Targeting a Novel Epigenetic Signaling Nexus ACK1-pY88H4-AR/AR-V7 in Drug Resistant Metastatic Prostate Cancer

摘要 半个多世纪以来，前列腺癌的研究一直集中在雄激素受体（AR）上， 它在疾病的起始和进展到高转移阶段、去势中的作用 耐药前列腺癌（CRPC）。虽然AR拮抗剂如恩杂鲁胺或雄激素- 合成抑制剂阿比特龙抑制AR功能活性，这些通常长期无效 长期，因为复发性疾病在2-3年内复发。CRPC不仅在低或 阉割雄激素水平，但也严格维持功能性AR。因此，反 雄激素抗性已经成为前列腺癌治疗中最令人烦恼的问题之一。 尽管进行了大量的努力，靶向调节AR及其剪接变体AR-V7的辅因子 转录，直接和有效地，与小分子抑制剂尚未被 办妥了一批我们发现AR募集癌基因ACK 1，一种非受体酪氨酸激酶， 调节自身的表达-在雄激素中建立病理性正反馈回路 剥夺的条件。从机制上讲，激活的ACK 1通过磷酸化 组蛋白H4在一个新的位点，酪氨酸88（pY 88-H4），AR基因上游的三个位点 AREM 1 -3，以促进转录。相反，逆转这种pY 88-H4组蛋白修饰 通过用新的ACK 1抑制剂（R）-9bMS或H4 Y88 F突变体的过表达处理 显著抑制全长AR以及AR-V7剪接变体的转录， 从而下调AR靶基因（例如PSA）的表达。而且我们 证明了WDR 5/MLL 2组蛋白-赖氨酸N-甲基转移酶复合物， pY 88-H4表观遗传标记，存款转录激活H3 K4三甲基标记， 反式，从而揭示了AR基因座的表观遗传调控的新模式。基于这些 广泛的初步数据，我们假设抑制ACK 1表观遗传调节活性， 抑制AR转录和阻断AR剪接变体的产生将是关键， 对抗恩杂鲁胺和阿比特龙耐药CRPC。为了解决这个问题，我们将 追求以下目标： 目标1。评估pY 88-H4沉积位点AREM 1 -3对AR/AR-V7的功能作用 CRPC中的转录。 目标二。检查全基因组MLL 2/WDR 5/ASH 2L和pY 88-H4共缔合及其在细胞内的作用。 驱动AR/AR-V7表达和CRPC进展。 目标3：ACK 1抑制剂（R）-9bMS克服恩杂鲁胺和 阿比特龙耐药性和ADME/DMPK研究。

Abstract For over half a century, prostate cancer research has focused on the Androgen receptor (AR), its role in the initiation of the disease and progression to the highly metastatic stage, castration resistant prostate cancer (CRPC). While AR antagonists such as enzalutamide or androgen- synthesis inhibitor abiraterone dampen AR functional activity, these are often ineffective long term, as the recalcitrant disease recurs within 2-3 years. CRPCs not only thrive under low or castrate levels of androgen, but also fastidiously maintain functional AR. Consequently, anti- androgen-resistance has become one of the most vexing problems in prostate cancer therapy. Despite intensive efforts, targeting co-factors that regulate AR and its splice variant, AR-V7 transcription, directly and efficaciously, with small molecule inhibitors has not yet been achieved. We uncovered that AR recruits the oncogene ACK1, a non-receptor tyrosine kinase, to regulate its own expression- setting up a pathological positive feedback loop in androgen deprived condition. Mechanistically, activated ACK1 modifies chromatin via phosphorylation of the histone H4 at a novel site, tyrosine 88 (pY88-H4), upstream of the AR gene at three sites AREM1-3, to promote transcription. Conversely, reversal of this pY88-H4 histone modification by treatment with a novel ACK1 inhibitor, (R)-9bMS, or overexpression of the H4Y88F mutant significantly suppressed transcription of the full length AR as well as AR-V7 splice variant, consequently downregulating expression of AR-target genes, such as PSA. Moreover, we demonstrate that WDR5/MLL2 histone-Lysine N-Methyltransferase complex, interact with the pY88-H4 epigenetic marks, deposit the transcriptionally activating H3K4 tri-methyl marks in trans, thus uncovering a novel mode of epigenetic regulation at the AR locus. Based on these extensive preliminary data, we hypothesize that inhibition of ACK1 epigenetic regulator activity that suppresses AR transcription and block production of AR splice variants will be critical to combat enzalutamide- and abiraterone-resistant CRPCs. To address this hypothesis, we will pursue the following aims: Aim 1. Assess the functional role of pY88-H4 deposition sites AREM1-3 on AR/AR-V7 transcription in CRPCs. Aim 2. Examine genome wide MLL2/WDR5/ASH2L and pY88-H4 co-association and its role in driving AR/AR-V7 expression and CRPC progression. Aim 3. Detail characterization of ACK1 inhibitor (R)-9bMS to overcome enzalutamide & abiraterone-resistance and ADME/DMPK studies.