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cycle analysis, and two-way ANOVA for mutation rates with and without treatment as well as in vivo studies.
Results
Previously we noted relapse-specific heterozygous dele- tions in MSH6 in 4 out of 76 patients that were near iden- tical and deleted MSH6 only for 3 patients while one har- bored a larger deletion involving more genes within the region (Online Supplementary Figure S1). To begin to eluci- date the impact of MSH6 deletion on the development of relapsed disease, we knocked down expression of MSH6 using shRNA in 697 cells, a B-ALL cell line that expresses all four MMR proteins (Figure 1A) and is MMR profi- cient,25 and tested for changes in chemosensitivity. We observed approximately 80-90% (shRNA1) and 50-60% (shRNA2) knockdown of MSH6 expression, as well as decreased expression of MSH2, compared to non-target- ing (NT) control cells (Figure 1A), which is consistent with literature on the loss of protein stability of MSH2 and MSH6 when not dimerized.17,26 Knockdown of MSH6 with both shRNA1 and shRNA2 leads to a significant decrease in apoptotic cells when treated with thiopurines for five days (Figure 1A). A 26-fold increase in IC50 with 6-TG (NT: 0.027 vs. shRNA1: 0.716 μg/mL; P=0.007) and 8.5-fold increase for 6-MP (NT: 0.340 vs. shRNA1: 2.89 μg/mL; P=0.006) was observed for shRNA1 (Online Supplementary Figure S2). A 1.7-fold (NT: 0.015 vs. shRNA2: 0.025 μg/mL; P=0.015) and a 2.6-fold (NT: 0.143 vs. shRNA2: 0.373 μg/mL; P=0.032) increase in IC50 for 6- TG and 6-MP, respectively, were observed for shRNA2 cells compared to NT cells (Online Supplementary Figure
S2). However, no significant differences were observed when cells were treated with prednisolone (Pred), doxoru- bicin (Doxo), cytarabine (Ara-c), or methotrexate (MTX) (Online Supplementary Figure S3A). Interestingly, we found that knockdown of MSH6 also resulted in decreased sen- sitivity to temozolomide (TMZ), an alkylating agent used to treat glioblastomas, as reported previously (Online Supplementary Figure S3B).27,28
To further support the role of MSH6 in chemoresis- tance, we knocked down expression in UOCB1 cells, another B-ALL cell line that expresses all four MMR pro- teins (Figure 1B). Similar to the effect observed in 697 cells, depletion of MSH6 with either shRNA significantly reduced the induction of apoptosis upon treatment with thiopurines (Figure 1B) [fold increase in IC50 as compared to NT with 6TG: 4.8 for shRNA1 (P=0.007) and 3 for shRNA2 (P<0.001); 6MP: 8.3 for shRNA1 (P<0.001) and 9.2 shRNA2 (P<0.001)] (Online Supplementary Figure S4). Additionally, a similar impact on TMZ resistance was observed with UOCB1 MSH6 shRNA1 expressing cells compared to NT control cells, although shRNA2 did not show the same effect, possibly due to less depletion by shRNA2 (Online Supplementary Figure S3B).
To determine the specificity of the phenotype observed for MSH6 depletion versus defects in other MMR proteins, we assessed the effect of MSH6 knock- down in MMR deficient B-ALL cell lines Reh and RS4;11.25,29 Both Reh and RS4;11 have minimal to no expression of MLH1 and PMS2 (Figure 1C). Knockdown of MSH6 expression had no effect on the sensitivity of either Reh or RS4;11 to 6-TG or 6-MP (Figure 1C and Online Supplementary Figure S5).
To begin to elucidate the mechanism of resistance, we
MSH6 haploinsufficiency contributes to resistance
AB
C
Figure 2. Knockdown of MSH6 leads to increased tolerance of incorporated thioguanine (TGN). (A and B) TGN incorporation into DNA was measured over time after treatment with 0.1 μg/mL of 6-thiogua- nine (6-TG) in 697 (A) and Reh (B) cells using liquid chromatography-tandem mass spectrometry. A rep- resentative graph from 3 independent experiments is shown. (C) Combined ratio of TGN fmole/μg of DNA in MSH6 shRNA1 knockdown (KD) cells compared to non-targeting (NT) cells from 3 experiments. Bars indicate mean+Standard Deviation.
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