From 7db6489a64a15e197ff9fc0f29de482787fa0fe8 Mon Sep 17 00:00:00 2001 From: Ally Hawkins Date: Fri, 3 Jan 2025 09:27:52 -0600 Subject: [PATCH 1/7] reference file for genes in tumor cell states --- .../references/tumor-cell-state-markers.tsv | 18 ++++++++++++++++++ 1 file changed, 18 insertions(+) create mode 100644 analyses/cell-type-ewings/references/tumor-cell-state-markers.tsv diff --git a/analyses/cell-type-ewings/references/tumor-cell-state-markers.tsv b/analyses/cell-type-ewings/references/tumor-cell-state-markers.tsv new file mode 100644 index 000000000..a257e12fb --- /dev/null +++ b/analyses/cell-type-ewings/references/tumor-cell-state-markers.tsv @@ -0,0 +1,18 @@ +cell_state gene_symbol ensembl_gene_id source +proliferative MKI67 ENSG00000148773 https://doi.org/10.1101/2024.01.18.576251;https://doi.org/10.1016/j.celrep.2020.01.049 +proliferative PCNA ENSG00000132646 https://doi.org/10.1101/2024.01.18.576251 +EWS-low NT5E ENSG00000135318 https://doi.org/10.1158/1078-0432.CCR-23-1111 +EWS-low IGFBP3 ENSG00000146674 https://doi.org/10.1016/j.celrep.2020.01.049 +EWS-low IL8 ENSG00000169429 https://doi.org/10.1016/j.celrep.2020.01.049 +EWS-low LOX ENSG00000113083 https://doi.org/10.1016/j.celrep.2020.01.049 +EWS-low VIM ENSG00000026025 https://doi.org/10.1016/j.celrep.2020.01.049 +EWS-low TNC ENSG00000041982 https://doi.org/10.1158/1078-0432.CCR-23-1111 +EWS-low COL1A1 ENSG00000108821 https://doi.org/10.1158/1078-0432.CCR-23-1111 +EWS-low BGN ENSG00000182492 https://doi.org/10.1158/1078-0432.CCR-23-1111 +EWS-low S100A10 ENSG00000197747 https://doi.org/10.1158/1078-0432.CCR-23-1111 +EWS-low SPARC ENSG00000113140 https://doi.org/10.1158/1078-0432.CCR-23-1111 +EWS-low COL1A2 ENSG00000164692 https://doi.org/10.1158/1078-0432.CCR-23-1111 +EWS-high PRKCB ENSG00000166501 https://doi.org/10.1016/j.celrep.2020.01.049 +EWS-high LIPI ENSG00000188992 https://doi.org/10.1016/j.celrep.2020.01.049 +EWS-high CCND1 ENSG00000110092 https://doi.org/10.1016/j.celrep.2020.01.049 +EWS-high NR0B1 ENSG00000169297 https://doi.org/10.1016/j.celrep.2020.01.049 From dab5cdd21c49bf858eb7c235175c8f08cf14e50b Mon Sep 17 00:00:00 2001 From: Ally Hawkins Date: Fri, 3 Jan 2025 09:28:06 -0600 Subject: [PATCH 2/7] add info about gene lists to readme --- .../cell-type-ewings/references/README.md | 33 +++++++++++++++++++ 1 file changed, 33 insertions(+) diff --git a/analyses/cell-type-ewings/references/README.md b/analyses/cell-type-ewings/references/README.md index 23ced6920..2ffe31e12 100644 --- a/analyses/cell-type-ewings/references/README.md +++ b/analyses/cell-type-ewings/references/README.md @@ -49,3 +49,36 @@ The other required input for `InferCNV` is an annotation file with two columns a The first column contains the cell barcode and the second contains the annotation for that cell (either `reference` or `unknown`). These files are specific for each library and depend on which cells are denoted as the reference. Each library contains a folder with any annotations file used to run `InferCNV` for that library. + +## Marker gene sets for identifying tumor cell states + +The `tumor-cell-state-markers.tsv` file contains a list of marker genes that can be used to classify tumor cell states in Ewing samples. +The marker genes included here are specific to EWS-FLI1 high, EWS-FLI1 low, and proliferative tumor cells. +This list was obtained based on key genes mentioned in [Goodspeed _et al._](https://doi.org/10.1101/2024.01.18.576251), [Aynaud _et al._](https://doi.org/10.1016/j.celrep.2020.01.049), and [Wrenn _et al._](https://doi.org/10.1158/1078-0432.CCR-23-1111). + +### Gene signatures + +The `gene_signatures` folder contains any custom gene lists obtained from publications that can be used to identify tumor cell states: + +1. `anyaud-ews-targets.tsv`: A list of the 78 marker genes defined by [Aynaud _et al._](https://doi.org/10.1016/j.celrep.2020.01.049) to be EWS-FLI1 targets. +Figure 4 shows that expression of these targets is correlated with EWS-FLI1 levels at a single-cell level. + +2. `wrenn-nt5e-genes.tsv`: A list of 28 genes from [Wrenn _et al._](https://doi.org/10.1158/1078-0432.CCR-23-1111) that represent the overlap between the top 217 genes correlated with _NT5E_ expression in patient tumors and the top 200 markers of _NT5E+_ Ewing sarcoma cells _in vitro_. +These genes are shown in Figure 5D and 5E. + +The following gene sets from MsigDb were also used to define EWS-FLI1 targets and may be helpful in defining cell states: + +- [STAEGE_EWING_FAMILY_TUMOR](https://www.gsea-msigdb.org/gsea/msigdb/human/geneset/STAEGE_EWING_FAMILY_TUMOR.html) +- [MIYAGAWA_TARGETS_OF_EWSR1_ETS_FUSIONS_UP](https://www.gsea-msigdb.org/gsea/msigdb/human/geneset/MIYAGAWA_TARGETS_OF_EWSR1_ETS_FUSIONS_UP.html) +- [MIYAGAWA_TARGETS_OF_EWSR1_ETS_FUSIONS_DN](https://www.gsea-msigdb.org/gsea/msigdb/human/geneset/MIYAGAWA_TARGETS_OF_EWSR1_ETS_FUSIONS_DN.html) +- [ZHANG_TARGETS_OF_EWSR1_FLI1_FUSION](https://www.gsea-msigdb.org/gsea/msigdb/human/geneset/ZHANG_TARGETS_OF_EWSR1_FLI1_FUSION.html) +- [RIGGI_EWING_SARCOMA_PROGENITOR_UP](https://www.gsea-msigdb.org/gsea/msigdb/human/geneset/RIGGI_EWING_SARCOMA_PROGENITOR_UP.html) +- [RIGGI_EWING_SARCOMA_PROGENITOR_DN](https://www.gsea-msigdb.org/gsea/msigdb/human/geneset/RIGGI_EWING_SARCOMA_PROGENITOR_DN.html) +- [KINSEY_TARGETS_OF_EWSR1_FLI1_FUSION_UP](https://www.gsea-msigdb.org/gsea/msigdb/human/geneset/KINSEY_TARGETS_OF_EWSR1_FLII_FUSION_UP.html) +- [KINSEY_TARGETS_OF_EWSR1_FLI1_FUSION_DN](https://www.gsea-msigdb.org/gsea/msigdb/human/geneset/KINSEY_TARGETS_OF_EWSR1_FLII_FUSION_DN.html) + +Wrenn _et al._ also used found that the following additional gene sets were highly expressed in CD73 high, EWS-FLI1 low tumor cells: + +- [HALLMARK_EPITHELIAL_MESENCHYMAL_TRANSITION](https://www.gsea-msigdb.org/gsea/msigdb/human/geneset/HALLMARK_EPITHELIAL_MESENCHYMAL_TRANSITION.html) +GO:BP ECM Organization +- [GOBP_REGULATION_OF_EXTRACELLULAR_MATRIX_ORGANIZATION](https://www.gsea-msigdb.org/gsea/msigdb/human/geneset/GOBP_REGULATION_OF_EXTRACELLULAR_MATRIX_ORGANIZATION.html) From 2f27d6dc621e7d30277c2ea7414a1c49b1aeaf2e Mon Sep 17 00:00:00 2001 From: Ally Hawkins Date: Fri, 3 Jan 2025 09:58:15 -0600 Subject: [PATCH 3/7] add custom gene sets --- .../gene_signatures/aynaud-ews-targets.tsv | 79 +++++++++++++++++++ .../gene_signatures/wrenn-nt5e-genes.tsv | 29 +++++++ 2 files changed, 108 insertions(+) create mode 100644 analyses/cell-type-ewings/references/gene_signatures/aynaud-ews-targets.tsv create mode 100644 analyses/cell-type-ewings/references/gene_signatures/wrenn-nt5e-genes.tsv diff --git a/analyses/cell-type-ewings/references/gene_signatures/aynaud-ews-targets.tsv b/analyses/cell-type-ewings/references/gene_signatures/aynaud-ews-targets.tsv new file mode 100644 index 000000000..0cf15f85d --- /dev/null +++ b/analyses/cell-type-ewings/references/gene_signatures/aynaud-ews-targets.tsv @@ -0,0 +1,79 @@ +gene_symbol ensembl_gene_id +CAV1 ENSG00000105974 +APCDD1 ENSG00000154856 +CCND1 ENSG00000110092 +RNF141 ENSG00000110315 +RBM11 ENSG00000185272 +TSPAN13 ENSG00000106537 +FZD1 ENSG00000157240 +RCOR1 ENSG00000089902 +ATP1A1 ENSG00000163399 +TMEM47 ENSG00000147027 +GLCE ENSG00000138604 +LIPI ENSG00000188992 +RP13-16H11.2 NA +CLDN1 ENSG00000163347 +PAX9 ENSG00000198807 +TNFAIP6 ENSG00000123610 +UGT3A2 ENSG00000168671 +GPR64 NA +SLAIN1 ENSG00000139737 +RP11-1258F18.1 NA +MARCH1 NA +AASS ENSG00000008311 +ABHD6 ENSG00000163686 +AC022311.1 NA +ADAMTS4 ENSG00000158859 +AK3 ENSG00000147853 +AKAP7 ENSG00000118507 +CDH8 ENSG00000150394 +CES1 ENSG00000198848 +COL21A1 ENSG00000124749 +CTTNBP2 ENSG00000077063 +ATP11C ENSG00000101974 +BARX2 ENSG00000043039 +CACHD1 ENSG00000158966 +CADPS2 ENSG00000081803 +CAPRIN1 ENSG00000135387 +CAV2 ENSG00000105971 +CDC37L1 ENSG00000106993 +DCC ENSG00000187323 +DCDC2 ENSG00000146038 +DLGAP1 ENSG00000170579 +DNAL4 ENSG00000100246 +EBF3 ENSG00000108001 +ENPP1 ENSG00000197594 +FAM46A NA +FAM49A NA +FNBP1 ENSG00000187239 +FOS ENSG00000170345 +HES1 ENSG00000114315 +HMCN1 ENSG00000143341 +IGF1 ENSG00000017427 +JARID2 ENSG00000008083 +KCNE4 ENSG00000152049 +KDSR ENSG00000119537 +KIAA1456 NA +KIAA1462 NA +KIT ENSG00000157404 +LRRC4C ENSG00000148948 +MAN2A1 ENSG00000112893 +MLLT3 ENSG00000171843 +MYC ENSG00000136997 +NMI ENSG00000123609 +NPTXR ENSG00000221890 +NTNG1 ENSG00000162631 +PCDH7 ENSG00000169851 +PCSK2 ENSG00000125851 +PRKCB ENSG00000166501 +RP11-521M14.1 NA +RP11-6C14.1 NA +RP13-16H11.1 NA +SH2B3 ENSG00000111252 +SH3PXD2A ENSG00000107957 +SLC26A2 ENSG00000155850 +SLCO5A1 ENSG00000137571 +SPATA6L ENSG00000106686 +TRAV1-2 ENSG00000256553 +TRPM4 ENSG00000130529 +ZC3H13 ENSG00000123200 diff --git a/analyses/cell-type-ewings/references/gene_signatures/wrenn-nt5e-genes.tsv b/analyses/cell-type-ewings/references/gene_signatures/wrenn-nt5e-genes.tsv new file mode 100644 index 000000000..1664a47df --- /dev/null +++ b/analyses/cell-type-ewings/references/gene_signatures/wrenn-nt5e-genes.tsv @@ -0,0 +1,29 @@ +gene_symbol ensembl_gene_id +NT5E ENSG00000135318 +FBN1 ENSG00000166147 +COL5A2 ENSG00000204262 +COL6A2 ENSG00000142173 +TNC ENSG00000041982 +COL1A1 ENSG00000108821 +COL1A2 ENSG00000164692 +SPARC ENSG00000113140 +BGN ENSG00000182492 +S100A10 ENSG00000197747 +FSTL1 ENSG00000163430 +CAMK2D ENSG00000145349 +CTSB ENSG00000164733 +THY1 ENSG00000154096 +GPNMB ENSG00000136235 +NID1 ENSG00000116962 +PPIC ENSG00000168938 +PRSS23 ENSG00000150687 +EMP1 ENSG00000134531 +CD63 ENSG00000135404 +S100A11 ENSG00000163191 +LGMN ENSG00000100600 +S100A13 ENSG00000189171 +S100A4 ENSG00000196154 +MYOF ENSG00000138119 +PLAC9 ENSG00000189129 +TGFB1I1 ENSG00000140682 +CTSZ ENSG00000101160 From 092e5a9e63b78f455f2ed382825a0384f929122c Mon Sep 17 00:00:00 2001 From: Ally Hawkins Date: Fri, 3 Jan 2025 10:27:00 -0600 Subject: [PATCH 4/7] fix doi error --- .../cell-type-ewings/references/tumor-cell-state-markers.tsv | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/analyses/cell-type-ewings/references/tumor-cell-state-markers.tsv b/analyses/cell-type-ewings/references/tumor-cell-state-markers.tsv index a257e12fb..15cee5a87 100644 --- a/analyses/cell-type-ewings/references/tumor-cell-state-markers.tsv +++ b/analyses/cell-type-ewings/references/tumor-cell-state-markers.tsv @@ -1,5 +1,5 @@ cell_state gene_symbol ensembl_gene_id source -proliferative MKI67 ENSG00000148773 https://doi.org/10.1101/2024.01.18.576251;https://doi.org/10.1016/j.celrep.2020.01.049 +proliferative MKI67 ENSG00000148773 https://doi.org/10.1101/2024.01.18.576251;https://doi.org/10.1158/1078-0432.CCR-23-1111 proliferative PCNA ENSG00000132646 https://doi.org/10.1101/2024.01.18.576251 EWS-low NT5E ENSG00000135318 https://doi.org/10.1158/1078-0432.CCR-23-1111 EWS-low IGFBP3 ENSG00000146674 https://doi.org/10.1016/j.celrep.2020.01.049 From 8e6dbf12e9928ac02bb4dd1a43cc9fc81c8d26e0 Mon Sep 17 00:00:00 2001 From: Ally Hawkins <54039191+allyhawkins@users.noreply.github.com> Date: Mon, 6 Jan 2025 11:52:33 -0600 Subject: [PATCH 5/7] Apply suggestions from code review Co-authored-by: Stephanie Spielman --- analyses/cell-type-ewings/references/README.md | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/analyses/cell-type-ewings/references/README.md b/analyses/cell-type-ewings/references/README.md index 2ffe31e12..8dcd005e0 100644 --- a/analyses/cell-type-ewings/references/README.md +++ b/analyses/cell-type-ewings/references/README.md @@ -66,7 +66,7 @@ Figure 4 shows that expression of these targets is correlated with EWS-FLI1 leve 2. `wrenn-nt5e-genes.tsv`: A list of 28 genes from [Wrenn _et al._](https://doi.org/10.1158/1078-0432.CCR-23-1111) that represent the overlap between the top 217 genes correlated with _NT5E_ expression in patient tumors and the top 200 markers of _NT5E+_ Ewing sarcoma cells _in vitro_. These genes are shown in Figure 5D and 5E. -The following gene sets from MsigDb were also used to define EWS-FLI1 targets and may be helpful in defining cell states: +The following gene sets from MSigDB were also used to define EWS-FLI1 targets and may be helpful in defining cell states: - [STAEGE_EWING_FAMILY_TUMOR](https://www.gsea-msigdb.org/gsea/msigdb/human/geneset/STAEGE_EWING_FAMILY_TUMOR.html) - [MIYAGAWA_TARGETS_OF_EWSR1_ETS_FUSIONS_UP](https://www.gsea-msigdb.org/gsea/msigdb/human/geneset/MIYAGAWA_TARGETS_OF_EWSR1_ETS_FUSIONS_UP.html) From 53355b941535ca75247b56cff89f0eacd30df236 Mon Sep 17 00:00:00 2001 From: Ally Hawkins Date: Mon, 6 Jan 2025 12:07:16 -0600 Subject: [PATCH 6/7] add CD44 to marker gene list --- .../cell-type-ewings/references/tumor-cell-state-markers.tsv | 1 + 1 file changed, 1 insertion(+) diff --git a/analyses/cell-type-ewings/references/tumor-cell-state-markers.tsv b/analyses/cell-type-ewings/references/tumor-cell-state-markers.tsv index 15cee5a87..ca0ad997e 100644 --- a/analyses/cell-type-ewings/references/tumor-cell-state-markers.tsv +++ b/analyses/cell-type-ewings/references/tumor-cell-state-markers.tsv @@ -12,6 +12,7 @@ EWS-low BGN ENSG00000182492 https://doi.org/10.1158/1078-0432.CCR-23-1111 EWS-low S100A10 ENSG00000197747 https://doi.org/10.1158/1078-0432.CCR-23-1111 EWS-low SPARC ENSG00000113140 https://doi.org/10.1158/1078-0432.CCR-23-1111 EWS-low COL1A2 ENSG00000164692 https://doi.org/10.1158/1078-0432.CCR-23-1111 +EWS-low CD44 ENSG00000026508 https://doi.org/10.1101/2024.01.18.576251 EWS-high PRKCB ENSG00000166501 https://doi.org/10.1016/j.celrep.2020.01.049 EWS-high LIPI ENSG00000188992 https://doi.org/10.1016/j.celrep.2020.01.049 EWS-high CCND1 ENSG00000110092 https://doi.org/10.1016/j.celrep.2020.01.049 From 5f1b8785f4b5c8f279390fd6f23c8007552757e0 Mon Sep 17 00:00:00 2001 From: Ally Hawkins Date: Mon, 6 Jan 2025 12:11:41 -0600 Subject: [PATCH 7/7] document expectations for custom gene signatures --- analyses/cell-type-ewings/references/README.md | 4 +++- 1 file changed, 3 insertions(+), 1 deletion(-) diff --git a/analyses/cell-type-ewings/references/README.md b/analyses/cell-type-ewings/references/README.md index 8dcd005e0..d0270e5da 100644 --- a/analyses/cell-type-ewings/references/README.md +++ b/analyses/cell-type-ewings/references/README.md @@ -62,9 +62,11 @@ The `gene_signatures` folder contains any custom gene lists obtained from public 1. `anyaud-ews-targets.tsv`: A list of the 78 marker genes defined by [Aynaud _et al._](https://doi.org/10.1016/j.celrep.2020.01.049) to be EWS-FLI1 targets. Figure 4 shows that expression of these targets is correlated with EWS-FLI1 levels at a single-cell level. +We expect these targets to have increased expression in cells with high EWS-FLI1 activity. 2. `wrenn-nt5e-genes.tsv`: A list of 28 genes from [Wrenn _et al._](https://doi.org/10.1158/1078-0432.CCR-23-1111) that represent the overlap between the top 217 genes correlated with _NT5E_ expression in patient tumors and the top 200 markers of _NT5E+_ Ewing sarcoma cells _in vitro_. -These genes are shown in Figure 5D and 5E. +These genes are shown in Figure 5D and 5E. +We expect these targets to have increased expression in cells with low EWS-FLI1 activity. The following gene sets from MSigDB were also used to define EWS-FLI1 targets and may be helpful in defining cell states: