Subclonal CHD1 deletion is more frequent in African American prostate cancers and associated with worse clinical outcome.
CHD1 is frequently subclonally deleted in prostate cancer (18). Our initial analysis on the SNP array data from TCGA comparing AA and EA PCa cases suggested that the subclonal loss of CHD1 may be a more frequent event in AA men (Suppl. Figures 1 and 2). To independently validate this observation, we assessed CHD1 copy number by FISH (for probe design see Suppl. Figure 3) in tissue microarrays (TMAs) sampling multiple tissue cores from each tumor focus. Sampling included index tumors and non-index tumors per whole mounted radical prostatectomy sections in a matched cohort of 91 AA and 109 EA patients from the equal-access military healthcare system (Fig. 1a). Key clinico-pathological features including age at the diagnosis, serum PSA levels at diagnosis, pathological T-stages, Gleason sums, Grade groups, margin status, biochemical recurrence (BCR) and metastasis had no significant differences between AA and EA cases (Suppl. Table 1a). Consistent with the cohort design and long-term follow up (median: 14.5 years), we observed a 40% biochemical recurrence (BCR) and 16% metastasis rate (20). For each case up to four cancerous foci were analyzed, each sampled by two TMA punch cores on average (for details see methods and Suppl. Table 1a, 1b and 1c). We detected monoallelic CHD1 loss in 27 out of 91 AA cases (29.7%), and 14 out of 109 (11%) EA cases indicating that CHD1 deletion is about three times more frequent in prostate tumors of AA men. Our FISH data showed only 3 (2 AA cases and 1 EA case) cases where all TMA punch cores in a single tumor focus harbored CHD1 deletion in the entire samples areas of a given tumor. (Fig. 1b and see the methods and materials “FISH assay part” for details.) In most cases CHD1 deletion was present in only a subset of tumor glands within a 1 mm TMA punch, which further confirmed the subclonal nature of CHD1 deletion in prostate cancer. As a control, we performed FISH staining for PTEN deletion and immunohistochemistry (IHC) staining for ERG overexpression in a subset of the cohort (42 AA and 59 EA prostate cancer cases) confirming previously described frequency differences between AA and EA PCa (4, 5) (Suppl. Table 1e). There was a frequent exclusivity between CHD1 deletion, PTEN deletion and ERG expression both when individual tumor cores or when all tumor cores from a given patient were considered (Suppl. Figures 4a and 4b). In general, the genomic defects including CHD1 deletion, PTEN deletion and ERG expression were mainly detected in index tumors.
Further analyses revealed a significant association between CHD1 deletion and pathologic stages and Gleason sum. Higher frequency of CHD1 deletion was detected in T3-4 pathological stage compared to T2 stage (P = 0.043, Suppl. Table 1d). Prostate cancer cases with higher Gleason sum scores (3 + 4, 4 + 3, 8–10) were seen more frequently in the CHD1 deletion group than in the non-deletion group (P < 0.001). In contrast, lower Gleason sum score (3 + 3) was more often seen in non-deletion cases (P < 0.001, Suppl. Table 1d). The CHD1 deletion was more commonly detected in the cases with higher grade group (GG3 and GG4-GG5) (P = 0.024, Suppl. Table 1d). CHD1 deletion was more strongly associated with rapid biochemical recurrence in AA cases (P < 0.0001, Fig. 1c) than in EA cases (P = 0.051, Suppl Fig. 5b). The univariable survival analysis was conducted to determine the association of the clinical features including CHD1 deletion to BCR and metastasis for further multivariable model analysis (Suppl. Figures 5a and c, respectively). The multivariate Cox model analysis showed that CHD1 deletion was an independent predictor of BCR (P = 0.012 and P = 0.032, Suppl. Figure 5b) after adjusting for age at diagnosis, PSA at diagnosis, race, pathological tumor stage, grade group and surgical margins. Moreover, a significant correlation between CHD1 deletion and metastasis was also detected in both AA (P = 0.0055, Fig. 1d) and EA (P = 0.023, Suppl. Figure 5d) patients with Kaplan-Meier analysis. Following multivariable adjustment in the Cox proportional hazards model, CHD1 deletion was significantly associated with metastasis (P = 0.032 and P = 0.048, Suppl. Figure 5d). Taken together, our data strongly support the association of CHD1 deletions with aggressive prostate cancer and worse clinical outcomes in AA PCa.
Estimating the frequency of subclonal CHD1 loss in next generation sequencing data of AA and EA prostate cancer.
Previous publications characterizing the genome of AA prostate cancer cases (10, 21) did not report an increased frequency of CHD1 loss as we observed in the FISH-based analysis presented above. Methods to detect copy number variations from WGS or WES data have at least two major limitations. First, subclonal copy number variations (sCNV) can be missed if they are present in fewer than 30%, of the sampled cells (19). Second, copy number loss can be underestimated with smaller deletions (e.g., < 10 kb). Although various tools are available for inferring sCNVs from WES, WGS or SNP array data, such as TITAN (19), THetA (22), and Sclust (23), they are designed to work on the entire genome, and likely miss small (~ 1-10kb) CNVs during the data segmentation process. To maximize the accuracy of our analysis we performed a gene focused analysis of the copy number loss in CHD1. We considered several factors such as the change in the normalized coverage in the tumors relative to their normal pairs’, the cellularity of the tumor genome, and the approximate proportion of tumor cells exhibiting the loss. We also evaluated whether the deletion was heterozygous or homozygous using a statistical method designed for calling subclonal loss of heterozygosity (LOH) events within a confined genomic region (details are available in the Materials and Methods section, and in the Supplementary Material).
Using this approach in a large cohort (N = 530 cases; 59 AA WES, 18AA WGS, 408 EA WES and 45 EA WGS, for details see supplementary material and Suppl. Figures 6–25), we observed that CHD1 is more frequently deleted in AA tumors (N = 20; 26%) than in EA tumors (N = 73 EA; 16%). Taken together, when next generation sequencing based copy number variations were analyzed with a more sensitive method, on the combined cohorts of whole exomes and whole genomes, CHD1 loss was detected more frequently in AA cases than in EA cases (p = 0.029, Fisher exact test), which is consistent with our observations with FISH method in the TMA cohort.
Subclonal CHD1 loss is present in a significant subset of prostate cancer cases without SPOP mutations.
SPOP mutations and CHD1 deletions often occur together in prostate cancer, with SPOP mutation as an early event and CHD1 loss is a later, subclonal event during tumor progression (18). However, as we pointed out above, subclonal CHD1 loss is often missed by routine next generation sequencing analysis. Therefore, we reanalyzed the next generation sequencing cohorts for SPOP mutations and found that CHD1 loss and SPOP mutations frequently occur independently from each other as well. In the 530 cases analyzed, we identified 61 SPOP mutant cases and 95 subclonal CHD1 deletions, but only 42 cases (about 68% of SPOP mutants and 44% of CHD1 deleted cases) had both genomic aberration present.
CHD1 loss is not associated with genomic aberration features that are usually observed in HR-deficient cancers.
CHD1 loss was proposed to be associated with reduced HR competence in cell line model systems (15, 24). Detecting and quantifying HR deficiency in tumor biopsies is currently best achieved by analyzing next generation sequencing data for specific HR deficiency associated mutational signatures. Those include: 1) A single nucleotide variation based mutational signature (“COSMIC signatures 3 (25) and SBS3 (26)); 2) a short insertions/deletions based mutational profile, often dominated by deletions with microhomology, a sign of alternative repair mechanisms joining double-strand breaks in the absence of HR, which is also captured by COSMIC indel signatures ID6 and ID8 (26); 3) large scale rearrangements such as non-clustered tandem duplications in the size range of 1-100kb (mainly associated with BRCA1 loss of function) (27). Some of these signatures can be efficiently induced by the inactivation of BRCA1, BRCA2 or several other key downstream HR genes (Suppl. Figures 26–44) (28) .
HR deficiency is also assessed in the clinical setting by a large scale genomic aberration based signature, namely the HRD score (29), which is also approved as companion diagnostic for PARP inhibitor therapy. A composite mutational signature, HRDetect (30), combining several of the mutational features listed above was also evaluated as an alternative method to detect HR deficiency in prostate adenocarcinoma (31). In order to investigate whether an association between CHD1 loss and HR deficiency exists in prostate cancer biopsies, we performed a detailed analysis on the mutational signature profiles of CHD1 deficient prostate cancer.
We analyzed whole exome and whole genome sequencing data of several prostate adenocarcinoma cohorts (For the detailed results see the Supplementary Material) containing samples both from AA (52 WES and 18 WGS cases) and EA (387 WES and 45 WGS) individuals in order to determine whether CHD1 loss is associated with the HRD mutational signatures.
We divided the cohorts into three groups: 1) BRCA2 deficient cases that served as positive controls for HR deficiency, 2) CHD1 deleted cases without mutations in HR genes, and 3) cases without BRCA gene aberration or CHD1 deletion (for details see Supplementary Material).
In the WGS cohorts CHD1 deficient cases showed a limited increase of the HRD score relative to the control cases but significantly lower than the BRCA2 deficient cases and none of the CHD1 deficient cases had an HRD score above the threshold currently accepted in the clinic as an indicator of HR deficiency (Fig. 2a). Since CHD1 deletions tend to be subclonal, we investigated whether the low levels of HRD score is due to a “dilution” effect, where the HR proficient regions without CHD1 deletion reduce the intensity of the HRD score. The HRD score did not show a statistically significant correlation with the estimated fraction of the subclonal loss of CHD1 (Fig. 2a, Suppl. Figure 26–27), and even cases where all cells had CHD1 deletion did not have a high enough HRD score indicating HR deficiency. Similarly, the most characteristic HRD associated single nucleotide variation signature (signature 3, SBS3), was significantly increased in the BRCA2 deficient cases but only slightly increased in the CHD1 deficient cases (Fig. 2b).
The increase of the relative contribution of short indel signatures ID6 and ID8 to the total number of indels characteristic of loss of function on BRCA2 biallelic mutants was not observed in the CHD1 loss cases (Suppl. Figure 32–34). This suggests, that the alternative end-joining repair pathways do not dominate the repair of DSBs in CHD1 deleted tumors.
In the WGS cohort we also determined the number of structural variants (SVs) as previously defined (Suppl. Figure 35)(32). The SV signature associated with HR deficiency (SV3) was not elevated in the CHD1 deficient tumors. Interestingly, an SV signature characterized by an increase in the number of non-clustered 1kb-1Mb deletions (termed RS5 (27)) was significantly increased both in the BRCA2 mutant and CHD1 deficient cases (Fig. 2c), with the latter showing a less significant increase. Notably, this signature also displayed a strong subclonal dilution. This signature was described to be associated with BRCA2 deficiency previously (27, 32) but it is also present in tumors without BRCA2 deficiency and the current version of this signature, SV5 (https://cancer.sanger.ac.uk/signatures/sv/sv5/) is not associated with HR deficiency.
Finally, the BRCA2 deficient cases showed high HRDetect scores (Suppl. Figures 36–38). However, since the HRDetect scores arise from a logistic regression, which involves the non-linear transformation of the weighted sum of its attributes, even slightly lower linear sums in the CHD1 loss cases compared to the BRCA2 mutant cases can result in substantially lower HRDetect scores (Suppl. Figure 38).
We have previously processed WES prostate adenocarcinoma data for the various HR deficiency associated mutational signatures (31). When the CHD1 deficient cases were compared to the BRCA1/2 deficient and BRCA1/2 intact cases we obtained results that were consistent with the WGS based results outlined above (Suppl. Figures 39–44).
Deleting CHD1 in prostate cancer cell lines does not induce homologous recombination deficiency as detected by the RAD51 foci formation assay or mutational signatures.
In order to investigate the functional impact of the biallelic loss of CHD1 we created several CRISPR-Cas9 edited clones of the AR- PC-3 and AR + 22Rv1 cell lines (Fig. 4a, Suppl Fig. 47a). RAD51 foci formation was induced by 4Gy irradiation. The CHD1 deficient prostate cancer cell lines did not show reduction of RAD51 foci formation. (Fig. 3a). As controls, non-irradiated cells were used (Suppl Fig. 46)
DNA repair pathway aberration induced mutational signatures can also be detected in cell lines by whole genome sequencing (28, 33). We grew single cell clones from the PC-3 and 22Rv1 cell lines for 45 generations to accumulate the genomic aberrations induced by CHD1 loss (Suppl. Figure 45). Two of such late passage clones and an early passage clone were subjected to WGS analysis. All the clones retained the BRCA2 wild type background of their parental clone.
Furthermore, CHD1 elimination did not induce any of the mutational signatures commonly associated with HR deficiency (Fig. 3b-d).
Taken together, CHD1 loss in prostate cancer cell line model systems did not induce any signs of HR deficiency.
CHD1 deficient cell lines show limited sensitivity to PARP inhibitors, with talazoparib more effective in some model systems.
CHD1 deficient cancer cells were reported to have moderately increased sensitivity to the PARP inhibitor Olaparib (15), which is consistent with the lack of observed HR deficiency described in the previous section. PARP inhibitors were initially thought to exert their therapeutic activity by inhibiting the enzymatic activity of PARP, but it was later revealed that trapped PARP on DNA may have a more significant contribution to cytotoxicity (reviewed in (34)). Therefore, in addition to olaparib, we also determined the efficacy of the strong PARP trapping agent talazoparib in several prostate cancer cell lines in which CHD1 was either knocked out or suppressed. In addition to the PC-3, 22Rv1 and LNCaP cells with CRISPR-Cas9-mediated CHD1 deletion we also suppressed CHD1 by shRNA in the C4-2b, Du145 and MDA-PCa-2b prostate cancer cell lines, the last one is one of the few AA derived prostate cancer cell line models. Consistent with previous reports, deleting CHD1 induced a maximum of approximately 5-fold increase in olaparib sensitivity with minimal or no change in some cell lines (Fig. 4 panels c, e, i, k, o, q) (15). The increase in talazoparib sensitivity was similar to that of olaparib for most cell lines with a few notable exceptions. Talazoparib sensitivity increased by about 15-20-fold in the CHD1 deficient PC-3 cells (Fig. 4d), and, notably in the CHD1 deficient AA derived cell line (MDA-PCa-2b), talazoparib sensitivity increased by 4-fold (Fig. 4p), while the increase in olaparib sensitivity was approximately 1.5-fold (Fig. 4o). In summary, in four of the six cell lines (Fig. 4d,j,l,p), CHD1 suppression was associated with a talazoparib sensitivity consistent with therapeutically achievable concentrations (around 10nM or less.)
These data suggest that trapped PARP may have a more toxic effect in cells with CHD1 deficiency.
The impact of SPOP mutations on the clonality of CHD1 deletions and HR deficiency associated mutational signatures.
Although less frequent, SPOP mutations and CHD1 deletions may co-exist in a subset of prostate cancer (35) and SPOP mutations have been shown to suppress key HR genes (17). Therefore, we investigated whether the presence of SPOP mutation in a CHD1 deficient prostate cancer is associated with a further increase of HR deficiency associated mutational signatures. We identified cases with SPOP mutations or CHD1 deletions only, cases with both SPOP mutations and CHD1 deletions and cases without either of those aberrations (Fig. 5a). Cases with both mutations showed significantly higher levels of signature SBS3, RS5 and the total number of large-scale structural rearrangements relative to cases with either mutation alone. It should be noted, however, that the proportion of cells in a given tumor with CHD1 deletions tended to be significantly higher in SPOP mutant cases than those with CHD1 deletions without SPOP mutations. Therefore, it is possible that the presence of SPOP will intensify HR deficiency associated mutational signatures by enhancing the proportion of CHD1 deficient cells in a tumor (Fig. 5b).
Finally, we investigated whether adding SPOP mutations to a CHD1 deficient background increases PARP inhibitor sensitivity. We overexpressed the SPOP mutant SPOPF102C in the CHD1 deleted PC3 cells (Suppl. Figure. 48), but we could not detect a further increase either in the olaparib or talazoparib sensitivity (Suppl Fig. 47)