Lapatinib and Obatoclax combination therapy

描述(申请人提供)：本实验室以前的研究表明，在癌细胞中，对FDA批准的ErbB1/ERBB2抑制剂Lapatinib致死效应的抵抗是通过促进生存的bcl2家族蛋白如mcl1和bclxl的表达增加而介导的。最近，我们进一步证明，使用分子方法或使用BCL-2家族抑制剂obatoclax(GX15-070)抑制MCL-1和BCL-XL，这是一种处于II期试验的药物，可以促进拉帕替尼的毒性并逆转拉帕替尼的耐药性。这些研究是使用广泛的乳腺和结肠癌细胞在体外进行的；药物组合的毒性与ErbB1/2的过度表达并不简单相关。此外，突变的活性K-RAS表达或P53功能的丧失也不能抑制毒性。这项应用有三个假设：我们假设拉帕替尼+奥贝克拉克斯治疗会导致线粒体自噬的一种有毒形式。我们假设拉帕替尼+奥托克拉克斯治疗在体内的相互作用大于相加的方式，以促进乳腺肿瘤细胞的杀伤；减少肿瘤的生长和转移扩散。我们假设，通过将肿瘤暴露在低剂量的电离辐射下，(拉帕替尼+奥托克拉克斯)在体内的毒性将以大于相加的方式被放大。具体目的1.确定奥曲克雷和拉帕替尼相互作用引起毒性自噬的分子机制。线粒体介导的自噬的分子机制还不完全清楚。我们将确定obtoclax是否通过BAK和NOXA(以及它们与bcl2家族蛋白的改变关联)导致线粒体快速自噬降解，导致细胞ATP水平迅速下降，而不是内质网降解。我们将评估ATG8和ATG6(Beclin1)与线粒体(如HSP60)和ER标记物(如钙网蛋白)的相关性。具体目的2.利用裸鼠和免疫正常小鼠的原位系统，确定奥贝克拉克斯和拉帕替尼是否相互作用，以杀灭肿瘤细胞，抑制肿瘤生长，延长动物存活。我们建议使用多种肿瘤细胞类型来明确测试拉帕替尼和奥托克拉克斯是否在体内相互作用以减少肿瘤生长。我们将使用生长在裸鼠乳房脂肪垫中的人乳腺癌BT474细胞。我们将鼠类乳腺癌MMTV-HER2细胞和4T1细胞分别接种于免疫活性同基因FVB/NJ和BALB/c小鼠的乳房脂肪垫中；对于4T1细胞，我们将通过生物发光成像来确定药物治疗对转移瘤细胞扩散和生长的影响。具体目的3.确定(拉帕替尼+奥托克拉克斯)疗法在体内是否具有放射增敏作用。我们将在体外和体内确定(拉帕替尼+奥巴克拉克斯)治疗是否增强肿瘤细胞的放射敏感性。根据在AIM 2中获得的肿瘤生长/存活数据，将在AIM 3中选择其中一个肿瘤细胞株进行(Lapatinib+obatoclax)治疗和辐射照射的体内评估。拉帕替尼是FDA批准的ErbB1/ERBB2抑制剂。Obtoclax正在进行第二阶段的试验。申请中建议的研究的成功完成将迅速促进这种联合疗法在乳腺癌患者中的I期翻译。 公共卫生相关性： 公共卫生相关声明/项目叙述。每年有200,000名患者被诊断为乳腺癌，每年约有40,000人死亡。因此，尽管许多乳腺癌患者有很好的5年生存率，但仍有必要将患者的生存率提高到更高的水平。拉帕替尼是FDA批准的用于治疗乳腺癌患者的ErbB1/ERBB2抑制剂。Obatoclax(GX15-070)是一种保护性bcl2家族蛋白的抑制剂，目前处于II期试验。我们发现，拉帕替尼+奥巴克拉克斯的组合可以迅速杀死耐拉帕替尼和拉帕替尼的乳腺癌细胞。从新开始或对癌症治疗产生抵抗力，通常是通过过度表达保护性的bcl2家族蛋白，这是获得更好的患者预后的主要障碍。由于Lapatinib已经获得FDA批准，本申请中建议的研究的成功完成将迅速促进该药物组合在复发乳腺癌患者中的I期翻译。

DESCRIPTION (provided by applicant): Prior studies from this laboratory have demonstrated that resistance to the lethal effects of the FDA approved ERBB1 / ERBB2 inhibitor Lapatinib in carcinoma cells is mediated by increased expression of pro-survival BCL-2 family proteins e.g. MCL-1 and BCL-XL. More recently we have gone on to demonstrate that inhibition of MCL-1 and BCL-XL using molecular approaches or using the BCL-2 family inhibitor Obatoclax (GX15-070), a drug in phase II trials, promotes Lapatinib toxicity and reverts Lapatinib resistance. These studies were performed in vitro using a wide range breast and colon cancer cells; drug combination toxicity did not simplistically correlate with ERBB1/2 over-expression. Toxicity, also, was not suppressed by mutant active K- RAS expression or loss of p53 function in the colon cancer cells. This application has three hypotheses: We hypothesize that Lapatinib + Obatoclax treatment causes a toxic form of mitochondrial autophagy. We hypothesize that Lapatinib + Obatoclax treatment interact in vivo in a greater than additive fashion to promote mammary tumor cell killing; reducing tumor growth and metastatic spread. We hypothesize that (Lapatinib + Obatoclax) toxicity in vivo will be magnified in a greater than additive fashion by exposure of the tumor to low doses of ionizing radiation. Specific Aim 1. Determine the molecular mechanisms by which Obatoclax and Lapatinib interact to cause toxic autophagy. The molecular mechanisms of mitochondrial mediated autophagy are not fully understood. We will determine whether Obatoclax causes rapid autophagic degradation of mitochondria via BAK and NOXA (and their altered association with BCL-2 family proteins), with a rapid drop in cellular ATP levels, rather than degradation of the endoplasmic reticulum. We will assess the association of ATG8 and ATG6 (Beclin1) with mitochondrial e.g. HSP60, and ER markers e.g. calreticulin. Specific Aim 2. Determine using orthotopic systems in athymic and immune competent mice whether Obatoclax and Lapatinib interact to kill tumor cells, suppress tumor growth and prolong animal survival. We propose to use multiple tumor cell types to definitively test whether Lapatinib and Obatoclax interact in vivo to reduce tumor growth. We will use human mammary carcinoma BT474 cells growing in the mammary fat pad of athymic mice. We will grow rodent mammary carcinoma MMTV-HER2 cells and 4T1in the mammary fat pad of immune competent syngeneic FVB/NJ and BALB/c mice, respectively; for 4T1 cells we will determine the impact of drug treatment on metastatic tumor cell spread and growth via bioluminescent imaging. Specific Aim 3. Determine whether (Lapatinib + Obatoclax) therapy acts as a radiosensitizer in vivo. We will determine whether (Lapatinib + Obatoclax) treatment enhances tumor cell radiosensitivity in vitro and in vivo. Based on tumor growth / viability data obtained in Aim 2, one of the tumor cell lines will be selected for in vivo evaluation with (Lapatinib + Obatoclax) treatment and radiation exposure in aim 3. Lapatinib is an FDA approved ERBB1 / ERBB2 inhibitor. Obatoclax is in phase II trials. The successful completion of the studies proposed in the application would rapidly facilitate phase I translation of this combination therapy in breast cancer patients. PUBLIC HEALTH RELEVANCE: Public Health Relevance Statement / Project Narrative. Breast cancer is diagnosed in > 200,000 patients per annum, with ~40,000 deaths / year. Thus, although many breast cancer patients have a good 5 year survival rate there is still a major need to improve patient survival to higher levels. Lapatinib is an FDA approved ERBB1 / ERBB2 inhibitor used to treat breast cancer patients. Obatoclax (GX15-070) is an inhibitor of protective BCL-2 family proteins and is in phase II trials. We have discovered that a combination of Lapatinib + Obatoclax rapidly kills naove and Lapatinib resistant breast cancer cells. De novo or developed resistance to cancer therapies, frequently through over-expression of protective BCL- 2 family proteins, and this is a major impediment in obtaining better patient outcomes. As Lapatinib is already FDA approved, the successful completion of the studies proposed in this application would rapidly facilitate phase I translation of the drug combination in patients with recurrent breast cancer.