7831 Unveiling Drug Resistance Mechanisms In Triple-Positive Breast Cancer: Insights From Long-Term Estrogen Deprivation Models And Innovative Mutation Detection Strategies
Authors: Bahnassy Shaymaa , Andrews Hillary S , Jin Lu , Kohrn Brendan F , Tam Stanley , Mobin Dua , Dunn Yasmin J , Balachandran Manasi , Podar Mircea , He Wei , McCoy Matthew D , Beckman Robert A , Riggins Rebecca B 
Published: October 2024

Disclosure: S. Bahnassy: None. H.S. Andrews: None. L. Jin: None. B.F. Kohrn: None. S. Tam: None. D. Mobin: None. Y.J. Dunn: None. M. Balachandran: None. M. Podar: None. W. He: None. M.D. McCoy: None. R.A. Beckman: Consulting Fee; Self; AstraZeneca, Boehringer Ingelheim. Other; Self; Chief Scientific Officer of OncoMind, LLC, which owns patents on dynamic precision medicine. R.B. Riggins: None.

Compelling evidence from preclinical and clinical data suggests that triple-positive (TP; HER2+/HR+) breast cancer (BC) has lower response rates to HER2-targeted therapy and a higher risk of recurrence to endocrine therapy (ET) than HER2+/HR−. Molecular mechanisms underpinning drug resistance in TP-BC are poorly understood, thereby limiting our ability to improve patient outcomes and prevent metastatic progression. In our recent publication, we established and characterized long-term estrogen deprivation (LTED) ET-resistant (ETR) TP-BC models that mimic resistance to aromatase inhibitors without using genetic manipulation approaches. We reported intrinsic subtype shifts towards non-luminal phenotypes, metabolic reprogramming, and acquiring mutations in genes encoding for transcription factors and chromatin modifiers, as potential mechanisms of ETR in our LTED models. The emergence of drug-resistant mutator subclones under treatment-induced selective pressures, coupled with limitations in shallow-depth whole genome sequencing coverage, highlight an unmet need to develop a strategy for visually monitoring, capturing, and mapping resistant subclones through a mutational phenotypic screen, an approach offering valuable insights for optimizing treatment schedules. To address this, we developed and applied an innovative dual fluorescence mutation detection system to detect mutator cells expressing both mCherry and green fluorescence protein (GFP). First, we validated if our system is working properly. Compared to parental cells, transduced BT-474 and MDA-MB-361 LTEDs showed a drastic increase in GFP+ cells, suggesting the presence of resistant mutator cells. Consistent with the increase in GFP signal, TP-LTEDs showed increased mutation number and frequency in two Duplex Sequencing targeted capture panels (neutral loci and DNA polymerase-focused), used to perform ultra-deep, highly accurate sequencing. We then treated parental cells with single, dual, and triple combinations of anti-HER2, ET and CDK4/6 inhibitors to track the emergence of GFP+ mutator cells. Nine-day treatments with triple combinatorial therapies were best at inhibiting the growth of both BT-474 and MDA-MB-361 parental cell lines, compared to single- and double-agent targeted therapies. However, no GFP signal was detected within treated cells, suggesting that longer treatment durations are needed for drug-resistance mutator subclones to be discovered. Ongoing studies monitoring TP-drug resistance mutator subclones with long-term conventional targeted-therapies will improve our understanding of drug resistance mechanisms in this understudied subtype of BC. Importantly, these studies will enable dynamic precision medicine simulations to empower for a more tailored and effective therapeutic administration to prevent drug resistance and metastatic progression of TP-BC.

Presentation: 6/3/2024