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Brain metastases are refractory to therapies that control systemic disease in patients with human epidermal growth factor receptor 2 (HER2+) breast cancer, and the brain microenvironment contributes to this therapy resistance. Nutrient availability can vary across tissues, therefore metabolic adaptations required for brain metastatic breast cancer growth may introduce liabilities that can be exploited for therapy. Here, we assessed how metabolism differs between breast tumors in brain versus extracranial sites and found that fatty acid synthesis is elevated in breast tumors growing in brain. We determine that this phenotype is an adaptation to decreased lipid availability in brain relative to other tissues, resulting in a site-specific dependency on fatty acid synthesis for breast tumors growing at this site. Genetic or pharmacological inhibition of fatty acid synthase (FASN) reduces HER2+ breast tumor growth in the brain, demonstrating that differences in nutrient availability across metastatic sites can result in targetable metabolic dependencies.

Cancer-associated fibroblasts (CAFs) are highly heterogeneous. With the lack of a comprehensive understanding of CAFs' functional distinctions, it remains unclear how cancer treatments could be personalized based on CAFs in a patient's tumor. We have established a living biobank of CAFs derived from biopsies of patients' non-small lung cancer (NSCLC) that encompasses a broad molecular spectrum of CAFs in clinical NSCLC. By functionally interrogating CAF heterogeneity using the same therapeutics received by patients, we identify three functional subtypes: (1) robustly protective of cancers and highly expressing HGF and FGF7; (2) moderately protective of cancers and highly expressing FGF7; and (3) those providing minimal protection. These functional differences among CAFs are governed by their intrinsic TGF-β signaling, which suppresses HGF and FGF7 expression. This CAF functional classification correlates with patients' clinical response to targeted therapies and also associates with the tumor immune microenvironment, therefore providing an avenue to guide personalized treatment.

Brain metastases are refractory to therapies that otherwise control systemic disease in patients with human epidermal growth factor receptor 2 (HER2+) breast cancer, and the unique brain microenvironment contributes to this therapy resistance. Nutrient availability can vary across tissues, therefore metabolic adaptations required for breast cancer growth in the brain microenvironment may also introduce liabilities that can be exploited for therapy. Here, we assessed how metabolism differs between breast tumors growing in the brain versus extracranial sites and found that fatty acid synthesis is elevated in breast tumors growing in the brain. We determine that this phenotype is an adaptation to decreased lipid availability in the brain relative to other tissues, which results in a site-specific dependency on fatty acid synthesis for breast tumors growing at this site. Genetic or pharmacological inhibition of fatty acid synthase (FASN) reduces HER2+ breast tumor growth in the brain, demonstrating that differences in nutrient availability across metastatic sites can result in targetable metabolic dependencies.

Introduction Tumor immunotherapy have broadened therapeutic options for tumor treatment. The role of immune function in juvenile recurrent respiratory papillomatosis (JRRP) has not been investigated. Applying immunoblockade inhibitors as a novel disease treatment is unclear. Our study, for the first time, evaluates immune infiltration and immuno-suppressive molecule expression in JRRP. Our study provides insights in possibly treating this disease with tumor immunotherapies. We aimed to determine expression of programmed death-ligand 1 (PD-L1), a cancer escape protein, and presence of CD8+ T cell infiltration in tumor microenvironment. Material and methods Seven patients with JRRP (mean age: 7.43; age range 3–17) in this study routinely have their tumors surgical debulked at Massachusetts Eye and Ear Infirmary. Following surgery, samples were de-identified and sent to pathology where they were stained and analyzed. Results Six out of seven patients expressed PD-L1 on tumor cells to various extents. Three patients showed concurrent PD-L1 expression on tumor cells and abundant CD8+ tumor infiltrating lymphocytes as well as PD-L1+ stromal lymphocytes, while PD-L1 expression on tumor cells were not associated with CD8+ tumor infiltrating T cells nor PD-L1+ stromal lymphocytes in the other three patients. HPV 6/11 and p16 was detected in all the patients. There appeared to be no correlation between either PD-L1 expression and CD8+ infiltration and clinical severity as measured by both the number of surgeries per year or Derkay score. Conclusions Despite a small cohort, the expression of p16 and HPV 6/11 in all of the patients confirms the tissues were HPV tumor cells. PD-L1 expression was detected in the vast majority of tumor samples, while inflammatory cell compartments showed a higher degree of variation. Expression of PD-L1 on tumor cells but not inflammatory cells raises the possibility of a tumor cell intrinsic manner of PD-L1 expression. In contrast, a group of patients showed PD-L1 positivity in both tumor and inflammatory cells along with abundant CD8+ tumor infiltrating lymphocytes, suggesting adoptive immune resistance in these tumors and potential benefits from tumor immunotherapy.

Brain metastases are refractory to therapies that otherwise control systemic disease in patients with human epidermal growth factor receptor 2 (HER2+) breast cancer, and the unique brain microenvironment contributes to this therapy resistance. Nutrient availability can vary across tissues, therefore metabolic adaptations required for breast cancer growth in the brain microenvironment may also introduce liabilities that can be exploited for therapy. Here, we assessed how metabolism differs between breast tumors growing in the brain versus extracranial sites and found that fatty acid synthesis is elevated in breast tumors growing in the brain. We determine that this phenotype is an adaptation to decreased lipid availability in the brain relative to other tissues, which results in a site-specific dependency on fatty acid synthesis for breast tumors growing at this site. Genetic or pharmacological inhibition of fatty acid synthase (FASN) reduces HER2+ breast tumor growth in the brain, demonstrating that differences in nutrient availability across metastatic sites can result in targetable metabolic dependencies. Citation Format: Gino B. Ferraro, Ahmed Ali, Alba Luengo, David P. Kodack, Amy Deik, Keene L. Abbott, Divya Bezwada, Landry Blanc, Brendan Prideaux, Xin Jin, Jessica M. Possada, Jiang Chen, Christopher R. Chin, Zohreh Amoozgar, Raphael Ferreira, Ivy Chen, Kamila Naxerova, Christopher Ng, Anna M. Westermark, Mark Duquette, Sylvie Roberge, Costas A. Lyssiotis, Dan G. Duda, Todd R. Golub, Shawn M. Davidson, Dai Fukumura, Véronique A. Dartois, Clary B. Clish, Matthew G. Vander Heiden, Rakesh K. Jain. Fatty acid synthesis is required for breast cancer brain metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 90.

The effective treatment of cerebral metastases from HER2-positive breast cancer remains an unmet need. Recent studies indicate that activated astrocytes and brain endothelial cells exert chemoprotective effects on cancer cells through direct physical interaction. Here we report that the endothelin axis mediates protection of HER2-amplified brain metastatic breast cancers to the anti-HER2 antibody–drug conjugate ado-trastuzumab emtansine (T-DM1). Macitentan, a dual inhibitor of endothelin receptors A and B, improves the efficacy of T-DM1 against breast cancers grown in the brain. We show that direct contact of brain stroma with cancer cells is required for protection to T-DM1. Our data suggest that targeting the endothelin axis may be beneficial when anti-signaling agent and cytotoxic agent are combined. These findings may contribute to the development of therapeutic approaches with enhanced efficacy in the brain microenvironment.

Background: Brain metastases represent a major problem in the treatment of HER2-positive breast cancer (1). The antibody-drug conjugate ado-trastuzumab emtansine (T-DM1) has shown efficacy in trastuzumab-resistant systemic breast cancer. Here, we tested the hypothesis that T-DM1 could overcome trastuzumab resistance in murine models of brain metastases. Methods: We used previously established animal models of HER2-positive breast cancer brain metastases and organotypic brain slice cultures that recapitulate clinical scenarios (2). We treated mice bearing HER2-positive breast cancer brain metastases with trastuzumab or T-DM1 at equivalent or equipotent doses. Using intravital imaging, molecular techniques and histological analysis we determined tumor growth, mouse survival, cancer cell apoptosis and proliferation, tumor drug distribution, gene expression, and HER2 downstream signaling. Results: T-DM1 significantly delayed the growth of HER2-positive breast cancer brain metastases compared to trastuzumab. These findings were consistent between HER2-driven and PI3K-driven breast tumors. The activity of T-DM1 resulted in a striking survival benefit compared to trastuzumab (median survival for BT474 tumors: 28d for trastuzumab vs 112d for T-DM1, HR=6.2, P Conclusions: Our findings suggest that T-DM1 can overcome resistance to HER2-targeted therapies in the CNS, and warrants clinical investigation for the effective treatment of HER2-positive breast cancer brain metastases. References: 1. Kodack DP, Askoxylakis V, Ferraro GB, et al. Emerging strategies for treating brain metastases from breast cancer. Cancer Cell 2015, 27(2):163-175. 2. Kodack DP, Chung E, Yamashita H, et al. Combined targeting of HER2 and VEGFR2 for effective treatment of HER2-amplified breast cancer brain metastases. Proc Natl Acad Sci U S A 2012, 109(45):E3119-3127. Citation Format: Askoxylakis V, Ferraro G, Kodack D, Badeaux M, Jain R. Ado-trastuzumab emtansine (T-DM1) is effective against established HER2-positive breast cancer brain metastases in mice. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-17-02.

SummaryWe report the preclinical evaluation of PF-06463922, a potent and brain-penetrant ALK/ROS1 inhibitor. Compared with other clinically available ALK inhibitors, PF-06463922 displayed superior potency against all known clinically acquired ALK mutations, including the highly resistant G1202R mutant. Furthermore, PF-06463922 treatment led to regression of EML4-ALK-driven brain metastases, leading to prolonged mouse survival, in a superior manner. Finally, PF-06463922 demonstrated high selectivity and safety margins in a variety of preclinical studies. These results suggest that PF-06463922 will be highly effective for the treatment of patients with ALK-driven lung cancers, including those who relapsed on clinically available ALK inhibitors because of secondary ALK kinase domain mutations and/or brain metastases.

Although targeted therapies are often effective systemically, they fail to adequately control brain metastases. In preclinical models of breast cancer that faithfully recapitulate the disparate clinical responses in these micro-environments, we observed that brain metastases evade phosphatidylinositide 3-kinase (PI3K) inhibition despite drug accumulation in the brain lesions. In comparison to extracranial disease, we observed increased HER3 expression and phosphorylation in brain lesions. HER3 blockade overcame the resistance of HER2-amplified and/or PIK3CA-mutant breast cancer brain metastases to PI3K inhibitors, resulting in marked tumor growth delay and improvement in mouse survival. These data provide a mechanistic basis for therapeutic resistance in the brain microenvironment and identify translatable treatment strategies for HER2-amplified and/or PIK3CA-mutant breast cancer brain metastases.

Approximately 10% of patients with colorectal cancer (CRC) harbor the BRAF V600E driver mutation. Unlike melanoma, the response rate of BRAF-mutant CRC to the combination of BRAF and MEK inhibitors is limited. In order to target the MAPK signaling pathway more effectively by blocking EGFR-mediated re-activation of the pathway, triple combination trials of BRAF, MEK and EGFR inhibitors are on-going, but the response is underwhelming. To find alternative combination strategies that could deepen therapeutic responses driven by a BRAFi and MEKi combination, we performed pooled shRNA screens under the treatment pressure of the dual combination of the BRAF inhibitor dabrafenib and MEK inhibitor trametinib. In some of the BRAF-mutant CRC models, we observed marked discrepancies in the therapeutic responses between in vitro and in vivo conditions. Therefore, shRNA screens were conducted in cancer cell lines grown both in vitro (i.e. 2D and 3D culture conditions) and in vivo in xenograft tumor models. The aim of the study was to identify novel targets to combine with BRAFi/MEKi, and to compare the results of the screens preformed in vitro and in vivo. The biggest technical challenge for an in vivo pooled screening approach is achieving adequate library representation after the bottleneck of cell implantation and engraftment in mice. Our in vivo screen had an additional bottleneck due to the dabrafenib/trametinib combination treatment. Therefore, by performing a pilot screen with the BRAF-mutant cell line model HT29 we aimed to address two questions: 1) whether the in vivo screen under treatment pressure would be technically feasible and 2) if novel combination partners to dabrafenib/trametinib would be identified to potentially improve efficacy beyond that observed with the triple combination with EGFR inhibitors. We were able to achieve comparable intra-group variability and repeatability between in vitro and in vivo conditions, whereby gene level analysis revealed several differential hits between the two conditions, which were both sensitizers and activators to the dabrafenib/trametinib combination treatment. We identified targets specific for the in vivo condition that had not been identified in vitro and vice versa. Thus, in vivo screening may identify powerful hits that would not be realized by in vitro investigations. With success of this pilot effort, the screen is currently being expanded into additional BRAF-mutant CRC models. Citation Format: Hyo-eun C. Bhang, Matthew T. DiMare, David P. Kodack, Lujian Tan, Grainne Kerr, Viveksagar Krishnamurthy Radhakrishna, Javad Golji, David A. Ruddy, Tina Yuan, Matthew J. Niederst, Joshua M. Korn, Diana Graus Porta, Peter S. Hammerman, Jeffrey A. Engelman, Tinya Abrams, Juliet Williams. In vivo shRNA screens under treatment pressure by BRAF and MEK inhibitors to identify novel combination treatment strategies for BRAF-mutant colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 394.

Juvenile Recurrent Respiratory Papillomatosis is a Human Papillomavirus (HPV) infection associated tumor that primarily affects children. Currently, there are no effective therapeutic reagents to treat this disease and patients often receive repeated surgeries at papilloma recurrence. Previous studies have shown promising results in treating HPV related tumors with immunological approaches. However, immune cell infiltration and immune response in JRRP has not yet to be investigated. Using immunochemistry staining as well as flow cytometry, we evaluated expression of program death ligand 1 (PD-L1) on tumor cells and presence of lymphocytes in these tumors. In our small cohort of samples (n=7), we detected PD-L1 positive tumor cells in all the samples with varying degree of signal strength and percentage of positivity. We also detected presence of immune cells including CD4 and CD8 T cells in these tumors. Our results suggest the possibility of managing this disease through modulation of immune cells. Citation Format: Tingyu Liu, Carissa Wentland, Max Greenberg, Brandon Sepe, Tiffany Huynh, David Kodack, Mari Mino-Kenudson, Cyril Benes, Christopher Hartnick, Jeffrey Engelman. Detection of PD-L1 and lymphocytes infiltration in Juvenile Recurrent Respiratory Papillomatosis (JRRP). [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A22.

Background: Tyrosine kinase inhibitors (TKI) have yielded promising responses in non-small-cell lung cancer (NSCLC) with EGFR mutations and ALK translocations. However, these and other targeted therapies are limited by intrinsic and acquired drug resistance. The previous study from our group investigated tumor autonomous resistance mechanisms by developing patient-derived cancer models (PDCs). In this study, we aimed to decipher the non-autonomous resistance mechanisms via tumor microenvironment by developing patient-derived fibroblast (PDF) models. Method: Cancer-associated fibroblast cell lines are established directly from individual EGFR mutant NSCLC biopsies. These cell lines, as representative of each patient's tumor microenvironment, are further subjected to functional analysis. An imaging-based high-throughput platform is developed to screen for non-autonomous resistance by co-culturing PDC and PDF models in vitro. In the parallel, two independent approaches are performed to further identify mechanisms underlying the non-autonomous resistance. These include a drug screen to determine the pathway maintaining the cancer cells' survival, and a secretomic analysis on the PDFs to identify the plausible cytokine(s) responsible for the resistance. Result: By co-culturing screening, non-autonomous resistance can be found in a wide spectrum of models. The subsequent drug screen reveals both a canonical HGF dependent and novel HGF independent mechanisms contributing to EGFR TKI resistance. Both of these can be explained by the PDF's variable cytokine secretion and can be overcome by specific therapeutic combinations. Moreover, the microenvironment-driven EGFR TKI resistance has also been validated in vivo. And the prevalence of the identified cytokine is further tested in clinical specimens. Conclusion: PDFs provide a new avenue to explore non-autonomous resistance for targeted therapy. Applying this approach, we identified both the canonical HGF dependent and novel HGF independent mechanisms that putatively conferring EGFR TKI resistance. Taking EGFR TKI therapy as a paradigm, these findings will be valuable to optimize targeted therapy and to inform the design of personalized pharmaceutical interventions. Citation Format: Haichuan Hu, Lecia Sequist, Zosia Piotrowska, David Kodack, Aaron Hata, Matt Niederst, Cyril Benes, Jeffrey Engelman. Decoding tumor microenvironment to enhance NSCLC targeted therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4954.

BACKGROUND Central nervous system (CNS) metastases represent a major problem in the treatment of human epidermal growth factor receptor 2 (HER2)-positive breast cancer because of the disappointing efficacy of HER2-targeted therapies against brain lesions. The antibody-drug conjugate ado-trastuzumab emtansine (T-DM1) has shown efficacy in trastuzumab-resistant systemic breast cancer. Here, we tested the hypothesis that T-DM1 could overcome trastuzumab resistance in murine models of brain metastases. METHODS We treated female nude mice bearing BT474 or MDA-MB-361 brain metastases (n = 9-11 per group) or cancer cells grown in organotypic brain slice cultures with trastuzumab or T-DM1 at equivalent or equipotent doses. Using intravital imaging, molecular techniques and histological analysis we determined tumor growth, mouse survival, cancer cell apoptosis and proliferation, tumor drug distribution, and HER2 signaling. Data were analyzed with one-way analysis of variance (ANOVA), Kaplan-Meier analysis, and Coefficient of Determination. All statistical tests were two-sided. RESULTS T-DM1 delayed the growth of HER2-positive breast cancer brain metastases compared with trastuzumab. These findings were consistent between HER2-driven and PI3K-driven tumors. The activity of T-DM1 resulted in a survival benefit (median survival for BT474 tumors: 28 days for trastuzumab vs 112 days for T-DM1, hazard ratio = 6.2, 95% confidence interval = 6.1 to 85.84, P < .001). No difference in drug distribution or HER2-signaling was revealed between the two groups. However, T-DM1 led to a statistically significant increase in tumor cell apoptosis (one-way ANOVA for ApopTag, P < .001), which was associated with mitotic catastrophe. CONCLUSIONS T-DM1 can overcome resistance to trastuzumab therapy in HER2-driven or PI3K-driven breast cancer brain lesions due to the cytotoxicity of the DM1 component. Clinical investigation of T-DM1 for patients with CNS metastases from HER2-positive breast cancer is warranted.

Astrocytes are emerging as essential regulators of brain metastasis progression. In a current issue of Nature, Chen et al. identify a novel mechanism of astrocyte-carcinoma interaction and exploit vulnerabilities therein to slow brain metastatic growth in pre-clinical models.

Brain metastases represent a devastating progression of luminal breast cancer. While targeted therapies are often effective systemically, they fail to adequately control brain metastases. In preclinical models that faithfully recapitulate the disparate clinical responses in these microenvironments, we observed that brain metastases evade PI3K inhibition despite efficient drug delivery. In comparison to extracranial disease, there is increased HER3 expression and phosphorylation in the brain lesions. HER3 blockade overcomes the resistance of both HER2-amplified and/or PIK3CA-mutant breast cancer brain metastases to PI3K inhibitors, leading to striking tumor growth delay and significant improvement of mouse survival. Collectively, these data provide a mechanistic basis underlying therapeutic resistance in the brain microenvironment and identify rapidly translatable treatment strategiesfor HER2-amplified and/or PIK3CA-mutant breast cancer brain metastases. Citation Format: Gino B. Ferraro, David P. Kodack, Vasileios Askoxylakis, Qing Sheng, Mark Badeaux, Shom Goel, Xiaolong Qi, Ram Shankaraiah, Alexander Z. Cao, Rakesh R. Ramjiawan, Divya Bezwada, Bhushankumar Patel, Youngchul Song, Carlotta Costa, Kamila Naxerova, Christina Wong, Jonas Kloepper, Rita Das, Angela Tam, Jantima Tanboon, Dan G. Duda, Ryan C. Miller, Marni B. Siegel, Carey K. Anders, Melinda Sanders, Valeria M. Estrada, Robert Schlegel, Carlos L. Arteaga, Elena Brachtel, Alan Huang, Dai Fukumura, Jeffrey A. Engelman, Rakesh K. Jain. The brain microenvironment mediates resistance in luminal breast cancer to PI3K inhibition through HER3 activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5008. doi:10.1158/1538-7445.AM2017-5008

Our labs have previously described a pharmacogenomic approach to identify therapeutic strategies in cancer cells derived directly from the biopsies of patients. These findings were made in pure cancer cell populations derived on the order of months. Our ultimate goal is to utilize one's own cancer cells for a personalized in vitro diagnostic test. Therefore, we aimed to develop a reliable method to analyze a high-throughput pharmacological screen in mixed cell populations with minimal cancer cells since this is the reality of fresh samples within weeks of the biopsy. The necessity for this is two-fold: first, the culture of patient biopsies is more successful on an irradiated fibroblast feeder layer and, second, noncancerous patient cells, including stromal fibroblasts, often survive biopsy culture. We identified a cocktail of two monoclonal antibodies, one against cytokeratin 8 and another against cytokeratin 18, as a consistent identifier of lung cancer cells that could be used in a high-throughput immunofluorescence-based assay. Drug sensitivity experiments with the immunofluorescence-based assay on patient-derived lung cancer cells mixed with feeder or stromal fibroblasts produced dose-response curves consistent with a pure cancer cell viability assay. We plan to utilize this assay to test the accuracy of patient-derived tumor models, obtained within weeks of biopsy, in mimicking patients' responses to targeted therapy. Ultimately, we hope this approach could help determine therapeutic choices for individual patients. Citation Format: David P. Kodack, Matthew Held, Leah Damon, Dana Lee, Melissa Parks, Richard Dicecca, Max Greenberg, Jeffrey A. Engelman, Cyril H. Benes. Development of a drug response assessment platform for biopsy-derived tumor models. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr A13.

Brain metastases represent a major problem in the treatment of HER2-positive breast cancer due to the poor efficacy of HER2-targeted therapies in the brain microenvironment. The antibody drug conjugate ado-trastuzumab emtansine (T-DM1) has shown efficacy in trastuzumab-resistant systemic breast cancer. We tested the hypothesis that T-DM1 could overcome trastuzumab resistance in murine models of brain metastases. Methods: We optimized established animal models of HER2-positive breast cancer brain metastases. We treated mice bearing BT474 (intracranial and intracarotid injections) or MDA-MB-361 (intracranial injection) tumors in the CNS with trastuzumab or T-DM1 at equivalent or equipotent doses. Using intravital imaging, molecular techniques and histological analysis we determined tumor growth, mouse survival, cancer cell apoptosis and proliferation, tumor drug distribution, and HER2 signaling. Results: Treatment with T-DM1 delayed tumor growth in comparison to trastuzumab and control IgG and improved survival. These findings were consistent between HER2-driven and PI3K-driven breast tumors. In BT474 tumors, median survival was 112 days for T-DM1 and 28 days for trastuzumab (p Conclusions: T-DM1 can overcome resistance to trastuzumab therapy in HER2-driven or PI3K-driven breast cancer brain lesions due to the cytotoxicity of the DM1 component. The results of our studies indicate that T-DM1 is effective in the brain microenvironment and will directly inform clinical trials in patients with HER2+ breast cancer brain metastases. Citation Format: Gino B. Ferraro, Vasileios Askoxylakis, David P. Kodack, Mark Badeaux, Dai Fukumura, Jeffrey A. Engelman, Rakesh K. Jain. Ado-trastuzumab emtansine (T-DM1) controls tumor progression of established HER2-positive breast cancer brain metastases in mice. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr A48.

Overcoming resistance to targeted kinase inhibitors is a major clinical challenge in oncology. For 1st and 2nd generation ALK inhibitors acquired resistance due to ALK kinase domain mutations and/or pharmacological drug resistance are major causes for disease relapse. Here, we report the preclinical evaluation of PF-06463922, a potent and brain penetrant ALK/ROS1 inhibitor with sub to low nanomolar cell potency against ALK fusions and all known clinically-acquired resistant mutations. PF-06463922 exhibited marked cytoreductive activity in tumor xenografts driven by various ALK mutants. Furthermore, PF-06463922 led to significant regression of EML4-ALK driven lung cancer brain metastasis and prolonged mouse survival. Compared to other clinically available ALK inhibitors, PF-06463922 is unique in its superior potency against a broad spectrum of acquired ALK mutations, including the highly resistant G1202R mutant and its robust antitumor activity in the brain. Furthermore, PF-06463922 demonstrated remarkable selectivity and safety margins in a variety of preclinical studies. These results suggest that PF-06463922 may be highly effective for the treatment of patients with ALK-driven lung cancers, including those who relapsed on clinically available ALK inhibitors due to ALK secondary mutations and/or brain metastases. Citation Format: Luc Friboulet, Helen Zou, David P. Kodack, Lars D. Engstrom, Qiuhua Li, Melissa West, Ruth W. Tang, Hui Wang, Konstantinos Tsaparikos, Jinwei Wang, Sergei Timofeevski, Dac M. Dinh, Hieu Lam, Justine L. Lam, Shinji Yamazaki, Wenyue Hu, Bhushankumar Patel, Divya Bezwada, Sidra Mahmood, Eugene Lifshits, Timothy Affolter, Patrick B. Lappin, Hovhannes Gukasyan, Nathan Lee, Shibing Deng, Rakesh K. Jain, Ted W. Johnson, Alice T. Shaw, Valeria R. Fantin, Tod Smeal. PF-06463922, a novel next generation ALK/ROS1 inhibitor, overcomes resistance to 1st and 2nd generation ALK inhibitors in pre-clinical models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 130. doi:10.1158/1538-7445.AM2015-130