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, 37 (32), 4372-4384

A Novel Three-Dimensional High-Throughput Screening Approach Identifies Inducers of a Mutant KRAS Selective Lethal Phenotype

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A Novel Three-Dimensional High-Throughput Screening Approach Identifies Inducers of a Mutant KRAS Selective Lethal Phenotype

Smitha Kota et al. Oncogene.

Abstract

The RAS proteins are the most frequently mutated oncogenes in cancer, with highest frequency found in pancreatic, lung, and colon tumors. Moreover, the activity of RAS is required for the proliferation and/or survival of these tumor cells and thus represents a high-value target for therapeutic development. Direct targeting of RAS has proven challenging for multiple reasons stemming from the biology of the protein, the complexity of downstream effector pathways and upstream regulatory networks. Thus, significant efforts have been directed at identifying downstream targets on which RAS is dependent. These efforts have proven challenging, in part due to confounding factors such as reliance on two-dimensional adherent monolayer cell cultures that inadequately recapitulate the physiologic context to which cells are exposed in vivo. To overcome these issues, we implemented a high-throughput screening (HTS) approach using a spheroid-based 3-dimensional culture format, thought to more closely reflect conditions experienced by cells in vivo. Using isogenic cell pairs, differing in the status of KRAS, we identified Proscillaridin A as a selective inhibitor of cells harboring the oncogenic KRasG12V allele. Significantly, the identification of Proscillaridin A was facilitated by the 3D screening platform and would not have been discovered employing standard 2D culturing methods.

Conflict of interest statement

Conflict of interest declaration

The authors declare no conflicts of interest with respect to the research, authorship, and/or publication of this article.

Figures

Figure 1
Figure 1. Characterization of the BxPC-3 isogenic cell pair
(A) Analysis of KRAS expression levels in BxPC-3-KRASG12V and BxPC-3-KRASWT stable cell lines. Individual clones were isolated and evaluated for the expression of KRAS by western blotting analysis using anti-KRAS or anti-Vinculin (loading control) antibodies. (B) Analysis of KRAS expression levels in selected BxPC-3-KRASG12V and BxPC-3-KRASWT stable cell lines and BxPC-3-parental cell line. Confirmation of spheroidicity of (C) BxPC-3-KRASWT or (D) BxPC-3-KRASG12V cells by confocal imaging. Z-stack images were taken at 10 μm increments from the equator of Hoechst-stained spheroids of BxPC-3-KRASG12V and BxPC-3-KRASWT on a GE IN Cell 6000 Analyzer (10× objective, f=1.18AU). Maximum intensity projection along the z-axis of the 12 individual planes aligned in Image J to generate an intensity projection biased by color scale are shown in the left panel. (E–F) Determination of cell viability assay conditions using CellTiter-Glo 3D (CTG3D). BxPC-3 cells were seeded at increasing numbers in a 384-well spheroid plate, grown for 24 hours and treated with CTG3D to assess viability. Relative luminescence of cells was determined at 48 hours post-seeding, using a ViewLux microplate imager (PerkinElmer). Error Bars = S.D. The data shown represent the mean of 3 independent replicates with triplicate data points.
Figure 2
Figure 2. Spectrum Library Screen of BxPC-3-KRASG12V and BxPC-3-KRASWT cells in 3D and 2D formats
(A–B) 2400 compounds from the Spectrum Library were screened in duplicate on 3D against BxPC-3-KRASG12V to validate the 3D assay. (A) The activity of the compounds was plotted (duplicate data but showing single point percent response), with high control, low control and hit cutoff (dashed line) shown. (B) Correlation plot for the two replicate screening datasets. (C–F) Activity of 2,400 compounds on BxPC-3-KRASWT and BxPC-3-KRASG12V cells in 3D and 2D formats (singlicate showing single point percent response along with high control, low control and hit cutoff shown): (C) 3D format BxPC-3-KRASG12V, (D) 3D format BxPC-3-KRASWT, (E) 2D format BxPC-3-KRASG12V, (F) 2D BxPC-3 KRASWT.
Figure 3
Figure 3. Comparison of performance of compounds in BxPC-3-KRASG12V and BxPC-3-KRASWT cells in 3D and 2D formats
Primary screening results of Spectrum library against BxPC-3-KRASWT and BxPC-3-KRASG12V in 3D and 2D formats. (A) Four-way Venn diagram of active compounds identified from the four screens. A hit was identified as any compound with % inhibition > the corresponding screen hit cutoff. The numbers in parentheses are the numbers of hits specific for that cell line. The numbers in the boxes represent the number of compounds found to active in those overlapping assays. (B–E) Correlation plots of the % inhibition values of compounds in each of the screens: (B) BxPC-3-KRASWT, 2D vs. 3D. (C) BxPC-3-KRASG12V, 2D vs. 3D. (D) BxPC-3-KRASG12V vs. BxPC-3-KRASWT, 2D. (E) BxPC-3-KRASG12V vs. BxPC-3-KRASWT, 3D.
Figure 4
Figure 4. Validation of specificity of select inhibitors toward KRAS mutant cells
(A) Top 15 compounds from the Spectrum library screen were analyzed at 12.4 μM in triplicate against BxPC-3-KRASG12V and BxPC-3-KRASWT cells in 3D format. Statistical significance was determined by unpaired t-test. NS = Non significant, * = p < 0.001, ** = p < 0.0001. (B–D) Concentration-response curves of Proscillaridrin A (SR-841251) on different 3D cell models: (B) BxPC-3-KRASG12V and BxPC-3-KRASWT; (C) Pancreatic ductal adenocarcinoma cell lines AspC1, HPAF11, and PANC-1. (D) Parental BxPC3 or hTERT-HPNE-E6/E7 immortalized pancreatic ductal cells. The data shown represent the mean of 3 independent experiments with triplicate data points in each. Error bars = S.D.
Figure 5
Figure 5. Characterization of select inhibitors in 2D and 3D formats
(A–B) Validation and specificity of select hits towards KRAS mutant cells in 3D (A) or 2D (B) formats. The top seven compounds from the Spectrum library screen were analyzed at ~ 12.4 μM in triplicate on BxPC-3-KRASG12V and BxPC-3-KRASWT cells in 3D and 2D formats. (C–D) Evaluation of BxPC-3-KRASG12V and BxPC-3-KRASWT cell growth rates in 3D (C) and 2D (D) formats. Cells were plated at 2500 cells/well in 3D and 2D formats and the growth rate was evaluated at 24, 48, and 72 hour time points using CTG3D or CTG, respectively. Statistical significance was determined by unpaired t-test. NS = Non significant, * = p < 0.001, ** = p < 0.0001. The data shown represent the mean of 3 independent experiments with triplicate data points in each. Error bars = S.D. (E–F) Effects of Proscillaridin A on apoptosis of BxPC-3-KRASG12V and BxPC-3-KRASWT cells. Proscillaridin A (PA) was tested at 12.4 μM against BxPC-3-KRASG12V and BxPC-3-KRASWT in 3D (E) and 2D (F) formats, and monitored at different treatment time points by RT-Glo Annexin V. Statistical significance was determined by unpaired t-test. All points represent the mean of 8 independent replicates. Error bars = S.D., *P <0.05.
Figure 6
Figure 6. Assessing the Na+/K+-ATPase as the potential target of Proscillaridin A
(A–D) BxPC-3-KRASG12V and BxPC-3-KRASWT were transfected with siRNA targeting ATP1A1 or control siRNA and knockdown of the ATP1A1 subunit was confirmed by western blotting in cells grown in (A) 3D format or (B) 2D format. Viability of BxPC-3-KRASG12V and BxPC-3-KRASWT cells transfected with siRNA targeting ATP1A1 or control siRNA grown in grown in (C) 3D format or (D) 2D format was determined at 48 hours post-transfection using CTG3D or CTG, respectively. Statistical significance was determined by unpaired t-test. NS = Non significant, ** = p < 0.0001. The data shown represent the mean of 3 independent experiments with triplicate data points in each. Error bars = S.D. Levels of (E) pAKT, AKT and (F) pERK1/2, ERK1/2 were determined in BxPC-3-KRASG12V and BxPC-3-KRASWT cells grown in 3D format. Vinculin was used as a loading control.

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