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Background: CDK4/6 inhibitors have been approved in combination with endocrine therapy for the treatment of estrogen receptor positive (ER+) metastatic breast cancer. The goal of this study was to discover mechanisms of resistance to ER antagonists alone and in combination with CDK4/6 inhibitors. Results: p21-activated kinase 1 (PAK1) is a serine/threonine kinase amplified in several human cancer types. Specifically, the survey of TCGA for copy number alterations and/or expression showed PAK1 amplification and/or overexpression in ~23% of ER+ breast cancer. To test the hypothesis that PAK1-amplification is related to CDK4/6 inhibitors resistance, we generated two ER+ breast cancer cell lines T47D (harbouring PAK1 amplification) and MCF7 (non PAK1-amplified) resistant to the combination of fulvestrant (ER downregulator) and abemaciclib (CDK4/6 inhibitor), namely T47DFAR and MCF7FAR. T47DFAR and MCF7FAR were obtained, exposing the parental cells to increasing doses of drugs. Interestingly, only the T47DFAR but not the MCF7FAR showed higher level of PAK1 expression and activation compared to the parental cell line. PAK1 pathway activation was evaluated measuring by immunoblot the levels of phosphorylation of PAK1, MEK, ERK and Beta-Catenin. Moreover, the genomic knockdown of PAK1 through RNAi causes a decrease in cell proliferation of T47DFAR, but not of MCF7FAR and parental cells, suggesting the role of PAK1 as a possible mediator of drug resistance. Conversely, overexpression of PAK1 in MCF7 and T47D parental cells was able to generate resistance to the drug combination. PAK-1 has shown to have a different role in the cytosol as pathways mediator and in the nucleus as transcriptional factor. Our data obtained by subcellular fractionation showed that after treatment with fulvestrant-abemaciclib, PAK1 protein accumulates into the cytosol in T47D-FAR compared to parental T47D, probably mediating an enhancement in cytoplasmatic cell signalling and pathway activation. To univocally demonstrate that PAK1 mediates tumor resistance to fulvestrant-abemaciclib combination, we are generating data from RNA-Seq and whole exome sequencing from T47DFAR and MCF7FAR and the relative parental cell lines. Also, we will assess the efficacy of PAK pharmacological inhibition in overcoming such resistance. Conclusions: In conclusion, our data suggest the role of PAK1 as a novel therapeutic target in ER+/PAK1-amplified breast cancer model resistant to endocrine therapy and CDK4/6 inhibitors. Citation Format: Stefania Belli, Alberto Servetto, Concetta Di Mauro, Daniela Esposito, Ada Pesapane, Fabiana Napolitano, Antonio Santaniello, Pietro De Placido, Priscilla Cascetta, Annachiara Carratù, Eleonora Mozzillo, Roberta Marciano, Roberto Bianco, Luigi Formisano. Pak1 as a novel mediator of resistance to endocrine therapy and CDK4/6 inhibitors in ER+/PAK-1amplified breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-06-08.

Background: Recently, immune check-point inhibitors have shown efficacy in triple-negative breast cancer (TNBC). Since Hedgehog (Hh) signalling mediates crosstalk between breast cancer cells and tumor-infiltrating immune cells, we investigated the mechanisms by which Hh can modulate PDL1 expression and the tumor-immune microenvironment. Methods: TNBC tumors from untreated patients were subjects to PDL1 and Gli1 expression analysis by IHC. The correlation of Hh pathway activation and PDL1 expression was assessed in TNBC cells treated with the SMO-inhibitor NVP-LDE225. The main aim was to study the PDL1/Gli1 cross-talk. Results: The expression of PDL1 and Gli1, the major indicator for the canonical Hh signaling activation, were assessed by IHC in a tissue microarray (TMA) of TNBC samples from 237 untreated patients. In 203/237 cases we could analyze both PDL1 and Gli1 protein expression. A significant correlation between PDL1 and Gli1 expression was found. Indeed, of 77/203 (38%) PDL1 positive tumors 42/77 (54%) expressed Gli1. In order to explore correlations between other Hh pathway members and PDL1 in TNBCs, we interrogated the open-access database TCGA; PDL1 positive TNBCs showed high levels of SMO and PTCH1, Hh pathway receptors (Q-value 0.018 and 0.010). To better understand the link between Hh pathway and PDL1, we selected a panel of TNBC cell lines; the pharmacological Hh pathway inhibition led to a reduction of PDL1 protein and mRNA. On the other hand, engineered cells harbouring Gli1 overexpression showed higher levels of PDL1. Therefore, we hypothesized that Hh pathway could be involved in the PDL1 transcriptional modulation. To address this issue, we performed a luciferase reporter assay and a ChIP analysis following by PCR amplification of the PDL1 gene promoter. We found that Gli1 binds the PDL1 promoter, indicating that Gli1 transcriptionally enhances PDL1 expression. Gli1 expression was evaluated also in 4T1 cells, a TNBC murine model; furthermore, in 4T1 cells PDL1 protein expression was inhibited by NVP-LDE225. In vivo experiment will be performed in Balb/C mice orthotopically xenografted with 4T1 cells to confirm the role of Hh pathway to modulate the PDL1 expression and the tumor microenviroment in vivo. Conclusions: Our results suggest that Hh pathway has a crucial role in cancer immune evasion trough PDL-1 modulation. Due to their ability to target both tumor cells and the tumor microenvironment, Hh inhibitors could represent promising therapeutics to be clinically investigated in Gli1 overexpressing TNBC patients. Citation Format: Pietro De Placido, Concetta Di Mauro, Daniela Esposito, Ada Pesapane, Stefania Belli, Fabiana Napolitano, Antonio Santaniello, Priscilla Cascetta, Annachiara Carratù, Eleonora Mozzillo, Roberta Marciano, Alberto Servetto, Roberto Bianco, Luigi Formisano. Hedgehog pathway is involved in cancer immune surveillance through PDL1 modulation [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-04-17.