Molecular ontology of drug tolerant persisters in HER2 positive breast cancer - Resubmission - 1

摘要：针对“致癌基因”的“靶向治疗”在许多肿瘤的治疗中发挥着关键作用。 包括由编码受体酪氨酸激酶HER2的基因扩增引起的乳腺癌 (HER2+BC)。然而，在转移性HER2+BC中，甚至对抗癌药物的组合也会产生耐药性 药物，并防止其出现，对于将缓解转化为治疗至关重要。“耐药的持久者” (DTP)在暴露于酪氨酸激酶抑制剂(TKI)和其他抗肿瘤药物的非增殖性、 通过可逆的、非遗传机制的静止(休眠)状态。DTP与抵抗猪瘟有关 EGFR突变的非小细胞肺癌(NSCLC)、BRAFmut黑色素瘤和其他癌症，但在 在HER2+BC中，还没有得到彻底的探索。我们发现用HER2-TKI处理的HER2+BC细胞系 (拉帕替尼、图卡替尼)产生两种类型的DTP，它们有腔转录体或间充质转录体。AS 在其他细胞系中，部分HER2+BC细胞在退出有丝分裂后随机通过G0 直接进入G1。值得注意的是，管腔DTP独特地出现在这些短暂的G0细胞(“Pre-Pre- DTP“)，也表达DTP基因的一个子集。HER2+BC高度增殖，但像其他肿瘤一样， 它们也有大量的G0(Ki67-)细胞，我们对TKI治疗后的样本进行了初步分析 HER2+BC患者可从G0细胞产生DTP。因此，与目前的模型相比，它 争辩说，耐药、静止状态是由TKI和其他有针对性的 治疗/化疗，我们的数据表明DTP前可能已经存在于药物中--幼稚 人口。 我们假设这种瞬时的G0/Pre-DTP细胞的染色质具有全局不同的 启动它们以诱导完整的DTP转录组并对其产生耐药性的组织 TKI曝光。这种“G0选择/诱导”模型是否适用于间充质样HER2+DTP 由其他因素诱导，和/或更广泛地说，到真正的HER2肿瘤仍不清楚。因为DTP 包括潜在的细胞储存库，用于播种对HER2 TKIs和其他 靶向/常规治疗，描绘其个体发生机制，可能揭示新的癌症 脆弱性和最终的新疗法。我们关注的是G0细胞基因调控的独特特征 也可能对肿瘤休眠有影响。这项建议加入了表观遗传学/染色体专家的行列 组织(SKOK)和癌症生物学/细胞信号(NEEL)来解决这些及时、相关的问题 具体地说，我们将：(1)表征HER2-TKI Pre-DTPs和HER2-TKI Pre-DTPs中的染色质结构和基因调控 来自管腔样HER2+BC系的DTP，(2)检验来自间充质HER2-TKI DTP的假设 HER2+BC系也起源于类似G0的Pre-DTP，并且具有凝集素II组织的染色质，以及(3)测试 拉帕替尼DTPs在HER2-TKI DTPs体内模型中的病理生理学意义

SUMMARY: “Targeted therapies” against “driver oncogenes” play key roles in the therapy of many tumors, including breast cancers caused by amplification of the gene encoding the receptor tyrosine kinase HER2 (HER2+ BC). In metastatic HER2+ BC, however, resistance occurs even to combinations of anti-cancer drugs, and preventing its emergence is essential for converting remissions to cures. “Drug-tolerant persisters” (DTPs) survive exposure to tyrosine kinase inhibitors (TKIs) and other anti-neoplastics in a non-proliferative, quiescent (dormant) state via reversible, non-genetic mechanisms. DTPs are implicated in resistance of EGFRmut non-small cell lung cancer (NSCLC), BRAFmut melanoma, and other cancers, but a potential role in in HER2+ BC had not been explored thoroughly. We found that HER2+ BC lines treated with HER2-TKIs (lapatinib, tucatinib) give rise to two types of DTPs, which have luminal or mesenchymal transcriptomes. As in other cell lines, a fraction of HER2+ BC cells transit stochastically through G0 after exiting mitosis, instead of proceeding directly into G1. Remarkably, luminal DTPs arose uniquely from these transient G0 cells (“pre- DTPs”), which also express a subset of DTP genes. HER2+ BCs are highly proliferative, yet like other tumors, they also have significant numbers of G0 (Ki67-) cells, and our initial analyses of samples from TKI-treated HER2+ BC patients comport with DTP generation from G0 cells. Thus, in contrast to current models, which contend that the drug-tolerant, quiescent state is induced by TKIs and other targeted therapies/chemotherapies, our data indicate that pre-DTPs might already be present in the drug-naïve population. We hypothesize that the chromatin of such transient, G0/pre-DTP cells has a globally distinct organization that primes them to induce the complete DTP transcriptome and become drug-tolerant upon TKI exposure. Whether this “G0 selection/induction” model applies to mesenchymal-like HER2+ DTPs, DTPs induced by other agents, and/or more broadly to bona fide HER2 tumors remains unclear. Because DTPs comprise a potential cellular reservoir for seeding stable resistance to HER2 TKIs and other targeted/conventional therapies, delineating their ontogenetic mechanisms could reveal novel cancer vulnerabilities and ultimately, new therapies. Our focus on the unique features of gene regulation in G0 cells could also have implications for tumor dormancy. This proposal joins experts in epigenetics/chromosome organization (SKOK) and cancer biology/cell signaling (NEEL) to address these timely, relevant questions Specifically, we will: (1) Characterize chromatin architecture and gene regulation in HER2-TKI pre-DTPs and DTPs from luminal-like HER2+ BC lines, (2) Test the hypothesis that HER2-TKI DTPs from mesenchymal HER2+ BC lines also arise from G0-like pre-DTPs and have condensin II-organized chromatin, and (3) Test the pathophysiological significance of lapatinib DTPs in in vivo models of HER2-TKI DTPs.