Lapatinib and Obatoclax combination therapy

描述（由申请人提供）：该实验室先前的研究表明，FDA批准的ERBB1 / ERBB2抑制剂拉帕替尼在癌细胞中对致死效应的抗性是通过促生存BCL-2家族蛋白如MCL-1和BCL-XL的表达增加介导的。最近，我们继续证明，使用分子方法或使用BCL-2家族抑制剂Obatoclax (GX15-070)（一种处于II期试验的药物）抑制MCL-1和BCL-XL，可促进拉帕替尼毒性并恢复拉帕替尼耐药性。这些研究是在体外进行的，使用了广泛的乳腺癌和结肠癌细胞；药物联合毒性与ERBB1/2过表达无关。在结肠癌细胞中，活性K- RAS表达的突变或p53功能的丧失也不会抑制毒性。该应用有三个假设：我们假设拉帕替尼+ Obatoclax治疗引起线粒体自噬的毒性形式。我们假设拉帕替尼+ Obatoclax治疗在体内的相互作用比相加的方式更能促进乳腺肿瘤细胞的杀伤；减少肿瘤生长和转移扩散。我们假设（拉帕替尼+ Obatoclax）在体内的毒性会因肿瘤暴露于低剂量电离辐射而以大于相加的方式被放大。具体目标确定Obatoclax和拉帕替尼相互作用导致毒性自噬的分子机制。线粒体自噬的分子机制尚不完全清楚。我们将确定Obatoclax是否通过BAK和NOXA（以及它们与BCL-2家族蛋白的改变关联）引起线粒体的快速自噬降解，并导致细胞ATP水平的快速下降，而不是内质网的降解。我们将评估ATG8和ATG6 （Beclin1）与线粒体（如HSP60）和内质网标志物（如钙网蛋白）的关联。具体目标2。在胸腺和免疫功能正常的小鼠中使用原位系统确定Obatoclax和拉帕替尼是否相互作用杀死肿瘤细胞，抑制肿瘤生长并延长动物存活。我们建议使用多种肿瘤细胞类型来明确测试拉帕替尼和Obatoclax是否在体内相互作用以减少肿瘤生长。我们将使用生长在胸腺小鼠乳腺脂肪垫中的人乳腺癌BT474细胞。我们将在免疫正常的同源FVB/NJ和BALB/c小鼠乳腺脂肪垫中分别培养啮齿动物乳腺癌MMTV-HER2细胞和4t1细胞；对于4T1细胞，我们将通过生物发光成像确定药物治疗对转移性肿瘤细胞扩散和生长的影响。具体目标3。确定（拉帕替尼+ Obatoclax）治疗是否在体内作为放射增敏剂。我们将确定（拉帕替尼+ Obatoclax）治疗是否在体外和体内增强肿瘤细胞的放射敏感性。根据Aim 2中获得的肿瘤生长/活力数据，将在Aim 3中选择一种肿瘤细胞系进行（拉帕替尼+ Obatoclax）治疗和辐射暴露的体内评估。拉帕替尼是FDA批准的ERBB1 / ERBB2抑制剂。Obatoclax正在进行II期临床试验。申请中提出的研究的成功完成将迅速促进该联合疗法在乳腺癌患者中的I期转化。

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.