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Combination Inhibition of PI3K and Mtorc1 Yields Durable Remissions

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Combination Inhibition of PI3K and Mtorc1 Yields Durable Remissions BRIEF COMMUNICATIONS We also verified the expression of epithelial marker cytokeratin 7 Combination inhibition of PI3K (CK7) and the absence of glial markers (GFAP and OLIG2) (Fig. 1b). We subsequently established PDXs by using BCBMs from three other and mTORC1 yields durable patients with HER2-positive BCBM. None of the five PDXs expressed remissions in mice bearing detectable levels of the tumor suppressor protein PTEN (Fig. 1b and Supplementary Table 1). Of 27 clinical specimens of HER2-posi- orthotopic patient-derived tive BCBMs, 67% showed no PTEN staining (Fig. 1c), which further confirms that the loss of PTEN is widespread in BCBMs1,2. xenografts of HER2-positive To assess the response of HER2-positive BCBMs to targeted therapy, we treated DF-BM355 PDXs with a HER2 kinase inhibitor lapatinib. breast cancer brain metastases DF-BM355 PDXs showed no response to lapatinib, which is consis­ tent with the resistance of the donor’s tumor to HER2-directed therapy Jing Ni1,2,11, Shakti H Ramkissoon3,4,11, Shaozhen Xie1,2,11, (Supplementary Fig. 2a,b). Because DF-BM355 lacks PTEN—a key reg- Shom Goel1,3, Daniel G Stover3, Hanbing Guo1,2, Victor Luu1,2, ulator of the PI3K pathway—we tested the combination of lapatinib with Eugenio Marco5, Lori A Ramkissoon3, Yun Jee Kang3, BKM120, a pan-PI3K inhibitor that penetrates the blood–brain barrier Marika Hayashi3, Quang-De Nguyen6, Azra H Ligon4, Rose Du7, (BBB)3–5. Again, no response was observed (Supplementary Fig. 2a,b). Elizabeth B Claus7,8, Brian M Alexander9,10, Guo-Cheng Yuan5, To understand the lack of response to combined inhibition with Zhigang C Wang1,3, J Dirk Iglehart1,3, Ian E Krop3, HER2 and PI3K, we assessed tumor PI3K-pathway signaling in Thomas M Roberts1,2, Eric P Winer3,12, Nancy U Lin3, response to lapatinib and/or BKM120. Although, as compared to the Keith L Ligon3,4 & Jean J Zhao1,2,12 control, these treatments reduced the phosphorlyation of AKT and S6RP—the downstream effectors of PI3K and mTOR, respectively— Brain metastases represent the greatest clinical challenge we observed little change in p-4EBP1, an mTORC1 effector that medi- in treating HER2-positive breast cancer. We report the ates translation (Supplementary Fig. 2c,d). Even combined HER2 development of orthotopic patient-derived xenografts (PDXs) and PI3K inhibition therefore did not completely suppress mTORC1 of HER2-expressing breast cancer brain metastases (BCBM), activity in the DF-BM355 model. and their use for the identification of targeted combination Notably, persistent mTOR activity in breast cancers can mediate therapies. Combined inhibition of PI3K and mTOR resulted in resistance to PI3K inhibition, and this can be overcome by inhibi- Nature America, Inc. All rights reserved. Inc. Nature America, durable tumor regressions in three of five PDXs, and therapeutic tion with mTORC1 (ref. 6). However, the brain microenvironment is 6 response was correlated with a reduction in the phosphorylation unique, and brain metastases are notoriously refractory to systemic of 4EBP1, an mTORC1 effector. The two nonresponding PDXs therapies that are effective against extracranial metastases. In keeping © 201 showed hypermutated genomes with enrichment of mutations with this, it is not known whether mTOR inhibition might overcome in DNA-repair genes, which suggests an association of genomic PI3K-inhibitor resistance in BCBMs. To explore this, we combined instability with therapeutic resistance. These findings suggest either lapatinib or BKM120 with RAD001, an mTORC1 inhibitor that that a biomarker-driven clinical trial of PI3K inhibitor in penetrates the BBB7,8. combination with an mTOR inhibitor should be conducted Whereas DF-BM355 PDXs showed limited response to the combi- for patients with HER2-positive BCBM. nation of lapatinib and RAD001 (Supplementary Fig. 2e), the admin- istration of BKM120 and RAD001 together resulted in marked tumor To develop therapeutic strategies for HER2-positive BCBM, we estab- regression, as measured by bioluminescence (Fig. 2a). Owing to the lished a panel of orthotopic PDXs (Fig. 1a). Fresh BCBMs from two unprecedented nature of this response, we removed two mice from patients, Dana-Farber Brain Metastasis (DF-BM)354 and DF-BM355, the control group bearing large tumors, and introduced BKM120 were grafted directly into the brains of SCID mice with a median survival and RAD001. The large tumors also regressed over time (Fig. 2a). of roughly 2–3 months (Supplementary Fig. 1). The PDXs resembled Magnetic resonance imaging (MRI) before and after treatment con- the parental BCBMs histologically, as well in their estrogen receptor firmed these results (Fig. 2b). The remaining mice in the control (ER), progesterone receptor (PR) and HER2 expression (Fig. 1b). group quickly reached the study endpoint (developing systemic 1Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA. 2Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, USA. 3Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, USA. 4Department of Pathology, Brigham and Women’s Hospital, Boston, Massachusetts, USA. 5Department of Biostatistics and Computational Biology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA. 6Lurie Family Imaging Center, Dana-Farber Cancer Institute, Boston, Massachusetts, USA. 7Department of Neurosurgery, Brigham and Women’s Hospital, Boston, Massachusetts, USA. 8School of Public Health, Yale University, New Haven, Connecticut, USA. 9Department of Radiation Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA. 10Department of Radiation Oncology, Brigham and Women’s Hospital, Boston, Massachusetts, USA. 11These authors contributed equally to this work. 12These authors jointly directed this work. Correspondence should be addressed to J.J.Z. ([email protected]), K.L.L. ([email protected]), and N.U.L. ([email protected]). Received 2 February; accepted 9 May; published online 6 June 2016; doi:10.1038/nm.4120 NATURE MEDICINE 1 BRIEF COMMUNICATIONS Figure 1 Establishment of orthotopic a Intracranial HER2-positive BCBM PDXs. (a) Schematic injection depicting the process of generating orthotopic + Luciferase Evaluation of PDX BCBM models for use in preclinical DF-BM354 anticancer studies. Fresh brain metastatic tissues from drugs individuals with BCBM were grafted directly Patient BCBM ≤P5 into the brains of female SCID mice. The DF-BM355 xenografts in the brain were explanted, P0–P1 dissociated and transduced with a luciferase gene, and then re-injected into new cohorts b H&E HER2 ER PR CK7 GFAP OLIG2 Ki67 PTEN of mice. P0, primary graft; P1–P5, passage number in mice. Scale bars, 5 cm (left); Patient 500 µm (middle). (b) Representative histologic and immunophenotypic analyses of two patient DF-BM354 PDX surgical biopsies and the corresponding PDXs. Scale bars, 25 µm. (c) Compiled result of PTEN immunohistochemistry (IHC) scores performed on 27 human HER2-positive BCBM samples. Patient 0, no staining in >90% of tumor cells; 1+, weak DF-BM355 staining in >75% of tumor cells; 2+, strong PDX staining in >75% of tumor cells. c PTEN IHC score (7.4%) 2+, symptoms of morbidity or neurologic phenotypes) with high luci- 1+, (25.9%) ferase signals (Fig. 2c). Notably, the luciferase signal in BKM120- and 0, RAD001-treated tumors declined to a nearly undetectable level over (66.7%) the treatment period of 14 weeks, and mice remained healthy and luciferase-signal-free for weeks after treatment cessation (Fig. 2c and Total = 27 Supplementary Fig. 2e). All mice treated survived the 210-d period of observation, whereas all mice in the control group died after approximately 90 d (Fig. 2d). To avoid potential confounding symp- combination might be effective for HER2-positive BCBMs, regardless toms from aging-associated disease phenotypes, we stopped treatment of hormone-receptor expression. The DF-BM463 model also exhibited around 210 d, at which point the recipient mice were 270–280 d old. durable responses to BKM120–RAD001 therapy, along with similar The unique efficacy of this combination is underscored by results changes in p-4EBP1, Ki67 and cleaved caspase-3 levels (Fig. 2f and from additional experiments; neither a combination of BKM120 with Supplementary Fig. 5c,d). the MEK inhibitor MEK162—chosen because of high p-ERK levels By contrast, BKM120–RAD001 combination therapy had little effect in the PDX tumors—nor a combination of BKM120 with the BET on the survival of mice bearing DF-BM507 and DF-BM590 PDXs, and Nature America, Inc. All rights reserved. Inc. Nature America, the amount of p-S6RP and p-4EBP1 was also not reduced in these mod- 6 bromodomain inhibitor JQ1, which downregulates MYC expression— chosen owing to MYC amplifications in the PDX tumors—showed els (Fig. 2f and Supplementary Fig. 5e,f). To understand the molecular efficacy (Supplementary Figs. 3 and 4). basis for the differential therapeutic responses between models, we © 201 To understand the mechanism underlying the synergy between performed transcriptome analyses on tumors from untreated mice. BKM120 and RAD001, we harvested tumors from mice after 4 d of The three responding models showed significantly higher expression of treatment for pharmacodynamic assessment. Although both mono- AKT–mTOR-dependent signature genes10 than did two nonresponding therapies reduced p-S6RP, as compared to control, neither drug models (Fig. 2g), which suggests that some, but not all, HER2-positive alone significantly suppressed p-4EBP1 (Fig. 2e), which suggests that BCBMs depend on the AKT–mTOR pathway. mTORC1 was not completely inhibited. These observations are con- We also performed whole-exome sequencing (WES) of all five PDX sistent with reports indicating that although rapamycin (an mTORC1 tumors and matched samples from the donor patients’ blood (patient inhibitor) stably inhibits p-S6RP, its effect on p-4EBP1 is short lived, blood was unavailable for DF-BM355). Copy-number variations and that a rapid re-emergence of p-4EBP1 contributes to rapamycin (CNVs) were frequent in all five of the tumor models (Supplementary resistance9.
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