Proliferative control in cancer cells is frequently disrupted by mutations in the retinoblastoma protein (RB) pathway. Intriguingly, RB1 mutations can arise late in tumorigenesis in cancer cells whose RB pathway is already compromised by another mutation. In this study, we present evidence for increased DNA damage and instability in cancer cells with RB pathway defects when RB1 mutations are induced. We generated isogenic RB1 mutant genotypes with CRISPR/Cas9 in a number of cell lines. Cells with even one mutant copy of RB1 have increased basal levels of DNA damage and increased mitotic errors. Elevated levels of reactive oxygen species as well as impaired homologous recombination repair underlie this DNA damage. When xenografted into immunocompromised mice, RB1 mutant cells exhibit an elevated propensity to seed new tumors in recipient lungs. This study offers evidence that late-arising RB1 mutations can facilitate genome instability and cancer progression that are beyond the preexisting proliferative control deficit. FIG 1 CRISPR/Cas9-induced mutations in RB1 cause DNA damage. (A) Ethidium bromide-stained agarose gel showing examples of wild-type, heterozygous, and homozygous mutant RB1 genotypes that are detected by PCR amplification of exon 22 sequences. MW, molecular weight. (B, top) Representative Western blot showing RB expression in control, heterozygous, and homozygous mutant cells. (Bottom) Sp1 loading control.(C) Immunofluorescence microscopy was used to detect RB expression (green) in cultures of control, heterozygous, or homozygous mutants. Cells were counterstained with DAPI to visualize nuclei (blue). (D) Representative confocal microscopy images of ␥H2AX foci (red) in control, heterozygous, and homozygous RB1 mutant cells. Cells were counterstained with DAPI to visualize nuclei (blue). (E) Counts of ␥H2AX foci for each of the U2OS RB1 genotypes. The average proportions of cells with discrete numbers of foci are shown as histograms, while the cumulative frequency of foci for each genotype is shown in the inset. The average distributions of foci for RB1 wild-type (4 different clones), heterozygous (3 different clones), and knockout (4 different clones) cells were compared using the Kolmogorov-Smirnov test (*, P Ͻ 0.05). (F) U2OS cells were transfected with CRISPR/Cas9 constructs targeting either a safe-harbor site in the genome or exon 2 of the RB1 gene. Three clones were selected under both control and knockout conditions, and ␥H2AX foci were quantified by fluorescence microscopy. The average proportions of ␥H2AX foci for both RB1 wild-type and knockout genotypes are shown as histograms, while the cumulative relative frequency of foci is shown in the inset. Focus distributions were again compared by a Kolmogorov-Smirnov test. (G) H460 lung cancer cells were stained for ␥H2AX foci and analyzed as described above for panel F. (H) H1792 non-small cell lung cancer cells were analyzed as described above for panel F. All error bars are ϩ1 standard error of the mean (SEM). *, P Ͻ 0.05. RB1Deletion Causes ...

Organization of chromatin structure is indispensible to the maintenance of genome integrity. The retinoblastoma tumor suppressor protein (pRB) mediates both transcriptional repression and chromatin organization, but the independent contributions of these functions have been difficult to study. Here, we utilize a synthetic Rb1 mutant allele (F832A) that maintains pRB association at cell cycle gene promoters, but disrupts a cyclin-dependent kinase (CDK)-resistant interaction with E2F1 to reduce occupancy of pRB on intergenic chromatin. Reduced pRB chromatin association increases spontaneous γH2AX deposition and aneuploidy. Our data indicates that the CDK-resistant pRB-E2F1 scaffold recruits Condensin II to major satellite repeats to stabilize chromatin structure in interphase and mitosis through mechanisms that are distinct from silencing of repetitive sequence expression.

Introduction: Castration resistant prostate cancer (CRPC) cells can acquire resistance to the androgen receptor (AR) inhibitor enzalutamide (EZ). These cells can switch lineages from an adenocarcinoma to a neuroendocrine (NE) cell type that proliferate independently of the AR signaling pathway. Cancer genomic and molecular studies identified that co-deletion of the retinoblastoma (RB1) and TP53 genes can promote the acquisition of EZ resistance and neuroendocrine features. However, RB and p53 are both tumour suppressor and are therefore difficult to target pharmacologically. The purpose of this study is to identify an actionable molecular factor downstream of RB and p53 that drives EZ resistance in CRPC. Methods and Results: To characterize functional and molecular features of RB and p53 loss, CRISPR-Cas9 was used to generate a double knockout (DKO) line in LNCaP prostate cancer cells. Compared with LNCaP wild type (WT) cells, only DKOs formed distinct colonies over a 4-week colony forming assay under EZ treatment. RNA sequencing of DKO and WT cells, followed by gene ontology (GO) analysis, revealed NE and stemness genes, including HOXA9, were significantly upregulated in DKO cells. To categorize gene loss events in EZ-treated LNCaP cells, a genome-wide CRISPR knockout screen was performed. Pools of KO cells were treated with either EZ or DMSO and then analyzed by next generation sequencing to identify gene mutations that confer increased resistance or sensitivity to EZ. GO analysis of de-enriched genes following EZ treatment identified stemness genes, including HOXA9, highlighting the potential importance of a stem-like phenotype for acquiring EZ resistance EZ. To further investigate the functional significance of HOXA9 we analyzed EZ-resistant prostate tumour genomic data. HOXA9 is mutated or mis-expressed in 10% of cases. Importantly, HOXA9 is either amplified or overexpressed in virtually all these cases, and is associated with poorer prognosis, suggesting an oncogenic role for HOXA9 in CRPC. HOXA9 transcript levels were positively correlated with neuroendocrine features and negatively correlated with RB1expression in these tumour samples. LNCaP WT and DKO cells we then engineered to overexpress HOXA9 displayed increased IC50 values following a 6-day EZ treatment, compared with either parental line. DKO cells that overexpress HOXA9 also formed significantly more colonies following a 4-week EZ treatment, compared with parentals. In contrast, shRNA knockdown of HOXA9 caused a reduction in IC50 values and formed fewer drug resistant colonies compared with control cells. Finally, DKO and WT cells were co-treated with varying concentrations of the HOXA9 inhibitor DB818 and EZ. DKO cells were more sensitive to DB818 alone at high concentrations and displayed higher synergy as measured by a ZIP synergy score when co-treated with EZ. Conclusions: Overall, these results suggest that HOXA9 regulates EZ resistance in prostate cancer. Furthermore, HOXA9 inhibition may be of therapeutic benefit for treating EZ-resistant CRPC. Citation Format: Michael V. Roes, Fred A. Dick. HOXA9 promotes enzalutamide resistance in RB-p53 deficient prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B015.

Proliferative control in cancer cells is frequently disrupted by mutations in the RB-pathway.Intriguingly, RB1 mutations can arise late in tumorigenesis in cancer cells whose RB-pathway is already compromised by another mutation. In this study, we present evidence for increased DNA damage and instability in CDKN2A silenced cancer cells when RB1 mutations are induced.We generated isogenic RB1 mutant genotypes with CRISPR in a number of cell lines. Cells with even one mutant copy of RB1 have increased basal levels of DNA damage and increased mitotic errors. Elevated levels of reactive oxygen species as well as impaired homologous recombination repair underlie this DNA damage. When xenografted into immune compromised mice RB1 mutant cells exhibit an elevated propensity to seed new tumors in recipient lungs. This study offers evidence that late arising RB1 mutations can facilitate genome instability and cancer progression that are beyond the pre-existing proliferative control deficit.