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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 wild-type ETV6 with potentially dominant-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 acute myeloid leukemia 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 assay for transposase-accessible chromatin sequencing profiling. ETV6-mediated ALL predisposition exemplifies the intricate interactions between inherited and acquired genomic variations in leukemia pathogenesis.

Acute lymphoblastic leukemia (ALL) is the most common malignancy in children. Characterized by high levels of Native American ancestry, Hispanics are disproportionally affected by this cancer with high incidence and inferior survival. However, the genetic basis for this disparity remains poorly understood because of a paucity of genome-wide investigation of ALL in Hispanics. Performing a genome-wide association study (GWAS) in 940 Hispanic children with ALL and 681 ancestry-matched non-ALL controls, we identified a novel susceptibility locus in the ERG gene (rs2836365; P = 3.76 × 10−8; odds ratio [OR] = 1.56), with independent validation (P = .01; OR = 1.43). Imputation analyses pointed to a single causal variant driving the association signal at this locus overlapping with putative regulatory DNA elements. The effect size of the ERG risk variant rose with increasing Native American genetic ancestry. The ERG risk genotype was underrepresented in ALL with the ETV6-RUNX1 fusion (P < .0005) but enriched in the TCF3-PBX1 subtype (P < .05). Interestingly, ALL cases with germline ERG risk alleles were significantly less likely to have somatic ERG deletion (P < .05). Our results provide novel insights into genetic predisposition to ALL and its contribution to racial disparity in this cancer.

Publisher's Note: There is an [Inside Blood Commentary][1] on this article in this issue. To the editor: Our understanding of acute lymphoblastic leukemia (ALL) has expanded tremendously in the past few years because of large-scale genomic studies.[1][2],[2][3] ALL is characterized by a relatively

Acute lymphoblastic leukemia (ALL) is the most common malignancy in children. Characterized by high levels of Native American ancestry, Hispanics are disproportionally affected by this cancer with high incidence and inferior survival. However, the genetic basis for this disparity remains poorly understood because of a paucity of genome-wide investigation of ALL in Hispanics. Performing a genome-wide association study (GWAS) in 940 Hispanic children with ALL and 681 ancestry-matched non-ALL controls, we identified a novel susceptibility locus in the

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.

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.

Abstract 3866 Immunoproteasome is a special form of proteasome which contains three unique interferonγ (IFNγ) induced catalytic subunits, i.e. PSMB8, PSMB9 and PSMB10. Immunoproteasome plays a pivotal role in generating certain peptide antigens for MHC class I presentation. Dysregulation of the immunoproteasome system may contribute to the pathogenesis of certain types of malignancies, including leukemia. Our previous study has identified the target genes of PML/RARa, the initiating factor of acute promyelocytic leukemia (APL) on the genome-wide scale, and demonstrated that PML/RARa could selectively target PU.1-regulated genes, which is a critical mechanism for the pathogenesis of APL. PSMB10, encoding an important composition of immunoproteasome, is one of the identified target genes which are regulated by PML/RARa in this manner. Here we revealed the detailed transcriptional regulation mechanism of PSMB10 in APL. Chromatin immunoprecipitation (ChIP)-PCR assay showed that PML/RARa and PU.1 could bind to the PSMB10 promoter in APL cells, including patient derived NB4 cells and Zn-treated PR9 cells. Re-ChIP assay further demonstrated that PML/RARa and PU.1 co-existed on the same DNA fragment of the PSMB10 promoter, which provided the possibility that PML/RARa and PU.1 could co-regulate the PSMB10 promoter. Using a transient luciferase reporter system, we found that PU.1 transactivated the PSMB10 promoter and PML/RARa repressed the PU.1-dependent transactivation. All-trans retinoic acid (ATRA) could relief the repression caused by PML/RARa. To further demonstrate that the PU.1 site (-37bp∼-29bp) and related retinoic acid response elements (RAREs) (-555bp∼-549bp, -258bp∼-252bp) were essential for PML/RARa to function as an effective repressor, we prepared a series of mutant constructs, including the PU.1-site mutant, the construct mutated on both RARE half (RAREh) sites and two constructs respectively mutated on one of the two RAREh sites, and then transfected them into myeloid U937 cells. From the results of luciferase reporter assays, we found that both PU.1 site and RAREh sites played important roles in PML/RARa-mediated transcriptional repression, moreover, the second RAREh site (-258bp∼-252bp) contributed more than the first one (-555bp∼-549bp). Through electrophoretic mobility shift assay (EMSA), we further determined that PML/RARa could interact with PU.1 through protein-protein interaction, and then bind to the PU.1 site on the PSMB10 promoter. Recent study has shown that ATRA treatment could induce the production of anti-PML/RARa in APL mouse, which implicates that ATRA plays an important role in activating immune system. As the essential elements for immune response, HLA class I antigens (A, B & C) present peptides, which are produced from digested proteins degraded by immunoproteasome, to the surface of antigen-presenting cells. We thus utilized real time RT-PCR to measure the expression of PSMB10 and HLA-A/B/C during ATRA-induced NB4 cells differentiation. We found the levels of PSMB10 and HLA-A/B/C expression were up-regulated in ATRA-treated NB4 cells. These results suggested that the enhanced expression of PSMB10 availed immunoproteasome restoration, which benefited the reactivation of immune system during ATRA treatment therapy. Our results not only demonstrate the detailed transcriptional regulation of PSMB10 in APL but imply the potential function of PSMB10 during ATRA treatment as well. Disclosures: No relevant conflicts of interest to declare.