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Abstract Defining genetic factors impacting chemotherapy failure can help to better predict response and identify drug resistance mechanisms. However, there is limited understanding of the contribution of inherited noncoding genetic variation on inter-individual differences in chemotherapy response in childhood acute lymphoblastic leukemia (ALL). Here we map inherited noncoding variants associated with treatment outcome and/or chemotherapeutic drug resistance to ALL cis-regulatory elements and investigate their gene regulatory potential and target gene connectivity using massively parallel reporter assays and three-dimensional chromatin looping assays, respectively. We identify 54 variants with transcriptional effects and high-confidence gene connectivity. Additionally, functional interrogation of the top variant, rs1247117, reveals changes in chromatin accessibility, PU.1 binding affinity and gene expression, and deletion of the genomic interval containing rs1247117 sensitizes cells to vincristine. Together, these data demonstrate that noncoding regulatory variants associated with diverse pharmacological traits harbor significant effects on allele-specific transcriptional activity and impact sensitivity to antileukemic agents.

Oncogenic gene fusions are frequent in childhood cancers but remain poorly understood and untargeted. Here, the authors identify 272 oncogenic fusions in transcriptomics data from 5190 childhood cancer patients, revealing their possible etiologies, their links with tumor progression and evolution, and their potential as therapeutic targets.

Vincristine is a widely used chemotherapeutic drug for the treatment of multiple malignant diseases that causes a dose-limiting peripheral neurotoxicity. There is no clinically effective preventative treatment for vincristine-induced sensory peripheral neurotoxicity (VIPN), and mechanistic details of this side effect remain poorly understood. We hypothesized that VIPN is dependent on transporter-mediated vincristine accumulation in dorsal root ganglion neurons. Using a xenobiotic transporter screen, we identified OATP1B3 as a neuronal transporter regulating the uptake of vincristine. In addition, genetic or pharmacological inhibition of the murine orthologue transporter OATP1B2 protected mice from various hallmarks of VIPN — including mechanical allodynia, thermal hyperalgesia, and changes in digital maximal action potential amplitudes and neuronal morphology — without negatively affecting plasma levels or antitumor effects of vincristine. Finally, we identified α-tocopherol from an untargeted metabolomics analysis as a circulating endogenous biomarker of neuronal OATP1B2 function, and it could serve as a companion diagnostic to guide dose selection of OATP1B-type transport modulators given in combination with vincristine to prevent VIPN. Collectively, our findings shed light on the fundamental basis of VIPN and provide a rationale for the clinical development of transporter inhibitors to prevent this debilitating side effect.

Abstract Vincristine (VCR) is one of the most widely prescribed medications for treating solid tumors and acute lymphoblastic leukemia (ALL) in children and adults. However, its major dose‐limiting toxicity is peripheral neuropathy that can disrupt curative therapy. Peripheral neuropathy can also persist into adulthood, compromising quality of life of childhood cancer survivors. Reducing VCR‐induced neurotoxicity without compromising its anticancer effects would be ideal. Here, we show that low expression of NHP2L1 is associated with increased sensitivity of primary leukemia cells to VCR, and that concomitant administration of VCR with inhibitors of NHP2L1 increases VCR cytotoxicity in leukemia cells, prolongs survival of ALL xenograft mice, but decreases VCR effects on human‐induced pluripotent stem cell‐derived neurons and mitigates neurotoxicity in mice. These findings offer a strategy for increasing VCR’s antileukemic effects while reducing peripheral neuropathy in patients treated with this widely prescribed medication.

Abstract Background Fluoroquinolone antibiotics are frequently utilized in pediatric oncology patients as prophylaxis or step‐down therapy following broad spectrum beta‐lactam therapy for febrile neutropenia. Concerns regarding neurotoxicity limit the use of these agents. No studies have evaluated the association between fluoroquinolone use and neurotoxicity in pediatric oncology patients receiving other neurotoxic agents such as vincristine. Methods An observational cohort study comprising patients aged 0‐18 at diagnosis enrolled on a prospective study for treatment of acute lymphoblastic leukemia (ALL) at a pediatric comprehensive cancer center between October 2007 and November 2018. Data for neuropathic pain and sensory or motor neuropathy were collected prospectively, and a Cox proportional hazards regression model was used to evaluate associations between administration of fluoroquinolone antibiotics during induction therapy and subsequent development of vincristine‐induced peripheral neurotoxicity (VIPN). Results A total of 598 participants were enrolled, including 338 (57%) who received fluoroquinolones during induction therapy; of these 470 (79%) were diagnosed with VIPN and 139 (23%) were diagnosed with high‐grade (Grade 3+) VIPN. On unadjusted analyses, and analyses adjusted for age and race, there was no evidence of an association between fluoroquinolone exposure and subsequent VIPN (hazard ratio [HR] 0.8, 95% CI 0.5‐1.04, P = .08) or high‐grade VIPN (HR 1.1, 95% CI 0.4‐2.2, P = .87). Conclusions The results of this observational study do not show an association between exposure to fluoroquinolone antibiotics during induction therapy for ALL and subsequent development of vincristine‐induced peripheral neuropathies, and suggest that a large increase in VIPN is unlikely.

Chemotherapy dosages are often compromised, but most reports lack data on dosages that are actually delivered. In two consecutive acute lymphoblastic leukemia trials that differed in their asparaginase formulation, native E. coli L-asparaginase in St. Jude Total 15 (T15, n=365) and pegaspargase in Total 16 (T16, n=524), we tallied the dose intensities for all drugs on the low-risk or standard-risk arms, analyzing 504,039 dosing records. The median dose intensity for each drug ranged from 61-100%. Dose intensities for several drugs were more than 10% higher on T15 than on T16: cyclophosphamide (P

B-cell acute lymphoblastic leukaemia (B-ALL) is a common childhood cancer. Here, the authors conducted a meta-analysis with four genome-wide association studies, totalling 5,321 cases and 16,666 controls of European descent, identifying B-ALL risk loci, whose integration with epigenomic profiling indicates cell-cycle and B-cell development deregulation as central mechanisms in B-ALL susceptibility, often in a subtype-specific fashion.

Abstract Background Despite its relatively low incidence, pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer deaths because of the aggressive growth/metastasis of the tumor, the lack of early symptoms, and the poor treatment options. Basic research to identify potential therapeutic targets for PDAC is greatly needed. Methods We used a negative-selection genome-wide CRISPR screen to identify essential genes in the PANC-1 human pancreatic carcinoma cell line. We validated the top hits with follow-up siRNA screens, using the HPNE, HPAF-II, AsPC-1, and Mia PaCa-2 cell lines. Results The PSMA6 gene was an identified candidate hit after the CRISPR screen, siRNA validation screen, and siRNA deconvolution screen. Spheroid formation assays and flow cytometry analysis showed that PSMA6 is critical for survival in many pancreatic ductal carcinoma cell models. Lastly, as PSMA6 protein is a proteosomal subunit of the 20S core complex, we showed that bortezomib, a proteasome inhibitor, was especially toxic in PANC-1 cells. Conclusions Further study of PSMA6 and the proteasome subunit that it encodes, along with other hits identified in our CRISPR screens, may provide valuable insights into potential therapeutic targets for PDAC.

Measuring the amount of methotrexate polyglutamates (MTXPG) in leukemia cells after high-dose methotrexate (HDMTX) revealed that molecular subtype and lineage of acute lymphoblastic leukemia (ALL), the ratio of expression of folate influx and efflux transporters, methotrexate (MTX) infusion time, folylpolyglutamate synthase mRNA expression, and MTX systemic clearance explain 42% of the variation in active MTXPGs accumulation in ALL cells in vivo, providing insights into mechanisms underlying interpatient differences in the antileukemic effects of HDMTX.

In vitro and in vivo resistance to prednisolone are predictive for an adverse prognosis in pediatric precursor B-acute lymphoblastic leukemia. Causes of resistance are still poorly understood. In this study, we observed that prednisolone exposure of prednisolone-sensitive patients’ leukemic cells decreased anti-apoptotic MCL1 protein levels by 2.9-fold, while MCL1 protein expression in prednisolone-resistant leukemic patients’ cells was unaffected (P

IntroductionCombination therapy for acute lymphoblastic leukemia (ALL) is highly effective but results in significant toxicity including osteonecrosis. Asparaginase is known to potentiate both the antileukemic and osteonecrosis-inducing effects of dexamethasone. The schedule of dexamethasone alters osteonecrosis risk. However, the effects of the interaction with asparaginase are unknown when dexamethasone is given on a discontinuous schedule.MethodsUsing the murine model of osteonecrosis, we compared the frequency of osteonecrosis in mice receiving discontinuous dexamethasone (3.5 days/ week) with mice receiving asparaginase and discontinuous dexamethasone. We then tested the effect on antileukemic efficacy using six pediatric ALL xenografts.ResultsThe addition of asparaginase to discontinuous dexamethasone did not alter the rate of osteonecrosis compared to dexamethasone alone (7/35 in dexamethasone with asparaginase combination vs. 10/36 in dexamethasone alone, p = 0.62) despite increasing steady-state plasma dexamethasone levels (103.9 nM vs. 33.4 nM, p = 9.2x10-7). Combination therapy with asparaginase and dexamethasone demonstrated synergistic antileukemic effects across all six xenografts studied.ConclusionsWhen discontinuous dexamethasone was given, its anti-leukemic activity synergized with asparaginase but the osteonecrosis-worsening effects of asparaginase (above dexamethasone alone) were not observed. Thus, there is a favorable drug interaction (unchanged toxicity, synergistic efficacy) between discontinuous dexamethasone and asparaginase.

MicroRNAs are important regulators of gene expression, acting primarily by binding to sequence-specific locations on already transcribed messenger RNAs (mRNA) and typically down-regulating their stability or translation. Recent studies indicate that microRNAs may also play a role in up-regulating mRNA transcription levels, although a definitive mechanism has not been established. Double-helical DNA is capable of forming triple-helical structures through Hoogsteen and reverse Hoogsteen interactions in the major groove of the duplex, and we show physical evidence (i.e., NMR, FRET, SPR) that purine or pyrimidine-rich microRNAs of appropriate length and sequence form triple-helical structures with purine-rich sequences of duplex DNA, and identify microRNA sequences that favor triplex formation. We developed an algorithm (Trident) to search genome-wide for potential triplex-forming sites and show that several mammalian and non-mammalian genomes are enriched for strong microRNA triplex binding sites. We show that those genes containing sequences favoring microRNA triplex formation are markedly enriched (3.3 fold, p

Osteonecrosis is one of the most common, serious, toxicities resulting from the treatment of acute lymphoblastic leukemia. In recent years, pediatric acute lymphoblastic leukemia clinical trials have used discontinuous rather than continuous dosing of dexamethasone in an effort to reduce the incidence of osteonecrosis. However, it is not known whether discontinuous dosing would compromise antileukemic efficacy of glucocorticoids. Therefore, we tested the efficacy of discontinuous dexamethasone against continuous dexamethasone in murine models bearing human acute lymphoblastic leukemia xenografts (n = 8 patient samples) or murine BCR-ABL+ acute lymphoblastic leukemia. Plasma dexamethasone concentrations (7.9 to 212 nM) were similar to those achieved in children with acute lymphoblastic leukemia using conventional dosages. The median leukemia-free survival ranged from 16 to 59 days; dexamethasone prolonged survival from a median of 4 to 129 days in all seven dexamethasone-sensitive acute lymphoblastic leukemias. In the majority of cases (7 of 8 xenografts and the murine BCR-ABL model) we demonstrated equal efficacy of the two dexamethasone dosing regimens; whereas for one acute lymphoblastic leukemia sample, the discontinuous regimen yielded inferior antileukemic efficacy (log-rank p = 0.002). Our results support the clinical practice of using discontinuous rather than continuous dexamethasone dosing in patients with acute lymphoblastic leukemia.

Methotrexate (MTX) is widely used for the treatment of childhood acute lymphoblastic leukemia (ALL). The accumulation of MTX and its active metabolites, methotrexate polyglutamates (MTXPG), in ALL cells is an important determinant of its antileukemic effects. We studied 194 of 356 patients enrolled on St. Jude Total XV protocol for newly diagnosed ALL with the goal of characterizing the intracellular pharmacokinetics of MTXPG in leukemia cells; relating these pharmacokinetics to ALL lineage, ploidy and molecular subtype; and using a folate pathway model to simulate optimal treatment strategies. Serial MTX concentrations were measured in plasma and intracellular MTXPG concentrations were measured in circulating leukemia cells. A pharmacokinetic model was developed which accounted for the plasma disposition of MTX along with the transport and metabolism of MTXPG. In addition, a folate pathway model was adapted to simulate the effects of treatment strategies on the inhibition of de novo purine synthesis (DNPS). The intracellular MTXPG pharmacokinetic model parameters differed significantly by lineage, ploidy, and molecular subtypes of ALL. Folylpolyglutamate synthetase (FPGS) activity was higher in B vs T lineage ALL (p

The degree to which gene expression covaries between different primary tissues within an individual is not well defined. We hypothesized that expression that is concordant across tissues is more likely influenced by genetic variability than gene expression which is discordant between tissues. We quantified expression of 11,873 genes in paired samples of primary leukemia cells and normal leukocytes from 92 patients with acute lymphoblastic leukemia (ALL). Genetic variation at >500,000 single nucleotide polymorphisms (SNPs) was also assessed. The expression of only 176/11,783 (1.5%) genes was correlated (p

Childhood acute lymphoblastic leukemia (ALL) is the most common cancer in children, and can now be cured in approximately 80% of patients. Nevertheless, drug resistance is the major cause of treatment failure in children with ALL. The drug methotrexate (MTX), which is widely used to treat many human cancers, is used in essentially all treatment protocols worldwide for newly diagnosed ALL. Although MTX has been extensively studied for many years, relatively little is known about mechanisms of de novo resistance in primary cancer cells, including leukemia cells. This lack of knowledge is due in part to the fact that existing in vitro methods are not sufficiently reliable to permit assessment of MTX resistance in primary ALL cells. Therefore, we measured the in vivo antileukemic effects of MTX and identified genes whose expression differed significantly in patients with a good versus poor response to MTX.We utilized measures of decreased circulating leukemia cells of 293 newly diagnosed children after initial "up-front" in vivo MTX treatment (1 g/m(2)) to elucidate interpatient differences in the antileukemic effects of MTX. To identify genomic determinants of these effects, we performed a genome-wide assessment of gene expression in primary ALL cells from 161 of these newly diagnosed children (1-18 y). We identified 48 genes and two cDNA clones whose expression was significantly related to the reduction of circulating leukemia cells after initial in vivo treatment with MTX. This finding was validated in an independent cohort of children with ALL. Furthermore, this measure of initial MTX in vivo response and the associated gene expression pattern were predictive of long-term disease-free survival (p < 0.001, p = 0.02).Together, these data provide new insights into the genomic basis of MTX resistance and interpatient differences in MTX response, pointing to new strategies to overcome MTX resistance in childhood ALL.Total XV, Therapy for Newly Diagnosed Patients With Acute Lymphoblastic Leukemia, http://www.ClinicalTrials.gov (NCT00137111); Total XIIIBH, Phase III Randomized Study of Antimetabolite-Based Induction plus High-Dose MTX Consolidation for Newly Diagnosed Pediatric Acute Lymphocytic Leukemia at Intermediate or High Risk of Treatment Failure (NCI-T93-0101D); Total XIIIBL, Phase III Randomized Study of Antimetabolite-Based Induction plus High-Dose MTX Consolidation for Newly Diagnosed Pediatric Acute Lymphocytic Leukemia at Lower Risk of Treatment Failure (NCI-T93-0103D).

Purpose Germline TP53 variation is the genetic basis of Li-Fraumeni syndrome, a highly penetrant cancer predisposition condition. Recent reports of germline TP53 variants in childhood hypodiploid acute lymphoblastic leukemia (ALL) suggest that this type of leukemia is another manifestation of Li-Fraumeni syndrome; however, the pattern, prevalence, and clinical relevance of TP53 variants in childhood ALL remain unknown. Patients and Methods Targeted sequencing of TP53 coding regions was performed in 3,801 children from the Children's Oncology Group frontline ALL clinical trials, AALL0232 and P9900. TP53 variant pathogenicity was evaluated according to experimentally determined transcriptional activity, in silico prediction of damaging effects, and prevalence in non-ALL control populations. TP53 variants were analyzed for their association with ALL presenting features and treatment outcomes. Results We identified 49 unique nonsilent rare TP53 coding variants in 77 (2.0%) of 3,801 patients sequenced, of which 22 variants were classified as pathogenic. TP53 pathogenic variants were significantly over-represented in ALL compared with non-ALL controls (odds ratio, 5.2; P < .001). Children with TP53 pathogenic variants were significantly older at ALL diagnosis (median age, 15.5 years v 7.3 years; P < .001) and were more likely to have hypodiploid ALL (65.4% v 1.2%; P < .001). Carrying germline TP53 pathogenic variants was associated with inferior event-free survival and overall survival (hazard ratio, 4.2 and 3.9; P < .001 and .001, respectively). In particular, children with TP53 pathogenic variants were at a dramatically higher risk of second cancers than those without pathogenic variants, with 5-year cumulative incidence of 25.1% and 0.7% ( P < .001), respectively. Conclusion Loss-of-function germline TP53 variants predispose children to ALL and to adverse treatment outcomes with ALL therapy, particularly the risk of second malignant neoplasms.

Purpose Children with acute lymphoblastic leukemia (ALL) are generally instructed to take mercaptopurine (6-MP) in the evening and without food or dairy products. This study examines the association between 6-MP ingestion habits and 6-MP adherence, red cell thioguanine nucleotide (TGN) levels, and risk of relapse in children with TMPT wild-type genotype. Methods Participants included 441 children with ALL receiving oral 6-MP for maintenance. Adherence was monitored over 48,086 patient-days using the Medication Event Monitoring System; nonadherence was defined as adherence rate < 95%. 6-MP ingestion habits examined included: takes 6-MP with versus never with food, takes 6-MP with versus never with dairy, and takes 6-MP in the evening versus morning versus varying times. Results Median age at study was 6 years (range, 2 to 20 years); 43.8% were nonadherent. Certain 6-MP ingestion habits were associated with nonadherence (taking 6-MP with dairy [odds ratio (OR), 1.9; 95% CI, 1.3 to 2.9; P = .003] and at varying times [OR, 3.4; 95% CI, 1.8 to 6.3; P = .0001]). After adjusting for adherence and other prognosticators, there was no association between 6-MP ingestion habits and relapse risk (6-MP with food: hazard ratio [HR], 0.7; 95% CI, 0.3 to 1.9; P = .5; with dairy: HR, 0.3; 95% CI, 0.07 to 1.5; P = .2; taken in evening/night: HR, 1.1; 95% CI, 0.2 to 7.8; P = .9; at varying times: HR, 0.3; 95% CI, 0.04 to 2.7; P = .3). Among adherent patients, there was no association between red cell TGN levels and taking 6-MP with food versus without (206.1 ± 107.1 v 220.6 ± 121.6; P = .5), with dairy versus without (220.1 ± 87.8 v 216.3 ± 121.3; P =.7), or in the evening/night versus morning/midday versus varying times (218.8 ± 119.7 v 195.5 ± 82.3 v 174.8 ± 93.4; P = .6). Conclusion Commonly practiced restrictions surrounding 6-MP ingestion might not influence outcome but may hinder adherence. Future recommendations regarding 6-MP intake during maintenance therapy for childhood ALL should aim to simplify administration.

Purpose: Acute pancreatitis is one of the common causes of asparaginase intolerance. The mechanism is unknown, and genetic predisposition to asparaginase-induced pancreatitis has not been previously identified., Methods: To determine clinical risk factors for asparaginase-induced pancreatitis, we studied a cohort of 5,185 children and young adults with acute lymphoblastic leukemia, including 117 (2.3%) who were diagnosed with at least one episode of acute pancreatitis during therapy. A genome-wide association study was performed in the cohort and in an independent case-control group of 213 patients to identify genetic risk factors., Results: Risk factors associated with pancreatitis included genetically defined Native American ancestry (P < .001), older age (P < .001), and higher cumulative dose of asparaginase (P < .001). No common variants reached genome-wide significance in the genome-wide association study, but a rare nonsense variant rs199695765 in CPA2, encoding carboxypeptidase A2, was highly associated with pancreatitis (hazard ratio, 587; 95% CI, 66.8 to 5166; P = 9.0 × 10(-9)). A gene-level analysis showed an excess of additional CPA2 variants in patients who did versus those who did not develop pancreatitis (P = .001). Sixteen CPA2 single-nucleotide polymorphisms were associated (P < .05) with pancreatitis, and 13 of 24 patients who carried at least one of these variants developed pancreatitis. Biologic functions that were overrepresented by common variants modestly associated with pancreatitis included purine metabolism and cytoskeleton regulation., Conclusion: Older age, higher exposure to asparaginase, and higher Native American ancestry were independent risk factors for pancreatitis in patients with acute lymphoblastic leukemia. Those who inherit a nonsense rare variant in the CPA2 gene had a markedly increased risk of asparaginase-induced pancreatitis., (© 2016 by American Society of Clinical Oncology.)

Purpose: Mercaptopurine (MP) is the mainstay of curative therapy for acute lymphoblastic leukemia (ALL). We performed a genome-wide association study (GWAS) to identify comprehensively the genetic basis of MP intolerance in children with ALL., Patients and Methods: The discovery GWAS and replication cohorts included 657 and 371 children from two prospective clinical trials. MP dose intensity was a marker for drug tolerance and toxicities and was defined as prescribed dose divided by the planned protocol dose during maintenance therapy; its association with genotype was evaluated using a linear mixed-effects model., Results: MP dose intensity varied by race and ethnicity and was negatively correlated with East Asian genetic ancestry (P < .001). The GWAS revealed two genome-wide significant loci associated with dose intensity: rs1142345 in TPMT (Tyr240Cys, present in *3A and *3C variants; P = 8.6 × 10(-9)) and rs116855232 in NUDT15 (P = 8.8 × 10(-9)), with independent replication. Patients with TT genotype at rs116855232 were exquisitely sensitive to MP, with an average dose intensity of 8.3%, compared with those with TC and CC genotypes, who tolerated 63% and 83.5% of the planned dose, respectively. The NUDT15 variant was most common in East Asians and Hispanics, rare in Europeans, and not observed in Africans, contributing to ancestry-related differences in MP tolerance. Of children homozygous for either TPMT or NUDT15 variants or heterozygous for both, 100% required ≥ 50% MP dose reduction, compared with only 7.7% of others., Conclusion: We describe a germline variant in NUDT15 strongly associated with MP intolerance in childhood ALL, which may have implications for treatment individualization in this disease., (© 2015 by American Society of Clinical Oncology.)