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Loss of Spindly sensitizes EML4-ALK v3 lung cancer cells to HSP90 inhibitors

ABSTRACT: Heat Shock Protein 90 inhibitors (HSP90i) have shown encouraging activity in EML4-ALK+ non-small cell lung cancer (NSCLC) patients but clinical responses have been heterogeneous. It has been suggested that distinct EML4-ALK variants may have a differential impact on the response to HSP90 inhibition. Here, we show that NSCLC cells harboring the most common EML4-ALK variant 1 (v1) or variant 3 (v3) are similarly sensitive to HSP90i. To discover new genetic alterations that could be involved in stratifying sensitivity, we performed a genome-wide CRISPR/Cas9 knockout screen and found that loss of Spindly increases the sensitivity of EML4-ALK v3, but not v1, NSCLC cells to low concentrations of HSP90i from three distinct chemical families. Upon loss of Spindly, prolonged exposure to low concentrations of HSP90i impairs chromosome congression and cellular fitness. Collectively, our data suggest that mutations leading to loss of Spindly in EML4-ALK v3 NSCLC patients may increase sensitivity to low doses of HSP90i.

ORGANISM(S): Homo sapiens

PROVIDER: GSE197655 | GEO | 2025/02/01

REPOSITORIES: GEO

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			Action	DRS
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	GSE197655_ProcessedData.xlsx	Xlsx

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Project description:Purpose: The goal of this study was to use enzyme activity as a proxy for profiling tumor progression and treatment response in an autochthonous mouse model of Alk-mutant non-small-cell lung cancer (NSCLC). The Eml4-Alk model was originally described in Maddalo et al., Nature 2014. Single-cell transcriptomic profiling of the Eml4-Alk NSCLC model was performed for unbiased discovery of the phenotypic landscape of Eml4-Alk and wild type mice. Methods: Single cell suspensions were prepared from excised lungs of Eml4-Alk and sex- and age-matched healthy, wild type C57/Bl6J mice. Suspensions were pooled from n=3 mice per condition, and then were enriched for cell viability and depleted for CD45+ cells. Single cells were processed using the 10X Genomics Single Cell 3’ platform using the Chromium Single Cell 3’ Library & Gel Bead Kit V2 kit (10X Genomics), per manufacturer’s protocol. Approximately 10,000 cells were loaded onto each channel and partitioned into Gel Beads in Emulsion (GEMs) in the 10x Chromium instrument. Following lysis of the captured cells, the released RNA was barcoded through reverse transcription in individual GEMs, and complementary DNA was generated and amplified. Libraries were constructed using a Single Cell 3’ Library and Gel Bead kit. The libraries were sequenced using an Illumina NovaSeq6000 sequencer on an Illumina NovaSeq SP flow cell. Results: Gene expression matrices were generated for each sample by the Cell Ranger (v.3.0.2) Pipeline coupled with mouse reference version GRCm38. The output filtered gene expression matrices were analyzed by Python software (v.3.9.0) with the scanpy package (v.1.7.2). For final analysis, genes expressed in at least three cells in the data and cells with > 200 genes detected were selected for further analyses, and low quality cells were removed based on number of total counts and percentage of mitochondrial genes expressed. Conclusions: This study provides the first (to the best of our knowledge) single-cell RNA-seq dataset of the Eml4-Alk autochthonous model of NSCLC.

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Loss of Spindly sensitizes EML4-ALK v3 lung cancer cells to HSP90 inhibitors

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