CRISPR screens in cancer spheroids identify 3D growth-specific vulnerabilities

Han, Ke; Pierce, Sarah E.; Li, Amy; Spees, Kaitlyn; Anderson, Grace R.; Seoane, José; Lo, Yuan–Hung; Dubreuil, Michael M.; Olivas, Micah; Kamber, Roarke A.; Wainberg, Michael; Kostyrko, Kaja; Kelly, Marcus R.; Yousefi, Maryam; Simpkins, Scott W.; Yao, David; Lee, Keonil; Kuo, Calvin J.; Jackson, Peter K.; Sweet‐Cordero, Alejandro; Kundaje, Anshul; Gentles, Andrew J.; Curtis, Christina; Winslow, Monte M.; Bassik, Michael C.

doi:10.1038/s41586-020-2099-x

Cited by 225 publications

(210 citation statements)

References 61 publications

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“…2). CRISPR screens performed in spheroid cultures of KRAS-and EGFR-mutated NSCLC cell lines more accurately reproduce in vivo findings and identify drivers of oncogenic growth compared to screens performed in 2D cultures (55). Intriguingly, in this study SOS1 was essential for 3D spheroid survival but not 2D spheroid growth of both EGFR-and KRAS-mutated cells.…”

Section: Discussionmentioning

confidence: 56%

Marked Synergy by Vertical Inhibition of EGFR signaling in NSCLC Spheroids: SOS1 as a therapeutic target in EGFR-mutated cancer

Theard

Sheffels

Sealover

et al. 2020

Preprint

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Drug treatment of 3D cancer spheroids more accurately reflects in vivo therapeutic responses compared to adherent culture studies. In EGFR-mutated lung adenocarcinoma, EGFR-TKIs show enhanced efficacy in spheroid cultures. Simultaneous inhibition of multiple parallel RTKs further enhances EGFR-TKI effectiveness. We show that the common RTK signaling intermediate SOS1 was required for 3D spheroid growth of EGFR-mutated NSCLC cells. Using two distinct measures of pharmacologic synergy, we demonstrated that SOS1 inhibition strongly synergized with EGFR-TKI treatment only in 3D spheroid cultures. Combined EGFR-and SOS1-inhibition markedly inhibited Raf/MEK/ERK and PI3K/AKT signaling. Finally, broad assessment of the pharmacologic landscape of drug-drug interactions downstream of mutated EGFR revealed synergy when combining an EGFR-TKI with inhibitors of proximal signaling intermediates SOS1 and SHP2, but not inhibitors of downstream RAS effector pathways. These data indicate that vertical inhibition of proximal EGFR signaling should be pursued as a potential therapy to treat EGFR-mutated tumors.of RAS, co-targeting intermediates of the RAF/MEK/ERK and PI3K/AKT pathways enhances of osimertinib effectiveness, however, signaling through the uninhibited effector pathway may drive resistance (17)(18)(19)(20). Thus, it may be important for therapeutic combinations including osimertinib to stifle all downstream RTK/RAS signaling to be effective.Recent studies suggest that pharmacologic assessments of targeted therapeutics should be performed under 3D culture conditions rather than in 2D adherent cultures (21,22). 3D spheroids show altered growth characteristics, changes in cell surface proteins, altered metabolism, changes in activation of signaling pathways or altered responses to targeted pathway inhibitors, and are more resistant to drug-induced apoptosis compared to 2D adherent cultures signaling (23-26). These differences may be particularly relevant in EGFR-mutated NSCLC. EGFR-mutated cells show differential RTK expression and phosphorylation in 3D versus 2Dconditions (27). Further, EGFR-mutated cells respond more robustly to first-generation EGFR-TKIs in 3D cultures, and these responses more closely resemble responses seen in vivo (28).These data highlight the need for pharmacologic assessment of therapeutics designed to treat EGFR-mutated NSCLC under 3D culture conditions. The ubiquitously expressed RasGEFs (guanine nucleotide exchange factors) SOS1 and SOS2 (son of sevenless 1 and 2) are common signaling intermediates of RTK-mediated RAS activation. Although not initially considered as drug targets because of the low oncogenic potential of SOS (29), there has been renewed interest in SOS proteins as therapeutic targets for cancer treatment. We and others have shown that SOS1 and SOS2 may be important therapeutic targets in KRAS-mutated cancer cells (30-32), and a specific SOS1 inhibitor (BAY-293) has recently been identified (33). Here, we investigate SOS1 and SOS2 as potential therapeutic targets in EGFR-mutated l...

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Section: Discussionmentioning

confidence: 56%

Marked Synergy by Vertical Inhibition of EGFR signaling in NSCLC Spheroids: SOS1 as a therapeutic target in EGFR-mutated cancer

Theard

Sheffels

Sealover

et al. 2020

Preprint

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“…Reads were aligned to the sgRNA library and the log2 fold change was calculated for each sgRNA. Median phenotypes for each gene were calculated as previously described (24). P-values were calculated using a Mann-Whitney U-test and adjusted FDRs were computed using the Benjamini–Hochberg procedure.…”

Section: Supporting Informationmentioning

confidence: 99%

Metabolic precision labeling enables selective probing of O-linkedN-acetylgalactosamine glycosylation

Debets¹,

Tastan

Wisnovsky³

et al. 2020

Preprint

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38Protein glycosylation events that happen early in the secretory pathway are often dysregulated during 39 tumorigenesis. These events can be probed, in principle, by monosaccharides with bioorthogonal tags that 40 would ideally be specific for distinct glycan subtypes. However, metabolic interconversion into other 41 monosaccharides drastically reduces such specificity in the living cell. Here, we use a structure-based 42 design process to develop the monosaccharide probe GalNAzMe that is specific for cancer-relevant 43

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“…3m), and carboxypeptidase D ( Fig. 3n), two proteins directly involved in IGF1R maturation and activity [42][43] .…”

Section: Integrative Analysis Of Isogenic Crispr Screen Data Revealsmentioning

confidence: 99%

An integrative oncogene-dependency map identifies unique vulnerabilities of oncogenic EGFR, KRAS, and RIT1 in lung cancer

Vichas

Nkinsi

Riley

et al. 2020

Preprint

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ABSTRACTAdvances in precision oncology have transformed cancer therapy from broadly-applied cytotoxic therapy to personalized treatments based on each tumor’s unique molecular alterations. Here we investigate the oncogene-specific dependencies conferred by lung cancer driver variants of KRAS, EGFR, and RIT1. Integrative analysis of genome-wide CRISPR screens in isogenic cell lines identified shared and unique vulnerabilities of each oncogene. The non-identical landscape of dependencies underscores the importance of genotype-guided therapies to maximize tumor responses. Combining genetic screening data with small molecule sensitivity profiling, we identify a unique vulnerability of RIT1-mutant cells to loss of spindle assembly checkpoint regulators. This sensitivity may be related to a novel role of RIT1 in mitosis; we find that oncogenic RIT1M90I alters mitotic timing via weakening of the spindle assembly checkpoint. In addition, we uncovered a specific cooperation of mutant RIT1 with loss of Hippo pathway genes. In human lung cancer, RIT1 mutations and amplifications frequently co-occur with loss of Hippo pathway gene expression. These results provide the first genome-wide atlas of oncogenic RIT1-cooperating factors and genetic dependencies and identify components of the RAS pathway, spindle assembly checkpoint, and Hippo/YAP1 network as candidate therapeutic targets in RIT1-mutant lung cancer.

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CRISPR screens in cancer spheroids identify 3D growth-specific vulnerabilities

Cited by 225 publications

References 61 publications

Marked Synergy by Vertical Inhibition of EGFR signaling in NSCLC Spheroids: SOS1 as a therapeutic target in EGFR-mutated cancer

Marked Synergy by Vertical Inhibition of EGFR signaling in NSCLC Spheroids: SOS1 as a therapeutic target in EGFR-mutated cancer

Metabolic precision labeling enables selective probing of O-linkedN-acetylgalactosamine glycosylation

An integrative oncogene-dependency map identifies unique vulnerabilities of oncogenic EGFR, KRAS, and RIT1 in lung cancer

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