RB constrains lineage fidelity and multiple stages of tumour progression and metastasis - PubMed (original) (raw)

. 2019 May;569(7756):423-427.

doi: 10.1038/s41586-019-1172-9. Epub 2019 May 1.

Travis J Yates # 1, Miguel Ruiz-Torres # 1, Caroline Kim-Kiselak 1, A Andrea Gudiel 1, Charuhas Deshpande 3 4, Walter Z Wang 5, Michelle Cicchini 1, Kate L Stokes 1, John W Tobias 4 6, Elizabeth Buza 7, David M Feldser 8 9 10 11

Affiliations

RB constrains lineage fidelity and multiple stages of tumour progression and metastasis

David M Walter et al. Nature. 2019 May.

Abstract

Mutations in the retinoblastoma (RB) tumour suppressor pathway are a hallmark of cancer and a prevalent feature of lung adenocarcinoma1-3. 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 unclear4-6. 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 adenocarcinoma7-9. 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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Conflict of interest statement

Author information: The authors disclose no potential conflicts of interest.

Figures

Extended Data Fig.1:

Extended Data Fig.1:. The RB pathway is frequently altered in human lung adenocarcinoma.

(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 dataset,. (b) RB pathway components and their corresponding mutation frequencies in the provisional lung adenocarcinoma TCGA dataset. (c) Kaplan-Meier survival analysis of lung adenocarcinoma patients whose tumours do (n=456 patients) or do not (n=987 patients) contain alterations in RB pathway members. Patient data was 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 cBioPortal.

Extended Data Fig. 2:

Extended Data Fig. 2:. Rb XTR allows Cre-dependent inactivation and FlpO-dependent reactivation of RB.

(a) Top: Rb XTR (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 Rb XTR/XTR mice (Ref. 10). Middle: Rb TR (Trapped): Inhalation of Cre-expressing adenoviral or lentiviral vectors induces the permanent conversion of the Rb XTR allele to Rb TR allele that inactivates RB 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: Rb R (Restored): The Rosa26FlpO-ERT2 allele enables tamoxifen-dependent conversion of trapped Rb TR to its restored Rb R allelic state via excision of the gene trap. (b) Western blot analysis of 3 KP and 3 KPRb TR tumour derived cell lines. (c) Western blot analysis of 2 KPRb 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 KPRb TR tumour-derived cell lines treated with Adeno-FlpO to restore RB expression. data are normalized to Adeno-Cre treated cells for control. Log2 fold change +/− standard deviation is shown. n=3 technical replicates for each cell line. (e) Volcano plot of differentially expressed genes from RNA-seq data obtained from KP (n=4) versus KPRb TR/TR (n=4) cell lines. The RB Signature, defined by genes whose expression change is ≥ 2-fold and p-value adjusted for multiple testing ≤ 0.05, is boxed. Statistical significance determined by two-sided Wald’s test using Benjamini-Hochberg correction via DESEQ2. (f) Kaplan-Meier survival analysis of lung adenocarcinoma patients 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:

Extended Data Fig. 3:. RB deficiency is associated with low MAPK pathway signalling in mouse and human lung adenocarcinomas.

(a) H&E images of KP and KPRb TR/TR tumours 8 weeks post tumour initiation. Corresponding IHC for ERK(P) (bottom). (b) IHC for Ki67 in KP and KPRb TR/TR tumours 8 and 14 weeks post tumour initiation. (c) Quantification of Ki67 positive cells from (b). Symbols represent individual tumours (_KP_-8 week: n=9 tumours from 2 mice; KPRb _TR/TR_-8 week: n=9 tumours from 2 mice; _KP_-14 week: n=31 tumours from 3 mice; KPRb _TR/TR_-14 week: n=31 tumours from 3 mice). Significance was determined by two-sided unpaired t-test with Welch’s correction for 8 week (p=0.0034) and 14 week analyses (p=0.2686). Line indicates mean±SD. (d) Plot showing the relationship of ERK(P) positive fraction versus Ki67 positive fraction in KP (n=15 tumours from 2 mice) and KPRb TR/TR (n=16 tumours from 2 mice) tumours at 14 weeks after tumour initiation. Significance was determined by linear regression analysis (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 RB(P)807/811 depicting two examples of cores with low expression (left), and high expression (right). (h) Immunohistochemistry for ERK(P) depicting a core with an ERK(P) staining score of 0 (left), a score of 1 (middle-left), a score of 2 (middle-right), and a score of 3 (right). Scale bars=100μM, inset images are magnified 5x further.

Extended Data Fig. 4:

Extended Data Fig. 4:. RB and p27 phosphorylation are enhanced by MAPK signal amplification and suppressed by MEK1/2 inhibition.

(a) Representative images of IHC for ERK(P) and RB(P)807/811 in KP tumours treated with vehicle control or Braf inhibitor PLX4720. (b) Contingency analysis of ERK(P) and RB(P)807/811 from (a) (n=40 tumours from 4 mice treated with vehicle control and n=66 tumours from 5 mice treated with PLX4720). Significance was determined by chi square test (two-sided) for vehicle treated group (p=6.4e-5) and for PLX4720 treated group (p=0.0003). (c) Representative images of IHC for ERK(P), p27, p27(P)S10 and p27(P)T187 in KP tumours treated with vehicle control, Braf inhibitor PLX4720, or MEK inhibitor PD0325901. Tumour sections were stained with p27(P)Ser10 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 ERK(P) low (n=28) and ERK(P) high (n=68) tumours as determined by immunohistochemistry in KP and KPRbTR mice. n=2 KP mice and n=2 KPRb TR/TR mice. Centre value is the mean +/− SD. Significance was determined by two-tailed unpaired t-test (p=0.0350). (e) Analysis of p27(P)S10 levels in ERK(P) low (n=43) and ERK(P) high (n=70) tumours as determined by immunohistochemistry in KP and KPRbTR mice. n=2 KP mice and n=2 KPRb TR/TR mice. Significance was determined by two-tailed unpaired t-test (p=0.6937). (f) Analysis of p27(P)T187 levels in ERK(P) low (n=40) and ERK(P) high (n=73) tumours as determined by immunohistochemistry in KP and KPRbTR mice. n=2 KP mice and n=2 KPRb TR/TR mice. Significance was determined by two-tailed unpaired t-test (p=0.0127). (g) Analysis of p27 levels in KP (n=85) and KPRbTR (n=54) tumours as determined by immunohistochemistry. n=2 KP mice and n=2 KPRb TR/TR mice. Significance was determined by two-tailed unpaired t-test (p=0.0018). (h) Analysis of p27(P)S10 levels in KP (n=82) and KPRbTR (n=74) tumours as determined by immunohistochemistry. n=2 KP mice and n=2 KPRb TR/TR mice. Significance was determined by two-tailed unpaired t-test (p=0.4372). (i) Analysis of p27(P)T187 levels in KP (n=86) and KPRbTR (n=72) tumours as determined by immunohistochemistry. n=2 KP mice and n=2 KPRb TR/TR mice. Centre value is the mean +/− SD. Significance was determined by two-tailed unpaired t-test (p=3.1e-6). (j) Representative images of IHC for ERK(P), and RB(P)807/811 in KP tumours treated with vehicle control or MEK1/2 inhibitor PD0325901. (k) Contingency analysis of ERK(P) and RB(P)807/811 from (c) (n=42 tumours from 2 mice treated with vehicle control and n=60 tumours from 3 mice treated with PD0325901). Significance was determined by chi square test (two-sided) for vehicle treated group (p=1.1e-7) and for PD0325901 treated group (p=2.7e-5).

Extended Data Fig. 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 labeling 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). Bars indicate mean±SD where appropriate. (b) Cell cycle analysis (BrdU/7AAD double labeling) of KP clones expressing GFP (n=1 clone per cell line) or Cdk2 (n=2 clones per cell line) targeting sgRNAs. Percentage of cells in each stage of the cell cycle are graphically represented (bottom). (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 KO clones and Control GFP cells are shown. Bars indicate the mean. (e,f) Cell proliferation assay performed in triplicate showing number of cells 24, 48, and 72 hours after initial plating for KP54 (e) or KP62 (f) cells. Three independent Cdk2 KO clones and one control is shown. Bars indicate mean±SD. Significance was determined by ANOVA, Dunnett’s multiple comparisons test (n=3 for all). (e) 24 hour GFP compared to CDK2–2 (p=0.0013), CDK2–3 (p=0.0029), CDK2–8 (p=0.0302). 48 hour GFP compared to CDK2–2 (p<0.0001), CDK2–3 (p<0.0001), CDK2–8 (p=0.0052). 72 hour GFP compared to CDK2–2 (p<0.0001), CDK2–3 (p<0.0001), CDK2–8 (p<0.0001). (**f**) 48 hour GFP compared to CDK2–2 (p=0.0415). 72 hour GFP compared to CDK2–2 (p<0.0001), CDK2–5 (p<0.0001). (**g**) Proliferation of _KP_ (left) and _KPRb_ _TR/TR_ (right) cells in quadruplicate, 72 hours 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) only controls (white). Bars indicate mean±SD. Significance was determined by ANOVA, 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 + 0.1 μM Palbociclib (p<0.0001) or 6 μM Roscovitine + 1 μM Palbociclib (p<0.0001). _KPRb_ _TR/TR_ control compared to 6 μM Roscovitine (p=0.0098). _KPRb_ _TR/TR_ 6 μM Roscovitine compared to 6 μM Roscovitine + 0.1 μM Palbociclib (p=0.9811) or 6 μM Roscovitine + 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 hours after addition doxycyclin (red) and/or palbociclib (increasing grey tones). Cell numbers normalized to the average of the vehicle (DMSO) only controls (white). Bars indicate mean±SD. Significance was determined by ANOVA, 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 + 0.1 μM Palbociclib (p=0.4184) or 1 μM Doxycycline + 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, RB(P)807/811, CDK2, CDK4, CDK6, and RB(P)780. Actin controls for loading. (**j**) Effects of CDK4/6 inhibition and CDK2 knock out on human lung adenocarcinoma cell lines. data mined from the Sanger Center’s COSMIC database showing the relative sensitivity (IC50) of independent human lung adenocarcinoma cells lines to palbociclib. (**k**) Western blot showing CDK2 loss following CRISPR-mediated knock out in indicated human lung adenocarcinoma cell lines. Hsp90 controls for loading. (**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. Bars indicate mean±SD. Significance was determined by ANOVA, Sidak’s multiple comparisons test (n=3 for all). A549 with inert sgRNA compared to CDK2 sgRNA in combination with either 0 μM palbociclib (p>0.9999), 0.1 μM palbociclib (p=1.0e-6) or 1 μM palbociclib (p=0.0023). H1993 with inert sgRNA compared to CDK2 sgRNA in combination with either 0 μM palbociclib (p=2.7e-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 either 0 μM palbociclib (p=0.5412), 0.1 μM palbociclib (p=0.0003) or 1 μM palbociclib (p=0.5929).

Extended Data Fig. 6:

Extended Data Fig. 6:. Loss of RB promotes alternative pathways toward gaining metastatic competency.

(a) H&E photomicrographs of metastases that formed from KPRb TR/TR tumours. (b) Immunofluorescence analysis of KP and KPRb TR/TR tumours for co-expression of HMGA2 and NKX2–1. (c) IHC staining of serial sections from KP and KPRb TR/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 percentage of HMGA2 positive tumours from KP (n=29 tumours from 3 mice) or KPRb TR/TR (n=37 tumours from 3 mice) mice that are positive or negative for FOXA2. Significance was determined by chi-square test (two-sided, p=1.1e-5). (d) Histological analysis of KPRb TR/TR metastases. H&E and IHC for NKX2–1, HMGA2 and FOXA2 on serial sections from representative metastases that are NKX2–1 positive or negative. Results are tabulated in the adjacent graph. (e) IHC staining of KP and KPRb TR/TR tumours for club (CC10) and neuroendocrine (synaptophysin) cell markers. For control and comparison, a synaptophysin positive small cell lung cancer from a p_53_ flox/flox; Rb flox/flox; p130 flox/flox mouse model is shown46.

Extended Data Fig. 7:

Extended Data Fig. 7:. Loss of p16 or RB is associated with increased metastatic proclivity.

(a) Western blot analysis of KPRbTR and KP (TMet and TnonMet) tumour-derived cell lines examining Cyclin D1 and p16 expression. HSP90 controls for loading. (b) RNA-Sequencing reads at the Cdkn2a locus for KP TNonMet (n=2) and TMet (n=2) cell lines. Reads from Exon 1α coding for p16 (left) and Exon 1β coding for 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 TNonmet tumour (red), TMet tumour (blue), and metastasis (green). The number 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 upper left corner. (d) Quantification of the ratio of p16 to Arf reads from the Cdkn2a locus. RNA-sequencing results examining TnonMet tumours (n=8), TMet tumours (n=8) and extrapulmonary metastases (n=19) were obtained from GEO (Chuang et al., accession GSE84447). Significance for each comparison was determined by two-tailed unpaired t-test (TnonMet vs TMet: p=0.0682; TMet vs metastases: p=0.0504; TnonMet vs metastases: p=0.0006). Data is represented with 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 KPRb TR/TR tumour-derived cell lines for NKX2–1, HMGA2, and RB. Actin controls for protein loading. (f,g) Analysis of subcutaneous tumour growth (f) and associated lung metastases (g) of KP and KPRb TR/TR tumour-derived cell lines from (e). Symbols represent individual mice injected with either one of 2 KP-TMet cell lines (n=4 and 5 mice per cell line), 2 KPRb TR/TR NKX2–1Pos./HMGA2Pos. cell lines (n=3 and 4 mice per cell line) or 2 KPRb TR/TR NKX2–1Neg./HMGA2Pos. cell lines (n=4 mice per cell line). Significance was determined by unpaired t-test with Welch’s correction (two-tailed). (f) Primary tumour weight: KP vs KPRb TR/TR NKX2–1Pos./HMGA2Pos. (p=0.2508), and KP vs KPRb TR/TR NKX2–1Neg./HMGA2Pos. (p=0.2727). (g) Lung metastases: KP vs KPRb TR/TR NKX2–1Pos./HMGA2Pos. (p=4.8e-5), and KP vs KPRb TR/TR NKX2–1Neg./HMGA2Pos. (p=0.0027). Centre lines indicate the mean +/− SD. (h) H&E photomicrographs of representative metastases from KPRb TR/TR cell line allografts. (i) Liver metastases after intrasplenic injection of KP and KPRb TR/TR tumour-derived cell lines (top) from Extended Data Fig. 5i. Symbols represent individual mice injected with either a _KP_-TNonMet cell line (n=2 mice), a _KP_-TMet cell line (n=5 mice), 2 KPRb TR/TR NKX2–1Pos./HMGA2Pos. cell lines (n=4 mice for each cell line) or 2 KPRb TR/TR NKX2–1Neg./HMGA2Pos. cell lines (n=5 mice for each cell line). Significance was determined by two-tailed unpaired t-test with Welch’s correction (p=0.0007). Centre lines indicate the mean +/− SD. Scale bars=100μM.

Extended Data Fig. 8:

Extended Data Fig. 8:. Growth of lung adenocarcinomas after RB reactivation

(a) Imaging of individual tumour growth by μCT. Images taken weekly starting 11 weeks post tumour initiation. RB reactivation (tamoxifen treatment) initiated at week 12. Average fold change after week 12 is shown. KPRb TR/TR:n=6 mice; KPRb R/R:n=6 mice. data points indicate the mean±SD. Significance at 14 weeks was determined by unpaired t-test (two-tailed, p=0.2617). (b) Representative μCT images quantified in (a). (c) Low power scans of sections through tumour lobes showing relative tumour burden 2 weeks after RB reactivation in KPRb TR/TR and KPRb R/R cohorts. (d) Quantitative PCR detection of the GFP cDNA within the Rb TR allele. DNA templates for PCR were isolated by laser capture microdissection of individual tumours. KPRb TR/TR: n=8 tumours from 2 mice, KPRb R/R: n=8 tumours from 2 mice. Significance was determined by two-tailed unpaired t-test (p=0.0023). Bar indicates the mean±SD.

Extended Data Fig. 9:

Extended Data Fig. 9:. Impact of RB reactivation of proliferation, MAPK signaling, and RB phosphorylation.

(a) Ki67 IHC of KPRb TR/TR, and KPRb R/R tumours 3, 7, and 14 days after Rb restoration. (b) Quantification of (a) (n=10 tumours from 3 mice for 0, 3 and 7 day time points, and n=15 tumours from 3 mice for the 14 day time point). Significance was determined by chi-square test for trend (p=0.3289). (c) Quantification of the percentage of total tumour cells that are Ki67 positive in KPRb TR/TR (n=14 tumours from 1 mouse) and KPRb R/R (n=14 tumours from 1 mouse) tumours 14 days post Rb restoration. Significance was determined by two-tailed unpaired t test with Welch’s correction (p=0.0110). Line indicates the mean±SD. (d) IHC for RB, RB(P)807/811, and ERK(P) in KPRb TR/TR, and KPRb R/R tumours 3, 7, and 14 days after Rb restoration. Scale bars=100μM, insets are magnified 5x further. (e) Contingency test for Nkx2–1Pos./Hmga2Pos. KPRb TR/TR (n=23 tumours from 2 mice) and KPRb R/R (n=12 tumours from 2 mice) tumours two weeks post Rb restoration. Significance was determined by chi-square test (two-sided, p=0.0019).

Extended Data Fig. 10:

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). Down regulation of p16INK4a expression is associated with the metastatic cell state (grey). (b) The additional deletion of RB in the KPRb TR model alters the molecular trajectory of these tumours by first abrogating the ‘Early Barrier’ through eliminating the requirement for MAPK signal amplification (lack of red), and then by facilitating loss of lineage fidelity to overcome and blur the ‘Late Barrier’. Carcinomatous KPRb TR tumours can rapidly derepress HMGA2 (yellow) and lose lineage identity marker SPC (green) however, loss of lineage fidelity is unlinked from NKX2–1 and FOXA2 that normally enforce lung cell identity in these tumours. Interestingly, 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, MAPK signal amplification activates CDK2-dependent hyper-phosphorylation 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 (e.g. through MEK inhibition) to fully reactivate RB-mediated tumour suppression. These data emphasize the need for the development of selective CDK2 inhibitors.

Figure 1:

Figure 1:. Inactivation of RB abrogates the requirement for MAPK signal amplification during carcinoma progression.

(a) Experimental scheme. (b) XTR cassette at the Rb1 locus. (c) Lungs from KP and KPRb XTR/XTR mice 8 and 14 weeks after tumour initiation. Immunohistochemistry for RB. (d) Immunohistochemistry for MEK(P), ERK(P) and BrdU in KP and KPRb TR/TR tumours 8 and 14 weeks after tumour initiation. (e) Grades for individual tumours. _KP_-8 Week: (n=73 tumours/5 mice), KPRb _TR/TR_-8 Week: (n=112 tumours/5 mice), _KP_-14 Week: (n=380 tumours/9 mice), KPRb _TR/TR_-14 Week: (n=824 tumours/12 mice). Significance by chi-square test (two-sided) for 8 (p=8.6e-10) and 14 week (p=0.8262) time points. (f,g,h,i) Tumor burden and positivity for (g) BrdU, (h) MEK(P), and (i) ERK(P) at 8 and 14 weeks. Symbols represent individual tumours, bar is mean±SD. Significance by unpaired t-test with Welch’s correction (two-tailed). Number of mice analysed as (e). Number of tumours and p values shown. Scale bars=100μM, insets magnified 5x.

Figure 2:

Figure 2:. CDK2 inactivation overcomes intrinsic resistance to CDK4/6 inhibition.

(a) ERK(P) and RB(P)807/811 immunohistochemistry on KP and KPRb TR/TR tumors. (b) Contingency for RB(P)807/811 and tumour grade (top), and RB(P)807/811 and ERK(P) (bottom). Significance by Chi-square test (two-sided), tumour number and p values shown. n=3 mice. (c-e) IHC on human TMAs. (c) ERK(P) in RB-expressing tumours with low or high RB(P)807/811, tumour grade (d) and ERK(P) (e) where RB pathway is lost or intact. Significance by Chi-square test for trend. Number of samples and p values shown. (f) Clonogenic growth of KP54 and KP62 cells with (sgRNA-GFP) or without (sgRNA-CDK2) CDK2 +/− palbociclib. (g) Area covered from (f). Bars indicate mean±SD. Significance determined by ANOVA, Sidak’s multiple comparisons (n=2 biological replicates, n=3 technical replicates), p values (shown) calculated from technical replicates. (h) Clonogenic growth of human cell lines with (sgRNA-Inert) or without (sgRNA-CDK2) CDK2 +/− palbociclib. (i) Area covered from (h). Bars indicate mean±SD. Significance determined by ANOVA, Sidak’s multiple comparisons (n=3 technical replicates), significant p values shown. (j) Xenograft growth of A549 (top) and H838 (bottom) expressing inert or CDK2-targeting sgRNAs. Formation of palpable masses set to day 0 and treatments initiated (t=0). Points indicate mean±SD. Tumour volume significance determined by unpaired t-test (two-tailed) at d16. Mouse number and p values shown. Combination of CDK2-KO and palbociclib treatment is synergistic (A549: CI=0.274 and H838: CI=0.000002237).

Figure 3:

Figure 3:. RB inactivation accelerates onset of metastasis and enables alternative pathways to gaining metastatic competency.

(a) (Left) Grades for carcinomas 14 weeks after tumour initiation. KP:n=258 tumours/9 mice, KPRb TR/TR:n=580 tumours/12 mice. Significance by Chi-square test for trend (p=2.9e-7). Bars indicate mean±SD. (Right) Metastases per mouse KP:n=9 mice, KPRb TR/TR:n=12 mice. Significance by two-tailed unpaired t-test with Welch’s correction (p=0.0201). Line indicates mean. (b) Tumour stage, lymph node, and distant metastasis in lung adenocarcinoma patients where RB pathway is lost or intact. Significance by Chi-square test for trend (tumour stage) and Chi-square test (two-sided) (lymph node and distant metastasis). Patient number and p values shown. Data from TCGA. (c) Tumour grade and tumour size of human lung adenocarcinomas where RB pathway is lost or intact. Centre value indicates mean, error bars indicate SD. Significance of tumour grade by Chi-square test for trend, and significance for tumour size by two-tailed unpaired t-test. p values and number of samples shown. Data from Weir et al. (Ref 1) (d) NKX2–1, HMGA2, and SPC immunohistochemistry from KP and KPRb TR/TR tumours. Results summary indicated at bottom. Red dashes separate lower and higher grade regions. (e) Percentage of HMGA2Pos. tumours co-staining for NKX2–1; KP (n=22 tumours/4 mice) and KPRb TR/TR (n=84 tumours/4 mice). Significance by two-sided Chi-square test (p=4.2e-6). (f) Percentage of Grade 3 tumours positive for HMGA2; KP (n=88 tumours/4 mice) and KPRb TR/TR (n=86 tumours/4 mice). Significance by Fisher’s exact test (two-sided, p=6.2e-6). (g) Percentage of SPCPos./SPCNeg. areas by grade in NKX2–1Pos./HMGA2Pos. tumours from individual KPRb TR/TR mice (n=28 tumours/3 mice). Significance by Chi-square test for trend (p=0.0021).

Figure 4:

Figure 4:. RB reactivation reprograms tumours toward a less advanced state.

(a) Experimental scheme. (b) Conversion of Rb TR (Trapped) allele to Rb R (Restored). (c) Kaplan–Meier survival analysis; KP, KPRb TR/TR, KPRbR/R (n=6 mice each), and KPRb flox/flox (n=5 mice). Significance by two-sided Log-rank test (p=0.0005). (d) RB, Ki67, NKX2–1, HMGA2, SPC, and ERK(P) IHC in KPRb TR/TR and KPRb R/R tumours two weeks post RB restoration. Quantification in Extended Data Fig. 9e. (e) Expression patterns for RB, RB(P)807/811, NKX2–1, HMGA2, SPC, and ERK(P) in KPRb TR/TR from Fig.4d (n=35 tumours/3 mice) and KPRb R/R tumours on day(d) d3 (n=30 tumours/4 mice), d7 (n=30 tumours/4 mice) and d14 (n=34 tumours/3 mice) post RB restoration. RB(P)807/811 analysis from n=20 tumours/2 mice for 0, 7 and 14 day time points, and n=18 tumours/2 mice for 3 day time point. Significance determined by chi-square test for trend, p values shown. (f) Percentage of ERK(P) positive cells two weeks post RB restoration (n=21 KPRb TR/TR, n=20 KPRb R/R tumours, 1 mouse each). Significance by unpaired t-test with Welch’s correction (two-tailed, p=2.3e-8). Bar indicates mean±SD. (g) KPRb TR/TR (n=224 tumours/4 mice) and KPRb R/R (n=131 tumours/4 mice) tumour grades. Significance by chi-square test for trend (p=3.6e-13). (h) Metastases in KPRb TR/TR and KPRb R/R mice (n=6 each) two weeks post RB restoration. Significance by two-sided unpaired t-test (p=0.0344). Bar indicates mean±SD. Scale bars=100μM, insets magnified 5x.

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