Genome-Wide CRISPR-Cas9 Knockout Screening Identifies NUDCD2 Depletion as Sensitizer for Bortezomib, Carfilzomib and Ixazomib in Multiple Myeloma
Abstract
1. Introduction
2. Materials and Methods
2.1. Cell Culture
2.2. Compounds
2.3. Genome-Wide CRISPR-Cas9 Knockout Screening
2.4. Candidate Hit Validation
2.5. Proteasome Activity Assay
2.6. RNA Sequencing
2.7. Statistical Analysis
3. Results
3.1. Genome-Wide CRISPR-Cas9 Knockout Screens Identify Genetic Determinants for Bortezomib and Carfilzomib Sensitivity in MM Cells
3.1.1. Genome-Wide CRISPR-Cas9 Knockout Screens
3.1.2. Gene Set Enrichment Analysis of CRISPR-Cas9 Knockout Screens
3.1.3. Identification of Top Candidate Hits
3.1.4. Validation of Top Candidate Hits
3.2. NUDCD2 Knockout Does Not Sensitize Myeloma Cells by Modulating Proteasome Activity
3.3. RNA Sequencing Identifies Potential Mechanisms of NUDCD2 KO Sensitization
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
MM | Multiple myeloma |
KO | Knockout |
CPT1A | Carnitine Palmitoyltransferase 1A |
OS | Overall survival |
IMiDs | Immunomodulatory drugs |
PIs | Proteasome inhibitors |
PFS | Progression-free survival |
UPS | Ubiquitin proteasome system |
PSMB5 | Proteasome subunit beta type-5 |
ER | Endoplasmic reticulum |
UPR | Unfolded protein response |
JCRB | Japanese Collection of Research Bioresources |
FBS | Fetal bovine serum |
DMSO | Dimethyl sulfoxide |
sgRNAs | Single guide RNAs |
MOI | Multiplicity of infection |
PBS | Phosphate-buffered saline |
CC2 | CRISPRCloud2 |
Log2FC | Log2 fold change |
TKOv3 | Toronto KnockOut CRISPR library |
AAVS1 | Adeno-associated virus integration site 1 |
CTL | Chymotrypsin-like activities |
TL | Trypsin-like activities |
CL | Caspase-like activities |
SD | Standard deviation |
ANOVA | One-way analysis of variance |
IC50 | Inhibitory concentration of 50% |
GSEA | Gene Set Enrichment Analysis |
NES | Normalized enrichment score |
Padj. | Adjusted p-values |
ECPAS | Proteasome adapter and scaffold protein ECM29 |
PSMF1 | Proteasome inhibitor PI31 subunit |
OSER1 | Oxidative stress-responsive serine-rich protein 1 |
PCA | Principal Component Analysis |
DEG | Differentially expressed gene |
NS | Non-significant |
HMMR | Hyaluronan Mediated Motility Receptor |
FAO | Fatty acid oxidation |
LCFA | Long chain fatty acids |
MAST1 | Microtubule Associated Serine/Threonine Kinase 1 |
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Vlayen, S.; Dierckx, T.; Caruso, M.; Sieben, S.; De Keersmaecker, K.; Daelemans, D.; Delforge, M. Genome-Wide CRISPR-Cas9 Knockout Screening Identifies NUDCD2 Depletion as Sensitizer for Bortezomib, Carfilzomib and Ixazomib in Multiple Myeloma. Hemato 2025, 6, 21. https://doi.org/10.3390/hemato6030021
Vlayen S, Dierckx T, Caruso M, Sieben S, De Keersmaecker K, Daelemans D, Delforge M. Genome-Wide CRISPR-Cas9 Knockout Screening Identifies NUDCD2 Depletion as Sensitizer for Bortezomib, Carfilzomib and Ixazomib in Multiple Myeloma. Hemato. 2025; 6(3):21. https://doi.org/10.3390/hemato6030021
Chicago/Turabian StyleVlayen, Sophie, Tim Dierckx, Marino Caruso, Swell Sieben, Kim De Keersmaecker, Dirk Daelemans, and Michel Delforge. 2025. "Genome-Wide CRISPR-Cas9 Knockout Screening Identifies NUDCD2 Depletion as Sensitizer for Bortezomib, Carfilzomib and Ixazomib in Multiple Myeloma" Hemato 6, no. 3: 21. https://doi.org/10.3390/hemato6030021
APA StyleVlayen, S., Dierckx, T., Caruso, M., Sieben, S., De Keersmaecker, K., Daelemans, D., & Delforge, M. (2025). Genome-Wide CRISPR-Cas9 Knockout Screening Identifies NUDCD2 Depletion as Sensitizer for Bortezomib, Carfilzomib and Ixazomib in Multiple Myeloma. Hemato, 6(3), 21. https://doi.org/10.3390/hemato6030021