Multivalent state transitions shape the intratumoral composition of small cell lung carcinoma

Abstract

Studies to date have not resolved how diverse transcriptional programs contribute to the intratumoral heterogeneity of small cell lung carcinoma (SCLC), an aggressive tumor associated with a dismal prognosis. Here, we identify distinct and commutable transcriptional states that confer discrete functional attributes in individual SCLC tumors. We combine an integrative approach comprising the transcriptomes of 52,975 single cells, high-resolution measurement of cell state dynamics at the single-cell level, and functional and correlative studies using treatment naïve xenografts with associated clinical outcomes. We show that individual SCLC tumors contain distinctive proportions of stable cellular states that are governed by bidirectional cell state transitions. Using drugs that target the epigenome, we reconfigure tumor state composition in part by altering individual state transition rates. Our results reveal new insights into how single-cell transition behaviors promote cell state equilibrium in SCLC and suggest that facile plasticity underlies its resistance to therapy and lethality.

INTRODUCTION

The use of nongenetic means to diversify phenotypes provides organisms with the ability to adapt quickly to environmental changes (1). This diversity can promote survival, especially in the context of extreme environmental shifts (2). Similarly, cancers maintain stable proportions of cells in several distinct transcriptionally defined states categorized on the basis of developmental programs (e.g., “stem”-like, epithelial, mesenchymal, neural, and neuroendocrine) or additional undefined gene expression states (3, 4). Within individual tumors, cell state transitions could be modulated by environmental cues, cell-cell signaling, and/or stochastic switches in gene expression programs (5), with the potential to skew state proportions, at least transiently. Critically, anticancer therapies appear to preferentially kill specific cancer cell states, resulting in predictable changes in phenotypic proportions (4, 6, 7). Despite some preliminary observations regarding cell-cell switches and state equilibrium in some cancers, very little is known about the regulation of the proportions of cancer cells in the various cell states, the types and rates of transitions across and within cancer types, and if multistate tumors can be modulated to enhance the prospect of a tumor’s extinction (8).

Small cell lung carcinoma (SCLC) is an aggressive disease that accounts for ~15% of all lung cancers and affects ~200,000 patients annually worldwide (9). A diagnosis of SCLC is typically associated with a dismal prognosis, with most patients succumbing within 1 year and a 5-year survival rate of only 6%. First-line treatments include the combination of cisplatin and etoposide alone or with radiotherapy (10). Although most patients respond to first-line therapy, most patients also experience disease recurrence. In contrast to other types of lung cancer, there have been no major advances in the use of targeted therapies, and the benefits of immunotherapy have been modest (11–13). New paradigms and approaches focused on advancing therapeutic strategies in patients with SCLC are urgently needed.

Large-scale sequencing of SCLC has not revealed consistent cooccurrence or mutual exclusivity of genetic alterations, frustrating efforts to stratify patients on the basis of the genome (14). On the other hand, there are indications of transcriptionally distinct subtypes of SCLC (15–20). These studies have indicated at least three transcriptional clusters, with the largest representing an ASCL1^high NEUROD1^low subtype, the second largest representing a NEUROD1^high subtype, and a small number of tumors expressing low levels of both ASCL1 and NEUROD1, but high levels of YAP1. ASCL1 is a basic helix-loop-helix (bHLH) transcription factor (TF) that is a marker of neuroendocrine differentiation (21). NEUROD1 is also a bHLH TF that is required for neural development (18). YAP1, a regulator of transcription that is inhibited by the Hippo growth signaling pathway, is expressed in mesenchymal cells but is undetectable or low in most SCLC cell lines (22). A fourth TF, POU2F3, was recently identified as defining a previously unappreciated tuft cell variant of SCLC (23). Despite a proposed quaternary taxonomy (16, 24), some immunohistochemical (IHC) analyses have not confirmed a Yap1 subtype (25). Therefore, SCLC transcriptional classification remains, at least in part, discordant and incomplete.

Further confounding the attempts to stratify SCLC tumors into discrete categories are suggestions of intratumoral heterogeneity. Morphologically different cell types have been noted in a number of SCLC cell lines, including some with mixtures of cells in suspension in combination with variably adherent monolayers (26). Expression profiling of these distinct populations suggests that the suspended cells are more likely to have neuroendocrine features, whereas the adherent population adopts a less neuroendocrine, more mesenchymal expression profile. Mesenchymal cells, expressing VIM, CD44, and YAP1 have a reduced proliferative rate, relatively greater chemoresistance, and support the survival, growth, and migration of the neuroendocrine subtype of cells within admixed tumors (27, 28). Additional data support a role for Notch signaling, generally suppressed in the predominant ASCL1^high subtype, in inducing a neuroendocrine to mesenchymal fate switch in both mouse and human SCLC cells (29–31). Studies conducted in genetically engineered mouse models of SCLC and driven by heterologous MYC^T58A demonstrate a unidirectional transition from one cell fate (ASCL1) to another (YAP1), resulting in ultimately homogeneous populations of cell types (32, 33). Recent work using circulating tumor cell–derived xenografts suggests increased intratumoral heterogeneity following treatment resistance, attributed to subtype switching, in human SCLC (34, 35). Overall, these results, combined with a recent single-cell atlas of 21 SCLC tumors, provide indications that distinct cell types can exist within the same SCLC tumor. However, the extent of this heterogeneity in treatment naïve tumors (i.e., not induced by therapeutic stress), the direct observation of subtype switching, the frequency and single-cell kinetics of state transitions, the functional specialization of additional subpopulations, and the impact of tumor state proportions on clinical outcomes have yet to be thoroughly examined. Here, we characterize the proportions and single-cell dynamics of distinct intratumoral subpopulations in SCLC and use epigenetic modifiers to reconfigure tumor composition, with the aim of advancing new therapeutic strategies to improve clinical outcomes.

RESULTS

Distinct gene coexpression networks and the taxonomy of SCLC cell lines

To find categories of SCLC in established cell lines, we performed k-means clustering using gene expression data from 53 SCLC cell lines. Four groups were robust to sampling variability (Fig. 1A and fig. S1). We identified differentially expressed genes and gene sets in each group (data S1 and S2). Consistent with the previous identification of some SCLC subtypes (15, 36, 37), we found differential expression of fate-determining TFs (Fig. 1B). Namely, ASCL1, ISL1, NEUROD1, INSM1, and YAP1 either individually or in aggregate differentially marked each cluster. We selected representative cell lines based on silhouette scores from each group and assessed the concordance between transcript levels and protein levels (Fig. 1C). We found that protein expression was generally associated with gene transcript levels across the representative cell lines…