A curative combination cancer therapy achieves high fractional cell killing through low cross-resistance and drug additivity
- Laboratory of Systems Pharmacology, Harvard Medical School, United States
- Harvard Medical School, United States
Figures
Figure 1 with 1 supplement
Pairs of drugs in R-CHOP exhibit little synergy, but some strong antagonism, in a Diffuse Large B-Cell Lymphoma cell line.
(a) Pfeiffer cells grown in microtiter plates were treated with drug combinations for 72 hr followed by a luminescence-based assay for cell viability. ‘Excess over Bliss’ measures the observed …
Figure 1—figure supplement 1
Measuring cytotoxic responses to R-CHOP drugs in DLBCL cultures.
(a) Human serum complement enhances the cytotoxicity of rituximab to DLBCL cell lines. The DLBCL cell lines Pfeiffer, SU-DHL-4, SU-DHL-6 were each seeded in 384-well plates at 105 cells/mL in …
Figure 2 with 1 supplement
Higher order drug combinations do not exhibit synergistic cell killing.
(a) Experimental design: two or more drugs were mixed in equipotent ratios such that they similarly contributed to cytotoxicity as the dose of the mixture was increased. Dose gradients of drug …
Figure 2—figure supplement 1
Measuring high-order interactions among R-CHOP drugs with equipotent combinations.
(a) Combining the drugs in R-CHOP at equipotent ratio. In order to measure high-order interactions in R-CHOP (Figure 2 and Figure 2—figure supplement 1B,C), it was necessary to combine agents in …
Figure 3 with 1 supplement
Strategy for measuring cross-resistance between drugs.
(a) Cells were mutagenized and barcoded using one of three approaches: (i) random mutagenesis and clone tracing, (ii) knockdown by CRISPRi or (iii) overexpression by CRISPRa. 106 mutagenized clones …
Figure 3—figure supplement 1
Dosing schedule and replication strategy for all three approaches taken to isolate cells resistant to single cytotoxic drugs in the R-CHOP combination.
(a) Experimental design for measuring drug resistance in DNA-barcoded Pfeiffer clones. 1 million unique mutagenized Pfeiffer cells individually tagged with DNA barcodes were expanded. Replicate …
Figure 4 with 1 supplement
In mutagenized clones single-drug resistance is common but multi-drug resistance is rare.
(a) Reproducibility of DNA barcode enrichment among triplicate drug treatments. Horizontal axis is the highest value for each barcode’s enrichment scores in any replicate. Vertical axis is the …
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Figure 4—source data 1
Barcode counts for all clone tracing experiments.
- https://cdn.elifesciences.org/articles/50036/elife-50036-fig4-data1-v2.txt
Figure 4—figure supplement 1
Prednisolone-resistant clones show low cross-resistance to other drugs in R-CHOP, and repeats of vincristine show high cross-resistance.
(a) Enriched barcodes in the cultures treated with prednisolone show a high degree of reproducibility between three replicate drug selections. As prednisolone did not show single-agent cytotoxicity …
Figure 5 with 1 supplement
Identification of mechanisms of single drug resistance by genome-wide CRISPRi and CRISPRa screening.
Volcano plots of gene phenotype and p-value for CRISPRi (left) and CRISPRa (right) screens of single R-CHOP drugs. Phenotype of 1 is full resistance, 0 is parental sensitivity,<0 is …
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Figure 5—source data 1
sgRNA counts for all CRISPR screens.
- https://cdn.elifesciences.org/articles/50036/elife-50036-fig5-data1-v2.xlsx
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Figure 5—source data 2
sgRNA phenotype scores for all CRISPR screens.
- https://cdn.elifesciences.org/articles/50036/elife-50036-fig5-data2-v2.xlsx
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Figure 5—source data 3
Gene scores for all CRISPR screens.
- https://cdn.elifesciences.org/articles/50036/elife-50036-fig5-data3-v2.xlsx
Figure 5—figure supplement 1
CRISPRi/a cell lines strongly alter gene expression of targeted genes and additional cyclophosphamide CRISPRi screen identifies hypersensitive hits in the DNA interstrand crosslink pathway.
(a) CRISPRi targeting of 3 control genes results in knockdown in Pfeiffer cells (left) and CRISPRa targeting of 3 control genes results in overexpression in K562 cells (left). On-target effect is …
Figure 6 with 1 supplement
Validation of CRISPRi and CRISPRa screen results by individual drug sensitivity measurements.
(a) Gene knockdown by CRISPRi produces changes in drug sensitivity (IC50) that are correlated with resistance phenotypes from the genome-wide CRISPRi screen (Pearson correlation r = 0.66, p<10–5). …
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Figure 6—source data 1
Data obtained in the CRISPR screen validation experiments.
- https://cdn.elifesciences.org/articles/50036/elife-50036-fig6-data1-v2.xlsx
Figure 6—figure supplement 1
Changing transcript abundance with CRISPRi and CRISPRa.
(a–b) Quantification of changes in gene expression in validation CRISPRi and CRISPRa cell lines and (c–d) Gene overexpression by CRISPRa produces changes in drug resistance that are correlated …
Figure 7 with 2 supplements
Cross-resistance analysis of the CRISPRi and CRISPRa screens reveals a small number of multi-drug resistance mechanisms.
(a) Scatter plots of resistance scores obtained in CRISPRi screens for each pair of drugs in RCHO; each dot represents a gene. Resistance scores were calculated from the product of the gene …
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Figure 7—source data 1
Gamma growth scores for triple-resistant genes identified in CRISPRi screens.
This data was extracted from Figure 5—source data 3.
- https://cdn.elifesciences.org/articles/50036/elife-50036-fig7-data1-v2.xlsx
Figure 7—figure supplement 1
Determination of a cutoff threshold for cross-resistance analysis and identity of all cross-resistant genes for CRISPRi and CRISPRa screens.
(a) Plots of the total number of double-, triple- and quadruple-resistant genes as a function of the resistance score determined in the CRISPRi screens. Whole genome sets of negative control genes …
Figure 7—figure supplement 2
Determination of a cutoff threshold for cross-hypersensitivity analysis and identity of all cross-hypersensitive genes for CRISPRi and CRISPRa screens.
(a) Plots of the total number of double-, triple- and quadruple-hypersensitive genes as a function of the hypersensitivity score determined in the CRISPRi screens. Whole genome sets of negative …
Figure 8
Cross-resistance between drugs in R-CHOP is close to its theoretical minimum.
(a) Fraction of clones or genetic perturbations resistant to one or more drugs in RCHO. Gray shading spans the range for different sets of drugs (e.g. six different pairs), and black points mark the …
Figure 9
The role of multiple drug mechanisms in increasing the probability of cure by combination therapy.
(a) Conceptual schematic of the role of multiple drug mechanisms, each subject to different mechanisms of resistance, in the eradication of drug resistant clones and cure of a patient’s cancer. When …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (Homo-sapiens) | Pfeiffer | ATCC (Cat# CRL-2632) | RRID:CVCL_3326 | Diffuse Large B-Cell Lymphoma |
| Cell line (Homo-sapiens) | SU-DHL-4 | ATCC (Cat# CRL-2957) | RRID:CVCL_0539 | Diffuse Large B-Cell Lymphoma |
| Cell line (Homo-sapiens) | SU-DHL-6 | ATCC (Cat# CRL-2959) | RRID:CVCL_2206 | Diffuse Large B-Cell Lymphoma |
| Biological sample (Homo-sapiens) | Pooled human complement serum | Innovative Research | IPLA-CSER | |
| Peptide, recombinant protein | Rituximab | Dana Farber Cancer Institute | 8 mg/mL in the clinical formulation + 10% Glycerol | |
| Chemical compound, drug | 4-hydroperoxy-cyclophosphamide (4-H-Cyclo.) | Niomech | D-18864 | Pre-activated form of cyclophosphamide |
| Chemical compound, drug | Doxorubicin | Selleck Chemicals | S1208 | |
| Chemical compound, drug | Vincristine | Selleck Chemicals | S1241 | |
| Chemical compound, drug | Prednisolone | Selleck Chemicals | S1737 | Pre-activated form of prednisone |
| Commercial assay or kit | CellTiter-Glo | Promega | G7573 | Luminescent cell viability assay |
| Chemical compound, drug | N-ethyl-N-nitrosourea (ENU) | Sigma Aldrich | N3385 | Mutation-inducing agent |
| Recombinant DNA reagent | ClonTracer Barcoding library | Addgene (Cat# 67267) | RRID:Addgene_67267 | (Bhang et al., 2015) |
| Software, algorithm | clonTracer_analyze v1.0 | (Bhang et al., 2015) | Script for analysis of barcode composition | |
| Cell line (Homo-sapiens) | HEK293T | ATCC (Cat# CRL-3216) | RRID:CVCL_0063 | For lentivirus production |
| Recombinant DNA reagent | psPAX2 | Addgene (Cat# 12260) | RRID:Addgene_12260 | Lentiviral packaging plasmid |
| Recombinant DNA reagent | pCMV-VSV-G | Addgene (Cat# 8454) | RRID:Addgene_8454 | VSV-G envelope expressing plasmid for lentivirus production |
| Cell line (Homo-sapiens) | Pfeiffer CRISPRi | This paper | See Materials and methods, Section ‘Generation of dCas9-expressing cell lines’ | |
| Recombinant DNA reagent | pMH0001 | Addgene (Cat# 85969) | RRID:Addgene_85969 | Lentiviral construct for expression of dCas9-BFP-KRAB |
| Cell line (Homo-sapiens) | K562 | ATCC (Cat# CCL-243) | RRID:CVCL_0004 | Chronic myeloid leukemia (CML) cell line |
| Cell line (Homo-sapiens) | K562 CRISPRa | This paper | See Materials and methods, Section ‘Generation of dCas9-expressing cell lines’ | |
| Recombinant DNA reagent | pHRdSV40-dCas9-10xGCN4_v4-P2A-BFP | Addgene (Cat# 60903) | RRID:Addgene_60903 | Lentiviral construct for expression of dCas9-SunTag |
| Recombinant DNA reagent | pHRdSV40-scFv-GCN4-sfGFP-VP64-GB1-NLS | Addgene (Cat# 60904) | RRID:Addgene_60904 | Lentiviral construct for expression of scFv-sfGFP-VP64 |
| Recombinant DNA reagent | pU6-sgRNA EF1Alpha-puro-T2A-BFP | Addgene (Cat# 60955) | RRID:Addgene_60955 | Lentiviral construct for expression of sgRNAs |
| Recombinant DNA reagent | hCRISPRi_v2 | Addgene (Cat#83969 and 83970) | RRID:Addgene_83969 | Genome-wide human library of sgRNAs for CRISPRi |
| Recombinant DNA reagent | hCRISPRa_v2 | Addgene (Cat# 83978 and 83979) | RRID:Addgene_83978 | Genome-wide human library of sgRNAs for CRISPRa |
| Software, algorithm | ScreenProcessing pipeline | (Horlbeck et al., 2016) | https://github.com/mhorlbeck/ScreenProcessing | |
| Cell line (Homo-sapiens) | Pfeiffer CRISPRa | This paper | See Materials and methods, Section ‘Validation of CRISPR screens’ |
Additional files
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Supplementary file 1
List of oligonucleotides used in this study and sequence of sgRNA protospacers used in individual CRISPRi/a cell line construction.
- https://cdn.elifesciences.org/articles/50036/elife-50036-supp1-v2.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/50036/elife-50036-transrepform-v2.pdf
