are allowed for personal or inter- cancer mortality amongst children. Development of chemoresistance is a crucial nal use. Sharing published material for non-commercial pur- factor contributing to relapse, therefore understanding the biological mechanisms poses is subject to the following conditions: https://creativecommons.org/licenses/by-nc/4.0/legalcode, underlying this resistance is imperative for discovering innovative treatment strate- 1 sect. 3. Reproducing and sharing published material for com- gies. Recent work has begun to highlight the direct role of relapse mercial purposes is not allowed without permission in writing specific/enriched genetic alterations in the emergence of clones that have gained a from the publisher. selective advantage under the pressure of specific chemotherapeutics, such as NT5C2, TBL1XR1, PRPS1, and CREBBP.1-5 Many of these mutations cause resist- ance specifically to thiopurines, which are the backbone of maintenance therapy 830 haematologica | 2018; 103(5) MSH6 haploinsufficiency contributes to resistance and have proven vital for achieving cures.6 Our analysis of RPMI1640 medium. HEK293T (ATCC) cells were grown in copy number alterations (CNAs) in diagnosis/relapse pairs DMEM medium. All media were supplemented with 10% FBS, revealed a relapse specific hemizygous deletion on chro- 1% penicillin/streptomycin under 5% CO2 at 37°C. mosome 2p16.3 involving MSH6 in 4-10% of patients.7,8 MutS homolog 6 (MSH6) is a major component of the Drug preparation, viral preparation, immunoblotting, mismatch repair (MMR) system, which is a highly con- apoptosis assays, and cell cycle served biological process that recognizes and repairs Standard protocols were followed and have been previously errors in nascent DNA strands during replication to main- described.3,21 More detailed information is provided in the Online tain genomic integrity. Initial recognition of replicative Supplementary Appendix. errors is carried out by protein heterodimers consisting of either MSH6 and MSH2 (hMutSa), or MSH3 and MSH2 Patients’ samples (hMutSβ). Upon recognition of a mismatch, hMutSa Cryopreserved pediatric B-ALL specimens were obtained from recruits MutLa (MLH1-PMS2) which engages down- the Children's Oncology Group (COG) ALL cell bank. All patients stream proteins and enzymes involved in DNA repair.9,10 were treated on COG protocols for newly diagnosed ALL. All sub- Constitutional defects in MMR, including monoallelic jects provided consent for banking and future research use of these mutations in Lynch syndrome and biallelic loss in consti- specimens in accordance with the regulations of the institutional tutional mismatch repair deficiency (CMMRD), are review boards of all participating institutions. strongly linked to carcinogenesis, where loss of MMR functionality causes increased mutability and predisposi- Microsatellite instability analysis tion to malignancy.11-15 Previous work has linked defects Microsatellite instability (MSI) analysis was performed using in MMR to drug resistance, including thiopurines, in var- MSI Analysis System, v.1.2 (Promega, Madison, WI, USA) follow- ious cancers.16-19 However, it is uncertain if resistance in ing the manufacturer’s protocol. Detailed information is provided MMR defective clones occurs through the acquisition of in the Online Supplementary Appendix. secondary mutations as a consequence of mutagenic ther- apy, or the outgrowth of clones that have intrinsic drug Measurement of mutation rate resistance. Our lab previously demonstrated that lower Spontaneous mutation rate was measured using a flow cytom- expression of MSH6 in patient samples was associated etry assay previously described by Araten et al.22 that detects the with increased ex vivo resistance to 6-mercaptopurine and presence of numerous glycosylphosphatidylinositol-linked (GPI) prednisone,7 highlighting the clinical importance of under- membrane proteins (see Online Supplementary Appendix). Briefly, standing the role of this genetic alteration in B-ALL. GPI(+) isolated clones from the NT and MSH6-KD cell lines were The mechanism of action of thiopurines is based upon expanded either untreated or treated with 6-TG (0.040 μg/mL and the insertion of a false nucleotide, namely a thioguanine 0.100 μg/mL, respectively, based on IC50 values determined for (TGN), into DNA that when thiomethylated pairs with a clones). Cells were then stained for GPI-dependent markers thymine instead of a cytosine.18 Cytotoxicity is thought to including FLAER-Alexa 488 (Pinewood), CD48, CD52, and CD59 be dependent on the MMR machinery recognizing the (Serotec),23 and analyzed by flow cytometry. The mutant frequen- mismatch and attempting to match the TGN on the cy (f) was calculated as the number of GPI(-) events divided by the parental
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