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MutLa, is what initiates apoptosis. Our data demonstrat- ing the involvement of the ATR-Chk1-H2AX signaling cascade is supported by the work of Eich et al. which demonstrated activation of this pathway upon treatment with temozolomide.32 Interestingly, understanding how MMR deficient cells respond to thiopurines in terms of TGN incorporation could prove essential given the emerg- ing idea of measuring these parameters in patients on maintenance therapy.44
The data presented here do not support the hypothesis of increased mutation burden, genomic instability, or MSI when MSH6 is reduced. Our inability to demonstrate MSI-high, which is considered a standard method for clin- ical testing of MMR deficiencies in tumors,45 in MSH6 depleted cell lines and clinical samples is consistent with the lack of MSI in glioma samples with MSH6 deletions or mutations.46,47 Haploinsufficiency of MSH6 or compensa- tion by MSH2/MSH3 may account for this observation.25,48 In addition, ALL clonal evolution from diagnosis to relapse is not associated with increased mutation burden support- ing our mutation rate analysis, although Ma et al. reported a subset of hypermutated relapse cases.49 Of these, one had a bialleic mutation of PMS2, another had multiple damaging MSH6 mutations as well as an MLH1 splice site mutation, while the others harbored no MMR muta- tions.49 Furthermore, one case demonstrated that a het- erozygous deletion of MSH6 at diagnosis was not suffi- cient to cause a hypermutator phenotype, but the acquisi- tion of a second hit in the WT allele at relapse was.49 Likewise, the majority of hypermutated gliomas at relapse show defects in multiple MMR genes or loss of heterozy-
gosity.50 Thus, our work supports a model whereby hap- loinsufficiency of MSH6 results in TGN tolerance and resistance directly rather than by generation of secondary mutations. However, it does not rule out the possibility that haploinsufficiency, along with other defects in the MMR pathway, may result in a mutator phenotype.
Overall, it has become increasingly evident that the genetic and epigenetic landscape of cancer cells is vital to the overall effectiveness of treatment. These studies illus- trate yet another example of a mutation/deletion found at relapse that directly influences the response to a therapeu- tic agent that is currently heavily relied on. Continuous efforts to elucidate the potential functions and mecha- nisms of genes found mutated at relapse will help lead us to novel treatment strategies.
Funding
This work was supported by the Leukemia and Lymphoma Society SCOR grant: 7010-14 (WLC, JY, DT, SPH), the US National Institutes of Health (NIH) funded grant RO1 CA140729 (WLC), and the Perlmutter Cancer Center Support Grant: P30 DA016087.
Acknowledgments
We gratefully acknowledge the Children’s Oncology Group (COG) Specimen Bank for samples. Support for flow cytometry was provided by NYU School of Medicine’s Cytometry and Cell Sorting Laboratory, which is supported in part by grant P30CA016087 from the NIH/NCI, and the CHOP Flow Cytometry Core. We acknowledge the VA-Mertid award 1I01BX-000670, which helped support this work.
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