Targeting RET Kinase in Neuroendocrine Prostate Cancer

Abstract

The increased treatment of metastatic castration-resistant prostate cancer (mCRPC) with second-generation antiandrogen therapies (ADT) has coincided with a greater incidence of lethal, aggressive variant prostate cancer (AVPC) tumors that have lost dependence on androgen receptor (AR) signaling. These AR-independent tumors may also transdifferentiate to express neuroendocrine lineage markers and are termed neuroendocrine prostate cancer (NEPC). Recent evidence suggests kinase signaling may be an important driver of NEPC. To identify targetable kinases in NEPC, we performed global phosphoproteomics comparing several AR-independent to AR-dependent prostate cancer cell lines and identified multiple altered signaling pathways, including enrichment of RET kinase activity in the AR-independent cell lines. Clinical NEPC patient samples and NEPC patient-derived xenografts displayed upregulated RET transcript and RET pathway activity. Genetic knockdown or pharmacologic inhibition of RET kinase in multiple mouse and human models of NEPC dramatically reduced tumor growth and decreased cell viability. Our results suggest that targeting RET in NEPC tumors with high RET expression could be an effective treatment option. Currently, there are limited treatment options for patients with aggressive neuroendocrine prostate cancer and none are curative. IMPLICATIONS: Identification of aberrantly expressed RET kinase as a driver of tumor growth in multiple models of NEPC provides a significant rationale for testing the clinical application of RET inhibitors in patients with AVPC.

Figure 1.. Global phosphorylation and kinase signaling pathways are differentially regulated in AVPC cell lines compared to AdCa cell lines.

A and B. Supervised hierarchical clustering heatmap of 4,235 unique phosphoserine/threonine (pS/T) enriched peptides (Figure 1A) and 115 unique phosphotyrosine (pY) enriched peptides (Figure 1B) from AdCa cell lines (Blue: C4–2, 22Rv1, LNCaP, and VCaP) and AVPC cell lines (Red: cMyc/myrAKT, LASCPC-01, EF-1, PARCB-1, PARCB-2, PARCB-3, PARCB-5, NCI-H660, DU145, and PC3). Yellow = hyperphosphorylation; Blue = hypophosphorylation. C and D. Kinase substrate enrichment analysis (KSEA) performed on the 10 AVPC and 4 AdCa cell lines in A and B, showed multiple predicted alterations to kinase signaling. (Figure 1C) KSEA for pS/T analysis used a false discovery rate (FDR) <0.05, substrate hits > 5, and normalized K score >2.0. (Figure 1D) KSEA for pY analysis used an FDR <0.1, substrate hits >4, and normalized K score >1.1. E. Phosphorylated residues identified in the global phosphoproteomics (from Figure 1A and 1B) or F. human phosphoproteome data (23) were mapped onto signaling pathways downstream of RET kinase. Yellow = Enriched in AVPC relative to AdCa; Blue = Reduced in AVPC relative to AdCa. Thick black outline = activating phosphorylation; white outline = inactivating phosphorylation; thin outline = no defined function.

Figure 2.. RET kinase along with other neuroendocrine transcripts are upregulated in NEPC relative to AdCa patient samples.

A. Microarray data from the University of Washington rapid autopsy data of metastatic prostate cancer biopsies (32) were clustered based on gene expression of RET, neuroendocrine markers: CHGA and ASCL1, as well as androgen regulated genes: KLK3, AR, and NKX3–1. Upregulation of expression is represented by yellow, while downregulated genes are represented by blue. Patient samples were classified by AR and NE markers as AR+NE- (green, n=134), AR-NE- (blue, n=10), AR-NE+ (red, n=20), and AR+NE+ (purple, n=7). B. Pearson correlation matrix of gene expression from Figure 2A showing a correlation of RET gene expression with neuroendocrine markers and negative correlation with AR responsive markers. C. Box and whisker plot of average transcript measurements of CHGA, SYP, or RET in Adenocarcinoma (AR+NE-) versus the NEPC (AR-NE+) patients. The data is represented in Tukey plots and expression values were analyzed by Student’s t test. D. Agilent oligo array expression analysis of four neuroendocrine AR-negative LuCaP patient derived xenografts (PDX) and 20 LuCaP adenocarcinoma PDX published in Zhang et al. 2015. Clinical Cancer Research. (33) were clustered as in Figure 2A. E. Pearson correlation matrix of expression data represented in Figure 2D. F. Box and whisker plot shows an upregulation in CHGA, SYP, and RET kinase in NEPC versus AdCa PDX samples. Data is represented as in Figure 2C.

Figure 3.. RET expression correlates with NE markers in prostate cancer cell lines and is important for NEPC cell line growth.

A. RET expression dependency profiling for 11280 genes across eight prostate cell lines (PRECLH, LNCaP, VCaP, DU145, MDA PCa 2b, 22Rv1, NCI-H660, and PC3). RET expression was positively correlated with NEPC driver genes (blue) and negatively correlated with AR and AR regulators (cyan). B. Relative RET dependency scores reflect the ability of 503 cancer cell lines to maintain proliferation after RET knockdown (taken from the Project Achilles 2.201). Among the 8 prostate cancer cell lines, PC3 and NCI-H660 cells showed the greatest dependency on RET. C. RET protein expression in NCI-H660 cells stably transduced with scrambled (Scr), anti-GFP or two unique anti-RET shRNA. RET protein levels were reduced in two RET knockdown NCI-H660 cell lines and β-Actin serves as a loading control. D. RET knockdown reduces cellular proliferation in H660 cell lines. The line graph represents relative cellular proliferation as measured by WST assay of one biological replicate. Cell proliferation was analyzed by linear regression of log transformed data to determine statistical significance and error bars represent the standard deviation of five technical replicates.

Figure 4.. NCI-H660 cells are sensitive to RET inhibition and show sensitivity to RET inhibitors.

A. Immunoprecipitation of RET kinase from H660 cells shows that 4 hour treatment with 1 μM AD80, LOXO-292, or BLU-667 reduces RET tyrosine phosphorylation, as assayed with a total phosphotyrosine antibody 4G10. B. NCI-H660 cells treated for 4 hours with DMSO (Con) the indicated concentrations (nM) of AD80, LOXO-292, or BLU-667, showed reduced activity of the MAPK and AKT signaling cascades downstream of RET. Activity was analyzed by western blot for phosphorylation of ERK1/2 at Tyr202/Tyr204 and phosphorylation of AKT1/2 at Ser473. The AD80 treatment reduced phosphorylation of both downstream targets, while LOXO-292 and BLU-667 reduced the activity of ERK1/2. In all treatments the total ERK1/2, total AKT1/2 and Actin loading control remained unaffected. C. The activity of pERK1/2 (Tyr202/Tyr204) and pAKT1/2 (Ser 473) in NCI-H660 scrambled control and RET knockdown cells was assayed after a 4 hour treatment with DMSO (D), or 1μM of AD80 (A), LOXO-292 (L), or BLU-667 (B). D. The relative ERK1/2 activity was measured by comparing pERK1/2 (Tyr202/Tyr204) to total ERK1/2 protein and normalized to the scrambled DMSO treated sample. The ERK1/2 activity is reduced by both RET knockdown and after treatment with RET inhibitors. The bars represent the average values from three experiments and the error bars are standard deviation. E. Quantification of AKT1/2 activity (pAKT1/2 S473 relative to total AKT protein and normalized DMSO treated Scr cells) shows AD80 potently inhibits AKT1/2 activity while knockdown may reduce activity slightly. Bars represent the mean from three experiments and the error bars are standard deviation.

Figure 5.. Organoid NEPC models are sensitive to treatment with multiple RET inhibitors.

A. A dose response curve of Pten^−/− and Rb^−/− prostate specific double knockout (DKO) organoids treated with increasing concentrations of AD80, LOXO-292, and BLU-667. Viability was measured by staining for dead cells. Circles represent mean and error bars ± standard deviation. B. Bright field images and corresponding fluorescence images of GFP labeled-DKO organoids treated with the indicated concentrations of AD80. Blue=DAPI staining of nuclei, Red=Propidium iodide staining of dead cells. Scale bar =100μm. C and D. Representative brightfield and fluorescent images of LOXO-292 (C) and BLU-667 (D) DKO organoids treated with the indicated concentrations of drugs stained as described in E with the GFP channel omitted.

Figure 6.. AD80 reduces NCI-H660 xenograft tumor growth.

A. Schematic of in vivo study in which NCI-H660 cells were injected subcutaneously into the right flank of NOD-SCID mice and tumors were allowed to grow to approximately 100 to 200mm³ before being randomly assigned into two treatment groups: Control (DMSO alone, n=6) or AD80 (10mg/kg/day, n=6). B. The fold change in tumor volume by treatment group was plotted as a function of the number of days of treatment. Means and confidence intervals (CIs) were calculated on the log scale and reported in terms of geometric means after exponentiation with error bars ± 95% confidence interval. There was evidence of an overall treatment effect on tumor growth rate (p=0.006) with a significantly lower tumor volume at day 22 (p=0.006). C. Average animal weights were measured at the same time as tumor volumes and no differences in average animal weight between treatment groups was observed over the duration of the study. Symbols represent means with error bars ± standard error. D. Following the termination of the xenograft tumor experiment, tumors were excised from animals that survived to the end of the study and photographed with a centimeter scale ruler. Separate images from the same group are divided by a white line. E. Representative images of H&E (2.5X and 20X), RET IHC (20X), Ki67 IHC (20X), and TUNEL (2.5X and 20X) stained sections of tumors from each group. White scale bars are 500μm. Yellow and black scale bars are 50μm. F and G. Average optical density of (F) RET staining and (G) Ki67 staining from five distinct fields of view in each tumor are represented by symbols with a horizontal bar representing the mean. Quantification was analyzed by one way ANOVA. H. Quantification of the average TUNEL positive area (2.5X) was analyzed with the Kruskal-Wallis test (p=0.1727). Symbols represent averages for individual tumors with a horizontal line representing the mean. Bars represent the mean with error bars represent ± standard error.