Stephen Fawell

Tankyrase inhibition stabilizes axin and antagonizes Wnt signalling

The stability of the Wnt pathway transcription factor β-catenin is tightly regulated by the multi-subunit destruction complex. Deregulated Wnt pathway activity has been implicated in many cancers, making this pathway an attractive target for anticancer therapies. However, the development of targeted Wnt pathway inhibitors has been hampered by the limited number of pathway components that are amenable to small molecule inhibition. Here, we used a chemical genetic screen to identify a small molecule, XAV939, which selectively inhibits β-catenin-mediated transcription. XAV939 stimulates β-catenin degradation by stabilizing axin, the concentration-limiting component of the destruction complex. Using a quantitative chemical proteomic approach, we discovered that XAV939 stabilizes axin by inhibiting the poly-ADP-ribosylating enzymes tankyrase 1 and tankyrase 2. Both tankyrase isoforms interact with a highly conserved domain of axin and stimulate its degradation through the ubiquitin-proteasome pathway. Thus, our study provides new mechanistic insights into the regulation of axin protein homeostasis and presents new avenues for targeted Wnt pathway therapies.

Targeting the Lymphotoxin-β Receptor with Agonist Antibodies as a Potential Cancer Therapy

The lymphotoxin-beta receptor (LT beta R) is a tumor necrosis factor receptor family member critical for the development and maintenance of various lymphoid microenvironments. Herein, we show that agonistic anti-LT beta R monoclonal antibody (mAb) CBE11 inhibited tumor growth in xenograft models and potentiated tumor responses to chemotherapeutic agents. In a syngeneic colon carcinoma tumor model, treatment of the tumor-bearing mice with an agonistic antibody against murine LT beta R caused increased lymphocyte infiltration and necrosis of the tumor. A pattern of differential gene expression predictive of cellular and xenograft response to LT beta R activation was identified in a panel of colon carcinoma cell lines and when applied to a panel of clinical colorectal tumor samples indicated 35% likelihood a tumor response to CBE11. Consistent with this estimate, CBE11 decreased tumor size and/or improved long-term animal survival with two of six independent orthotopic xenografts prepared from surgical colorectal carcinoma samples. Targeting of LT beta R with agonistic mAbs offers a novel approach to the treatment of colorectal and potentially other types of cancers.

Activity of deacetylase inhibitor panobinostat (LBH589) in cutaneous T-cell lymphoma models: Defining molecular mechanisms of resistance.

Panobinostat (LBH589) is a highly potent deacetylase inhibitor that has demonstrated clinical efficacy in patients with advanced cutaneous T‐cell lymphoma (CTCL). To gain a better understanding of the compound activity in this tumor type, we investigated the cellular and molecular effects of panobinostat using both in vitro and in vivo models of CTCL. All 4 tested CTCL cell lines exhibited very high sensitivity to panobinostat‐induced growth inhibition. However, only 2 of 4 lines exhibited significant response to the cytotoxic activity of panobinostat. In a CTCL xenograft mouse tumor model, panobinostat treatment resulted in complete tumor regression. The difference in cell sensitivity to panobinostat‐induced death enabled us to further investigate potential mechanisms responsible for tumor sensitivity or resistance. In CTCL cell lines that were insensitive to panobinostat‐induced apoptosis, constitutively activated NF‐κB and high levels of Bcl‐2 were observed. Inhibition of Bcl‐2 sensitized cells to the cytotoxic activity of panobinostat. Conversely, knockdown of Bax diminished the CTCL cell sensitivity. Interestingly, panobinostat could induce cytotoxicity in vorinostat‐resistant CTCL cells by downregulating phosphorylated STAT3 and STAT5 proteins. These studies suggest distinct mechanisms responsible for resistance to different deacetylase inhibitors. We show that the intrinsic apoptotic signaling plays an essential role in mediating panobinostat anticancer activity. Moreover, cancer cell sensitivity to panobinostat treatment may be further improved by combination with inhibition of anti‐apoptotic factors. These data provide preclinical support that panobinostat, as a single agent or in combination with other anticancer agents, is a promising therapy for CTCL.

Abstract 3853: TAS266, a novel tetrameric nanobody agonist targeting death receptor 5 (DR5), elicits superior antitumor efficacy than conventional DR5-targeted approaches

Preferential induction of apoptosis in cancer cells has been the objective of therapeutic strategies targeting apoptotic pathways. To this end, multiple therapeutic agonists of Death Receptors 4 and 5 (DR4, DR5), have been developed and are under clinical evaluation. Although these agonists, including antibodies and soluble ligand TRAIL, demonstrate significant anti-tumor activity in preclinical models, the clinical efficacy in human cancer patients has been notably disappointing. One possible explanation for the discrepant pre-clinical and clinical results is that DR5 may play a more prominent role in in vitro model systems as opposed to cancers in humans. Alternatively, these results might indicate that the current classes of therapeutic molecules are not sufficiently potent to elicit significant response in patients. In particular, naturally dimeric antibody agonists require secondary cross-linking via Fcα receptors expressed on immune cells present in the tumor microenvironment to achieve optimal clustering of DR5 into a ternary active state. Because immune cell content in the tumor can be heterogeneous, reliance on this secondary mechanism for activity may limit the potency of these antibodies. To overcome this limitation, a novel nanobody approach was taken to eliminate the need for cross-linking and improve receptor activation with the goal of generating a significantly more potent DR5 agonist. Nanobodies are a class of therapeutic proteins based on single, high affinity heavy chain domain (VHH) antibodies that naturally occur in camelid species, and these VHH domains can be linked to form multivalent structures (di-, tri-, tetra-, etc). This approach led to the development of a tetrameric DR5 targeted agonist, TAS266, with significantly greater avidity for DR5 binding. TAS266 activates downstream caspases with more rapid kinetics and is up to 1000-fold more potent in cell death assays when compared to a cross-linked DR5 antibody or TRAIL. In vivo, TAS266 elicits single dose tumor regressions in multiple tumor xenograft models and sustained tumor regressions after treatment cessation. TAS266 showed superior anti-tumor activity compared to a DR5 agonist antibody and TRAIL, including the ability to induce tumor regression in a patient-derived primary pancreatic tumor model that is insensitive to the agonist antibody. Thus, TAS266 has the potential for superior clinical activity in settings insensitive to the conventional therapeutic approaches to DR5. First-in-man trials are expected to begin in 2012. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3853. doi:1538-7445.AM2012-3853

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.