PRL3-zumab, a first-in-class humanized antibody for cancer therapy

Novel, tumor-specific drugs are urgently needed for a breakthrough in cancer therapy. Herein, we generated a first-in-class humanized antibody (PRL3-zumab) against PRL-3, an intracellular tumor-associated phosphatase upregulated in multiple human cancers, for unconventional cancer immunotherapies. We focused on gastric cancer (GC), wherein elevated PRL-3 mRNA levels significantly correlated with shortened overall survival of GC patients. PRL-3 protein was overexpressed in 85% of fresh-frozen clinical gastric tumor samples examined but not in patient-matched normal gastric tissues. Using human GC cell lines, we demonstrated that PRL3-zumab specifically blocked PRL-3(+), but not PRL-3(–), orthotopic gastric tumors. In this setting, PRL3-zumab had better therapeutic efficacy as a monotherapy, rather than simultaneous combination with 5-fluorouracil or 5-fluorouracil alone. PRL3-zumab could also prevent PRL-3(+) tumor recurrence. Mechanistically, we found that intracellular PRL-3 antigens could be externalized to become “extracellular oncotargets” that serve as bait for PRL3-zumab binding to potentially bridge and recruit immunocytes into tumor microenvironments for killing effects on cancer cells. In summary, our results document a comprehensive cancer therapeutic approach to specific antibody-targeted therapy against the PRL-3 oncotarget as a case study for developing antibodies against other intracellular targets in drug discovery

Understanding mammographic density and breast cancer risk: from histology to genomics

© 2012 Dr. Suling Joyce LinThis thesis describes a novel multi-disciplinary strategy that advances our understanding of mammographic density (MD) biology and genetics and its relationship to breast cancer risk. Studies have indicated that women with high MD are at 4 to 6 fold increased risk of getting breast cancer. MD is also highly heritable. Hence, a greater understanding of MD biology and genetics would improve our knowledge about breast biology and potentially identify new cancer predisposition genes. Nonetheless, MD biology remains controversial and much is unknown about the genetic basis of this trait. This thesis revisits MD histopathology using a novel methodology of sampling high and low MD tissue within the breast of an individual. This method samples tissue guided by its true MD, as determined by real-time mammography imaging - in contrast to previous studies that examined MD histopathology relying on “random” sampling of breast tissue. Studies have suggested that dense regions of the breast are potentially associated with breast cancer risk on the basis that percent MD (PMD) adjusted for body mass index (BMI) and age is a better measure of breast cancer risk. Hence, this method also allows assessment of tissue most pertinent to MD associated breast cancer risk. Furthermore, the use of sampling within an individual allows control of all potential confounders that helps improve the power of analysis. The histopathological analysis revealed that high MD regions were significantly associated with greater composition of dense connective tissue stroma and lesser composition of adipose tissue while no difference in glandular areas between high and low MD tissue was observed. However, it was noted that high MD tissue tended to have smaller and lower complexity glands compared to low MD tissue. This raises the possibility of high MD regions being associated with stem-like cells and their niche compared to low MD regions. Whole-genome expression profiling of accrued high and low MD tissue were interrogated to further our understanding of the genetic and biological bases of MD. Currently, only two other studies have investigated gene expression in MD tissue and these studies have generally failed to account for all potential unwanted variation in the data that could impact on the validity of the analysis outcomes. The present work differs from previous studies in that careful quality assessments and analyses were performed to improve sensitivity and power of the study. The use of precisely sampled tissue also allowed a better representation of the MD expression profile. Both single-gene and gene-set based analyses of adjusted MD expression dataset concurred with the histopathological correlates of high versus low MD tissue. Interestingly, these analyses also showed that high MD regions correlated with a cancer-signature and a CD24 (i.e. luminal epithelial)-signature; while the observed anti-correlation with the CD44-signature was postulated to be of stromal origin. The association of high versus low MD tissue with a cancer-signature agrees with the general direction of MD associated breast cancer risk. Furthermore, immunohistochemical examination of the tissue uncovered a trend of potential “stemness” in high versus low MD tissue that suggests the CD24-signature being of progenitor origin. This supports the hypothesis of high MD tissue being associated with “stemness” (and their niche), as proposed from the initial histopathological examination of MD. To further understand MD biology and its genetic basis, both univariate and pathway-based analyses of genome-wide association (GWA) study data were performed. One major issue with such pathway-based analysis is the assignment of SNPs to their respective gene(s). Increasingly, studies suggest that conventional assignment of SNPs to their nearest gene may be inaccurate. This was the impetus to develop a novel framework using expression profiling analysis of high and low MD tissue to guide mapping of SNPs to their genes. The methodology helped improve the biological relevance and reproducibility of the significant pathways, and also identified the mitogen-activated protein kinase (MAPK) signalling pathway as the most significantly associated with the MD trait. This outcome corresponds to the results obtained from a recent study of breast cancer GWA studies. Taken together, this multi-disciplinary approach has given potential insight into the biological and genetic basis of MD, allowing inference to be made about the increased risk associated with high MD

A randomised double-blind, cross-over trial of 4-aminopyridine for downbeat nystagmus--effects on slowphase eye velocity, postural stability, locomotion and symptoms

Objective The effects of 4-aminopyridine (4-AP) on downbeat nystagmus (DBN) were analysed in terms of slow-phase velocity (SPV), stance, locomotion, visual acuity (VA), patient satisfaction and side effects using standardised questionnaires. Methods Twenty-seven patients with DBN received 5 mg 4-AP four times a day or placebo for 3 days and 10 mg 4-AP four times a day or placebo for 4 days. Recordings were done before the first, 60 min after the first and 60 min after the last drug administration. Results SPV decreased from 2.42 deg/s at baseline to 1.38 deg/s with 5 mg 4-AP and to 2.03 deg/s with 10 mg 4-AP (p<0.05; post hoc: 5 mg 4-AP: p=0.04). The rate of responders was 57%. Increasing age correlated with a 4-AP-related decrease in SPV (p<0.05). Patients improved in the ‘get-up-and-go test’ with 4-AP (p<0.001; post hoc: 5 mg: p=0.025; 10 mg: p<0.001). Tandem-walk time (both p<0.01) and tandem-walk error (4-AP: p=0.054; placebo: p=0.059) improved under 4-AP and placebo. Posturography showed that some patients improved with the 5 mg 4-AP dose, particularly older patients. Near VA increased from 0.59 at baseline to 0.66 with 5 mg 4-AP (p<0.05). Patients with idiopathic DBN had the greatest benefit from 4-AP. There were no differences between 4-AP and placebo regarding patient satisfaction and side effects. Conclusions 4-AP reduced SPV of DBN, improved near VA and some locomotor parameters. 4-AP is a useful medication for DBN syndrome, older patients in particular benefit from the effects of 5 mg 4-AP on nystagmus and postural stability

VHL Deficiency Drives Enhancer Activation of Oncogenes in Clear Cell Renal Cell Carcinoma

Protein-coding mutations in clear cell renal cell carcinoma (ccRCC) have been extensively characterized, frequently involving inactivation of the von Hippel-Lindau (VHL) tumor suppressor. Roles for noncoding cis-regulatory aberrations in ccRCC tumorigenesis, however, remain unclear. Analyzing 10 primary tumor/normal pairs and 9 cell lines across 79 chromatin profiles, we observed pervasive enhancer malfunction in ccRCC, with cognate enhancer-target genes associated with tissue-specific aspects of malignancy. Superenhancer profiling identified ZNF395 as a ccRCC-specific and VHL-regulated master regulator whose depletion causes near-complete tumor elimination in vitro and in vivo. VHL loss predominantly drives enhancer/superenhancer deregulation more so than promoters, with acquisition of active enhancer marks (H3K27ac, H3K4me1) near ccRCC hallmark genes. Mechanistically, VHL loss stabilizes HIF2 alpha-HIF1 beta heterodimer binding at enhancers, subsequently recruiting histone acetyltransferase p300 without overtly affecting preexisting promoter-enhancer interactions. Subtype-specific driver mutations such as VHL may thus propagate unique pathogenic dependencies in ccRCC by modulating epigenomic landscapes and cancer gene expression. SIGNIFICANCE: Comprehensive epigenomic profiling of ccRCC establishes a compendium of somatically altered cis-regulatory elements, uncovering new potential targets including ZNF395, a ccRCC master regulator. Loss of VHL, a ccRCC signature event, causes pervasive enhancer malfunction, with binding of enhancer-centric HIF2 alpha and recruitment of histone acetyltransferase p300 at preexisting lineage-specific promoter-enhancer complexes. (C) 2017 AACR

Genomic and epigenomic <i>EBF1</i> alterations modulate<i> TERT</i> expression in gastric cancer

Transcriptional reactivation of telomerase catalytic subunit (TERT) is a frequent hallmark of cancer, occurring in 90% of human malignancies. However, specific mechanisms driving TERT reactivation remain obscure for many tumor types and in particular gastric cancer (GC), a leading cause of global cancer mortality. Here, through comprehensive genomic and epigenomic analysis of primary GCs and GC cell lines, we identified the transcription factor early B cell factor 1 (EBF1) as a TERT transcriptional repressor and inactivation of EBF1 function as a major cause of TERT upregulation. Abolishment of EBF1 function occurs through 3 distinct (epi)genomic mechanisms. First, EBF1 is epigenetically silenced via DNA methyltransferase, polycomb-repressive complex 2 (PRC2), and histone deacetylase activity in GCs. Second, recurrent, somatic, and heterozygous EBF1 DNA-binding domain mutations result in the production of dominant-negative EBF1 isoforms. Third, more rarely, genomic deletions and rearrangements proximal to the TERT promoter remobilize or abolish EBF1-binding sites, derepressing TERT and leading to high TERT expression. EBF1 is also functionally required for various malignant phenotypes in vitro and in vivo, highlighting its importance for GC development. These results indicate that multimodal genomic and epigenomic alterations underpin TERT reactivation in GC, converging on transcriptional repressors such as EBF1

Genomic and epigenomic EBF1 alterations modulate TERT expression in gastric cancer

Transcriptional reactivation of telomerase catalytic subunit (TERT) is a frequent hallmark of cancer, occurring in 90% of human malignancies. However, specific mechanisms driving TERT reactivation remain obscure for many tumor types and in particular gastric cancer (GC), a leading cause of global cancer mortality. Here, through comprehensive genomic and epigenomic analysis of primary GCs and GC cell lines, we identified the transcription factor early B cell factor 1 (EBF1) as a TERT transcriptional repressor and inactivation of EBF1 function as a major cause of TERT upregulation. Abolishment of EBF1 function occurs through 3 distinct (epi)genomic mechanisms. First, EBF1 is epigenetically silenced via DNA methyltransferase, polycomb-repressive complex 2 (PRC2), and histone deacetylase activity in GCs. Second, recurrent, somatic, and heterozygous EBF1 DNA-binding domain mutations result in the production of dominant-negative EBF1 isoforms. Third, more rarely, genomic deletions and rearrangements proximal to the TERT promoter remobilize or abolish EBF1-binding sites, derepressing TERT and leading to high TERT expression. EBF1 is also functionally required for various malignant phenotypes in vitro and in vivo, highlighting its importance for GC development. These results indicate that multimodal genomic and epigenomic alterations underpin TERT reactivation in GC, converging on transcriptional repressors such as EBF1