we initially studied 90 patients ( age  21 years ) who had newly diagnosed all and were enrolled on the st .
, we subsequently studied an additional 170 patients with all enrolled on the same treatment protocol , using publicly available snp data we had previously reported .
jude children s research hospital , and informed consent was obtained from patients , their guardians , or both before enrollment .
leukemia cells were isolated by applying a ficoll - hypaque gradient to bone marrow aspirates obtained at diagnosis ( median , 97% blast cells ) .
normal leukocytes were isolated from peripheral blood samples obtained after the successful completion of remission induction therapy ( on days 4548 after the start of treatment ) . to assess gene copy number loss in msh2-l leukemia cells ,
dna was extracted from leukemia cells and normal peripheral blood leukocytes ( obtained when patients were in complete remission ) and genotyped for 600k snps using the affymetrix genechip human mapping 50k - hind-240 , 50k - xba-240 , 250k - sty and 250k nsp snp arrays .
dna was restriction enzyme digested , pcr - amplified , purified , labeled , fragmented and hybridized to the arrays according to the manufacturer s instructions .
snp array data were analyzed using dchip for chromosomal abnormalities . to improve the accuracy of copy number inference using dchip , we applied a normalization procedure that uses snps exclusively from regions shown to be diploid in the normal leukocytes and maps signals from those snps to a common target probability distribution .
the snps with an estimated copy number lower than 1.40 were considered as evidence of deletions .
we initially performed snp analysis on prkcz , because it is known to regulate msh2 stability .
this led us to subsequently perform a broader pathway analysis , to interrogate additional genes upstream of prkcz ( figure 1c ) .
for each snp , we evaluated the over - representation of deletions in msh2-l leukemia cells compared to msh - h leukemias , using fisher s exact test .
the significance of each snp was then adjusted for multiple testing using 100,000 permutations . at each permutation
, we recorded the smallest p - value among all the 122 snps based on randomly assigned msh2 status .
the adjusted p - values were computed as the proportion of permutations whose smallest p - values were lower than or equal to the observed .
the human t - lineage leukemia cell line ccrf - cem was obtained from the american type culture collection .
the human pre - b leukemia cell lines 697 and nalm-6 were obtained from the german collection of microorganisms and cell cultures .
cells were cultured in rpmi-1640 medium containing 2 mm glutamine and 10% fetal bovine serum at 37 c with 5% co2 .
ccrf - cem cells were infected with mission lentiviral transduction particles ( sigma - aldrich ) produced from a library of sequence - verified shrnas targeting human pik3c2b , frap1/mtor , herc1 or prkcz transcripts .
non - target shrna control particles ( shc002v ) were also purchased from sigma - aldrich .
fisher s exact test was used to test the overrepresentation of losses among msh2 low patients versus msh2 positive patients .
a linear regression model was used to test the correlation between the levels of msh2 mrna and protein .
the expression levels of the probe sets were analyzed by applying a general linear model in which the effect of msh2 status ( positive vs. negative ) was adjusted for the all genetic subtypes .
the cox proportional hazards model was used to identify independent prognostic effect of msh2 status . for patients who achieved complete remission , cumulative incidence of hematological relapse
were constructed by the method of kalbfleisch and prentice , and compared with gray s test .
we initially studied 90 patients ( age  21 years ) who had newly diagnosed all and were enrolled on the st .
, we subsequently studied an additional 170 patients with all enrolled on the same treatment protocol , using publicly available snp data we had previously reported .
jude children s research hospital , and informed consent was obtained from patients , their guardians , or both before enrollment .
leukemia cells were isolated by applying a ficoll - hypaque gradient to bone marrow aspirates obtained at diagnosis ( median , 97% blast cells ) .
normal leukocytes were isolated from peripheral blood samples obtained after the successful completion of remission induction therapy ( on days 4548 after the start of treatment ) .
to assess gene copy number loss in msh2-l leukemia cells , dna was extracted from leukemia cells and normal peripheral blood leukocytes ( obtained when patients were in complete remission ) and genotyped for 600k snps using the affymetrix genechip human mapping 50k - hind-240 , 50k - xba-240 , 250k - sty and 250k nsp snp arrays .
dna was restriction enzyme digested , pcr - amplified , purified , labeled , fragmented and hybridized to the arrays according to the manufacturer s instructions .
snp array data were analyzed using dchip for chromosomal abnormalities . to improve the accuracy of copy number inference using dchip , we applied a normalization procedure that uses snps exclusively from regions shown to be diploid in the normal leukocytes and maps signals from those snps to a common target probability distribution .
the snps with an estimated copy number lower than 1.40 were considered as evidence of deletions .
we initially performed snp analysis on prkcz , because it is known to regulate msh2 stability .
this led us to subsequently perform a broader pathway analysis , to interrogate additional genes upstream of prkcz ( figure 1c ) .
, we evaluated the over - representation of deletions in msh2-l leukemia cells compared to msh - h leukemias , using fisher s exact test .
the significance of each snp was then adjusted for multiple testing using 100,000 permutations . at each permutation
, we recorded the smallest p - value among all the 122 snps based on randomly assigned msh2 status .
the adjusted p - values were computed as the proportion of permutations whose smallest p - values were lower than or equal to the observed .
the human t - lineage leukemia cell line ccrf - cem was obtained from the american type culture collection . the human pre - b leukemia cell lines
cells were cultured in rpmi-1640 medium containing 2 mm glutamine and 10% fetal bovine serum at 37 c with 5% co2 .
ccrf - cem cells were infected with mission lentiviral transduction particles ( sigma - aldrich ) produced from a library of sequence - verified shrnas targeting human pik3c2b , frap1/mtor , herc1 or prkcz transcripts .
non - target shrna control particles ( shc002v ) were also purchased from sigma - aldrich .
fisher s exact test was used to test the overrepresentation of losses among msh2 low patients versus msh2 positive patients .
a linear regression model was used to test the correlation between the levels of msh2 mrna and protein .
the expression levels of the probe sets were analyzed by applying a general linear model in which the effect of msh2 status ( positive vs. negative ) was adjusted for the all genetic subtypes .
the cox proportional hazards model was used to identify independent prognostic effect of msh2 status . for patients who achieved complete remission , cumulative incidence of hematological relapse
were constructed by the method of kalbfleisch and prentice , and compared with gray s test .
 dna mismatch repair enzymes ( e.g. , msh2 ) maintain genomic integrity , and their deficiency predisposes to several human cancers and to drug resistance . 
 we found that leukemia cells from a substantial proportion of patients ( ~11% ) with newly diagnosed acute lymphoblastic leukemia ( all ) have low or undetectable msh2 protein levels ( msh2-l ) , despite abundant wild - type msh2 mrna . 
 msh2-l leukemia cells contained partial or complete somatic deletions of 14 genes that regulate msh2 degradation ( frap1 , herc1 , prkcz , pik3c2b ) ; these deletions were also found in adult all ( 16% ) and sporadic colorectal cancer ( 13.5% ) . 
 knockdown of these genes in human leukemia cells recapitulated the msh2 protein deficiency by enhancing msh2-degradation , leading to significant reduction in dna mismatch repair ( mmr ) and increased resistance to thiopurines . 
 these findings reveal a previously unrecognized mechanism whereby somatic deletions of genes regulating msh2 degradation result in undetectable levels of msh2 protein in leukemia cells , mmr deficiency and drug resistance . 
Methods
Patients with ALL (initial discovery and validation cohorts)
Affymetrix 600K SNP array analyses
Cell culture
Stable Short Hairpin RNA (shRNA) knockdowns
Statistical analyses
Supplementary Material