Cryptic recurrent ACIN1-NUTM1 fusions in non-KMT2A-rearranged infant acute lymphoblastic leukemia.

Infant acute lymphoblastic leukemias (ALL) are rare hematological malignancies occurring in children younger than 1 year of age, most frequently associated with KMT2A rearrangements (KMT2A-r). The smaller subset without KMT2A-r, which represents 20% of infant ALL cases, is poorly characterized. Here we report two cases of chemotherapy-sensitive non-KMT2A-r infant ALL. Transcriptome analyses revealed identical ACIN1-NUTM1 gene fusions in both cases, derived from cryptic chromosomal rearrangements undetected by standard cytogenetic approaches. Two isoforms of the gene fusion, joining exons 3 or 4 of ACIN1 to exon 3 of NUTM1, were identified. Both fusion transcripts contained the functional DNA-binding SAP (SAF-A/B, Acinus, and PIAS) domain of ACIN1 and most of NUTM1. The detection of the ACIN1-NUTM1 fusion by RT-PCR allowed the molecular monitoring of minimal residual disease in a clinical setting. Based on publicly available genomic datasets and literature review, we predict that NUTM1 gene fusions are recurrent events in infant ALL. As such, we propose two clinically relevant assays to screen for NUTM1 rearrangements in bone marrow cells, independent of the fusion partner: NUMT1 immunohistochemistry and NUTM1 RNA expression. In sum, our study identifies ACIN1-NUTM1 as a recurrent and possibly cryptic fusion in non-KMT2A-r infant ALL, provides clinical tools to screen for NUTM1-rearranged leukemia and contributes to the refinement of this new subgroup.

A universal RNA polymerase II CTD cycle is orchestrated by complex interplays between kinase, phosphatase, and isomerase enzymes along genes.

Transcription by RNA polymerase II (RNAPII) is coupled to mRNA processing and chromatin modifications via the C-terminal domain (CTD) of its largest subunit, consisting of multiple repeats of the heptapeptide YSPTSPS. Pioneering studies showed that CTD serines are differentially phosphorylated along genes in a prescribed pattern during the transcription cycle. Genome-wide analyses challenged this idea, suggesting that this cycle is not uniform among different genes. Moreover, the respective role of enzymes responsible for CTD modifications remains controversial. Here, we systematically profiled the location of the RNAPII phosphoisoforms in wild-type cells and mutants for most CTD modifying enzymes. Together with results of in vitro assays, these data reveal a complex interplay between the modifying enzymes, and provide evidence that the CTD cycle is uniform across genes. We also identify Ssu72 as the Ser7 phosphatase and show that proline isomerization is a key regulator of CTD dephosphorylation at the end of genes.

NUP98-BPTF gene fusion identified in primary refractory acute megakaryoblastic leukemia of infancy.

The advent of large scale genomic sequencing technologies significantly improved the molecular classification of acute megakaryoblastic leukaemia (AMKL). AMKL represents a subset (∼10%) of high fatality pediatric acute myeloid leukemia (AML). Recurrent and mutually exclusive chimeric gene fusions associated with pediatric AMKL are found in 60%-70% of cases and include RBM15-MKL1, CBFA2T3-GLIS2, NUP98-KDM5A and MLL rearrangements. In addition, another 4% of AMKL harbor NUP98 rearrangements (NUP98r), with yet undetermined fusion partners. We report a novel NUP98-BPTF fusion in an infant presenting with primary refractory AMKL. In this NUP98r, the C-terminal chromatin recognition modules of BPTF, a core subunit of the NURF (nucleosome remodeling factor) ATP-dependent chromatin-remodeling complex, are fused to the N-terminal moiety of NUP98, creating an in frame NUP98-BPTF fusion, with structural homology to NUP98-KDM5A. The leukemic blasts expressed two NUP98-BPTF splicing variants, containing one or two tandemly spaced PHD chromatin reader domains. Our study also identified an unreported wild type BPTF splicing variant encoding for 2 PHD domains, detected both in normal cord blood CD34+ cells and in leukemic blasts, as with the fly BPTF homolog, Nurf301. Disease course was marked by rapid progression and primary chemoresistance, with ultimately significant tumor burden reduction following treatment with a clofarabine containing regimen. In sum, we report 2 novel NUP98-BPTF fusion isoforms that contribute to refine the NUP98r subgroup of pediatric AMKL. Multicenter clinical trials are critically required to determine the frequency of this fusion in AMKL patients and explore innovative treatment strategies for a disease still plagued with poor outcomes.

CBFA2T3::GLIS2 pediatric acute megakaryoblastic leukemia is sensitive to BCL-XL inhibition by navitoclax and DT2216.

ABSTRACT: Acute megakaryoblastic leukemia (AMKL) is a rare, developmentally restricted, and highly lethal cancer of early childhood. The paucity and hypocellularity (due to myelofibrosis) of primary patient samples hamper the discovery of cell- and genotype-specific treatments. AMKL is driven by mutually exclusive chimeric fusion oncogenes in two-thirds of the cases, with CBFA2T3::GLIS2 (CG2) and NUP98 fusions (NUP98r) representing the highest-fatality subgroups. We established CD34+ cord blood-derived CG2 models (n = 6) that sustain serial transplantation and recapitulate human leukemia regarding immunophenotype, leukemia-initiating cell frequencies, comutational landscape, and gene expression signature, with distinct upregulation of the prosurvival factor B-cell lymphoma 2 (BCL2). Cell membrane proteomic analyses highlighted CG2 surface markers preferentially expressed on leukemic cells compared with CD34+ cells (eg, NCAM1 and CD151). AMKL differentiation block in the mega-erythroid progenitor space was confirmed by single-cell profiling. Although CG2 cells were rather resistant to BCL2 genetic knockdown or selective pharmacological inhibition with venetoclax, they were vulnerable to strategies that target the megakaryocytic prosurvival factor BCL-XL (BCL2L1), including in vitro and in vivo treatment with BCL2/BCL-XL/BCL-W inhibitor navitoclax and DT2216, a selective BCL-XL proteolysis-targeting chimera degrader developed to limit thrombocytopenia in patients. NUP98r AMKL were also sensitive to BCL-XL inhibition but not the NUP98r monocytic leukemia, pointing to a lineage-specific dependency. Navitoclax or DT2216 treatment in combination with low-dose cytarabine further reduced leukemic burden in mice. This work extends the cellular and molecular diversity set of human AMKL models and uncovers BCL-XL as a therapeutic vulnerability in CG2 and NUP98r AMKL.

DSIF and RNA polymerase II CTD phosphorylation coordinate the recruitment of Rpd3S to actively transcribed genes.

Histone deacetylase Rpd3 is part of two distinct complexes: the large (Rpd3L) and small (Rpd3S) complexes. While Rpd3L targets specific promoters for gene repression, Rpd3S is recruited to ORFs to deacetylate histones in the wake of RNA polymerase II, to prevent cryptic initiation within genes. Methylation of histone H3 at lysine 36 by the Set2 methyltransferase is thought to mediate the recruitment of Rpd3S. Here, we confirm by ChIP-Chip that Rpd3S binds active ORFs. Surprisingly, however, Rpd3S is not recruited to all active genes, and its recruitment is Set2-independent. However, Rpd3S complexes recruited in the absence of H3K36 methylation appear to be inactive. Finally, we present evidence implicating the yeast DSIF complex (Spt4/5) and RNA polymerase II phosphorylation by Kin28 and Ctk1 in the recruitment of Rpd3S to active genes. Taken together, our data support a model where Set2-dependent histone H3 methylation is required for the activation of Rpd3S following its recruitment to the RNA polymerase II C-terminal domain.

IDH1 as a Cooperating Mutation in AML Arising in the Context of Shwachman-Diamond Syndrome.

Shwachman-Diamond syndrome (SDS) is a rare and systemic disease mostly caused by mutations in the SBDS gene and characterized by pancreatic insufficiency, skeletal abnormalities, and a bone marrow dysfunction. In addition, SDS patients are predisposed to develop myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), typically during adulthood and associated with TP53 mutations. Although most SDS diagnoses are established in childhood, the nature and frequency of serial bone marrow cell investigations during the patients' lifetime remain a debatable topic. The precise molecular mechanisms leading to AML progression in SDS patients have not been fully elucidated because the patient cohorts are small and most disease monitoring is conducted using standard histological and cytogenetic approaches. Here we report a rare case of a patient with SDS who was diagnosed with AML at 5 years of age and survived. Intermittent neutropenia preceded the AML diagnostic but serial bone marrow monitoring according to the standard of care revealed no cytogenetic anomalies nor signs of clonal hematopoiesis. Using next generation sequencing approaches to find cytogenetically cryptic pathogenic mutations, we identified the cancer hotspot mutation c.394C>T/p.Arg132Cys in IDH1 with high variant allelic frequency in bone marrow cells, suggesting clonal expansion of a major leukemic clone karyotypically normal, in the SDS-associated AML. The mutation was somatic and likely occurred at the leukemic transformation stage, as it was not detected in a matched normal tissue nor in bone marrow smear prior to AML diagnosis. Gain-of-function mutations in IDH1, such as c.394C>T/p.Arg132Cys, create a neo-activity of isocitrate dehydrogenase 1 converting α-ketoglutarate into the oncometabolite D-2-hydroxyglutarate, inhibiting α-ketoglutarate-dependent enzymes, such as histone and DNA demethylases. Overall, our results suggest that along with previously described abnormalities such as TP53 mutations or monosomy7, 7q-, which are all absent in this patient, additional mechanisms including IDH1 mutations drive SDS-related AML and are likely associated with variable outcomes. Sensitive techniques complementary to standard cytogenetics, such as unbiased or targeted panel-based next generation sequencing approaches, warrant testing for monitoring of myelodysplasia, clonal hematopoiesis, and leukemia in the context SDS. Such analyses would also assist treatment decisions and allow to gain insight into the disease biology.

Human models of NUP98-KDM5A megakaryocytic leukemia in mice contribute to uncovering new biomarkers and therapeutic vulnerabilities.

Acute megakaryoblastic leukemia (AMKL) represents ∼10% of pediatric acute myeloid leukemia cases and typically affects young children (<3 years of age). It remains plagued with extremely poor treatment outcomes (<40% cure rates), mostly due to primary chemotherapy refractory disease and/or early relapse. Recurrent and mutually exclusive chimeric fusion oncogenes have been detected in 60% to 70% of cases and include nucleoporin 98 (NUP98) gene rearrangements, most commonly NUP98-KDM5A. Human models of NUP98-KDM5A-driven AMKL capable of faithfully recapitulating the disease have been lacking, and patient samples are rare, further limiting biomarkers and drug discovery. To overcome these impediments, we overexpressed NUP98-KDM5A in human cord blood hematopoietic stem and progenitor cells using a lentiviral-based approach to create physiopathologically relevant disease models. The NUP98-KDM5A fusion oncogene was a potent inducer of maturation arrest, sustaining long-term proliferative and progenitor capacities of engineered cells in optimized culture conditions. Adoptive transfer of NUP98-KDM5A-transformed cells into immunodeficient mice led to multiple subtypes of leukemia, including AMKL, that phenocopy human disease phenotypically and molecularly. The integrative molecular characterization of synthetic and patient NUP98-KDM5A AMKL samples revealed SELP, MPIG6B, and NEO1 as distinctive and novel disease biomarkers. Transcriptomic and proteomic analyses pointed to upregulation of the JAK-STAT signaling pathway in the model AMKL. Both synthetic models and patient-derived xenografts of NUP98-rearranged AMKL showed in vitro therapeutic vulnerability to ruxolitinib, a clinically approved JAK2 inhibitor. Overall, synthetic human AMKL models contribute to defining functional dependencies of rare genotypes of high-fatality pediatric leukemia, which lack effective and rationally designed treatments.

Effects of AGI-1067 and probucol after percutaneous coronary interventions.

BACKGROUND: AGI-1067, a metabolically stable modification of probucol, is an equipotent antioxidant to probucol but is pharmacologically distinct. In a multicenter trial, we studied whether AGI-1067 reduces restenosis assessed by intravascular ultrasound (IVUS) after percutaneous coronary intervention (PCI) compared with placebo and probucol used as a positive control. METHODS AND RESULTS: Two weeks before PCI, 305 patients were randomly assigned to 1 of 5 treatment groups: placebo, probucol 500 mg BID, or AGI-1067 70, 140, or 280 mg once daily. Patients were treated for 2 weeks before and 4 weeks after PCI. Baseline and 6-month follow-up IVUS were interpreted by a blinded core laboratory. Stents were used in 85% of patients. Luminal area at the PCI site at follow-up was 2.66+/-1.58 mm2 for placebo, 3.69+/-2.69 mm2 for probucol, 2.75+/-1.76 mm2 for AGI-1067 70 mg, 3.17+/-2.26 mm2 for AGI-1067 140 mg, and 3.36+/-2.12 mm2 for AGI-1067 280 mg (P=0.02 for the dose-response relationship; P< or =0.05 for AGI-1067 280 mg and probucol versus placebo). There was a mean narrowing of 5.3 mm3 of reference segment lumen in the placebo group and an enlargement in the AGI-1067 140- and 280-mg groups at follow-up (P=0.05 for 140 mg). An increase in QTc interval >60 ms occurred in 4.8% of placebo patients, 17.4% of probucol patients, and 4.8%, 2.4%, and 2.5% of patients in the AGI-1067 groups (P=0.02). CONCLUSIONS: AGI-1067 and probucol reduce restenosis after PCI. In contrast to probucol, AGI-1067 did not cause prolongation of the QTc interval and improved lumen dimensions of reference segments, suggestive of a direct effect on atherosclerosis.

Hospitalization costs of primary stenting versus thrombolysis in acute myocardial infarction: cost analysis of the Canadian STAT Study.

BACKGROUND: We previously showed that primary stenting was more effective than accelerated tPA in reducing the 6-month composite of death, reinfarction, stroke, or repeat revascularization for ischemia. This study looks at the hospitalization costs of primary stenting compared with accelerated tPA. METHODS AND RESULTS: Initial and 6-month hospitalization costs were computed for all patients randomly assigned to primary stenting (n=62) or accelerated tPA (n=61) in the Stenting versus Thrombolysis in Acute myocardial infarction Trial (STAT). Costs and resource usage were collected in detail for each patient. Physician fees were obtained directly from billings to the Ontario Health Insurance Plan. The length of initial hospitalization was 6.7+/-11.3 days in the stent group and 8.7+/-6.7 days in the tPA group (P<0.001). Total hospitalization days at 6 months were 8.3+/-13 days in the stent group and 12.1+/-14.0 days in the tPA group (P=0.001). Hospitalization costs were less in the stent group for the initial hospitalization, 6354 dollars +/-6382 versus 7893 dollars +/-4429 (P=0.001), and at 6 months, 7100 dollars +/-7111 versus 9559 dollars +/-6933 (P=0.001). CONCLUSIONS: In centers in which facilities and experienced interventionists are available, primary stenting is less costly and more effective than thrombolysis.

Systematic identification of fragile sites via genome-wide location analysis of gamma-H2AX.

Phosphorylation of histone H2AX is an early response to DNA damage in eukaryotes. In Saccharomyces cerevisiae, DNA damage or replication-fork stalling results in phosphorylation of histone H2A yielding gamma-H2A (yeast gamma-H2AX) in a Mec1 (ATR)- and Tel1 (ATM)-dependent manner. Here, we describe the genome-wide location analysis of gamma-H2A as a strategy to identify loci prone to engaging the Mec1 and Tel1 pathways. Notably, gamma-H2A enrichment overlaps with loci prone to replication-fork stalling and is caused by the action of Mec1 and Tel1, indicating that these loci are prone to breakage. Moreover, about half the sites enriched for gamma-H2A map to repressed protein-coding genes, and histone deacetylases are necessary for formation of gamma-H2A at these loci. Finally, our work indicates that high-resolution mapping of gamma-H2AX is a fruitful route to map fragile sites in eukaryotic genomes.