%0 Generic
%A Boucher, Matthew
%C Heidelberg
%D 2022
%F heidok:32427
%R 10.11588/heidok.00032427
%T Exploring combination treatment with a HER2 and a Pirin inhibitor in HER2-overexpressing breast cancer cells
%U https://archiv.ub.uni-heidelberg.de/volltextserver/32427/
%X HER2 over-expressing breast cancer (HER2+ BC) is an aggressive breast cancer subtype  that is responsible for approximately 20% of overall cases. Targeted therapies have been developed against the HER2 driver oncogene which has significantly improved patient outcomes. However, HER2+ BC still has comparatively high rates of relapses following first-line neoadjuvant or adjuvant therapy, compared to other breast cancer sub-types.    These relapses are characterised by a poor prognosis, and metastatic tumors (de novo  origin or relapsed) are considered incurable. This presents a continued need to identify therapeutic drugs that can be administered alongside first-line HER2-targeted therapy to kill or prevent re-growth of residually surviving tumor cells in order to prevent subsequent relapses.    Pirin (encoded by PIR) is a comparatively under-characterised iron-binding protein that serves as a transcriptional regulator through interactions with NF-κβ. It has been suggested in the literature as a potentially attractive target for cancer therapy. Preliminary functional characterisation of a pirin ligand generated and characterised by the EMBL Chemical Biology Core Facility (CBCF), named EMBL-703625, suggested anti-tumor efficacy and excellent in vivo tolerability in mice. Transcriptomic data from HeLa cells treated with EMBL-703625 from the EMBL-CBCF have identified multiple de-regulated cellular processes as a result of pirin inhibition, such as glycolysis and the expression of heat shock proteins. Many of these same processes have also been identified as mechanisms of treatment evasion or relapse in HER2-targeted therapy resistant breast cancer cells.    This project characterised treatment with EMBL-703625 alongside lapatinib, which is a clinically licensed HER2 tyrosine kinase inhibitor. Using a combination of biochemical assays, synergy modelling and transcriptomic experiments, we demonstrated that this combination synergistically reduces cell viability and increases toxicity in 3D grown, human, HER2-overexpressing breast cancer cell lines: BT-474 and SK-BR3. This appears to be conferred through their largely unique effects on the cell transcriptome. Additionally, it appears that this drug combination could also prevent re-growth of BT-474 cells following treatment removal. Experiments on tumor-inducible primary mouse mammary cells suggest that their combinatorial effect applies uniquely to neoplastically transformed cells. As well as this, it is possible that lapatinib and EMBL-703625 confer their synergy, in part, through glycolytic inhibition. One drug given after another may also enhance tumor  killing, which could allow patients to potentially avoid more severe side effects compared to simultaneous treatment.    Additionally, this project aimed to use light-sheet microscopy to characterize intercellular cell heterogeneity on a 3-dimensional spatial and temporal basis. This was performed through the light-sheet imaging of SK-BR3 cells transfected with a fluorescent sensor named SoNar, which reads out on the NAD+/NADH ratio of cells. Image analysis pipelines were established, with deconvolution of raw-light sheet images potentially allowing an understanding of cell-cell heterogeneity in the future.    Finally, as part of a parallel analysis with data generated in the Jechlinger Lab, an integrated metabolomic and transcriptomic analysis from experiments on tumor-inducible in vitro primary mouse mammary gland cells was performed. This highlighted potential cellular pathways and processes related to cell metabolism that could serve as nodes of vulnerability for residually surviving cells and/or tumor cells, which could inform on similar experimental strategies in the future.    Overall, these results provide pre-clinical data that could provide a rationale for the progression of combined HER2 and Pirin inhibition into a clinical setting, pending additional results from in vivo mouse experiments that are planned.