Project 2: Mechanisms Driving AR Full Length and Splice Variant Activities and Antagonist Resistance

我们的总体目标是解剖雄激素受体（AR）的转录激活 和AR剪接变体，以及在前列腺癌（PCa）中驱动这种活性的机制， 对包括阿比特龙在内的第二代AR靶向疗法产生耐药性， 恩杂鲁胺。我们最近发现AR募集蛋白磷酸酶1（PP 1 α）， 然后使CDK 9去磷酸化并使P-TEFb复合物移动。目标1： 与AR配体结合结构域相互作用的PP 1调节蛋白的鉴定 （独立于雄激素）介导这种相互作用，并在确定是否/如何 AR-V7剪接变体介导PP 1 α募集和随后的P-TEFb动员。我们 以前和现在的研究也表明，在AR N-末端的S81磷酸化 结构域（NTD）在驱动转录中起主要作用，并且是正反馈的枢纽 在低雄激素水平或存在AR拮抗剂的情况下，可能放大AR活性的环。 因此，目标2的重点是鉴定直接或间接与人的免疫反应有关的共激活因子相互作用。 受S81磷酸化调节，S81磷酸化在AR-V7活性中的作用，以及 CDK 9抑制剂的治疗潜力。目标3然后进一步聚焦于AR剪接变体。而 AR剪接变体同源二聚体可以驱动转录，来自其他人和我们的证据表明， AR-FL和AR剪接变体之间的异二聚体起主要作用。因此，我们将测试 假设AR-FL/V7异二聚体是AR活性的主要介质， Enzalutamide耐药PCa模型。然后，我们将重点讨论LBD在这些方面的作用 异二聚体，以及它们是否仍然是治疗靶点。最后，我们将确定 除了增加表达外，似乎还增强了AR-V7在细胞中的活性， Enzalutamide耐药PCa细胞。具体目标是：1）确定 通过AR全长和剪接变体募集PP 1 α，2）确定AR S81的功能 在驱动AR全长和剪接变体中的磷酸化，3）通过 AR-V7在抗酶模型中驱动AR活性。

Our overall objective has been to dissect transcriptional activation by androgen receptor (AR) and AR splice variants, and mechanisms driving this activity in prostate cancer (PCa) that becomes resistant to second generation AR targeted therapies including abiraterone and enzalutamide. We recently found that AR recruits protein phosphatase 1 (PP1α), which can then dephosphorylate CDK9 and mobilize the P-TEFb complex. In Aim 1 we focus on identification of the PP1 regulatory protein that interacts with the AR ligand binding domain (independently of androgen) that mediates this interaction, and on determining whether/how the AR-V7 splice variant mediates PP1α recruitment and subsequent P-TEFb mobilization. Our previous and current studies also indicate that phosphorylation of S81 in the AR N-terminal domain (NTD) plays a major role in driving transcription, and is a hub for a positive feedback loop that may amplify AR activity at low androgen levels or in the presence of AR antagonists. Therefore, Aim 2 focuses on identification of coactivator interactions that are directly or indirectly regulated by S81 phosphorylation, the role of S81 phosphorylation in AR-V7 activity, and the therapeutic potential of CDK9 inhibitors. Aim 3 then focuses further on AR splice variants. While AR splice variant homodimers can drive transcription, evidence from others and us indicate that heterodimers between AR-FL and AR splice variants play a major role. Therefore, we will test the hypothesis that AR-FL/V7 heterodimers are the major mediators of AR activity in enzalutamide-resistant PCa models. We then will focus on the role of the LBD in these heterodimers, and whether they it may still be a therapeutic target. Finally, we will identify mechanisms in addition to increased expression that appear to be enhancing AR-V7 activity in enzalutamide-resistant PCa cells. The Specific Aims are 1) Determine the molecular basis for PP1α recruitment by AR full length and splice variants, 2) Determine the function of AR S81 phosphorylation in driving AR full length and splice variants, 3) Identify mechanisms through which AR-V7 drives AR activity in ENZ-resistant models.