SUMMARY Prognostically relevant RNA expression states exist in pancreatic ductal adenocarcinoma (PDAC), but our understanding of their drivers, stability, and relationship to therapeutic response is limited. To examine these attributes systematically, we profiled metastatic biopsies and matched organoid models at single-cell resolution. In vivo , we identify a new intermediate PDAC transcriptional cell state and uncover distinct site- and state-specific tumor microenvironments (TMEs). Benchmarking models against this reference map, we reveal strong culture-specific biases in cancer cell transcriptional state representation driven by altered TME signals. We restore expression state heterogeneity by adding back in vivo -relevant factors and show plasticity in culture models. Further, we prove that non-genetic modulation of cell state can strongly influence drug responses, uncovering state-specific vulnerabilities. This work provides a broadly applicable framework for aligning cell states across in vivo and ex vivo settings, identifying drivers of transcriptional plasticity and manipulating cell state to target associated vulnerabilities.

ATP-competitive fi broblast 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 effi cacy in 4 patients with FGFR 2 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 profi les observed clinically for each inhibitor. Our fi ndings suggest that strategic sequencing of FGFR inhibitors, guided by serial biopsy and ctDNA analysis, may prolong the duration of benefi t from FGFR inhibition in patients with FGFR2 fusion-positive ICC. SIGNIFICANCE: ATP-competitive FGFR inhibitors (BGJ398, Debio 1347) show effi cacy 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 benefi t in patients with resistance to BGJ398 or Debio 1347 and overcomes several FGFR2 mutations in ICC models.

consults and holds stock in Ideaya, and cofounded and holds stock in Cedilla Therapeutics. G.G. receives research funding from IBM and Pharmacyclics and is an inventor on multiple patent applications related to bioinformatic tools, including applications related to MuTect, ABSOLUTE, MSMuTect, MSMutSig and MSIClass. Y.E.M. is an inventor on patent applications related to the bioinformatic tools, MSMuTect, MSMutSig and MSIClass. The Broad Institute filed a US patent application related to the target described in this manuscript.

Objective To evaluate the anti-microbial effects of photodynamic therapy (PDT) on infected human teeth ex vivo. Materials and Methods Fifty-two freshly extracted teeth with pulpal necrosis and associated periradicular radiolucencies were obtained from 34 subjects. Twenty-six teeth with 49 canals received chemomechanical debridement (CMD) with 6% NaOCl and twenty-six teeth with 52 canals received CMD plus PDT. For PDT, root canal systems were incubated with methylene blue (MB) at concentration of 50 µg/ml for 5 minutes followed by exposure to red light at 665 nm with an energy fluence of 30 J/cm2. The contents of root canals were sampled by flushing the canals at baseline and following CMD alone or CMD+PDT and were serially diluted and cultured on blood agar. Survival fractions were calculated by counting colony-forming units (CFU). Partial characterization of root canal species at baseline and following CMD alone or CMD+PDT was performed using DNA probes to a panel of 39 endodontic species in the checkerboard assay. Results The Mantel-Haenszel chi-square test for treatment effects demonstrated the better performance of CMD+PDT over CMD (P=0.026). CMD+PDT significantly reduced the frequency of positive canals relative to CMD alone (P=0.0003). Following CMD+PDT, 45 of 52 canals (86.5%) had no CFU as compared to 24 of 49 canals (49%) treated with CMD (canal flush samples). The CFU reductions were similar when teeth or canals were treated as independent entities. Post-treatment detection levels for all species were markedly lower for canals treated by CMD+PDT than were for those treated by CMD alone. Bacterial species within dentinal tubules were detected in 17/22 (77.3%) and 15/29 (51.7%) of canals in the CMD and CMD+PDT group, respectively (P= 0.034). Conclusion Data indicate that PDT significantly reduces residual bacteria within the root canal system, and that PDT, if further enhanced by technical improvements, holds substantial promise as an adjunct to CMD.

Allele-specific signaling by different KRAS alleles remains poorly understood. The KRAS G12R mutation displays uneven prevalence among cancers that harbor the highest occurrence of KRAS mutations: it is rare in lung and colorectal cancers (~1%), yet relatively common (~20%) in pancreatic ductal adenocarcinoma (PDAC), suggesting context-specific properties. We evaluated whether KRAS G12R is functionally distinct from the more common KRAS G12D or KRAS G12V mutant proteins (KRAS G12D/V ). We found that KRAS G12D/V but not KRAS G12R drives macropinocytosis and that MYC is essential for macropinocytosis in KRAS G12D/V but not KRAS G12R -mutant PDAC. Surprisingly, we found that KRAS G12R is defective for interaction with a key effector, p110α phosphoinositide 3-kinase (PI3Kα), due to structural perturbations in switch II. Instead, upregulated KRAS-independent PI3Kγ activity was able to support macropinocytosis in KRAS G12R -mutant PDAC. Finally, we determined that KRAS G12R -mutant PDAC displayed a distinct drug sensitivity profile compared with KRAS G12D -mutant PDAC but is still responsive to the combined inhibition of ERK and autophagy.Hobbs et al.

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 leveraged chemoproteomic profiling and structure-based design to develop the first 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.

Graphical Abstract Highlights d RTK-RAS-MAPK pathway members score strongly in genome-scale MEKi modifier screens d Depletion of SHOC2 potently sensitizes RAS-driven cells to MEK inhibition d SHOC2 loss impairs RTK-mediated adaptive reactivation of MAPK signaling induced by MEKi d A model of SHOC2 degradation suggests a combination therapeutic strategy with MEKi 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. 118 Cell Reports 29, 118-134, October 1, 2019 ª 2019 The Author(s). This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).(A) Schematic of pooled CRISPR-Cas9 screening strategy. (B-D) Genome-scale screen results in pancreatic cancer, CFPAC-1 (B), and lung cancer lines, A549 (C) and NCI-H23 (D). Red points have FDR < 0.25 (STARS algorithm). Mean trametinib sensitivity (x axis) is calculated as the difference in the log2(fold-change) in sgRNA representation between cells treated with trametinib for 14 days and the initial pool of sgRNAs. Differential sensitivity indicates the difference log2(fold-change) in sgRNA representation between the trametinib-treated and DMSO-treated arms of the screen. Scores represent the average of all guides for a given gene. (E) Venn diagram summarizes the overlap of genes that are depleted in all three screens with an FDR < 0.25.

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