SUMMARY We define here the activity and mechanisms of action of a small molecule lead compound for cancer targeting. We show that the compound, BMH-21, has wide and potent antitumorigenic activity across NCI60 cancer cell lines and represses tumor growth in vivo. BMH-21 binds GC-rich sequences, which is present at high frequency in ribosomal DNA genes, and potently and rapidly represses RNA polymerase I (Pol I) transcription. Strikingly, we find that BMH-21 causes proteasome-dependent destruction of RPA194, the large catalytic subunit protein of Pol I holocomplex, and this correlates with cancer cell killing. Our results show that Pol I activity is under proteasome-mediated control, which reveals an unexpected therapeutic opportunity.

Genetic instability, a hallmark feature of human cancers including prostatic adenocarcinomas, is considered a driver of metastasis. Somatic copy number alterations are found in most aggressive primary human prostate cancers, and the overall number of such changes is increased in metastases. Chromosome 10q23 deletions, encompassing PTEN, and amplification of 8q24, harboring MYC, are frequently observed, and the presence of both together portends a high risk of prostate cancer-specific mortality. In extant genetically engineered mouse prostate cancer models (GEMMs), isolated MYC overexpression or targeted Pten loss can each produce early prostate adenocarcinomas, but are not sufficient to induce genetic instability or metastases with high penetrance. While a previous study showed that combining Pten loss with focal MYC overexpression in a small fraction of prostatic epithelial cells exhibits cooperativity in GEMMs, additional targeted Tp53 disruption was required for formation of metastases. We hypothesized that driving combined MYC overexpression and Pten loss using recently characterized Hoxb13 transcriptional control elements that are active in prostate luminal epithelial cells would induce the development of genomic instability and aggressive disease with metastatic potential. Neoplastic lesions that developed with either MYC activation alone (Hoxb13-MYC) or Pten loss alone (Hoxb13-Cre|PtenFl/Fl) failed to progress beyond PIN and did not harbor genomic copy number alterations. By contrast, mice with both alterations (Hoxb13-MYC|Hoxb13-Cre|PtenFl/Fl or BMPC) developed lethal adenocarcinoma with distant metastases and widespread genome copy number alterations that were independent of forced disruption of Tp53 and telomere shortening. BMPC cancers lacked neuroendocrine or sarcomatoid differentiation, features uncommon in human disease but common in other models of prostate cancer that metastasize. These data show that combined MYC activation and Pten loss driven by the Hoxb13 regulatory locus synergize to induce genomic instability and aggressive prostate cancer that phenocopies the human disease at the histological and genomic levels.

Background Advanced prostate cancers depend on protein synthesis for continued survival and accelerated rates of metabolism for growth. RNA polymerase I (Pol I) is the enzyme responsible for ribosomal RNA (rRNA) transcription and a rate‐limiting step for ribosome biogenesis. We have shown using a specific and sensitive RNA probe for the 45S rRNA precursor that rRNA synthesis is increased in prostate adenocarcinoma compared to nonmalignant epithelium. We have introduced a first‐in‐class Pol I inhibitor, BMH‐21, that targets cancer cells of multiple origins, and holds potential for clinical translation. Methods The effect of BMH‐21 was tested in prostate cancer cell lines and in prostate cancer xenograft and mouse genetic models. Results We show that BMH‐21 inhibits Pol I transcription in metastatic, castration‐resistant, and enzalutamide treatment‐resistant prostate cancer cell lines. The genetic abrogation of Pol I effectively blocks the growth of prostate cancer cells. Silencing of p53, a pathway activated downstream of Pol I, does not diminish this effect. We find that BMH‐21 significantly inhibited tumor growth and reduced the Ki67 proliferation index in an enzalutamide‐resistant xenograft tumor model. A decrease in 45S rRNA synthesis demonstrated on‐target activity. Furthermore, the Pol I inhibitor significantly inhibited tumor growth and pathology in an aggressive genetically modified Hoxb13‐MYC|Hoxb13‐Cre|Ptenfl/fl (BMPC) mouse prostate cancer model. Conclusion Taken together, BMH‐21 is a novel promising molecule for the treatment of castration‐resistant prostate cancer.

<p>Supplementary Figure 1 Morphology of PIN in B13-MYC mice. Supplementary Figure 2 Phenotypic analysis of PIN in a B13-MYC mouse (ventral lobe, 52 weeks old). Supplementary Figure 3 Additional phenotypic analysis of B-MYC/Pten mouse PIN (anterior lobe, 8 weeks). Supplementary Figure 4 Invasive adenocarcinoma lesion in a 14 week old B-MYC/Pten animal. Supplementary Figure 5 Chromogranin (a) staining in a primary tumor and Foxa2 (b) staining in a lymph node metastasis from a 24 week-old B-MYC/Pten animal shows focal scattered tumor cells staining positively (arrows). Supplementary Figure 6 Morphological features of castrated B-MYC/Pten animals. Supplementary Figure 7. Genes within regions of copy number gains and losses are enriched in specific gene ontology (GO) gene sets. Supplementary Table 1. Prostate Phenotype in B-MYC/PtenFl/Fl mice and wild type (FVB/N) mice. Supplementary Table 2. Metastatic Spread in B-MYC/PtenFl/Fl mice and wild type (FVB/N) mice. Supplementary Table 3. Prostate Phenotype in B-MYC/PtenFl/+ mice and wild type mice. Supplementary Table 4. Effects of androgen withdrawal/castration in B-MYC/PtenFl/Fl mice and wild type (FVB/N) mice. Supplementary Table 5. Metastatic Spread in B-MYC/PtenFl/Fl mice after androgen withdrawal. Supplementary Table 6. Somatic copy number gains and losses in each sample. Supplementary Table 7. Regions of Copy Number alterations in each sample. Supplementary Table 8. Regions with recurrent gains and losses in at least two mice. Supplementary Table 9. Regions of high copy number gain and low copy number loss in any tumor specimen.</p>

<div>Abstract<p>Genetic instability, a hallmark feature of human cancers including prostatic adenocarcinomas, is considered a driver of metastasis. Somatic copy number alterations (CNA) are found in most aggressive primary human prostate cancers, and the overall number of such changes is increased in metastases. Chromosome 10q23 deletions, encompassing <i>PTEN</i>, and amplification of 8q24, harboring <i>MYC</i>, are frequently observed, and the presence of both together portends a high risk of prostate cancer-specific mortality. In extant genetically engineered mouse prostate cancer models (GEMM), isolated <i>MYC</i> overexpression or targeted <i>Pten</i> loss can each produce early prostate adenocarcinomas, but are not sufficient to induce genetic instability or metastases with high penetrance. Although a previous study showed that combining <i>Pten</i> loss with focal <i>MYC</i> overexpression in a small fraction of prostatic epithelial cells exhibits cooperativity in GEMMs, additional targeted <i>Tp53</i> disruption was required for formation of metastases. We hypothesized that driving combined <i>MYC</i> overexpression and <i>Pten</i> loss using recently characterized <i>Hoxb13</i> transcriptional control elements that are active in prostate luminal epithelial cells would induce the development of genomic instability and aggressive disease with metastatic potential. Neoplastic lesions that developed with either <i>MYC</i> activation alone (<i>Hoxb13-MYC</i>) or <i>Pten</i> loss alone (<i>Hoxb13-Cre</i>∣<i>Pten</i>^Fl/Fl) failed to progress beyond prostatic intraepithelial neoplasia and did not harbor genomic CNAs. By contrast, mice with both alterations (<i>Hoxb13-MYC</i>∣<i>Hoxb13-Cre</i>∣<i>Pten</i>^Fl/Fl, hereafter, BMPC mice) developed lethal adenocarcinoma with distant metastases and widespread genome CNAs that were independent of forced disruption of <i>Tp53</i> and telomere shortening. BMPC cancers lacked neuroendocrine or sarcomatoid differentiation, features uncommon in human disease but common in other models of prostate cancer that metastasize. These data show that combined <i>MYC</i> activation and Pten loss driven by the <i>Hoxb13</i> regulatory locus synergize to induce genomic instability and aggressive prostate cancer that phenocopies the human disease at the histologic and genomic levels. <i>Cancer Res; 76(2); 283–92. ©2015 AACR</i>.</p></div>