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 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.
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