Rebecca Mosher

Contribution of Polycomb Homologues Bmi-1 and Mel-18 to Medulloblastoma Pathogenesis

ABSTRACT Bmi-1 and Mel-18 are structural homologues that belong to the Polycomb group of transcriptional regulators and are believed to stably maintain repression of gene expression by altering the state of chromatin at specific promoters. While a number of clinical and experimental observations have implicated Bmi-1 in human tumorigenesis, the role of Mel-18 in cancer cell growth has not been investigated. We report here that short hairpin RNA-mediated knockdown of either Bmi-1 or Mel-18 in human medulloblastoma DAOY cells results in the inhibition of proliferation, loss of clonogenic survival, anchorage-independent growth, and suppression of tumor formation in nude mice. Furthermore, overexpression of both Bmi-1 and Mel-18 significantly increases the clonogenic survival of Rat1 fibroblasts. In contrast, stable downregulation of Bmi-1 or Mel-18 alone does not affect the growth of normal human WI38 fibroblasts. Proteomics-based characterization of Bmi-1 and Mel-18 protein complexes isolated from cancer cells revealed substantial similarities in their respective compositions. Finally, gene expression analysis identified a number of cancer-relevant pathways that may be controlled by Bmi-1 and Mel-18 and also showed that these Polycomb proteins regulate a set of common gene targets. Taken together, these results suggest that Bmi-1 and Mel-18 may have overlapping functions in cancer cell growth.

Maintenance of adenomatous polyposis coli (APC)-mutant colorectal cancer is dependent on Wnt/β-catenin signaling

Persistent expression of certain oncogenes is required for tumor maintenance. This phenotype is referred to as oncogene addiction and has been clinically validated by anticancer therapies that specifically inhibit oncoproteins such as BCR-ABL, c-Kit, HER2, PDGFR, and EGFR. Identifying additional genes that are required for tumor maintenance may lead to new targets for anticancer drugs. Although the role of aberrant Wnt pathway activation in the initiation of colorectal cancer has been clearly established, it remains unclear whether sustained Wnt pathway activation is required for colorectal tumor maintenance. To address this question, we used inducible β-catenin shRNAs to temporally control Wnt pathway activation in vivo. Here, we show that active Wnt/β-catenin signaling is required for maintenance of colorectal tumor xenografts harboring APC mutations. Reduced tumor growth upon β-catenin inhibition was due to cell cycle arrest and differentiation. Upon reactivation of the Wnt/β-catenin pathway colorectal cancer cells resumed proliferation and reacquired a crypt progenitor phenotype. In human colonic adenocarcinomas, high levels of nuclear β-catenin correlated with crypt progenitor but not differentiation markers, suggesting that the Wnt/β-catenin pathway may also control colorectal tumor cell fate during the maintenance phase of tumors in patients. These results support efforts to treat human colorectal cancer by pharmacological inhibition of the Wnt/β-catenin pathway.

An antibody that locks HER3 in the inactive conformation inhibits tumor growth driven by HER2 or neuregulin.

HER2/HER3 dimerization resulting from overexpression of HER2 or neuregulin (NRG1) in cancer leads to HER3-mediated oncogenic activation of phosphoinositide 3-kinase (PI3K) signaling. Although ligand-blocking HER3 antibodies inhibit NRG1-driven tumor growth, they are ineffective against HER2-driven tumor growth because HER2 activates HER3 in a ligand-independent manner. In this study, we describe a novel HER3 monoclonal antibody (LJM716) that can neutralize multiple modes of HER3 activation, making it a superior candidate for clinical translation as a therapeutic candidate. LJM716 was a potent inhibitor of HER3/AKT phosphorylation and proliferation in HER2-amplified and NRG1-expressing cancer cells, and it displayed single-agent efficacy in tumor xenograft models. Combining LJM716 with agents that target HER2 or EGFR produced synergistic antitumor activity in vitro and in vivo. In particular, combining LJM716 with trastuzumab produced a more potent inhibition of signaling and cell proliferation than trastuzumab/pertuzumab combinations with similar activity in vivo. To elucidate its mechanism of action, we solved the structure of LJM716 bound to HER3, finding that LJM716 bound to an epitope, within domains 2 and 4, that traps HER3 in an inactive conformation. Taken together, our findings establish that LJM716 possesses a novel mechanism of action that, in combination with HER2- or EGFR-targeted agents, may leverage their clinical efficacy in ErbB-driven cancers.

The Polycomb Group Protein Bmi-1 Is Essential for the Growth of Multiple Myeloma Cells

Bmi-1 is a member of the Polycomb group family of proteins that function in the epigenetic silencing of genes governing self-renewal, differentiation, and proliferation. Bmi-1 was first identified through its ability to accelerate c-Myc-induced lymphomagenesis. Subsequent studies have further supported an oncogenic role for Bmi-1 in several cancers including those of the breast, lung, prostate, and brain. Using a stable and inducible shRNA system to silence Bmi-1 gene expression, we show a novel role for Bmi-1 in regulating the growth and clonogenic capacity of multiple myeloma cells both in vitro and in vivo. Moreover, to elucidate novel gene targets controlled by Bmi-1, global transcriptional profiling studies were performed in the setting of induced loss of Bmi-1 function. We found that the expression of the proapoptotic gene Bim is negatively regulated by Bmi-1 and that Bim knockdown functionally rescues the apoptotic phenotype induced upon loss of Bmi-1. Therefore, these studies not only highlight Bmi-1 as a cancer-dependent factor in multiple myeloma, but also elucidate a novel antiapoptotic mechanism for Bmi-1 function involving the suppression of Bim.

Multivalent nanobodies targeting death receptor 5 elicit superior tumor cell killing through efficient caspase induction

Multiple therapeutic agonists of death receptor 5 (DR5) have been developed and are under clinical evaluation. Although these agonists demonstrate significant anti-tumor activity in preclinical models, the clinical efficacy in human cancer patients has been notably disappointing. One possible explanation might be that the current classes of therapeutic molecules are not sufficiently potent to elicit significant response in patients, particularly for dimeric antibody agonists that require secondary cross-linking via Fcγ receptors expressed on immune cells to achieve optimal clustering of DR5. To overcome this limitation, a novel multivalent Nanobody approach was taken with the goal of generating a significantly more potent DR5 agonist. In the present study, we show that trivalent DR5 targeting Nanobodies mimic the activity of natural ligand, and furthermore, increasing the valency of domains to tetramer and pentamer markedly increased potency of cell killing on tumor cells, with pentamers being more potent than tetramers in vitro. Increased potency was attributed to faster kinetics of death-inducing signaling complex assembly and caspase-8 and caspase-3 activation. In vivo, multivalent Nanobody molecules elicited superior anti-tumor activity compared to a conventional DR5 agonist antibody, including the ability to induce tumor regression in an insensitive patient-derived primary pancreatic tumor model. Furthermore, complete responses to Nanobody treatment were obtained in up to 50% of patient-derived primary pancreatic and colon tumor models, suggesting that multivalent DR5 Nanobodies may represent a significant new therapeutic modality for targeting death receptor signaling.

Patterns of HER2 Gene Amplification and Response to Anti-HER2 Therapies

A chromosomal region that includes the gene encoding HER2, a receptor tyrosine kinase (RTK), is amplified in 20% of breast cancers. Although these tumors tend to respond to drugs directed against HER2, they frequently become resistant and resume their malignant progression. Gene amplification in double minutes (DMs), which are extrachromosomal entities whose number can be dynamically regulated, has been suggested to facilitate the acquisition of resistance to therapies targeting RTKs. Here we show that ~30% of HER2-positive tumors show amplification in DMs. However, these tumors respond to trastuzumab in a similar fashion than those with amplification of the HER2 gene within chromosomes. Furthermore, in different models of resistance to anti-HER2 therapies, the number of DMs containing HER2 is maintained, even when the acquisition of resistance is concomitant with loss of HER2 protein expression. Thus, both clinical and preclinical data show that, despite expectations, loss of HER2 protein expression due to loss of DMs containing HER2 is not a likely mechanism of resistance to anti-HER2 therapies.

Discovery and Optimization of HKT288, a Cadherin-6 Targeting ADC for the Treatment of Ovarian and Renal Cancer.

Despite an improving therapeutic landscape, significant challenges remain in treating the majority of patients with advanced ovarian or renal cancer. We identified the cell-cell adhesion molecule cadherin-6 (CDH6) as a lineage gene having significant differential expression in ovarian and kidney cancers. HKT288 is an optimized CDH6-targeting DM4-based antibody-drug conjugate (ADC) developed for the treatment of these diseases. Our study provides mechanistic evidence supporting the importance of linker choice for optimal antitumor activity and highlights CDH6 as an antigen for biotherapeutic development. To more robustly predict patient benefit of targeting CDH6, we incorporate a population-based patient-derived xenograft (PDX) clinical trial (PCT) to capture the heterogeneity of response across an unselected cohort of 30 models-a novel preclinical approach in ADC development. HKT288 induces durable tumor regressions of ovarian and renal cancer models in vivo, including 40% of models on the PCT, and features a preclinical safety profile supportive of progression toward clinical evaluation.Significance: We identify CDH6 as a target for biotherapeutics development and demonstrate how an integrated pharmacology strategy that incorporates mechanistic pharmacodynamics and toxicology studies provides a rich dataset for optimizing the therapeutic format. We highlight how a population-based PDX clinical trial and retrospective biomarker analysis can provide correlates of activity and response to guide initial patient selection for first-in-human trials of HKT288. Cancer Discov; 7(9); 1030-45. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 920.

Abstract B27: Correlation between TNFα and LCL161 anti‐tumor activity in patient derived xenograft models of human cancer

LCL161, a small molecule antagonist of inhibitor of apoptosis proteins (IAPs), induces TNF ‐mediated apoptosis in a subset of tumor cell lines including the MDA‐MB‐231 breast cancer line. To investigate the in vivo activity of LCL161, MDA‐MB‐231 tumor bearing mice were treated with a once weekly oral dose. We observed anti‐tumor activity that was associated with pharmacodynamic responses including degradation of CIAP1 and induction of cleaved caspase 3. The loss of CIAP1, specifically the E3 ligase activity of this protein, has been shown to directly impact the stability of a number of client proteins including NIK (NF‐κB inducing kinase), Mad1 (MAX‐binding protein), and others. Gene expression analysis on LCL161 treated MDA‐MB‐231 tumors revealed a striking upregulation of NF‐κB regulated target genes. These data were further supported by the observation that TNFα, a direct target of NF‐κB, was induced in LCL161 treated MDAMB‐231 tumor lysates. To explore whether TNFα expression levels could be used to predict single agent efficacy in a more clinically relevant context, a series of human primary tumor xenografts were profiled for baseline TNFα mRNA levels and evaluated for response to LCL161 in vivo. We observed that tumors with the highest TNFα expression levels showed the greatest sensitivity to LCL161. These studies demonstrate an essential role of the NF‐κB pathway in IAP antagonist anti‐tumor activity. Ongoing efforts focus on delineating the contribution of the canonical and non‐canonical NF‐κB pathways to efficacy and on leveraging this mechanism for LCL161 patient selection. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B27.

Abstract 138: Therapeutic targeting of inhibitor of apoptosis proteins

Inhibitor of Apoptosis Proteins (IAP) proteins negatively regulate cell death through a variety of mechanisms. The prototypical IAP family member XIAP binds and inhibits the catalytic activity of caspases 3/7 and caspase 9 via the BIR2-linker region and BIR3 domains, respectively. CIAP1 and CIAP2 do not directly inhibit caspases but negatively regulate death receptor mediated apoptosis via intrinsic E3 ligase activity towards RIPK and NIK among other client proteins. IAP inhibitors (IAPi) are low molecular weight compounds that mimic Smac and bind to the IAP binding motif in the BIR3 domain of XIAP, CIAP1 and CIAP2. Smac mimetics induce apoptosis as a single agent in a subset of tumor cell lines in vitro. Cell death is preceded by the rapid proteosome-mediated degradation of CIAP1 followed by activation of both canonical and non-canonical NFKB pathway activation, TNF production and robust activation of caspase 3/7 activity. Multiple nodes in the NFKB signaling pathway were interrogated following IAPi treatment in sensitive and resistant cancer cells to delineate the basis for differential responses. Although canonical and non-canonical NFKB signaling was activated in both sensitive and resistant cells, TNF was induced only in the former. LCL161 is a second generation orally bioavailable IAPi with nM affinity for XIAP, CIAP1, CIAP2. Consistent with above, tumor cell lines with high baseline TNF levels are predisposed to IAPi sensitivity. Curiously, addition of exogenous TNF can sensitize many otherwise resistant tumor cell lines, but not normal cells, to LCL161. We undertook an unbiased study of the entire TNF super family to determine what other TNF-like cytokines could sensitize tumor cells to LCL161- induced cell death. In addition to TNF, several cytokines synergized with LCL161 and in each case RIPK appeared to play a central role. LCL161 showed potent single agent activity in the MDA-MB-231 tumor xenograft model. In vivo efficacy was accompanied by a series of tumor pharmacodynamic readouts including CIAP1 elimination, activation of an NFKB transcriptional program and caspase activation. In primary patient derived human tumor xenograft models of triple negative breast cancer and NSCLC, LCL161 had a range of responses from no effect to tumor stasis. Consistent with in vitro mechanistic studies, tumor models which were sensitive had high basal TNF levels. LCL161 lacked single agent activity in the A2058 melanoma model but significantly enhanced the anti-tumor activity of paclitaxel. The LCL161-Taxol combination triggered synergistic activation of caspases and near complete regressions in xenograft tumors. Clinical trials in man with LCL161 are ongoing in patients with solid tumors. A range of pharmacodynamic readouts have been observed which are consistent with preclinical observations. These findings show promise for IAP inhibitor therapy in humans. 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 138.

Abstract 4443: Molecular mechanistic study of ASA404 (vadimezan)-induced endothelial cell death

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC The tumor vasculature is a key component in maintaining tumor growth and regulating the tumor microenvironment through the supply of nutrients and oxygen. ASA404 (vadimezan, 5,6-dimethylxanthenone-4-acetic acid) is a flavonoid, non-tubulin-binding Tumor-Vascular Disrupting Agent (Tumor-VDA) that induces breakdown of the established tumor vasculature through the induction of tumor endothelial cell apoptosis, resulting in the inhibition of tumor blood supply, leading to tumor ischemia and extensive necrosis of the tumor core. In contrast to antiangiogenics, ASA404 has limited effects on angiogenesis at the tumor periphery and so the remaining peripheral tumor rim represents a source of viable cells for regrowth of the tumor upon cessation of ASA404 treatment. Preclinical and clinical studies have highlighted the effectiveness of combining chemotherapies, especially taxanes, with ASA404 for marked tumor inhibition. Currently, the benefits of adding ASA404 to the standard of care in first- and second-line NSCLC indications are being evaluated in the ATTRACT-1 and ATTRACT-2 Phase III trials respectively. Despite the prior clinical efficacy observed with ASA404, the detailed molecular mechanism by which ASA404 selectively targets the tumor vasculature still remains to be defined. To investigate this, we initiated studies to explore the function of ASA404 both in vitro using human endothelial cells (HUVEC) and in ASA404-treated MDA-MB-231 breast cancer xenografts examined ex vivo. Consistent with previous reports, we observed inhibition of HUVEC cell proliferation along with rapid cell death in vitro as assessed using FACS analysis and newly demonstrated the rapid induction of caspase 3 assessed by immunocytochemistry. Treatment of the MDA-MB-231 breast xenografts in vivo with ASA404 resulted in significant tumor growth inhibition, tumor necrosis and elevation of the hypoxia marker carbonic anhydrase 9 (CAIX). The nature of ASA404-induced endothelial cell death is likely to be due to apoptosis since rapid caspase 3 cleavage was observed in the endothelial cells both in vitro and in vivo. In addition, the mitochondrial potential of cultured HUVEC cells in vitro was found to be seriously compromised after 1-2 hours treatment with ASA404 as monitored by Mito-tracker staining. This was accompanied by cytochrome C release into the cytosol as evidenced by immunohistochemical co-localization of cytochrome C and mitochondria markers. We also detected a moderate, but concentration-dependent increase in ceramide levels in HUVEC cells following 2 hours of ASA404 treatment, which may serve as a link to the observed mitochondrial depolarization phenotype. Lastly, a pooled-based shRNA screen was also conducted to search for genes that could modulate ASA404 activity in HUVEC cells. Some of the candidates identified may provide new insights into the pathways that are critical in the ASA404 mechanism of action. 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 4443.