Sujatmi Hariono

Genome scanning with array CGH delineates regional alterations in mouse islet carcinomas.

Carcinomas that develop in the pancreatic islets of transgenic mice expressing the SV40 T-antigens (Tag) under transcriptional control of the rat insulin II promoter (RIP) progress through well-characterized stages that are similar to aspects of human tumor progression, including hyperplastic growth, increased angiogenesis and reduced apoptosis. The latter two stages have been associated with recurrent loss of heterozygosity (LOH) and reduced genome copy number on chromosomes 9 (LOH9) and 16 (LOH16), aberrations which we believe contribute to these phenotypes. Earlier analyses localized LOH9 to approximately 3 Mb and LOH16 to approximately 30 Mb (both syntenic with human 3q21–q25) but were limited by low throughput and a lack of informative polymorphic markers. Here we show that comparative genomic hybridization to DNA microarrays (array CGH) overcomes these limitations by allowing efficient, genome-wide analyses of relative genome copy number. The CGH arrays used in these experiments carried BACs distributed at 2–20-MB intervals across the mouse genome and at higher density in regions of interest. Using array CGH, we further narrowed the loci for LOH9 and LOH16 and defined new or previously unappreciated recurrent regions of copy-number decrease on chromosomes 6, 8 and 14 (syntenic with human chromosomes 12p11–p13, 16q24.3 and 13q11–q32, respectively) and regions of copy-number increase on chromosomes 2 and 4 (syntenic to human chromosomes 20q13.2 and 1p32–p36, respectively). Our analyses of human genome sequences syntenic to these regions suggest that CYP24, PFDN4, STMN1, CDKN1B, PPP2R3 and FSTL1 are candidate oncogenes or tumor-suppressor genes. We also show that irradiation and genetic background influence the spectrum of aberrations present in these tumors.

Targeted Therapy for BRAFV600E Malignant Astrocytoma

Purpose: Malignant astrocytomas (MA) are aggressive central nervous system tumors with poor prognosis. Activating mutation of BRAF (BRAFV600E) has been reported in a subset of these tumors, especially in children. We have investigated the incidence of BRAFV600E in additional pediatric patient cohorts and examined the effects of BRAF blockade in preclinical models of BRAFV600E and wild-type BRAF MA. Experimental Design: BRAFV600E mutation status was examined in two pediatric MA patient cohorts. For functional studies, BRAFV600E MA cell lines were used to investigate the effects of BRAF shRNA knockdown in vitro, and to investigate BRAF pharmacologic inhibition in vitro and in vivo. Results: BRAFV600E mutations were identified in 11 and 10% of MAs from two distinct series of tumors (six of 58 cases total). BRAF was expressed in all MA cell lines examined, among which BRAFV600E was identified in four instances. Using the BRAFV600E-specific inhibitor PLX4720, pharmacologic blockade of BRAF revealed preferential antiproliferative activity against BRAFV600E mutant cells in vitro, in contrast to the use of shRNA-mediated knockdown of BRAF, which inhibited cell growth of glioma cell lines regardless of BRAF mutation status. Using orthotopic MA xenografts, we show that PLX4720 treatment decreases tumor growth and increases overall survival in mice-bearing BRAFV600E mutant xenografts, while being ineffective, and possibly tumor promoting, against xenografts with wild-type BRAF. Conclusions: Our results indicate a 10% incidence of activating BRAFV600E among pediatric MAs. With regard to implications for therapy, our results support evaluation of BRAFV600E-specific inhibitors for treating BRAFV600E MA patients. Clin Cancer Res; 17(24); 7595–604. ©2011 AACR.

Comparative analyses of gene copy number and mRNA expression in glioblastoma multiforme tumors and xenografts

Development of model systems that recapitulate the molecular heterogeneity observed among glioblastoma multiforme (GBM) tumors will expedite the testing of targeted molecular therapeutic strategies for GBM treatment. In this study, we profiled DNA copy number and mRNA expression in 21 independent GBM tumor lines maintained as subcutaneous xenografts (GBMX), and compared GBMX molecular signatures to those observed in GBM clinical specimens derived from the Cancer Genome Atlas (TCGA). The predominant copy number signature in both tumor groups was defined by chromosome-7 gain/chromosome-10 loss, a poor-prognosis genetic signature. We also observed, at frequencies similar to that detected in TCGA GBM tumors, genomic amplification and overexpression of known GBM oncogenes, such as EGFR, MDM2, CDK6, and MYCN, and novel genes, including NUP107, SLC35E3, MMP1, MMP13, and DDX1. The transcriptional signature of GBMX tumors, which was stable over multiple subcutaneous passages, was defined by overexpression of genes involved in M phase, DNA replication, and chromosome organization (MRC) and was highly similar to the poor-prognosis mitosis and cell-cycle module (MCM) in GBM. Assessment of gene expression in TCGA-derived GBMs revealed overexpression of MRC cancer genes AURKB, BIRC5, CCNB1, CCNB2, CDC2, CDK2, and FOXM1, which form a transcriptional network important for G2/M progression and/or checkpoint activation. Our study supports propagation of GBM tumors as subcutaneous xenografts as a useful approach for sustaining key molecular characteristics of patient tumors, and highlights therapeutic opportunities conferred by this GBMX tumor panel for testing targeted therapeutic strategies for GBM treatment.

Copy number aberrations in mouse breast tumors reveal loci and genes important in tumorigenic receptor tyrosine kinase signaling

Receptor tyrosine kinase (RTK) signaling plays a key role in the development of breast cancer. Defining the genes and pathways in the RTK signaling network that are important regulators of tumorigenesis in vivo will unveil potential candidates for targeted therapeutics. To this end, we used microarray comparative genomic hybridization to identify and compare copy number aberrations in five mouse models of breast cancer induced by wild-type and mutated forms of oncogenic ErbB2 or the polyomavirus middle T antigen (PyMT). We observed distinct genomic alterations among the various models, including recurrent chromosome 11 amplifications and chromosome 4 deletions, syntenic with human 17q21-25 and 1p35-36, respectively. Expression of oncogenic Erbb2 (NeuNT) under control of the endogenous Erbb2 promoter results in frequent (85%) amplification at the Erbb2 locus with striking structural similarity to the human amplicon, resulting in overexpression of at least two of the genes, Erbb2 and Grb7. Chromosome 11 amplicons distal to Erbb2 arise in a model (DB) overexpressing a mutant variant of PyMT (Y315/322F) unable to activate phosphatidylinositol 3-kinase. These amplicons are not observed in DB hyperplasias or in tumors overexpressing wild-type PyMT and result in overexpression of Grb2 and Itgb4. Distal chromosome 4 deletions occur in a significantly higher proportion of Erbb2 than PyMT tumors and encompass 14-3-3sigma (Stratifin), which is expressed at low or undetectable levels in the majority of NeuNT tumors. Our studies highlight loci and genes important in the regulation of tumorigenic RTK signaling in mammary epithelial cells in vivo.

Kinetics of Inhibitor Cycling Underlie Therapeutic Disparities between EGFR-Driven Lung and Brain Cancers

UNLABELLED Although mutational activation of the epidermal growth factor receptor (EGFR) features prominently in glioma and non-small cell lung cancer (NSCLC), inhibitors of EGFR improve survival only in patients with NCSLC. To understand how mutations in EGFR influence response to therapy, we generated glioma cells expressing either glioma- or NSCLC-derived alleles and quantified kinase-site occupancy by clinical inhibitors with the use of a novel affinity probe and kinetic methodology. At equivalent doses, erlotinib achieved lower kinase-site occupancy in glioma-derived EGFRvIII compared with NSCLC-derived EGFR mutants. Kinase-site occupancy correlated directly with cell-cycle arrest. EGFRvIII released erlotinib rapidly compared with wild-type EGFR, whereas NSCLC-derived mutants released erlotinib slowly. SIGNIFICANCE These data suggest that kinase-site occupancy is a biomarker for efficacy of EGFR inhibitors, that rapid binding and release of erlotinib in glioma-derived EGFRvIII opposes the blockade of downstream signaling, and that slower cycling of erlotinib within the active site of NSCLC-derived mutants underlies their improved clinical response.

Oncogene Expression and Genetic Background Influence the Frequency of DNA Copy Number Abnormalities in Mouse Pancreatic Islet Cell Carcinomas

Quantitative measurements of tumor genome composition show remarkable heterogeneity in tumors arising from the same anatomical location and/or histopathological class and stage. The factors that contribute to genomic heterogeneity are not clear, but germ-line allelic variation and timing of initiating oncogenic events are likely candidates. We investigated these factors by using array comparative genomic hybridization to measure genomic aberrations in genetically engineered mouse models of pancreatic islet cell carcinoma, in which oncogenic transformation is elicited by the SV40 T antigens expressed under the control of the rat insulin promoter (RIP-Tag). Two distinct transgenic RIP-Tag lines, and three polymorphic sublines of one, enabled us to investigate the effects of genetic background and differing age of oncogene induction. Both parameters were found to bias the spectrum of genomic copy number abnormalities. Specifically, the frequency of losing portions of chromosomes 9 and 16 was significantly modulated by genetic background, with the former being lost at highest rates in the FVB/N background and the latter being lost to greatest extent in both FVB/N and C57Bl/6 tumors compared with C3HeB/Fe tumors. The frequency of losing a region of chromosome 6 varied according to the age when tumorigenesis was initiated; loss of chromosome 6 was significantly higher when oncogene expression was first activated in adulthood. These studies illustrate the utility of transgenic animal models for investigation of factors influencing genomic heterogeneity despite the commonalty of target cell type and initiating oncogene.

Expression of miR-124 inhibits growth of medulloblastoma cells

Medulloblastoma is the most common malignant brain tumor in children, and a substantial number of patients die as a result of tumor progression. Overexpression of CDK6 is present in approximately one-third of medulloblastomas and is an independent poor prognostic marker for this disease. MicroRNA (miR)-124 inhibits expression of CDK6 and prevents proliferation of glioblastoma and medulloblastoma cells in vitro. We examined the effects of miR-124 overexpression on medulloblastoma cells both in vitro and in vivo and compared cell lines that have low and high CDK6 expression. MiR-124 overexpression inhibits the proliferation of medulloblastoma cells, and this effect is mediated mostly through the action of miR-124 upon CDK6. We further show that induced expression of miR-124 potently inhibits growth of medulloblastoma xenograft tumors in rodents. Further testing of miR-124 will help define the ultimate therapeutic potential of preclinical models of medulloblastoma in conjunction with various delivery strategies for treatment.

Combined BRAFV600E and MEK blockade for BRAFV600E-mutant gliomas

BRAFV600E is a common finding in glioma (about 10–60% depending on histopathologic subclassification). BRAFV600E monotherapy shows modest preclinical efficacy against BRAFV600E gliomas and also induces adverse secondary skin malignancies. Here, we examine the molecular mechanism of intrinsic resistance to BRAFV600E inhibition in glioma. Furthermore, we investigate BRAFV600E/MEK combination therapy that overcomes intrinsic resistance to BRAFV600E inhibitor and also prevents BRAFV600E inhibitor induced secondary malignancies. Immunoblotting and Human Phospho-Receptor Tyrosine Kinase Array assays were used to interrogate MAPK pathway activation. The cellular effect of BRAFV600E and MEK inhibition was determined by WST-1 viability assay and cell cycle analysis. Flanked and orthotopic GBM mouse models were used to investigate the in vivo efficacy of BRAFV600E/MEK combination therapy and the effect on secondary malignancies. BRAFV600E inhibition leads to recovery of ERK phosphorylation. Combined BRAFV600E and MEK inhibition prevents reactivation of the MAPK signaling, which correlates with decreased cell viability and augmented cell cycle arrest. Similarly, mice bearing BRAFV600E glioma showed reduced tumor growth when treated with a combination of BRAFV600E and MEK inhibitor compared to BRAFV600E inhibition alone. Additional benefit of BRAFV600E/MEK inhibition was reflected by reduced cutaneous squamous-cell carcinoma (cSCC) growth (a surrogate for RAS-driven secondary maligancies). In glioma, recovery of MAPK signaling upon BRAF inhibition accounts for intrinsic resistance to BRAFV600E inhibitor. Combined BRAFV600E and MEK inhibition prevents rebound of MAPK activation, resulting in enhanced antitumor efficacy and also reduces the risk of secondary malignancy development.

Abstract 2314: Erlotinib response in lung- and glioma-derived EGFR alleles: Correlating kinase site occupancy with efficacy

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL INTRODUCTION: Mutational activation of the Epidermal Growth Factor Receptor (EGFR) gene features prominently in malignant glioma (MG) and non-small-cell lung cancer (NSCLC). However, while NCSLC-derived mutants of EGFR exhibit high sensitivity to small molecule tyrosine kinase inhibitors, patients with MG-derived EGFR alleles respond poorly to TKIs. We tested MG- and NSCLC-derived alleles of EGFR to determine whether differences in occupancy within the mutant kinase domains determine response to therapy. METHODS: Human malignant glioma-derived cell lines expressing wild-type and mutant alleles of EGFR (derived from MG (EGFRvIII) or NSCLC (EGFR L858R and EGFRdel746-750)) were generated by viral transduction. Cell cycle analyses and viability assays were performed to confirm the in vitro recapitulation of the range of erlotinibs clinical efficacy in cells expressing MG- or NSCLC-specific alleles of EGFR. A novel affinity probe selective for the kinase site of EGFR was used to quantify the level of erlotinib binding in this panel of EGFR-allele expressing cell lines. Cell-signaling was analyzed through western-blotting and quantified with densitometry. RESULTS: Using the EGFR-specific affinity probe, erlotinibs kinase site occupancy was measured at clinically relevant doses. In cells expressing MG-derived EGFRvIII, significantly lower levels of kinase site occupancy were reached as compared to NSCLC-derived EGFR L858R and EGFRdel746-750. Surprisingly, these data did not correlate with levels of phospho-EGFR. Across all alleles, kinase site occupancy rather than levels of p-EGFR correlated with downstream signaling through Akt and mTOR, and with growth inhibition and cell cycle arrest. CONCLUSION: Erlotinib, a small molecule tyrosine kinase inhibitor, achieves higher levels of kinase site occupancy in NSCLC-derived mutations of EGFR, as compared with MG-derived mutations. Kinase site occupancy correlated directly with efficacy. In contrast, levels of p-EGFR correlated poorly with efficacy for these agents. These data suggest kinase site occupancy as a potential biomarker for efficacy of EGFR inhibitors. This also raises the intriguing possibility that the reason for the differential efficacy of erlotinib in lung- and MG-derived activating mutations in EGFR is due to differences in ATP binding affinity: the lung-derived activating mutations in EGFR have lower ATP affinity as compared to the wild-type kinase, resulting in higher relative erlotinib affinity, while MG-derived activating mutations in EGFR have a higher affinity for ATP, which outcompetes erlotinib and results in poor drug affinity for the kinase site. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2314. doi:10.1158/1538-7445.AM2011-2314

Abstract 1973: Targeting activated alleles of EGFR derived from glioma and lung cancer: Correlating kinase active-site occupancy with efficacy

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Introduction: The Epidermal Growth Factor Receptor (EGFR) features prominently in malignant glioma (MG), yet attempts to exploit this target in patients with MG have been somewhat disappointing. Unlike the highly sensitizing kinase site alleles of EGFR derived from patients with non-small cell lung cancer (NSCLCA), mutationally activated alleles of EGFR derived from patients with MG respond poorly to the currently available reversible clinical EGFR inhibitors. We hypothesize that the anti-tumor effects of EGFR inhibitors in MG-associated alleles of EGFR require more efficient blockade to achieve anti-tumor responses, in comparison with NSCLCA-associated alleles of EGFR, and that thisincreased efficiency can be achieved using irreversible inhibitors. Methods: We generated glioma and fibroblast cell lines that expressed wild-type or tumor-associated alleles of EGFR derived either from MG (EGFRvIII) or NSCLCA (L858R or del 747-753). Using an EGFR affinity probe (an irreversible EGFR inhibitor tethered to a flourophore), we quantified occupancy of the kinase active site in response to erotinib, a clinical reversible inhibitor of EGFR, correlating occupancy with proliferation and apoptosis. Next, we repeated these measurements using the tool compound and irreversible EGFR inhibitor PD-168394. Results: Using the affinity probe in pulse-chase experiments, we quantified active site occupancy of EGFR int response to increasing doses of inhibitors. In cells expressing MG-derived alleles, irreversible EGFR inhibitors were significantly more potent than reversible agents in abrogating signaling downstream of EGFR and in decreasing viability. Using the affinity probe, we found that irreversible inhibitors showed nearly complete occupancy of the active site, whereas treatment with reversible EGFR inhibitors resulted in only partial occupancy. Cells containing NSCLCA-derived EGFR alleles showed similar signaling and growth responses to both reversible and EGFR inhibitors, despite a large difference in EGFR kinase site occupancy with the two types of inhibitors. Flow cytometry studies correlating kinase site occupancy with downstream signaling in individual cells will also be presented. Conclusions: Activated alleles of EGFR derived from MG require significantly higher levels of kinase site blockade to achieve an anti-proliferative threshold, as compared with activated alleles derived from NSCLCA. This higher degree of kinase site blockade can be achieved using irreversible inhibitors of EGFR. These studies support the initiation of clinical trials using irreversible inhibitors of EGFR in MG. 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 1973.