Xingzhi Tan

Paclitaxel-resistant cells have a mutation in the paclitaxel-binding region of β-tubulin (Asp26Glu) and less stable microtubules

Resistance to paclitaxel-based therapy is frequently encountered in the clinic. The mechanisms of intrinsic or acquired paclitaxel resistance are not well understood. We sought to characterize the resistance mechanisms that develop upon chronic exposure of a cancer cell line to paclitaxel in the presence of the P-glycoprotein reversal agent, CL-347099. The epidermoid tumor line KB-3-1 was exposed to increasing concentrations of paclitaxel and 5 μmol/L CL-347099 for up to 1 year. Cells grown in 15 nmol/L paclitaxel plus CL-347099 (KB-15-PTX/099) developed 18-fold resistance to paclitaxel and were dependent upon paclitaxel for maximal growth. They grew well and retained resistance to paclitaxel when grown in athymic mice. Cross-resistance (3- to 5-fold) was observed in tissue culture to docetaxel, the novel taxane MAC-321, and epothilone B. Collateral sensitivity (∼3-fold) was observed to the depolymerizing agents vinblastine, dolastatin-10, and HTI-286. KB-15-PTX/099–resistant cells did not overexpress P-glycoprotein nor did they have an alteration of [14C]paclitaxel accumulation compared with parental cells. However, a novel point mutation (T to A) resulting in Asp26 to glutamate substitution in class I (M40) β-tubulin was found. Based on an electron crystallography structure of Zn-stabilized tubulin sheets, the phenyl ring of C-3′ NHCO-C6H5 of paclitaxel makes contact with Asp26 of β-tubulin, suggesting a ligand-induced mutation. Optimized model complexes of paclitaxel, docetaxel, and MAC-321 in β-tubulin show a novel hydrogen bonding pattern for the glutamate mutant and rationalize the observed resistance profiles. However, a mutation in the paclitaxel binding pocket does not explain the phenotype completely. KB-15-PTX/099 cells have impaired microtubule stability as determined by a reduced percentage of tubulin in microtubules and reflected by less acetylated tubulin. These results suggest that a mutation in tubulin might affect microtubule stability as well as drug binding and contribute to the observed resistance profile. [Mol Cancer Ther 2006;5(2):270–8]

Tumor Cells Chronically Treated with a Trastuzumab–Maytansinoid Antibody–Drug Conjugate Develop Varied Resistance Mechanisms but Respond to Alternate Treatments

Antibody–drug conjugates (ADC) are emerging as clinically effective therapy. We hypothesized that cancers treated with ADCs would acquire resistance mechanisms unique to immunoconjugate therapy and that changing ADC components may overcome resistance. Breast cancer cell lines were exposed to multiple cycles of anti-Her2 trastuzumab–maytansinoid ADC (TM-ADC) at IC80 concentrations followed by recovery. The resistant cells, 361-TM and JIMT1-TM, were characterized by cytotoxicity, proteomic, transcriptional, and other profiling. Approximately 250-fold resistance to TM-ADC developed in 361-TM cells, and cross-resistance was observed to other non–cleavable-linked ADCs. Strikingly, these 361-TM cells retained sensitivity to ADCs containing cleavable mcValCitPABC-linked auristatins. In JIMT1-TM cells, 16-fold resistance to TM-ADC developed, with cross-resistance to other trastuzumab-ADCs. Both 361-TM and JIMT1-TM cells showed minimal resistance to unconjugated mertansine (DM1) and other chemotherapeutics. Proteomics and immunoblots detected increased ABCC1 (MRP1) drug efflux protein in 361-TM cells, and decreased Her2 (ErbB2) in JIMT1-TM cells. Proteomics also showed alterations in various pathways upon chronic exposure to the drug in both cell models. Tumors derived from 361-TM cells grew in mice and were refractory to TM-ADC compared with parental cells. Hence, acquired resistance to trastuzumab–maytansinoid ADC was generated in cultured cancer cells by chronic drug treatment, and either increased ABCC1 protein or reduced Her2 antigen were primary mediators of resistance. These ADC-resistant cell models retain sensitivity to other ADCs or standard-of-care chemotherapeutics, suggesting that alternate therapies may overcome acquired ADC resistance. Mol Cancer Ther; 14(4); 952–63. ©2015 AACR.

Caveolae-Mediated Endocytosis as a Novel Mechanism of Resistance to Trastuzumab Emtansine (T-DM1)

Trastuzumab emtansine (T-DM1) is an antibody–drug conjugate (ADC) that has demonstrated clinical benefit for patients with HER2+ metastatic breast cancer; however, its clinical activity is limited by inherent or acquired drug resistance. The molecular mechanisms that drive clinical resistance to T-DM1, especially in HER2+ tumors, are not well understood. We used HER2+ cell lines to develop models of T-DM1 resistance using a cyclical dosing schema in which cells received T-DM1 in an “on-off” routine until a T-DM1–resistant population was generated. T-DM1–resistant N87 cells (N87-TM) were cross-resistant to a panel of trastuzumab-ADCs (T-ADCs) with non–cleavable-linked auristatins. N87-TM cells do not have a decrease in HER2 protein levels or an increase in drug transporter protein (e.g., MDR1) expression compared with parental N87 cells. Intriguingly, T-ADCs using auristatin payloads attached via an enzymatically cleavable linker overcome T-DM1 resistance in N87-TM cells. Importantly, N87-TM cells implanted into athymic mice formed T-DM1 refractory tumors that remain sensitive to T-ADCs with cleavable-linked auristatin payloads. Comparative proteomic profiling suggested enrichment in proteins that mediate caveolae formation and endocytosis in the N87-TM cells. Indeed, N87-TM cells internalize T-ADCs into intracellular caveolin-1 (CAV1)–positive puncta and alter their trafficking to the lysosome compared with N87 cells. T-DM1 colocalization into intracellular CAV1-positive puncta correlated with reduced response to T-DM1 in a panel of HER2+ cell lines. Together, these data suggest that caveolae-mediated endocytosis of T-DM1 may serve as a novel predictive biomarker for patient response to T-DM1. Mol Cancer Ther; 17(1); 243–53. ©2017 AACR.

Multivalent peptidic linker enables identification of preferred sites of conjugation for a potent thialanstatin antibody drug conjugate

Antibody drug conjugates (ADCs) are no longer an unknown entity in the field of cancer therapy with the success of marketed ADCs like ADCETRIS and KADCYLA and numerous others advancing through clinical trials. The pursuit of novel cytotoxic payloads beyond the mictotubule inhibitors and DNA damaging agents has led us to the recent discovery of an mRNA splicing inhibitor, thailanstatin, as a potent ADC payload. In our previous work, we observed that the potency of this payload was uniquely tied to the method of conjugation, with lysine conjugates showing much superior potency as compared to cysteine conjugates. However, the ADC field is rapidly shifting towards site-specific ADCs due to their advantages in manufacturability, characterization and safety. In this work we report the identification of a highly efficacious site-specific thailanstatin ADC. The site of conjugation played a critical role on both the in vitro and in vivo potency of these ADCs. During the course of this study, we developed a novel methodology of loading a single site with multiple payloads using an in situ generated multi-drug carrying peptidic linker that allowed us to rapidly screen for optimal conjugation sites. Using this methodology, we were able to identify a double-cysteine mutant ADC delivering four-loaded thailanstatin that was very efficacious in a gastric cancer xenograft model at 3mg/kg and was also shown to be efficacious against T-DM1 resistant and MDR1 overexpressing tumor cell lines.

Abstract 4837: Extracellular proteolytic cleavage of peptide-linked antibody-drug conjugates promotes bystander killing of cancer cells

Antibody drug conjugates (ADCs) are designed to deliver cytotoxics to tumor cells via binding to surface antigen followed by internalization and intracellular drug release. ADC linkers are typically categorized as non-cleavable or cleavable; a cleavable linker example is Y_mcValCitPABC_X, with antibody Y, a dipeptide sequence with self-immolative PABC spacer, and payload X. This linker is known to be cleaved by endosomal/lysosomal proteases such as cathepsins, releasing attached payload. In addition to intracellular processing of this linker, we report that conditioned media of cultured tumor cell lines is sufficient to promote extracellular cleavage of ADCs with peptide-linked payloads. Cultured cell lines N87 (gastric) and U87 (glioblastoma), and patient-derived xenograft PA0165 (pancreatic) adapted to in vitro culture, were plated either in standard 2D culture or in 3D Cultrex embedded culture. After 3 - 7 days, conditioned media from cells was transferred onto MDA-MB-468 or HT29 cells, and then ADCs (Y_mcValCitPABC_Aur) were added to cultures. ADCs were non-targeting IgG conjugated via cleavable dipeptide-PABC linker to auristatin tubulin inhibitor. Minimal cytotoxicity was observed with ADC alone on 468 or HT29 cells. However, in the presence of conditioned media from N87, U87, or PA0165 cells plus the ADC, cytotoxicity was observed in the recipient cells (up to 31, 22, 56% growth inhibition respectively at 100 nM ADC). Moreover, in all cases, the magnitude of the response was greatest when cells providing conditioned media were grown in 3D culture (up to 56, 48, 70%, respectively). In contrast, minimal response was observed using conditioned media from other cancer cell lines (ie HCC2429, 1 - 17%). Additional analyses were conducted by incubating conditioned media from these cells with a dipeptide-based cleavable substrate with fluorescent probe and measuring released product in a plate-based assay. Conditioned media promoted fluorescence, suggesting proteolytic enzymes secreted by cells. An ELISA confirmed the presence of cathepsins in conditioned media. Complementing these studies, proteolytic activity was detected in the interstitial fluid derived from tumors grown in athymic mice. Fluid extracted from xenograft tumors (cultured cancer lines and patient-derived tumors) was analyzed for proteolytic activity using cleavable-fluorescent linker-probe in a plate assay. The majority of samples demonstrated proteolytic activity. These data are consistent with reported secretion of cathepsins by cancer cells and we now show that these proteases may mediate extracellular release of cytotoxic payloads from ADCs containing peptide-based cleavable linkers. This activity is magnified when cells are grown in 3D culture and is observed in tumor xenografts grown in vivo. This response may provide a beneficial bystander effect of ADCs on antigen negative cells in a heterogenous tumor population. Citation Format: My-Hanh Lam, Judy Lucas, Andreas Maderna, Hallie Wald, Megan Wojciechowicz, Russell Dushin, Bryan Peano, Fang Wang, Jeremy Myers, Xingzhi Tan, Sylvia Musto, Manoj Charati, Hans-Peter Gerber, Frank Loganzo. Extracellular proteolytic cleavage of peptide-linked antibody-drug conjugates promotes bystander killing of cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4837. doi:10.1158/1538-7445.AM2014-4837

Abstract 2113: Caveolae-mediated endocytosis as a novel mechanism of resistance to T-DM1 ADC

Trastuzumab emtansine (T-DM1) is an antibody-drug conjugate (ADC) comprised of the HER2-targeting antibody trastuzumab and DM1, a microtubule depolymerizing agent covalently attached via a non-cleavable thioether linker. T-DM1 has demonstrated clinical benefit for patients with metastatic breast cancer, however activity may be limited by inherent or acquired resistance during prolonged treatment periods. The molecular mechanisms that drive clinical resistance to T-DM1, especially in HER2 positive tumors, are not well understood. We used the HER2+ cell line N87 to develop a model of T-DM1 resistance utilizing a cyclical dosing schema in which cells received T-DM1 in an “on-off” routine until a T-DM1 resistant population of N87 cells (N87-TM) was generated. N87-TM cells displayed 103-fold resistance toward T-DM1 treatment compared to the parental N87 cells. The N87-TM cells were cross-resistant to a panel of trastuzumab-ADCs (T-ADCs) with non-cleavable-linked auristatins. N87-TM cells do not have a decrease in HER2 protein levels or an increase in drug efflux pump (e.g. MDR1) protein expression compared to parental N87 cells. Comparative proteomic profiling suggested an enrichment in proteins (e.g. caveolin-1, CAV1) that mediate caveolae formation and endocytosis in the N87-TM cells. Indeed, N87-TM cells internalize ADCs into intracellular CAV1+ puncta and alter their trafficking to the lysosome compared to N87 cells. Intriguingly, T-ADCs utilizing auristatin payloads attached via an enzymatically cleavable linker (i.e. ValCit linker) overcome T-DM1 resistance in N87-TM cells. Importantly, N87-TM cells implanted into athymic mice in vivo formed T-DM1 refractory tumors which remain sensitive to T-ADCs with cleavable-linked auristatin payloads. When comparing antigen positive patient-derived xenograft models that were refractory to T-DM1 yet responded to T-ADCs with ValCit linker-payloads, CAV1 was found to be a predictive protein biomarker identifying T-DM1 refractory tumors. These data implicate caveolae-mediated endocytosis in ADC biology and suggest that alterations in this pathway may impact a tumor9s response profile to ADCs with non-cleavable linkers. We also propose CAV1 as a novel protein biomarker whose high tumoral expression predicts a refractory response to the T-DM1 ADC. Citation Format: Matthew S. Sung, Xingzhi Tan, Christine Hosselet, Michael Cinque, Erik Upeslacis, Jonathon Golas, Fang Wang, Bingwen Lu, Laurie Tylaska, Lindsay King, Jeremy Myers, Edward Rosfjord, Judy Lucas, Hans-Peter Gerber, Frank Loganzo. Caveolae-mediated endocytosis as a novel mechanism of resistance to T-DM1 ADC. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2113.

Abstract 1830: Antibody-drug conjugates with modified linker-payloads overcome resistance to a trastuzumab-maytansinoid conjugate in multiple cultured tumor cell models

Antibody-drug conjugates (ADCs) enable targeted delivery of therapeutics to cancer cells and offer potential for more selective therapy. Several 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. As a model system, we used a trastuzumab-maytansinoid ADC (TM) similar to T-DM1 to treat cultured tumor cell lines at high doses followed by recovery. We previously reported the generation of two breast cancer cell lines with acquired resistance to TM. These parental lines, MDA-MB-361 and JIMT1, express moderate levels of Her2. We now report the induction of resistance to TM-ADC in a high Her2 expressing gastric carcinoma cell line, N87. Chronic exposure of N87 cells to TM-ADC on a 3 day on/ 4 day off cycle results in ∼240 fold resistance. Two parallel flasks of N87 cells were treated with TM, and identical levels of resistance, cross-resistance, and sensitivity to other agents are observed, suggesting common selective pressure upon drug exposure in a single tumor cell type. Interestingly, both the N87 and 361 cell models developed similar patterns of cross-resistance (up to >1000X) to ADCs of trastuzumab containing non-cleavable linkers and microtubule inhibitors. In contrast, ADCs with proteolytic cleavable linkers which also release tubulin inhibitors completely overcome resistance. Moreover, the TM-resistant cell lines retain sensitivity to ADCs delivering payloads with non-tubulin mechanisms of action, including DNA inhibitor calicheamicin. Minimal or no resistance (1 - 6X) was observed to free drugs, including maytansine or other standard-of-care tubulin or DNA targeted chemotherapeutics. In 361-TM cells, ABCC1 (MRP1) is induced and may partially mediate resistance to TM-ADC and other non-cleavable ADCs. In contrast, N87-TM cells do not express ABCC1 (MRP1) or ABCB1 (MDR1), as demonstrated by immunoblot and flow cytometry, nor are these cells cross-resistant to free drugs which are substrates of such transporter proteins. Proteomic profiling of N87-TM cells is underway to fully characterize the protein changes in TM-ADC-resistant cells. These data suggest that chronic cycling treatment of TM-ADC at high doses can induce resistance in different tumor cell models. In 2 of 3 cell lines tested, high level resistance was observed, including nearly identical patterns of cross-resistance to other non-cleavable linked trastuzumab ADCs, yet sensitivity to cleavable linked ADCs. The same selection pressure may be inducing different mechanisms of drug resistance, dependent upon the cell background. These data suggest that ADC resistant cancers may be treated with conjugates targeting the same antigen but with modified linker payloads. Citation Format: Xingzhi Tan, Bingwen Lu, Guixian Jin, Fang Wang, Jeremy Myers, Sylvia Musto, My-Hanh Lam, William Hu, Kiran Khandke, Kim Arndt, Hans-Peter Gerber, Frank Loganzo. Antibody-drug conjugates with modified linker-payloads overcome resistance to a trastuzumab-maytansinoid conjugate in multiple cultured tumor cell models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1830. doi:10.1158/1538-7445.AM2014-1830

Abstract C161: Tumor cells selected for resistance to an antibody-drug conjugate modulate protein trafficking and signaling pathways.

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 IC 80 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 (>20× & >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.