Lineage-specific intolerance to oncogenic drivers restricts histological transformation

Abstract

Lung adenocarcinoma (LUAD) and small cell lung cancer (SCLC) are thought to originate from different epithelial cell types in the lung. Intriguingly, LUAD can histologically transform into SCLC after treatment with targeted therapies. In this study, we designed models to follow the conversion of LUAD to SCLC and found that the barrier to histological transformation converges on tolerance to Myc, which we implicate as a lineage-specific driver of the pulmonary neuroendocrine cell. Histological transformations are frequently accompanied by activation of the Akt pathway. Manipulating this pathway permitted tolerance to Myc as an oncogenic driver, producing rare, stem-like cells that transcriptionally resemble the pulmonary basal lineage. These findings suggest that histological transformation may require the plasticity inherent to the basal stem cell, enabling tolerance to previously incompatible oncogenic driver programs.

Figure 1.. A GEMM model to generate distinct histologic subtypes of lung cancer.

A) Nomenclature used for the ERPMT model with abbreviated alleles bolded/underlined and general strategy to use promoter-restricted adenovirus to initiate tumorigenesis in specific airway cells; B) Survival and histologic appearance of the ERPMT model initiated in alveolar type 2 cells (AT2; red cartoon cell) or C) pulmonary neuroendocrine cells (PNEC; blue cartoon cell) when mice are on doxycycline (DOX) containing (dashed line; n = 10 per group) or control diet (solid line; n = 10 per group). Representative sagittal H&E and tdTomato (“tdTom”) immunohistochemistry (IHC) lung sections alongside high powered (100um scalebar) H&E and synaptophysin (Syp) IHC from the LUAD model initiated from AT2 cells on DOX (above) or the SCLC model initiated from PNEC cells off DOX (below). D) Mean imputed expression of AT2 and PNEC lineage markers (table S1) (77) in single cells isolated from LUAD (red; n = 5,394 cells) or SCLC (blue; n = 4,371 cells) ERPMT models with overlaid kernel density estimates reflecting cell density; tdTom+ tumor cells sorted and pooled from n = 3 mice at 8wks post-infection.

Figure 2.. Tracing the origins and transitions between LUAD and SCLC in vivo.

A) Survival of AT2-derived ERPMT model on DOX (red), after DOX removal at 8wks (teal), and after restarting DOX diet one month following initial DOX removal (yellow; n = 10 mice per group); ***p<0.001, ****p<0.0001. B) Representative sagittal lung H&E sections from moribund mice in each group in A. C) tdTom+ burden in the airway after one month of EGFR inhibition via DOX removal (pink; n=9) or daily treatment with osimertinib (10mg/kg; PO d1–5 of 7; white; n = 9) as compared to 8wks timepoint before DOX removal (red; n = 12). DOX removal or osimertinib treatment [on DOX] were initiated at 8wks following the development of extensive ERPMT-derived LUAD tumorigenesis. For clarity, comparisons are effectively an 8wk pre-treatment cohort to ~12wk post-treatment cohorts (1mo of treatment); ***p<0.001, ****p<0.0001. D) Outline of single cell samples sequenced at distinct timepoints along the transition between LUAD and SCLC following DOX perturbations (as in A) E) Force-directed layout of cell states captured along the transition from at AT2 cell to ERPMT LUAD and finally towards a neuroendocrine fate as compared to de novo SCLC tumorigenesis (transparent blue); colored by sample (annotated in D; n = 15,828 cells pooled from 3 mice per sample). F) PCA projection of the lung epithelial lineage probability space (see Methods; same data as in E). Individual cells are colored by their max lineage probability and archetypes (see Methods) are overlaid as colored nodes (fig. S4, C to E). G) Flow plot showing relative abundance of cells assigned to their nearest lineage archetype, ordered by sampling time. Bar height is normalized by sample size (log-scale). H) Heatmap of scaled imputed and min-max normalized highly variable transition genes (see Methods), top ranked macrostate genes (n = 5, see Methods), and lineage markers along terminal cell state probabilities computed using CellRank (see Methods) for all cells in E. For each gene, expression was smoothed along the terminal state probability using a generalized additive model (GAM) as described in CellRank (34). The top 20 HVGs correlated with the bottleneck macrostate (table S2) and select lineage-specific markers are labeled on the right. A stacked KDE reflecting sample abundance (lower) and a KDE reflecting total cell frequency (upper, grey) are shown above the ranked heatmap. Above this, scaled imputed gene expression trends for the oncogenic drivers Myc and EGFR, modeled using a GAM along the terminal probability.

Figure 3.. Cell of origin and oncogenic driver incompatibility.

A) Frequency of tdTom+ cells in the airway of RPMT mice following infection with equivalent titers of adenovirus (~10⁶ pfu per mouse) using Ad5.Cgrp-Cre (blue) or Ad5.Spc-Cre (red) over a period of 8 weeks; n = 4 mice per timepoint); *p<0.01. B) Comparative histology of RPMT (no Tg.TetO-EGFR^L858R allele) mice 8wks post-infection following infection with Ad5.Cgrp-Cre (blue outline) or Ad5.Spc-Cre (red outline). C) Lineage tracing oncogenic Myc^T58A (down-pointing triangles) or EGFR^L858R (circles) on AT2 (Spc^CreERT2; red) or PNEC (Ascl1^CreERT2; blue) cells in the airway over time; n = 3 mice per timepoint. Control traces (tdTomato only; “wildtype”) are shown as grey squares; **p<0.001, ****p<0.0001. D) Long-term survival for cohorts shown in B; Ascl1^CreERT2 > EGFR^L858R (n = 13) or Myc^T58A (n = 15) and below, Spc^CreERT2 > EGFR^L858R (n = 14) or Myc^T58A (n = 12). Mice having a single copy of Rosa26^LSL-tdTom and Rosa26^LSL-rtTA3 were maintained on DOX chow throughout studies investigating lineage trace allele-mediated expression of Tg.TetO-EGFR^L858R.

Figure 4.. Loss of Pten in the AT2 lineage removes the barrier to Myc transformation.

A) Survival of mice where Myc (n = 12), Pten^Fl/wt (n = 5) or the combination of these alleles (n = 23) are initiated in AT2 cells using a single copy of Spc^CreERT2. Data were censored between 250–325d in the non-lethal arms. B) Comparative histology of whole lungs from representative Spc>Myc, Spc>Pten^Fl/+;Myc), Spc>Pten^Fl/Fl;Myc or Spc>PI(3)K^LSL-E545K;Myc mice at ~3mo post-labeling. Higher magnification regions (boxed) are provided at right; 200um scalebar. C) Bar plot showing fraction of each epithelial lineage archetype detected per sample as in (Fig. 2G). D) Bar plot of Hallmark gene sets significantly enriched (FDR < 0.01) within the undifferentiated cell state of the Spc>Pten^Fl/+;Myc sample pool, as in (fig. S4F). E) Effect of Pten deletion combined with deletion of p53, Rb1, and expression of Myc and tdTomato (RPPtenMT or RPMT) in neuroendocrine (blue) or AT2 (red) cells; n = 10 per arm with x-axis split for clarity, ***p<0.001. F) Clustered heatmap of imputed and z-normalized expression of AT2 and PNEC signature genes, model oncogenic drivers (Myc and Tg.EGFR), and SCLC subtype identifiers (NeuroD1, Pou2f3, and Yap1) for all tumor-epithelial cells from the RPPtenMT model (green) and the de novo LUAD (red) and SCLC (blue) models described (Fig. 1). Genes and cells are clustered using the Euclidean distance method.

Figure 5.. Rb1 loss cooperates with Myc expression to facilitate HT.

A) Survival of mice with the ERPT genotype (no Myc transgene) initiated with Ad5.Spc-Cre on DOX (ERPT on; dark green, n = 23), or on and then off DOX once mice developed advanced disease (ERPT on > off; light green, n = 20). As before, DOX diet was removed when individual mice exhibited signs of labored breathing and/or significant weight loss with a hunched appearance. For each model, cohorts of mice were followed until ~3X the median latency elapsed, at which point lungs were collected and the study was ended. B) Histologic appearance of “ERPT on” lungs by H&E and IHC staining for Ascl1 and Myc (scalebar 100 um). C) Similar to B, now with mice on then off DOX representative of a SCLC-like tumor. D) As in B, now with mice on then off DOX representative of a LUAD-like tumor. Pathologic interpretation provided below each representative example. E) Dot plot (left) showing frequency of expressing cells (node size) and log-transformed expression (node color) of AT1, AT2, and basal cell marker genes in normal AT2, ERPT LUAD, and ERPMT LUAD models. Genes shown are expressed in at least 20% of cells within at least one condition. Kernel density estimate (KDE, right) plots showing mean log-transformed expression by condition for select gene signatures. F) Volcano plot showing differentially expressed genes from bulk RNAseq of human transformed SCLC vs LUAD. Genes from the Hallmark MYC Targets V1 signature are colored according to conditional enrichment, top genes (abs(log2FC) > 1 and FDR < 1e-5) are labeled, and the pathway NES and FDR from GSEA are inset (bottom right). G) Tumorigenesis initiated using an adenoviral (n = 10; Ad5.Spc-Cre) or AT2 lineage trace allele (n = 19; Spc^CreERT2) in the EPMT model (Rb1^+/+) produces LUAD that does not relapse following DOX removal (n = 5 per group). H) Representative sagittal lung H&E sections from each group in G on DOX at point of moribund disease or one month following the removal of DOX from an otherwise moribund animal. I) Clustered heatmap of gene signatures differentially enriched between the tumor-epithelial cells of the EPMT and ERPMT models before and after DOX removal. All pathways are significantly enriched (NES > 0 and FDR < 1e-5) in at least one condition. Less significant signatures (FDR < 0.01) are transparent and signatures not meeting this threshold are blank. Rows and columns are clustered using the complete Manhattan distance method and metric. J) J) Bar plot showing fraction of each epithelial lineage archetype detected per sample (as in Fig. 2G) for the EPMT (left) and ERPMT (right) models before and after removal of DOX.