MSH6 haploinsufficiency at relapse contributes to the development of thiopurine resistance in pediatric B-lymphoblastic leukemia
Nikki A. Evensen,
P. Pallavi Madhusoodhan,
Julia Meyer,
Jason Saliba,
Ashfiyah Chowdhury,
David J. Araten,
Jacob Nersting,
Teena Bhatla,
Tiffaney L. Vincent,
David Teachey,
Stephen P. Hunger,
Jun Yang,
Kjeld Schmiegelow,
William L. Carroll
Affiliations
Nikki A. Evensen
Departments of Pediatrics and Pathology, Perlmutter Cancer Center, NYU-Langone Medical Center, New York, NY, USA
P. Pallavi Madhusoodhan
Departments of Pediatrics and Pathology, Perlmutter Cancer Center, NYU-Langone Medical Center, New York, NY, USA
Julia Meyer
Huntsman Cancer Institute, University of Utah Medical Center, Salt Lake City, USA
Jason Saliba
Departments of Pediatrics and Pathology, Perlmutter Cancer Center, NYU-Langone Medical Center, New York, NY, USA
Ashfiyah Chowdhury
Departments of Pediatrics and Pathology, Perlmutter Cancer Center, NYU-Langone Medical Center, New York, NY, USA
David J. Araten
Department of Medicine, Perlmutter Cancer Center, NYU-Langone Medical Center, New York NY, USA
Jacob Nersting
Department of Pediatrics and Adolescent Medicine, The University Hospital Rigshospitalet, Copenhagen, Denmark
Teena Bhatla
Departments of Pediatrics and Pathology, Perlmutter Cancer Center, NYU-Langone Medical Center, New York, NY, USA
Tiffaney L. Vincent
Department 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
David Teachey
Department 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
Stephen P. Hunger
Department 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
Jun Yang
St. Jude Children’s Research Hospital, Memphis, TN, USA
Kjeld Schmiegelow
Department of Pediatrics and Adolescent Medicine, The University Hospital Rigshospitalet, Copenhagen, Denmark
William L. Carroll
Departments of Pediatrics and Pathology, Perlmutter Cancer Center, NYU-Langone Medical Center, New York, NY, USA
Survival of children with relapsed acute lymphoblastic leukemia is poor, and understanding mechanisms underlying resistance is essential to developing new therapy. Relapse-specific heterozygous deletions in MSH6, a crucial part of DNA mismatch repair, are frequently 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) and showed significant increases in IC50s to 6-thioguanine and 6-mercaptopurine (697: 26- and 9-fold; UOCB1: 5- and 8-fold) in vitro, as well as increased resistance to 6-mercaptopurine treatment in vivo. No shift in IC50 was observed in deficient cells (Reh and RS4;11). 697 MSH6 knockdown resulted in increased DNA thioguanine nucleotide levels compared to non-targeted cells (3070 vs. 1722 fmol/μg DNA) with no difference observed in mismatch repair deficient cells. Loss of MSH6 did not 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 showed minimal activation of checkpoint regulator CHK1, γH2AX (DNA damage marker) and p53 levels upon treatment with thiopurines, consistent with intrinsic chemoresistance due to failure to recognize thioguanine nucleotide mismatching and initiate mismatch repair. Aberrant MSH6 adds to the list of alterations/mutations associated with acquired resistance to purine analogs emphasizing the importance of thiopurine therapy.
