Identification of New Vulnerabilities in Conjunctival Melanoma Using Image-Based High Content Drug Screening

Abstract

Recent evidence suggests that numerous similarities exist between the genomic landscapes of both conjunctival and cutaneous melanoma. Since alterations of several components of the MAP kinases, PI3K/mTOR, and cell cycle pathways have been reported in conjunctival melanoma, we decided to assess the sensitivity of conjunctival melanoma to targeted inhibition mostly of kinase inhibitors. A high content drug screening assay based on automated fluorescence microscopy was performed in three conjunctival melanoma cell lines with different genomic backgrounds with 489 kinase inhibitors and 53 other inhibitors. IC50 and apoptosis induction were respectively assessed for 53 and 48 compounds. The genomic background influenced the response to MAK and PI3K/mTOR inhibition, more specifically cell lines with BRAF ^V600E mutations were more sensitive to BRAF/MEK inhibition, while CRMM2 bearing the NRAS^Q61L mutation was more sensitive to PI3k/mTOR inhibition. All cell lines demonstrated sensitivity to cell cycle inhibition, being more pronounced in CRMM2, especially with polo-like inhibitors. Our data also revealed new vulnerabilities to Hsp90 and Src inhibition. This study demonstrates that the genomic background partially influences the response to targeted therapy and uncovers a large panel of potential vulnerabilities in conjunctival melanoma that may expand available options for the management of this tumor.

Keywords: Hsp90; MAPK pathway; PI3K/mTOR pathway; Tirbanibulin; aurora-kinase; conjunctival melanoma; cyclin dependent kinase; drug screening; kinase inhibitors; polo-like kinase.

Figure 1

Live/Dead viability/cytotoxicity assay screen. (A) Example images of the control conditions for all 3 conjunctival melanoma cell lines. Upper row: CRMM1; middle row: CRMM2; lower row: CM2005.1. Left: DMSO; Right: Gambogic Acid. All nuclei are stained blue with Hoechst 33342, live cells, green with Calcein AM, and dead nuclei red with Ethidium homodimer-1. Objective 4×/0.2. The full well area is contained in a single image per well. Scale bar 500 μm. (B) Full plate snapshot of the Z’ plate validating the assay for CRMM1 and CRMM2 cell lines. All images of the plate, acquired in the same conditions as in (A), are montaged together. (C) Image segmentation performed with CellProfiler. DMSO control image of CRMM1 cells stained as in (A) (top row), and with the overlays of segmented regions (bottom row). Cyan: all nuclei. Yellow: live Calcein AM-positive cells. Pink: dead Ethidium homodimer-1-positive nuclei. Right: zoomed crop of the area highlighted by a dashed rectangle. Scale bars 500 µm (left) and 100 µm (right). (D) Full plate snapshot example of one of the screen plates with CRMM1 cells, composed of 32 replicates of each of the controls and 320 screen compounds, montaged together after imaging as in (A).

Figure 2

Annexin V apoptosis assay screen. (A) Full plate snapshot example of the Annexin V screen with CM2005.1 cells, composed of 8 replicates of each of the controls and 80 screen compounds, montaged together. All nuclei are stained blue with Hoechst, apoptotic cells green with Annexin V, and dead nuclei red with Ethidium homodimer-1. Objective 10×/0.45. The snapshot is composed of a single field of view out of the 9 fields acquired per well. (B) Example images of the control conditions for CM2005.1 cells. Top: DMSO. Bottom: Staurosporine. Left panel: brightfield images. Middle panel: fluorescent images acquired as in A. Right panel: zoomed crop of the area highlighted by a dashed rectangle, with an overlay of image segmentation performed by CellProfiler. Brown: all nuclei. Yellow: Annexin V-positive apoptotic cells. Red: dead Ethidium homodimer-1 positive nuclei. Scale bar 200 µm.