Multiplex single-cell chemical genomics reveals the kinase dependence of the response to targeted therapy

Abstract

Chemical genetic screens are a powerful tool for exploring how cancer cells' response to drugs is shaped by their mutations, yet they lack a molecular view of the contribution of individual genes to the response to exposure. Here, we present sci-Plex-Gene-by-Environment (sci-Plex-GxE), a platform for combined single-cell genetic and chemical screening at scale. We highlight the advantages of large-scale, unbiased screening by defining the contribution of each of 522 human kinases to the response of glioblastoma to different drugs designed to abrogate signaling from the receptor tyrosine kinase pathway. In total, we probed 14,121 gene-by-environment combinations across 1,052,205 single-cell transcriptomes. We identify an expression signature characteristic of compensatory adaptive signaling regulated in a MEK/MAPK-dependent manner. Further analyses aimed at preventing adaptation revealed promising combination therapies, including dual MEK and CDC7/CDK9 or NF-κB inhibitors, as potent means of preventing transcriptional adaptation of glioblastoma to targeted therapy.

A heterogeneous pharmaco-transcriptomic landscape induced by targeting a single oncogenic kinase

“…However, we show that an inhibitor’s impact on viability is not always correlated with global transcriptional response, indicating that inhibitor responses extend beyond viability, consistent with previous chemical genomic profiling efforts ( 5 , 16 ). Moreover, we show that EGFRi differentially induce an adaptive resistance molecular response recently associated with MEK inhibition ( 31 ). The presence of an adaptive resistance may explain the disparity between promising preclinical results and broad clinical nonsuccess.…”

“…Relationship between the total number of differentially expressed genes as a function of EGFR inhibitor dose (quasi-poisson regression, Wald test FDR < 1%) for the specified EGFR inhibitor (top annotations) with the expression of the proliferation marker MKI67 across BT112 ( A ), BT228 ( B ) and BT333 ( C ) PDCLs. The scatter plots ( right of each heatmap) represent the Pearson correlation within a cell line of inhibitor impact on MKI67 (normalized beta coefficient from fitting a GLM of MKI67 as a function of dose) versus overall transcriptional changes (as total number of DEGs) D ) Heatmap of z-scored mean aggregate expression of genes previously associated with adaptation ( 31 ) to inhibition of the RTK pathway.…”

“…Given the minimal efficacy of EGFR inhibitors to treat GBM in the clinic, we next examined the expression of programs associated with acquired resistance to therapy. Recently, we identified an adaptive resistance signature across established GBM cell lines and glioma stem cell models in response to the inhibition of RTK signaling, most prominently by inhibition of MEK via trametinib ( 31 ). Across the EGFRi signatures from our pilot screen, we found that the shared EGFR signature ( Fig.…”

Building and analyzing metacells in single-cell genomics data

“…As single-cell sequencing becomes more affordable and widely used, both the number and the size of single-cell genomics datasets are growing exponentially 1,2 with no signs of slowing down. Current single-cell sequencing studies from tissues, organs and whole organisms can profile millions of cells [3][4][5][6][7][8][9] . Along with the development of integration algorithms, even larger atlases have been built, reaching tens of millions of cells 10…”

Prevalence of and gene regulatory constraints on transcriptional adaptation in single cells

“…Several mechanisms underpinning this plasticity have been proposed and more continue to be reported. These mechanisms-operational at multiple levels of biological organization-include protein feedback loops, adaptive mutations, and single-cell molecular variabilities [10][11][12][13][14][15][16] . One recently reported robustness mechanism is a type of transcriptional adaptation, wherein nonsense mutations, i.e., mutations that result in premature termination of protein synthesis, can remarkably trigger the transcription of related genes, including paralogs 14,15,[17][18][19] .…”