Abstract 307: Blocking LBH overexpression sensitizes triple negative breast cancer cells to ATR inhibition therapy
Authors: Garikapati Koteswararao , Young In-Chi , Briegel Karoline 
Published: April 2023

Abstract
Background: Triple Negative Breast Cancers (TNBC) have a poor prognosis, are difficult-to-treat and predominant in young, African American women. Specific treatments are lacking, as TNBC do not express targetable markers i.e., ER, PR and/or HER2. TNBC tumors exhibit high genomic instability due to frequent loss-of-function mutations in tumor suppressors (i.e., p53) and DNA repair enzymes (i.e., BRCA1, PALP2). Yet TNBC are highly proliferative and resist DNA damaging chemo/radiation therapy, leading to rapid disease relapse and mortality. The pro-survival factors that protect TNBC cells from genotoxic stress remain poorly understood. We identified, and study the LIMB-BUD-AND-HEART (LBH) protein, a stem cell transcription regulator in the WNT signaling pathway that is critical for breast cancer disease progression (Lindley L, et al., Development 2015, Ashad-Bishop, K. et al, BBRC 2019). We showed LBH is predominantly over expressed in TNBC, correlating with reduced patient survival. Functional analysis indicates LBH is critical for high cancer stem cell abundance, metastatic potential, and chemoresistance in TNBC (Rieger, M. et al, Mol. Cell Biol 2010; Garikapati, K. et al, bioRxiv preprint 2021). Key molecular functions are under investigation.
Results: Here, we identified that LBH is also an essential pro-survival factor in TNBC. Depletion of LBH in multiple TNBC cell lines (MDA-MB-231, HCC1806, and HCC1395) by RNAi reduced cell viability and induced apoptosis in vitro and in vivo. Contrarily, ectopic LBH expression in TNBC cells protected from cell death, as determined by caspase 3/7 assays, and Annexin V FACS analysis. To identify the mechanisms underlying the pro-survival function of LBH, we examined cell cycle progression and DNA integrity in LBH knockdown TNBC cells using EdU click, DNA comet, and DNA fiber assays, together with cell cycle and DNA damage marker analysis. LBH loss significantly attenuated S to G2 cell cycle progression due to increased DNA replicative stress, leading to increased DNA damage, upregulation of DNA damage markers (phospho-γH2AX, P53BP1), and activation of the ATR/CHK1 DNA damage response pathway. Notably, ATR inhibition (AZD6738) in combination with LBH down modulation had a synergistic effect on inducing TNBC cell death and blocking in vivo tumor growth.
Conclusions: Our results yield new mechanistic insight into treatment resistance in poor prognosis TNBC by identifying LBH as a key protector of DNA integrity. Importantly, they provide a novel rationale for inhibiting LBH in combination with ATR inhibitors to improve survival in TNBC patients.
Citation Format: Koteswararao Garikapati, In-Chi Young, Karoline Briegel. Blocking LBH overexpression sensitizes triple negative breast cancer cells to ATR inhibition therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 307.