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MSH6 haploinsufficiency contributes to resistance
and have proven vital for achieving cures.6 Our analysis of copy number alterations (CNAs) in diagnosis/relapse pairs revealed a relapse specific hemizygous deletion on chro- mosome 2p16.3 involving MSH6 in 4-10% of patients.7,8
MutS homolog 6 (MSH6) is a major component of the mismatch repair (MMR) system, which is a highly con- served biological process that recognizes and repairs errors in nascent DNA strands during replication to main- tain genomic integrity. Initial recognition of replicative errors is carried out by protein heterodimers consisting of either MSH6 and MSH2 (hMutSa), or MSH3 and MSH2 (hMutSβ). Upon recognition of a mismatch, hMutSa recruits MutLa (MLH1-PMS2) which engages down- stream proteins and enzymes involved in DNA repair.9,10
Constitutional defects in MMR, including monoallelic mutations in Lynch syndrome and biallelic loss in consti- tutional mismatch repair deficiency (CMMRD), are strongly linked to carcinogenesis, where loss of MMR functionality causes increased mutability and predisposi- tion to malignancy.11-15 Previous work has linked defects in MMR to drug resistance, including thiopurines, in var- ious cancers.16-19 However, it is uncertain if resistance in MMR defective clones occurs through the acquisition of secondary mutations as a consequence of mutagenic ther- apy, or the outgrowth of clones that have intrinsic drug resistance. Our lab previously demonstrated that lower expression of MSH6 in patient samples was associated with increased ex vivo resistance to 6-mercaptopurine and prednisone,7 highlighting the clinical importance of under- standing the role of this genetic alteration in B-ALL.
The mechanism of action of thiopurines is based upon the insertion of a false nucleotide, namely a thioguanine (TGN), into DNA that when thiomethylated pairs with a thymine instead of a cytosine.18 Cytotoxicity is thought to be dependent on the MMR machinery recognizing the mismatch and attempting to match the TGN on the parental strand with an appropriate base on the daughter strand.19,20 Whether the DNA damage induced by the repetitive, futile cycles of DNA excision and repair, or simply the recognition of mismatches by hMutSa is enough to initiate a signaling cascade culminating in cell cycle arrest and apoptosis is not entirely understood.
We sought to delineate whether reduced expression of MSH6 could give rise to chemoresistance in B-precursor ALL and elucidate the mechanism responsible for the resistance. Our data here support the view that reduced MSH6 directly results in an increased tolerance to incor- porated TGN and subsequent mismatches through a fail- ure to initiate MMR, thus allowing cells to proliferate and survive under thiopurine treatment both in vitro and in vivo. We demonstrate that ALL cell lines with a functional MMR trigger a CHK1-mediated cell cycle arrest in response to thiopurines that is followed by DNA damage and apoptosis. In contrast, upon reduction of MSH6, the MMR signaling cascade is not fully activated and cells do not undergo apoptosis.
Methods
Cells and reagents
The B-lineage leukemia cell lines RS4;11 (ATCC, Manassas, VA, USA), Reh (ATCC), 697 (DSMZ, Braunschweig, Germany), and UOCB1 (a kind gift from Dr. Terzah Horton at Texas Children’s Cancer Center/Baylor College of Medicine) were grown in
RPMI1640 medium. HEK293T (ATCC) cells were grown in DMEM medium. All media were supplemented with 10% FBS, 1% penicillin/streptomycin under 5% CO2 at 37°C.
Drug preparation, viral preparation, immunoblotting, apoptosis assays, and cell cycle
Standard protocols were followed and have been previously described.3,21 More detailed information is provided in the Online Supplementary Appendix.
Patients’ samples
Cryopreserved pediatric B-ALL specimens were obtained from the Children's Oncology Group (COG) ALL cell bank. All patients were treated on COG protocols for newly diagnosed ALL. All sub- jects provided consent for banking and future research use of these specimens in accordance with the regulations of the institutional review boards of all participating institutions.
Microsatellite instability analysis
Microsatellite instability (MSI) analysis was performed using MSI Analysis System, v.1.2 (Promega, Madison, WI, USA) follow- ing the manufacturer’s protocol. Detailed information is provided in the Online Supplementary Appendix.
Measurement of mutation rate
Spontaneous mutation rate was measured using a flow cytom- etry assay previously described by Araten et al.22 that detects the presence of numerous glycosylphosphatidylinositol-linked (GPI) membrane proteins (see Online Supplementary Appendix). Briefly, GPI(+) isolated clones from the NT and MSH6-KD cell lines were expanded either untreated or treated with 6-TG (0.040 μg/mL and 0.100 μg/mL, respectively, based on IC50 values determined for clones). Cells were then stained for GPI-dependent markers including FLAER-Alexa 488 (Pinewood), CD48, CD52, and CD59 (Serotec),23 and analyzed by flow cytometry. The mutant frequen- cy (f) was calculated as the number of GPI(-) events divided by the total number of live events, and mutation rate (μ) was calculated as f divided by cell divisions.22
Thioguanine quantification assay
Cells were treated with 6-thioguanine (6TG) and collected every day for four days. DNA was extracted using Puregene Core Kit A (QIAGEN). DNA TGN levels were measured using liquid chro- matography-tandem mass spectrometry as described previously.24
In vivo mouse model of chemoresistance
All experiments were conducted on protocols approved by
the Institutional Animal Care and Use Committee and Institutional Review Board of the Children's Hospital of Philadelphia. Briefly, 1 million UOCB1 NT GFP-CBG or MSH6 shRNA1 GFP-CBR cells were injected into NSG mice via tail vein on day 0 (total 20 mice; 10 per cell line). On day 6 leukemic bur- den was confirmed via bioluminescence imaging (BLI) (IVIS Spectrum imaging system, Perkin Elmer) and animals were ran- domized to treatment groups [PBS vehicle or Purixan (50 mg/kg) diluted in PBS]. Mice were treated on day 7 by gavage (0.2 mL/mouse). For BLI, 3 mg of luciferin was injected intraperi- toneally and mice were imaged ten minutes post injection. Quantification of total flux was determined by analyzing the BLI images using Living Image Software (Perkin Elmer) (see Online Supplementary Appendix).
Statistical analysis
Statistical significance was calculated using unpaired t-test for IC50s, paired t-test for mutation rates, one-way ANOVA for cell
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