Mutations in the retinoblastoma (RB) tumour suppressor pathway are a hallmark of cancer and a prevalent feature of lung adenocarcinoma1,2,3. Although RB was the first tumour suppressor to be identified, the molecular and cellular basis that underlies selection for persistent RB loss in cancer remains unclear4,5,6. Methods that reactivate the RB pathway using inhibitors of cyclin-dependent kinases CDK4 and CDK6 are effective in some cancer types and are currently under evaluation for the treatment of lung adenocarcinoma7,8,9. Whether RB pathway reactivation will have therapeutic effects and whether targeting CDK4 and CDK6 is sufficient to reactivate RB pathway activity in lung cancer remains unknown. Here we model RB loss during lung adenocarcinoma progression and pathway reactivation in established oncogenic KRAS-driven tumours in mice. We show that RB loss enables cancer cells to bypass two distinct barriers during tumour progression. First, RB loss abrogates the requirement for amplification of the MAPK signal during malignant progression. We identify CDK2-dependent phosphorylation of RB as an effector of MAPK signalling and critical mediator of resistance to inhibition of CDK4 and CDK6. Second, RB inactivation deregulates the expression of cell-state-determining factors, facilitates lineage infidelity and accelerates the acquisition of metastatic competency. By contrast, reactivation of RB reprograms advanced tumours towards a less metastatic cell state, but is nevertheless unable to halt cancer cell proliferation and tumour growth due to adaptive rewiring of MAPK pathway signalling, which restores a CDK-dependent suppression of RB. Our study demonstrates the power of reversible gene perturbation approaches to identify molecular mechanisms of tumour progression, causal relationships between genes and the tumour suppressive programs that they control and critical determinants of successful cancer therapy.
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All data generated or analysed during this study are included in this published Letter (and its Supplementary Information) with the exception of raw RNA sequencing data associated with Extended Data Fig. 2e, f, which have been deposited publicly in the Gene Expression Omnibus (GEO) under accession number GSE128506. Source Data associated with Figs. 1e–i, 2b–e, g, i, j, 3a–c, e–g, 4c, e–h and Extended Data Figs. 2d–f, 3c, d, 4b, d–i, k, 5a–h, j, m, 6c, d, 7d, f, g, i, 8a, d, 9b, c, e are available in the online version of the paper. Raw images from western blots are shown in Supplementary Fig. 1. In gels with multiple bands per lane or in which specific lanes were selected, the locations at which gels were cropped are also shown. For Extended Data Figs. 2b, c, 5i, k, 7e, controls were run on separate gels as sample-processing controls; for Extended Data Fig. 7a, loading controls for each gel are provided in the Supplementary Information.
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We thank J. Wang and A. Bedenbaugh for histology, E. Blankemeyer for computed tomography, Plexxikon for the gift of PLX4720, I. Asangani for help with sequencing, H. Ehly and ULAR staff for animal husbandry, and R. Greenberg, M. Winslow and members of the Feldser Laboratory for manuscript critique. This work is supported by: NIH grants (R00CA158581, R21CA205340, R01CA193602 and R01CA222503 to D.M.F., T32ES019851 to M.C. and T32CA115299 to K.S.), American Lung Association (LCD400095 to D.M.F.), American Cancer Society, (PF-16-22401TBE to T.Y.) and the Penn Abramson Cancer Center grant NIH P30-CA016520.
The authors declare no competing interests.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
a, Oncoprint from CBioPortal showing frequency and co-occurrence of mutations and copy number alterations in RB pathway components, KRAS and TP53 in the provisional lung adenocarcinoma TCGA dataset38. b, RB pathway components and their corresponding mutation frequencies in the provisional lung adenocarcinoma TCGA dataset. c, Kaplan–Meier survival analysis of patients with lung adenocarcinoma whose tumours do (n = 456 patients) or do not (n = 987 patients) contain alterations in RB pathway members. Patient data were obtained from the MSK-IMPACT clinical sequencing cohort. Significance was determined by two-sided log-rank test (P = 0.0015). Source Data can be found at the cBioPortal38.
Extended Data Fig. 2 RbXTR allows Cre-dependent inactivation and FlpO-dependent reactivation of Rb1.
a, Top, RbXTR (expressed): XTR gene trap cassette consists of a splice acceptor (SA), GFP complementary DNA ‘GeneTrap’, and the polyadenylation transcriptional terminator sequence (pA). Stable inversion is achieved by the use of two pairs of mutually incompatible mutant loxP sites (Lox2272 and Lox5171) arranged in the ‘double-floxed’ configuration. In the germline and in normal somatic cells, RB expression is normal in RbXTR/XTR mice10. Middle, RbTR (trapped): inhalation of Cre-expressing adenoviral or lentiviral vectors induces the permanent conversion of the RbXTR allele to the RbTR allele that inactivates Rb1 gene expression. Transcripts are spliced from the upstream exon to the GFP reporter gene and downstream transcription is terminated to functionally inactivate gene function. RB expression is inactivated only in the tumour cells. Bottom, RbR (restored): the Rosa26FlpO-ERT2 allele enables tamoxifen-dependent conversion of trapped RbTR to its restored RbR allelic state via excision of the gene trap. b, Western blot analysis of 3 KP and 3 KP;RbTR/TR tumour-derived cell lines. c, Western blot analysis of 2 KP;RbTR/TR tumour-derived cell lines treated with Adeno-Cre as a control, or Adeno-FlpO to restore RB expression. d, Quantitative RT–PCR analysis of 3 KP;RbTR/TR tumour-derived cell lines treated with Adeno-FlpO to restore RB expression. Data are normalized to Adeno-Cre-treated cells (control). The log2-normalized fold change in expression ± s.d. is shown. n = 3 technical replicates for each cell line. e, Volcano plot of differentially expressed genes from RNA sequencing data obtained from KP (n = 4) versus KP;RbTR/TR (n = 4) cell lines. The RB signature, defined by genes for which the change in expression is ≥2-fold and the P value adjusted for multiple testing is ≤ 0.05, is boxed. Statistical significance was determined by two-sided Wald’s test using Benjamini–Hochberg correction using DESeq2. f, Kaplan–Meier survival analysis of patients with lung adenocarcinoma whose tumours exhibit a high (n = 114 patients) or low (n = 189 patients) Rb signature. Significance was determined by two-sided log-rank test. P = 0.0175.
Extended Data Fig. 3 RB deficiency is associated with low MAPK pathway signalling in mouse and human lung adenocarcinomas.
a, Top, H&E staining images of tumours from KP and KP;RbTR/TR mice 8 weeks after tumour initiation. Bottom, corresponding immunohistochemistry for p-ERK. b, Immunohistochemistry for Ki67 in tumours from KP and KP;RbTR/TR mice 8 and 14 weeks after tumour initiation. c, Quantification of Ki67+ cells from b. Symbols represent individual tumours. KP 8 weeks, n = 9 tumours from 2 mice; KP;RbTR/TR 8 weeks, n = 9 tumours from 2 mice; KP 14 week, n = 31 tumours from 3 mice; KP;RbTR/TR 14 weeks, n = 31 tumours from 3 mice. Significance was determined by two-sided unpaired Student’s t-test with Welch’s correction for 8-week (P = 0.0034) and 14-week (P = 0.2686) analyses. Data are mean ± s.d. d, Plot showing the relationship of p-ERK+ fraction versus Ki67+ fraction in KP and KP;RbTR/TR tumours at 14 weeks after tumour initiation. KP, n = 15 tumours from 2 mice; KP;RbTR/TR, n = 16 tumours from 2 mice. Significance was determined by linear regression analysis and the line of best fit for each is shown (P = 0.0376). e, Immunohistochemistry for RB depicting an RB-negative core (left) and an RB-positive core (right). f, Immunohistochemistry for p16 depicting a p16-negative core (left), a core with low staining (middle) and a core with high staining (right). g, Immunohistochemistry for p-RB S807/S811 depicting two examples of cores with low expression (left) and high expression (right). h, Immunohistochemistry for p-ERK depicting a core with an p-ERK staining score of 0 (left), 1 (middle left), 2 (middle right) and 3 (right). Scale bars, 100 μm; inset images are magnified 5×.
Extended Data Fig. 4 RB and p27 phosphorylation are enhanced by amplification of the MAPK signal and suppressed by inhibition of MEK1/2.
a, Representative images of immunohistochemistry for p-ERK and p-RB S807/S811 in KP tumours treated with vehicle control or BRAF inhibitor PLX4720. b, Contingency analysis of p-ERK and p-RB S807/S811 from a. n = 40 tumours from 4 mice treated with vehicle control; n = 66 tumours from 5 mice treated with PLX4720. Significance was determined by two-sided χ2 test for the vehicle-treated group (P = 6.4 × 10−5) and for the PLX4720-treated group (P = 0.0003). c, Representative images of immunohistochemistry for p-ERK, p27, p-p27 S10 and p-p27 T187 in KP tumours treated with vehicle control, BRAF inhibitor PLX4720 or MEK inhibitor PD0325901. Tumour sections were stained with anti-p-p27 S10 antibody as a measure of non-CDK2-dependent suppression of p27. Significant changes in phosphorylation at this site were not observed. d, Analysis of p27 levels in p-ERK low (n = 28) and p-ERK high (n = 68) tumours as determined by immunohistochemistry in KP and KP;RbTR/TR mice. n = 2 KP mice and n = 2 KP;RbTR/TR mice. Data are mean ± s.d. Significance was determined by two-tailed unpaired Student’s t-test. P = 0.0350. e, Analysis of p-p27 S10 levels in p-ERK low (n = 43) and p-ERK high (n = 70) tumours as determined by immunohistochemistry in KP and KP;RbTR/TR mice. n = 2 KP mice and n = 2 KP;RbTR/TR mice. Significance was determined by two-tailed unpaired Student’s t-test. P = 0.6937. f, Analysis of p-p27 T187 levels in p-ERK low (n = 40) and p-ERK high (n = 73) tumours as determined by immunohistochemistry in KP and KP;RbTR/TR mice. n = 2 KP mice and n = 2 KP;RbTR/TR mice. Significance was determined by two-tailed unpaired Student’s t-test. P = 0.0127. g, Analysis of p27 levels in KP (n = 85) and KP;RbTR/TR (n = 54) tumours as determined by immunohistochemistry. n = 2 KP mice and n = 2 KP;RbTR/TR mice. Significance was determined by two-tailed unpaired Student’s t-test. P = 0.0018. h, Analysis of p-p27 S10 levels in KP (n = 82) and KP;RbTR/TR (n = 74) tumours as determined by immunohistochemistry. n = 2 KP mice and n = 2 KP;RbTR/TR mice. Significance was determined by two-tailed unpaired Student’s t-test. P = 0.4372. i, Analysis of p-p27 T187 levels in KP (n = 86) and KP;RbTR/TR (n = 72) tumours as determined by immunohistochemistry. n = 2 KP mice and n = 2 KP;RbTR/TR mice. Data are mean ± s.d. Significance was determined by two-tailed unpaired Student’s t-test. P = 3.1 × 10−6. j, Representative images of immunohistochemistry for p-ERK and p-RB S807/S811 in KP tumours treated with vehicle control or MEK1/2 inhibitor PD0325901. k, Contingency analysis of p-ERK and p-RB S807/S811 from c. n = 42 tumours from 2 mice treated with vehicle control; n = 60 tumours from 3 mice treated with PD0325901. Significance was determined by two-sided χ2 test for vehicle-treated group (P = 1.1 × 10−7) and for PD0325901-treated group (P = 2.7 × 10−5).
Extended Data Fig. 5 CDK2 blockade enhances the effects of CDK4/6 inhibition in mouse KP and human lung adenocarcinoma cell lines.
a, BrdU/7AAD double labelling of KP clones expressing GFP (n = 1 biological replicate) or Cdk2 (n = 2 biological replicates) targeting sgRNAs with 0, 0.1 or 1.0 μM palbociclib. Percentage of cells in G1, S or G2/M phase are shown (2 technical replicates for each sample). Data are mean ± s.d. where appropriate. b, Top, cell cycle analysis (BrdU/7AAD double labelling) of KP clones expressing GFP (n = 1 clone per cell line) or Cdk2 (n = 2 clones per cell line) targeting sgRNAs. Bottom, the percentage of cells in each stage of the cell cycle is shown. c, d, Cell proliferation assay performed in duplicate showing number of cells 3 days after initial plating treated with 0, 0.1 or 1.0 μM palbociclib for either KP54 (c) or KP62 (d) cells. Two independent Cdk2 knockout clones and control GFP cells are shown. Data are means. e, f, Cell proliferation assay performed in triplicate showing number of cells 24, 48 and 72 h after initial plating for KP54 (e) or KP62 (f) cells. Three independent Cdk2 KO clones and one control is shown. Data are mean ± s.d. Significance was determined by ANOVA with Dunnett’s multiple comparisons test (n = 3 for all). e, Treatment for 24 h with GFP shRNA compared to CDK2-2 (P = 0.0013), CDK2-3 (P = 0.0029) or CDK2-8 (P = 0.0302) shRNA. Treatment for 48 h with GFP shRNA compared to CDK2-2 (P < 0.0001), CDK2-3 (P < 0.0001) or CDK2-8 (P = 0.0052) shRNA. Treatment for 72 h with GFP shRNA compared to CDK2-2 (P < 0.0001), CDK2-3 (P < 0.0001) or CDK2-8 (P < 0.0001) shRNA. f, Treatment for 48 h with GFP shRNA compared to CDK2-2 shRNA (P = 0.0415). Treatment for 72 h with GFP shRNA compared to CDK2-2 (P < 0.0001) or CDK2-5 (P < 0.0001) shRNA. g, Proliferation of KP (left) and KP;RbTR/TR (right) cells in quadruplicate, 72 h after addition of roscovitine (red outlines) and/or palbociclib (increasing grey tones) at the indicated concentrations. Cell numbers normalized to the average of the vehicle (DMSO) controls (white). Data are mean ± s.d. Significance was determined by ANOVA with Dunnett’s multiple comparisons test (n = 4 for all). KP control compared to 6 μM roscovitine (P < 0.0001) or 1 μM Palbociclib (P = 0.0016). KP 6 μM roscovitine compared to 6 μM roscovitine and 0.1 μM palbociclib (P < 0.0001) or 6 μM roscovitine and 1 μM palbociclib (P < 0.0001). KP;RbTR/TR control compared to 6 μM roscovitine (P = 0.0098). KP;RbTR/TR 6 μM roscovitine compared to 6 μM roscovitine and 0.1 μM palbociclib (P = 0.9811) or 6 μM roscovitine and 1 μM palbociclib (P = 0.0160). h, Proliferation of KP cells in quadruplicate, stably transduced with a tet-regulated dominant negative Cdk2 allele (CDK2DN) 72 h after addition doxycyclin (red) and/or palbociclib (increasing grey tones). Cell numbers normalized to the average of the vehicle (DMSO) controls (white). Data are mean ± s.d. Significance was determined by ANOVA with Dunnett’s multiple comparisons test (n = 4 for all). Control compared to 1 μM doxycycline to induce CDK2DN (P < 0.0001) or 1 μM palbociclib (P = 0.0194). 1 μM doxycycline compared to 1 μM doxycycline and 0.1 μM palbociclib (P = 0.4184) or 1 μM doxycycline and 1 μM palbociclib (P = 0.0020). i, Analysis of KP clones targeted with GFP-targeting or Cdk2-targeting sgRNAs. Western blot for Rb pathway component expression: RB, p-RB S807/S811, CDK2, CDK4, CDK6 and p-RB 780. Actin was used as loading control. j, Effects of CDK4/6 inhibition and CDK2 knockout on human lung adenocarcinoma cell lines. Data mined from the Sanger Center’s COSMIC database21 showing the relative sensitivity (IC50) of independent human lung adenocarcinoma cells lines to palbociclib. k, Western blot showing CDK2 loss following CRISPR-mediated knockout in indicated human lung adenocarcinoma cell lines. HSP90 was used as loading control. l, Representative images of clonogenic survival analysis of human lung adenocarcinoma cell lines performed in triplicate, treated every 3 days for 1.5 weeks with 0, 0.1 or 1.0 μM palbociclib. Cell lines were targeted with either an inert sgRNA or one targeting CDK2. m, Quantification of culture area covered by cells in l. Dark grey bars indicate inert sgRNA and red bars indicate CDK2 sgRNA. Data are mean ± s.d. Significance was determined by ANOVA with Sidak’s multiple comparisons test (n = 3 for all). A549 with inert sgRNA compared to CDK2 sgRNA in combination with 0 μM palbociclib (P > 0.9999), 0.1 μM palbociclib (P = 1.0 × 10−6) or 1 μM palbociclib (P = 0.0023). H1993 with inert sgRNA compared to CDK2 sgRNA in combination with 0 μM palbociclib (P = 2.7 × 10−7), 0.1 μM palbociclib (P = 0.0331) or 1 μM palbociclib (P = 0.6557). EKVX with inert sgRNA compared to CDK2 sgRNA in combination with 0 μM palbociclib (P = 0.5412), 0.1 μM palbociclib (P = 0.0003) or 1 μM palbociclib (P = 0.5929).
Extended Data Fig. 6 Loss of RB promotes alternative pathways towards gaining metastatic competency.
a, H&E photomicrographs of metastases that formed from KP;RbTR/TR tumours. b, Immunofluorescence analysis of KP and KP;RbTR/TR tumours for co-expression of HMGA2 and NKX2-1. c, Immunohistochemistry staining of serial sections from KP and KP;RbTR/TR tumours for HMGA2 and FOXA2. Orange dotted lines outline mutually exclusive staining and red dotted lines outline co-expressing staining patterns. Quantification of staining pattern (right) showing percentages of HMGA2+ tumours from KP or KP;RbTR/TR mice that are FOXA2+ or FOXA2−. KP, n = 29 tumours from 3 mice; KP;RbTR/TR, n = 37 tumours from 3 mice. Significance was determined by two-sided χ2 test. P = 1.1 × 10−5. d, Left, histological analysis of KP;RbTR/TR metastases. H&E staining and immunohistochemistry for NKX2-1, HMGA2 and FOXA2 of serial sections from representative metastases that are NKX2-1+ or NKX2-1−. Right, quantification. e, Immunohistochemistry staining of KP and KP;RbTR/TR tumours for club (CC10) and neuroendocrine (synaptophysin) cell markers. For control and comparison, a synaptophysin-positive small-cell lung cancer from a Trp53flox/flox;Rbflox/flox;p130flox/flox mouse model is shown.
a, Western blot analysis of KP;RbTR/TR and KP (NKX2-1−HMGA2+ (TMet) and NKX2-1+HMGA2− (TnonMet)) tumour-derived cell lines examining cyclin D1 and p16 expression. HSP90 was used as loading control. b, RNA sequencing reads at the Cdkn2a locus for NKX2-1+HMGA2− KP (n = 2) and NKX2-1−HMGA2+ KP (n = 2) cell lines. Reads from exon 1α encoding p16 (left) and exon 1β encoding Arf (right) are shown. c, Representative Sashimi plots comparing the number of p16 and Arf exon-spanning reads from the Cdkn2a locus. Plots are shown for a representative NKX2-1+HMGA2− tumour (red), NKX2-1−HMGA2+ tumour (blue) and metastasis (green). The number of reads that span each exon–exon junction is displayed. The range of minimum to maximum read count for the given plot is displayed in the top left corner. d, Quantification of the ratio of p16 to Arf reads from the Cdkn2a locus. RNA sequencing results examining NKX2-1+HMGA2− tumours (n = 8), NKX2-1−HMGA2+ tumours (n = 8) and extrapulmonary metastases (n = 19) were obtained from the GEO (accession GSE84447)25. Significance for each comparison was determined by two-tailed unpaired Student’s t-test. NKX2-1+HMGA2− versus NKX2-1−HMGA2+, P = 0.0682; NKX2-1−HMGA2+ versus metastases, P = 0.0504; NKX2-1+HMGA2− versus metastases, P = 0.0006. Data are represented by box and whisker plots, with the line indicating the median and whiskers indicating the minimum and maximum values. e, Western blot analysis of KP and KP;RbTR/TR tumour-derived cell lines for NKX2-1, HMGA2 and RB. Actin was used as loading control. f, g, Analysis of subcutaneous tumour growth (f) and associated lung metastases (g) of KP and KP;RbTR/TR tumour-derived cell lines from e. Symbols represent individual mice injected with either one of two NKX2-1−HMGA2+ KP cell lines (n = 4 and 5 mice per cell line), two NKX2-1+HMGA2+ KP;RbTR/TR cell lines (n = 3 and 4 mice per cell line) or two NKX2-1−HMGA2+ KP;RbTR/TR cell lines (n = 4 mice per cell line). Significance was determined by unpaired two-tailed Student’s t-test with Welch’s correction. f, Primary tumour weight. NKX2-1–HMGA2+ KP versus NKX2-1+HMGA2+ KP;RbTR/TR (P = 0.2508) and KP versus NKX2-1−HMGA2+ KP;RbTR/TR (P = 0.2727). g, Lung metastases. KP versus NKX2-1+HMGA2+ KP;RbTR/TR (P = 4.8 × 10−5) and KP versus NKX2-1−HMGA2+ KP;RbTR/TR (P = 0.0027). Data are mean ± s.d. h, H&E photomicrographs of representative metastases from KP;RbTR/TR cell line allografts. Insets show magnification of boxed areas. Scale bars, 100 μm. i, Liver metastases after intrasplenic injection of KP and KP;RbTR/TR tumour-derived cell lines (top) from Extended Data Fig. 5i. Symbols represent individual mice injected with a NKX2-1+HMGA2− KP cell line (n = 2 mice), a NKX2-1−HMGA2+ KP cell line (n = 5 mice), two NKX2-1+HMGA2+ KP;RbTR/TR cell lines (n = 4 mice for each cell line) or two NKX2-1−HMGA2+ KP;RbTR/TR cell lines (n = 5 mice for each cell line). Significance was determined by two-tailed unpaired Student’s t-test with Welch’s correction. P = 0.0007. Data are mean ± s.d.
a, Imaging of individual tumour growth by μCT. Images were taken weekly starting 11 weeks after tumour initiation. RB reactivation (tamoxifen treatment) initiated at week 12. Average fold change after week 12 is shown. KP;RbTR/TR, n = 6 mice; KP;RbR/R, n = 6 mice. Data are mean ± s.d. Significance at 14 weeks was determined by unpaired two-tailed Student’s t-test. P = 0.2617. b, Representative μCT images quantified in a. c, Low-magnifiation scans of sections through tumour lobes showing relative tumour burden 2 weeks after RB reactivation in KP;RbTR/TR and KP;RbR/R cohorts. d, Quantitative PCR detection of the GFP cDNA within the RbTR allele. DNA templates for PCR were isolated by laser capture microdissection of individual tumours. KP;RbTR/TR, n = 8 tumours from 2 mice; KP;RbR/R, n = 8 tumours from 2 mice. Significance was determined by two-tailed unpaired Student’s t-test. P = 0.0023. Data are mean ± s.d.
Extended Data Fig. 9 Impact of RB reactivation on proliferation, MAPK signalling and RB phosphorylation.
a, Ki67 immunohistochemistry of KP;RbTR/TR and KP;RbR/R tumours 3, 7 and 14 days after Rb1 restoration. b, Quantification of a. n = 10 tumours from 3 mice for 0-, 3- and 7-day time points; n = 15 tumours from 3 mice for the 14-day time point. Significance was determined by χ2 test for trend. P = 0.3289. c, Quantification of the percentage of total tumour cells that are Ki67+ in KP;RbTR/TR and KP;RbR/R tumours 14 days after Rb1 restoration. KP;RbTR/TR, n = 14 tumours from 1 mouse; KP;RbR/R, n = 14 tumours from 1 mouse. Significance was determined by two-tailed unpaired Student’s t-test with Welch’s correction. P = 0.0110. Data are mean ± s.d. d, Immunohistochemistry for RB, p-RB S807/S811 and p-ERK in KP;RbTR/TR and KP;RbR/R tumours 3, 7 and 14 days after Rb1 restoration. Scale bars, 100 μm; insets are magnified 5×. e, Contingency test for NKX2-1+HMGA2+ KP;RbTR/TR and KP;RbR/R tumours 2 weeks after Rb1 restoration. NKX2-1+HMGA2+ KP;RbTR/TR, n = 23 tumours from 2 mice; KP;RbR/R, n = 12 tumours from 2 mice. Significance was determined by two-sided χ2 test. P = 0.0019.
Extended Data Fig. 10 RB controls multiple barriers to tumour progression and is repressed by multiple pathways that will require multiple pharmacological interventions to reverse.
a, In the KP model, adenomas transit through an ‘early barrier’ that limits progression to the carcinoma state by amplifying the MAPK signalling cascade (red). The ‘late barrier’ limits the onset of metastatic ability and is characterized by the loss of lineage fidelity marked by lost expression of lineage-specific transcription factors, NKX2-1 (blue) and FOXA2 (purple), and the differentiation marker of alveolar type 2 cells, SPC (green). Loss of these lineage commitment factors precedes the derepression of the embryonic-restricted chromatin factor HMGA2 that functionally drives metastasis and marks the metastatic cell state (yellow). Downregulation of p16INK4a expression is associated with the metastatic cell state (grey). b, The additional suppression of Rb1 in the KP;RbTR/TR model alters the molecular trajectory of these tumours by first abrogating the early barrier through the elimination of the requirement for amplification of the MAPK signal (lack of red) and then by facilitating loss of lineage fidelity to overcome and blur the late barrier. Carcinomatous KP;RbTR/TR tumours can rapidly derepress HMGA2 (yellow) and lose the lineage identity marker SPC (green); however, loss of lineage fidelity is unlinked from NKX2-1 and FOXA2, which normally enforce lung cell identity in these tumours. Notably, metastatic primary tumours and distant metastases can sometimes maintain expression of NKX2-1 (blue) and FOXA2 (purple). Expression of p16INK4a is maintained in RB-deficient metastatic cell states (grey). c, In lung adenomas that maintain RB, RB tumour suppressor activity blocks progression to carcinomatous stages and the onset of metastatic cell states and enforces lineage fidelity. d, In lung adenocarcinomas that maintain RB expression, amplification of the MAPK signal activates CDK2-dependent hyperphosphorylation of RB to promote carcinoma progression. e, Inactivation of RB removes early barriers that limit carcinoma progression, removes constraints that reinforce lineage fidelity and disrupts late barriers that suppress metastatic competency. f, Reactivation of RB in tumours that lack Rb pathway function highlights a need for a multi-pronged approach to inhibit CDK4/6 as well as CDK2 and/or MAPK pathway signalling (for example, through MEK inhibition) to fully reactivate RB-mediated tumour suppression. These data emphasize the need for the development of selective CDK2 inhibitors.
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Walter, D.M., Yates, T.J., Ruiz-Torres, M. et al. RB constrains lineage fidelity and multiple stages of tumour progression and metastasis. Nature 569, 423–427 (2019). https://doi.org/10.1038/s41586-019-1172-9
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