Abstract A131: Rapid constitutive internalization and degradation of prolactin receptor (PRLR) is associated with potent cell killing by PRLR antibody drug conjugates (ADC)
Authors: Andreev Julian , Thambi NIthya , Delfino Frank , Martin Joel , Kelly Marcus P. , Kirshner Jessica R. , MacDonald Douglas , Popadopoulos Nicholas , Olson Willian , Thurston Gavin 
Published: May 2016

Abstract
Ado-trastuzumab-emtansine or T-DM1, an antibody drug conjugate (ADC) targeting the well-characterized breast cancer oncogene HER2, has shown benefit for breast cancer patients. However, treatment is not indicated for patients whose tumors express low or intermediate levels of HER2. Thus, additional targets for ADC are needed in breast cancer. The lineage-restricted marker prolactin receptor (PRLR) is also expressed in a subset of breast cancers. Unexpectedly, we found that, unlike HER2, low levels of cell-surface PRLR are sufficient to mediate efficient ADC killing of breast ductal carcinoma cells, including T47D. To understand what properties of PRLR vs HER2 allow for efficient cell killing, we compared intracellular trafficking of these two receptors. We found that approximately 90% of a PRLR antibody was internalized by T47D cells within 1h after treatment, and the internalized PRLR Ab co-localized with the lysosomal marker, Lysotracker Red. In contrast, trastuzumab was restricted to the plasma membrane and did not co-localize with Lysotracker Red. Overnight incubation of T47D cells with PRLR Ab, but not trastuzumab, resulted in accumulation in a late lysosomal compartment, as detected using the pH-sensitive marker, pHrodo. Inhibiting protein synthesis with cycloheximide resulted in almost complete degradation of PRLR after 2h, whereas HER2 was degraded only slightly after 4h. The rapid turnover of PRLR was not significantly affected by adding exogenous ligand (prolactin), or by PRLR antibodies, or by proteasome inhibitors, but was blocked by lysosomal inhibitors including bafilomycin A1, and monensin. The signals for this constitutive PRLR internalization and degradation appear to be contained within its cytoplasmic domain, since substitution of the PRLR extracellular domain by that of HER2 still resulted in degradation rates similar to those of full length PRLR. Moreover, simultaneous substitution of two dileucine lysosomal sorting signals contained in the PRLR cytoplasmic domain (e.g. 283LL and 292LL) to alanine significantly diminished constitutive PRLR turnover. In accordance with these data, PRLR ADC, but not T-DM1, induced cell cycle arrest (proportional to PRLR ADC-induced cell killing) in T47D cells, which was completely abolished by lysosomal inhibitors. Taken together, these data indicate that rapid constitutive ligand-independent turnover of PRLR, but not Her2, can deliver high amounts of ADC to lysosomes, resulting in efficient tumor cell killing.
Citation Format: Julian Andreev, NIthya Thambi, Frank Delfino, Joel Martin, Marcus P. Kelly, Jessica R. Kirshner, Douglas MacDonald, Nicholas Popadopoulos, Willian Olson, Gavin Thurston. Rapid constitutive internalization and degradation of prolactin receptor (PRLR) is associated with potent cell killing by PRLR antibody drug conjugates (ADC). [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A131.