Targeting STAT3 for the Treatment of CDK4/6 Inhibitor Resistant Advanced Estrogen Receptor Positive Breast Cancer Patients

摘要 雌激素受体阳性(ER+)/HER2阴性乳腺癌占所有乳腺癌病例的70%。 手术和辅助/新辅助内分泌治疗(ET)是早期疾病治疗的主要手段。 然而，一些接受ET治疗的早期ER阳性乳腺癌患者只有部分减少 他们复发和死亡的风险，以及那些患有晚期乳腺癌(ABC)的人在不久之后就会进展 启动治疗(内在抵抗)，或最终经历随时间进展(获得性抵抗)。 CDK4/6抑制剂(CDK4/6iS)联合ET目前被认为是晚期ER患者的标准护理 +/HER2阴性乳腺癌。CDK4/6抑制的一个关键特征是它引起的细胞周期抑制反应 通过诱导衰老，可以逃脱，导致细胞很容易重新进入细胞周期 一旦药物被撤除。衰老细胞分泌白介素类、炎性细胞因子和生长因子， 包括影响周围细胞的衰老相关分泌表型(SASP)和 促进肿瘤生长。最突出的SASP细胞因子是白介素6(IL-6)，它与 转移、肿瘤侵袭性和生存率下降。IL-6激活STAT3，它与一种 更具侵袭性的表型和对多种疗法的耐药性[化疗、靶向治疗和免疫 检查点抑制疗法]。我们建立了CDK4/6I(即Palbociclib)耐药乳腺肿瘤细胞系模型，并 他们的分子分析表明，耐药细胞通过上调IL-6和 STAT3的激活(STAT3在Y705上的磷酸化，Py-STAT3)。对耐药细胞的处理 口服STAT3小分子抑制剂(TTI-101)使细胞活力降低25倍，并导致细胞活力下降 在(I)干状(CD44高/CD24低)群体中，Py-STAT3水平及其伴随的降低，(Ii)原发和 二次乳房形成，(Iii)EMT途径。此外，TTI-101对荷瘤小鼠的治疗作用 患者来源的异种移植(PDX)表达与Palbociclib耐药细胞相似的基因表达特征 LINES导致肿瘤体积显著缩小，延长无瘤生存期，并下调血清水平 IL-6水平。我们假设抑制IL-6和/或STAT3可以在体内逆转获得性CDK4/6I耐药 转基因和PDX模型，以及基于CDK4/6I的治疗进展的患者。我们提出了一个 协调机制、临床前和早期临床测试策略以开发生物标记物合格 临床治疗需要克服CDK4/6I耐药。为了实现这些目标，我们将1)：确定 长期抑制CDK4/6体内诱导IL-6的机制及IL-6对小鼠肿瘤发生的影响 转基因小鼠模型；2)对帕罗西利耐药小鼠和转基因小鼠进行临床前试验 抑制STAT3和IL-6能否提高CDK4/6I耐药小鼠的存活率 肿瘤；以及3)进行IB/II期临床试验，将TTI-101添加到标准护理Palbociclib和芳香酶中 进展时的抑制物。

ABSTRACT Estrogen receptor-positive (ER+)/HER2-negative breast cancer represents 70% of all breast cancer cases. Surgery and adjuvant/neo-adjuvant endocrine therapy (ET) are mainstays of treatment in early stage disease. However, some patients receiving ET for early stage ER-positive breast cancer only have a partial reduction in their risk of recurrence and mortality, and those with advanced breast cancer (ABC) either progress shortly after initiating therapy (intrinsic resistance), or ultimately experience progression over time (acquired resistance). CDK4/6 inhibitors (CDK4/6is) with ET are currently considered standard of care for patients with advanced ER +/HER2 negative breast cancer. A key feature of CDK4/6 inhibition is the cell cycle inhibitory response it elicits through induction of senescence, which can be escaped resulting in cells readily re-entering the cell cycle as soon as drug is withdrawn. Senescent cells secrete interleukins, inflammatory cytokines, and growth factors, which comprise the senescence-associated secretory phenotype (SASP) that affects surrounding cells and promotes tumor growth. The most prominent SASP cytokine is interleukin-6 (IL-6), which is associated with metastasis, tumor aggressiveness and decreased survival. IL-6 activates STAT3, which is associated with a more aggressive phenotype and resistance to many therapies [chemotherapy, targeted therapy, and immune checkpoint inhibitor therapy]. We developed CDK4/6i (i.e. palbociclib) resistant breast tumor cell line models and their molecular analysis showed that resistant cells adapt to palbociclib treatment by upregulation of IL-6 and activation of STAT3 (phosphorylation of STAT3 on Y705, pY-STAT3). Treatment of the resistant cells with an oral, small-molecule inhibitor of STAT3 (TTI-101) decreased cell viability by >25-fold and resulted in decreased levels of pY-STAT3 with concomitant decreases in (i) stem-like (CD44high/CD24low) population, (ii) primary and secondary mammosphere formation, (iii) the EMT pathway. Furthermore, TTI-101 treatment of mice bearing patient derived xenografts (PDX) that express a similar gene expression signature as palbociclib-resistant cell lines resulted in a marked decrease in tumor volume, prolonged tumor-free survival and downregulation of serum IL-6 levels. We hypothesize that inhibition of IL-6 and/or STAT3 can reverse acquired CDK4/6i resistance in vivo transgenic and PDX models and in patients who have progressed on CDK4/6i based therapy. We propose a coordinated mechanistic, preclinical and early phase clinical testing strategy to develop biomarker-qualified therapy for the clinical need to overcome CDK4/6i resistance. To address these goals we will 1): Determine the mechanism of IL-6 induction by long term CDK4/6 inhibition in vivo and the impact of IL-6 on tumorigenesis in transgenic mouse models; 2) Conduct pre-clinical trials in palbociclib resistant PDX and transgenic mouse models to determine if inhibition of STAT3 and IL-6 can improve the survival of mice with CDK4/6i resistant tumors; and 3) Perform a Phase IB/II clinical trial of adding TTI-101 to standard of care palbociclib and aromatase inhibitor upon progression.