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Background There is growing evidence for the inherited basis of susceptibility to childhood acute lymphoblastic leukemia (ALL). Genome-wide association studies have identified non-coding ALL risk variants at the ARID5B gene locus, but their exact functional effects and the molecular mechanism linking ARID5B to B-cell ALL leukemogenesis remain largely unknown. Methods We performed targeted sequencing of ARID5B in germline DNA of 5008 children with ALL. Variants were evaluated for association with ALL susceptibility using 3644 patients from the UK10K cohort as non-ALL controls, under an additive model. Cis-regulatory elements in ARID5B were systematically identified using dCas9-KRAB–mediated enhancer interference system enhancer screen in ALL cells. Disruption of transcription factor binding by ARID5B variant was predicted informatically and then confirmed using chromatin immunoprecipitation and coimmunoprecipitation. ARID5B variant association with hematological traits was examined using UK Biobank dataset. All statistical tests were 2-sided. Results We identified 54 common variants in ARID5B statistically significantly associated with leukemia risk, all of which were noncoding. Six cis-regulatory elements at the ARID5B locus were discovered using CRISPR-based high-throughput enhancer screening. Strikingly, the top ALL risk variant (rs7090445, P = 5.57 × 10–45) is located precisely within the strongest enhancer element, which is also distally tethered to the ARID5B promoter. The variant allele disrupts the MEF2C binding motif sequence, resulting in reduced MEF2C affinity and decreased local chromosome accessibility. MEF2C influences ARID5B expression in ALL, likely via a transcription factor complex with RUNX1. Using the UK Biobank dataset (n = 349 861), we showed that rs7090445 was also associated with lymphocyte percentage and count in the general population (P = 8.6 × 10–22 and 2.1 × 10–18, respectively). Conclusions Our results indicate that ALL risk variants in ARID5B function by modulating cis-regulatory elements at this locus.

Relapse remains a formidable challenge for acute lymphoblastic leukemia (ALL). Recently, recurrent mutations in NT5C2 were identified as a common genomic lesion unique in relapsed ALL and were linked to acquired thiopurine resistance. However, molecular mechanisms by which NT5C2 regulates thiopurine cytotoxicity were incompletely understood. To this end, we sought to comprehensively characterize the biochemical and cellular effects of NT5C2 mutations. Compared with wild-type NT5C2, mutant proteins showed elevated 5′-nucleotidase activity with a stark preference of thiopurine metabolites over endogenous purine nucleotides, suggesting neomorphic effects specific to thiopurine metabolism. Expression of mutant NT5C2 mutations also significantly reduced thiopurine uptake in vitro with concomitant increase in efflux of 6-mercaptopurine (MP) metabolites, plausibly via indirect effects on drug transporter pathways. Finally, intracellular metabolomic profiling revealed significant shifts in nucleotide homeostasis induced by mutant NT5C2 at baseline; MP treatment also resulted in global changes in metabolomic profiles with completely divergent effects in cells with mutant versus wild-type NT5C2. Collectively, our data indicated that NT5C2 mutations alter thiopurine metabolism and cellular disposition, but also influence endogenous nucleotide homeostasis and thiopurine-induced metabolomic response. These complex mechanisms contributed to NT5C2-mediated drug resistance in ALL and pointed to potential opportunities for therapeutic targeting in relapsed ALL.

This supplementary table describes metabolite levels and statistics from metabolite experiments

Supplementary figure 1 and 2 describe protein purity and expression. Supplementary Figure 3 shows cell-based cytotoxic assays. Supplementary Figure 4 shows protein activity against several substrates. Supplementary Figure 5 describes levels of individual metabolites.

Table S1 shows that downregulation of ARID5B led to more increases in LC50 of MTX and 6-MP than other antileukemic drugs

Figure S1 shows the knock-down effect of ARID5B with different shRNAs in Nalm6 cells; Figure S2 shows the correlation between MTX and 6-MP sensitivity with different levels of ARID5B knock-down in Nalm6 cells; Figure S3 shows that down-regulation of ARID5B mediated MTX and 6-MP drug resistance can be reversed by re-expression of ARID5B; Figure S4 shows that the levels of MTX and 6-MP active metabolites were reduced significantly after ARID5B knockdown; Figure S5 shows similar MTX and 6-MP drug resistance after CRISPR-mediated ARID5B downregulation; Figure S6 shows that decreased cell growth was detected by BrdU-uptake assay; Figure S7 shows treatment with increased concentrations of MTX and 6-MP led to gradually decreased p21 level; Figure S8 shows that a potential ARID5B binding site 25kb distal to CDKN1A locus was identified by ATAC-seq signal and Histone modification marks; Figure S9 shows an enrichment of components of the p53 signaling pathway was differentially expressed upon ARID5B knockdown.

Purpose:Treatment outcomes for childhood acute lymphoblastic leukemia (ALL) have improved steadily, but a significant proportion of patients still experience relapse due to drug resistance, which is partly explained by inherited and/or somatic genetic alternations. Recently, we and others have identified genetic variants in the ARID5B gene associated with susceptibility to ALL and also with relapse. In this study, we sought to characterize the molecular pathway by which ARID5B affects antileukemic drug response in patients with ALL.Experimental Design:We analyzed association of ARID5B expression in primary human ALL blasts with molecular subtypes and treatment outcome. Subsequent mechanistic studies were performed in ALL cell lines by manipulating ARID5B expression isogenically, in which we evaluated drug sensitivity, metabolism, and molecular signaling events.Results:ARID5B expression varied substantially by ALL subtype, with the highest level being observed in hyperdiploid ALL. Lower ARID5B expression at diagnosis was associated with the risk of ALL relapse, and further reduction was noted at ALL relapse. In isogenic ALL cell models in vitro, ARID5B knockdown led to resistance specific to antimetabolite drugs (i.e., 6-mercaptopurine and methotrexate), without significantly affecting sensitivity to other antileukemic agents. ARID5B downregulation significantly inhibited ALL cell proliferation and caused partial cell-cycle arrest. At the molecular level, the cell-cycle checkpoint regulator p21 (encoded by CDKN1A) was most consistently modulated by ARID5B, plausibly as its direct transcription regulation target.Conclusions:Our data indicate that ARID5B is an important molecular determinant of antimetabolite drug sensitivity in ALL, in part, through p21-mediated effects on cell-cycle progression.

Blinatumomab is an efficacious immunotherapeutic agent in B cell acute lymphoblastic leukemia (B-ALL). However, the pharmacogenomic basis of leukemia response to blinatumomab is unclear. Using genome-wide CRISPR, we comprehensively identified leukemia intrinsic factors of blinatumomab sensitivity, i.e., the loss ofCD58as a top driver for resistance, in addition toCD19. Screening 1639 transcription factor genes, we then identified PAX5 as the key activator of CD58. ALL with thePAX5P80R mutation also expressed the lowest level ofCD58among 20 ALL molecular subtypes in 1988 patients. Genome editing confirmed the effects of this mutation on CD58 expression and blinatumomab sensitivity in B-ALL, with validation in patient leukemic blasts. We described a PAX5-driven enhancer at theCD58locus, which was disrupted by PAX5 P80R, and the loss of CD58 abolished blinatumomab-induced T cell activation with global changes in transcriptomic/epigenomic program. In conclusion, we identified previously unidentified genetic mechanisms of blinatumomab resistance in B-ALL, suggesting strategies for genomics-guided treatment individualization.

Background A phase 1 study was conducted to determine the maximum tolerated dose of bendamustine when given in combination with clofarabine, etoposide, and dexamethasone daily for 5 days in children and adolescents with relapsed or refractory hematologic malignancies. Methods Patients younger than 22 years with second or greater relapsed or refractory acute leukemia or lymphoma after 2 or more prior regimens were eligible. With the rolling 6 design, participants received escalating doses of bendamustine (30, 40, or 60 mg/m2 /d) in combination with clofarabine (40 mg/m2 ), etoposide (100 mg/m2 ), and dexamethasone (8 mg/m2 ) daily for 5 days. Optional pharmacokinetic studies were performed in cycle 1 on day 1 and day 5. Results Sixteen patients were enrolled. Six patients were treated at the dose level of 30 mg/m2 /d, 6 were treated at the dose level of 40 mg/m2 /d, and 4 were treated at the dose level of 60 mg/m2 /d. The dose-limiting toxicity was prolonged myelosuppression. The combination was otherwise well tolerated. The recommended dose of bendamustine in this combination was 30 mg/m2 /d for 5 days. Ten responses were observed after 1 cycle: 6 complete remissions, 1 durable minimal residual disease-negative complete remission without platelet recovery in a patient with early T-cell precursor leukemia, and 3 partial remissions. Six patients proceeded to transplantation. The event-free survival rate was 40.6% (95% confidence interval [CI], 17.5%-63.7%) at 1 year and 33.9% (95% CI, 11.9%-55.9%) at 3 years. Conclusions Bendamustine is well tolerated in combination with clofarabine, etoposide, and dexamethasone. The combination administered over 5 days is effective for multiple relapsed and refractory hematologic malignancies. This trial is registered with ClinicalTrials.gov (NCT01900509). Lay summary Improvements to the existing chemotherapy regimen are still needed for patients who relapse after targeted therapies and immunotherapies and for those who are not eligible for or have no access to such therapies. A regimen combining cyclophosphamide, clofarabine, and etoposide has been used in relapsed and refractory pediatric patients with hematologic malignancies. This study shows that substituting bendamustine for cyclophosphamide in combination with clofarabine and etoposide is safe and effective.

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.

Financial extreme jumps in asset price may propagate across stock markets and lead to the market-wide crashes, which severely threatens the stability of the financial system. In order to analyzing the contagion features of jump tail risk, this paper proposes a mutually exciting contagion model based on Hawkes process with intraday high frequency data. We use a simple two-stage method that first extracts the jump component nonparametrically from the high frequency data and then models the intraday jump tail using mutually exciting Hawkes process. Moreover, we take both the occurrence time and magnitude of jump into account in modeling the conditional intensity of Hawkes process. The proposed method is applied to the five-minute high frequency data of the Chinese stock market. The empirical results show that, for the two main Chinese stock markets, only background intensity is significant in the Shanghai stock market, while mutually exciting effect is significant in the Shenzhen stock market. Both the location and size of jump in the Shanghai stock market have significant stimulation to the next occurrences of jump in the Shenzhen stock market. Furthermore, the proposed model performs very well in predicting the future jump tail events.

Purpose: Treatment outcomes for childhood acute lymphoblastic leukemia (ALL) have improved steadily, but a significant proportion of patients still experience relapse due to drug resistance, which is partly explained by inherited and/or somatic genetic alternations. Recently, we and others have identified genetic variants in the ARID5B gene associated with susceptibility to ALL and also with relapse. In this study, we sought to characterize the molecular pathway by which ARID5B affects antileukemic drug response in patients with ALL. Experimental Design: We analyzed association of ARID5B expression in primary human ALL blasts with molecular subtypes and treatment outcome. Subsequent mechanistic studies were performed in ALL cell lines by manipulating ARID5B expression isogenically, in which we evaluated drug sensitivity, metabolism, and molecular signaling events. Results: ARID5B expression varied substantially by ALL subtype, with the highest level being observed in hyperdiploid ALL. Lower ARID5B expression at diagnosis was associated with the risk of ALL relapse, and further reduction was noted at ALL relapse. In isogenic ALL cell models in vitro, ARID5B knockdown led to resistance specific to antimetabolite drugs (i.e., 6-mercaptopurine and methotrexate), without significantly affecting sensitivity to other antileukemic agents. ARID5B downregulation significantly inhibited ALL cell proliferation and caused partial cell-cycle arrest. At the molecular level, the cell-cycle checkpoint regulator p21 (encoded by CDKN1A) was most consistently modulated by ARID5B, plausibly as its direct transcription regulation target. Conclusions: Our data indicate that ARID5B is an important molecular determinant of antimetabolite drug sensitivity in ALL, in part, through p21-mediated effects on cell-cycle progression.

Background Previous clinical studies have suggested that trimethylamine-N-oxide (TMAO) could contribute to the development of atherosclerosis cardiovascular disease. However, the synthetic analysis in coronary heart disease (CHD) was not yet performed. We aimed to clarify the relationship between elevated plasma concentrations of TMAO and the incidence of major adverse cardiovascular events (MACE) in CHD patients. Methods Meta-analysis and dose-response analysis of hazard ratio data from prospective observational studies reporting on the association between TMAO plasma concentrations and the incidence of MACE in patients with CHD were conducted. Results Of the 2369 published articles identified in the search, seven papers, with data from nine cohort studies (10,301 patients), were included in the meta-analysis. Combined data showed that elevated plasma TMAO concentrations could increase 58% higher risk of MACE in patients with CHD (hazard ratios [HR]: 1.58; 95% confidence interval [CI] = 1.35–1.84, P = 0.000). For follow-up ≥ 1 year, it was associated with 62% higher risk of MACE in patients with longer-term than shorter-term (HR for follow-up ≥ 4 years: 1.96; 95% CI = 1.52–2.52 vs one to 3 years: 1.34; 95% CI = 1.26–1.43, P = 0.004). The dose-response analysis revealed a ‘J’ shaped association between TMAO concentration and the incidence of MACE (P = 0.033), with the concentration above 5.1 μmol/L being associated with HR of > 1. Conclusions Elevated levels of TMAO are associated with an increased incidence of MACE in patients with CHD. TMAO concentration of 5.1 μmol/L may be a cut-off value for prognosis.

Loss of function of CDKN2A / B , also known as INK4 / ARF [encoding p16 INK4A , p15 INK4B , and p14 ARF (mouse p19 Arf )], confers susceptibility to cancers, whereas its up-regulation during organismal aging provokes cellular senescence and tissue degenerative disorders. To better understand the transcriptional regulation of p16 INK4A , a CRISPR screen targeting open, noncoding chromatin regions adjacent to p16 INK4A was performed in a human p16 INK4A-P2A-mCherry reporter cell line. We identified a repressive element located in the 3′ region adjacent to the ARF promoter that controls p16 INK4A expression via long-distance chromatin interactions. Coinfection of lentiviral dCas9-KRAB with selected single-guide RNAs against the repressive element abrogated the ARF / p16 INK4A chromatin contacts, thus reactivating p16 INK4A expression. Genetic CRISPR screening identified candidate transcription factors inhibiting p16 INK4A regulation, including ZNF217, which was confirmed to bind the ARF / p16 INK4A interaction loop. In summary, direct physical interactions between p16 INK4A and ARF genes provide mechanistic insights into their cross-regulation.

FLT3 is an attractive therapeutic target in acute lymphoblastic leukemia (ALL) but the mechanism for its activation in this cancer is incompletely understood. Profiling global gene expression in large ALL cohorts, we identify over-expression of FLT3 in ZNF384-rearranged ALL, consistently across cases harboring different fusion partners with ZNF384. Mechanistically, we discover an intergenic enhancer element at the FLT3 locus that is exclusively activated in ZNF384-rearranged ALL, with the enhancer-promoter looping directly mediated by the fusion protein. There is also a global enrichment of active enhancers within ZNF384 binding sites across the genome in ZNF384-rearranged ALL cells. Downregulation of ZNF384 blunts FLT3 activation and decreases ALL cell sensitivity to FLT3 inhibitor gilteritinib in vitro. In patient-derived xenograft models of ZNF384-rearranged ALL, gilteritinib exhibits significant anti-leukemia efficacy as a monotherapy in vivo. Collectively, our results provide insights into FLT3 regulation in ALL and point to potential genomics-guided targeted therapy for this patient population.

Genetic alterations in the RUNX1 gene are associated with benign and malignant blood disorders, particularly of megakaryocyte and myeloid lineages. The role of RUNX1 in acute lymphoblastic leukemia (ALL) is less clear, particularly in terms of how germline genetic variation influences the predisposition to this type of leukemia. Sequencing DNA of 4836 children with B cell ALL (B-ALL) and 1354 with T cell ALL (T-ALL), we identified 31 and 18 germline RUNX1 variants, respectively. RUNX1 variants in B-ALL consistently showed minimal damaging effects. In contrast, 6 T-ALL–related variants resulted in drastic loss of RUNX1 activity as a transcription activator in vitro. Ectopic expression of dominant-negative RUNX1 variants in human CD34(+) cells repressed differentiation into erythroid cells, megakaryocytes, and T cells, while promoting myeloid cell development. Chromatin immunoprecipitation sequencing of T-ALL models showed distinctive patterns of RUNX1 binding by variant proteins. Further whole-genome sequencing identified the JAK3 mutation as the most frequent somatic genomic abnormality in T-ALL with germline RUNX1 variants. Cointroduction of RUNX1 variant and JAK3 mutation in hematopoietic stem and progenitor cells in mice gave rise to T-ALL with the early T cell precursor phenotype. Taken together, these results indicate that RUNX1 is an important predisposition gene for T-ALL and point to biology of RUNX1-mediated leukemogenesis in the lymphoid lineages.

Nucleobase and nucleoside analogs (NNA) are widely used as anti-viral and anti-cancer agents, and NNA phosphorylation is essential for the activity of this class of drugs. Recently, diphosphatase NUDT15 was linked to thiopurine metabolism with NUDT15 polymorphism associated with drug toxicity in patients. Profiling NNA drugs, we identify acyclovir (ACV) and ganciclovir (GCV) as two new NNAs metabolized by NUDT15. NUDT15 hydrolyzes ACV and GCV triphosphate metabolites, reducing their effects against cytomegalovirus (CMV) in vitro. Loss of NUDT15 potentiates cytotoxicity of ACV and GCV in host cells. In hematopoietic stem cell transplant patients, the risk of CMV viremia following ACV prophylaxis is associated with NUDT15 genotype (P = 0.015). Donor NUDT15 deficiency is linked to graft failure in patients receiving CMV-seropositive stem cells (P = 0.047). In conclusion, NUDT15 is an important metabolizing enzyme for ACV and GCV, and NUDT15 variation contributes to inter-patient variability in their therapeutic effects., Nucleoside analogs (NNA), such as acyclovir (ACV) and ganciclovir (GCV), are widely used as anti-virals to treat herpes virus infection. Here, Nishii et al. show that diphosphatase NUDT15 hydrolyzes ACV and GCV, therewith reducing NNA activity in vitro and link NUDT15 variation to inter-patient variability in ACV and GCV therapeutic effects.

Introduction. Advances in genomic profiling and sequencing studies have identified germline and somatic variations that are associated with childhood ALL, improving our understanding of the genetic basis of childhood acute lymphoblastic leukemia (ALL). Recent genome-wide association studies (GWAS) have identified germline genetic variations of ARID5B and, more recently, IGF2BP1 that are associated with susceptibility to ALL. Genome-wide sequencing studies also discovered a new ALL subtype characterized of ZNF384-mediated chromosomal translocations, providing new insights into genetic heterogeneity in childhood ALL. However, the underlying mechanism by which these genetic variants contribute to the transcriptional regulatory circuitries of ALL is still poorly understood. We tested these hypotheses: 1) A low ARID5B expression will increase the relapse risk of ALL, 2) Genetic variants of ARID5B will affect its expression and thus influence susceptibility to childhood ALL, 3) IGF2BP1 is transcriptionally suppressed by ETV6, 4) ZNF384-mediated fusion genes transcriptionally upregulate FLT3 expression as being a therapeutic target. Specific aims in this study include: 1) identifying the causal variant of ARID5B, 2) identifying molecular mechanism underlying drug resistance, 3) identifying molecular mechanism of transcriptional regulation of IGF2BP1 by ETV6, and 4) identifying molecular mechanism of transcriptional regulation of FLT3 by EP300-ZNF384 fusion protein. Methods. We analyzed association of ARID5B expression in primary human ALL blasts with different molecular subtypes and treatment outcomes. Subsequent mechanistic studies were performed in ALL cell lines by manipulating ARID5B expression isogenically, in which we evaluated drug sensitivity, metabolism, and molecular signaling events. We performed ARID5B targeted sequencing in 5,008 children with ALL and conducted high throughput CRISPR/dCas9 screening in an engineered ARID5B mCherry knock-in cell line. Effects of genetic polymorphism on binding affinity of transcription factor and chromatin accessibility were subsequently assessed. We applied CRISPR/dCas9 to investigate transcriptional regulation of IGF2BP1 by ETV6 in ALL cell lines. We stably knocked down EP300-ZNF384 fusion gene by CRISPR editing in ALL cell line, in which we analyzed FLT3 expression and drug sensitivity. Results. ARID5B expression varied substantially by ALL subtype, with the highest level being observed in hyperdiploid ALL. Lower ARID5B expression at diagnosis was associated with the risk of ALL relapse, and further reduction was noted at ALL relapse. In isogenic ALL cell models in vitro, ARID5B knockdown led to resistance specific to antimetabolite drugs. ARID5B downregulation significantly inhibited ALL cell proliferation and caused partial cell-cycle arrest partially through upregulating expression the cell-cycle checkpoint regulator p21 (encoded by CDKN1A). Using targeted sequencing in germline DNA of 5,008 children with ALL and high throughput CRISPR/dCas9 screening in an engineered ARID5B mCherry knock-in cell line, we nominated ALL risk variant (rs7090445, P = 1.82 × 10-10) as the causal variant. And its polymorphisms disrupted binding of transcription factor MEF2C and local chromosome accessibility as confirmed by ChIP-Sanger-seq and ATAC-seq. Although it was previously reported that IGF2BP1 expression was significantly higher in ETV6-RUNX1 ALL as well as other cancers, the underlying transcriptional regulatory mechanism remains elusive. In ALL cell models, we identified a cis-regulatory element (CRE) blocking of which by dCas9-KRAB strongly influenced transcription of IGF2BP1. Moreover, we presented a CRISPR-based approach to comprehensively investigate the transcriptional regulatory mechanism of IGF2BP1 by identifying its CREs and upstream transcriptional regulators. In tissue-specific overexpression mouse models, we demonstrated that role of Igf2bp1 in B-cell development was stage-specific. In a novel ALL subtype characterized of ZNF384-mediated rearrangements, for the first time we reported overexpression of FLT3 in this new ALL subtype, providing a novel therapeutic target for ALL patient with high expression of FLT3. Furthermore, we defined EP300-ZNF384 fusion protein as a transcriptional activator of FLT3 gene with direct binding at its 5’UTRand knocking down this fusion gene led to downregulation of FLT3 expression as well as decreased sensitivity to FLT3 inhibitor in vitro. Conclusions. Our studies have demonstrated that a causal variant of ARID5B affected its transcription in-cis and that a low expression of ARID5B increased ALL relapse risk. As a downstream effector of ETV6, IGF2BP1 expression influenced B-cell development in vivo in a stage-specific manner. Moreover, expression of FLT3 was transcriptionally upregulated by ZNF384-mediated fusion genes. This study sheds light on the underlying mechanism by which genetic variations altered transcriptional programs in childhood ALL and refined our understanding of the genetic basis of childhood ALL, providing new molecular targets which can be harnessed for development of new therapies for patients with ALL.

RUNX1 is a transcription factor critical for definitive hematopoiesis and genetic alterations in RUNX1 have been implicated in both benign and malignant blood disorders, particularly of the megakaryocyte and myeloid lineages. Somatic RUNX1 mutations are reported in B- and T-cell acute lymphoblastic leukemia (B-ALL and T-ALL), but germline genetic variation of RUNX1 in these lymphoid malignancies have not been comprehensively investigated. Sequencing 4,836 children with B-ALL and 1,354 cases of T-ALL, we identified 31 and 18 unique germline RUNX1 variants in these two ALL subtypes, respectively. RUNX1 variants in B-ALL were predicted to have minimal impact. By contrast, 54.5% of variants in T-ALL result in complete or partial loss of RUNX1 activity as a transcription activator in vitro, with dominant negative effects for 4 variants. Ectopic expression of dominant negative deleterious RUNX1 variants in human CD34+ cells repressed differentiation into erythroid, megakaryocytes, and T cells, while promoting differentiation towards myeloid cells. We then performed chromatin immunoprecipitation profiling in isogenic T-ALL models with variants introduced by genome editing of endogenous RUNX1. We observed highly distinctive patterns of DNA binding and target genomic loci by RUNX1 proteins encoded by the truncating vs missense variants. The p.G365R RUNX1 variant resulted in a novel methylation site in RUNX1 and alteration in its interaction with CBFβ. Further whole genome sequencing showed that JAK3 mutation was the most frequent somatic genomic abnormality in T-ALL with germline RUNX1 variants. Consistently, co-introduction of RUNX1 variant and JAK3 mutation in hematopoietic stem and progenitor cells in mouse gave rise to T-ALL with early T-cell precursor phenotype in vivo, compared to thymic T-ALL seen in mice with JAK3 mutation alone. Taken together, these results indicated that RUNX1 is an important predisposition gene for ALL, especially in T-ALL and also pointed to novel biology of RUNX1-mediated leukemogenesis in the lymphoid lineages.

Relapse remains a formidable challenge for acute lymphoblastic leukemia (ALL). Recently, recurrent mutations in NT5C2 were identified as a common genomic lesion unique in relapsed ALL and were linked to acquired thiopurine resistance. However, molecular mechanisms by which NT5C2 regulates thiopurine cytotoxicity were incompletely understood. To this end, we sought to comprehensively characterize the biochemical and cellular effects of NT5C2 mutations. Compared with wild-type NT5C2, mutant proteins showed elevated 5′-nucleotidase activity with a stark preference of thiopurine metabolites over endogenous purine nucleotides, suggesting neomorphic effects specific to thiopurine metabolism. Expression of mutant NT5C2 mutations also significantly reduced thiopurine uptake in vitro with concomitant increase in efflux of 6-mercaptopurine (MP) metabolites, plausibly via indirect effects on drug transporter pathways. Finally, intracellular metabolomic profiling revealed significant shifts in nucleotide homeostasis induced by mutant NT5C2 at baseline; MP treatment also resulted in global changes in metabolomic profiles with completely divergent effects in cells with mutant versus wild-type NT5C2. Collectively, our data indicated that NT5C2 mutations alter thiopurine metabolism and cellular disposition, but also influence endogenous nucleotide homeostasis and thiopurine-induced metabolomic response. These complex mechanisms contributed to NT5C2-mediated drug resistance in ALL and pointed to potential opportunities for therapeutic targeting in relapsed ALL.

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.

Formaldehyde (FA), as a reactive carbonyl species, is extremely hazardous to human health if its concentration is above normal level. In live cells, lysosome is a main organelle to generate endogenous FA. Thus, the design of facile, stable, and sensitive probes for the detection of FA in lysosome is essential. Herein, a self-assembled fluorescent nanoprobe based on homoallylamino substituted perylene (P-FA) has been developed for FA detection in lysosome. P-FA can react with FA along with emission color change from blue to green. P-FA exhibited high sensitivity and selectivity to FA in DMSO solution. In aqueous solution, P-FA self-assembled into uniform sphere-like nanoparticle as a fluorescent nanoprobe. Furthermore, the reaction between the nanoprobe and FA was greatly facilitated at pH 4-5, which led to a lower detection limit (0.96 μM at pH 5) than that in DMSO. In live cells, P-FA nanoprobe achieved long-term tracking of lysosome (over 12 h). The fluorescent nanoprobe was then used for both exogenous and endogenous FA detection. Our work provides a facile and effective strategy for the detection of FA in lysosome.

Aberrant HOXA9 expression is a hallmark of most aggressive acute leukemias, notably those with KMT2A (MLL) gene rearrangements. HOXA9 overexpression not only predicts poor diagnosis and outcome but also plays a critical role in leukemia transformation and maintenance. However, our current understanding of HOXA9 regulation in leukemia is limited, hindering development of therapeutic strategies. Here, we generated the HOXA9-mCherry knock-in reporter cell lines to dissect HOXA9 regulation. By utilizing the reporter and CRISPR/Cas9 screens, we identified transcription factors controlling HOXA9 expression, including a novel regulator, USF2, whose depletion significantly down-regulated HOXA9 expression and impaired MLLr leukemia cell proliferation. Ectopic expression of Hoxa9 rescued impaired leukemia cell proliferation upon USF2 loss. Cut and Run analysis revealed the direct occupancy of USF2 at HOXA9 promoter in MLLr leukemia cells. Collectively, the HOXA9 reporter facilitated the functional interrogation of the HOXA9 regulome and has advanced our understanding of the molecular regulation network in HOXA9-driven leukemia.

Sodium tanshinone IIA sulfonate (STS) has been widely used by Chinese medicine practitioners for chronic cardiovascular diseases. However, its direct clinical efficacy in patients with acute coronary syndrome following percutaneous coronary intervention (PCI) has not been reported yet. The present trial aimed to investigate potential cardioprotection of STS in patients undergoing PCI for non-ST elevation acute coronary syndrome (NSTE-ACS). In a randomized, double-blind, placebo-controlled trial, 372 patients with NSTE-ACS were randomly assigned to receive STS (n = 192) or saline (n = 180) for 2 days before and 3 days after PCI along with standard therapy. The primary endpoint was the composite incidence of major adverse cardiac events (MACEs), including death, non-fatal myocardial infarction, repeated revascularization of the target vessel, and stent thrombosis, within 30 days after PCI. The 30-day MACEs occurred in 18.8% of the patients in the STS group and in 27.2% of the patients in the control group (P = 0.038); this difference was mostly driven by reduction of myocardial infarction incidence (17.2% vs. 26.7%, P = 0.027). Post-procedural elevation of troponin-I was also significantly lower in the STS group (26.56% vs. 47.78%, P

Sepsis-induced myocardial dysfunction (SIMD) contributes significantly to cardiovascular dysfunction during septic shock. We aimed to evaluate the potential role of Xinmailong injection (XMLI), a polypeptide medicine extracted from Periplaneta americana, in reversing the progression of myocardial damage to SIMD in sepsis patients. This was a multicenter, randomized, double-blind, parallel-group trial. We recruited all patients consecutively admitted to intensive care units (ICUs) who were aged 18 to 85 years old and met the sepsis 3.0 criteria. The primary outcome measure was the incidence of sepsis-induced myocardial dysfunction while in the ICU. Of the 192 patients, 96 were assigned to the treatment group, and 96 to the control group. Subsequently, 41 patients [41/96 (42.7%)] in the XMLI group and 61 patients in the placebo group [61/96 (63.5%)] were confirmed to have diastolic dysfunction on the fifth day (D5). The incidence of diastolic SIMD was significantly different between the two groups (P = 0.004). There were 36 deaths in the two groups during the 28-day follow-up, with a general mortality rate of 18.8% (36/192). The 28-day mortality rates were not significantly different between the groups (P = 0.45). However, the brain natriuretic peptide (BNP) plasma concentration trends on D0, D2, and D5 significantly differed between the two groups (P = 0.049). In septic patients, XMLI decreased the occurrence rate of diastolic SIMD more effectively than the placebo. The improvement in serum BNP concentration was also greater in the XMLI group. XMLI may, therefore, effectively and safely improve cardiac function in patients with sepsis.

Aberrant HOXA9 expression is a hallmark of most aggressive acute leukemias, including human acute myeloid leukemia (AML) and subtypes of acute lymphoblastic leukemia (ALL). HOXA9 overexpression not only predicts poor diagnosis and outcome but also plays a critical role in leukemia transformation and maintenance. However, our current understanding of HOXA9 regulation in leukemia is limited, hindering development of therapeutic strategies to treat HOXA9-driven leukemia. To mitigate these challenges, we generated the first HOXA9-mCherry knock-in reporter in an MLL-rearranged (MLLr) B-ALL cell line to dissect HOXA9 regulation. By utilizing the reporter and CRISPR/Cas9 mediated screens, we identified transcription factors controlling HOXA9 expression, including a novel regulator, USF2 and its homolog USF1. USF1/USF2 depletion significantly down-regulated HOXA9 expression and impaired MLLr leukemia cell proliferation. Ectopic expression of HOXA9-MEIS1 fusion protein rescued the impaired leukemia cell proliferation upon USF2 loss. Cut&Run analysis revealed the direct occupancy of USF2 onto HOXA9 promoter in MLLr leukemia cells. Collectively, the HOXA9 reporter facilitated the functional interrogation of the HOXA9 regulome and has advanced our understanding of the molecular regulation network in HOXA9-driven leukemia.

The PML‐RARα fusion gene, generated by the t(15;17) chromosome translocation, is regarded as the initiating factor of acute promyelocytic leukaemia (APL). In addition to the well‐known effects on blocking myeloid differentiation at the promyelocytic stage, promyelocytic leukaemia‐retinoic acid receptor α (PML‐RARα) has also been reported to interfere with multiple differentiation processes, including erythroid differentiation. However, the detailed molecular mechanism by which PML‐RARα impairs erythropoiesis has not yet been fully addressed. By chromatin immunoprecipitation‐PCR assay, we found that PML‐RARα bound to the distal promoter region of LMO2 (LIM‐only protein 2), a critical erythroid‐specific transcription factor. Luciferase reporter assays and qRT‐PCR results demonstrated that PML‐RARα down‐regulated the expression of the LMO2 distal transcript through transrepressing its promoter activity. Analysis of gene expression profiling data from large cohorts of acute myeloid leukaemia (AML) patients confirmed that LMO2 expressed at a markedly lower level in APL patients in comparison to non‐APL AML patients. Further flow cytometry analysis demonstrated that PML‐RARα inhibited erythropoietin‐induced erythroid differentiation by down‐regulating LMO2 expression. Our findings reveal a previously unidentified mechanism, by which PML‐RARα interferes with erythropoiesis through directly targeting and transrepressing LMO2 expression in the development of APL.

Gaseous formaldehyde (FA), a common indoor pollutant, presents a serious threat to human health. As an efficient tool for FA detection, fluorescent probes exhibit the advantages of low cost, ease of use, facile operation, etc. However, previously developed FA fluorescent probes are mostly based on fluorophores with aggregation-caused quenching features and thus require dispersion in solvent to detect FA. In this study, a fluorescent probe (TPE-FA) based on an aggregation-induced emission (AIE) fluorophore (tetraphenylethylene) has been developed for facile detection of gaseous FA through a fluorescence “turn-on” response. TPE-FA reacts with FA through 2-aza-Cope sigmatropic rearrangement. Based on the AIE features of TPE-FA, we fabricated a portable solid sensor, FA test plate, by directly loading TPE-FA on high performance thin-layer chromatography silica gel plate. The FA test plate achieved sensitive, selective, and quantitative detection of gaseous FA. The detection limit (0.036 mg/m3) of the FA test ...

Loss of function of CDKN2A/B, also known as INK4/ARF [encoding p16(INK4A), p15(INK4B), and p14(ARF) (mouse p19(Arf))], confers susceptibility to cancers, whereas its up-regulation during organismal aging provokes cellular senescence and tissue degenerative disorders. To better understand the transcriptional regulation of p16(INK4A), a CRISPR screen targeting open, noncoding chromatin regions adjacent to p16(INK4A) was performed in a human p16(INK4A-P2A-mCherry) reporter cell line. We identified a repressive element located in the 3′ region adjacent to the ARF promoter that controls p16(INK4A) expression via long-distance chromatin interactions. Coinfection of lentiviral dCas9-KRAB with selected single-guide RNAs against the repressive element abrogated the ARF/p16(INK4A) chromatin contacts, thus reactivating p16(INK4A) expression. Genetic CRISPR screening identified candidate transcription factors inhibiting p16(INK4A) regulation, including ZNF217, which was confirmed to bind the ARF/p16(INK4A) interaction loop. In summary, direct physical interactions between p16(INK4A) and ARF genes provide mechanistic insights into their cross-regulation.

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

This paper proposes a simple two-step nonparametric procedure to estimate the intraday jump tail and measure the jump tail risk in asset price with noisy high frequency data. We first propose the pre-averaging threshold approach to estimate the intraday jumps occurred, and then use the peaks-over-threshold (POT) method and generalized Pareto distribution (GPD) to model the intraday jump tail and further measure the jump tail risk. Finally, an empirical example further demonstrates the power of the proposed method to measure the jump tail risk under the effect of microstructure noise.

Ethnopharmacological relevance Tongguan Capsules (TGC), a patented Chinese herbal remedy containing Salvia miltiorrhiza, Astragalus membranaceus, Borneolum syntheticum and Grasshopper, has been previously tested in the experimental model of animal hearts subjected to ischemia/reperfusion injury and its cardioprotective effect has been described. Aim of the study This clinical trial was aimed at investigation whether the administration of TGC to patients suffered myocardial infarction (MI), would diminish dilation of the left ventricular (LV) and reduce development of the adverse clinical consequences. Methods Eligible patients were enrolled and randomized 1:1 to TGC (4.5 g/d for 6 months) superimposed on standard treatment for MI, or the control group receiving the standard protocol alone. The outcomes of this trial were valued after 6 months and reported as a mean change from the baseline in LV end-systolic volume index (LVESVI) and as a frequency of MI recurrence, target-vessel revascularization, severity of heart failure or significant arrhythmia that required the additional therapy within 6 months. In addition, arrays with a panel of specific antibodies were used to assess levels of major cytokines and other pathophysiologic markers, that prompted conclusions about the mechanisms of the ultimate clinical outcomes in both patient's subgroups. Results Meaningfully, obtained results indicated that MI patients randomly assigned to the TGC treatment, demonstrated a significant reduction of LVESVI (−4.03 ± 0.73 vs. 1.59 ± 0.43 mL/m2, P Conclusion Addition of TGC to the current conventional treatment of MI patients, significantly reduced their adverse LV remodeling and contributed to the more positive clinical outcome. Trial registration ChiCTR-IPR-17011618.