Ryan Chuang

Rewiring of Genetic Networks in Response to DNA Damage

DNA Damage Pathways Revealed Despite the dynamic nature of cellular responses, the genetic networks that govern these responses have been mapped primarily as static snapshots. Bandyopadhyay et al. (p. 1385; see the Perspective by Friedman and Schuldiner) report a comparison of large genetic interactomes measured among all yeast kinases, phosphatases, and transcription factors, as the cell responded to DNA damage. The interactomes revealed were highly dynamic structures that changed dramatically with changing conditions. These dynamic interactions reveal genetic relationships that can be more effective than classical “static” interactions (for example, synthetic lethals and epistasis maps) in identifying pathways of interest. A network comparison of genetic interactions mapped at two conditions reveals genetic responses to DNA damage in yeast. Although cellular behaviors are dynamic, the networks that govern these behaviors have been mapped primarily as static snapshots. Using an approach called differential epistasis mapping, we have discovered widespread changes in genetic interaction among yeast kinases, phosphatases, and transcription factors as the cell responds to DNA damage. Differential interactions uncover many gene functions that go undetected in static conditions. They are very effective at identifying DNA repair pathways, highlighting new damage-dependent roles for the Slt2 kinase, Pph3 phosphatase, and histone variant Htz1. The data also reveal that protein complexes are generally stable in response to perturbation, but the functional relations between these complexes are substantially reorganized. Differential networks chart a new type of genetic landscape that is invaluable for mapping cellular responses to stimuli.

A Pan-BCL2 Inhibitor Renders Bone-Marrow-Resident Human Leukemia Stem Cells Sensitive to Tyrosine Kinase Inhibition

Leukemia stem cells (LSCs) play a pivotal role in the resistance of chronic myeloid leukemia (CML) to tyrosine kinase inhibitors (TKIs) and its progression to blast crisis (BC), in part, through the alternative splicing of self-renewal and survival genes. To elucidate splice-isoform regulators of human BC LSC maintenance, we performed whole-transcriptome RNA sequencing, splice-isoform-specific quantitative RT-PCR (qRT-PCR), nanoproteomics, stromal coculture, and BC LSC xenotransplantation analyses. Cumulatively, these studies show that the alternative splicing of multiple prosurvival BCL2 family genes promotes malignant transformation of myeloid progenitors into BC LSCS that are quiescent in the marrow niche and that contribute to therapeutic resistance. Notably, sabutoclax, a pan-BCL2 inhibitor, renders marrow-niche-resident BC LSCs sensitive to TKIs at doses that spare normal progenitors. These findings underscore the importance of alternative BCL2 family splice-isoform expression in BC LSC maintenance and suggest that the combinatorial inhibition of prosurvival BCL2 family proteins and BCR-ABL may eliminate dormant LSCs and obviate resistance.

ΔNp63 Versatilely Regulates a Broad NF-κB Gene Program and Promotes Squamous Epithelial Proliferation, Migration, and Inflammation

Head and neck squamous cell carcinoma (HNSCC) and many other epithelial malignancies exhibit increased proliferation, invasion, and inflammation, concomitant with aberrant nuclear activation of TP53 and NF-κB family members ΔNp63, cRel, and RelA. However, the mechanisms of cross-talk by which these transcription factors coordinate gene expression and the malignant phenotype remain elusive. In this study, we showed that ΔNp63 regulates a cohort of genes involved in cell growth, survival, adhesion, and inflammation, which substantially overlaps with the NF-κB transcriptome. ΔNp63 with cRel and/or RelA are recruited to form novel binding complexes on p63 or NF-κB/Rel sites of multitarget gene promoters. Overexpressed ΔNp63- or TNF-α-induced NF-κB and inflammatory cytokine interleukin-8 (IL-8) reporter activation depended on RelA/cRel regulatory binding sites. Depletion of RelA or ΔNp63 by small interfering RNA (siRNA) significantly inhibited NF-κB-specific, or TNF-α-induced IL-8 reporter activation. ΔNp63 siRNA significantly inhibited proliferation, survival, and migration by HNSCC cells in vitro. Consistent with these data, an increase in nuclear ΔNp63, accompanied by increased proliferation (Ki-67) and adhesion (β4 integrin) markers, and induced inflammatory cell infiltration was observed throughout HNSCC specimens, when compared with the basilar pattern of protein expression and minimal inflammation seen in nonmalignant mucosa. Furthermore, overexpression of ΔNp63α in squamous epithelial cells in transgenic mice leads to increased suprabasilar cRel, Ki-67, and cytokine expression, together with epidermal hyperplasia and diffuse inflammation, similar to HNSCC. Our study reveals ΔNp63 as a master transcription factor that, in coordination with NF-κB/Rels, orchestrates a broad gene program promoting epidermal hyperplasia, inflammation, and the malignant phenotype of HNSCC.

YAP Dysregulation by Phosphorylation or ΔNp63-mediated Gene Repression Promotes Proliferation, Survival and Migration in Head and Neck Cancer Subsets

Overexpression of the Yes-associated protein (YAP), and TP53 family members ΔNp63 and p73, have been independently detected in subsets of head and neck squamous cell carcinomas (HNSCCs). YAP may serve as a nuclear cofactor with ΔNp63 and p73, but the functional role of YAP and their potential relationship in HNSCCs are unknown. In this study, we show that in a subset of HNSCC lines and tumors, YAP expression is increased but localized in the cytoplasm in association with increased AKT and YAP phosphorylation, and with decreased expression of ΔNp63 and p73. In another subset, YAP expression is decreased but detectable in the nucleus in association with lower AKT and YAP phosphorylation, and with increased ΔNp63 and p73 expression. Inhibiting AKT decreased serine-127 phosphorylation and enhanced nuclear translocation of YAP. ΔNp63 bound to the YAP promoter and suppressed its expression. Transfection of a YAP-serine-127-alanine phosphoacceptor-site mutant or ΔNp63 knockdown significantly increased nuclear YAP and cell death. Conversely, YAP knockdown enhanced cell proliferation, survival, migration and cisplatin chemoresistance. Thus, YAP function as a tumor suppressor may alternatively be dysregulated by AKT phosphorylation at serine-127 and cytoplasmic sequestration, or by transcriptional repression by ΔNp63, in different subsets of HNSCC. AKT and/or ΔNp63 are potential targets for enhancing YAP-mediated apoptosis and chemosensitivity in HNSCCs.

Evolution of Laparoscopic Donor Nephrectomy Technique and Outcomes: A Single-center Experience With More Than 1300 Cases

OBJECTIVE To describe and illustrate the evolution of surgical technique, emphasizing technical modifications of laparoscopic donor nephrectomy (LDN) and the impact on complication outcome. METHODS This is a retrospective observational study of prospectively collected data on all consecutive purely LDN surgeries performed at a tertiary academic medical center (n = 1325), performed between March 2000 and October 2013. RESULTS Over time, LDN was performed on older patients, changing from a mean of 35.7 years in 2000 to 41.2 years in 2013 (P <.001). Additionally, mean blood loss decreased from 75 mL in 2000 to 21.6 mL in 2013 (P <.001). However, body mass index, operative time, and length of stay remained similar. Overall, there were 105 (7.9%) complications: Clavien grade 1 (n = 81, 6.1%) and grade 2 or higher (n = 23, 1.8%). Procedure duration, blood loss, surgeon, year of procedure, laterality, body mass index, age, and gender did not significantly predict complications. There was no significant difference for Clavien complication rates between the early learning period (first 150 cases) and the rest of the series. CONCLUSION With continual refinement with LDN techniques based on intraoperative observations and technological advances, complication rates remain consistently low, despite increasing donor age.

Drug Target Optimization in Chronic Myeloid Leukemia Using Innovative Computational Platform

Chronic Myeloid Leukemia (CML) represents a paradigm for the wider cancer field. Despite the fact that tyrosine kinase inhibitors have established targeted molecular therapy in CML, patients often face the risk of developing drug resistance, caused by mutations and/or activation of alternative cellular pathways. To optimize drug development, one needs to systematically test all possible combinations of drug targets within the genetic network that regulates the disease. The BioModelAnalyzer (BMA) is a user-friendly computational tool that allows us to do exactly that. We used BMA to build a CML network-model composed of 54 nodes linked by 104 interactions that encapsulates experimental data collected from 160 publications. While previous studies were limited by their focus on a single pathway or cellular process, our executable model allowed us to probe dynamic interactions between multiple pathways and cellular outcomes, suggest new combinatorial therapeutic targets, and highlight previously unexplored sensitivities to Interleukin-3.

The Cost Burden of Complications after Major Surgery for Urological Cancer: Opportunities for Value Creation in Urology

Introduction While improving patient outcomes and controlling costs have become primary pursuits in health care, priority areas for value creation remain unclear. In urology operative morbidity serves as a major barrier to high value care. To guide improvement efforts we assessed the prevalence and cost of inpatient complications among patients undergoing major surgery for urological cancer. Methods Using the Nationwide Inpatient Sample from 2009 to 2011 we identified hospital admissions for cancer related prostatectomy, nephrectomy and cystectomy among adults age 18 years or older. We then measured the occurrence of inpatient complications, medical and surgical, and used multivariable, mixed effect models to estimate the associated marginal cost. Results Among weighted samples of 229,743 prostatectomies, 111,683 nephrectomies and 31,213 cystectomies, inpatient complications occurred in 9.4% (95% CI 8.6–10.2), 32.0% (95% CI 30.7–33.4) and 57.7% (95% CI 54.7–60.6) of hospital admissions, respectively. For these respective samples an adverse event added

Health Insurance Status and Disparities in Kidney Cancer Care

4,947 (95% CI 4,523–5,454),

Technique and outcomes of bladder neck intussusception during robot-assisted laparoscopic prostatectomy: A parallel comparative trial

6,782 (95% CI 6,336–7,293) and

A Case of an Invisible Ureteral Stent

10,756 (95% CI 9,999–11,759) to the cost of inpatient care. While surgical events occurred most frequently, medical complications generated