Brian T. Golitz

MicroRNA 9-3p Targets β1 Integrin To Sensitize Claudin-Low Breast Cancer Cells to MEK Inhibition

ABSTRACT MEK1/2 inhibitors such as AZD6244 are in clinical trials for the treatment of multiple cancers, including breast cancer. Targeted kinase inhibition can induce compensatory kinome changes, rendering single therapeutic agents ineffective. To identify target proteins to be used in a combinatorial approach to inhibit tumor cell growth, we used a novel strategy that identified microRNAs (miRNAs) that synergized with AZD6244 to inhibit the viability of the claudin-low breast cancer cell line MDA-MB-231. Screening of a miRNA mimic library revealed the ability of miR-9-3p to significantly enhance AZD6244-induced extracellular signal-regulated kinase inhibition and growth arrest, while miR-9-3p had little effect on growth alone. Promoter methylation of mir-9 genes correlated with low expression of miR-9-3p in different breast cancer cell lines. Consistent with miR-9-3p having synthetic enhancer tumor suppressor characteristics, miR-9-3p expression in combination with MEK inhibitor caused a sustained loss of c-MYC expression and growth inhibition. The β1 integrin gene (ITGB1) was identified as a new miR-9-3p target, and the growth inhibition seen with small interfering RNA knockdown or antibody blocking of ITGB1 in combination with MEK inhibitor phenocopied the growth inhibition seen with miR-9-3p plus AZD6244. The miRNA screen led to identification of a druggable protein, ITGB1, whose functional inhibition synergizes with MEK inhibitor.

Enhancer Remodeling during Adaptive Bypass to MEK Inhibition Is Attenuated by Pharmacologic Targeting of the P-TEFb Complex

Targeting the dysregulated BRAF-MEK-ERK pathway in cancer has increasingly emerged in clinical trial design. Despite clinical responses in specific cancers using inhibitors targeting BRAF and MEK, resistance develops often involving nongenomic adaptive bypass mechanisms. Inhibition of MEK1/2 by trametinib in patients with triple-negative breast cancer (TNBC) induced dramatic transcriptional responses, including upregulation of receptor tyrosine kinases (RTK) comparing tumor samples before and after one week of treatment. In preclinical models, MEK inhibition induced genome-wide enhancer formation involving the seeding of BRD4, MED1, H3K27 acetylation, and p300 that drives transcriptional adaptation. Inhibition of the P-TEFb-associated proteins BRD4 and CBP/p300 arrested enhancer seeding and RTK upregulation. BRD4 bromodomain inhibitors overcame trametinib resistance, producing sustained growth inhibition in cells, xenografts, and syngeneic mouse TNBC models. Pharmacologic targeting of P-TEFb members in conjunction with MEK inhibition by trametinib is an effective strategy to durably inhibit epigenomic remodeling required for adaptive resistance.Significance: Widespread transcriptional adaptation to pharmacologic MEK inhibition was observed in TNBC patient tumors. In preclinical models, MEK inhibition induces dramatic genome-wide modulation of chromatin, in the form of de novo enhancer formation and enhancer remodeling. Pharmacologic targeting of P-TEFb complex members at enhancers is an effective strategy to durably inhibit such adaptation. Cancer Discov; 7(3); 302-21. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 235.

SWI/SNF Chromatin-Remodeling Factor Smarcd3/Baf60c Controls Epithelial-Mesenchymal Transition by Inducing Wnt5a Signaling

ABSTRACT We previously identified a gene signature predicted to regulate the epithelial-mesenchymal transition (EMT) in both epithelial tissue stem cells and breast cancer cells. A phenotypic RNA interference (RNAi) screen identified the genes within this 140-gene signature that promoted the conversion of mesenchymal epithelial cell adhesion molecule-negative (EpCAM−) breast cancer cells to an epithelial EpCAM+/high phenotype. The screen identified 10 of the 140 genes whose individual knockdown was sufficient to promote EpCAM and E-cadherin expression. Among these 10 genes, RNAi silencing of the SWI/SNF chromatin-remodeling factor Smarcd3/Baf60c in EpCAM− breast cancer cells gave the most robust transition from the mesenchymal to epithelial phenotype. Conversely, expression of Smarcd3/Baf60c in immortalized human mammary epithelial cells induced an EMT. The mesenchymal-like phenotype promoted by Smarcd3/Baf60c expression resulted in gene expression changes in human mammary epithelial cells similar to that of claudin-low triple-negative breast cancer cells. These mammary epithelial cells expressing Smarcd3/Baf60c had upregulated Wnt5a expression. Inhibition of Wnt5a by either RNAi knockdown or blocking antibody reversed Smarcd3/Baf60c-induced EMT. Thus, Smarcd3/Baf60c epigenetically regulates EMT by activating WNT signaling pathways.

The E3 ligase PARC mediates the degradation of cytosolic cytochrome c to promote survival in neurons and cancer cells

Neurons and cancer cells rely on the protein PARC to survive mitochondrial stress. Putting the PARC’ing Break on Cell Death Release of cytochrome c (Cyt c) from the mitochondria in response to cell stress or mitochondrial damage triggers cell death. Cancer cells and cells that have ceased dividing, such as neurons, can be less susceptible to Cyt c–induced cell death. The inability of neurons to cope with mitochondrial stress or damage contributes to some neurodegenerative diseases. Gama et al. induced mitochondrial stress in cultures of human glioma or neuroblastoma cell lines and in cultures of sympathetic neurons from mice and found that the E3 ligase PARC ubiquitylated cytosolic Cyt c, inducing its degradation. Cyt c accumulated in PARC-deficient cells, and mitochondrial stress produced an increased cell death response in these cells. The findings indicate that PARC may be important for promoting neuronal survival in diseases associated with mitochondrial damage and might be therapeutically targeted to enhance the cytotoxicity of cancer treatments. The ability to withstand mitochondrial damage is especially critical for the survival of postmitotic cells, such as neurons. Likewise, cancer cells can also survive mitochondrial stress. We found that cytochrome c (Cyt c), which induces apoptosis upon its release from damaged mitochondria, is targeted for proteasome-mediated degradation in mouse neurons, cardiomyocytes, and myotubes and in human glioma and neuroblastoma cells, but not in proliferating human fibroblasts. In mouse neurons, apoptotic protease-activating factor 1 (Apaf-1) prevented the proteasome-dependent degradation of Cyt c in response to induced mitochondrial stress. An RNA interference screen in U-87 MG glioma cells identified p53-associated Parkin-like cytoplasmic protein (PARC, also known as CUL9) as an E3 ligase that targets Cyt c for degradation. The abundance of PARC positively correlated with differentiation in mouse neurons, and overexpression of PARC reduced the abundance of mitochondrially-released cytosolic Cyt c in various cancer cell lines and in mouse embryonic fibroblasts. Conversely, neurons from Parc-deficient mice had increased sensitivity to mitochondrial damage, and neuroblastoma or glioma cells in which PARC or ubiquitin was knocked down had increased abundance of mitochondrially-released cytosolic Cyt c and decreased viability in response to stress. These findings suggest that PARC-mediated ubiquitination and degradation of Cyt c is a strategy engaged by both neurons and cancer cells to prevent apoptosis during conditions of mitochondrial stress.

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

Application of Integrated Drug Screening/Kinome Analysis to Identify Inhibitors of Gemcitabine-Resistant Pancreatic Cancer Cell Growth

Continuous exposure of a pancreatic cancer cell line MIA PaCa-2 (MiaS) to gemcitabine resulted in the formation of a gemcitabine-resistant subline (MiaR). In an effort to discover kinase inhibitors that inhibited MiaR growth, MiaR cells were exposed to kinase inhibitors (PKIS-1 library) in a 384-well screening format. Three compounds (UNC10112721A, UNC10112652A, and UNC10112793A) were identified that inhibited the growth of MiaR cells by more than 50% (at 50 nM). Two compounds (UNC10112721A and UNC10112652A) were classified as cyclin-dependent kinase (CDK) inhibitors, whereas UNC10112793A was reported to be a PLK inhibitor. Dose–response experiments supported the efficacy of these compounds to inhibit growth and increase apoptosis in 2D cultures of these cells. However, only UNC10112721A significantly inhibited the growth of 3D spheroids composed of MiaR cells and GFP-tagged cancer-associated fibroblasts. Multiplexed inhibitor bead (MIB)–mass spectrometry (MS) kinome competition experiments identified CDK9, CLK1-4, DYRK1A, and CSNK1 as major kinase targets for UNC10112721A in MiaR cells. Another CDK9 inhibitor (CDK-IN-2) replicated the growth inhibitory effects of UNC10112721A, whereas inhibitors against the CLK, DYRK, or CSNK1 kinases had no effect. In summary, these studies describe a coordinated approach to discover novel kinase inhibitors, evaluate their efficacy in 3D models, and define their specificity against the kinome.

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.