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1.

Abstract
Individuals vary in their responses to therapeutics and susceptibility to disease. Genetic
factors that contribute to variable responses to therapeutics remain largely unknown. We probed

the response of 140 individual C. elegans strains to a panel of commonly used and newly
developed topoisomerase II beta poisons and identified 19 unique quantitative trait loci (QTL).
Chromosomes II, IV,and V all contain QTL hotspots across different compounds, suggesting a
common mechanism of resistance. Etoposide, teniposide, and XK469 resistance is associated
with the right arm of chromosome II. A strong candidate gene in this region is top-2, which is the
C. elegans version of topoisomerase and contains genetic variation in the known drug binding
pocket. Our work shows that Caenorhabditis elegans provides an excellent starting point for
testing the effects of genetic variation on therapeutic efficacy/toxicity.
2. Question

Does genetic variation between individuals in the Caenorhabditis elegans population
contribute to variable responses to chemotherapeutics?
3. Hypothesis
Genetic differences between individuals in the C. elegans population contribute to
variable responses to chemotherapeutics.
4. Background
During DNA replication the DNA double helix must be unwound for DNA polymerase to
access the genetic material. This unwinding causes torsional strain to accumulate in front of and
behind the polymerase, which, if left unchecked, will cause the polymerase to not make forward
progress. Topoisomerase II enzymes mediate the relaxing of DNA supercoils by generating
double stranded DNA breaks and unlooping DNA, which allows for polymerase progression [4].
Because DNA replication is a necessary step for cellular division, topoisomerase function is
essential for cell cycle progression.
Cancer cells have six distinct characteristics: sustaining proliferative signaling, evading
growth suppressors, activating invasion and metastasis, enabling replicative immortality,
inducing angiogenesis, and resisting cell death [5]. Cancer cells replicative immortality and
ability to evade growth suppressors allows for unfettered cell division. As mentioned above,
cancer cells must replicate their DNA in order to divide and in order to replicate DNA,
topoisomerases are needed. Chemotherapeutics, specifically topoisomerase 2 inhibitors, target
the characteristic of replicative immortality by inhibiting the cancer cells topoisomerases,
therefore controlling rapid cell division [6].
Clinical trials are both inefficient and expensive, with a low success rate of drug
approval. According to Hay et al., the likeliness of approval of oncology drugs from phase 1 of
clinical trials was 7% [2]. The cost of developing a new drug through the FDA drug approval
process $350 million [3]. Importantly, genetic variation present in test subjects is rarely
investigated due to the added complexity this variable presents.
Caenorhabditis elegans (C. elegans) provides an excellent model to probe how genetic
variation affects the response to therapeutics because of its short generation time, low cost of
experimentation, levels of genetic variation present in the species similar to humans, and
conservation of molecular machinery [7]. For this project, we were interested in studying the
effect of a class of chemotherapeutics that inhibit topoisomerase II activity on the C. elegans
population. This work has to potential to identify genetic factors that lead to differential
susceptibility in C. elegans, which can then help guide studies in in mammalian models and
clinical trials.

5. Materials and Methods

Phenotyping Pipeline

1. Propagate animals for five generations to avoid


transgenerational effects that result from starvation. 2.
Bleach synchronize generation four in order to isolate
embryos. 3. Embryos hatch into the L1 larval stage and
are then aliquoted into 96-well plates with growth media.
Bacterial lysate (@ 5mg/ml) is added to each well
containing worms to initiate growth. 4. Two days later,
three L4 larval stage are sorted into assay plates
containing drug or control conditions using the COPAS
large-particle sorter. 5. Animals are incubated at 20C
for four days and scored using the COPAS large-particle
sorter. The COPAS large-particle sorter will measure
brood size, worm length, and worm optical density.
Cegwas Pipeline
1. Process phenotypes
a. process_pheno(): outputs a list consisting of a column with strain names and a
column of traits corresponding to the strains.

2. Perform GWAS mappings


a. Use output data from process_pheno() as the input data for the GWAS function
from the rrBLUP package.
3. Process mapping data frame

ex)
processed_phenotypes <- process_pheno(data)
mapping_df <- gwas_mappings(processed_phenotypes, cores = 4, only_sig = TRUE)
processed_mapping_df <- process_mappings(mapping_df, snp_df = snps,
processed_phenotypes, CI_size = 50, snp_grouping = 200)

Plotting Functions:
- manplot(): uses GWAS mapping results to output a manhattan plot
- pxg_plot(): a boxplot at a QTL peak position that shows different phenotypes per
genotype
- gene_variants(): a plot comparing strain and variant to isolate genes of interest

Phenotype data from easysorter is input into the cegwas pipeline. 1. Phenotypes are put into the
correct structure for data analysis. 2. GWAS mappings are performed using algorithm developed
by Kang et. al., which accounts for population structure present in inbred populations, which can
cause false-positive associations.. For example, if a subset of the assayed population is closely
related and susceptible to a topoisomerase poison, then genomic regions corresponding to that
subsets relatedness will show up as significantly associated with the susceptibility phenotype.
Only mappings that have significance values above the Bonferroni corrected value are kept for
further processing. Confidence intervals are calculated by taking +/- 50 SNVs from the peak
position, which was determined by simulation studies. Finally, fine-mapping is performed on all
variants present in the confidence interval to identify the variants that are most highly correlated
with the phenotype of interest.
Easysorter Pipeline

read_data():
Preps data from COPAS
sorter for further processing
remove_contamination():
Removes data from
contaminated wells that are
listed in the contamination
files
sumplate():
Summarizes the data from
plates
bioprune():
Removes biologically
impossible wells (population
of wells > 1000 or population
of wells < 5)
regress(..., assay=TRUE):
Takes a pruned or unpruned
data frame and organizes the
data to account for
differences between assays
bamf_prune():
Takes a summarized plate
and removes outliers from
data
regress():
Takes a pruned or unpruned
data frame and organizes the
data to account for
differences between control
and drug.
Pipeline for processing data generated by COPUS large-particle sorter. 1. Individual worm
size (time of flight or TOF) and optical density (extinction or EXT) measurements are first
prepared for statistical analysis. Contaminated wells are flagged in the processing, number of
animals sorted is added to the score data set, and plates that were stopped in the middle of the run
are stitched together. 2. Contaminated wells and biologically impossible wells are eliminated.
Wells are marked as contaminated while running the plates. Biologically impossible wells are
those that contain fewer than five objects or greater than 1000 objects because each individual
worm that is sorted can have a maximum of 300 progeny. 3. Summary statistics for each well are
generated - normalized brood size (number of objects in score well divided by the number of
objects that were sorted to that well), the 10th quantile, 25th quantile, median, 75th quantile, and
the 90th quantile for TOF and EXT. 4. Linear regression is done to correct for the differences
between assays, which allows for the correct identification of outlier strains. 5. Outlier strains are
removed. 6. A final regression step is performed to account for any variation present in the
control conditions (DMSO (solvent) + bacterial lysate)

Drug Concentrations

Drug Concentration
Amsacrine 50 M
Etoposide 250 M
Mitoxantrone
Low 200 M
Mitoxantrone
High 250 M
Symadex 500 M
Teniposide 120 M
XK469 1000 M

Table 1) Concentrations of drugs used. Two concentrations of mitoxantrone were used because
dose response data (not shown) were not clear. All drugs were dissolved in DMSO. DMSO only
controls were run to account for any response to this solvent
6. Figures
QTL Overview

Figure 1) Unique Quantitative Trait Loci (QTL) for each drug. A QTL is a specific location of a
genome that contributes to phenotypic differences. Phenotypic traits are shown on the y-axis.
Physical genomic position (Mb) is shown on the x-axis. The 48 QTL identified in this
experiment have been condensed to 19 unique QTL for individual treatments. Unique QTL for a
given drug were identified to have less than 50% overlap in their confidence intervals. A
majority of the QTL on chromosome II and chromosome IV are in the same genomic position in
response to four different drugs, suggesting a common mechanism contributing to topoisomerase
II poison sensitivity. Overlapping of between-drug QTL is also observed on chromosomes I and
V, albeit to a lesser extent.

Manhattan Plots
Figure 2) Variation in the fraction of L1s in response to XK469 maps to two loci, one on
chromosome II and one on chromosome IV. This is a representative plot that highlights the to
main topoisomerase II inhibitor QTL hotspots. Each dot corresponds to a genetic difference
present in the tested population (single-nucleotide variant or SNV). The y-axis is the log
transformed p-value obtained from the linear mixed model [8] and the x-axis corresponds to
physical genomic position (Mb). The gray line indicates the Bonferroni-corrected p-value, which
is the most conservative significance threshold for a QTL. Blue dots correspond to SNVs
significantly associated with the fraction of L1 worms in response to XK469 treatment and black
to those below. Red bars highlight the extent of the 95% confidence interval (i.e. we can say with
95% certainty that the causal genetic variant is present within this region).

PxG Plots

Figure 3) Phenotypic difference between the alternate (red box) and the reference genotype
(blue box) for peak QTL SNVs on chromosome II and IV. C. elegans strain N2 serves as the
reference genotype, whereas strains with differences are categorized as the alternate genotype.
The alternate genotype has a higher fraction of L1s compared to the reference genotype for both
QTL, suggesting that genetic factors are contributing to this phenotype in response to XK469
treatment. Having a larger fraction of L1s in the well suggests that the animals were more
strongly affected by the drug. Other drugs that mapped to the same location had similar trends.

Gene Name Gene ID Condition Absolute Spearman Correlation


npp-3 WBGene00003789 Etoposide 0.5769719789
top-2 WBGene00010785 Etoposide 0.5769719789
kel-1 WBGene00002184 Etoposide 0.4272566591
xpf-1 WBGene00008140 Etoposide 0.4170199308
Y17G7B.21
WBGene00012472 Etoposide 0.4074332167
npp-3 WBGene00003789 XK469 0.5333494669
top-2 WBGene00010785 XK470 0.5333494669
tag-180 WBGene00007041 XK471 0.431021521
din-1 WBGene00008549 XK472 0.4194982376
jmjd-1.1 WBGene00005013 XK473 0.3895144475
ubxn-2 WBGene00022381 Mitoxantrone High 0.4397527053
clec-85 WBGene00021872 Mitoxantrone High 0.3884503872
smf-3 WBGene00004878 Mitoxantrone High 0.3389386961
cdh-10 WBGene00000402 Mitoxantrone High 0.3362296169
gpx-4 WBGene00022377 Mitoxantrone High 0.3306811416
col-163 WBGene00000736 Amsacrine 0.4135455241
grd-2 WBGene00001691 Amsacrine 0.4135455241
ttr-12 WBGene00009759 Amsacrine 0.4135455241
clec-263 WBGene00009171 Amsacrine 0.4097860193
abf-4 WBGene00000015 Amsacrine 0.4094350138
fbxb-112 WBGene00021716 Mitoxantrone Low 0.3388836384
fbxb-90 WBGene00044694 Mitoxantrone Low 0.3142012346
nep-10 WBGene00017555 Mitoxantrone Low 0.3086823448
irld-27 WBGene00077751 Mitoxantrone Low 0.3017851456
bath-29 WBGene00017452 Mitoxantrone Low 0.3010109039
bra-1 WBGene00000262 Teniposide 0.3716017065
col-9 WBGene00000598 Teniposide 0.3716017065
flr-1 WBGene00001465 Teniposide 0.3214218877
hlb-1 WBGene00011904 Teniposide 0.3094734099
cwp-5 WBGene00009844 Teniposide 0.2973011866

Table 2) Top five most highly correlated genes for each drug tested. The QTL on chromosome II
in response to XK469, etoposide, and teniposide were highly correlated with eight genes:
F44E5.15, ZK930.5, Y17G7B.21, vps-15, top-2, xpf-1, npp-3, and kel-1. Three genestop-2,
npp-3, and ZK930.5have the same correlation value for a specific phenotype and are in close
proximity to each other, which indicates linkage disequilibrium in that area of the chromosome.
Though QTL appeared on chromosome IV, no genes caused apparent correlation for any
drug/phenotype test. Similar to chromosome II, chromosome V also had QTL, but in response to
different drugsamsacrine and etoposideand were highly correlated with five genes:
Y39B6A.32, fbxb-1, asp-16, nhr-145, and fbxb-63.

Figure 4) Positions of top-2 variants strongly correlated with etoposide, teniposide, and XK469.
The glutamine to methionine mutation (Q797M) is in the known etoposide-DNA-topoisomerase
interaction pocket and is potentially contributing to differential drug binding affinity.
Interestingly, this residue also varies between the two human versions of topoisomerase
(hTOP2alpha and hTOP2beta) that are known to have differential drug binding[9]. E1217A,
D138N, and G1418D are all present in a region of the enzyme known to have little conservation
between species.

7. Conclusion
The purpose of the experiment was to investigate the effects of genetic variation present
in the Caenorhabditis elegans species on chemotherapeutic susceptibility in tightly controlled
environmental conditions. By phenotyping C. elegans, using the COPAS large particle cytometer
to collect data, and using R to process the data, connections between genetic variance and
chemotherapeutic toxicity was discovered. Our approach identified quantitative trait loci on all
six chromosomes. The QTL on chromosome II in response to XK469, etoposide, and teniposide
were highly correlated with eight genes. The hypothesis that genetic variation present in the
C. elegans population contribute to variable responses to chemotherapeutics was supported by
the 19 QTL we identified. The key correlation between drug/phenotype and the gene
topoisomerase II on chromosome II also suggested genetic variance contribution to
chemotherapeutic responses. Genetic variation corresponding with chemotherapeutic response is
the main focus of graduate student Stefan Zdraljevic in the Andersen Lab. Eventually this
experiment will expand, studying additional C. elegans strains and chemotherapeutics. The
end-goal of this experiment is to define a correlation between genetic variation and drug
responses in order to discover the same correlation in humans. Knowing this information will
help personalize medicine for individuals, minimizing side effects while maximizing drug
efficiency.
8. Citations
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[3] Drugs.com,. Drug Approval Process Information. (2015). at
<http://www.drugs.com/fda-approval-process.html>
[4] Burden, D.Andrew, and Neil Osheroff. "Mechanism Of Action Of Eukaryotic Topoisomerase
II And Drugs Targeted To The Enzyme". Biochimica et Biophysica Acta (BBA) - Gene Structure
and Expression1400.1-3 (1998): 139-154. Web.
[5] Hanahan, D. & Weinberg, R. Hallmarks of Cancer: The Next Generation. Cell 144, 646-674
(2011).
[6] Shewach, D. & Kuchta, R. Introduction to Cancer Chemotherapeutics. Chemical Reviews
109, 2859-2861 (2009).
[7] Andersen, E. et al. Chromosome-scale selective sweeps shape Caenorhabditis elegans
genomic diversity. Nature Genetics 44, 285-290 (2012).
[8] Kang, H. et al. Efficient Control of Population Structure in Model Organism Association
Mapping. Genetics 178, 1709-1723 (2008).
[9] Structural Basis of Type II Topoisomerase Inhibition by the Anticancer Drug Etoposide.
Wu, C.-C., T.-K. Li, L. Farh, L.-Y. Lin, T.-S. Lin, Y.-J. Yu, T.-J. Yen, C.-W. Chiang, and N.-L.
Chan. Science (2011)