Abstract Background: HER2+ breast cancer is routinely treated with neoadjuvant HER2-targeted therapy but the molecular mechanisms driving residual disease (RD) remain unclear. We previously reported that RD after HER2 genetic inhibition in an inducible mouse model is associated with reduced proliferation and upregulation of the leucyl tRNA synthetase (LARS2). We also found that LARS2 was negatively correlated with ERBB2 expression and originated in a ‘HER2-low’ subpopulation in primary HER2+ mouse tumors. Here we describe a preliminary mechanism by which LARS2 promotes RD after trastuzumab deruxtecan treatment (T-DXd) in vitro and characterize the expression of LARS2 in HER2 high and HER2 low patient tissues. Methods: We used HER2+ve and HER2-ve cell lines, SK-BR-3 and HCC-1806, respectively treated with T-DXd at 3 concentrations of 1mM, 100mM and 10mM for 21 days. Total protein lysates were collected at five different time points - day 1, 3, 7, 14, and 21 post shRNA transfection (ERBB2, LARS2, HEXB, non-targeting control) as well as post-tDXD drug treatment. For the cell proliferation / survival assay (Cell-Titer Glo) cells were plated in 24 well plates and assayed at three different time points - day 1, 7, 14 To examine LARS2 and HER2 expression at single-cell resolution in patient tissues we used cyclic immunofluorescence on a tissue a microarray of 41 patients (81 cores). 48 cores were HER2+ (IHC 3+) and 33 were HER2-low (30 HER2 2+ FISH negative, 3 HER2 IHC 1+). We used anti-human HER2 (29D8), which has been previously validated against clinical grade antibodies. We also examined the expression of LARS2 between proliferating/quiescent cancer cells using our previously described multivariate proliferation index (MPI). Results: We found that T-DXd treatment reduces ERBB2 expression at doses 10 nM starting at day 1 by immunoblotting in SK-BR-3 but not HCC1806 cells. T-DXd treatment increased LARS2 expression by immunoblotting in SK-BR-3 cells between day 1-7 that returned to baseline by day 14. This change was not seen in HCC-1806. We saw a similar transient increase in LARS2 expression by immunoblotting over day 1-7 in response to shRNA mediated ERBB2 knockdown. Compared to control, T-DXd treatment, ERBB2 knockdown and LARS2 knockdown reduced cell proliferation in SK-BR-3 but not HCC1806 cell lines, as measured by Cell-Titer-Glo. Experiments to test whether T-DXd-mediated reduction in cell numbers can be abrogated by LARS2 knock down will be reported at the conference. In our TMA, mean LARS2 intensity per tumor cell as measured by t-CyCIF was higher in HER2-low tumors (0.62 ± 0.07 in HER2 1+ and 0.61 ± 0.09 in HER2 2+) compared to HER2+ (IHC 3+) tumors (0.59 ± 0.09, p1.00e-04, paired t-tests), consistent with our mouse data showing a negative correlation between Erbb2 and Lars2 at the mRNA level. Mean LARS2 intensity in tumor cells from our TMA of HER2 expressing tumors was higher in MPI 0 (QCC) cells than MPI 1 (proliferating) tumor cells (0.41 ± 0.15 vs 0.38 ± 0.14, p ≤ 1.00e-04). Conclusion: LARS2 expression is higher in HER2-low vs HER2+ human tumors and in quiescent vs proliferating tumor cells at baseline. Genetic (ERBB2 knockdown) or pharmacologic (T-DXd) HER2 inhibition increases LARS2 expression in vitro in HER2+ but not HER2- cell lines, while LARS2 knockdown reduced proliferation in HER2+ but not HER2- cell lines. These data suggest that LARS2 is specifically upregulated after HER2 inhibition and may promote RD after T-DXd by regulating the quiescence to proliferation switch. Further functional studies will be reported at the conference. Citation Format: V. Kurisetty, M. Homsi, S. Kabraji. Residual disease after trastuzumab deruxtecan treatment of HER2+ breast tumors is promoted by leucyl tRNA synthetase 2 (LARS2) in HER2-low tumor cells abstract. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS2-11-14.
Kurisetty et al. (Tue,) studied this question.