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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.

Brain metastasis is an end stage in breast cancer progression. Traditional treatment options have minimal efficacy, and overall survival is on the order of months. The incidence of brain metastatic disease is increasing with the improved management of systemic disease and prolongation of survival. Unfortunately, the targeted therapies that control systemic disease have diminished efficacy against brain lesions. There are reasons to be optimistic, however, as emerging therapies have shown promise in preclinical and early clinical settings. This review discusses recent advances in breast cancer brain metastasis therapy and potential approaches for successful treatment.

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.

Colorectal cancers harboring KRAS or BRAF mutations are refractory to current targeted therapies. Using data from a high-throughput drug screen, we have developed a novel therapeutic strategy that targets the apoptotic machinery using the BCL-2 family inhibitor ABT-263 (navitoclax) in combination with a TORC1/2 inhibitor, AZD8055. This combination leads to efficient apoptosis specifically in KRAS- and BRAF-mutant but not wild-type (WT) colorectal cancer cells. This specific susceptibility results from TORC1/2 inhibition leading to suppression of MCL-1 expression in mutant, but not WT, colorectal cancers, leading to abrogation of BIM/MCL-1 complexes. This combination strategy leads to tumor regressions in both KRAS-mutant colorectal cancer xenograft and genetically engineered mouse models of colorectal cancer, but not in the corresponding KRAS-WT colorectal cancer models. These data suggest that the combination of BCL-2/BCL-XL inhibitors with TORC1/2 inhibitors constitutes a promising targeted therapy strategy to treat these recalcitrant cancers. Significance: Effective targeted therapies directed against colorectal cancer with activating mutations in KRAS remain elusive. We have leveraged drug-screen data from a large panel of human colorectal cancers to uncover an effective, rational targeted therapy strategy that has preferential activity in colorectal cancers with KRAS or BRAF mutations. This combination may be developed for clinical testing. Cancer Discov; 4(1); 42–52. ©2013 AACR. See related commentary by Russo et al., p. 19 This article is highlighted in the In This Issue feature, p. 1

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.

Brain metastases remain a serious obstacle in the treatment of patients with human epidermal growth factor receptor-2 (HER2)-amplified breast cancer. Unlike HER2-amplified breast tumors growing in extra-cranial locations, brain metastases do not respond well to HER2 inhibitors and are often the reason for treatment failure. One of the major challenges in studying brain metastases is the lack of preclinical models. We developed a HER2-amplified mouse model of brain metastasis using an orthotopic xenograft of BT474 cells in mice. As seen in patients, the HER2 inhibitors trastuzumab and lapatinib failed to contain brain metastatic tumor growth. Based on previous findings from our laboratory suggesting a role of vascular endothelial growth factor (VEGF) in the resistance of HER2-overexpressing breast cancer brain metastases to trastuzumab, we combined HER2 inhibitors with the anti-VEGFR2 antibody DC101. The combination of either trastuzumab and DC101 or lapatinib and DC101 significantly slowed metastatic tumor growth in the brain, and resulted in a striking improvement in overall survival. The benefit is due largely to an anti-angiogenic effect. The combination of anti-HER2 and anti-VEGFR2 therapy reduced both the total and functional microvascular density in the brain metastatic tumors. Moreover, tumor tissues under combination therapy showed a marked increase in necrosis. Preclinical and clinical evidence suggest that the combination of trastuzumab and lapatinib is superior to either agent alone – though this has never been tested in the brain metastatic setting. We consistently observed increased phosphorylation of HER2 in breast tumor cells growing in the brain compared with the mammary fat pad. In addition, while short-term lapatinib treatment significantly reduced HER2 activation in the brain, it could do so only to the level of that observed in the untreated mammary fat pad - and this effect disappeared over time. We hypothesized that more pronounced HER2 inhibition would be beneficial to these brain metastases with increased HER2 activation. We show here a significant growth delay with the combination of the two HER2 inhibitors compared with monotherapy. Moreover, we found a dramatic brain metastatic tumor growth delay in mice treated with both HER2 inhibitors, trastuzumab and lapatinib, and DC101. The triple combination prolonged overall survival 5 times longer than control-treated mice. Brain metastasis from breast cancer is considered the “final frontier” of breast cancer research and treatment. Our findings support the clinical development of a three-drug regimen of trastuzumab, lapatinib and a VEGF pathway inhibitor for the treatment of HER2-amplified breast cancer brain metastases. While the anti-VEGF antibody bevacizumab in combination with trastuzumab and chemotherapy has shown some promise in HER2-positive metastatic breast cancer patient, there are no data on its efficacy in the context of brain metastases. A clinical trial is now recruiting patients to evaluate the efficacy of bevacizumab in breast cancer patients with active brain metastases, including its combination with trastuzumab in patients with HER2-positive disease. This trial may provide clinical evidence for the approach presented here. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P3-12-03.

Brain metastases are a serious obstacle in the treatment of patients with solid tumors and contribute to the morbidity and mortality of these cancers. It is speculated that the frequency of brain metastasis is increasing for several reasons, including improved systemic therapy and survival, and detection of metastases in asymptomatic patients. The lack of preclinical models that recapitulate the clinical setting and the exclusion of patients with brain metastases from most clinical trials have slowed progress. Molecular factors contributing to brain metastases are being elucidated, such as genes involved in cell adhesion, extravasation, metabolism, and cellular signaling. Furthermore, the role of the unique brain microenvironment is beginning to be explored. Although the presence and function of the blood–brain barrier in metastatic tumors is still poorly understood, it is likely that some tumor cells are protected from therapeutics by the blood–tumor barrier, creating a sanctuary site. This Review discusses what is known about the biology of brain metastases, what preclinical models are available to study the disease, and which novel therapeutic strategies are being studied in patients.

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.

Despite rapid advances in the stem cell field, the ability to identify and track transplanted or migrating stem cells in vivo is limited. To overcome this limitation, we used magnetic resonance imaging (MRI) to detect and follow transplanted stem cells over a period of 28 days in mice using an established myocardial infarction model. Pluripotent mouse embryonic stem (mES) cells were expanded and induced to differentiate into beating cardiomyocytes in vitro. The cardiac-differentiated mES cells were then loaded with superparamagnetic fluorescent microspheres (1.63 μm in diameter) and transplanted into ischemic myocardium immediately following ligation and subsequent reperfusion of the left anterior descending coronary artery. To identify the transplanted stem cells in vivo, MRI was performed using a Varian Inova 4.7 Tesla scanner. Our results show that (a) the cardiac-differentiated mES were effectively loaded with superparamagnetic microspheres in vitro, (b) the microsphere-loaded mES cells continued to beat in culture prior to transplantation, (c) the transplanted mES cells were readily detected in the heart in vivo using noninvasive MRI techniques, (d) the transplanted stem cells were detected in ischemic myocardium for the entire 28-day duration of the study as confirmed by MRI and post-mortem histological analyses, and (e) concurrent functional MRI indicated typical loss of cardiac function, although significant amelioration of remodeling was noted after 28 days in hearts that received transplanted stem cells. These results demonstrate that it is feasible to simultaneously track transplanted stem cells and monitor cardiac function in vivo over an extended period using noninvasive MRI techniques. Disclosure of potential conflicts of interest is found at the end of this article.

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.

Matrix metalloproteinases (MMPs) facilitate cancer progression (1–3). However, broad-spectrum MMP inhibitors failed in part because MMPs mediate both pro- and anticancer effects (4–7) and because off-target, dose-limiting toxicity impeded efficacy (7). To counter these deficiencies, agents targeting specific MMPs have been developed. For breast cancer (BC), MMP14 (membrane type 1-MMP; MT1-MMP) is an especially attractive target (2,8). Genetic knockdown of MMP14 in BC cells impedes their migration and metastases but does not affect their in vitro viability or primary tumor growth (3,8). In contrast, antibody inhibition—which blocks both cancer and stromal MMP14 activity—slows primary tumor growth (9,10). Indeed, a considerable number of reactive stromal cells also express MMP14 (2), illustrating the potential importance of stromal MMP14. MMP14 facilitates angiogenesis (11–13), and MMP14 blockade can inhibit tumor angiogenesis (9,10). Various antiangiogenic agents, however, can also transiently normalize the tumor vasculature, improving tumor perfusion and oxygenation, leading to enhanced efficacy of chemo- and/or radiation-therapy (14–16). In preclinical models, blockade of MMP14 could enhance the response to cytotoxic therapies (9,10). These findings prompted us to determine a potential improvement in tumor vascular function by MMP14 blockade. The antiangiogenic effect of MMP14 inhibition is thought to result from reduced activation of MMP2 by MMP14 (9). However, other MMP14 targets have not been studied, including transforming growth factor β (TGFβ), a mediator of vascular response and a potent immunosuppressor. TGFβ is associated with poor clinical outcome in BC (17). TGFβ has proangiogenic activities and mediates vessel stabilization (18–20). TGFβ inhibitors increase antitumor immunity associated with increasing interferon (IFN)-γ and granzyme B production from natural killer (NK) cells and cytotoxic CD8+ T cells, reducing T regulatory cells, and shifting macrophages toward an inducible nitric oxide synthase (iNOS)–expressing antitumor M1-like phenotype and away from tumor-supportive M2-type (21–27). In this study we investigated if DX-2400 (9), a highly selective MMP14 inhibitory antibody, could decrease TGFβ levels and alter the macrophage phenotype in tumors. We also aimed to determine if DX-2400 could improve tumor vessel function and thus provide additional benefits when combined with radiation therapy.

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.

Brain metastases are a serious obstacle in the treatment of patients with human epidermal growth factor receptor-2 ( HER2 )–amplified breast cancer. Although extracranial disease is controlled with HER2 inhibitors in the majority of patients, brain metastases often develop. Because these brain metastases do not respond to therapy, they are frequently the reason for treatment failure. We developed a mouse model of HER2 -amplified breast cancer brain metastasis using an orthotopic xenograft of BT474 cells. As seen in patients, the HER2 inhibitors trastuzumab and lapatinib controlled tumor progression in the breast but failed to contain tumor growth in the brain. We observed that the combination of a HER2 inhibitor with an anti–VEGF receptor-2 (VEGFR2) antibody significantly slows tumor growth in the brain, resulting in a striking survival benefit. This benefit appears largely due to an enhanced antiangiogenic effect: Combination therapy reduced both the total and functional microvascular density in the brain xenografts. In addition, the combination therapy led to a marked increase in necrosis of the brain lesions. Moreover, we observed even better antitumor activity after combining both trastuzumab and lapatinib with the anti-VEGFR2 antibody. This triple-drug combination prolonged the median overall survival fivefold compared with the control-treated group and twofold compared with either two-drug regimen. These findings support the clinical development of this three-drug regimen for the treatment of HER2 -amplified breast cancer brain metastases.

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

The activity of growth factors is crucial for tumor progression. We previously characterized a secreted fibroblast growth factor–binding protein (FGF-BP1) as a chaperone molecule, which enhances the biological functions of FGFs by releasing FGFs from the extracellular matrix. Here, we characterize the frequency and pattern of FGF-BP1 expression during the malignant progression of pancreas and colorectal carcinoma. For this, we generated monoclonal antibodies that detect FGF-BP1 protein in formalin-fixed, paraffin-embedded tissues and applied in situ hybridization to detect FGF-BP1 mRNA in adjacent tissue sections. FGF-BP1 protein and mRNA were found up-regulated (>70% positive) in parallel (r = 0.70, P < 0.0001) in colon adenoma (n = 9) as well as primary (n = 46) and metastatic (n = 71) colorectal cancers relative to normal colon epithelia (all P < 0.0001, versus normal). Similarly, pancreatitis (n = 17), pancreatic intraepithelial neoplasia (n = 80), and pancreatic adenocarcinoma (n = 67) showed a significant up-regulation of FGF-BP1 compared with normal pancreas (n = 42; all P < 0.0001, relative to normal). Furthermore, the biological activity of FGF-BP1 is neutralized by one of the antibodies, suggesting the potential for antibody-based therapeutic targeting. We propose that the up-regulation of the secreted FGF-BP1 protein during initiation of pancreas and colon neoplasia could make this protein a possible serum marker indicating the presence of high-risk premalignant lesions. (Cancer Res 2006; 66(2): 1191-8)

Colorectal cancers (CRCs) harboring KRAS or BRAF mutations are refractory to current targeted therapies. Using data from a high-throughput drug screen, we have developed a novel therapeutic strategy that combines targeting of the apoptotic machinery using the BCL-2 family inhibitor ABT-263 (navitoclax) in combination with a TORC1/2 inhibitor, AZD8055. This combination leads to efficient apoptosis specifically in KRAS mutant (MT) and BRAF MT but not wild-type (WT) CRC cells. This specific susceptibility results from TORC1/2 inhibition leading to suppression of MCL-1 expression in mutant, but not wild-type CRCs, leading to abrogation of BIM/MCL-1 complexes. This combination strategy leads to tumor regressions in both KRAS MT colorectal cancer xenograft and genetically-engineered mouse models of CRC, but not in the corresponding KRAS WT CRC models. These data suggest that the combination of BCL-2/XL inhibitors with TORC1/2 inhibitors constitutes a promising targeted therapy strategy to treat these recalcitrant cancers. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C263. Citation Format: Erin M. Coffee, Anthony C. Faber, Carlotta Costa, Anahita Dastur, Hiromichi Ebi, Aaron N. Hata, Alan T. Yeo, Elena J, Youngchul Song, Ah Ting Tam, Jessica L. Boisvert, Randy J. Milano, Jatin Roper, David P. Kodack, Rakesh K. Jain, Ryan B. Corcoran, Miguel N. Rivera, Sridhar Ramaswamy, Kenneth E. Hung, Cyril H. Benes, Jeffrey A. Engelman. mTOR inhibition specifically sensitizes colorectal cancers with KRAS or BRAF mutations to BCL-2/BCL-XL inhibition by suppressing MCL-1. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C263.

Pleiotrophin (PTN) is a heparin-binding growth factor that can support tumor growth, invasion, angiogenesis, and metastasis. The mechanism of action (receptor signaling pathway) responsible for mediating PTN's biological effects, however, is still controversial. Here we show that an antibody targeting the ligand-binding domain of anaplastic lymphoma kinase (ALK) is capable of blocking the signaling effects of PTN in a glioblastoma cell line that expresses high levels of ALK. This corroborates recent findings by Stylianou, et al. (2009), who showed disruption of PTN activity with a single chain variable IgG fragment that binds to the ALK ligand-binding domain. Furthermore, expression of protein tyrosine phosphatase receptor ζ (Ptprz) in cells resulted in crosstalk with the PTN-ALK signaling axis at the level of p38 MAPK. While PTN increased p38 MAPK phosphorylation, the presence of Ptprz decreased its phosphorylation, both at its basal as well as PTN-enhanced levels. Enhancement of p38 MAPK phosphorylation by PTN is possibly mediated through activation of MKK4, as it was phosphorylated in the presence of PTN. On the other hand, the mechanism for Ptprz-mediated suppression of p38 MAPK is not yet clear, as there was no change in phosphorylation of MKK3, MKK4, or MKK6 after its over-expression. These findings suggest that ALK is playing a direct role in PTN activity, and that the Ptprz signaling pathway may act in a separate but inter-connecting fashion. Parallel targeting of both the ALK and Ptprz signaling pathway may be necessary for the treatment of tumors (glioblastoma) expressing both membrane proteins. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4018.