Faculty Bibliography

Approximately 90% of the 2,000 children, adolescents, and young adults enrolled each year in Children's Oncology Group acute lymphoblastic leukemia (ALL) trials will be cured. However, high-risk subsets with significantly inferior survival remain, including infants, newly diagnosed patients with age >/=10 years, white blood count >/=50,000/microl, poor early response or T-cell ALL, and relapsed ALL patients. Effective strategies to improve survival include better risk stratification, optimizing standard chemotherapy and combining targeted therapies with cytotoxic chemotherapy, the latter of which is dependent upon identification of key driver mutations present in ALL. Pediatr Blood Cancer (c) 2012 Wiley Periodicals, Inc.

Background: The poor prognosis of relapsed ALL warrants the need for new insights into drug resistance mechanisms. We have previously shown that relapsed blasts can be re-sensitized to chemotherapy by the reversal of their gene expression signature using epigenetic agents.[1] Objectives: We hypothesize that aberrant epigenetic mechanisms may play a role in chemoresistance. To assess the degree to which the histone code evolves from diagnosis to relapse, we have used an unbiased, whole genome approach to examine changes in the epigenomic landscape by mapping the genome-wide location of key histone marks in diagnosis/relapse patient pairs with ALL enrolled on Children's Oncology Group protocols. Design/Method: We have performed Chromatin-immunoprecipitation sequencing (ChIP-seq) on two cryopreserved matched diagnosis/relapse pairs using active (H3K4me3, H3K9ac) and repressive (H3K9me3, H3K27me3) histone "mark" antibodies, with non-immunoprecipitated DNA (input) as control. Libraries for each patient pair were multiplexed in a single lane and sequenced in duplicate. 51-cycle single-end sequencing was performed using the Illumina HiSeq2000 Analyzer. Reads were aligned to reference genome using BWA and filtered using Samtools to remove multiple mapping reads. Peaks were called using MACS (v2.0.9) with default settings for histone marks. Results: 94.2% of the sequence reads passed filter (PF) and 96.4% of PF reads had quality score of more than 30. Using a p-value<0.00001, we identified 83,380 and 74,453 peaks (enriched regions) for activating and, 37,483 and 46,143 peaks for repressive histone marks at diagnosis and relapse respectively; suggesting that relapsed blasts are enriched with repressive marks and depleted of activating marks compared to those at diagnosis. Sixty-six genes had shared peaks for various histone marks between the two patients. Upon analyzing the top most differentially expressed transcripts at relapse from our previous cohort[2],64% genes showed concordant histone modificatio!

BACKGROUND: Acute lymphoblastic leukemia (ALL) is the most common cancer in children and the incidence of ALL varies by ethnicity. Although accumulating evidence indicates inherited predisposition to ALL, the genetic basis of ALL susceptibility in diverse ancestry has not been comprehensively examined. METHODS: We performed a multiethnic genome-wide association study in 1605 children with ALL and 6661 control subjects after adjusting for population structure, with validation in three replication series of 845 case subjects and 4316 control subjects. Association was tested by two-sided logistic regression. RESULTS: A novel ALL susceptibility locus at 10p12.31-12.2 (BMI1-PIP4K2A, rs7088318, P = 1.1 x 10(-11)) was identified in the genome-wide association study, with independent replication in European Americans, African Americans, and Hispanic Americans (P = .001, .009, and .04, respectively). Association was also validated at four known ALL susceptibility loci: ARID5B, IKZF1, CEBPE, and CDKN2A/2B. Associations at ARID5B, IKZF1, and BMI1-PIP4K2A variants were consistent across ethnicity, with multiple independent signals at IKZF1 and BMI1-PIP4K2A loci. The frequency of ARID5B and BMI1-PIP4K2A variants differed by ethnicity, in parallel with ethnic differences in ALL incidence. Suggestive evidence for modifying effects of age on genetic predisposition to ALL was also observed. ARID5B, IKZF1, CEBPE, and BMI1-PIP4K2A variants cumulatively conferred strong predisposition to ALL, with children carrying six to eight copies of risk alleles at a ninefold (95% confidence interval = 6.9 to 11.8) higher ALL risk relative to those carrying zero to one risk allele at these four single nucleotide polymorphisms. CONCLUSIONS: These findings indicate strong associations between inherited genetic variation and ALL susceptibility in children and shed new light on ALL molecular etiology in diverse ancestry.

Relapsed childhood acute lymphoblastic leukemia (ALL) carries a poor prognosis, despite intensive retreatment, owing to intrinsic drug resistance. The biological pathways that mediate resistance are unknown. Here, we report the transcriptome profiles of matched diagnosis and relapse bone marrow specimens from ten individuals with pediatric B-lymphoblastic leukemia using RNA sequencing. Transcriptome sequencing identified 20 newly acquired, novel nonsynonymous mutations not present at initial diagnosis, with 2 individuals harboring relapse-specific mutations in the same gene, NT5C2, encoding a 5'-nucleotidase. Full-exon sequencing of NT5C2 was completed in 61 further relapse specimens, identifying additional mutations in 5 cases. Enzymatic analysis of mutant proteins showed that base substitutions conferred increased enzymatic activity and resistance to treatment with nucleoside analog therapies. Clinically, all individuals who harbored NT5C2 mutations relapsed early, within 36 months of initial diagnosis (P = 0.03). These results suggest that mutations in NT5C2 are associated with the outgrowth of drug-resistant clones in ALL.

The bone marrow environment (BME) has been shown to promote resistance of leukemia cells towards chemotherapy, which has been attributed to several proteins, including integrins. Our analysis of 207 children with high-risk (BCR/ABL1-) pre-B ALL revealed that high expression of the integrin alpha6 (alpha6beta1) portends poor clinical outcomes in patients with minimal residual disease (MRD+) on day 29 of induction. Therefore, we hypothesized that alpha6-mediated adhesion of ALL cells to the bone marrow stromal niche contributes to drug resistance. We evaluated the role of alpha6 in BCR-ABL1+ leukemia using two of our established models of leukemia, a conditional knockout model of alpha6 in murine BCR-ABL1+ leukemia and a xenograft model of human BCR-ABL1+ leukemia. alpha6fl/fl cells were oncogenically transformed using BCR-ABL1 (p210). Subsequent transduction with CreERT2 or EmptyERT2 generated leukemia cells in which alpha6 ablation could be induced (CreERT2) or not (EmptyERT2) by addition of Tamoxifen. Conditional ablation of alpha6 in vitro decreased adhesion significantly compared to undeleted controls. Moreover, alpha6 deletion sensitized murine ALL to a tyrosine kinase inhibitor (TKI), Nilotinib. To test the effect of alpha6 deletion on leukemic progression in vivo, alpha6 BCR/ABL1+ pre-B CreERT2+ or control ALL cells were transferred into NOD/SCIDmice. 3 days thereafter, in vivo deletion of alpha6 alone delayed leukemia progression compared to controls (Median survival time, MST, 43 days versus 30 days post-leukemia injection; p=0.008). Nilotinib-treated recipients of to alpha6-competent cells have uniformly died with aMSTof 39.5 days, however the Nilotinib treated alpha6-deleted recipients stayed completely alive. Moreover, we show that alpha6 blockade de-adheres primary ALL cells from their cognate counter receptor laminin in vitro, and sensitizes them to TKI. Taken together, inhibition of alpha6 offers a new approach to overcome drug resistance in pre-B ALL

One recently identified subtype of pediatric B-precursor acute lymphoblastic leukemia (ALL) has been termed BCR-ABL1-like or Ph-like because of similarity of the gene expression profile to BCR-ABL1 positive ALL suggesting the presence of lesions activating tyrosine kinases, frequent alteration of IKZF1, and poor outcome. Prior studies demonstrated that approximately half of these patients had genomic lesions leading to CRLF2 overexpression, with half of such cases harboring somatic mutations in the Janus kinases JAK1 and JAK2. To determine whether mutations in other tyrosine kinases might also occur in ALL, we sequenced the tyrosine kinome and downstream signaling genes in 45 high-risk pediatric ALL cases with either a Ph-like gene expression profile or other alterations suggestive of activated kinase signaling. Aside from JAK mutations and 1 FLT3 mutation, no somatic mutations were found in any other tyrosine kinases, suggesting that alternative mechanisms are responsible for activated kinase signaling in high-risk ALL.

The Sox4 transcription factor mediates early B-cell differentiation. Compared with normal pre-B cells, SOX4 promoter regions in Ph(+) ALL cells are significantly hypomethylated. Loss and gain-of-function experiments identified Sox4 as a critical activator of PI3K/AKT and MAPK signaling in ALL cells. ChIP experiments confirmed that SOX4 binds to and transcriptionally activates promoters of multiple components within the PI3K/AKT and MAPK signaling pathways. Cre-mediated deletion of Sox4 had little effect on normal pre-B cells but compromised proliferation and viability of leukemia cells, which was rescued by BCL2L1 and constitutively active AKT and p110 PI3K. Consistent with these findings, high levels of SOX4 expression in ALL cells at the time of diagnosis predicted poor outcome in a pediatric clinical trial (COG P9906). Collectively, these studies identify SOX4 as a central mediator of oncogenic PI3K/AKT and MAPK signaling in ALL.