Andrew S. Chi

Genomewide Analysis of PRC1 and PRC2 Occupancy Identifies Two Classes of Bivalent Domains

In embryonic stem (ES) cells, bivalent chromatin domains with overlapping repressive (H3 lysine 27 tri-methylation) and activating (H3 lysine 4 tri-methylation) histone modifications mark the promoters of more than 2,000 genes. To gain insight into the structure and function of bivalent domains, we mapped key histone modifications and subunits of Polycomb-repressive complexes 1 and 2 (PRC1 and PRC2) genomewide in human and mouse ES cells by chromatin immunoprecipitation, followed by ultra high-throughput sequencing. We find that bivalent domains can be segregated into two classes—the first occupied by both PRC2 and PRC1 (PRC1-positive) and the second specifically bound by PRC2 (PRC2-only). PRC1-positive bivalent domains appear functionally distinct as they more efficiently retain lysine 27 tri-methylation upon differentiation, show stringent conservation of chromatin state, and associate with an overwhelming number of developmental regulator gene promoters. We also used computational genomics to search for sequence determinants of Polycomb binding. This analysis revealed that the genomewide locations of PRC2 and PRC1 can be largely predicted from the locations, sizes, and underlying motif contents of CpG islands. We propose that large CpG islands depleted of activating motifs confer epigenetic memory by recruiting the full repertoire of Polycomb complexes in pluripotent cells.

GC-rich sequence elements recruit PRC2 in mammalian ES cells

Polycomb proteins are epigenetic regulators that localize to developmental loci in the early embryo where they mediate lineage-specific gene repression. In Drosophila, these repressors are recruited to sequence elements by DNA binding proteins associated with Polycomb repressive complex 2 (PRC2). However, the sequences that recruit PRC2 in mammalian cells have remained obscure. To address this, we integrated a series of engineered bacterial artificial chromosomes into embryonic stem (ES) cells and examined their chromatin. We found that a 44 kb region corresponding to the Zfpm2 locus initiates de novo recruitment of PRC2. We then pinpointed a CpG island within this locus as both necessary and sufficient for PRC2 recruitment. Based on this causal demonstration and prior genomic analyses, we hypothesized that large GC-rich elements depleted of activating transcription factor motifs mediate PRC2 recruitment in mammals. We validated this model in two ways. First, we showed that a constitutively active CpG island is able to recruit PRC2 after excision of a cluster of activating motifs. Second, we showed that two 1 kb sequence intervals from the Escherichia coli genome with GC-contents comparable to a mammalian CpG island are both capable of recruiting PRC2 when integrated into the ES cell genome. Our findings demonstrate a causal role for GC-rich sequences in PRC2 recruitment and implicate a specific subset of CpG islands depleted of activating motifs as instrumental for the initial localization of this key regulator in mammalian genomes.

Improved tumor oxygenation and survival in glioblastoma patients who show increased blood perfusion after cediranib and chemoradiation

Significance This study demonstrates that antiangiogenic therapy increases tumor blood perfusion in a subset of newly diagnosed glioblastoma patients, and that it is these patients who survive longer when this expensive and potentially toxic therapy is combined with standard radiation and chemotherapy. This study provides fresh insights into the selection of glioblastoma patients most likely to benefit from antiangiogenic treatments. Antiangiogenic therapy has shown clear activity and improved survival benefit for certain tumor types. However, an incomplete understanding of the mechanisms of action of antiangiogenic agents has hindered optimization and broader application of this new therapeutic modality. In particular, the impact of antiangiogenic therapy on tumor blood flow and oxygenation status (i.e., the role of vessel pruning versus normalization) remains controversial. This controversy has become critical as multiple phase III trials of anti-VEGF agents combined with cytotoxics failed to show overall survival benefit in newly diagnosed glioblastoma (nGBM) patients and several other cancers. Here, we shed light on mechanisms of nGBM response to cediranib, a pan-VEGF receptor tyrosine kinase inhibitor, using MRI techniques and blood biomarkers in prospective phase II clinical trials of cediranib with chemoradiation vs. chemoradiation alone in nGBM patients. We demonstrate that improved perfusion occurs only in a subset of patients in cediranib-containing regimens, and is associated with improved overall survival in these nGBM patients. Moreover, an increase in perfusion is associated with improved tumor oxygenation status as well as with pharmacodynamic biomarkers, such as changes in plasma placenta growth factor and sVEGFR2. Finally, treatment resistance was associated with elevated plasma IL-8 and sVEGFR1 posttherapy. In conclusion, tumor perfusion changes after antiangiogenic therapy may distinguish responders vs. nonresponders early in the course of this expensive and potentially toxic form of therapy, and these results may provide new insight into the selection of glioblastoma patients most likely to benefit from anti-VEGF treatments.

ANTITUMOR ACTIVITY OF HYALURONIC ACID SYNTHESIS INHIBITOR 4-METHYLUMBELLIFERONE IN PROSTATE CANCER CELLS

4-Methylumbelliferone (4-MU) is a hyaluronic acid (HA) synthesis inhibitor with anticancer properties; the mechanism of its anticancer effects is unknown. We evaluated the effects of 4-MU on prostate cancer cells. 4-MU inhibited proliferation, motility, and invasion of DU145, PC3-ML, LNCaP, C4-2B, and/or LAPC-4 cells. At IC(50) for HA synthesis (0.4 mmol/L), 4-MU induced >3-fold apoptosis in prostate cancer cells, which could be prevented by the addition of HA. 4-MU induced caspase-8, caspase-9, and caspase-3 activation, PARP cleavage, upregulation of Fas-L, Fas, FADD and DR4, and downregulation of bcl-2, phosphorylated bad, bcl-XL, phosphorylated Akt, phosphorylated IKB, phosphorylated ErbB2, and phosphorylated epidermal growth factor receptor. At IC(50), 4-MU also caused >90% inhibition of NF-kappaB reporter activity, which was prevented partially by the addition of HA. With the exception of caveolin-1, HA reversed the 4-MU-induced downregulation of HA receptors (CD44 and RHAMM), matrix-degrading enzymes (MMP-2 and MMP-9), interleukin-8, and chemokine receptors (CXCR1, CXCR4, and CXCR7) at the protein and mRNA levels. Expression of myristoylated-Akt rescued 4-MU-induced apoptosis and inhibition of cell growth and interleukin-8, RHAMM, HAS2, CD44, and MMP-9 expression. Oral administration of 4-MU significantly decreased PC3-ML tumor growth (>3-fold) when treatment was started either on the day of tumor cell injection or after the tumors became palpable, without organ toxicity, changes in serum chemistry, or body weight. Tumors from 4-MU-treated animals showed reduced microvessel density ( approximately 3-fold) and HA expression but increased terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive cells and expression of apoptosis-related molecules. Therefore, the anticancer effects of 4-MU, an orally bioavailable and relatively nontoxic agent, are primarily mediated by inhibition of HA signaling.

Angiogenesis as a Therapeutic Target in Malignant Gliomas

Currently, adult glioblastoma (GBM) patients have poor outcomes with conventional cytotoxic treatments. Because GBMs are highly angiogenic tumors, inhibitors that target tumor vasculature are considered promising therapeutic agents in these patients. Encouraging efficacy and tolerability in preliminary clinical trials suggest that targeting angiogenesis may be an effective therapeutic strategy in GBM patients. However, the survival benefits observed to date in uncontrolled trials of antiangiogenic agents have been modest, and several obstacles have limited their effectiveness. This article reviews the rationale for antiangiogenic agents in GBM, their potential mechanisms of action, and their clinical development in GBM patients. Although challenges remain with this approach, ongoing studies may improve upon the promising initial benefits already observed in GBM patients.

Alectinib Salvages CNS Relapses in ALK-Positive Lung Cancer Patients Previously Treated with Crizotinib and Ceritinib

Background: Leptomeningeal metastases (LM) are an increasingly frequent and devastating complication of anaplastic lymphoma kinase (ALK)-rearranged non–small-cell lung cancer (NSCLC). Currently, the optimal management of LM in ALK-positive patients remains poorly understood as these patients have been routinely excluded from clinical trials. Methods: We describe four ALK-positive patients with LM who were treated with the next-generation ALK inhibitor alectinib through single-patient, compassionate use protocols at two institutions. All patients had previously been treated with both FDA-approved ALK inhibitors—crizotinib and ceritinib. Patients received alectinib at a starting dose of 600 mg twice daily. Results: Four ALK-positive NSCLC patients with symptomatic leptomeningeal disease were identified. Three of four patients experienced significant clinical and radiographic improvements in LM upon treatment with alectinib. A fourth patient had stable intracranial disease for 4 months before eventual systemic disease progression. Overall, alectinib was well tolerated. One patient required dose reduction due to grade 2 hyperbilirubinemia. Conclusions: Alectinib is active in ALK-rearranged NSCLC patients with LM, including in patients previously treated with crizotinib and ceritinib. Additional prospective studies of alectinib in ALK-positive patients with LM are warranted.

Rapid Radiographic and Clinical Improvement After Treatment of a MET-Amplified Recurrent Glioblastoma With a Mesenchymal-Epithelial Transition Inhibitor

Case Report A 62-year-old right-handed white woman presented with periodic motion-related nausea and vertigo. On physical examination, she had only mild high-frequency hearing loss on the right side. Cranial magnetic resonance imaging (MRI) revealed an unresectable contrast-enhancing lesion measuring 3.6 cm in maximal horizontal diameter within the genu of the corpus callosum, with surrounding abnormal T2/fluid-attenuated inversion recovery (FLAIR) hyperintensity. Stereotactic biopsy of the contrast-enhancing lesion established the diagnosis of glioblastoma (GBM), WHO grade 4. Her tumor tissue was tested for genetic abnormalities using a comprehensive panel that included screening for commonly mutated loci in 15 cancer-related genes using a multiplex polymerase chain reaction platform (SNaPshot, Version 3; Applied Biosystems, Carlsbad, CA); amplification of the mesenchymal-epithelial transition (MET), epidermal growth factor receptor (EGFR), and plateletderived growth factor receptor A (PDGFRA) genes using fluorescence in situ hybridization (FISH); status of chromosomes 1p and 19q by FISH; and O-6-methylguanine-DNA methyltransferase (MGMT) promoter DNA methylation status by methylationspecific polymerase chain reaction. Her tumor was found to have MET gene amplification by a dual-color FISH assay using probes for the MET locus (Fig 1, pink dots) and centromere 7 (Fig 1, aqua dots), which served as a copy-number control. In addition, the MGMT promoter was methylated. She was enrolled onto a clinical trial for patients with newly diagnosed GBM that combined the standard regimen of concurrent radiation and temozolomide followed by monthly temozolomide with cediranib, an oral small-molecule pan–vascular endothelial growth factor (VEGF) receptor tyrosine kinase inhibitor. After five postradiation cycles of temozolomide and cediranib, she developed headaches, fatigue, disorientation, and short-term memory loss. A cranial MRI examination demonstrated increased size of the contrast-enhancing lesion and significantly increased surrounding T2/FLAIR hyperintensity consistent with progressive disease. Administration of dexamethasone was started at 4 mg per day, and subsequently, her headache intensity mildly decreased. Her MET amplification status enabled her to enroll onto the MET-amplification/mutation enriched molecular cohort of a phase I trial of crizotinib (PF-02341066), an oral small-molecule inhibitor of the MET and anaplastic lymphoma kinase (ALK) tyrosine kinases, in patients with advanced cancer (A8081001). This cohort includes patients with tumors harboring specific genetic alterations in MET or ALK independent of tumor type, including MET gene amplification, kinase domain activating mutation, or other chromosomal translocations leading to altered transcriptional regulation of MET as well as ALK chromosomal translocation or gene amplification. She began receiving crizotinib 250 mg twice daily, and within 2 weeks, her headaches and short-term memory had significantly improved, and she was able to decrease her dexamethasone dose to 1 mg daily. Her first scheduled cranial MRI examination performed after two 28-day cycles of crizotinib demonstrated a 40% reduction (using two-dimensional measurements) of the contrast-enhancing lesion (Fig 2A) and decreased surrounding T2/FLAIR hyperintensity (Fig 2B). After four cycles, a restaging cranial MRI examination confirmed a stable 40% reduction in size of the contrast-enhancing lesion. She was neurologically stable, and dexamethasone was discontinued. One week before her sixth cycle, she developed increased disorientation, short-term memory loss, headaches, and nausea. At that time, a scheduled restaging cranial MRI examination demonstrated interval increase in size of the contrast-enhancing lesion consistent with progressive disease. Administration of crizotinib was stopped 6 months after initiation of treatment. The only adverse effects attributed to crizotinib were asymptomatic elevated ALT (grade 2) and hypophosphatemia (grade 2). During the third crizotinib cycle, she developed left popliteal venous thrombosis (grade 2) and began receiving enoxaparin. She subsequently developed small, asymptomatic peritumoral hemorrhages (grade 1) that were noted incidentally on her cranial MRI examination at the end of cycle four. The hemorrhages were attributed to enoxaparin therapy. Enoxaparin was then discontinued, and an inferior vena cava filter was placed. Crizotinib was postponed for 1 week and then restarted after a cranial computed tomography scan showed no extension of the hemorrhages. At the time of crizotinib discontinuation, a cranial MRI confirmed stability of the peritumoral hemorrhages since their discovery. Fig 1. JOURNAL OF CLINICAL ONCOLOGY D I A G N O S I S I N O N C O L O G Y VOLUME 30 NUMBER 3 JANUARY 2

Detection of oncogenic IDH1 mutations using magnetic resonance spectroscopy of 2-hydroxyglutarate

The investigation of metabolic pathways disturbed in isocitrate dehydrogenase (IDH) mutant tumors revealed that the hallmark metabolic alteration is the production of D-2-hydroxyglutarate (D-2HG). The biological impact of D-2HG strongly suggests that high levels of this metabolite may play a central role in propagating downstream the effects of mutant IDH, leading to malignant transformation of cells. Hence, D-2HG may be an ideal biomarker for both diagnosing and monitoring treatment response targeting IDH mutations. Magnetic resonance spectroscopy (MRS) is well suited to the task of noninvasive D-2HG detection, and there has been much interest in developing such methods. Here, we review recent efforts to translate methodology using MRS to reliably measure in vivo D-2HG into clinical research.

Increase in tumor-associated macrophages after antiangiogenic therapy is associated with poor survival among patients with recurrent glioblastoma

Antiangiogenic therapy is associated with increased radiographic responses in glioblastomas, but tumors invariably recur. Because tumor-associated macrophages have been shown to mediate escape from antiangiogenic therapy in preclinical models, we examined the role of macrophages in patients with recurrent glioblastoma. We compared autopsy brain specimens from 20 patients with recurrent glioblastoma who received antiangiogenic treatment and chemoradiation with 8 patients who received chemotherapy and/or radiotherapy without antiangiogenic therapy or no treatment. Tumor-associated macrophages were morphologically and phenotypically analyzed using flow cytometry and immunohistochemistry for CD68, CD14, CD163, and CD11b expression. Flow cytometry showed an increase in macrophages in the antiangiogenic-treated patients. Immunohistochemical analysis demonstrated an increase in CD68+ macrophages in the tumor bulk (P < .01) and infiltrative areas (P = .02) in antiangiogenic-treated patients. We also observed an increase in CD11b+ cells in the tumor bulk (P < .01) and an increase in CD163+ macrophages in infiltrative tumor (P = .02). Of note, an increased number of CD11b+ cells in bulk and infiltrative tumors (P = .05 and P = .05, respectively) correlated with poor overall survival among patients who first received antiangiogenic therapy at recurrence. In summary, recurrent glioblastomas showed an increased infiltration in myeloid populations in the tumor bulk and in the infiltrative regions after antiangiogenic therapy. Higher numbers of CD11b+ cells correlated with poor survival among these patients. These data suggest that tumor-associated macrophages may participate in escape from antiangiogenic therapy and may represent a potential biomarker of resistance and a potential therapeutic target in recurrent glioblastoma.

Targetable signaling pathway mutations are associated with malignant phenotype in IDH-mutant gliomas

Purpose: Isocitrate dehydrogenase (IDH) gene mutations occur in low-grade and high-grade gliomas. We sought to identify the genetic basis of malignant phenotype heterogeneity in IDH-mutant gliomas. Methods: We prospectively implanted tumor specimens from 20 consecutive IDH1-mutant glioma resections into mouse brains and genotyped all resection specimens using a CLIA-certified molecular panel. Gliomas with cancer driver mutations were tested for sensitivity to targeted inhibitors in vitro. Associations between genomic alterations and outcomes were analyzed in patients. Results: By 10 months, 8 of 20 IDH1-mutant gliomas developed intracerebral xenografts. All xenografts maintained mutant IDH1 and high levels of 2-hydroxyglutarate on serial transplantation. All xenograft-producing gliomas harbored “lineage-defining” mutations in CIC (oligodendroglioma) or TP53 (astrocytoma), and 6 of 8 additionally had activating mutations in PIK3CA or amplification of PDGFRA, MET, or N-MYC. Only IDH1 and CIC/TP53 mutations were detected in non–xenograft-forming gliomas (P = 0.0007). Targeted inhibition of the additional alterations decreased proliferation in vitro. Moreover, we detected alterations in known cancer driver genes in 13.4% of IDH-mutant glioma patients, including PIK3CA, KRAS, AKT, or PTEN mutation or PDGFRA, MET, or N-MYC amplification. IDH/CIC mutant tumors were associated with PIK3CA/KRAS mutations whereas IDH/TP53 tumors correlated with PDGFRA/MET amplification. Presence of driver alterations at progression was associated with shorter subsequent progression-free survival (median 9.0 vs. 36.1 months; P = 0.0011). Conclusion: A subset of IDH-mutant gliomas with mutations in driver oncogenes has a more malignant phenotype in patients. Identification of these alterations may provide an opportunity for use of targeted therapies in these patients. Clin Cancer Res; 20(11); 2898–909. ©2014 AACR.