Computational modeling of ERBB2-amplified breast cancer identifies combined ErbB2/3 blockade as superior to the combination of MEK and AKT inhibitors.

Crosstalk and compensatory circuits within cancer signaling networks limit the activity of most targeted therapies. For example, altered signaling in the networks activated by the ErbB family of receptors, particularly in ERBB2-amplified cancers, contributes to drug resistance. We developed a multiscale systems model of signaling networks in ERBB2-amplified breast cancer to quantitatively investigate relationships between biomarkers (markers of network activity) and combination drug efficacy. This model linked ErbB receptor family signaling to breast tumor growth through two kinase cascades: the PI3K/AKT survival pathway and the Ras/MEK/ERK growth and proliferation pathway. The model predicted molecular mechanisms of resistance to individual therapeutics. In particular, ERBB2-amplified breast cancer cells stimulated with the ErbB3 ligand heregulin were resistant to growth arrest induced by inhibitors of AKT and MEK or coapplication of two inhibitors of the receptor ErbB2 [Herceptin (trastuzumab) and Tykerb (lapatinib)]. We used model simulations to predict the response of ErbB2-positive breast cancer xenografts to combination therapies and verified these predictions in mice. Treatment with trastuzumab, lapatinib, and the ErbB3 inhibitor MM-111 was more effective in inhibiting tumor growth than the combination of AKT and MEK inhibitors and even induced tumor regression, indicating that targeting both ErbB3 and ErbB2 may be an improved therapeutic approach for ErbB2-positive breast cancer patients.

Antitumor activity of a novel bispecific antibody that targets the ErbB2/ErbB3 oncogenic unit and inhibits heregulin-induced activation of ErbB3.

The prevalence of ErbB2 amplification in breast cancer has resulted in the heavy pursuit of ErbB2 as a therapeutic target. Although both the ErbB2 monoclonal antibody trastuzumab and ErbB1/ErbB2 dual kinase inhibitor lapatinib have met with success in the clinic, many patients fail to benefit. In addition, the majority of patients who initially respond will unfortunately ultimately progress on these therapies. Activation of ErbB3, the preferred dimerization partner of ErbB2, plays a key role in driving ErbB2-amplified tumor growth, but we have found that current ErbB2-directed therapies are poor inhibitors of ligand-induced activation. By simulating ErbB3 inhibition in a computational model of ErbB2/ErbB3 receptor signaling, we predicted that a bispecific antibody that docks onto ErbB2 and subsequently binds to ErbB3 and blocks ligand-induced receptor activation would be highly effective in ErbB2-amplified tumors, with superior activity to a monospecific ErbB3 inhibitor. We have developed a bispecific antibody suitable for both large scale production and systemic therapy by generating a single polypeptide fusion protein of two human scFv antibodies linked to modified human serum albumin. The resulting molecule, MM-111, forms a trimeric complex with ErbB2 and ErbB3, effectively inhibiting ErbB3 signaling and showing antitumor activity in preclinical models that is dependent on ErbB2 overexpression. MM-111 can be rationally combined with trastuzumab or lapatinib for increased antitumor activity and may in the future complement existing ErbB2-directed therapies to treat resistant tumors or deter relapse.

Cocaine-induced chromatin remodeling increases brain-derived neurotrophic factor transcription in the rat medial prefrontal cortex, which alters the reinforcing efficacy of cocaine.

Cocaine self-administration alters patterns of gene expression in the brain that may underlie cocaine-induced neuronal plasticity. In the present study, male Sprague Dawley rats were allowed to self-administer cocaine (0.25 mg/infusion) 2 h/d for 14 d, followed by 7 d of forced abstinence. Compared with yoked saline control rats, cocaine self-administration resulted in increased brain-derived neurotrophic factor (BDNF) protein levels in the rat medial prefrontal cortex (mPFC). To examine the functional relevance of this finding, cocaine self-administration maintained under a progressive ratio schedule of reinforcement was assessed after short hairpin RNA-induced suppression of BDNF expression in the mPFC. Decreased BDNF expression in the mPFC increased the cocaine self-administration breakpoint. Next, the effect of cocaine self-administration on specific BDNF exons was assessed; results revealed selectively increased BDNF exon IV-containing transcripts in the mPFC. Moreover, there were significant cocaine-induced increases in acetylated histone H3 (AcH3) and phospho-cAMP response element binding protein (pCREB) association with BDNF promoter IV. In contrast, there was decreased methyl-CpG-binding protein 2 (MeCP2) association with BDNF promoter IV in the mPFC of rats that previously self-administered cocaine. Together, these results indicate that cocaine-induced increases in BDNF promoter IV transcript in the mPFC are driven by increased binding of AcH3 and pCREB as well as decreased MeCP2 binding at this BDNF promoter. Collectively, these results indicate that cocaine self-administration remodels chromatin in the mPFC, resulting in increased expression of BDNF, which appears to represent a compensatory neuroadaptation that reduces the reinforcing efficacy of cocaine.

Altered histone monoubiquitylation mediated by mutant huntingtin induces transcriptional dysregulation.

Although transcriptional dysregulation is a critical pathogenic mechanism in Huntington's disease (HD), it is still not known how mutant huntingtin causes it. Here we show that alteration of histone monoubiquitylation is a key mechanism. Disrupted interaction of huntingtin with Bmi-1, a component of the hPRC1L E3 ubiquitin ligase complex, increases monoubiquityl histone H2A (uH2A) levels in a cell culture model of HD. Genes with expression that is repressed in transgenic R6/2 mouse brain have increased uH2A and decreased uH2B at their promoters, whereas actively transcribed genes show the opposite pattern. Reduction in uH2A reverses transcriptional repression and inhibits methylation of histone H3 at lysine 9 in cell culture. In contrast, reduction in uH2B induces transcriptional repression and inhibits methylation of histone H3 at lysine 4. This is the first report to demonstrate hPRC1L as a huntingtin-interacting histone modifying complex and a crucial role for histone monoubiquitylation in mammalian brain gene expression, which broadens our understanding of histone code. These findings also provide a rationale for targeting histone monoubiquitylation for therapy in HD.

Histones associated with downregulated genes are hypo-acetylated in Huntington's disease models.

Transcriptional dysregulation plays a major role in the pathology of Huntington's disease (HD). However, the mechanisms causing selective downregulation of genes remain unknown. Histones regulate chromatin structure and thereby control gene expression; recent studies have demonstrated a therapeutic role for histone deacetylase (HDAC) inhibitors in polyglutamine diseases. This study demonstrates that despite no change in overall acetylated histone levels, histone H3 is hypo-acetylated at promoters of downregulated genes in R6/2 mice, ST14a and STHdh cells, as demonstrated by in vivo chromatin immunoprecipitation. In addition, HDAC inhibitor treatment increases association of acetylated histones with downregulated genes and corrects mRNA abnormalities. In contrast, there is a decrease in mRNA levels in wild-type cells following treatment with a histone acetyltransferase inhibitor. Although changes in histone acetylation correlate with decreased gene expression, histone hypo-acetylation may be a late event, as no hypo-acetylation is observed in 4-week-old R6/2 mice. Nevertheless, treatment with HDAC inhibitors corrects mRNA abnormalities through modification of histone proteins and may prove to be of therapeutic value in HD.

BMP-2 mediates retinoid-induced apoptosis in medulloblastoma cells through a paracrine effect.

The mechanisms of retinoid activity in tumors remain largely unknown. Here we establish that retinoids cause extensive apoptosis of medulloblastoma cells. In a xenograft model, retinoids largely abrogated tumor growth. Using receptor-specific retinoid agonists, we defined a subset of mRNAs that were induced by all active retinoids in retinoid-sensitive cell lines. We also identified bone morphogenetic protein-2 (BMP-2) as a candidate mediator of retinoid activity. BMP-2 protein induced medulloblastoma cell apoptosis, whereas the BMP-2 antagonist noggin blocked both retinoid and BMP-2-induced apoptosis. BMP-2 also induced p38 mitogen-activated protein kinase (MAPK), which is necessary for BMP-2- and retinoid-induced apoptosis. Retinoid-resistant medulloblastoma cells underwent apoptosis when treated with BMP-2 or when cultured with retinoid-sensitive medulloblastoma cells. Retinoid-induced expression of BMP-2 is thus necessary and sufficient for apoptosis of retinoid-responsive cells, and expression of BMP-2 by retinoid-sensitive cells is sufficient to induce apoptosis in surrounding retinoid-resistant cells.