Dejan Juric

Functional Network Analysis Reveals Extended Gliomagenesis Pathway Maps and Three Novel MYC-Interacting Genes in Human Gliomas

Gene expression profiling has proven useful in subclassification and outcome prognostication for human glial brain tumors. The analysis of biological significance of the hundreds or thousands of alterations in gene expression found in genomic profiling remains a major challenge. Moreover, it is increasingly evident that genes do not act as individual units but collaborate in overlapping networks, the deregulation of which is a hallmark of cancer. Thus, we have here applied refined network knowledge to the analysis of key functions and pathways associated with gliomagenesis in a set of 50 human gliomas of various histogenesis, using cDNA microarrays, inferential and descriptive statistics, and dynamic mapping of gene expression data into a functional annotation database. Highest-significance networks were assembled around the myc oncogene in gliomagenesis and around the integrin signaling pathway in the glioblastoma subtype, which is paradigmatic for its strong migratory and invasive behavior. Three novel MYC-interacting genes (UBE2C, EMP1, and FBXW7) with cancer-related functions were identified as network constituents differentially expressed in gliomas, as was CD151 as a new component of a network that mediates glioblastoma cell invasion. Complementary, unsupervised relevance network analysis showed a conserved self-organization of modules of interconnected genes with functions in cell cycle regulation in human gliomas. This approach has extended existing knowledge about the organizational pattern of gene expression in human gliomas and identified potential novel targets for future therapeutic development.

Convergent loss of PTEN leads to clinical resistance to a PI(3)Kα inhibitor

Broad and deep tumour genome sequencing has shed new light on tumour heterogeneity and provided important insights into the evolution of metastases arising from different clones. There is an additional layer of complexity, in that tumour evolution may be influenced by selective pressure provided by therapy, in a similar fashion to that occurring in infectious diseases. Here we studied tumour genomic evolution in a patient (index patient) with metastatic breast cancer bearing an activating PIK3CA (phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha, PI(3)Kα) mutation. The patient was treated with the PI(3)Kα inhibitor BYL719, which achieved a lasting clinical response, but the patient eventually became resistant to this drug (emergence of lung metastases) and died shortly thereafter. A rapid autopsy was performed and material from a total of 14 metastatic sites was collected and sequenced. All metastatic lesions, when compared to the pre-treatment tumour, had a copy loss of PTEN (phosphatase and tensin homolog) and those lesions that became refractory to BYL719 had additional and different PTEN genetic alterations, resulting in the loss of PTEN expression. To put these results in context, we examined six other patients also treated with BYL719. Acquired bi-allelic loss of PTEN was found in one of these patients, whereas in two others PIK3CA mutations present in the primary tumour were no longer detected at the time of progression. To characterize our findings functionally, we examined the effects of PTEN knockdown in several preclinical models (both in cell lines intrinsically sensitive to BYL719 and in PTEN-null xenografts derived from our index patient), which we found resulted in resistance to BYL719, whereas simultaneous PI(3)K p110β blockade reverted this resistance phenotype. We conclude that parallel genetic evolution of separate metastatic sites with different PTEN genomic alterations leads to a convergent PTEN-null phenotype resistant to PI(3)Kα inhibition.

mTORC1 Inhibition Is Required for Sensitivity to PI3K p110α Inhibitors in PIK3CA-Mutant Breast Cancer

Persistent mTORC1 signaling correlates with resistance to PI3K p110α inhibition in breast cancer, which can be overcome by inhibiting mTORC1. Caveat mTOR In recent years, numerous new drugs have been developed to take advantage of specific molecular changes in cancer cells. Unfortunately, tumors are often a step ahead of the scientists, becoming resistant to these targeted drugs just when they seem to be working perfectly. Now, two groups of researchers have developed rational combination treatments that block resistance to targeted cancer drugs by inhibiting mTOR. Elkabets and coauthors were working on breast cancer, where the PIK3CA gene is frequently mutated. Inhibitors of PI3K (the protein product of PIK3CA) are currently in clinical trials, but some of the cancers are resistant to these drugs. The authors have discovered that breast cancers resistant to the PI3K inhibitor BYL719 had persistently active mTOR signaling, both in cultured cell lines and in patient tumors. Adding an mTOR inhibitor to the treatment regimen could reverse the resistance and kill the tumor cells. Corcoran et al. found a similar mTOR-dependent drug resistance mechanism to be active in melanoma as well. BRAF-mutant melanomas, the most common type, are frequently treated with RAF and MEK inhibitors, but only with mixed results, because melanomas quickly develop resistance to these drugs. Now, the authors have shown that drug-resistant melanomas also have persistent activation of mTOR, and adding an mTOR inhibitor to the treatment regimen can block drug resistance and kill the cancer cells. In both studies, the activation of mTOR in drug-resistant tumors has been confirmed in human patients, but the combination treatments have only been tested in cells and in mouse models thus far. Thus, the next critical step would be to confirm that adding mTOR inhibition to treatment regimens for these cancers is effective in the clinical setting as well. Some mTOR inhibitors are already available for use in patients, so hopefully soon mTOR activation will not be something to beware of, but something to monitor and target with specific drugs. Activating mutations of the PIK3CA gene occur frequently in breast cancer, and inhibitors that are specific for phosphatidylinositol 3-kinase (PI3K) p110α, such as BYL719, are being investigated in clinical trials. In a search for correlates of sensitivity to p110α inhibition among PIK3CA-mutant breast cancer cell lines, we observed that sensitivity to BYL719 (as assessed by cell proliferation) was associated with full inhibition of signaling through the TORC1 pathway. Conversely, cancer cells that were resistant to BYL719 had persistently active mTORC1 signaling, although Akt phosphorylation was inhibited. Similarly, in patients, pS6 (residues 240/4) expression (a marker of mTORC1 signaling) was associated with tumor response to BYL719, and mTORC1 was found to be reactivated in tumors from patients whose disease progressed after treatment. In PIK3CA-mutant cancer cell lines with persistent mTORC1 signaling despite PI3K p110α blockade (that is, resistance), the addition of the allosteric mTORC1 inhibitor RAD001 to the cells along with BYL719 resulted in reversal of resistance in vitro and in vivo. Finally, we found that growth factors such as insulin-like growth factor 1 and neuregulin 1 can activate mammalian target of rapamycin (mTOR) and mediate resistance to BYL719. Our findings suggest that simultaneous administration of mTORC1 inhibitors may enhance the clinical activity of p110α-targeted drugs and delay the appearance of resistance.

High-resolution genome-wide mapping of genetic alterations in human glial brain tumors.

High-resolution genome-wide mapping of exact boundaries of chromosomal alterations should facilitate the localization and identification of genes involved in gliomagenesis and may characterize genetic subgroups of glial brain tumors. We have done such mapping using cDNA microarray-based comparative genomic hybridization technology to profile copy number alterations across 42,000 mapped human cDNA clones, in a series of 54 gliomas of varying histogenesis and tumor grade. This gene-by-gene approach permitted the precise sizing of critical amplicons and deletions and the detection of multiple new genetic aberrations. It has also revealed recurrent patterns of occurrence of distinct chromosomal aberrations as well as their interrelationships and showed that gliomas can be clustered into distinct genetic subgroups. A subset of detected alterations was shown predominantly associated with either astrocytic or oligodendrocytic tumor phenotype. Finally, five novel minimally deleted regions were identified in a subset of tumors, containing putative candidate tumor suppressor genes (TOPORS, FANCG, RAD51, TP53BP1, and BIK) that could have a role in gliomagenesis.

Tumor Necrosis Factor-α–Induced Protein 3 As a Putative Regulator of Nuclear Factor-κB–Mediated Resistance to O6-Alkylating Agents in Human Glioblastomas

Purpose Pre-existing and acquired drug resistance are major obstacles to the successful treatment of glioblastomas. Methods We used an integrated resistance model and genomics tools to globally explore molecular factors and cellular pathways mediating resistance to O6-alkylating agents in glioblastoma cells. Results We identified a transcriptomic signature that predicts a common in vitro and in vivo resistance phenotype to these agents, a proportion of which is imprinted recurrently by gene dosage changes in the resistant glioblastoma genome. This signature was highly enriched for genes with functions in cell death, compromise, and survival. Modularity was a predominant organizational principle of the signature, with functions being carried out by groups of interacting molecules in overlapping networks. A highly significant network was built around nuclear factor-κB (NF-κB), which included the persistent alterations of various NF-κB pathway elements. Tumor necrosis factor-α–induced protein 3 (TNFAIP3) was...

PI3K regulates MEK/ERK signaling in breast cancer via the Rac-GEF, P-Rex1

Significance Genetic alterations targeting the PI3K pathway are highly prevalent in breast cancers. Although breast cancers harboring PIK3CA mutation and HER2 amplification have enhanced sensitivity to PI3K inhibitors, the mechanism underlying this sensitivity is unknown. This study shows that PI3K inhibitors suppress MEK/ERK pathway in these cancers, and inhibition of both AKT and ERK pathways is necessary for maximal antitumoral activity. We elucidate a unique mechanistic link between PI3K and ERK via PI3K-dependent regulation of P-Rex1, which in turn regulates the Rac1/PAK/c-RAF/MEK/ERK pathway. Importantly, expression levels of the Rac-GEF, P-Rex1, correlate with sensitivity to PI3K inhibitors among these breast cancer cell lines, indicating its potential utility as a biomarker to identify cancers that will respond to PI3K inhibitors. The PI3K pathway is genetically altered in excess of 70% of breast cancers, largely through PIK3CA mutation and HER2 amplification. Preclinical studies have suggested that these subsets of breast cancers are particularly sensitive to PI3K inhibitors; however, the reasons for this heightened sensitivity are mainly unknown. We investigated the signaling effects of PI3K inhibition in PIK3CA mutant and HER2 amplified breast cancers using PI3K inhibitors currently in clinical trials. Unexpectedly, we found that in PIK3CA mutant and HER2 amplified breast cancers sensitive to PI3K inhibitors, PI3K inhibition led to a rapid suppression of Rac1/p21-activated kinase (PAK)/protein kinase C-RAF (C-RAF)/ protein kinase MEK (MEK)/ERK signaling that did not involve RAS. Furthermore, PI3K inhibition led to an ERK-dependent up-regulation of the proapoptotic protein, BIM, followed by induction of apoptosis. Expression of a constitutively active form of Rac1 in these breast cancer models blocked PI3Ki-induced down-regulation of ERK phosphorylation, apoptosis, and mitigated PI3K inhibitor sensitivity in vivo. In contrast, protein kinase AKT inhibitors failed to block MEK/ERK signaling, did not up-regulate BIM, and failed to induce apoptosis. Finally, we identified phosphatidylinositol 3,4,5-trisphosphate-dependent Rac exchanger 1 (P-Rex1) as the PI(3,4,5)P3-dependent guanine exchange factor for Rac1 responsible for regulation of the Rac1/C-RAF/MEK/ERK pathway in these cells. The expression level of P-Rex1 correlates with sensitivity to PI3K inhibitors in these breast cancer cell lines. Thus, PI3K inhibitors have enhanced activity in PIK3CA mutant and HER2 amplified breast cancers in which PI3K inhibition down-regulates both the AKT and Rac1/ERK pathways. In addition, P-Rex1 may serve as a biomarker to predict response to single-agent PI3K inhibitors within this subset of breast cancers.

AXL Mediates Resistance to PI3Kα Inhibition by Activating the EGFR/PKC/mTOR Axis in Head and Neck and Esophageal Squamous Cell Carcinomas

Phosphoinositide-3-kinase (PI3K)-α inhibitors have shown clinical activity in squamous cell carcinomas (SCCs) of head and neck (H&N) bearing PIK3CA mutations or amplification. Studying models of therapeutic resistance, we have observed that SCC cells that become refractory to PI3Kα inhibition maintain PI3K-independent activation of the mammalian target of rapamycin (mTOR). This persistent mTOR activation is mediated by the tyrosine kinase receptor AXL. AXL is overexpressed in resistant tumors from both laboratory models and patients treated with the PI3Kα inhibitor BYL719. AXL dimerizes with and phosphorylates epidermal growth factor receptor (EGFR), resulting in activation of phospholipase Cγ (PLCγ)-protein kinase C (PKC), which, in turn, activates mTOR. Combined treatment with PI3Kα and either EGFR, AXL, or PKC inhibitors reverts this resistance.

Differential Gene Expression Patterns and Interaction Networks in BCR-ABL–Positive and –Negative Adult Acute Lymphoblastic Leukemias

PURPOSE To identify gene expression patterns and interaction networks related to BCR-ABL status and clinical outcome in adults with acute lymphoblastic leukemia (ALL). PATIENTS AND METHODS DNA microarrays were used to profile a set of 54 adult ALL specimens from the Medical Research Council UKALL XII/Eastern Cooperative Oncology Group E2993 trial (21 p185BCR-ABL-positive, 16 p210BCR-ABL-positive and 17 BCR-ABL-negative specimens). RESULTS Using supervised and unsupervised analysis tools, we detected significant transcriptomic changes in BCR-ABL-positive versus -negative specimens, and assessed their validity in an independent cohort of 128 adult ALL specimens. This set of 271 differentially expressed genes (including GAB1, CIITA, XBP1, CD83, SERPINB9, PTP4A3, NOV, LOX, CTNND1, BAALC, and RAB21) is enriched for genes involved in cell death, cellular growth and proliferation, and hematologic system development and function. Network analysis demonstrated complex interaction patterns of these genes, and identified FYN and IL15 as the hubs of the top-scoring network. Within the BCR-ABL-positive subgroups, we identified genes overexpressed (PILRB, STS-1, SPRY1) or underexpressed (TSPAN16, ADAMTSL4) in p185BCR-ABL-positive ALL relative to p210BCR-ABL-positive ALL. Finally, we constructed a gene expression- and interaction-based outcome predictor consisting of 27 genes (including GRB2, GAB1, GLI1, IRS1, RUNX2, and SPP1), which correlated with overall survival in BCR-ABL-positive adult ALL (P = .0001), independent of age (P = .25) and WBC count at presentation (P = .003). CONCLUSION We identified prominent molecular features of BCR-ABL-positive adult ALL, which may be useful for developing novel therapeutic targets and prognostic markers in this disease.

Cancer cell profiling by barcoding allows multiplexed protein analysis in fine-needle aspirates.

Barcoding technology enabled measurement of hundreds of cellular proteins from cancer patients with single-cell resolution. Fine-Tuning Single Cancer Cell Protein Analysis Fine-needle aspirates (FNAs) use thin needles to obtain cells from tumor masses. FNAs can give tremendous insight into malignancy, but the number of cells is so small that current technologies for protein analysis, such as immunohistochemistry, are insufficient. To address this technological gap, Ullal and colleagues developed the antibody barcoding with photocleavable DNA (ABCD) platform that allows for simultaneous analysis of many surface proteins on cells from cancer patients. The authors first isolated cancer cells within the FNAs of patients. These cells were then exposed to a cocktail of 90 antibodies, covering the hallmark processes in cancer (for example, apoptosis and DNA damage), and each containing a unique “barcode”—a single strand of DNA that could be released by light (photocleaved) and quantified using fluorescent complementary probes. After validating protein expression on human cancer cell lines with known protein composition, Ullal et al. moved to FNA samples from patients with lung adenocarcinoma. The protein profiles of 11 single cells taken from one patient showed low correlation with the bulk tumor sample, indicating high intratumor heterogeneity. The authors also noted high intertumor heterogeneity, because six patients with tumors that looked identical under a microscope had different proteomic profiles. By clustering the protein expression results and comparing to the patients’ genetic makeup, the authors suggest that therapies could be better personalized. The ABCD platform could help researchers to better understand tumor heterogeneity, as well as allow clinicians to personalize therapies and perhaps even track therapeutic response less invasively, as the authors demonstrated in vitro and in four patients taking kinase inhibitors. Technological challenges remain in making this platform validated and ready for the bedside, but successful early demonstrations in patients with lung cancer suggest that larger-scale clinical trials are in the near future. Immunohistochemistry-based clinical diagnoses require invasive core biopsies and use a limited number of protein stains to identify and classify cancers. We introduce a technology that allows analysis of hundreds of proteins from minimally invasive fine-needle aspirates (FNAs), which contain much smaller numbers of cells than core biopsies. The method capitalizes on DNA-barcoded antibody sensing, where barcodes can be photocleaved and digitally detected without any amplification steps. After extensive benchmarking in cell lines, this method showed high reproducibility and achieved single-cell sensitivity. We used this approach to profile ~90 proteins in cells from FNAs and subsequently map patient heterogeneity at the protein level. Additionally, we demonstrate how the method could be used as a clinical tool to identify pathway responses to molecularly targeted drugs and to predict drug response in patient samples. This technique combines specificity with ease of use to offer a new tool for understanding human cancers and designing future clinical trials.

Antagonism of EGFR and HER3 Enhances the Response to Inhibitors of the PI3K-Akt Pathway in Triple-Negative Breast Cancer

Predictions regarding drug resistance mechanisms and treatment strategies in triple-negative breast cancer are confirmed in tumors from patients. From Models to Breast Cancer Treatments Patients with triple-negative breast cancer (TNBC), a particularly aggressive form, have few treatment options. Targeting either the phosphatidylinositol 3-kinase to Akt (PI3K-Akt) pathway or epidermal growth factor receptor (EGFR) inhibits tumor growth in some patients, but durable responses are rare. Modeling studies using cell lines predict that the EGFR family member HER3 (human epidermal growth factor receptor 3) may confer drug resistance. Now, Tao et al. provide evidence from patient tumors to support those predictions. Treatment with PI3K-Akt pathway inhibitors increased the abundance of both total and activated HER3 in TNBC cells in culture and TNBC xenografts in mice. Residual tumors from patients treated with EGFR inhibitors had increased abundance and activation of HER3. Combining inhibitors of the PI3K-Akt pathway with a dual inhibitor of EGFR and HER3 substantially suppressed tumor growth in mice with TNBC xenografts derived from either cell lines or patients, suggesting that this combined strategy may improve therapeutic outcome in TNBC patients. Both abundant epidermal growth factor receptor (EGFR or ErbB1) and high activity of the phosphatidylinositol 3-kinase (PI3K)–Akt pathway are common and therapeutically targeted in triple-negative breast cancer (TNBC). However, activation of another EGFR family member [human epidermal growth factor receptor 3 (HER3) (or ErbB3)] may limit the antitumor effects of these drugs. We found that TNBC cell lines cultured with the EGFR or HER3 ligand EGF or heregulin, respectively, and treated with either an Akt inhibitor (GDC-0068) or a PI3K inhibitor (GDC-0941) had increased abundance and phosphorylation of HER3. The phosphorylation of HER3 and EGFR in response to these treatments was reduced by the addition of a dual EGFR and HER3 inhibitor (MEHD7945A). MEHD7945A also decreased the phosphorylation (and activation) of EGFR and HER3 and the phosphorylation of downstream targets that occurred in response to the combination of EGFR ligands and PI3K-Akt pathway inhibitors. In culture, inhibition of the PI3K-Akt pathway combined with either MEHD7945A or knockdown of HER3 decreased cell proliferation compared with inhibition of the PI3K-Akt pathway alone. Combining either GDC-0068 or GDC-0941 with MEHD7945A inhibited the growth of xenografts derived from TNBC cell lines or from TNBC patient tumors, and this combination treatment was also more effective than combining either GDC-0068 or GDC-0941 with cetuximab, an EGFR-targeted antibody. After therapy with EGFR-targeted antibodies, some patients had residual tumors with increased HER3 abundance and EGFR/HER3 dimerization (an activating interaction). Thus, we propose that concomitant blockade of EGFR, HER3, and the PI3K-Akt pathway in TNBC should be investigated in the clinical setting.