Resistance to Androgen receptor (AR) antagonists is a significant problem in the treatment of Castration resistant prostate cancers (CRPCs). Identification of the mechanisms by which CRPCs evade Androgen Deprivation Therapies (ADT) is critical to develop novel therapeutics. We uncovered that CRPCs rely on BRD4-HOXB13 epigenetic reprogramming for androgen-independent cell proliferation. Mechanistically, BRD4, a member of the BET bromodomain family epigenetically promotes HOXB13 expression. Consistently, genetic disruption or pharmacological suppression of HOXB13 mRNA and protein expression by the novel dual activity BET bromodomain-kinase inhibitors directly correlates with rapid induction of apoptosis, potent inhibition of tumor cell proliferation, inhibits cell migration and suppresses CRPC growth. Integrative analysis revealed that the BRD4-HOXB13 transcriptome comprises a proliferative gene network implicated in cell cycle progression, nucleotide metabolism and chromatin assembly. Notably, while the core HOXB13 target genes responsive to BET inhibitors (HOTBIN10) are overexpressed in metastatic cases, in ADT treated CRPC cell lines and patient derived circulating tumor cells (CTCs) they are insensitive to AR depletion or blockade. Among the HOTBIN10 genes, AURKB and MELK expression correlate with HOXB13 expression in CTCs of mCRPC patients who did not respond to Abiraterone (ABR), suggesting that AURKB inhibitors could be used additionally against high-risk HOXB13 positive metastatic PCs. Combined, our study demonstrates that BRD4-HOXB13-HOTBIN10 regulatory circuit maintains the malignant state of CRPCs and identifies a core pro-proliferative network driving ADT resistance that is targetable with potent dual activity bromodomain-kinase inhibitors.

HOXB13, a homeodomain transcription factor, is linked to recurrence following radical prostatectomy. While HOXB13 regulates Androgen Receptor (AR) functions in a context dependent manner, its critical effectors in prostate cancer (PC) metastasis remain largely unknown. To identify HOXB13 transcriptional targets in metastatic PCs, we performed integrative bioinformatics analysis of differentially expressed genes (DEGs) in the proximity of the human prostate tumor-specific AR binding sites. Unsupervised Principal Component Analysis (PCA) led to a focused core HOXB13 target gene-set referred to as HOTPAM9 ( HO XB13 T argets separating P rimary A nd M etastatic PCs). HOTPAM9 comprised 7 mitotic kinase genes overexpressed in metastatic PCs, TRPM8 , and the heat shock protein HSPB8 , whose levels were significantly lower in metastatic PCs compared to the primary disease. The expression of a two-gene set, CIT and HSPB8 with an overall balanced accuracy of 98.8% and a threshold value of 0.2347, was sufficient to classify metastasis. HSPB8 mRNA expression was significantly increased following HOXB13 depletion in multiple metastatic CRPC models. Increased expression of HSPB8 by the microtubule inhibitor Colchicine or by exogenous means suppressed migration of mCRPC cells. Collectively, our results indicate that HOXB13 promotes metastasis of PCs by coordinated regulation of mitotic kinases and blockade of a putative tumor suppressor gene.

Overall survival rates for patients with advanced osteosarcoma have remained static for over three decades. An in vitro analysis of osteosarcoma cell lines for sensitivity to an array of approved cancer therapies revealed that panobinostat, a broad spectrum histone deacetalyase (HDAC) inhibitor, is highly effective at triggering osteosarcoma cell death. Using in vivo models of orthotopic and metastatic osteosarcoma, here we report that panobinostat impairs the growth of primary osteosarcoma in bone and spontaneous metastasis to the lung, the most common site of metastasis for this disease. Further, pretreatment of mice with panobinostat prior to tail vein inoculation of osteosarcoma prevents the seeding and growth of lung metastases. Additionally, panobinostat impaired the growth of established lung metastases and improved overall survival, and these effects were also manifest in the lung metastatic SAOS2-LM7 model. Mechanistically, the efficacy of panobinostat was linked to high expression of HDAC1 and HDAC2 in osteosarcoma, and silencing of HDAC1 and 2 greatly reduced osteosarcoma growth in vitro. In accordance with these findings, treatment with the HDAC1/2 selective inhibitor romidepsin compromised the growth of osteosarcoma in vitro and in vivo. Analysis of patient-derived xenograft osteosarcoma cell lines further demonstrated the sensitivity of the disease to panobinostat or romidepsin. Collectively, these studies provide rationale for clinical trials in osteosarcoma patients using the approved therapies panobinostat or romidepsin.

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