Background
Materials and methods
Cell culture
Small molecule inhibitors
Cloning, library amplification, and DNA production
Production of lentivirus
Functional titration of lentivirus
Generation of T24 SAM cells with lentivirus transduction
Palbociclib resistance screen
Genomic DNA extraction and next-generation sequencing (NGS)
Analysis of NGS data
Gene set enrichment analysis
CRISPR/Cas9 synergistic activation mediator (SAM) for individual gene targeting
Cell viability assay and cell proliferation assay
Caspase-Glo 3/7 assay (apoptosis assay)
Cell cycle analysis
Clonogenic assay
Chicken chorioallantoic membrane (CAM) assay
Immunoblotting and antibodies
Combination index analysis
Statistical analysis
Results
Identification of sgRNAs that confer resistance to CDK4/6 inhibition
Validation of sgRNA candidates
sgRNA | Gene | LFC | FDR | Rank |
---|---|---|---|---|
NM_000458_1073 | HNF1B | 3.0416 | 4.39E-69 | 1 |
NM_013441_42667 | RCAN3 | 2.9917 | 1.57E-21 | 3 |
NM_001142595_12383 | P4HA1 | 1.954 | 2.21E-08 | 13 |
NM_001033930_5799 | UBA52 | 1.4143 | 2.60E-08 | 15 |
NM_152411_62833 | ZNF786 | 1.2864 | 0.004702 | 252 |
NM_199336_69028 | FAHD2B | 1.8803 | 0.006676 | 306 |
NM_001042454_6659 | TGFB1I1 | 1.1487 | 0.01617 | 425 |
NM_016653_47440 | MAP 3 K20 | 1.1754 | 0.023672 | 499 |
Activation of multiple signaling pathways confer resistance to Palbociclib
Inhibitors/agonists | Target | Pathway |
---|---|---|
PIK-90 | PI3Kα/γ/δ | PI3K-AKT |
MK2206 | Akt1/2/3 | PI3K-AKT |
Everolimus | mTOR | PI3K-AKT |
NVP-BEZ235 | p110α/γ/δ/β and mTOR | PI3K-AKT |
Axitinib | VEGFR1/2/3, PDGFRβ and c-Kit | Ras/MAPK |
Erdafitinib | FGFR1/2/3/4 | Ras/MAPK |
CI-1040 | MEK1/2 | Ras/MAPK |
Ruxolitinib | JAK1/2 | JAK-STAT |
Stattic | STAT3 | JAK-STAT |
SH-4-54 | STAT3/5 | JAK-STAT |
Roscovitine | CDK2/7/9 | Cell cycle |
Validation of synergistic combination therapies
IC50 (95% CI,nM) | Effect (IC50 + Palbociclib 1000 nM) | Combination Index | |
---|---|---|---|
Axitinib | 919.1 (754.8–1119) | 0.63 | 0.67 |
Erdafitinib | 3752 (3296–4271) | 0.79 | 0.43 |
NVP-BEZ235 | 9.12 (6.755–12.31) | 0.67 | 0.33 |
CI-1040 | 1661 (1225–2254) | 0.87 | 0.2 |
MK2206 | 1260 (407.6–3897) | 0.55 | 0.44 |
PIK90 | 1581 (908.4–2753) | 0.74 | 0.16 |
Everolimus | 3.501 (1.605–7.637) | 0.65 | 0.15 |
Roscovitine | 4815 (3094–7492) | 0.81 | 0.72 |
SH-4-54 | 2324 (2129–2537) | 0.57 | 2.93 |
Stattic | 2384 (2015–2820) | 0.53 | 0.88 |
Ruxolitinib | 21221 (18381–24500) | 0.54 | 3.85 |