Esther A. Obeng

Pediatric Heparin-Induced Thrombocytopenia: prevalence, thrombotic risk, and application of the 4Ts scoring system

OBJECTIVE To characterize heparin-induced thrombocytopenia (HIT) at a single pediatric center including the prevalence and the accuracy of the 4Ts scoring system as a predictor of HIT. STUDY DESIGN In this retrospective cohort study, we identified 155 consecutive patients <21 years old with sufficient data for 4Ts scoring. The 4Ts scoring system is a validated pretest tool in adults that predicts the likelihood of HIT using clinical features. Hospital-wide exposure to unfractionated and low molecular weight heparin was determined by querying the hospital pharmacy database. RESULTS The majority of patients with suspected HIT (61.2%) were on surgical services. Prediction of HIT risk using initial 4Ts scoring found 3 (2%) had high risk 4Ts scores, 114 (73%) had intermediate risk 4Ts scores, and the remaining 38 (25%) had low risk 4Ts scores. HIT was confirmed in 0/38 patients with low risk 4Ts scores, 2/114 patients with intermediate-risk 4Ts scores, and all 3 patients with high-risk 4Ts scores presented with HIT with thrombosis. Of 12 positive HIT screening tests, results were falsely positive in 66.6% of patients with intermediate risk 4Ts scores and 100% of patients with low risk 4Ts scores. The prevalence of HIT was 0.058% and HIT with thrombosis was 0.046% in pediatric patients on unfractionated heparin. CONCLUSIONS The prevalence of HIT appears significantly lower in pediatric patients compared with adults. Application of the 4Ts system as a pretest tool may reduce laboratory evaluation for HIT in heparin-exposed children with low risk 4Ts scores, decreasing unnecessary further testing, intervention, and cost.

Copy-number and gene dependency analysis reveals partial copy loss of wild-type SF3B1 as a novel cancer vulnerability

Genomic instability is a hallmark of human cancer, and results in widespread somatic copy number alterations. We used a genome-scale shRNA viability screen in human cancer cell lines to systematically identify genes that are essential in the context of particular copy-number alterations (copy-number associated gene dependencies). The most enriched class of copy-number associated gene dependencies was CYCLOPS (Copy-number alterations Yielding Cancer Liabilities Owing to Partial losS) genes, and spliceosome components were the most prevalent. One of these, the pre-mRNA splicing factor SF3B1, is also frequently mutated in cancer. We validated SF3B1 as a CYCLOPS gene and found that human cancer cells harboring partial SF3B1 copy-loss lack a reservoir of SF3b complex that protects cells with normal SF3B1 copy number from cell death upon partial SF3B1 suppression. These data provide a catalog of copy-number associated gene dependencies and identify partial copy-loss of wild-type SF3B1 as a novel, non-driver cancer gene dependency. DOI: http://dx.doi.org/10.7554/eLife.23268.001

Congenital macrothrombocytopenia with focal myelofibrosis due to mutations in human G6b-B is rescued in humanized mice.

Unlike primary myelofibrosis (PMF) in adults, myelofibrosis in children is rare. Congenital (inherited) forms of myelofibrosis (cMF) have been described, but the underlying genetic mechanisms remain elusive. Here we describe 4 families with autosomal recessive inherited macrothrombocytopenia with focal myelofibrosis due to germ line loss-of-function mutations in the megakaryocyte-specific immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing receptor G6b-B (G6b, C6orf25, or MPIG6B). Patients presented with a mild-to-moderate bleeding diathesis, macrothrombocytopenia, anemia, leukocytosis and atypical megakaryocytes associated with a distinctive, focal, perimegakaryocytic pattern of bone marrow fibrosis. In addition to identifying the responsible gene, the description of G6b-B as the mutated protein potentially implicates aberrant G6b-B megakaryocytic signaling and activation in the pathogenesis of myelofibrosis. Targeted insertion of human G6b in mice rescued the knockout phenotype and a copy number effect of human G6b-B expression was observed. Homozygous knockin mice expressed 25% of human G6b-B and exhibited a marginal reduction in platelet count and mild alterations in platelet function; these phenotypes were more severe in heterozygous mice that expressed only 12% of human G6b-B. This study establishes G6b-B as a critical regulator of platelet homeostasis in humans and mice. In addition, the humanized G6b mouse will provide an invaluable tool for further investigating the physiological functions of human G6b-B as well as testing the efficacy of drugs targeting this receptor.

A Prospective Cohort Quality Improvement Study to Reduce the Time to Antibiotics for New Fever in Neutropenic Pediatric Oncology Inpatients.

Fever and neutropenia (F&N) is a pediatric oncology emergency due to the risk of disseminated infection. Quality improvement (QI) efforts to improve time to antibiotics for F&N in the emergency department have been documented, but the issue has not been studied in the established inpatient setting.

Abstract B125: Mutant SF3B1 downregulates proteins involved in differentiation, including ABCB7

Refractory Anemia with Ringed Sideroblasts (RARS), a subtype of Myelodysplatic Syndrome (MDS), occurs with a high frequency of hotspot mutations in HEAT (Huntingtin, Elongation factor 3, protein phosphatase 2A, Targets of rapamycin 1 domains) domains of SF3B1. This protein component of the U2 snRNP complex of the spliceosome is essential in the proper selection and usage of 39 splice sites. RNAseq analysis of MDS and other tumor types in which SF3B1 hotspot mutations have been found show that alternative 39 splice site usage is the predominant cause of RNA transcript aberration. These modifications can result in mRNAs encoding novel peptides, or they can introduce premature termination codons into the pre-mRNA, most likely directing it to the Nonsense Mediated Decay (NMD) pathway for degradation. Using a predictive tool to determine the likelihood of a given aberrant transcript to be targeted for NMD, we determined that nearly 50% of the SF3B1-mutant-associated aberrant transcripts were candidates for degradation. We confirmed this experimentally by treating isogenic Nalm-6 cells (engineered by AAV homology to express SF3B1 K700E or K700K) with or without cycloheximide, an agent known to inhibit translation and RNA degradation by NMD. Investigation of the resulting RNAseq data showed significant rescue of gene expression only for the transcripts predicted to be NMD targets. Ingenuity Pathway Analysis indicated that many of the downregulated genes in SF3B1 mutant samples were involved in differentiation, which has been shown to be dysregulated in MDS. We tested the idea that such modifications in the transcriptome confer selective advantage or impair differentiation in SF3B1 mutant cells. We began by manipulating the expression of ABCB7, one of the genes identified in our RNAseq analysis to be downregulated by aberrant splicing and subsequent NMD. ABCB7 is a mitochondrial transporter important in cellular iron metabolism and, indirectly, in heme production. Additionally, loss of function of ABCB7 is causal in X-linked sideroblastic anemia and has been implicated in RARS MDS. We discovered in our SILAC proteomic analysis that ABCB7 protein was dramatically decreased in K700E SF3B1 Nalm-6 cells relative to K700K Nalm-6, in agreement with our RNAseq analysis. Using doxycycline-inducible shRNA expression, we knocked down ABCB7 mRNA and protein expression in TF-1 erythroblasts. These cells show significant decreases in erythropoeitin (EPO)-induced differentiation when expressing exogenous K700E SF3B1, but not K700R (a very conservative mutation) or WT SF3B1. With direct knock down of ABCB7, we observed a similar phenotype - impairment of EPO-induced differentiation in ABCB7 shRNA-induced cells by Day 7, with no overall decline in cell viability. Interestingly, knock down of SF3B1 expression with shRNA also reduces ABCB7 mRNA. However, it also promotes cell death. This is consistent with the heterozygous nature of SF3B1 hotspot mutations; severe loss of SF3B1 function is deleterious. We propose that hotspot SF3B1 mutants promote aberrant splicing of multiple genes, inducing a general “spliceosomal sickness” in addition to downregulating key genes (e.g. ABCB7) responsible for erythroid differentiation impairment, such as that observed in RARS. Citation Format: Rachel B. Darman, Samantha A. Perino, Michael Seiler, Shouyong Peng, Jacob Feala, Peter Fekkes, Gregg F. Keaney, Kaiko Kunii, Linda Lee, Kian Huat Lim, Yoshiya Oda, Khin Myint, Esther A. Obeng, Ermira Pazolli, Eun Sun Park, John Yuan Wang, Markus Warmuth, Lihua Yu, Ping Zhu, Yoshiharu Mizui, Benjamin L. Ebert, Peter G. Smith, Silvia Buonamici. Mutant SF3B1 downregulates proteins involved in differentiation, including ABCB7. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B125.

Abstract 2040: Mutations in SF3B1 lead to aberrant splicing through cryptic 3′ splice site selection and impair hematopoietic cell differentiation

Heterozygous mutations in SF3B1, a component of the U2 complex involved in the recognition of 3′ splice sites (ss), have been reported with high frequency in refractory anemia with ring sideroblasts (RARS, a subtype of myelodysplastic syndrome, MDS) and have also been observed in chronic lymphocytic leukemia (CLL) and several solid tumors. To study the impact of SF3B1 mutations on splicing, RNAseq data obtained from breast cancer, melanoma, CLL and MDS samples with mutant (SF3B1MUT) or wild-type SF3B1 (SF3B1WT) were compared. The majority of aberrant junctions identified in the samples with mutant SF3B1 utilized an alternative 3′ss, suggesting its neomorphic function. Motif analysis of the sequences used by SF3B1MUT revealed the usage of a cryptic AG with a shorter and weaker polypyrimidine tract. Minigenes with modifications of these sites revealed the importance of both intronic and exonic sequence features for the recognition of the cryptic AG by SF3B1MUT. Of the aberrant junctions identified, several were common across all hotspot mutations and diseases; however, a unique aberrant splicing profile was found for each disease suggesting lineage and disease specific effects. The majority of splicing defects introduced a premature termination codon downstream of the cryptic AG leading to nonsense mediated decay (NMD) of aberrant transcripts and downregulation of gene expression, such as ABCB7. Gene-set enrichment analysis of aberrantly spliced and differentially expressed genes in SF3B1MUT MDS samples identified genes involved in cell differentiation and epigenetic pathways which are known to be deregulated in MDS. The impact on erythroid differentiation by SF3B1MUT was studied in transduced TF-1 cells following erythropoietin (EPO) stimulation. As expected, TF-1 SF3B1WT cells were able to differentiate normally after EPO treatment; however, expression of SF3B1K700E (the most common hotspot mutation found in RARS and CLL) induced a block in erythoid differentiation. This differentiation block was not observed with the expression of SF3B1G742D, a mutation found in CLL but not RARS, suggesting a context dependent role for SF3B1 mutations. Interestingly, the differentiation block observed in SF3B1K700E was associated with cytokine independent growth. Initial mining of RNAseq data from SF3B1MUT TF-1 cells highlighted several aberrantly spliced and NMD-downregulated genes previously implicated in MDS. Finally, a xenograft model was developed by subcutaneous implantation of transduced TF-1 cells. After several passages, an enrichment of TF-1 SF3B1K700E cells was observed, suggesting a growth advantage for SF3B1MUT cells over SF3B1WT cells. This data suggests that the K700E SF3B1 mutation can lead to a block in differentiation and competitive advantage as observed in human RARS. Citation Format: Silvia Buonamici, Samantha Perino, Kian Huat Lim, Jacob Feala, Rachel Darman, Esther Obeng, Richard R. Furman, Suzanna Bailey, Gregg Keaney, Pavan Kumar, Yoshiharu Mizui, Eunice Park, John Wang, Markus Warmuth, Lihua Yu, Ping Zhu, Benjamin L. Ebert, Peter Smith. Mutations in SF3B1 lead to aberrant splicing through cryptic 3′ splice site selection and impair hematopoietic cell differentiation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2040. doi:10.1158/1538-7445.AM2015-2040