Journal: Cancer Research

Article Title: CRISPR-Cas9 Screening Identifies Resistance Mechanisms to KRAS Inhibition in Pancreatic Cancer

doi: 10.1158/0008-5472.CAN-25-1835

Figure Lengend Snippet: RMC-7977 synergizes with erlotinib in a subset of PDAC cell lines. A, KRAS-mutant PDAC cell lines were treated with RMC-7977 and erlotinib, and proliferation assays were performed after 72 hours of treatment; axis values are in nM. B, PDAC cell lines were treated with RMC-7977 for 24 hours, and ERK activity was evaluated. C and D, KRAS Q61H - and KRAS G12D -mutant PDAC cell lines were treated with BI-2865 ( C ) or AMG 410 ( D ), and proliferation assays were performed. Error bars, ±SD. A one-tailed Student t test was performed. *, P < 0.05; ****, P < 0.0001; ns, nonsignificant, 0.0587.

Article Snippet: PDM-36 PDAC organoids were purchased from the ATCC.

Techniques: Mutagenesis, Activity Assay, One-tailed Test

Journal: Cancer Research

Article Title: CRISPR-Cas9 Screening Identifies Resistance Mechanisms to KRAS Inhibition in Pancreatic Cancer

doi: 10.1158/0008-5472.CAN-25-1835

Figure Lengend Snippet: Q61H-mutant PDAC cell lines exhibit rapid SOS1-driven pERK rebound. A, KRAS Q61H -mutant PDAC cell lines were treated with RMC-7977, and ERK activity/rebound was evaluated. B, PDAC cell lines were treated with RMC-7977 for 2 hours (T3M4) or 4 hours (Pa02C, Pa14C) or MRTX1133 for 4 hours, and active RAS levels were assessed with RAF-RBD pull-downs (PD). C, KRAS Q61H -mutant PDAC cell lines stably expressing shRNAs targeting SOS1 (or a nonspecific control) were treated with RMC-7977, and RAS-GTP levels were assessed with RAF-RBD pull-downs. D and E, HRAS, NRAS, the HRAS/NRAS, and SOS1 were genetically suppressed with shRNA in KRAS Q61H -mutant PDAC cells ( D ), and proliferation assays ( E ) were performed after 72 hours of treatment. Error bars, ±SD. A two-tailed Student t test was performed. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. F and G, T3M4 ( F ) or Pa14C ( G ) cells expressing shRNAs targeting HRAS, NRAS, HRAS/NRAS, and SOS1 were treated with a dose response of RMC-7977 for 72 hours, and proliferation assays were performed. Error bars, ±SD. WCL, whole-cell lysate.

Article Snippet: PDM-36 PDAC organoids were purchased from the ATCC.

Techniques: Mutagenesis, Activity Assay, Stable Transfection, Expressing, Control, shRNA, Two Tailed Test

Journal: Cancer Research

Article Title: CRISPR-Cas9 Screening Identifies Resistance Mechanisms to KRAS Inhibition in Pancreatic Cancer

doi: 10.1158/0008-5472.CAN-25-1835

Figure Lengend Snippet: RMC-7977 synergizes with erlotinib in KRAS Q61H -mutant PDAC and in PDAC cells with high EGFR signaling. A, Working model. B, T3M4 cells were treated with RMC-7977, erlotinib, or the combinations, and active ERK was assessed after 72 hours. C, RNA-seq was performed on a panel of PDAC cell lines, and EGFR expression was quantified and plotted for KRAS G12X lines that showed synergy with RMC-7977 + erlotinib vs. the other cell lines. A Mann–Whitney U test was performed. *, P = 0.0182. D, EGFR and ERK activity were assessed in a panel of PDAC cell lines. Red, KRAS G12X lines in which synergy was observed with RMC-7977 and erlotinib cotreatment. E, KRAS G12X synergistic cell lines were treated with RMC-7977, erlotinib, or the combinations, and active ERK was assessed after 72 hours.

Article Snippet: PDM-36 PDAC organoids were purchased from the ATCC.

Techniques: Mutagenesis, RNA Sequencing, Expressing, MANN-WHITNEY, Activity Assay

Journal: Cancer Research

Article Title: CRISPR-Cas9 Screening Identifies Resistance Mechanisms to KRAS Inhibition in Pancreatic Cancer

doi: 10.1158/0008-5472.CAN-25-1835

Figure Lengend Snippet: Pharmacologic validation of novel hits. A–C, KRAS-mutant PDAC cell lines were treated with MRTX1133 and SGC-CK2-1 ( A ), RMC-7977 and SGC-CK2-1 ( B ), or RMC-7977 and the CK2i silmitasertib ( C ), and proliferation assays were performed after 72 hours; axis values are in nmol/L. D, Pa14C cells were treated with 5 μmol/L silmitasertib for 16 hours, 10 μg/mL cycloheximide (CHX) was added, and MYC protein was assessed. Data are representative of two independent biological replicates, and protein half-life was determined by averaging the replicates. E, HPAC cells expressing shRNAs targeting LEF1 were treated with RMC-7977 and SGC-CK2-1, and proliferation assays were performed after 72 hours; axis values are in nmol/L.

Article Snippet: PDM-36 PDAC organoids were purchased from the ATCC.

Techniques: Biomarker Discovery, Mutagenesis, Expressing

Journal: Cancer Research

Article Title: CRISPR-Cas9 Screening Identifies Resistance Mechanisms to KRAS Inhibition in Pancreatic Cancer

doi: 10.1158/0008-5472.CAN-25-1835

Figure Lengend Snippet: PDAC cell lines without RMC-7977 + erlotinib intrinsic synergy do not develop acquired resistance to erlotinib upon developing RMC-7977 resistance. A, Pa14C, Pa16C, and PANC-1 cells were treated with increasing doses of RMC-7977 for 10 weeks, followed by a dose-response assessment of RMC-7977, and proliferation assays were performed after 72 hours of treatment in the parental and RMC-7977–resistant (indicated by R affixed to the end of the cell line) cell lines. Error bars, ±SD. B, EGFR, ERK, Akt, and MYC were assessed in parental and RMC-7977–resistant cell lines at 4, 6, and 10 weeks. C, EGFR, ERK, and MYC were assessed in parental and MRTX1133-resistant cell lines (indicated by MR affixed to the end of the cell line) after 4 weeks. D, Parental and RMC-7977–resistant Pa14C, Pa16C, and PANC-1 cells were treated with a dose response of erlotinib for 72 hours. Error bars, ±SD. E, Parental and RMC-7977–resistant cell lines were assessed for active (GTP-bound) RAS levels with RAF-RBD pull-downs (PD). WCL, whole-cell lysate. F, Parental and RMC-7977–resistant Pa14C cells were imaged microscopically. G, Parental and RMC-7977–resistant cell lines were assessed for EMT markers. KRASi-resistant cells were cultured in the presence of KRASi for the duration of the experiments.

Article Snippet: PDM-36 PDAC organoids were purchased from the ATCC.

Techniques: Cell Culture

Journal: Cancer Research

Article Title: CRISPR-Cas9 Screening Identifies Resistance Mechanisms to KRAS Inhibition in Pancreatic Cancer

doi: 10.1158/0008-5472.CAN-25-1835

Figure Lengend Snippet: RMC-7977–resistant PDAC cell lines retain partial sensitivity to ERK MAPK inhibition. A and B, Parental and RMC-7977–resistant cell lines were treated with a dose response of LY3009120 ( A ) or SCH772984 ( B ) for 72 hours. Error bars, ±SD. C, Parental cell lines (black), RMC-7977–resistant cell lines (blue), RMC-7977–resistant cell lines with RMC-7977 removed (blue), RMC-7977–resistant cell lines with the compound removed for 30 days (green), and RMC-7977–resistant cell lines with the compound removed for 30 days and then rechallenged with RMC-7977 (purple) were plated, harvested each day, and counted for 5 days. D, Parental or RMC-7977–resistant cell lines with the compound removed for 30 days were rechallenged with DMSO or RMC-7977, and active ERK, MYC, and active Akt were assessed. E, Parental, RMC-7977–resistant, and RMC-7977–resistant Pa14C cells with the compound removed for 30 days were imaged microscopically. Resistant cells were treated with RMC-7977 for the duration of the experiments.

Article Snippet: PDM-36 PDAC organoids were purchased from the ATCC.

Techniques: Inhibition

Journal: Lab on a Chip

Article Title: Accelerated maturation of branched organoids confined in collagen droplets

doi: 10.1039/d5lc00287g

Figure Lengend Snippet: Branched organoids in large droplets display similar developmental stages to those in bulk. (a) PDAC organoids cultured in bulk display local heterogeneous expression levels of E-cadherin and F-actin that indicate lumen formation sites on day 13 ( n = 3 independent experiments). (b) PDAC organoids grown in large droplets display local heterogeneous expression levels of E-cadherin and F-actin, indicating lumen formation sites already on day 6 ( n = 3 independent experiments). (c) A fully formed lumen in a branched organoid grown in a large droplet on day 7 ( n = 3 independent experiments).

Article Snippet: For nuclei visualisation, PDAC organoids were incubated with SiRDNA (Spirochrome SPY650-DNA SC501) for a minimum of 1 h before imaging at 1–2 μg ml −1 .

Techniques: Cell Culture, Expressing

Journal: Lab on a Chip

Article Title: Accelerated maturation of branched organoids confined in collagen droplets

doi: 10.1039/d5lc00287g

Figure Lengend Snippet: PDAC organoids expression pattern in different growth conditions. (a) K -means clustering with five clusters ( k = 5) using 2000 genes with the most variable expression level between different growth conditions: droplets day 4, 7, 9 and bulk day 7, 9, 13 ( n = 4 individual experiments), FDR = 0.05. (b) GO biological processes enrichment analysis from cluster 3 for selected conditions (droplets day 4, 9 and bulk day 7, 13) highlights genes involved in MAPK signaling and apoptosis-related processes. (c) Gene expression heatmap (cluster 2) for selected genes regulating tube morphogenesis and cell migration. (d) Gene expression heatmap (cluster 1) of a subset of genes involved in lipid metabolism and small molecule metabolism. (e) Heat map for selected genes (cluster 4) involved in transcription regulation and anti-apoptotic pathways facilitating cancer survival.

Article Snippet: For nuclei visualisation, PDAC organoids were incubated with SiRDNA (Spirochrome SPY650-DNA SC501) for a minimum of 1 h before imaging at 1–2 μg ml −1 .

Techniques: Expressing, Gene Expression, Migration