Abstract 3165: Elucidation and pharmacological targeting of master regulator proteins representing mechanistic determinants of breast cancer stem-like tumor initiating cell state
Authors: Worley Jeremy , Ding Hongxu , Noh Heeju , Paull Evan , Griffin Aaron T. , Grunn Adina , You Daoqi , Guillan Kristina , Bush Erin , Dalerba Piero , Sims Peter , Dela Cruz Filemon S. , Kung Andrew L. , Califano Andrea 
Published: June 2022

Abstract
Although breast cancer Stem-Like Tumor Initiating Cells (SLTIC) represent only a minute fraction of the total tumor mass, they are resistant to standard of care treatment and play a key role in tumor initiation, maintenance, and progression. Unequivocal SLTIC isolation, using surface markers, has proven highly elusive, thus impeding characterization and targeting of their mechanistic dependencies. To address this challenge, we applied a systems biology approach to effectively characterize SLTIC biology and to prioritize drugs that can reprogram them to a more differentiated state that is sensitive to chemotherapy.
To isolate breast cancer cells enriched for SLTICs, we performed flow cytometry-based sorting of tumor cells from 7 metastatic breast cancer patients, based on the expression of Epcam and CD49F, which are established epithelial and SLTIC-enriched cell markers, respectively. Activity-based clustering of single cell RNASeq profiles using the VIPER (Alvarez et al. Nat Genet 2017) algorithm identified two cell states, comprising cells presenting high activity of either SLTIC (i.e., BMI1, NOTCH1, etc.) or differentiated, proliferative epithelial cell markers (i.e., PCNA, CCNB1, etc.). Analysis of RNASeq profiles of BT20 cells treated with ~400 FDA approved and late-stage experimental drugs identified albendazole as the drug inducing the most significant activity inversion of SLTIC Master Regulator proteins (Alvarez et al. Nat Genet 2018) (p=4.21x10-5). This was experimentally confirmed in vivo by single cell analysis of metastatic TNBC PDX models at 14 days after treatment with albendazole, paclitaxel (a drug known to kill differentiated but not SLTIC cells), and vehicle control. As expected, paclitaxel induced dramatic decrease of the differentiated vs. SLTIC cell ratio, while albendazole had the opposite effect, inducing equally dramatic increase in that ratio, as assessed by a combination of CytoTRACE and established SLTIC marker analysis. Sequential therapy, based on a 30-day treatment with albendazole with 3 rounds of paclitaxel at day 15, 22, and 30, repeated after a 15-day drug holiday produced highly synergistic tumor volume reduction, compared to individual monotherapies (p=0.00869 by Bliss independence analysis). Indeed, while albendazole had little effect compared to vehicle control, as monotherapy, it induced &gt;50% additional tumor viability reduction when combined with paclitaxel. The approach is highly generalizable and can be used to identify potential combination therapy approaches for any tumor in which molecular distinct subpopulations co-exist with different drug sensitivities, thus offering a practical solution to addressing tumor heterogeneity.
Citation Format: Jeremy Worley, Hongxu Ding, Heeju Noh, Evan Paull, Aaron T. Griffin, Adina Grunn, Daoqi You, Kristina Guillan, Erin Bush, Piero Dalerba, Peter Sims, Filemon S. Dela Cruz, Andrew L. Kung, Andrea Califano. Elucidation and pharmacological targeting of master regulator proteins representing mechanistic determinants of breast cancer stem-like tumor initiating cell state [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3165.