Integrated CRISPR screening and drug profiling identifies combination opportunities for EGFR, ALK, and BRAF/MEK inhibitors

Tiedt, Ralph; King, Frederick J.; Stamm, Christelle; Niederst, Matthew J.; Delach, Scott; Zumstein-Mecker, Sabine; Meltzer, Jodi; Mulford, Iain J.; Labrot, Emma; Engstler, Barbara Schacher; Baltschukat, Sabrina; Kerr, Gráinne; Golji, Javad; Wyss, Daniel F.; Schnell, Christian; Ainscow, Edward; Engelman, Jeffrey A.; Sellers, William R.; Barretina, Jordi; Caponigro, Giordano; Porta, Diana Graus

doi:10.1016/j.celrep.2023.112297

Cited by 7 publications

(7 citation statements)

References 62 publications

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“…Thus, it would have been surprising to see these hits or other similar ones identified across both screens. The convergence of non-overlapping hits from both screens onto the same overarching biological process of apoptosis confirms the validity of our findings and is not uncommon in studies CRISPR screening in multiple cell types 72 , 73 . Taken together, these screens support our findings that apoptosis is a major mediator of cfDNA release.…”

Section: Discussionsupporting

confidence: 86%

An in vitro CRISPR screen of cell-free DNA identifies apoptosis as the primary mediator of cell-free DNA release

Davidson,

Miranda,

Reed

et al. 2024

Commun Biol

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AbtractClinical circulating cell-free DNA (cfDNA) testing is now routine, however test accuracy remains limited. By understanding the life-cycle of cfDNA, we might identify opportunities to increase test performance. Here, we profile cfDNA release across a 24-cell line panel and utilize a cell-free CRISPR screen (cfCRISPR) to identify mediators of cfDNA release. Our panel outlines two distinct groups of cell lines: one which releases cfDNA fragmented similarly to clinical samples and purported as characteristic of apoptosis, and another which releases larger fragments associated with vesicular or necrotic DNA. Our cfCRISPR screens reveal that genes mediating cfDNA release are primarily involved with apoptosis, but also identify other subsets of genes such as RNA binding proteins as potential regulators of cfDNA release. We observe that both groups of cells lines identified primarily produce cfDNA through apoptosis. These results establish the utility of cfCRISPR, genetically validate apoptosis as a major mediator of DNA release in vitro, and implicate ways to improve cfDNA assays.

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Section: Discussionsupporting

confidence: 86%

An in vitro CRISPR screen of cell-free DNA identifies apoptosis as the primary mediator of cell-free DNA release

Davidson,

Miranda,

Reed

et al. 2024

Commun Biol

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“…The mainstay of systemic therapy for cancer is the combination of anticancer drugs with nonoverlapping mechanisms and toxicities to increase anticancer efficacy, decrease toxicity, address tumor heterogeneity, and prevent chemoresistance (3). Achieving these goals requires a comprehensive understanding of mechanisms of action for single drugs/combinations, and dissecting the genetic dependencies for pharmacological interactions (4). Although the basis for such knowledge is being established for the combinations of conventional chemotherapeutics (5,6), the mechanisms by which molecular-targeted agents interact with chemotherapeutics are yet to be discovered.…”

Section: Discussionmentioning

confidence: 99%

“…However, distinct modes of action do not guarantee a synergistic effect or, at least, an additive effect on cancer cell death (3). A rational selection of the combinatorial agents based on a clear understanding of the genetic dependencies and vulnerabilities for synergistic action is valuable to improve patient outcomes while minimizing the risk of treatment failure from empirical combination approaches (4,5). Such an understanding also paves the way for discovering novel agents with potent synergy in anticancer action.…”

Section: Introductionmentioning

confidence: 99%

Functional genomics reveals an off-target dependency of drug synergy in gastric cancer therapy

Leylek,

Honeywell,

Lee

et al. 2023

Preprint

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The rational combination of anticancer agents is critical to improving patient outcomes in cancer. Nonetheless, most combination regimens in the clinic result from empirical methodologies disregarding insight into the mechanism of action and missing the opportunity to improve therapy outcomes incrementally. Deciphering the genetic dependencies and vulnerabilities responsible for synergistic interactions is crucial for rationally developing effective anticancer drug combinations. Hence, we screened pairwise pharmacological interactions between molecular-targeted agents and conventional chemotherapeutics and examined the genome-scale genetic dependencies in gastric adenocarcinoma cell models. Since this type of cancer is mainly chemoresistant and incurable, clinical situations demand effective combination strategies. Our pairwise combination screen revealed SN38/erlotinib as the drug pair with the most robust synergism. Genome-wide CRISPR screening and a shRNA-based signature assay indicated that the genetic dependency/vulnerability signature of SN38/erlotinib was not divergent from that of SN38 alone. Additional investigation revealed that the enhanced cell death with improved death kinetics caused by the SN38/erlotinib combination is surprisingly due to erlotinib’s off-target effect that inhibits ABCG2, not its on-target effect on EGFR. These findings demonstrate the importance of assessing off-target effects in addition to the perceived on-target effects of a drug. Our results confirm that a genetic dependency signature different from the single-drug application may not be necessary for the synergistic interaction of molecular-targeted agents with conventional chemotherapeutics in gastric adenocarcinoma. The findings also demonstrated the efficacy of functional genomics approaches in unveiling biologically validated mechanisms of pharmacological interactions.SignificanceFunctional genomics approaches efficiently demonstrated that a divergence in genetic dependency/vulnerability signature is not essential for the synergistic interaction of molecular-targeted agents and conventional chemotherapeutic combinations in gastric adenocarcinoma.

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“…Pooled library CRISPR screens have revolutionized mammalian functional genomics. DepMap teams have screened over a thousand cancer cell lines with CRISPR knockout libraries to identify background-specific genetic vulnerabilities 1 – 3 , while dozens of genetic modifier screens with small molecules have explored biomarkers and mechanisms of drug sensitivity and resistance 4 – 10 . However, initial efforts to assay genetic interactions (GIs) – that is, the manipulation of multiple genes in the same cell to identify nonlinear combinatorial phenotypes – have proven complex and costly 11 – 15 .…”

Section: Introductionmentioning

confidence: 99%

Efficient gene knockout and genetic interaction screening using the in4mer CRISPR/Cas12a multiplex knockout platform

Esmaeili Anvar,

Lin,

et al. 2024

Nat Commun

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Genetic interactions mediate the emergence of phenotype from genotype, but technologies for combinatorial genetic perturbation in mammalian cells are challenging to scale. Here, we identify background-independent paralog synthetic lethals from previous CRISPR genetic interaction screens, and find that the Cas12a platform provides superior sensitivity and assay replicability. We develop the in4mer Cas12a platform that uses arrays of four independent guide RNAs targeting the same or different genes. We construct a genome-scale library, Inzolia, that is ~30% smaller than a typical CRISPR/Cas9 library while also targeting ~4000 paralog pairs. Screens in cancer cells demonstrate discrimination of core and context-dependent essential genes similar to that of CRISPR/Cas9 libraries, as well as detection of synthetic lethal and masking/buffering genetic interactions between paralogs of various family sizes. Importantly, the in4mer platform offers a fivefold reduction in library size compared to other genetic interaction methods, substantially reducing the cost and effort required for these assays.

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Integrated CRISPR screening and drug profiling identifies combination opportunities for EGFR, ALK, and BRAF/MEK inhibitors

Cited by 7 publications

References 62 publications

An in vitro CRISPR screen of cell-free DNA identifies apoptosis as the primary mediator of cell-free DNA release

An in vitro CRISPR screen of cell-free DNA identifies apoptosis as the primary mediator of cell-free DNA release

Functional genomics reveals an off-target dependency of drug synergy in gastric cancer therapy

Efficient gene knockout and genetic interaction screening using the in4mer CRISPR/Cas12a multiplex knockout platform

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