Individualized Pooled CRISPR/Cas9 Screenings Identify CDK2 as a Druggable Vulnerability in a Canine Mammary Carcinoma Patient
Simple Summary
Abstract
1. Introduction
2. Materials and Methods
2.1. Sample Collection and Tissue Processing
2.2. Histology
2.3. Maintenance of CMT Organoid Cultures
2.4. Lentivirus Production, Lentiviral Transduction
2.5. Pooled CRISPR/Cas9 Screening
2.6. Proliferation Assay
2.7. Drug Testing and Cell Viability Assays
2.8. RNA Isolation and Sequencing of the Primary Tumors and Non-Neoplastic Mammary Tissues
2.9. RNA Sequencing of the Organoids (Paired Carcinoma and Non-Neoplastic Mammary Tissue)
2.10. Statistical Analysis and Data Representation
3. Results
3.1. Design and Lentiviral Transduction of Two Custom Canine CRISPR/Cas9 Libraries to Identify Therapeutic Vulnerabilities in CMTs
3.2. Pooled CRISPR/Cas9 Screening Targeting the Canine Epigenome in PDOs to Better Understand CMT Biology and Tumorigenesis
- Outline of the screens performed with a custom canine CRISPR/Cas9 library
- Volcano plot representing depleted (LFC < 0) and enriched (LFC > 0) genes for ORG-63-C forty days (D40) after D0 for the druggable screen. LFC and p-values were calculated from two technical replicates with MAGeCK analysis. Each dot represents one gene for which at least four sgRNAs (out of six) were enrolled in the analysis. Selected hits are color-coded.
- Volcano plot representing depleted (LFC < 0) and enriched (LFC > 0) genes for ORG-63-C forty days (D40) after D0 for the epigenome screen. LFC and p-values were calculated from two technical replicates with MAGeCK analysis. Each dot represents one gene for which at least four sgRNAs (out of six) were enrolled in the analysis. Selected hits are color-coded.
- Scatter plot of the log fold change (LFC) of ORG-63-C and ORG-63-N for the druggable screen. Each dot represents one gene for which at least four sgRNAs (out of six) were enrolled in the analysis, and the p-value < 0.05 for at least one of the two ORG lines. Selected hits are color-coded.
- Scatter plot of the log fold change (LFC) of ORG-63-C and ORG-63-N for the epigenome screen. Each dot represents one gene for which at least four sgRNAs (out of six) were enrolled in the analysis, and the p-value < 0.05 for at least one of the two ORG lines. Selected hits are color-coded.
Gene | Gene Description | p-Value_N | lfc_N | p-Value_C | lfc_C |
---|---|---|---|---|---|
SPEN | Msx2-interacting protein | 0.9141 | 1.531 | 0.02454 | −2.126 |
VPS72 | Vacuolar protein sorting-associated protein 72 homolog | 0.8979 | 0.4675 | 0.01602 | −2.577 |
PLOD2 | Procollagen-lysine,2-oxoglutarate 5-dioxygenase 2 | 0.8599 | 1.6872 | 0.01308 | −3.333 |
NCOR1 | Nuclear receptor co-repressor 1 | 0.7682 | 0.25754 | 0.021244 | −1.889 |
SRSF2 | Serine/arginine-rich splicing factor 2 | 0.6809 | 0.46804 | 0.015419 | −2.955 |
HDAC1 | Histone deacetylase 1 | 0.6639 | 0.50999 | 0.045327 | −1.647 |
ACTL6A | Actin-like protein 6A | 0.60347 | 0.48882 | 0.009406 | −1.670 |
TBL1X | F-box-like/WD repeat-containing protein TBL1X | 0.60168 | −0.4438 | 0.046566 | −2.318 |
BAZ1A | Bromodomain adjacent to zinc finger domain protein 1A | 0.59139 | −0.2259 | 0.014092 | −2.371 |
NOC2L | Nucleolar complex protein 2 homolog | 0.58843 | −0.4693 | 0.018451 | −2.221 |
TAF8 | Transcription initiation factor TFIID subunit 8 | 0.58046 | −0.3521 | 0.018177 | −1.594 |
TFDP1 | Transcription factor Dp-1 | 0.46931 | 0.3727 | 0.033807 | −2.012 |
CBX5 | Chromobox protein homolog 5 | 0.45174 | 0.40584 | 0.039584 | −1.473 |
SMARCA5 | SWI/SNF-related matrix-associated actin-dependent | 0.44409 | 0.87841 | 0.013878 | −2.98 |
SRRM1 | Serine/arginine repetitive matrix protein 1 | 0.43516 | −0.4318 | 0.019006 | −1.859 |
COPS5 | Information COP9 signalosome complex subunit 5 | 0.43285 | 0.75487 | 0.015724 | −3.111 |
NAP1L1 | Nucleosome assembly protein 1-like 1 | 0.40623 | −0.3009 | 0.024866 | −2.719 |
HASPIN | Serine/threonine-protein kinase haspin | 0.40398 | −0.5946 | 0.007778 | −3.185 |
RCC1 | Regulator of chromosome condensation | 0.39469 | 0.23547 | 0.01764 | −1.503 |
OIP5 | Protein Mis18-beta | 0.38418 | −0.7262 | 0.018399 | −2.218 |
SRSF5 | Splicing factor, arginine/serine-rich 4/5/6 | 0.34631 | −0.8543 | 0.04839 | −1.951 |
MIS18BP1 | Mis18-binding protein 1 | 0.34095 | −1.0051 | 0.019045 | −2.84 |
RBPMS | RNA binding protein, mRNA processing factor | 0.33435 | −0.9347 | 0.047674 | −1.959 |
SETD1A | Histone-lysine N-methyltransferase SETD1A | 0.32603 | −0.6491 | 0.020014 | −1.490 |
CDK2 | Cyclin-dependent kinase 2 | 0.31995 | −0.7495 | 0.024024 | −1.354 |
USP7 | Ubiquitin carboxyl-terminal hydrolase 7 | 0.31791 | −1.0529 | 0.027406 | −1.860 |
SUZ12 | SUZ12 Polycomb repressive complex 2 subunit | 0.31684 | −0.4537 | 0.019363 | −3.057 |
DHX38 | Pre-mRNA-splicing factor ATP-dependent RNA helicase PRP16 | 0.31248 | −0.6269 | 0.025534 | −2.534 |
KDM8 | JmjC-domain-containing protein 5 | 0.30885 | −0.8832 | 0.007473 | −2.061 |
OTUB1 | Ubiquitin thioesterase | 0.30217 | −0.9042 | 0.017099 | −1.689 |
GTF3C4 | General transcription factor 3C polypeptide 4 | 0.29667 | −0.1029 | 0.048652 | −2.046 |
CENPA | Histone H3-like centromeric protein A | 0.2798 | −0.8915 | 0.008441 | −2.949 |
ZZZ3 | ZZ-type zinc finger-containing protein 3 | 0.26212 | −0.8331 | 0.017243 | −3.213 |
CHEK1 | Serine/threonine-protein kinase Chk1 | 0.25733 | 1.0502 | 0.02631 | −2.563 |
DDX17 | Probable ATP-dependent RNA helicase DDX17 | 0.24814 | 0.56402 | 0.031922 | −2.85 |
SNAPC4 | snRNA-activating protein complex subunit 4 | 0.24499 | −0.9796 | 0.021048 | −2.847 |
MED30 | Mediator Complex Subunit 30 | 0.17747 | −0.6267 | 8.51 × 10−5 | −2.719 |
PCNA | Proliferating cell nuclear antigen | 0.14632 | −0.6846 | 0.012683 | −3.062 |
ECD | Protein ecdysoneless homolog | 0.12707 | −0.2861 | 0.026677 | −3.026 |
RSF1 | Remodeling and spacing factor 1 | 0.11897 | −0.7464 | 0.032301 | −2.881 |
UBE2A | Ubiquitin-conjugating enzyme E2 A | 0.11589 | −0.9619 | 0.010972 | −1.337 |
GTF2B | Transcription initiation factor IIB | 0.1149 | −0.7796 | 0.023854 | −2.791 |
SMC2 | Structural maintenance of chromosomes protein 2 | 0.10679 | −0.3809 | 0.006639 | −3.191 |
RUVBL1 | RuvB-like 1 | 0.096328 | 0.44603 | 0.0039294 | −3.093 |
AURKB | Aurora kinase B | 0.068751 | −0.9429 | 0.0055614 | −3.071 |
3.3. Pooled CRISPR/Cas9 Screening in PDOs to Identify Potential Drug Targets for CMTs
3.4. Candidates from the CRISPR/Cas9 Screens Are Expressed in Primary Tumor Tissues and PDOs but Very Rarely Differentially Expressed Compared to the Non-Neoplastic Mammary Tissues
- Volcano plot representing downregulated (log fold change = LFC < 0) and upregulated (LFC 0) genes between all CMT samples compared with all normal mammary tissue samples. Significantly (p-value < 0.05 and |LFC|> = 1.0) downregulated (green) and upregulated (red) are color coded. Keratins and keratin-associated proteins are labeled in blue.
- Volcano plot representing significantly downregulated and upregulated (p-value < 0.05 and |LFC| > 1.0) genes between D63_3_CMT and D63_nor. Candidates from the CRISPR/Cas9 screens that are differentially expressed between D63_3_CMT and D63_nor are labeled in orange.
- Volcano plot representing downregulated (LFC < 0) and upregulated (LFC > 0) genes between three organoid lines derived from carcinomas compared with their matched organoids derived from normal mammary tissues. Significantly (p-value < 0.05 and |LFC| > 1.0) downregulated (green) and upregulated (red) are color coded.
- Bar plot showing Gene Ontology (biological processes) analysis results of all differentially expressed genes between three organoid lines derived from carcinomas compared with their matched organoids derived from normal mammary tissues. The color of each bar represents the p-value of each term involved in the analysis, and the bar size represents the gene counts for this term.
3.5. CDK2 May Be a Druggable Vulnerability for CMTs
- Dose-response curves illustrating the effect of PF3600 on cell viability in organoid lines ORG-63-C and ORG-63-N. Error bars represent the standard deviation (SD) of three independent experiments. Statistical analysis of the log-transformed IC50 values was performed using an unpaired t-test, **** p < 0.0001. IC50s are indicated in the figure legend.
- Dose-response curves illustrating the effect of doxorubicin on cell viability in organoid lines ORG-63-C and ORG-63-N. Error bars represent the standard deviation (SD) of three independent experiments. Statistical analysis of the log-transformed IC50 values was performed using an unpaired t-test, **** p < 0.0001. IC50s are indicated in the figure legend.
- Dose-response curves depicting the effect of reversine on cell viability in organoid lines ORG-63-C and ORG-63-N. Error bars represent the standard deviation (SD) of three independent experiments. Statistical analysis of the log-transformed IC50 values was performed using an unpaired t-test, ns = non-significant. IC50s are indicated in the figure legend.
- Dose-response curves depicting the effect of luminespib on cell viability in organoid lines ORG-63-C and ORG-63-N. Error bars represent the standard deviation (SD) of three independent experiments. Statistical analysis of the log-transformed IC50 values was performed using an unpaired t-test, ns = non-statistical. IC50s are indicated in the figure legend.
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
CMT | Canine Mammary Tumor |
CRISPR | Clustered Regularly Interspaced Short Palindromic Repeats |
D0 | Day 0 |
D40 | Day 40 |
HBC | Human Breast Cancer |
IC50 | Half Maximal Inhibitory Concentration |
LFC | Log Fold Change |
mRNA | Messenger RNA |
pDNA | Plasmid DNA |
PDO | Patient-Derived Organoid |
sgRNA | Single Guide RNA |
TCGA | The Cancer Genome Atlas |
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Gene | Gene Description | p-Value_N | lfc_N | p-Value_C | lfc_C |
---|---|---|---|---|---|
HSP90AA1 | Heat shock protein HSP 90-alpha | 0.70337 | 0.18138 | 0.02346 | −1.547 |
FANCD2 | Fanconi anemia group D2 protein | 0.603 | −0.22887 | 0.04947 | −1.1426 |
CDK2 | Cyclin-dependent kinase 2 | 0.53595 | −0.27009 | 0.039806 | −1.4028 |
TOP2B | DNA topoisomerase 2-beta | 0.39481 | −0.0917 | 0.0087646 | −1.5298 |
RPTOR | Regulatory-associated protein of mTOR | 0.39167 | −0.45626 | 0.048055 | −1.0535 |
BCL2L1 | Bcl-2-like protein 1 | 0.31849 | −0.26447 | 0.013289 | −1.2187 |
MYBL1 | Myb proto-oncogene like 1 | 0.31416 | −0.5715 | 0.024879 | −1.6852 |
TTK | Serine/threonine-protein kinase ttk/mps1 | 0.28795 | −0.61574 | 0.013661 | −1.584 |
FANCC | Fanconi anemia group C protein | 0.24945 | −0.54887 | 0.0073124 | −1.9841 |
POLE | DNA polymerase ε catalytic subunit A | 0.24504 | −0.27922 | 0.013083 | −1.3574 |
CDK12 | Cyclin-dependent kinase 12 | 0.23879 | −0.16167 | 0.008662 | −1.9851 |
GMPS | GMP synthase [glutamine-hydrolyzing] | 0.23684 | −0.25904 | 0.035056 | −1.4074 |
CCND3 | G1/S-specific cyclin-D3 | 0.18021 | −0.86676 | 0.017937 | −1.3585 |
USP5 | Ubiquitin carboxyl-terminal hydrolase 5 | 0.16569 | −0.87191 | 0.023271 | −1.2658 |
NFKBIA | NF-kappa-B inhibitor alpha | 0.14329 | −0.99465 | 0.0078846 | −2.0144 |
PI4KB | Phosphatidylinositol 4-kinase beta | 0.10617 | −0.86682 | 0.036475 | −1.4132 |
FANCE | Fanconi anemia group E protein | 0.09315 | −0.67103 | 0.026213 | −1.027 |
Library | Gene | p-Value_N | lfc_N | p-Value_C | lfc_C |
---|---|---|---|---|---|
CP1736 | CDK2 | 0.53595 | −0.27009 | 0.039806 | −1.4028 |
CDK4 | 0.92691 | 0.28251 | 0.16707 | −0.84773 | |
CDK6 | 0.10149 | −1.1315 | 0.0024321 | −1.6764 | |
CP1737 | CDK2 | 0.31995 | −0.74951 | 0.024024 | −1.3541 |
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Inglebert, M.; Dettwiler, M.; He, C.; Markkanen, E.; Opitz, L.; Naguleswaran, A.; Rottenberg, S. Individualized Pooled CRISPR/Cas9 Screenings Identify CDK2 as a Druggable Vulnerability in a Canine Mammary Carcinoma Patient. Vet. Sci. 2025, 12, 183. https://doi.org/10.3390/vetsci12020183
Inglebert M, Dettwiler M, He C, Markkanen E, Opitz L, Naguleswaran A, Rottenberg S. Individualized Pooled CRISPR/Cas9 Screenings Identify CDK2 as a Druggable Vulnerability in a Canine Mammary Carcinoma Patient. Veterinary Sciences. 2025; 12(2):183. https://doi.org/10.3390/vetsci12020183
Chicago/Turabian StyleInglebert, Marine, Martina Dettwiler, Chang He, Enni Markkanen, Lennart Opitz, Arunasalam Naguleswaran, and Sven Rottenberg. 2025. "Individualized Pooled CRISPR/Cas9 Screenings Identify CDK2 as a Druggable Vulnerability in a Canine Mammary Carcinoma Patient" Veterinary Sciences 12, no. 2: 183. https://doi.org/10.3390/vetsci12020183
APA StyleInglebert, M., Dettwiler, M., He, C., Markkanen, E., Opitz, L., Naguleswaran, A., & Rottenberg, S. (2025). Individualized Pooled CRISPR/Cas9 Screenings Identify CDK2 as a Druggable Vulnerability in a Canine Mammary Carcinoma Patient. Veterinary Sciences, 12(2), 183. https://doi.org/10.3390/vetsci12020183