Abstract
Tumor heterogeneity is a primary cause of treatment failure and acquired resistance in cancer patients. Even in cancers driven by a single mutated oncogene, variability in response to targeted therapies is well known. The existence of additional genomic alterations among tumor cells can only partially explain this variability. As such, nongenetic factors are increasingly seen as critical contributors to tumor relapse and acquired resistance in cancer. Here, we show that both genetic and nongenetic factors contribute to targeted drug response variability in an experimental model of tumor heterogeneity. We observe significant variability to epidermal growth factor receptor (EGFR) inhibition among and within multiple versions and clonal sublines of PC9, a commonly used EGFR mutant nonsmall cell lung cancer (NSCLC) cell line. We resolve genetic, epigenetic, and stochastic components of this variability using a theoretical framework in which distinct genetic states give rise to multiple epigenetic "basins of attraction," across which cells can transition driven by stochastic noise. Using mutational impact analysis, single-cell differential gene expression, and correlations among Gene Ontology (GO) terms to connect genomics to transcriptomics, we establish a baseline for genetic differences driving drug response variability among PC9 cell line versions. Applying the same approach to clonal sublines, we conclude that drug response variability in all but one of the sublines is due to epigenetic differences; in the other, it is due to genetic alterations. Finally, using a clonal drug response assay together with stochastic simulations, we attribute subclonal drug response variability within sublines to stochastic cell fate decisions and confirm that one subline likely contains genetic resistance mutations that emerged in the absence of drug treatment.
Highlights
Cancer is a complex and dynamic disease characterized by intertumoral and intratumoral heterogeneities that have been implicated in treatment avoidance and acquired resistance to therapy [1,2]
In response to the EGFR inhibitor (EGFRi) erlotinib, the 3 cell line versions display drastically different drug sensitivities (Fig 2A): PC9-MGH exhibits substantial cell death after an initial equilibration phase, PC9-VU settles into a near-zero rate of growth, and PC9-BR1 displays insensitivity to EGFRi
These observations are consistent with the high sensitivity of PC9-MGH to erlotinib reported in Sharma and colleagues [54] and the lower sensitivity of PC9-VU that we reported previously [55,56]
Summary
Cancer is a complex and dynamic disease characterized by intertumoral and intratumoral heterogeneities that have been implicated in treatment avoidance and acquired resistance to therapy [1,2]. Genomic instability is a hallmark of cancer [9,10] and is considered to be the primary source of this genetic heterogeneity [11,12]. Researchers are, increasingly looking to nongenetic sources of tumor heterogeneity for explanations. These include factors such as cell type of origin, microenvironmental differences between primary and metastatic sites, spatial variations in the microenvironment of an individual tumor, cell plasticity, cell–cell interactions, probabilistic cell fate decisions, and noise in gene expression [17]. Nongenetic heterogeneity has been linked to drug tolerance and decreased drug sensitivity in vitro [2,30,31,32,33], in vivo [30,31,34], and clinically [35,36]
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