Amanda E.D. Van Swearingen

Efficacy of Carboplatin Alone and in Combination with ABT888 in Intracranial Murine Models of BRCA-Mutated and BRCA–Wild-Type Triple-Negative Breast Cancer

Patients with breast cancer brain metastases have extremely limited survival and no approved systemic therapeutics. Triple-negative breast cancer (TNBC) commonly metastasizes to the brain and predicts poor prognosis. TNBC frequently harbors BRCA mutations translating to platinum sensitivity potentially augmented by additional suppression of DNA repair mechanisms through PARP inhibition. We evaluated brain penetrance and efficacy of carboplatin ± the PARP inhibitor ABT888, and investigated gene-expression changes in murine intracranial TNBC models stratified by BRCA and molecular subtype status. Athymic mice were inoculated intracerebrally with BRCA-mutant: SUM149 (basal), MDA-MB-436 (claudin-low); or BRCA–wild-type (wt): MDA-MB-468 (basal), MDA-MB-231BR (claudin-low). TNBC cells were treated with PBS control [intraperitoneal (IP), weekly], carboplatin (50 mg/kg/wk, IP), ABT888 (25 mg/kg/d, oral gavage), or their combination. DNA damage (γ-H2AX), apoptosis (cleaved caspase-3, cC3), and gene expression were measured in intracranial tumors. Carboplatin ± ABT888 significantly improved survival in BRCA-mutant intracranial models compared with control, but did not improve survival in BRCA-wt intracranial models. Carboplatin + ABT888 revealed a modest survival advantage versus carboplatin in BRCA-mutant models. ABT888 yielded a marginal survival benefit in the MDA-MB-436, but not in the SUM149 model. BRCA-mutant SUM149 expression of γ-H2AX and cC3 proteins was elevated in all treatment groups compared with control, whereas BRCA-wt MDA-MB-468 cC3 expression did not increase with treatment. Carboplatin treatment induced common gene-expression changes in BRCA-mutant models. Carboplatin ± ABT888 penetrates the brain and improves survival in BRCA-mutant intracranial TNBC models with corresponding DNA damage and gene-expression changes. Combination therapy represents a potential promising treatment strategy for patients with TNBC brain metastases warranting further clinical investigation. Mol Cancer Ther; 14(4); 920–30. ©2015 AACR.

Combination therapy with potent PI3K and MAPK inhibitors overcomes adaptive kinome resistance to single agents in preclinical models of glioblastoma

Background Glioblastoma (GBM) is the most common and aggressive primary brain tumor. Prognosis remains poor despite multimodal therapy. Developing alternative treatments is essential. Drugs targeting kinases within the phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) effectors of receptor tyrosine kinase (RTK) signaling represent promising candidates. Methods We previously developed a non-germline genetically engineered mouse model of GBM in which PI3K and MAPK are activated via Pten deletion and KrasG12D in immortalized astrocytes. Using this model, we examined the influence of drug potency on target inhibition, alternate pathway activation, efficacy, and synergism of single agent and combination therapy with inhibitors of these 2 pathways. Efficacy was then examined in GBM patient-derived xenografts (PDX) in vitro and in vivo. Results PI3K and mitogen-activated protein kinase kinase (MEK) inhibitor potency was directly associated with target inhibition, alternate RTK effector activation, and efficacy in mutant murine astrocytes in vitro. The kinomes of GBM PDX and tumor samples were heterogeneous, with a subset of the latter harboring MAPK hyperactivation. Dual PI3K/MEK inhibitor treatment overcame alternate effector activation, was synergistic in vitro, and was more effective than single agent therapy in subcutaneous murine allografts. However, efficacy in orthotopic allografts was minimal. This was likely due to dose-limiting toxicity and incomplete target inhibition. Conclusion Drug potency influences PI3K/MEK inhibitor-induced target inhibition, adaptive kinome reprogramming, efficacy, and synergy. Our findings suggest that combination therapies with highly potent, brain-penetrant kinase inhibitors will be required to improve patient outcomes.

Efficacy and pharmacokinetics of a modified acid-labile docetaxel-PRINT® nanoparticle formulation against non-small-cell lung cancer brain metastases

AIM Particle Replication in Nonwetting Templates (PRINT(®)) PLGA nanoparticles of docetaxel and acid-labile C2-dimethyl-Si-Docetaxel were evaluated with small molecule docetaxel as treatments for non-small-cell lung cancer brain metastases. MATERIALS & METHODS Pharmacokinetics, survival, tumor growth and mice weight change were efficacy measures against intracranial A549 tumors in nude mice. Treatments were administered by intravenous injection. RESULTS Intracranial tumor concentrations of PRINT-docetaxel and PRINT-C2-docetaxel were 13- and sevenfold greater, respectively, than SM-docetaxel. C2-docetaxel conversion to docetaxel was threefold higher in intracranial tumor as compared with nontumor tissues. PRINT-C2-docetaxel increased median survival by 35% with less toxicity as compared with other treatments. CONCLUSION The decreased toxicity of the PRINT-C2-docetaxel improved treatment efficacy against non-small-cell lung cancer brain metastasis.

Breast cancer brain metastases: evidence for neuronal-like adaptation in a 'breast-to-brain' transition?

Brain metastases remain a significant challenge in the treatment of breast cancer patients due to the unique environment posed by the central nervous system. A better understanding of the biology of breast cancer cells that have metastasized to the brain is required to develop improved therapies. A recent Proceedings of the National Academy of Sciences article demonstrates that breast cancer cells in the brain microenvironment express γ-aminobutyric acid (GABA)-related genes, enabling them to utilize GABA as an oncometabolite, thus gaining a proliferative advantage. In this viewpoint, we highlight these findings and their potential impact on the treatment of breast cancer brain metastases.

Abstract 2579: Combination therapy with MEK inhibition is efficacious in intracranial triple negative breast cancer models

Introduction: Nearly half of metastatic triple negative breast cancer (TNBC) patients develop brain metastases (BM) and face a poor prognosis. The blood-brain barrier (BBB) prevents many treatments from reaching intracranial tumors, and there are no FDA-approved systemic therapies to treat TNBC BM. In this study, we evaluated the efficacy of BBB-permeable, clinically-available inhibitors of MEK and identified rational co-target pathways in preclinical models of intracranial (IC) TNBC. Methods: In vitro IC50s, synergy, and siRNA screens (700 kinase genes) were conducted in 4 human-derived TNBC lines (SUM149, MDA-MB-468, MDA-MB-436, MDA-MB-231Br). We evaluated the efficacy of the MEK1/2 inhibitor AZD6244 (AZD), the pan-PI3K inhibitor BKM120 (BKM), and the PDGFR inhibitor Pazopanib (Pazo) in IC TNBC mouse models. Tumor burden was monitored via bioluminescence, and IC tumors were frozen for gene expression analyses using custom human 4×44K Agilent microarrays or of kinome activity profiles using multiplex kinase inhibitor beads and mass spectrometry. To determine drivers of AZD sensitivity, DNA copy number data (Broad CCLE) was analyzed using SWITCHplus to identify copy number alterations that differ between sensitive (n = 8) vs. resistant (n = 12) TNBC lines based on their IC50s (Sanger Cancerxgene). Results: In vitro, SUM149 and 231Br TNBC cells exhibited lower ( 40 uM). Several genes synthetically enhanced lethality in SUM149 and 231Br cells: PI3K genes and PDGFRα/β with AZD, and MAPK/MAP2K/MAP3K genes with BKM, suggesting MEK+PI3K and MEK+PDGFR inhibition as rational combinations. AZD plus BKM or Pazo were synergistic in vitro in sensitive cell lines. In vivo, AZD reduced tumor burden and improved survival in the SUM149 (72 vs. 45 days in controls, p Several DNA segments were significantly altered in sensitive vs. resistant TNBC cell lines. Notably, MEK-pathway genes were lost in the resistant lines. Ongoing work will complete characterization of therapies in all models in vitro and in vivo and will compare genetic, transcriptional, and kinome activity alterations. Conclusions: TNBC models exhibit different innate sensitivities to the BBB-permeable MEK inhibitor AZD6244. In sensitive models, AZD improves survival and reduces intracranial tumor burden, and rational combined inhibition of PI3K or PDGFR further increases survival. Identification of predictive biomarkers will enable translation of our results to biomarker-driven clinical trials for patients with TNBC BM. Citation Format: Amanda E.D. Van Swearingen, Marni B. Siegel, Maria J. Sambade, Shivani Sud, Samantha M. Miller, Grace Silva, Ryan E. Bash, Charlene M. Santos, David B. Darr, Brian Golitz, Joel S. Parker, C. Ryan Miller, Gary L. Johnson, Carey K. Anders. Combination therapy with MEK inhibition is efficacious in intracranial triple negative breast cancer 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 2579. doi:10.1158/1538-7445.AM2015-2579

Abstract 5449A: PI3K and MEK inhibition in intracranial triple negative breast cancer: Efficacy of BKM120 and AZD6244 in preclinical mouse models

Introduction: Triple-negative breast cancer (TNBC) is an aggressive subtype which lacks the classical clinical biomarkers for hormone receptors (HR) and HER2. Nearly half of metastatic TNBC patients develop brain metastases and these patients face a poor prognosis. Most new effective treatments for breast cancer target hyperactivated HER2 and HR pathways, which TNBC tumors lack. The blood-brain barrier (BBB) also prevents many treatments from reaching the brain and any developing tumors therein. There is currently no FDA-approved systemic chemotherapy for the treatment of TNBC brain metastases. Tissue-based studies show activation of the PI3K pathway in breast cancer brain metastases and in vivo models of extracranial TNBC are sensitive to PI3K and MEK inhibitors. BBB-permeable inhibitors targeting the PI3K and MEK pathways are in clinical development. In this study, we evaluated the efficacy of two such inhibitors, the pan-PI3K inhibitor BKM120 and the MEK1/2 inhibitor AZD6244, in preclinical models of intracranial (IC) TNBC. Methods: The efficacy of PI3K and MEK inhibition by BKM120 and AZD6244, respectively, alone and in combination was determined in orthotopic mouse models of basal-like TNBC through IC implantation of SUM149 or MDA-MB-468 cell lines. Drugs were administered at the maximum tolerated doses via chow: 30 mg/kg BKM, 37 mg/kg AZD, or 25 mg/kg BKM + 18 mg/kg AZD. Tumor burden was monitored via weekly bioluminescence imaging, and brain tumors were frozen at sacrifice for gene expression analyses. To explore potential resistance mechanisms and inform rational combination therapies, in vitro IC50s, combination synergy determinations, and single-agent synthetic lethal siRNA screens were conducted. Results: In the SUM149 model, median survival was 45 days (control), 53.5 days (BKM), 53.5 days (BKM+AZD), and 76 days (AZD). AZD alone and with BKM reduced tumor burden via bioluminescence imaging. In vitro, 3-day dose response curves showed that BKM and AZD were similarly potent but BKM was more effective (IC50; maximal % cell reduction: BKM: 1.3 uM, >90%; AZD: 816 nM, >50%). Several genes were identified as synthetically lethal “hits” in an initial kinome screen by two-class SAM. AURKA was a hit with BKM120, while BRAF and several PI3K, AMPK, and CDK genes were synthetically lethal with AZD6244. Ongoing work will include survival studies in the 468 model and gene expression changes in IC tumors in response to therapy. Conclusions: BKM120 and AZD6244 both improved survival in an IC TNBC SUM149 mouse model, with single agent AZD6244 being most efficacious. The siRNA screens indicate that combined treatment with BKM120 and AZD6244 should be synthetically lethal, suggesting that combination therapy may have underperformed due to toxicity. Ongoing in vitro and in vivo studies (including dosing schedules) will further characterize the effects of these drugs in intracranial TNBC. Citation Format: Amanda E.D. Van Swearingen, Marni B. Siegel, Ryan Bash, Brian Golitz, Charlene Santos, David Darr, Joel Parker, Gary L. Johnson, C. Ryan Miller, Carey K. Anders. PI3K and MEK inhibition in intracranial triple negative breast cancer: Efficacy of BKM120 and AZD6244 in preclinical mouse models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5449A. doi:10.1158/1538-7445.AM2014-5449A

Integrated RNA and DNA sequencing reveals early drivers of metastatic breast cancer

Breast cancer metastasis remains a clinical challenge, even within a single patient across multiple sites of the disease. Genome-wide comparisons of both the DNA and gene expression of primary tumors and metastases in multiple patients could help elucidate the underlying mechanisms that cause breast cancer metastasis. To address this issue, we performed DNA exome and RNA sequencing of matched primary tumors and multiple metastases from 16 patients, totaling 83 distinct specimens. We identified tumor-specific drivers by integrating known protein-protein network information with RNA expression and somatic DNA alterations and found that genetic drivers were predominantly established in the primary tumor and maintained through metastatic spreading. In addition, our analyses revealed that most genetic drivers were DNA copy number changes, the TP53 mutation was a recurrent founding mutation regardless of subtype, and that multiclonal seeding of metastases was frequent and occurred in multiple subtypes. Genetic drivers unique to metastasis were identified as somatic mutations in the estrogen and androgen receptor genes. These results highlight the complexity of metastatic spreading, be it monoclonal or multiclonal, and suggest that most metastatic drivers are established in the primary tumor, despite the substantial heterogeneity seen in the metastases.

LCCC 1025: a phase II study of everolimus, trastuzumab, and vinorelbine to treat progressive HER2-positive breast cancer brain metastases

PurposeHER2 + breast cancer (BC) is an aggressive subtype with high rates of brain metastases (BCBM). Two-thirds of HER2 + BCBM demonstrate activation of the PI3K/mTOR pathway driving resistance to anti-HER2 therapy. This phase II study evaluated everolimus (E), a brain-permeable mTOR inhibitor, trastuzumab (T), and vinorelbine (V) in patients with HER2 + BCBM.Patients and methodsEligible patients had progressive HER2 + BCBM. The primary endpoint was intracranial response rate (RR); secondary objectives were CNS clinical benefit rate (CBR), extracranial RR, time to progression (TTP), overall survival (OS), and targeted sequencing of tumors from enrolled patients. A two-stage design distinguished intracranial RR of 5% versus 20%.Results32 patients were evaluable for toxicity, 26 for efficacy. Intracranial RR was 4% (1 PR). CNS CBR at 6 mos was 27%; at 3 mos 65%. Median intracranial TTP was 3.9 mos (95% CI 2.2–5). OS was 12.2 mos (95% CI 0.6–20.2). Grade 3–4 toxicities included neutropenia (41%), anemia (16%), and stomatitis (16%). Mutations in TP53 and PIK3CA were common in BCBM. Mutations in the PI3K/mTOR pathway were not associated with response. ERBB2 amplification was higher in BCBM compared to primary BC; ERBB2 amplification in the primary BC trended toward worse OS.ConclusionWhile intracranial RR to ETV was low in HER2 + BCBM patients, one-third achieved CNS CBR; TTP/OS was similar to historical control. No new toxicity signals were observed. Further analysis of the genomic underpinnings of BCBM to identify tractable prognostic and/or predictive biomarkers is warranted. Clinical Trial: (NCT01305941).

Abstract A03: Several rational combination kinase inhibitor treatments identified by synthetic lethality screens are efficacious in intracranial triple negative breast cancer models

Introduction: Nearly half of metastatic triple negative breast cancer (TNBC) patients develop brain metastases (BMs) and face a poor prognosis. There are no FDA-approved systemic therapies to treat TNBC BM, due in part to the blood-brain barrier. TNBC and breast cancer BMs exhibit both activation of the PI3K and MEK pathways, but attempts to target them in preclinical models have shown limited efficacy due to innate and acquired resistance to kinase inhibition in TNBC. In this study, we identify several clinically relevant rational combination therapies based on synthetic lethality and evaluate the efficacy of combined brain-penetrant, clinically-available kinase inhibitors in intracranial TNBC models. Methods: An siRNA screen against 720 kinase genes was used to identify synthetic enhancers of lethality with pan-PI3K inhibitor (BKM120) or MEK1/2 inhibitor (AZD6244) treatment in vitro using TNBC models capable of growing in mouse brain (SUM149, MDA-MB-231Br). The efficacy of these and other brain-penetrant drugs of interest based on the screen were assessed for efficacy in vitro, alone (IC50s) and combined (synergy). Some combinations were evaluated in vivo in mice bearing intracranial TNBC tumors for their effects on survival and tumor burden. Tumor burden was monitored via bioluminescence. IC tumors from treated mice were extracted, fresh frozen, and analyzed for the activation state of the kinome using multiplexed kinase inhibitor bead (MIB) enriched mass spectrometry (MS). Results: The screen identified the following combinations as synthetic lethal pairs: PI3K+MEK, MEK+PDGFR, PI3K+AURKA, MEK+BRAF. Pharmacological synergy of combined treatments was confirmed in vitro between PI3K(/mTOR)+MEK, MEK+PDGFR, and PI3K+AURKA, in TNBC cells (SUM149, 231Br) using brain-penetrant drugs in clinical development (mTOR inhibitor Everolimus, dual PI3K/mTOR inhibitor GNE317, PDGFR inhibitor Pazopanib, AURKA inhibitor MLN8237, BRAF inhibitor Dabrafenib). For some combinations, particularly PI3K+AURKA inhibition, sequencing of the drugs significantly altered the combined effects and synergy. Combinations which were synthetically lethal and synergistic at physiologically relevant doses in vitro demonstrated enhanced efficacy in vivo, including PI3K+MEK, MEK+PDGFR, and PI3K+AURKA. In contrast, other combinations (i.e. PI3K+mTOR) did not significantly improve survival or tumor burden in vivo. Despite improved survival with some combination treatments, mice eventually succumbed to tumor burden as tumors eventually grew. Kinome analysis of IC tumors treated with PI3K (BKM120) and/or MEK1/2 (AZD6244) inhibitors for 2 weeks identified several potential resistance markers, including INSR, IGF1R, and FGFR2, which may be targetable clinically. Conclusions: Synthetic lethality screens identified multiple rational combination therapies based on PI3K and/or MEK inhibition in TNBC cells, particularly PI3K+MEK, MEK+PDGFR, and PI3K+AURKA. Combined use of brain-penetrant, clinically available inhibitors against these targets showed promising efficacy in intracranial TNBC mouse models. Rational combinations of brain-penetrant kinase inhibitors are promising strategies for a patient population with few options. In vivo studies assessing the efficacy of other identified combinations, as well as more extensive characterization of potential resistance mechanisms, in intracranial TNBC mouse models are warranted to provide the translational foundation for future clinical studies. Citation Format: Amanda E.D. Van Swearingen, Maria J. Sambade, Marni B. Siegel, Shivani Sud, Samantha M. Bevill, Brian T. Golitz, Ryan E. Bash, Charlene M. Santos, David B. Darr, Joel S. Parker, C. Ryan Miller, Gary L. Johnson, Carey K. Anders. Several rational combination kinase inhibitor treatments identified by synthetic lethality screens are efficacious in intracranial triple negative breast cancer models [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr A03.

Abstract 3867: Combined PI3K and AURKA inhibition are efficacious in triple-negative breast cancer models

Introduction: Nearly half of metastatic triple negative breast cancer (TNBC) patients develop brain metastases (BMs) and face a poor prognosis. There are no FDA-approved systemic therapies to treat TNBC BM, due in part to the blood-brain barrier. BCBMs exhibit both activation of the PI3K pathway and AURKA amplification/overexpression relative to primary breast cancers. In this study, we evaluate the efficacy of brain-penetrant, clinically-available inhibitors of PI3K and AURKA in TNBC cell lines that are capable of growing in the mouse brain. Methods: In vitro characterization of the pan-PI3K inhibitor BKM120 and AURKA inhibitor MLN8237 were conducted in 2 human-derived TNBC cell lines, SUM149 and (MDA-MB-)231Br. A siRNA screen (720 kinase genes) was used to identify synthetic enhancers of lethality with BKM120 treatment. To assess the efficacy of these drugs, the IC50s of BKM120 and MLN8237 and synergy of the combination were determined. To compare the effects of BKM120 and/or MLN8237 treatment on cell cycle progression, FACS analysis was conducted at 24, 48, and 72 hours in parent cells and in cells continuously cultured in MLN8237-treated media for 12 weeks. Results: The screen confirmed that combined PI3K and AURKA inhibition synthetically enhanced lethality in SUM149 and 231Br cells. SUM149 and 231Br cells and two additional TNBC cell lines (MDA-MB-468 and MDA-MB-436) exhibited similar IC50s (1.3-21 μM) to BKM120. However, there was a >2.5 fold range (26.5-69 μM) in IC50s for MLN8237, with the greatest potency in the 231Br line. Concurrent treatment with BKM120+MLN8237 was synergistic or additive in 231Brs at most doses, whereas the combination was additive to antagonistic in SUM149s. Pretreatment with MLN8237 prior to concurrent BKM120+MLN8237 improved synergy in SUM149s, while BKM120 pretreatment improved synergy in 231Brs. FACS analysis of BKM120 in the SUM149 and 231Br cells induced a slight G1 arrest from 24 to 72 hours, while MLN8237 initially induced a G2 arrest at 24 hours, polyploidy at 48 hours, and a mixed polypoid/G2 arrested population at 72 hours. These effects were more pronounced in the 231Brs than the SUM149s. Combined BKM120+MLN8237 in both cell lines yielded results similar to MLN8237 alone. Cells continuously exposed to increasing MLN8237 concentrations from 50 nM to 300 nM for 12 weeks were resistant to MLN8237-induced cell cycle changes as compared to passage-matched controls. Conclusions: Combined PI3K+AURKA inhibition using brain-penetrant compounds is a promising strategy for a patient population with few options. In vivo studies evaluating the efficacy of BKM120+MLN8237 in intracranial TNBC mouse models to provide the translational foundation for future clinical studies are warranted. Citation Format: Amanda E.D. Van Swearingen, Maria J. Sambade, Shivani Sud, Brian Golitz, Gary L. Johnson, Carey K. Anders. Combined PI3K and AURKA inhibition are efficacious in triple-negative breast cancer models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3867.