Last updated: 2023-09-28

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Knit directory: Cardiotoxicity/

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library(tidyverse)
library(BiocGenerics)
library(data.table)
library(cowplot)
library(ggsignif)
library(RColorBrewer)
library(broom)
library(ggVennDiagram)
library(paletteer)
toplistall <- readRDS("data/toplistall.RDS")
siglist <- readRDS("data/siglist_final.RDS")
list2env(siglist, envir = .GlobalEnv)
<environment: R_GlobalEnv>
cpmcounts <- readRDS("data/cpmcount.RDS")

my_exp_genes <- read.csv("data/backGL.txt")
GTEx_genes <- read.csv("data/GTEx_gene_list.csv",row.names = 1)
not_eqtls <- read.csv("output/not_eqtls_GTEX.csv",row.names = 1)
heart_gtex <- read.csv("output/heart_gtex.csv",row.names = 1)
egenes <- read.csv("output/egenes.csv",row.names = 1)
plot_venn <- function (x, show_intersect, set_color, set_size, label, label_geom,
                       label_alpha, label_color, label_size, label_percent_digit,
                       label_txtWidth, edge_lty, edge_size, ...)  {
  venn <- Venn(x)
  data <- process_data(venn)
  p <- ggplot() + geom_sf(aes_string(fill = "count"), data = data@region) +
    geom_sf(aes_string(color = "name"), data = data@setEdge,
            show.legend = F, lty = edge_lty, size = edge_size, color = set_color) +
    geom_sf_text(aes_string(label = "name"), data = data@setLabel,
                 size = set_size, color = set_color) + theme_void()
  if (label != "none" & show_intersect == FALSE) {
    region_label <- data@region %>% dplyr::filter(.data$component ==
                                                    "region") %>% dplyr::mutate(percent = paste(round(.data$count *
                                                                                                        100/sum(.data$count), digits = label_percent_digit),
                                                                                                "%", sep = "")) %>% dplyr::mutate(both = paste(.data$count,
                                                                                                                                               paste0("(", .data$percent, ")"), sep = "\n"))
    if (label_geom == "label") {
      p <- p + geom_sf_label(aes_string(label = label),
                             data = region_label, alpha = label_alpha, color = label_color,
                             size = label_size, lineheight = 0.85, label.size = NA)
    }
    if (label_geom == "text") {
      p <- p + geom_sf_text(aes_string(label = label),
                            data = region_label, alpha = label_alpha, color = label_color,
                            size = label_size, lineheight = 0.85)
    }
  }
  if (show_intersect == TRUE) {
    items <- data@region %>% dplyr::rowwise() %>% dplyr::mutate(text = stringr::str_wrap(paste0(.data$item,
                                                                                                collapse = " "), width = label_txtWidth)) %>% sf::st_as_sf()
    label_coord = sf::st_centroid(items$geometry) %>% sf::st_coordinates()
    p <- ggplot(items) + geom_sf(aes_string(fill = "count")) +
      geom_sf_text(aes_string(label = "name"), data = data@setLabel,
                   inherit.aes = F) + geom_text(aes_string(label = "count",
                                                           text = "text"), x = label_coord[, 1], y = label_coord[,
                                                                                                                 2], show.legend = FALSE) + theme_void()
    ax <- list(showline = FALSE)
    p <- plotly::ggplotly(p, tooltip = c("text")) %>% plotly::layout(xaxis = ax,
                                                                     yaxis = ax)
  }
  p
}

Figure 8: DOX response eGenes are enriched amongst TOP2i response genes​.

A. 24 hour DEG enrichment in GTEx genes

## create GTEx data set from my data
GTEx <- intersect(GTEx_genes$entrezgene_id,my_exp_genes$ENTREZID)
## exclude GTEX and create nQTL set with other expressed genes
nQTLmy <- my_exp_genes %>%
   dplyr:: filter(!ENTREZID %in%GTEx)
drug_palspc <- c("darkblue","cornflowerblue","darkblue","cornflowerblue")
drug_pal_fact <- c("#8B006D" ,"#DF707E", "#F1B72B" ,"#3386DD", "#707031","#41B333")
#GET nQTL umbers
nQTLsum <- toplistall %>%
  mutate(id =dplyr::case_match(id, "Daunorubicin"~"DNR",
                               "Doxorubicin"~"DOX",
                               "Epirubicin"~"EPI",
                               "Mitoxantrone"~"MTX",
                               "Trastuzumab"~"TRZ",
                               "Vehicle"~"VEH",
                               .default = id)) %>% 
  dplyr::filter(time=="24_hours") %>%
  dplyr::filter(adj.P.Val <0.05) %>%
  mutate(nQTL=if_else(ENTREZID %in% nQTLmy$ENTREZID,'nQTL_y','nQTL_no')) %>% 
  group_by(id,nQTL) %>% 
  tally() %>% 
  separate(nQTL, into=c('set', 'group')) %>% 
  mutate(total=length(nQTLmy$ENTREZID) - n) %>% 
  dplyr::filter(group=="y")
#GETx GTEX numbers
GTExsum <- toplistall %>%
  mutate(id =dplyr::case_match(id, "Daunorubicin"~"DNR",
                               "Doxorubicin"~"DOX",
                               "Epirubicin"~"EPI",
                               "Mitoxantrone"~"MTX",
                               "Trastuzumab"~"TRZ",
                               "Vehicle"~"VEH", 
                               .default = id)) %>% 
  dplyr::filter(time=="24_hours") %>%
  dplyr::filter(adj.P.Val <0.05) %>%
  mutate(GTEx=if_else(ENTREZID %in%GTEx,"GTEx_y","GTEx_no")) %>% 
  group_by(id,GTEx) %>% 
  tally() %>% 
  separate(GTEx, into=c('set', 'group')) %>% 
  mutate(total=length(GTEx) - n) %>% 
  dplyr::filter(group=="y")

##combine and create long data frame for plot
GTEXcr8z <- GTExsum %>% 
  rbind(., nQTLsum) %>% 
  dplyr::select(id,set, n,total) %>%
  mutate(id=factor(id, levels = c('DOX','EPI','DNR','MTX','TRZ','VEH'))) %>%
  pivot_longer(cols=n:total, 
               names_to="group",
               values_to="total") %>%
   mutate(group=factor(group,levels = c("total", "n"),labels=c("not DE","DE"))) %>% 
  mutate(set=factor(set,levels = c("GTEx","nQTL"), labels =c("eGene", "not eGene")))

 
GTEXcr8z %>% 
  ggplot(., aes(x=set,y=total, fill=group))+
  geom_col(position='fill')+
  facet_wrap(~id,nrow=2,ncol=4)+
  theme_classic()+
  scale_fill_manual(values=drug_palspc)+
  ylab("Heart: left ventricle eGenes")+
  xlab("")+
  scale_y_continuous( expand = expansion(c(0, 0.01))) +
  theme(strip.background = element_rect(fill = "white",
                                        linetype=1, 
                                        linewidth = 0.5),
        plot.title = element_text(size=12,
                                  hjust = 0.5,
                                  face="bold"),
        axis.title = element_text(size = 12, 
                                  color = "black"),
        axis.ticks = element_line(linewidth = 1.0),
        axis.text = element_text(color = "black"),
        panel.background = element_rect(colour = "black",
                                        size=1),
        strip.text.x = element_text(size=12,
                                    face = "bold"))#+

Version Author Date
4bb0b80 reneeisnowhere 2023-07-28
c40aced reneeisnowhere 2023-07-26
e20fc1d reneeisnowhere 2023-07-10
fa1dc68 reneeisnowhere 2023-07-06
b0dd36d reneeisnowhere 2023-07-04
272159f reneeisnowhere 2023-06-28

Additonal GTEx analysis and code is found here

B. 24 hour DEG enrichment in DOX-iPSC-DM eGenes

knowles4 <-readRDS("output/knowles4.RDS")
knowles5 <-readRDS("output/knowles5.RDS")
Knowles_count <- 
  toplistall %>%
  mutate(id = dplyr::case_match(id, "Daunorubicin"~"DNR",
                                "Doxorubicin"~"DOX",
                                "Epirubicin"~"EPI",
                                "Mitoxantrone"~"MTX",
                                "Trastuzumab"~"TRZ",
                                "Vehicle"~"VEH", 
                                .default = id)) %>%
  filter(id!='TRZ') %>% 
  mutate(id=factor(id, levels = c('DOX','EPI','DNR','MTX','TRZ','VEH'))) %>%
  mutate(time=factor(time, levels=c("3_hours","24_hours"))) %>%
  group_by(time, id) %>%
  mutate(K4 = if_else(ENTREZID %in% knowles4$entrezgene_id,1,0))%>%
  mutate(K5 = if_else(ENTREZID %in% knowles5$entrezgene_id,1,0))%>%
  filter(adj.P.Val<0.05) %>%
  dplyr::summarize(n=n(), K4=sum(K4), K5=sum(K5)) %>% 
  as.tibble() %>% 
  dplyr::select(time,id,K4,K5) %>% 
  rename("K4_y"='K4',"K5_y"='K5') %>% 
  mutate(time = case_match(time, '3_hours'~'3 hrs',
                           '24_hours'~'24 hrs',
                           .default = time)) %>% 
  mutate(K4_n= 417-K4_y, K5_n=273-K5_y) %>% 
  pivot_longer(!c(time,id), 
               names_to='QTL',
               values_to="gene_count") %>%
  separate(QTL,into=c("QTL_type",'group'),sep = '_') %>% 
  mutate(QTL_type =case_match(QTL_type, 
                              'K4'~'base\neGenes',
                              'K5'~'response\neGenes',.default = QTL_type)) %>%
  mutate(time=factor(time, levels=c("3 hrs","24 hrs"))) %>% 
  group_by(id,time,QTL_type) %>% 
  mutate(percent=gene_count/sum(gene_count)*100)  %>% 
  ungroup() %>% 
  filter(time=="24 hrs") %>% 
  mutate(id=factor(id, levels = c('DOX','EPI','DNR','MTX','TRZ','VEH'))) %>%  mutate(group=factor(group, levels= c("n","y"), labels=c("not DE","DE")))
  
  
  ggplot(Knowles_count, aes(x=QTL_type,y=gene_count, group=group,  fill=group))+
    geom_col(position='fill')+
    facet_wrap(~id,nrow=1,ncol=4)+
    theme_classic()+
    ylab("iPSC-CM DOX eGenes ")+
    xlab(" ")+
    scale_color_manual(values=drug_palspc)+
    scale_fill_manual(values=drug_palspc)+
    scale_y_continuous( expand = expansion(c(0, 0.01))) +
    theme(strip.background = element_rect(fill = "white",
                                        linetype=1, 
                                        linewidth = 0.5),
        plot.title = element_text(size=12,
                                  hjust = 0.5,
                                  face="bold"),
        axis.title = element_text(size = 12, 
                                  color = "black"),
        axis.ticks = element_line(linewidth = 1.0),
        axis.text = element_text(color = "black"),
        panel.background = element_rect(colour = "black",
                                        size=1),
        strip.text.x = element_text(size=12,
                                    face = "bold"))

Version Author Date
4bb0b80 reneeisnowhere 2023-07-28
c40aced reneeisnowhere 2023-07-26
e20fc1d reneeisnowhere 2023-07-10
fa1dc68 reneeisnowhere 2023-07-06
b0dd36d reneeisnowhere 2023-07-04
e81dfd3 reneeisnowhere 2023-06-28
272159f reneeisnowhere 2023-06-28

Additonal analysis and code for the Knowles data is found here

C. DOXreQTLS strongly overlap other TOP2i drugs

DOXreQTLs <- readRDS("output/DOXreQTLs.RDS")

assignInNamespace(x="plot_venn", value=plot_venn, ns="ggVennDiagram") 

reQTL_overlapDE24 <- list(DOXreQTLs$ENTREZID,sigVDA24$ENTREZID, sigVEP24$ENTREZID, sigVMT24$ENTREZID)
re_incommon <- c(DOXreQTLs$ENTREZID,sigVDA24$ENTREZID, sigVEP24$ENTREZID, sigVMT24$ENTREZID)

names(reQTL_overlapDE24) <- c("Dox_reQTLS", "DNR DEGs","EPI DEGs","MTX DEGs")

DEG_incommon <-c(sigVDA24$ENTREZID, sigVEP24$ENTREZID, sigVMT24$ENTREZID)
uniqueDEGs_incommon <- unique(DEG_incommon)
two_venn <- list( DOXreQTLs$ENTREZID,uniqueDEGs_incommon)

ggVennDiagram::ggVennDiagram(two_venn,
                             category.names = c("DOX egenes\nn = 142","union of TOP2i DEGs\n     n = 7838"),
              show_intersect = FALSE,
              set_color = "black",
              category_size = c(6,6),
              label = "count",
              label_percent_digit = 1,
              label_size = 4,
              label_alpha = 0,
              label_color = "black",
              edge_lty = "solid", set_size = 4.5)+
  scale_x_continuous(expand = expansion(mult = .3))+
  scale_y_continuous(expand = expansion(mult = .1))+
   scale_fill_distiller(palette="Spectral", direction = -1, limits= c(1,200),oob=scales::squish)

Version Author Date
c40aced reneeisnowhere 2023-07-26
fa1dc68 reneeisnowhere 2023-07-06
b0dd36d reneeisnowhere 2023-07-04
e81dfd3 reneeisnowhere 2023-06-28
272159f reneeisnowhere 2023-06-28

D. Stringently idetified DOX-only DE genes and DOX egenes only share JPH3

cpm_boxplot24h <-
  function(cpmcounts,
           GOI,
           brewer_palette,
           fill_colors,
           ylab) {
    ##GOI needs to be ENTREZID
    df <- cpmcounts
    df_plot <- df %>%
      dplyr::filter(rownames(.) == GOI) %>%
      pivot_longer(everything(),
                   names_to = "treatment",
                   values_to = "counts") %>%
      separate(treatment, c("drug", "indv", "time")) %>%
      mutate(time = case_match(time, "24h" ~ "24 hours", "3h" ~ "3 hours")) %>%
      mutate(indv = factor(indv, levels = c(1, 2, 3, 4, 5, 6))) %>%
      mutate(
        drug = case_match(
          drug,
          "Da" ~ "DNR",
          "Do" ~ "DOX",
          "Ep" ~ "EPI",
          "Mi" ~ "MTX",
          "Tr" ~ "TRZ",
          "Ve" ~ "VEH",
          .default = drug
        )
      ) %>%
      mutate(drug = factor(drug, levels = c('DOX', 'EPI', 'DNR', 'MTX', 'TRZ', 'VEH'))) %>%
      dplyr::filter(time == "24 hours")
    plot <- ggplot2::ggplot(df_plot, aes(x = drug, y = counts)) +
      geom_boxplot(position = "identity", aes(fill = drug)) +
      geom_point(aes(
        col = indv,
        size = 2,
        alpha = 0.5
      )) +
      guides(alpha = "none", size = "none") +
      scale_color_brewer(palette = brewer_palette, name = "Individual") +
      scale_fill_manual(values = fill_colors) +
      # facet_wrap("time", nrow=1, ncol=2)+
      theme_bw() +
      ylab(ylab) +
      xlab("") +
      ggtitle("24 hours") +
      theme(
        strip.background = element_rect(
          fill = "white",
          linetype = 1,
          linewidth = 0.5
        ),
        plot.title = element_text(
          size = 12,
          hjust = 0.5,
          face = "bold"
        ),
        axis.title = element_text(
          size = 15,
          color = "black",
          face = "bold"
        ),
        axis.ticks = element_line(linewidth = 1.0),
        panel.background = element_rect(colour = "black", size = 1),
        axis.text.x = element_blank(),
        strip.text.x = element_text(margin = margin(2, 0, 2, 0, "pt"), face = "bold")
      )
    print(plot)
  }
cpm_boxplot24h(cpmcounts,
               GOI = '57338',
               "Dark2",
               drug_pal_fact,
               ylab = (expression(atop(
                 " ", italic("JPH3") ~ log[2] ~ "cpm "
               ))))

Version Author Date
4bb0b80 reneeisnowhere 2023-07-28
c40aced reneeisnowhere 2023-07-26
fa1dc68 reneeisnowhere 2023-07-06
b0dd36d reneeisnowhere 2023-07-04
272159f reneeisnowhere 2023-06-28

For additional stringently identified genes, you can visit the supplementary data here or analysis located here.


sessionInfo()
R version 4.3.1 (2023-06-16 ucrt)
Platform: x86_64-w64-mingw32/x64 (64-bit)
Running under: Windows 10 x64 (build 19045)

Matrix products: default


locale:
[1] LC_COLLATE=English_United States.utf8 
[2] LC_CTYPE=English_United States.utf8   
[3] LC_MONETARY=English_United States.utf8
[4] LC_NUMERIC=C                          
[5] LC_TIME=English_United States.utf8    

time zone: America/Chicago
tzcode source: internal

attached base packages:
[1] stats     graphics  grDevices utils     datasets  methods   base     

other attached packages:
 [1] paletteer_1.5.0     ggVennDiagram_1.2.3 broom_1.0.5        
 [4] RColorBrewer_1.1-3  ggsignif_0.6.4      cowplot_1.1.1      
 [7] data.table_1.14.8   BiocGenerics_0.46.0 lubridate_1.9.2    
[10] forcats_1.0.0       stringr_1.5.0       dplyr_1.1.3        
[13] purrr_1.0.2         readr_2.1.4         tidyr_1.3.0        
[16] tibble_3.2.1        ggplot2_3.4.3       tidyverse_2.0.0    
[19] workflowr_1.7.1    

loaded via a namespace (and not attached):
 [1] gtable_0.3.4       xfun_0.40          bslib_0.5.1        processx_3.8.2    
 [5] callr_3.7.3        tzdb_0.4.0         vctrs_0.6.3        tools_4.3.1       
 [9] ps_1.7.5           generics_0.1.3     proxy_0.4-27       fansi_1.0.4       
[13] pkgconfig_2.0.3    KernSmooth_2.23-22 lifecycle_1.0.3    farver_2.1.1      
[17] compiler_4.3.1     git2r_0.32.0       munsell_0.5.0      getPass_0.2-2     
[21] httpuv_1.6.11      class_7.3-22       htmltools_0.5.6    sass_0.4.7        
[25] yaml_2.3.7         later_1.3.1        pillar_1.9.0       jquerylib_0.1.4   
[29] whisker_0.4.1      classInt_0.4-10    cachem_1.0.8       tidyselect_1.2.0  
[33] digest_0.6.33      stringi_1.7.12     sf_1.0-14          rematch2_2.1.2    
[37] labeling_0.4.3     rprojroot_2.0.3    fastmap_1.1.1      grid_4.3.1        
[41] colorspace_2.1-0   cli_3.6.1          magrittr_2.0.3     utf8_1.2.3        
[45] e1071_1.7-13       withr_2.5.0        RVenn_1.1.0        scales_1.2.1      
[49] promises_1.2.1     backports_1.4.1    timechange_0.2.0   rmarkdown_2.24    
[53] httr_1.4.7         hms_1.1.3          evaluate_0.21      knitr_1.44        
[57] rlang_1.1.1        Rcpp_1.0.11        DBI_1.1.3          glue_1.6.2        
[61] rstudioapi_0.15.0  jsonlite_1.8.7     R6_2.5.1           units_0.8-4       
[65] fs_1.6.3