Supplement: Synergy of Copanlisib with EZH2 inhibition in PIK3CA-driven NSCLCs

These funds are intended to be used for the funded Proposal. Describe how the Supplemental Funds will be used. Grantee may request to extend the term of the grant up to 12 months. Please limit to 2 pages including references. I would like to thank the AACR the opportunity to procure additional funding for the project “Synergy of Copanlisib with EZH2 inhibition in PIK3CA-driven NSCLCs”. For this additional funding ($5000) and time (1 year), the Aims of the grant will remain the same, and are: Specific Aim 1: to build isogenic PI3K-driven lines and test combination of EZH2 inhibition with PI3K inhibition Specific Aim 2: to develop a model of PI3K-driven lung cancer and combination of EZH2 inhibition with PI3K inhibition Progress for Aim 1 and Rationale for Additional Funds: To date, we have learned that PIK3CA-E545K is a weak oncogene that is able to sensitize tumors to a combination of Bayer’s PI3K inhibitor copanlisib, and EZH2 Inhibitors including GSK126 and EPZ6438. Here, we have included EPZ6438 (aka tazemetosat) because of its recent FDA approval (1). In contrast, in KRAS-driven lung adenocarcinomas, there is little synergy and sometimes antognism between these two drug classes. We have generated isogenic BEAS2B cell lines with dominant negative p53 (R175H) and either PIK3CA-E545K or KRAS-G12V mutations. Soft agar assays showed that PIK3CA-E545K is mildly transforming while KRAS-G12V is strongly transforming. Because PIK3CA-E545K is only mildly transforming, the cells that we have prepared do not form tumors in Nude mice as we expected. We will publish this finding so other investigators know this issue. In order to complete the drug synergy experiments in vivo, we plan to use a portion of additional funds for Nude mice experiments with established PI3K and KRAS driven human lung cancer cell lines including Calu3, Sw1573 and A549. Furthermore, our in vitro assays have shown that EPZ6438 as a single agent is not lethal, even to PI3K-driven lung cancers cells, but does synergize with copanlisib. Because EPZ6438, but not GSK126, is now clinically available, we would like to test both EPZ6438 and GSK126 in the in vivo models. Therefore, the additional funds will be used to purchase Nude mice, house the Nude mice and to purchase laboratory consumables including drugs for this work. Progress for Aim 2 and Rational for Additional Funds: The second Aim of our proposal was to develop a novel model of PI3K genetically induced autochthonous lung cancer in mice using the Pik3ca-FSF-E545K, p53 fl/fl mice (2). As planned, we induced mice with our standard titer of adeno-Cre virus and waited four to six months to start screening the mice by MRI. However, none of the MRI we did showed any tumor growth, which was confirmed after sacrificing a portion of the cohort. We did observe some hyperplasia by histology at 6 months, so decided to wait longer. Excitingly, at the 12 to 14 months time-point mice did have established tumors. We are hopeful this a new model of squamous lung cancer given the similar latency at the Pten fl/fl; Lkb1 fl/fl model (3). Histology on these tumors in pending, and we are now making a three-dimensional organoid cultures from these tumors, which should be transplantable into Nude mice. Therefore, we plan to use to remainder of the additional funds to complete histoogical analysis of these tumors, and to establish the three-dimensional tumor organoid cutlures and test if they can grow orthotopically in Nude mice after intratracheal transplantation. Therefore, the additional funds will be used to pay cage costs for additional mice, purchase Nude mice for transplant, purchase additional laboratory consummables for establishing organoids and histology costs.