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. 2023 Dec 11;27(1):108711.
doi: 10.1016/j.isci.2023.108711. eCollection 2024 Jan 19.

In situ modeling of acquired resistance to RTK/RAS-pathway-targeted therapies

Nancy E Sealover  1 , Patricia T Theard  1 , Jacob M Hughes  1 , Amanda J Linke  1 , Brianna R Daley  1 , Robert L Kortum  1
Affiliations

In situ modeling of acquired resistance to RTK/RAS-pathway-targeted therapies

Nancy E Sealover et al. iScience. .

Abstract

Intrinsic and acquired resistance limit the window of effectiveness for oncogene-targeted cancer therapies. Here, we describe an in situ resistance assay (ISRA) that reliably models acquired resistance to RTK/RAS-pathway-targeted therapies across cell lines. Using osimertinib resistance in EGFR-mutated lung adenocarcinoma (LUAD) as a model system, we show that acquired osimertinib resistance can be significantly delayed by inhibition of proximal RTK signaling using SHP2 inhibitors. Isolated osimertinib-resistant populations required SHP2 inhibition to resensitize cells to osimertinib and reduce MAPK signaling to block the effects of enhanced activation of multiple parallel RTKs. We additionally modeled resistance to targeted therapies including the KRASG12C inhibitors adagrasib and sotorasib, the MEK inhibitor trametinib, and the farnesyl transferase inhibitor tipifarnib. These studies highlight the tractability of in situ resistance assays to model acquired resistance to targeted therapies and provide a framework for assessing the extent to which synergistic drug combinations can target acquired drug resistance.

Keywords: Biochemistry; Biochemistry methods; Cancer; Cell biology.

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Conflict of interest statement

The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
Modeling osimertinib resistance in situ (A and B) Dose-response curves (A) and EC50–EC90 values (B) for the indicated EGFR-mutated LUAD cell lines treated with osimertinib under anchorage-dependent conditions. Data in (A) are mean ± SD from n = 3 independent experiments. (C) EGFR-mutated cell lines were plated at low density (250 cells/well) in replicate 96-well plates, and each plate was treated with the indicated dose of osimertinib. Wells were fed and assessed weekly for outgrowth; wells that were >50% confluent were scored as resistant to the given dose of osimertinib. Data are plotted as a Kaplan-Meyer survival curve. Plates treated with ≥ the EC80 osimertinib dose [see bold values in (B)] showed an extended inhibition of growth followed by outgrowth of individual colonies consistent with true drug resistance. Data in (C) are combined from n = 3 independent trials.
Figure 2
Figure 2
SHP2 inhibition limits the development of osimertinib resistance EGFR-mutated H1975 and PC9 cells were plated at low density (250 cells/well) in replicate 96-well plates, and each plate was either left untreated (black, dashed) or treated with osimertinib alone at 150 nM (dark gray) or 300 nM (black), a SHP2 inhibitor alone (RMC-4550 at 300 nM or SHP099 at 1 mM) (purple), or the combination of osimertinib + RMC-4550 or SHP099 (red). Wells were fed and assessed weekly for outgrowth; wells that were >50% confluent were scored as resistant to the given dose of osimertinib. Data are plotted as a Kaplan-Meyer survival curve. ∗∗∗p < 0.001 vs. osimertinib drug treatment. Data are combined from n = 3 independent trials.
Figure 3
Figure 3
Isolated osimertinib-resistant clones maintain osimertinib resistance upon continued culture (A) Dose-response curves for osimertinib in parental H1975 cells (black) and osimertinib-resistant H1975 populations (gray) isolated from osimertinib resistance assays. Data in (A) are mean ± SD from n = 3 independent experiments. (B) H1975 parental (black) or osimertinib-resistant populations (gray) were plated at low density (250 cells/well) in replicate 96-well plates; each plate was either left untreated (dashed line) or treated with 150 nM osimertinib (solid line). Wells were fed and assessed weekly for outgrowth; wells that were >50% confluent were scored as osimertinib resistant. Data are plotted as a Kaplan-Meyer survival curve. Data in (B) are combined from n = 3 independent trials.
Figure 4
Figure 4
Osimertinib-resistant populations show hyperactivation of multiple RTKs and are sensitive to SHP2 inhibition (A) Quantitation of tyrosine phosphorylation of the indicated RTKs in whole-cell lysates from osimertinib-resistant H1975 populations OR1-8 relative to parental controls; samples were assessed for RTK phosphorylation using a human RTK phosphorylation antibody array (RayBiotech, complete array data in Figure S2). Data are mean ± SD from n = 3 replicate wells of the RTK array. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001 vs. parental H1975 cells. (B–D) Quantitation of the change in % osimertinib efficacy (B, βobs), fold change in osimertinib potency (C, log α2), and a plot showing the change in osimertinib potency (log α2) versus efficacy (βobs) (D) from dose-response experiments assessing combined treatment of parental H1975 cells or OR1-8 populations with osimertinib +/− the indicated second RTK or RTK/RAS pathway inhibitor from Figure S4. For (D), cells in the upper right quadrant (positive log α2 and positive βobs) represent the most promising osimertinib combination for a given cell population. Data in (B) and (C) are shown as the mean and 95% confidence interval, and data in (D) are the mean from n = 3 independent experiments. (E–G) Quantitation of the change in % osimertinib efficacy (E, βobs), fold change in osimertinib potency (F, log α2), and a plot showing the change in osimertinib potency (log α2) versus efficacy (βobs) (G) from dose-response experiments assessing combined treatment of parental H1975 cells or OR1-8 populations with osimertinib +/− the SHP2 inhibitor RMC-4550. Data in E and F are shown as the mean and 95% confidence interval, and data in G are the mean from n = 3 independent experiments. (H) Parental and osimertinib-resistant H1975 populations were treated with a matrix of doses of osimertinib +/− the SHP2 inhibitor RMC-4550 for four days, and cell viability was assessed using CellTitre glo. Excess over Bliss was calculated for each dose combination as a measure of synergy. Data are the mean from n = 3 independent experiments. (I) Schematic showing hyperactivation of multiple RTKs driving acquired osimertinib resistance. Inhibition of proximal RTK signaling using a SHP2 inhibitor, but not individual RTK inhibitors, synergizes with osimertinib by enhancing osimertinib efficacy and potency. (J) Western blots for pERK, ERK, HSP90, and tubulin from whole-cell lysates of parental and osimertinib-resistant H1975 populations (OR2, OR3, OR5, and OR6) left untreated or treated with 300 nM or 1000 nM osimertinib ±300 nM RMC-4550. Western blots are representative of n = 2 independent experiments.
Figure 5
Figure 5
The in situ resistance assay can be used to model resistance to RTK/RAS-pathway-targeted therapies (A–D) KRASG12C-mutated H358 and H1373 cells (A and B), KRASG12C-mutated H727 and NF1-LOF H1838 cells (C), or HRAS-mutated H1915 and SMSCTR cells were plated at low density (250 cells/well) in replicate 96-well plates, and each plate was either left untreated or treated with the indicated dose of sotorasib (A), adagrasib (B), trametinib (C), or tipifarnib (D). Wells were fed and assessed weekly for outgrowth; wells that were >50% confluent were scored as resistant to the given dose of osimertinib-resistant. Data are plotted as a Kaplan-Meyer survival curve and are combined from n = 3 independent trials. Plates treated with ≥ the EC80 dose of a given targeted inhibitor (see bold values in E) showed an extended inhibition of growth followed by outgrowth of individual colonies consistent with true drug resistance. (E) EC50–EC90 values from dose-response curves assessing sotorasib (H358, H1373), adagrasib (H358, H1373), trametinib (H727, H1838), or tipifarnib (H1915, SMSCTR) sensitivity. Sotorasib, adagrasib, and trametinib sensitivities were assessed in 3D cultured spheroids; tipifarnib sensitivity was assessed in 2D adherent culture.
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