DataSheet_1_Hydrophobicity identifies false positives and false negatives in peptide-MHC binding.pdf

Major Histocompability Complex (MHC) Class I molecules allow cells to present foreign and endogenous peptides to T-Cells so that cells infected by pathogens can be identified and killed. Neural networks tools such as NetMHC-4.0 and NetMHCpan-4.1 are used to predict whether peptides will bind to variants of MHC molecules. These tools are trained on data gathered from binding affinity and eluted ligand experiments. However, these tools do not track hydrophobicity, a significant biochemical factor relevant to peptide binding, in their predictions. A previous study had concluded that the peptides predicted to bind to HLA-A*0201 by NetMHC-4.0 were much more hydrophobic than expected. This paper expands that study by also focusing on HLA-B*2705 and HLA-B*0801, which prefer binding hydrophilic and balanced peptides respectively. The correlation of hydrophobicity of 9-mer peptides with their predicted binding strengths to these various HLAs was investigated. Two studies were performed, one using the data that the two neural networks were trained on, and the other using a sample of the human proteome. NetMHC-4.0 was found to have a statistically significant bias towards predicting highly hydrophobic peptides as strong binders to HLA-A*0201 and HLA-B*2705 in both studies. Machine Learning metrics were used to identify the causes for this bias: hydrophobic false positives and hydrophilic false negatives. These results suggest that the retraining the neural networks with biochemical attributes such as hydrophobicity and better training data could increase the accuracy of their predictions. This would increase their impact in applications such as vaccine design and neoantigen identification.</p

Topoisomerase 2 Alpha Cooperates with Androgen Receptor to Contribute to Prostate Cancer Progression

<div><p>Overexpression of TOP2A is associated with risk of systemic progression in prostate cancer patients, and higher levels of TOP2A were found in hormone-resistant cases. To elucidate the mechanism by which high levels of TOP2A contribute to tumor progression we generated TOP2A overexpressing prostate cancer cell lines. We show that TOP2A promotes tumor aggressiveness by inducing chromosomal rearrangements of genes that contribute to a more invasive phenotype. Anti-androgen treatment alone was ineffective in killing TOP2A overexpressing cells due to activation of an androgen receptor network. TOP2A poisons killed tumor cells more efficiently early in the progression course, while at later stages they provided greater benefit when combined with anti-androgen therapy. Mechanistically, we find that TOP2A enhances androgen signaling by facilitating transcription of androgen responsive genes, thereby promoting tumor cell growth. These studies revealed a relationship between TOP2A and androgen receptor signaling pathway that contributes to prostate cancer progression and confers sensitivity to treatments.</p></div

TOP2A protein is co-recruited to promoters of androgen responsive elements to stimulate gene expression.

<p>Western blot showing total level of AR in TOP2A clones of different passages (as indicated), top panel. Quantification of AR expression normalized to beta actin level, bottom panel. <b>B)</b> Western blot showing induction of AR level (top) upon treatment of TOP2A clones.with 5 nM of R1881. Corresponding quantification of TOP2A levels normalized to TBP loading control is shown (bottom); nt is non-treated, t is treated; cell passages are as indicated. <b>C)</b> Schematic showing promoter area for <i>KLK3</i> (PSA) gene used in CHIP assay. DtFw and DtRv depict positions of forward and reverse primers respectively for distal promoter region; PxFw and PxRv -positions for forward and reverse primers of proximal promoter. <b>D)</b> CHIP analysis of TOP2A clones stimulated with 5 nM of R1881 for 3 hours. Enrichment of AR and TOP2A at distal and proximal promoter regions of KLK3 gene (top and middle panels) and their absence at coding region of GAPDH gene (bottom panel) are shown. M is DNA size marker, HG-is human genomic DNA used as a positive control for PCR amplification, El1 and El2 are consecutive elution fractions (buffers are described in Methods).</p

Genes affected in TOP2A overexpressing clone.

<p>Genes affected in TOP2A overexpressing clone.</p

TOP2A promotes accumulation of DNA rearrangements in proliferation PCa cells.

<p><b>A)</b> Total number of breakpoints present in T25 cells of different passages. LNCaP/casp is a derivative of LNCaP cell line stably expressing siRNA to knock down caspase 3. <b>B)</b> DNA alterations gained by TOP2A overexpressing T14 cells upon proliferation. Numbers represent alterations observed in addition to those present in control T25 clone. <b>C)</b> Schematic showing proposed role of TOP2A in prostate cancer progression and resistance to androgen ablation therapy.</p

Sensitivity of TOP2A overexpressing to treatment with TOP2A poison and anti-androgen.

<p><b>A)</b> Survival of TOP2A clones of early passage in response to treatment with doxorubicin (top panel) and casodex (bottom panel). <b>B)</b> Survival of TOP2A clones of late passage in response to treatment with doxorubicin (top panel) and casodex (bottom panel). <b>C)</b> Western blot showing levels of TOP2A in generated stable clones of early and late passages. Detection of nuclear protein TBP was used to verify equal loading. <b>D)</b> Quantification of TOP2A expression based on Western blot in C. Data are presented as mean ± SD, based on 3 independent experiments. P values are as indicated, n.s. is not significant.</p

TOP2A promotes aggressive invasive phenotype.

<p><b>A)</b> Proliferation of clones stably overexpressing TOP2A (T14) and matched control cells with endogenous level of TOP2A (T25 and T33). Proliferation was determined at 96 hours after cell plating. <b>B)</b> Motility of TOP2A clones of different passage and parental LNCaP cells determined using Boyden chamber assay. <b>C)</b> Images of cells with invasive phenotype after 72 hours of proliferation. P designates passage number; OD, au is measured optic density in arbitrary units. Data are presented as mean ± SD, based on 3 independent experiments. *p < 0.00001, and **p < 0.0001, n = 3.</p

Sensitivity of TOP2A overexpressing cells to combination treatment with TOP2A poison and anti-androgen.

<p><b>A)</b> Survival of TOP2A clones of early passage in response to combination treatment: top panel- constant dose of doxorubicin and increasing concentrations of casodex; bottom panel-constant dose of casodex and increasing concentrations of doxorubicin. <b>B)</b> Survival of TOP2A clones of late passage in response to combination treatment, drugs and concentrations are as in a. Data are presented as mean ± SD, based on 3 independent experiments. P values are as indicated, n.s. is not significant.</p

Genome plots showing landscape of DNA rearrangements in generated clones.

<p><b>A)</b> Late passage of T25 clone expressing endogenous level of TOP2A. <b>B)</b> Late passage of clone T14 overexpressing TOP2A. Grey dots (counts) show frequency of distribution of reads in 30KB windows and breakpoints for all chromosomes (numbers are indicated). The X axis spans the length of the chromosome, the Y axis shows the number of reads for each window. Window counts are shown according to the prediction by CNV algorithm. Grey points are normal, red points correspond to deletions and blue points show gains. Lines connect bioinformatically identified breakpoints. The widths of the lines correlate with number of associated mate-pair reads. Color of the connecting lines indicates polarity of the joined chromosome. For intra-chromosomal events red shows forward direction for both pieces, green indicates inversion for one partner and blue shows inversion for both. For inter-chromosomal events, red connects the p-side piece from the larger chromosome to the q-side piece of the smaller chromosome in forward direction, green connects the q-side piece from the larger chromosome to the p-side piece of the smaller chromosome in forward direction, blue connects the p-side piece from the larger chromosome to the p-side piece of the smaller chromosome in reverse direction and magenta connects the q-side piece from the larger chromosome to the q-side piece of the smaller chromosome in reverse direction. Black indicates balanced translocations. Blue arrowheads point to selected DNA rearrangements that were acquired upon proliferation (absent in corresponding cells of earlier passage.</p

Nanopore-Based Assay for Detection of Methylation in Double-Stranded DNA Fragments

DNA methylation is an epigenetic modification of DNA in which methyl groups are added at the 5-carbon position of cytosine. Aberrant DNA methylation, which has been associated with carcinogenesis, can be assessed in various biological fluids and potentially can be used as markers for detection of cancer. Analytically sensitive and specific assays for methylation targeting low-abundance and fragmented DNA are needed for optimal clinical diagnosis and prognosis. We present a nanopore-based direct methylation detection assay that circumvents bisulfite conversion and polymerase chain reaction amplification. Building on our prior work, we used methyl-binding proteins (MBPs), which selectively label the methylated DNA. The nanopore-based assay selectively detects methylated DNA/MBP complexes through a 19 nm nanopore with significantly deeper and prolonged nanopore ionic current blocking, while unmethylated DNA molecules were not detectable due to their smaller diameter. Discrimination of hypermethylated and unmethylated DNA on 90, 60, and 30 bp DNA fragments was demonstrated using sub-10 nm nanopores. Hypermethylated DNA fragments fully bound with MBPs are differentiated from unmethylated DNA at 2.1- to 6.5-fold current blockades and 4.5- to 23.3-fold transport durations. Furthermore, these nanopore assays can detect the <i>CpG</i> dyad in DNA fragments and could someday profile the position of methylated <i>CpG</i> sites on DNA fragments