Abstract C161: Tumor cells selected for resistance to an antibody-drug conjugate modulate protein trafficking and signaling pathways.
Authors: Tan Xingzhi , Whitney Lauren , Jin Guixian , Myers Jeremy , Diesl Veronica , Follettie Max , Musto Sylvia , Jiang Fan , Khandke Kiran , Arndt Kim , Gerber Hans-Peter , Loganzo Frank 
Published: June 2012

Abstract
Antibody-drug conjugates (ADCs) enable targeted delivery of therapeutics to cancer cells and offer potential for more selective therapy. Several new ADCs are demonstrating promising clinical efficacy, however due to the complexity of human cancer, tumors become refractory to most drug treatments. We hypothesized that cultured tumor cells chronically treated with an ADC would acquire mechanisms of resistance unique to ADC-based therapy. An anti-Her2 trastuzumab - maytansinoid conjugate (TM) was used. Human breast cancer cell lines sensitive (BT474) or insensitive (JIMT1 & MDA-MB-361) to unconjugated trastuzumab were used as models; all cells were highly responsive to TM conjugate. Cells were exposed to multiple cycles of TM at IC80 concentrations for 3 days followed by ∼1 week without treatment to simulate a maximally tolerated dose followed by recovery. After ∼2 months, minimal resistance was observed in BT474 cells, however significant resistance developed in JIMT and 361. The potency of TM conjugate on drug-selected cell lines was reduced to the activity observed on Her2-negative cells (&gt;20× & &gt;400× in JIMT & 361, respectively). In JIMT-TM cells, cross-resistance was observed to other trastuzumab-ADCs, including T-mcMMAD and T-vcMMAD (non-cleavable & cleavable linkers with dolastatin, respectively). In contrast, 361-TM cells retained partial sensitivity to ADCs containing vc-linked dolastatin and auristatins, including ADCs to other antigens. JIMT-TM & 361-TM cells showed low level resistance to free maytansine payload (∼ 4 − 6×). JIMT-TM was partially cross-resistant to free auristatins (∼4 − 5×), while 361-TM retained sensitivity to auristatins. Neither cell line showed marked resistance (1 − 3×) to other standard-of-care therapeutics such as paclitaxel, docetaxel, vinblastine, camptothecin, oxaliplatin, etoposide, doxorubicin, or 5FU. Interestingly, increased sensitivity was observed to Her2 kinase inhibitor neratinib in 361-TM cells. Flow cytometry revealed 58% and 25% decreases in Her2 receptor number in JIMT-TM & 361-TM, respectively, however partial resistance to free payloads suggests a complex resistance profile independent of Her2 levels. Proteomic profiling was conducted on surface proteins and organelle-enriched fractions. In JIMT-TM cells, significant increases were observed in proteins involved in post-translational modification, including ubiquitinating enzymes, kinases, and phosphatases. Interestingly, elevated levels of endosomal and vesicle proteins were noted, including RAB family members. Proteins mediating microtubule and actin dynamics were also increased in JIMT-TM resistant cells. Notably, ABC drug transporters were not altered in JIMT-TM cells. In 361-TM cells, an increase in ABCC1 (MRP1) was observed, but no changes in ABCB1 (MDR1) which typically effluxes tubulin inhibitors. Alterations in pathways involved in vesicle transport, cytoskeletal function, and phospho-signaling suggest unique ways by which cells may evade chronic ADC-based therapy. The observed sensitivity of 361-TM cells to other vc-linked-auristatin ADCs offers the potential for alternative conjugate-based therapy for cancers which fail TM treatment. Hence, these new ADC-refractory cell models will be valuable tools to interrogate molecular mechanisms underlying resistance to immunoconjugate therapy.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C161.