Aurora Cabrera

Pictilisib treatment impairs growth and PI3K signaling in PDAC cell lines

Pancreatic ductal adenocarcinoma (PDAC) is a highly KRAS-addicted cancer. Although KRAS-mutant PDAC is susceptible to treatment with direct RAS inhibitors, drug resistance is an ongoing challenge. KRAS-driven PDAC is dependent on nutrient scavenging pathways such as macropinocytosis to fuel increased metabolic demands. Here, we report on the dynamics of macropinocytosis in response to genetic suppression and pharmacologic inhibition of KRAS and the RAS–ERK mitogen-activated protein kinase pathway. We show that macropinocytosis is transiently suppressed following acute genetic suppression of KRAS but is rapidly restored and even enhanced under prolonged RAS pathway inhibition. Furthermore, we demonstrate that human PDAC cell lines that have acquired resistance to RAS inhibitors, as well as cell lines derived from KPC tumors with acquired resistance to RAS inhibitors display markedly elevated macropinocytosis. RAS-inhibitor resistant cell lines also display increased abundance of macropinocytosis-related proteins and enhanced albumin uptake and sensitivity to albumin-bound paclitaxel. Mechanistically, this adaptive phenotype is mediated through both phosphoinositide 3-kinase (PI3K)-dependent and -independent pathways that converge on the activation of RAC1. Our results demonstrate a previously unrecognized link between RAS pathway inhibitor resistance and upregulation of macropinocytosis, underscoring the importance of further elucidating metabolic reprogramming in RASi-R PDAC.Significance:Macropinocytosis is upregulated following prolonged RAS-inhibitor treatment and in RASi-R models of PDAC via several heterogeneous mechanisms that converge on RAC1 activation and render RASi-R PDAC cell lines more sensitive to nab-paclitaxel.

Pictilisib (PI3Ki) treatment decreases PI3K-mediated signaling in PDAC cell lines

Downregulation of macropinocytosis is not sustained following genetic loss of KRAS in KRAS-mutant PDAC cell lines. A, Representative images of macropinosomes labeled with FITC–dextran (green) and nuclear DAPI stain (blue) in indicated KRAS-mutant PDAC cells transiently transfected with an siRNA oligonucleotide (10 nmol/L) against KRAS (siKRAS) or a nonspecific control (siNS) for indicated time points. Images are representative of 10 fields of view analyzed in each of three independent experiments. Scale bars, 20 μm. B, Quantification of (A), in which the total area of macropinosomes (FITC–dextran immunofluorescence) was calculated and normalized to cell number [macropinocytic (MP) index]. The relative MP index is plotted, with each individual data point representing one field containing at least 10 analyzed cells. Data for MIA PaCa-2, Pa14C, and Pa16C are presented as the mean ± SD of one experiment that is representative of three independent experiments. *, ρ < 0.05 and ****, ρ < 0.0001, by the unpaired Student t test, comparing against NS. C, Immunoblots of indicated KRAS-mutant PDAC cell lines treated with siNS and siKRAS as in A. Vinculin levels were used to monitor equivalent total protein loading. Blots are representative of three independent experiments. D, Macropinocytosis was measured via flow cytometry in KRAS-mutant PDAC cell lines that were treated with siRNA oligonucleotides as in A. Macropinocytosis was quantified via TMR–dextran labeling. Data are presented as the mean ± SEM of three independent experiments. *, ρ < 0.05; **, ρ < 0.01; ***, ρ < 0.001, and ****, ρ < 0.0001, by the unpaired Student t test, comparing against NS. Dex, dextran; MFI, median fluorescence intensity; ns, not significant.

KRAS suppression results in a transient reduction in macropinocytosis

MEK inhibitor treatment enhances the sensitivity of PDAC cell lines to albumin-bound nab-paclitaxel but not free paclitaxel

Upregulated macropinocytosis in RAS inhibitor-resistant cell lines is associated with increased uptake of and sensitivity to albumin-bound paclitaxel compared with free drug. A, Schematic diagram highlighting the differential routes through which free paclitaxel and albumin-bound nab-paclitaxel enter the cell. B, Representative images of macropinosomes labeled with FITC-BSA (green) and nuclear DAPI stain (blue) in indicated parental or MRTX1133- (G12Di-R) or RMC7797-resistant (RASi-R) KRAS-mutant PDAC cells. Images are representative of 10 fields of view analyzed in each of two independent experiments. Scale bars, 20 μm. C, Quantification of median intensity of TMR-BSA fluorescence in indicated MRTX1133- (G12Di-R) or RMC7797-resistant (RASi-R) KRAS-mutant PDAC cell lines plotted relative to matched parental cell line, with each individual data point representing one field containing at least 10 analyzed cells. Data are presented as the mean ± SEM of three independent experiments. ***, ρ < 0.001, and ****, ρ < 0.0001, by the unpaired Student t test, comparing against the parental cell line. D, Representative images of clonogenic growth assays following treatment of matched parental and either MRTX1133- (G12Di-R) or RMC7977-resistant (RASi-R) PANC-1 or HPAC cell lines treated with nab-paclitaxel for 12–16 days. E, Relative colony number of matched parental and either MRTX1133- (G12Di-R) or RMC7977-resistant (RASi-R) PDAC cell lines treated with nab-paclitaxel (left) or paclitaxel (right) for 12–16 days. Data are presented as the mean ± SEM of three independent experiments. F, GI50 values from data plotted in (E) for nab-paclitaxel treated lines (top) and paclitaxel treated lines (bottom). *, ρ < 0.05; **, ρ < 0.01; ***, ρ < 0.001, by the unpaired Student t test, comparing against the parental cell line. [A, Created in BioRender. Robb, R. (2026) https://BioRender.com/nyleshf.]

Antibodies used for RPPA analysis.

PDAC cell lines with acquired resistance to pharmacologic inhibitors of RAS exhibit enhanced macropinocytosis. A, Schematic diagram showing the experimental design for generating cell lines with acquired resistance to MRTX1133 (G12Di) and RMC7797 (RASi). B, Representative images of macropinosomes labeled with FITC–dextran (green) and nuclear DAPI stain (blue) in indicated parental or MRTX849- (G12Ci-R), MRTX1133- (G12Di-R), or RMC7797-resistant (RASi-R) KRAS-mutant PDAC cells. Images are representative of 10 fields of view analyzed in each of three independent experiments. Scale bars, 20 μm. C, Quantification of relative macropinocytic (MP) index, with each individual data point representing one field containing at least 10 analyzed cells. Data are presented as the mean ± SD of one experiment that is representative of three independent experiments. ****, ρ < 0.0001, by the unpaired Student t test, comparing against the respective parental cell line. D, Macropinocytic uptake of TMR–dextran was measured by MFI via flow cytometry in indicated parental or MRTX849- (G12Ci-R), MRTX1133- (G12Di-R), or RMC7797-resistant (RASi-R) KRAS-mutant PDAC cell lines. Data are presented as the mean ± SEM of three independent experiments. **, ρ < 0.01 and ***, ρ < 0.001 by the unpaired Student t test, comparing against the respective parental cell line. E, Representative images of macropinosomes labeled with FITC–dextran (green) and nuclear DAPI stain (blue) in indicated treatment-naïve (naïve) or RMC7797-resistant (resistant) tumor-derived KPC cells. Images are representative of 10 fields of view analyzed in each of three independent experiments. Scale bars, 20 μm. F, Macropinocytic uptake of TMR–dextran was measured by MFI via flow cytometry in indicated treatment-naïve (naïve) or RMC7797-resistant (resistant) tumor-derived KPC cell lines. Data are presented as the mean ± SEM of three independent experiments. **, ρ < 0.01 by nested two-way ANOVA. G, Relative levels of selected macropinocytosis-related proteins in PANC-1 cells following 24 or 168 hours of RASi treatment, as well as in a RASi-R cell line. Line color represents the direction of change from 24 hours RASi treatment to RASi resistance, and line transparency represents the magnitude of change. Dex, dextran; MFI, median fluorescence intensity. [A, Created in BioRender. Robb, R. (2026) https://BioRender.com/rx7i90i.]

A subset of RASi-resistant PDAC cell lines activates YAP and/or TAZ to upregulate macropinocytosis