Published OnlineFirst March 25, 2020; DOI: 10.1158/2159-8290.CD-20-0215 RESEARCH ARTICLE HER2-Mediated Internalization of Cytotoxic Agents in ERBB2 Amplified or Mutant Lung Cancers Bob T. Li1,2, Flavia Michelini3,4, Sandra Misale5, Emiliano Cocco4, Laura Baldino3,4, Yanyan Cai3,4, Sophie Shifman4, Hai-Yan Tu1,6, Mackenzie L. Myers1, Chongrui Xu1,6, Marissa Mattar5,7, Inna Khodos5,7, Megan Little5,7, Besnik Qeriqi5,7, Gregory Weitsman8, Clare J. Wilhem1, Alshad S. Lalani9, Irmina Diala9, Rachel A. Freedman10, Nancy U. Lin10, David B. Solit1,2,4,11, Michael F. Berger3,4,11, Paul R. Barber8,12, Tony Ng8,12, Michael Offin1,2, James M. Isbell2,13, David R. Jones2,13, Helena A. Yu1,2, Sheeno Thyparambil14, Wei-Li Liao14, Anuja Bhalkikar14, Fabiola Cecchi15, David M. Hyman1,2, Jason S. Lewis2,16,17, Darren J. Buonocore3, Alan L. Ho1,2, Vicky Makker1,2, Jorge S. Reis-Filho3,4, Pedram Razavi1,2, Maria E. Arcila3, Mark G. Kris1,2, John T. Poirier1,5, Ronglai Shen18, Junji Tsurutani19, Gary A. Ulaner2,5,14, Elisa de Stanchina5,7, Neal Rosen5,20, Charles M. Rudin1,2, and Maurizio Scaltriti3,4,20 Downloaded from cancerdiscovery.aacrjournals.org on September 29, 2021. © 2020 American Association for Cancer Research. Published OnlineFirst March 25, 2020; DOI: 10.1158/2159-8290.CD-20-0215 ABSTRACT Amplification of and oncogenic mutations inERBB2 , the gene encoding the HER2 receptor tyrosine kinase, promote receptor hyperactivation and tumor growth. Here we demonstrate that HER2 ubiquitination and internalization, rather than its overexpression, are key mechanisms underlying endocytosis and consequent efficacy of the anti-HER2 antibody–drug conjugates (ADC) ado-trastuzumab emtansine (T-DM1) and trastuzumab deruxtecan (T-DXd) in lung cancer cell lines and patient-derived xenograft models. These data translated into a 51% response rate in a clinical trial of T-DM1 in 49 patients with ERBB2-amplified or -mutant lung cancers. We show that cotreatment with irreversible pan-HER inhibitors enhances receptor ubiquitination and consequent ADC internalization and efficacy. We also demonstrate that ADC switching to T-DXd, which harbors a different cytotoxic payload, achieves durable responses in a patient with lung cancer and correspond- ing xenograft model developing resistance to T-DM1. Our findings may help guide future clinical trials and expand the field of ADC as cancer therapy. SIGNIFicaNCE: T-DM1 is clinically effective in lung cancers with amplification of or mutations in ERBB2. This activity is enhanced by cotreatment with irreversible pan-HER inhibitors, or ADC switching to T-DXd. These results may help address unmet needs of patients with HER2-activated tumors and no approved targeted therapy. INTRODUCTION targeted therapy. HER2-targeted agents investigated in patients with lung cancers over the past two decades include Each year, thousands of patients with cancers bearing the mAbs trastuzumab and pertuzumab, and tyrosine kinase amplifications or mutations in the human epidermal growth inhibitors such as afatinib, dacomitinib, and neratinib (7, 8). factor receptor 2 (ERBB2, encoding HER2) are without effec- Although clinical activity was seen, neither class of drugs has tive treatments. HER2 is a transmembrane receptor tyrosine produced transformative results in clinical trials to date (9–13). kinase (RTK), and ERBB2 is amplified/overexpressed in 15% Antibody–drug conjugates (ADC) are emerging antitumoral to 20% of breast and gastroesophageal cancers, for which tar- agents that combine the unique binding capabilities of mAbs geted anti-HER2 therapy has transformed the standard of care with the cytotoxic activity of chemotherapy to specifically (1–3). In addition to gene amplification, HER2 is also hyper- target and harm cancer cells (14). The mechanism of action activated by specific mutations, most of which are located of ADCs involves the recognition and binding of the mAb either in its extracellular domain or in its kinase domain. The backbone to the extracellular domain of a cancer-specific trans- common denominator of both overexpression and mutation membrane protein, the internalization of the ADC–protein is receptor hyperactivation via an increased homo- or heter- complex via the endocytic pathway, and the release of the cyto- odimerization and activation of downstream signaling cas- toxic payload that ultimately kills or arrests the proliferation cades such as the PI3K and MAPK pathways (4). Mutations in of the malignant cell (14). To date, there are five FDA-approved or amplification ofERBB2 are also found in approximately 4% ADCs; three are recommended for hematologic malignancies, of lung tumors (5, 6), for which there is currently no approved and two, ado-trastuzumab emtansine (T-DM1, Genentech/ 1Department of Medicine, Memorial Sloan Kettering Cancer Center, New Kettering Cancer Center, New York, New York. 18Department of Epidemio- York, New York. 2Weill Cornell Medical College, New York, New York. logy and Biostatistics, Memorial Sloan Kettering Cancer Center, New York, 3Department of Pathology, Memorial Sloan Kettering Cancer Center, New New York. 19Advanced Cancer Translational Research Institute, Depart- York, New York. 4Human Oncology and Pathogenesis Program, Memorial ment of Medical Oncology, Showa University, Tokyo, Japan. 20Center for Sloan Kettering Cancer Center, New York, New York. 5Molecular Phar- Molecular-Based Therapy, Memorial Sloan Kettering Cancer Center, New macology Program, Memorial Sloan Kettering Cancer Center, New York, York, New York. 6 New York. Guangdong Lung Cancer Institute, Guangdong Provincial Peo- Note: Supplementary data for this article are available at Cancer Discovery ple’s Hospital and Guangdong Academy of Medical Sciences, Guangzhou, Online (http://cancerdiscovery.aacrjournals.org/). China. 7Antitumor Assessment Core Facility, Memorial Sloan Kettering Cancer Center, New York, New York. 8Richard Dimbleby Department of B.T. Li, F. Michelini, and S. Misale contributed equally to this article. Cancer Research, King’s College London, London, United Kingdom. 9Puma Corresponding Authors: Maurizio Scaltriti, Memorial Sloan Kettering Biotechnology, Los Angeles, California. 10Department of Medical Oncol- Cancer Center, 1275 York Avenue, Box 20, New York, NY 10065. Phone: ogy, Dana-Farber Cancer Institute, Boston, Massachusetts. 11Center for 646-888-3519; Fax: 646-422-0247; E-mail: [email protected]; Bob Molecular Oncology, Memorial Sloan Kettering Cancer Center, New York, T. Li, [email protected]; Flavia Michelini, [email protected]; and Sandra New York. 12UCL Cancer Institute, Paul O’Gorman Building, University Col- Misale, [email protected] 13 lege London, London, United Kingdom. Department of Surgery, Memorial Cancer Discov 2020;10:1–14 Sloan Kettering Cancer Center, New York, New York. 14mProbe Inc, Rockville, Maryland. 15AstraZeneca, Gaithersburg, Maryland. 16Department of Radi- doi: 10.1158/2159-8290.CD-20-0215 ology, Memorial Sloan Kettering Cancer Center, New York, New York. ©2020 American Association for Cancer Research. 17Radiochemistry and Molecular Imaging Probe Core, Memorial Sloan MAY 2020 CANCER DISCOVERY | OF2 Downloaded from cancerdiscovery.aacrjournals.org on September 29, 2021. © 2020 American Association for Cancer Research. Published OnlineFirst March 25, 2020; DOI: 10.1158/2159-8290.CD-20-0215 RESEARCH ARTICLE Li et al. Roche) and trastuzumab deruxtecan (T-DXd, formerly DS- show the highest pHrodo-T-DM1 signal at earlier time points 8201a, Daiichi-Sankyo/AstraZeneca; ref. 15), are indicated for and morphologic changes at later time points (Fig. 1A and HER2-positive breast cancer (16), with the latter also show- B), compatible with the onset of cell death, as confirmed by ing promising results in patients with HER2-positive gastric increased PARP cleavage (Fig. 1E). In both cell lines, express- cancer (17, 18). ing comparable WT or mutant HER2, receptor levels were In this work, we demonstrate that activated HER2, regard- reduced upon T-DM1 treatment, likely due to internalization less of its oncogenic potential or the addiction status of and subsequent degradation (Fig. 1E and F). the cancer cell to its downstream signaling pathways, can On the basis of these data, we posited that T-DM1 can serve as a vehicle to funnel potent chemotherapeutic agents accumulate in both mutant and amplified lung tumors. into lung tumors. Integrating parallel laboratory and clini- To evaluate this in the clinical setting, we performed cal data on the mechanisms of response, we also propose 89Zr-trastuzumab PET/CT functional imaging in patients two different strategies to improve the efficacy of anti-HER2 with lung cancer bearing HER2 alterations. We found that ADCs: cotreatment with irreversible pan-HER inhibitors that 89Zr-trastuzumab can accumulate in both ERBB2-amplified enhance receptor ubiquitination and internalization of ADCs and ERBB2-mutant tumors (Fig. 1G, H). or switching to an ADC bearing a different cytotoxic payload. We then tested the antitumoral activity of T-DM1 in different patient-derived xenografts (PDX) established from RESULTS patients with lung cancer with tumors bearing ERBB2 muta- tions or amplification. Treatment with T-DM1 resulted in HER2 Mutations Increase Receptor durable responses in both HER2-mutant (YVMA insertion) Internalization and T-DM1 Activity PDXs and in xenografts with co-occurrent amplification (2.8- We hypothesized that ERBB2-amplified or -mutated fold) and mutation (S310F) of ERBB2 (Fig. 1I and J). This tumors have exquisite susceptibility to HER2