ALBERT

Description: As a prototype of genomics-guided precision medicine, individualized thiopurine dosing based on pharmacogenetics is a highly effective way to mitigate hematopoietic toxicity of this class of drugs. Recently, NUDT15 deficiency was identified as a genetic cause of thiopurine toxicity, and NUDT15-informed preemptive dose reduction was quickly adopted in clinical settings. To exhaustively identify pharmacogenetic variants in this gene, we developed massively parallel NUDT15 function assays to determine the variants’ effect on protein abundance and thiopurine cytotoxicity. Of the 3,097 possible missense variants, we characterized the abundance of 2,922 variants and found 54 hotspot residues at which variants resulted in complete loss of protein stability. Analyzing 2,935 variants in the thiopurine cytotoxicity-based assay, we identified 17 additional residues where variants altered NUDT15 activity without affecting protein stability. We identified structural elements key to NUDT15 stability and/or catalytical activity with single amino acid resolution. Functional effects for NUDT15 variants accurately predicted toxicity risk alleles in patients treated with thiopurines with far superior sensitivity and specificity compared to bioinformatic prediction algorithms. In conclusion, our massively parallel variant function assays identified 1,152 deleterious NUDT15 variants, providing a comprehensive reference of variant function and vastly improving the ability to implement pharmacogenetics-guided thiopurine treatment individualization.

Description: There is growing evidence supporting an inherited basis for susceptibility to acute lymphoblastic leukemia (ALL) in children. In particular, we and others reported recurrent germline ETV6 variants linked to ALL risk, which collectively represent a novel leukemia predisposition syndrome. To understand the influence of ETV6 variation on ALL pathogenesis, we comprehensively characterized a cohort of 32 childhood leukemia cases arising from this rare syndrome. Of 34 nonsynonymous germline ETV6 variants in ALL, we identified 22 variants with impaired transcription repressor activity, loss of DNA binding, and altered nuclear localization. Missense variants retained dimerization with WT ETV6 with potentially dominate negative effects. Whole transcriptome and whole genome sequencing of this cohort of leukemia cases revealed a profound influence of germline ETV6 variants on leukemia transcriptional landscape, with distinct ALL subsets invoking unique patterns of somatic cooperating mutations. 70% of ALL cases with damaging germline ETV6 variants exhibited hyperdiploid karyotype with characteristic recurrent mutations in NRAS, KRAS, and PTPN11. In contrast, the remaining 30% cases had a diploid leukemia genome and an exceedingly high frequency of somatic copy number loss of PAX5 and ETV6, with a gene expression pattern that strikingly mirrored that of ALL with somatic ETV6-RUNX1 fusion. Two ETV6 germline variants gave rise to both AML and ALL, with lineage-specific genetic lesions in the leukemia genomes. ETV6 variants compromise its tumor suppressor activity in vitro with specific molecular targets identified by ATAC-seq profiling. ETV6-mediated ALL predisposition exemplifies the intricate interactions between inherited and acquired genomic variations in leukemia pathogenesis.

Description: Although somatically acquired genomic alterations have long been recognized as the hallmarks of acute lymphoblastic leukemia (ALL), the last decade has shown that inherited genetic variations (germline) are important determinants of interpatient variability in ALL susceptibility, drug response, and toxicities of ALL therapy. In particular, unbiased genome-wide association studies have identified germline variants strongly associated with the predisposition to ALL in children, providing novel insight into the mechanisms of leukemogenesis and evidence for complex interactions between inherited and acquired genetic variations in ALL. Similar genome-wide approaches have also discovered novel germline genetic risk factors that independently influence ALL prognosis and those that strongly modify host susceptibility to adverse effects of antileukemic agents (eg, vincristine, asparaginase, glucocorticoids). There are examples of germline genomic associations that warrant routine clinical use in the treatment of childhood ALL (eg, TPMT and mercaptopurine dosing), but most have not reached this level of actionability. Future studies are needed to integrate both somatic and germline variants to predict risk of relapse and host toxicities, with the eventual goal of implementing genetics-driven precision-medicine approaches in ALL treatment.

Description: Acute lymphoblastic leukemia (ALL) in children is a prototype of cancer that can be cured by chemotherapy alone. However, the molecular mechanisms for anti-leukemic drug sensitivity and genetic basis of inter-patient variability in treatment response are not fully understood. Taking a genome-wide approach, we recently identified genetic variants in the ARID5B gene that strongly predispose children to developing ALL and also a high risk of relapse following therapy (J Clin Oncol 2012 30:751, Nat Genet 2009 41:1001). To understand the mechanisms by which ARID5B is linked to treatment outcome in childhood ALL, we sought to 1) characterize ARID5B expression in different genetic subtypes of ALL, 2) determine the effects of ARID5B expression on cytotoxicity of chemotherapeutic agents commonly used in ALL therapy, and 3) describe molecular pathways linking ARID5B to anti-leukemic drug sensitivity. In 567 children with newly diagnosed ALL treated at St. Jude Children’s Research Hospital (GSE33315), ARID5B expression was highest in cases with hyperdiploid karyotype (〉50 chromosomes) and lowest in T-cell ALL and cases with MLL rearrangements. This pattern was validated in an independent cohort of 106 children from the Dutch Childhood Oncology Group (GSE13351). In 59 patients treated on the Children’s Oncology Group (COG) CCG1961 trial, lower ARID5B expression was associated with higher rates of relapse (P=0.01, GSE7440). Importantly, when we compared matched newly-diagnosed vs. relapsed ALL blasts from a cohort of 60 patients enrolled in COG trials (GSE28460), ARID5B expression was further downregulated at disease recurrence (P=0.0009). shRNA-mediated ARID5B knockdown in 3 ALL cell lines (Nalm6, SEM, and UOCB-1) substantially increased resistance to antimetabolites (an average of 5.16 and 35.3-fold increase in IC50 for methotrexate [MTX] and 6-mercaptopurine [6MP], respectively), with minimal effects on glucocorticoids, vincristine, asparaginase, and daunorubicin. Because cytotoxic effects of MTX and 6MP are highly dependent on the rate of cell proliferation, we postulate that ARID5B directly influences cell cycle entry. In all 3 cell lines, ARID5B knockdown led to significant blockade at the G1/S checkpoint, increasing the percent of cells in G0/G1 phase. At the molecular level, downregulation of ARID5B resulted in higher levels of p21 and reduction in phosphorylated Rb, consistent with the retention at G0/G1 phase. ARID5B expression was restricted to nucleus but affected both nuclear and cytoplasmic p21 expression in a time-dependent fashion. Interestingly, there was a highly significant negative correlation between p21 expression and MTX- and 6MP-induced apoptosis in all 3 ALL cell lines. Taken together, we hypothesize that lower expression of ARID5B impairs ALL cell cycling by upregulating p21, contributing to resistance to MTX and 6MP and eventually leukemia relapse. Finally, we compared global gene expression in ARID5B knockdown vs. control ALL cells, and via the Connectivity Map analysis we identified histone deacetylase (HDAC) inhibitors as promising agents for overcoming ARID5B-related drug resistance. Indeed, ARID5B knockdown cells were significantly more sensitive to panobinostat than controls, suggesting HDAC inhibitors as potential therapeutic options for patients with ARID5B-deficient and drug resistant ALL. Disclosures No relevant conflicts of interest to declare.

Description: Background Relapse remains as a formidable challenge for acute lymphoblastic leukemia (ALL), particularly because it is associated with dramatic drug resistance and thus dismal prognosis. It is crucial to understand the genetic basis of relapsed ALL to develop new therapeutic strategies. Recently, somatic mutations in NT5C2 were identified as one of the most common genomic lesions specific to relapsed ALL and were directly linked to thiopurine resistance (Nat Genet 2013, Nat Med 2013, Nat Commun 2015). A nucleotidase, NT5C2 dephosphorylates purine monophosphate nucleotides and is important for cellular nucleic acid homeostasis. However, the exact mechanisms by which NT5C2 mutations influence thiopurine resistance in ALL are not completely understood. Methods Wildtype and mutant human NT5C2 (p.R39Q, p.R238W, and p.R367Q) were expressed in E. Coli and purified by affinity chromatography followed by gel filtration. Nucleotidase activity was measured by quantifying the release of free phosphate with the Malachite Green assay. NT5C2 enzyme kinetics (Vmax and Km) were determined for endogenous purine nucleotide substrates (inosine monophosphate [IMP] and guanosine monophosphate [GMP]) and thiopurine drug metabolite substrates (thioinosine monophosphate [TIMP] and thioguanosine monophosphate [TGMP]). Human ALL cell lines (Nalm6 and REH) stably overexpressing wildtype or mutant NT5C2 were established by lentiviral transduction. Thiopurine transport was characterized in vitro by quantifying the influx and efflux of 14 C labeled mercaptopurine (MP) and its metabolites. Results To characterize NT5C2 substrate specificity, we first determined the nucleotidase activity of wildtype and mutant proteins with endogenous purine nucleotides (IMP and GMP) and thiopurine metabolites (TIMP and TGMP). Wildtype NT5C2 exhibited nucleotidase activity against all 4 nucleotide metabolites, but IMP and TIMP were clearly the preferred substrates with up to 4-fold higher reaction velocities compared to GMP and TGMP, respectively. All leukemia-derived NT5C2 mutations resulted in substantial increase in nucleotidase activity across substrates, with the p.R367Q mutant consistently exhibiting the greatest gain of activity. By comparison, mutant NT5C2 proteins showed similar substrate preference of IMP over GMP (e.g., reaction rate of 26.6 pmol/min and 10.4 pmol/min for IMP and GMP, respectively, with the p.R367Q NT5C2). However, this inosine over guanosine bias in substrate specificity was reversed with thiopurine metabolites: leukemia-derived mutant NT5C2s were substantially more efficient to dephosphorylate TGMP than TIMP (e.g., reaction rate of 27.8 pmol/min and 41.5 pmol/min for TIMP and TGMP, respectively, with the p.R367Q NT5C2). The "mutant to wildtype" ratio of NT5C2 nucleotidase activity (e.g., the relative increase of Vmax) was significantly greater for thiopurine metabolites than what was observed when endogenous purine nucleotides were used as substrates (e.g., 15.2- and 1.6-fold increase in Vmax for TIMP and IMP with the p.R367Q NT5C2). These results suggest that ALL relapse-associated NT5C2 mutations likely confer not only increase in endogenous nucleotidase activity but also novel enzymatic functions that specially affect thiopurine metabolism. In addition to the increase in thiopurine resistance, human ALL cell lines expressing mutant NT5C2 also showed significant reduction in intracellular levels of thiopurine metabolites after drug treatment, pointing to influence of NT5C2 mutations on thiopurine drug transport. Using 14 C labeled MP, we traced both the influx and efflux of drug metabolites in isogenic cell lines expressing wildtype or mutant NT5C2 s. In both Nalm6 and REH cells, intracellular uptake of MP was reduced by 51% in mutant cells compared to cells with wildtype protein. In contrast, the level of MP metabolites in the extracellular medium was 3 times higher with mutant cells, reflecting increased drug efflux. Therefore, expression of mutant NT5C2 resulted in a net reduction of intracellular exposure to thiopurine, suggesting disturbance of the drug transport pathway as a distinct mechanism for NT5C2-mediated thiopurine resistance. Conclusions NT5C2 mutations specifically influence thiopurine metabolism and transport, and therefore are critical determinants for drug resistance in relapsed ALL. Disclosures No relevant conflicts of interest to declare.

Description: Introduction 6-mercaptopurine (6-MP) is a main component of childhood acute lymphoblastic leukemia (ALL) therapy. The sensitivity of 6-MP is associated with genetic variant of 6-MP metabolism. Recently, the NUDT15 genetic variant has been identified as a risk factor of 6-MP intolerability, and its association with 6-MP-induced toxicities and 6-MP dose in ALL patients have been reported. The frequency of NUDT15 hypomorphic variant is higher in Asian populations than in European and African populations. However, the 6-MP tolerable dose and efficacy for NUDT15-deficient patients remains clear. Our study aimed to evaluate 6-MP tolerable dose, the frequencies of 6-MP induced toxicities, and outcome in 17 ALL patients with NUDT15-deficient genotype. Methods We genotyped NUDT15 genetic variants and evaluated the patients with NUDT15 homozygous variant in Japanese childhood ALL. The NUDT15 variants V18_V19insGV, V18I, R139C, and R139H were genotyped by Sanger sequencing, and the diplotype was precisely determined. The standard initiation dose of maintenance therapy was 6-MP 40 to 50 mg/m2/day and methotrexate 25 mg/m2/week. The 6-MP-induced toxicities were graded by CTCAE version 4.0. The survival rate was estimated by the log-rank test. Results A total of 17 patients with NUDT15 diplotype of *2/*2, *2/*3, *2/*5, *3/*3, *3/*5, and *5/*5 were genotyped as NUDT15 deficient. Fifteen patients were B cell-precursor (BCP) ALL and 2 patients were T-ALL. Of the 15 BCP ALL patients, 13 were standard risk and 2 were high risk patients according to National Cancer Institute/Rome criteria. Grade 3 leukopenia and grade 4 neutropenia were observed in all 17 patients, and the median observation time were 33 (range 3-95) days and 35 (20-137) days after initiating maintenance therapy, respectively. Grade 3 ALT elevation was observed in 6 patients (35%), and median observation time was 47 (range 19-427) days after initiating maintenance therapy. Moreover, during the early consolidation phase with 6-MP, severe myelosuppression was observed in 11 of these patients. The average 6-MP dose during maintenance therapy was 7.0 (range 2.7-18.3) mg/m2/day. Moreover, 16 of these 17 patients (94%) with NUDT15 deficiency required median 66 (range 5-376) days of therapy interruption. Notably, the average 6-MP dose was 18.3 mg/m2/day, and no therapy interruption occurred during maintenance therapy in patients with NUDT15 *5/*5 diplotype. Therefore, the degree of NUDT15 deficiency influenced 6-MP tolerable dose. The effect of NUDT15 deficiency on treatment outcome was evaluated in 14 patients, who completed treatment. Three patients relapsed at 124-388 days, and two of these three patients died at 877 and 959 days after the end of maintenance therapy, respectively. The overall and event-free survival rate at 4 years were 0.75 and 0.63, respectively. Neither the average 6-MP dose nor the interruption duration was associated with these events. Conclusions NUDT15-deficient genotypes strongly influence intolerability. Patients with NUDT15 deficiency did not tolerate standard 6-MP dose, and physicians should consider reducing 6-MP dose to 7 mg/m2 to avoid therapy interruption. Conversely, NUDT15 *5/*5 genotype displayed only mild NUDT15 deficiency, and the patients with this genotype tolerated 40% of the standard 6-MP dose. Further large-scale studies should be conducted to assess the NUDT15 variant's effect on survival. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

Description: Key Points We established a Nudt15 knockout mouse model with which to evaluate individualized thiopurine therapy. Preemptive NUDT15 genotype–guided thiopurine dosing can effectively prevent drug toxicity without compromising antileukemic efficacy.

Description: Key Points We identified 3 novel loss-of-function variants in NUDT15 linked to thiopurine intolerance. Our findings extended the importance of NUDT15 variation in thiopurine pharmacogenetics in diverse populations.

Description: While acute lymphoblastic leukemia (ALL) is a prototype of cancer that can be cured by chemotherapy alone, current ALL treatment regimens rely primarily on conventional cytotoxic agents with significant acute and long-term side effects. Better understanding of genomic landscape of ALL is critical for developing molecularly targeted therapy and implementing genomics-based precision medicine in this cancer. In particularly, sentinel chromosomal translocations are common in ALL and often involve key transcription factors important for hematopoiesis. Epigenetic regulator genes are also frequently targeted by somatic genomic alterations such as sequence mutations (e.g., CREBBP) and gene fusions (e.g., MLL, EP300). To comprehensively define transcriptomic abnormalities in childhood ALL, we performed RNA-seq of an unselected cohort of 231 children enrolled on the MaSpore frontline ALL protocols in Singapore or Malaysia. In total, we identified 58 putatively functional and predominant fusion genes in 125 patients (54.1%), the majority of which have not been reported previously. In particular, we described a distinct ALL subtype with a characteristic gene expression signature driven by chromosomal rearrangements of the ZNF384 gene with different partners (i.e., histone acetyl-transferases EP300 and CREBBP, TAF15, and TCF3). In 9 of 11 ALL cases with ZNF384 rearrangements, the breakpoint in this gene was invariably between exon 2 and exon 3, resulting in deletion of the 5'-UTR and then in-frame fusion of the entire ZNF384 coding sequence with the partner genes. The top two most significantly up-regulated genes in the ZNF384-rearranged group were CLCF1 and BTLA, whose expression levels were 15.5- and 15.0-fold higher than in ALL cases with wildtype ZNF384, respectively. In fact, ZNF384 binding was identified within the CLCF1 and BTLA loci (particularly the promoter regions) by chromatin immunoprecipitation sequencing in B lymphoblasoid cells. Using luciferase transcription driven by CLCF1 promoter in HEK293T cells as a model system, we observed significantly greater transcription activity with EP300-ZNF384 fusion compared to cells expressing wildtype ZNF384, suggesting that this chimeric gene resulted in gain of ZNF384 function. Similar results were obtained with luciferase transcription assay driven by the BTLA promoter. In human ALL cells, CLCF1 and BTLA promoter activities were consistently and significantly higher in ZNF384-rearranged ALL than in ALL cell line with wildtype ZNF384. To examine the effects of ZNF384 fusion on hematopoietic stem and progenitor cell (HSPCs) function, we also evaluated colony forming potential of HSPC in vitro upon ectopic expression of ZNF384 fusions. While there was marked suppression of colonies from myeloid and erythoid lineages, expression of EP300-ZNF384 or CREBBP-ZNF384 significantly stimulated preB cell colony formation. However, neither EP300- nor CREBBP-ZNF384 fusion was able to transform mouse hematopoietic precursor cell Ba/f3 in vitro, but instead increased the transforming potential of other oncogenic mutations (NRASG12D). EP300-ZNF384 and CREBBP-ZNF384 fusion proteins lacked the histone acetyltransferase (HAT) domain, and showed only 25% and 10% of HAT activity of full-length EP300 and CREBBP, respectively, with dominant-negative effects. Also, expression of EP300-ZNF384 led to significant decrease in global H3 acetylation in Ba/f3 cells in vitro. Finally, in NRASG12D-transformed Ba/f3 cells, co-expression of EP300-ZNF384 or CREBBP-ZNF384 substantially potentiated cytotoxic effects of histone deacetylase inhibitor vorinostat. Similarly, in a panel of human ALL cell lines, ZNF384-rearrangement was also associated with increased sensitivity to vorinostat, suggesting that some ZNF384-rearranged ALL may benefit from therapeutic agents targeting histone acetylation regulation. In conclusion, our results indicate that gene fusion is the major class of genomic abnormalities in childhood ALL and chromosomal rearrangements involving EP300 and CREBBP may cause global epigenetic deregulation in ALL with potentials for therapeutic targeting. Disclosures No relevant conflicts of interest to declare.