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High-throughput phenotypic screens leveraging biochemical perturbations, high-content readouts, and complex multicellular models could advance therapeutic discovery yet remain constrained by limitations of scale. To address this, we establish a method for compressing screens by pooling perturbations followed by computational deconvolution. Conducting controlled benchmarks with a highly bioactive small molecule library and a high-content imaging readout, we demonstrate increased efficiency for compressed experimental designs compared to conventional approaches. To prove generalizability, we apply compressed screening to examine transcriptional responses of patient-derived pancreatic cancer organoids to a library of tumor-microenvironment (TME)-nominated recombinant protein ligands. Using single-cell RNA-seq as a readout, we uncover reproducible phenotypic shifts induced by ligands that correlate with clinical features in larger datasets and are distinct from reference signatures available in public databases. In sum, our approach enables phenotypic screens that interrogate complex multicellular models with rich phenotypic readouts to advance translatable drug discovery as well as basic biology.

Pancreatic cancer is rapidly progressive and notoriously difficult to treat with cytotoxic chemotherapy and targeted agents. Recent demonstration of the efficacy of maintenance PARP inhibition in germline BRCA mutated pancreatic cancer has raised hopes that increased understanding of the DNA damage response pathway will lead to new therapies in both homologous recombination (HR) repair-deficient and proficient pancreatic cancer. Here, we review the potential mechanisms of exploiting HR deficiency, replicative stress, and DNA damage-mediated immune activation through targeted inhibition of DNA repair regulatory proteins.

We conducted next-generation DNA sequencing on 335 biliary tract cancers and characterized the genomic landscape by anatomic site within the biliary tree. In addition to frequent FGFR2 fusions among patients with intrahepatic cholangiocarcinoma (IHCC), we identified FGFR2 extracellular domain in-frame deletions (EID) in 5 of 178 (2.8%) patients with IHCC, including two patients with FGFR2 p.H167_N173del. Expression of this FGFR2 EID in NIH3T3 cells resulted in constitutive FGFR2 activation, oncogenic transformation, and sensitivity to FGFR inhibitors. Three patients with FGFR2 EIDs were treated with Debio 1347, an oral FGFR1/2/3 inhibitor, and all showed partial responses. One patient developed an acquired L618F FGFR2 kinase domain mutation at disease progression and experienced a further partial response for 17 months to an irreversible FGFR2 inhibitor, futibatinib. Together, these findings reveal FGFR2 EIDs as an alternative mechanism of FGFR2 activation in IHCC that predicts sensitivity to FGFR inhibitors in the clinic.Significance:FGFR2 EIDs are transforming genomic alterations that occur predominantly in patients with IHCC. These FGFR2 EIDs are sensitive to FGFR inhibition in vitro, and patients with these alterations benefited from treatment with FGFR inhibitors in the clinic.This article is highlighted in the In This Issue feature, p. 2355

Doublecortin like kinase 1 (DCLK1) is an understudied kinase that is upregulated in a wide range of cancers, including pancreatic ductal adenocarcinoma (PDAC). However, little is known about its potential as a therapeutic target. We used chemoproteomic profiling and structure-based design to develop a selective, in vivo-compatible chemical probe of the DCLK1 kinase domain, DCLK1-IN-1. We demonstrate activity of DCLK1-IN-1 against clinically relevant patient-derived PDAC organoid models and use a combination of RNA-sequencing, proteomics and phosphoproteomics analysis to reveal that DCLK1 inhibition modulates proteins and pathways associated with cell motility in this context. DCLK1-IN-1 will serve as a versatile tool to investigate DCLK1 biology and establish its role in cancer. A highly selective inhibitor of the DCLK1/2 kinases is used to uncover the consequences of DCLK1 inhibition on viability, phosphosignaling and the transcriptome in patient-derived organoid models of pancreatic ductal adenocarcinoma.

Summary: The mitogen-activated protein kinase (MAPK) pathway is a critical effector of oncogenic RAS signaling, and MAPK pathway inhibition may be an effective combination treatment strategy. We performed genome-scale loss-of-function CRISPR-Cas9 screens in the presence of a MEK1/2 inhibitor (MEKi) in KRAS-mutant pancreatic and lung cancer cell lines and identified genes that cooperate with MEK inhibition. While we observed heterogeneity in genetic modifiers of MEKi sensitivity across cell lines, several recurrent classes of synthetic lethal vulnerabilities emerged at the pathway level. Multiple members of receptor tyrosine kinase (RTK)-RAS-MAPK pathways scored as sensitizers to MEKi. In particular, we demonstrate that knockout, suppression, or degradation of SHOC2, a positive regulator of MAPK signaling, specifically cooperated with MEK inhibition to impair proliferation in RAS-driven cancer cells. The depletion of SHOC2 disrupted survival pathways triggered by feedback RTK signaling in response to MEK inhibition. Thus, these findings nominate SHOC2 as a potential target for combination therapy. : Sulahian, Kwon, and Walsh et al. performed several loss-of-function CRISPR-Cas9 screens in KRAS-mutant cancer cells treated with a MEK inhibitor and define the landscape of modifiers of MEK inhibitor sensitivity while highlighting that SHOC2 is a potent synthetic lethal target that serves as a critical signaling node to mediate MAP kinase pathway reactivation upon MEK inhibition. Keywords: Ras, KRAS, MEK inhibitor, synthetic lethal, SHOC2, CRISPR-Cas9 screen

Synthetic lethality-an interaction between two genetic events through which the co-occurrence of these two genetic events leads to cell death, but each event alone does not-can be exploited for cancer therapeutics1. DNA repair processes represent attractive synthetic lethal targets, because many cancers exhibit an impairment of a DNA repair pathway, which can lead to dependence on specific repair proteins2. The success of poly(ADP-ribose) polymerase 1 (PARP-1) inhibitors in cancers with deficiencies in homologous recombination highlights the potential of this approach3. Hypothesizing that other DNA repair defects would give rise to synthetic lethal relationships, we queried dependencies in cancers with microsatellite instability (MSI), which results from deficient DNA mismatch repair. Here we analysed data from large-scale silencing screens using CRISPR-Cas9-mediated knockout and RNA interference, and found that the RecQ DNA helicase WRN was selectively essential in MSI models in vitro and in vivo, yet dispensable in models of cancers that are microsatellite stable. Depletion of WRN induced double-stranded DNA breaks and promoted apoptosis and cell cycle arrest selectively in MSI models. MSI cancer models required the helicase activity of WRN, but not its exonuclease activity. These findings show that WRN is a synthetic lethal vulnerability and promising drug target for MSI cancers.

There is a critical need for the development of safe and efficient delivery technologies for CRISPR/Cas9 to advance translation of genome editing to the clinic. Non-viral methods that are simple, efficient, and completely based on biologically-derived materials could offer such potential. Here we report a simple and modular tandem peptide-based nanocomplex system with cell-targeting capacity that efficiently combines guide RNA (sgRNA) with Cas9 protein, and facilitates internalization of sgRNA/Cas9 ribonucleoprotein complexes to yield robust genome editing across multiple cell lines.

ATP-competitive fibroblast growth factor receptor (FGFR) kinase inhibitors, including BGJ398 and Debio 1347, show antitumor activity in patients with intrahepatic cholangiocarcinoma (ICC) harboring activating FGFR2 gene fusions. Unfortunately, acquired resistance develops and is often associated with the emergence of secondary FGFR2 kinase domain mutations. Here, we report that the irreversible pan-FGFR inhibitor TAS-120 demonstrated efficacy in 4 patients with FGFR2 fusion–positive ICC who developed resistance to BGJ398 or Debio 1347. Examination of serial biopsies, circulating tumor DNA (ctDNA), and patient-derived ICC cells revealed that TAS-120 was active against multiple FGFR2 mutations conferring resistance to BGJ398 or Debio 1347. Functional assessment and modeling the clonal outgrowth of individual resistance mutations from polyclonal cell pools mirrored the resistance profiles observed clinically for each inhibitor. Our findings suggest that strategic sequencing of FGFR inhibitors, guided by serial biopsy and ctDNA analysis, may prolong the duration of benefit from FGFR inhibition in patients with FGFR2 fusion–positive ICC.Significance:ATP-competitive FGFR inhibitors (BGJ398, Debio 1347) show efficacy in FGFR2-altered ICC; however, acquired FGFR2 kinase domain mutations cause drug resistance and tumor progression. We demonstrate that the irreversible FGFR inhibitor TAS-120 provides clinical benefit in patients with resistance to BGJ398 or Debio 1347 and overcomes several FGFR2 mutations in ICC models.This article is highlighted in the In This Issue feature, p. 983

Pancreatic ductal adenocarcinoma (PDAC) is driven by co-existing mutations in KRAS and TP53. However, how these mutations collaborate to promote this cancer is unknown. Here, we uncover sequence-specific changes in RNA splicing enforced by mutant p53 which enhance KRAS activity. Mutant p53 increases expression of splicing regulator hnRNPK to promote inclusion of cytosine-rich exons within GTPase-activating proteins (GAPs), negative regulators of RAS family members. Mutant p53-enforced GAP isoforms lose cell membrane association, leading to heightened KRAS activity. Preventing cytosine-rich exon inclusion in mutant KRAS/p53 PDACs decreases tumor growth. Moreover, mutant p53 PDACs are sensitized to inhibition of splicing via spliceosome inhibitors. These data provide insight into co-enrichment of KRAS and p53 mutations and therapeutics targeting this mechanism in PDAC.

Pancreatic cancer is one of the leading causes of cancer-related death, with 5-year survival of 8.5%. The lack of significant progress in improving therapy reflects our inability to overcome the desmoplastic stromal barrier in pancreatic ductal adenocarcinoma (PDAC) as well as a paucity of new approaches targeting its genetic underpinnings. RNA interference holds promise in targeting key mutations driving PDAC; however, a nucleic acid delivery vehicle that homes to PDAC and breaches the stroma does not yet exist. Noting that the cyclic peptide iRGD mediates tumor targeting and penetration through interactions with a v b 3/5 integrins and neuropilin-1, we hypothesized that "tandem" peptides combining a cell-penetrating peptide and iRGD can encapsulate siRNA to form tumor-penetrating nanocomplexes (TPN) capable of delivering siRNA to PDAC. The use of directly conjugated iRGD is justified by receptor expression patterns in human PDAC biopsies. In this work, we optimize iRGD TPNs with polyethylene glycol (PEG)peptide conjugates for systemic delivery to sites of disease. We show that TPNs effectively knockdown siRNA targets in PDAC cell lines and in an immunocompetent genetically engineered mouse model of PDAC. Furthermore, we validate their tumor-penetrating ability in three-dimensional organoids and autochthonous tumors. In murine therapeutic trials, TPNs delivering anti-Kras siRNA significantly delay tumor growth. Thus, iRGD TPNs hold promise in treating PDAC by not only overcoming physical barriers to therapy, but by leveraging the stroma to achieve knockdown of the gold-standard genetic target. Moreover, the modular construction of this delivery platform allows for facile adaptation to future genetic target candidates in pancreatic cancer., NCI (Grants P30-CA14051 and U54CA151884), NIH/NIGMS (Grant MSTP T32GM007753)

Angiogenesis is regulated by both soluble growth factors and cellular interactions with the extracellular matrix (ECM). While cell adhesion via integrins has been shown to be required for angiogenesis, the effects of quantitative changes in cell adhesion and spreading against the ECM remain less clear. Here, we show that angiogenic sprouting in natural and engineered three-dimensional matrices exhibited a biphasic response, with peak sprouting when adhesion to the matrix was limited to intermediate levels. Examining changes in global gene expression to determine a genetic basis for this response, we demonstrate a vascular endothelial growth factor (VEGF)-induced upregulation of genes associated with vascular invasion and remodeling when cell adhesion was limited, whereas cells on highly adhesive surfaces upregulated genes associated with proliferation. To explore a mechanistic basis for this effect, we turned to focal adhesion kinase (FAK), a central player in adhesion signaling previously implicated in angiogenesis, and its homologue, proline-rich tyrosine kinase 2 (Pyk2). While FAK signaling had some impact, our results suggested that Pyk2 can regulate both gene expression and endothelial sprouting through its enhanced activation by VEGF in limited adhesion contexts. We also demonstrate decreased sprouting of tissue explants from Pyk2-null mice as compared to wild type mice as further confirmation of the role of Pyk2 in angiogenic sprouting. These results suggest a surprising finding that limited cell adhesion can enhance endothelial responsiveness to VEGF and demonstrate a novel role for Pyk2 in the adhesive regulation of angiogenesis.

Focal adhesion kinase (FAK) transduces cell adhesion to the extracellular matrix into proliferative signals. We show that FAK overexpression induced proliferation in endothelial cells, which are normally growth arrested by limited adhesion. Interestingly, displacement of FAK from adhesions by using a FAK−/− cell line or by expressing the C-terminal fragment FRNK also caused an escape of adhesion-regulated growth arrest, suggesting dual positive and negative roles for FAK in growth regulation. Expressing kinase-dead FAK-Y397F in FAK−/− cells prevented uncontrolled growth, demonstrating the antiproliferative function of inactive FAK. Unlike FAK overexpression–induced growth, loss of growth control in FAK−/− or FRNK-expressing cells increased RhoA activity, cytoskeletal tension, and focal adhesion formation. ROCK inhibition rescued adhesion-dependent growth control in these cells, and expression of constitutively active RhoA or ROCK dysregulated growth. These findings demonstrate the ability of FAK to suppress and promote growth, and underscore the importance of multiple mechanisms, even from one molecule, to control cell proliferation.

A novel method to control three-dimensional cell cluster size and geometry using two-dimensional patterning techniques is described. Cells were first cultured on two-dimensional micropatterned collagen using conventional soft lithography techniques. Collagenase was used to degrade the micropatterned collagen and release cells from the micropatterns, forming clusters of cells which were then resuspended in a three-dimensional collagen matrix. This method facilitated the formation of uniformly sized clusters within a single sample. By systematically varying the geometry of the two-dimensional micropatterned islands, final cluster size and cell number in three dimensions could be controlled. Using this technique, we showed that proliferation of cells within collagen gels depended on the size of clusters, suggesting an important role for multicellular structure on biological function. Furthermore, by utilizing more complex two-dimensional patterns, non-spherical structures could be produced. This technique demonstrates a simple way to exploit two-dimensional micro-patterning in order to create complex and structured multicellular clusters in a three-dimensional environment.