Comparison of EUS-guided choledochoduodenostomy and percutaneous drainage for distal biliary obstruction: A multicenter cohort study.

Background and Objectives: Percutaneous transhepatic biliary drainage (PTBD) and EUS-guided choledochoduodenostomy (EUS-CD) are alternate therapies to endoscopic retrograde cholangiopancreatography with stent placement for biliary decompression. The primary outcome of this study is to compare the technical and clinical success of PTBD to EUS-CD in patients with distal biliary obstruction. Secondary outcomes were adverse events (AEs), need for reintervention, and survival. Methods: A multicenter retrospective cohort study from three different centers was performed. Cox regression was used to compare time to reintervention and survival and logistic regression to compare technical and clinical success and AE rates. Subgroup analysis was performed in patients with malignant biliary obstruction (MBO). Results: A total of 86 patients (58 PTBD and 28 EUS-CD) were included. The two groups were similar with respect to age, gender, and cause of biliary obstruction, with malignancy being the most common etiology (80.2%). EUS-CD utilized lumen-apposing metal stents in 15 patients and self-expandable metal biliary stents in 13 patients. Technical success was similar been EUS-CD (100%) and PTBD (96.6%; P = 0.3). EUS-CD was associated with higher clinical success compared to PTBD (84.6% vs. 62.1%; P = 0.04). There was a trend toward lower rates of AEs with EUS-CD 14.3% versus PTBD 29.3%, odds ratio: 0.40 (95% confidence interval [CI]: 0.12-1.33, P = 0.14). The need for reintervention was significantly lower among patients who underwent EUS-CD (10.7%) compared to PTBD (77.6%) (hazard ratio: 0.07, 95% CI: 0.02-0.24; P < 0.001). A sensitivity analysis of only patients with MBO demonstrated similar rate of reintervention between the groups in individuals who survived 50 days or less after the biliary decompression. However, reintervention rates were lower for EUS-CD in those with longer survival. Conclusion: EUS-CD is a technically and clinically highly successful procedure with a trend toward lower AEs compared to PTBD. EUS-CD minimizes the need for reintervention, which may enhance end-of-life quality in patients with MBO and expected survival longer than 50 days.

Tumorigenicity of Ewing sarcoma is critically dependent on the trithorax proteins MLL1 and menin.

Developmental transcription programs are epigenetically regulated by the competing actions of polycomb and trithorax (TrxG) protein complexes, which repress and activate genes, respectively. Ewing sarcoma is a developmental tumor that is associated with widespread de-regulation of developmental transcription programs, including HOX programs. Posterior HOXD genes are abnormally over-expressed by Ewing sarcoma and HOXD13, in particular, contributes to the tumorigenic phenotype. In MLL1 fusion-driven leukemia, aberrant activation of HOXA genes is epigenetically mediated by the TrxG complex and HOXA gene expression and leukemogenesis are critically dependent on the protein-protein interaction between the TrxG proteins MLL1 and menin. Based on these data, we investigated whether posterior HOXD gene activation and Ewing sarcoma tumorigenicity are similarly mediated by and dependent on MLL1 and/or menin. Our findings demonstrate that Ewing sarcomas express high levels of both MLL1 and menin and that continued expression of both proteins is required for maintenance of tumorigenicity. In addition, exposure of Ewing sarcoma cells to MI-503, an inhibitor of the MLL1-menin protein-protein interaction developed for MLL1-fusion driven leukemia, leads to loss of tumorigenicity and down-regulated expression of the posterior HOXD gene cluster. Together these data demonstrate an essential role for MLL1 and menin in mediating tumor maintenance and posterior HOXD gene activation in Ewing sarcoma. A critical dependency of these tumors on the MLL1-menin interaction presents a potentially novel therapeutic target.

Using genome-wide CRISPR library screening with library resistant DCK to find new sources of Ara-C drug resistance in AML.

Acute myeloid leukemia (AML) can display de novo or acquired resistance to cytosine arabinoside (Ara-C), a primary component of induction chemotherapy. To identify genes capable of independently imposing Ara-C resistance, we applied a genome-wide CRISPR library to human U937 cells and exposed to them to Ara-C. Interestingly, all drug resistant clones contained guide RNAs for DCK. To avoid DCK gene modification, gRNA resistant DCK cDNA was created by the introduction of silent mutations. The CRISPR screening was repeated using the gRNA resistant DCK, and loss of SLC29A was identified as also being capable of conveying Ara-C drug resistance. To determine if loss of Dck results in increased sensitivity to other drugs, we conducted a screen of 446 FDA approved drugs using two Dck-defective BXH-2 derived murine AML cell lines and their Ara-C sensitive parental lines. Both cell lines showed an increase in sensitivity to prednisolone. Guide RNA resistant cDNA rescue was a legitimate strategy and multiple DCK or SLC29A deficient human cell clones were established with one clone becoming prednisolone sensitive. Dck-defective leukemic cells may become prednisolone sensitive indicating prednisolone may be an effective adjuvant therapy in some cases of DCK-negative AML.

Using RNA-seq and targeted nucleases to identify mechanisms of drug resistance in acute myeloid leukemia.

The evolution from microarrays to transcriptome deep-sequencing (RNA-seq) and from RNA interference to gene knockouts using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) and Transcription Activator-Like Effector Nucleases (TALENs) has provided a new experimental partnership for identifying and quantifying the effects of gene changes on drug resistance. Here we describe the results from deep-sequencing of RNA derived from two cytarabine (Ara-C) resistance acute myeloid leukemia (AML) cell lines, and present CRISPR and TALEN based methods for accomplishing complete gene knockout (KO) in AML cells. We found protein modifying loss-of-function mutations in Dck in both Ara-C resistant cell lines. CRISPR and TALEN-based KO of Dck dramatically increased the IC50 of Ara-C and introduction of a DCK overexpression vector into Dck KO clones resulted in a significant increase in Ara-C sensitivity. This effort demonstrates the power of using transcriptome analysis and CRISPR/TALEN-based KOs to identify and verify genes associated with drug resistance.

Overexpression of HOX genes is prevalent in Ewing sarcoma and is associated with altered epigenetic regulation of developmental transcription programs.

The polycomb proteins BMI-1 and EZH2 are highly overexpressed by Ewing sarcoma (ES), a tumor of stem cell origin that is driven by EWS-ETS fusion oncogenes, most commonly EWS-FLI1. In the current study we analyzed expression of transcription programs that are controlled by polycomb proteins during embryonic development to determine if they are abnormal in ES. Our results show that polycomb target gene expression in ES deviates from normal tissues and stem cells and that, as expected, most targets are relatively repressed. However, we also discovered a paradoxical up regulation of numerous polycomb targets and these were highly enriched for homeobox (HOX) genes. Comparison of HOX profiles between malignant and non-malignant tissues revealed a distinctive HOX profile in ES, which was characterized by overexpression of posterior HOXD genes. In addition, ectopic expression of EWS-FLI1 during stem cell differentiation led to aberrant up regulation of posterior HOXD genes. Mechanistically, this up regulation was associated with altered epigenetic regulation. Specifically, ES and EWS-FLI1+ stem cells displayed a relative loss of polycomb-dependent H3K27me3 and gain of trithorax-dependent H3K4me3 at the promoters of posterior HOXD genes and also at the HOXD11.12 polycomb response element. In addition, a striking correlation was evident between HOXD13 and other genes whose regulation is coordinately regulated during embryonic development by distal enhancer elements. Together, these studies demonstrate that epigenetic regulation of polycomb target genes, in particular HOXD genes, is altered in ES and that these changes are mediated downstream of EWS-FLI1.