Novel Triple-Negative Breast cancer vulnerability governed by PNPT1-mediated RNA decay

项目总结/摘要 乳腺癌是最常见的癌症，也是女性癌症死亡的主要原因。三阴性 乳腺癌（TNBC）是具有有限治疗选择的高度侵袭性和致死性的乳腺癌亚型。 很大一部分TNBC具有转录因子MYC的超活化，其驱动基因表达。 调节异常并促进TNBC的生长和增殖。另一方面，它也被 表明癌症中的转录扩增，包括那些由MYC过度激活驱动的， 对参与RNA代谢的分子机器的特定负担。作为这样的例子之一，我们已经表明， 抑制参与RNA剪接的分子可导致显著的细胞应激和MYC+ TNBC的死亡。 然而，虽然突破性的研究集中在RNA合成和加工的失调， 然而，对于MYC等致癌基因如何解除RNA衰变途径的调控，从而驱动异常的RNA降解途径， 基因调控外切核糖核酸酶介导的RNA降解是RNA代谢的关键组成部分。虽然 存在于各种疾病环境中的核糖核酸外切酶的缺陷与细胞内 然而，由于RNA转录物的错误加工，这种与癌症的相关性还远未被理解。通过先前的全基因组 正向遗传筛选，我们确定了线粒体3'至5'核糖核酸外切酶PNPT 1的沉默是致命的， 特别是在MYC过度激活的情况下。癌症相关性地图（DepMap） 数据库进一步揭示了PNPT 1的功能在TNBC模型中是至关重要的。最近的证据 证明PNPT 1的缺陷可导致细胞内双链RNA（dsRNA）的积累， 以及在癌细胞中诱导dsRNA介导的抗病毒样免疫和凋亡应答。同样地， 我们已经证实，SUM 159（一种MYC依赖性人TNBC细胞系）中PNPT 1的沉默也导致了 细胞内dsRNA的积累。这些初步的发现使我们假设PNPT 1起作用， 在去除异常RNA转录物和防止dsRNA介导的细胞凋亡中起关键作用。 压力，从而赋予生存的好处，跨国公司。在此，我们建议利用尖端的化学- 遗传学（PROTAC）工具，RNA测序和蛋白质组学分析方法来阐明PNPT 1的作用 TNBC生存和进展。我们将1）评估PNPT 1扰动对dsRNA诱导的影响- 2）检查PNPT 1扰动对总RNA的影响 3）确定PNPT 1扰动对TNBC存活和进展的影响。 该项目的成功完成将为PNPT 1介导的RNA衰变提供新的见解 有助于维持TNBC的增长和生存。此外，这项研究将作为发展的基础， 靶向PNPT 1的新治疗方法，用于潜在的TNBC治疗。此外，该研究将 支持将其他核糖核酸外切酶和RNA衰变途径作为TNBC的潜在脆弱性进行评估 以及其他携带RNA代谢失调的癌症。

PROJECT SUMMARY / ABSTRACT Breast cancer is the most common cancer and the leading cause of cancer death in females. Triple negative breast cancer (TNBC) is a highly aggressive and lethal subtype of breast cancer with limited treatment options. A significant portion of TNBCs harbor hyperactivation of transcription factor MYC, which drives gene dysregulation and promote the elevated growth and proliferation of TNBCs. On the other hand, it has also been shown that transcriptional amplification in cancers, including those driven by MYC hyperactivation, places specific burdens on molecular machines involved in RNA metabolism. As one of such examples, we have shown that inhibiting molecules involved in RNA splicing can lead to significant cell stress and death of MYC+ TNBCs. However, while groundbreaking research has focused on dysregulation of RNA synthesis and processing in cancer, far less is known about how oncogenes such as MYC deregulate RNA decay pathways to drive aberrant gene regulation. Exoribonuclease-mediated RNA decay is a critical component of RNA metabolism. Although defects in exoribonucleases present in various disease settings have been linked to the cellular accumulation of misprocessed RNA transcripts, such relevance in cancer is far from understood. Through a prior genome-wide forward genetic screen, we identified that silencing of mitochondrial 3’ to 5’ exoribonuclease PNPT1 is lethal specifically in the context of MYC hyperactivation. Subsequent analysis of Cancer Dependency Map (DepMap) database further revealed that the function of PNPT1 is critical across models of TNBCs. Recent evidence demonstrated that defects in PNPT1 can lead to the accumulation of intracellular double-stranded RNA (dsRNA) and the induction of dsRNA-mediated antiviral-like immune and apoptotic responses in cancer cells. Similarly, we have confirmed that silencing of PNPT1 in SUM159, a MYC-dependent human TNBC line, also leads to the accumulation of intracellular dsRNA. These preliminary findings lead us to hypothesize that PNPT1 plays a critical role in the removal of aberrant RNA transcripts and prevents the induction of dsRNA-mediated cell stresses, thereby conferring survival benefits to TNBCs. Herein, we propose to leverage cutting-edge chemical- genetic (PROTAC) tools, RNA sequencing, and proteomic analysis approaches to elucidate the role of PNPT1 in TNBC survival and progression. We will 1) Assess the effects of PNPT1 perturbation on induction of dsRNA- mediated antiviral responses in TNBCs; 2) Examine the effects of PNPT1 perturbation on overall RNA metabolism in TNBCs; and 3) Determine the effects of PNPT1 perturbation on TNBC survival and progression. Successful completion of the proposed project will provide novel insights into how PNPT1-mediated RNA decay helps sustain TNBC growth and survival. Further, the study will serve as a foundation for the development of novel therapeutic approaches targeting PNPT1 for the potential treatment of TNBCs. Additionally, the study will support the evaluation of other exoribonucleases and RNA decay pathways as potential vulnerabilities of TNBCs and other cancers harboring dysregulated RNA metabolism.