Alwin Schuller

Targeting Wnt-driven cancer through the inhibition of Porcupine by LGK974

Significance Targeting the Wnt pathway in cancer is an attractive therapeutic approach. However, success has been limited because of the lack of effective therapeutic agents and the lack of biomarkers to define the patient population that would benefit from such a therapy. Herein, we report the discovery of LGK974, a drug that targets Porcupine, a Wnt-specific acyltransferase. We show that LGK974 potently inhibits Wnt signaling, has strong efficacy in rodent tumor models, and is well-tolerated. We also show that head and neck cancer cell lines with loss-of-function mutations in the Notch signaling pathway have a high response rate to LGK974. Together, these findings provide a strategy and tools for targeting Wnt-driven cancer. Wnt signaling is one of the key oncogenic pathways in multiple cancers, and targeting this pathway is an attractive therapeutic approach. However, therapeutic success has been limited because of the lack of therapeutic agents for targets in the Wnt pathway and the lack of a defined patient population that would be sensitive to a Wnt inhibitor. We developed a screen for small molecules that block Wnt secretion. This effort led to the discovery of LGK974, a potent and specific small-molecule Porcupine (PORCN) inhibitor. PORCN is a membrane-bound O-acyltransferase that is required for and dedicated to palmitoylation of Wnt ligands, a necessary step in the processing of Wnt ligand secretion. We show that LGK974 potently inhibits Wnt signaling in vitro and in vivo, including reduction of the Wnt-dependent LRP6 phosphorylation and the expression of Wnt target genes, such as AXIN2. LGK974 is potent and efficacious in multiple tumor models at well-tolerated doses in vivo, including murine and rat mechanistic breast cancer models driven by MMTV–Wnt1 and a human head and neck squamous cell carcinoma model (HN30). We also show that head and neck cancer cell lines with loss-of-function mutations in the Notch signaling pathway have a high response rate to LGK974. Together, these findings provide both a strategy and tools for targeting Wnt-driven cancers through the inhibition of PORCN.

Inhibition of tumorigenesis driven by different Wnt proteins requires blockade of distinct ligand-binding regions by LRP6 antibodies

Disregulated Wnt/β-catenin signaling has been linked to various human diseases, including cancers. Inhibitors of oncogenic Wnt signaling are likely to have a therapeutic effect in cancers. LRP5 and LRP6 are closely related membrane coreceptors for Wnt proteins. Using a phage-display library, we identified anti-LRP6 antibodies that either inhibit or enhance Wnt signaling. Two classes of LRP6 antagonistic antibodies were discovered: one class specifically inhibits Wnt proteins represented by Wnt1, whereas the second class specifically inhibits Wnt proteins represented by Wnt3a. Epitope-mapping experiments indicated that Wnt1 class-specific antibodies bind to the first propeller and Wnt3a class-specific antibodies bind to the third propeller of LRP6, suggesting that Wnt1- and Wnt3a-class proteins interact with distinct LRP6 propeller domains. This conclusion is further supported by the structural functional analysis of LRP5/6 and the finding that the Wnt antagonist Sclerostin interacts with the first propeller of LRP5/6 and preferentially inhibits the Wnt1-class proteins. We also show that Wnt1 or Wnt3a class-specific anti-LRP6 antibodies specifically block growth of MMTV-Wnt1 or MMTV-Wnt3 xenografts in vivo. Therapeutic application of these antibodies could be limited without knowing the type of Wnt proteins expressed in cancers. This is further complicated by our finding that bivalent LRP6 antibodies sensitize cells to the nonblocked class of Wnt proteins. The generation of a biparatopic LRP6 antibody blocks both Wnt1- and Wnt3a-mediated signaling without showing agonistic activity. Our studies provide insights into Wnt-induced LRP5/6 activation and show the potential utility of LRP6 antibodies in Wnt-driven cancer.

Preclinical Evaluation of the WEE1 Inhibitor MK-1775 as Single-Agent Anticancer Therapy

Inhibition of the DNA damage checkpoint kinase WEE1 potentiates genotoxic chemotherapies by abrogating cell-cycle arrest and proper DNA repair. However, WEE1 is also essential for unperturbed cell division in the absence of extrinsic insult. Here, we investigate the anticancer potential of a WEE1 inhibitor, independent of chemotherapy, and explore a possible cellular context underlying sensitivity to WEE1 inhibition. We show that MK-1775, a potent and selective ATP-competitive inhibitor of WEE1, is cytotoxic across a broad panel of tumor cell lines and induces DNA double-strand breaks. MK-1775–induced DNA damage occurs without added chemotherapy or radiation in S-phase cells and relies on active DNA replication. At tolerated doses, MK-1775 treatment leads to xenograft tumor growth inhibition or regression. To begin addressing potential response markers for MK-1775 monotherapy, we focused on PKMYT1, a kinase functionally related to WEE1. Knockdown of PKMYT1 lowers the EC50 of MK-1775 by five-fold but has no effect on the cell-based response to other cytotoxic drugs. In addition, knockdown of PKMYT1 increases markers of DNA damage, γH2AX and pCHK1S345, induced by MK-1775. In a post hoc analysis of 305 cell lines treated with MK-1775, we found that expression of PKMYT1 was below average in 73% of the 33 most sensitive cell lines. Our findings provide rationale for WEE1 inhibition as a potent anticancer therapy independent of a genotoxic partner and suggest that low PKMYT1 expression could serve as an enrichment biomarker for MK-1775 sensitivity. Mol Cancer Ther; 12(8); 1442–52. ©2013 AACR.

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.

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.

The MET inhibitor AZD6094 (Savolitinib, HMPL-504) induces regression in papillary renal cell carcinoma patient derived xenograft models

Purpose: Papillary renal cell carcinoma (PRCC) is the second most common cancer of the kidney and carries a poor prognosis for patients with nonlocalized disease. The HGF receptor MET plays a central role in PRCC and aberrations, either through mutation, copy number gain, or trisomy of chromosome 7 occurring in the majority of cases. The development of effective therapies in PRCC has been hampered in part by a lack of available preclinical models. We determined the pharmacodynamic and antitumor response of the selective MET inhibitor AZD6094 in two PRCC patient-derived xenograft (PDX) models. Experimental Design: Two PRCC PDX models were identified and MET mutation status and copy number determined. Pharmacodynamic and antitumor activity of AZD6094 was tested using a dose response up to 25 mg/kg daily, representing clinically achievable exposures, and compared with the activity of the RCC standard-of-care sunitinib (in RCC43b) or the multikinase inhibitor crizotinib (in RCC47). Results: AZD6094 treatment resulted in tumor regressions, whereas sunitinib or crizotinib resulted in unsustained growth inhibition. Pharmacodynamic analysis of tumors revealed that AZD6094 could robustly suppress pMET and the duration of target inhibition was dose related. AZD6094 inhibited multiple signaling nodes, including MAPK, PI3K, and EGFR. Finally, at doses that induced tumor regression, AZD6094 resulted in a dose- and time-dependent induction of cleaved PARP, a marker of cell death. Conclusions: Data presented provide the first report testing therapeutics in preclinical in vivo models of PRCC and support the clinical development of AZD6094 in this indication. Clin Cancer Res; 21(12); 2811–9. ©2015 AACR.

MCL1 and BCL-xL Levels in Solid Tumors Are Predictive of Dinaciclib-Induced Apoptosis

Dinaciclib is a potent CDK1, 2, 5 and 9 inhibitor being developed for the treatment of cancer. Additional understanding of antitumor mechanisms and identification of predictive biomarkers are important for its clinical development. Here we demonstrate that while dinaciclib can effectively block cell cycle progression, in vitro and in vivo studies, coupled with mouse and human pharmacokinetics, support a model whereby induction of apoptosis is a main mechanism of dinaciclibs antitumor effect and relevant to the clinical duration of exposure. This was further underscored by kinetics of dinaciclib-induced downregulation of the antiapoptotic BCL2 family member MCL1 and correlation of sensitivity with the MCL1-to-BCL-xL mRNA ratio or MCL1 amplification in solid tumor models in vitro and in vivo. This MCL1-dependent apoptotic mechanism was additionally supported by synergy with the BCL2, BCL-xL and BCL-w inhibitor navitoclax (ABT-263). These results provide the rationale for investigating MCL1 and BCL-xL as predictive biomarkers for dinaciclib antitumor response and testing combinations with BCL2 family member inhibitors.

AZ1366: An inhibitor of tankyrase and the canonical wnt pathway that limits the persistence of non-small cell lung cancer cells following EGFR inhibition

Purpose: The emergence of EGFR inhibitors such as gefitinib, erlotinib, and osimertinib has provided novel treatment opportunities in EGFR-driven non–small cell lung cancer (NSCLC). However, most patients with EGFR-driven cancers treated with these inhibitors eventually relapse. Recent efforts have identified the canonical Wnt pathway as a mechanism of protection from EGFR inhibition and that inhibiting tankyrase, a key player in this pathway, is a potential therapeutic strategy for the treatment of EGFR-driven tumors. Experimental Design: We performed a preclinical evaluation of tankyrase inhibitor AZ1366 in combination with multiple EGFR-inhibitors across NSCLC lines, characterizing its antitumor activity, impingement on canonical Wnt signaling, and effects on gene expression. We performed pharmacokinetic and pharmacodynamic profiling of AZ1366 in mice and evaluated its therapeutic activity in an orthotopic NSCLC model. Results: In combination with EGFR inhibitors, AZ1366 synergistically suppressed proliferation of multiple NSCLC lines and amplified global transcriptional changes brought about by EGFR inhibition. Its ability to work synergistically with EGFR inhibition coincided with its ability to modulate the canonical Wnt pathway. Pharmacokinetic and pharmacodynamic profiling of AZ1366-treated orthotopic tumors demonstrated clinically relevant serum drug levels and intratumoral target inhibition. Finally, coadministration of an EGFR inhibitor and AZ1366 provided better tumor control and improved survival for Wnt-responsive lung cancers in an orthotopic mouse model. Conclusions: Tankyrase inhibition is a potent route of tumor control in EGFR-dependent NSCLC with confirmed dependence on canonical Wnt signaling. These data strongly support further evaluation of tankyrase inhibition as a cotreatment strategy with EGFR inhibition in an identifiable subset of EGFR-driven NSCLC. Clin Cancer Res; 23(6); 1531–41. ©2016 AACR.

Acquired savolitinib resistance in non-small cell lung cancer arises via multiple mechanisms that converge on MET-independent mTOR and MYC activation

Lung cancer is the most common cause of cancer death globally with a significant, unmet need for more efficacious treatments. The receptor tyrosine kinase MET has been implicated as an oncogene in numerous cancer subtypes, including non-small cell lung cancer (NSCLC). Here we explore the therapeutic potential of savolitinib (volitinib, AZD6094, HMPL-504), a potent and selective MET inhibitor, in NSCLC. In vitro, savolitinib inhibits MET phosphorylation with nanomolar potency, which correlates with blockade of PI3K/AKT and MAPK signaling as well as MYC down-regulation. In vivo, savolitinib causes inhibition of these pathways and significantly decreases growth of MET-dependent xenografts. To understand resistance mechanisms, we generated savolitinib resistance in MET-amplified NSCLC cell lines and analyzed individual clones. We found that upregulation of MYC and constitutive mTOR pathway activation is a conserved feature of resistant clones that can be overcome by knockdown of MYC or dual mTORC1/2 inhibition. Lastly, we demonstrate that mechanisms of resistance are heterogeneous, arising via a switch to EGFR dependence or by a requirement for PIM signaling. This work demonstrates the efficacy of savolitinib in NSCLC and characterizes acquired resistance, identifying both known and novel mechanisms that may inform combination strategies in the clinic.

The novel tankyrase inhibitor (AZ1366) enhances irinotecan activity in tumors that exhibit elevated tankyrase and irinotecan resistance

Background Dysregulation of the canonical Wnt signaling pathway has been implicated in colorectal cancer (CRC) development as well as incipient stages of malignant transformation. In this study, we investigated the antitumor effects of AZ1366 (a novel tankyrase inhibitor) as a single agent and in combination with irinotecan in our patient derived CRC explant xenograft models. Results Six out of 18 CRC explants displayed a significant growth reduction to AZ1366. There was one CRC explant (CRC040) that reached the threshold of sensitivity (TGII ≤ 20%) in this study. In addition, the combination of AZ1366 + irinotecan demonstrated efficacy in 4 out of 18 CRC explants. Treatment effects on the WNT pathway revealed that tankyrase inhibition was ineffective at reducing WNT dependent signaling. However, the anti-tumor effects observed in this study were likely a result of alternative tankyrase effects whereby tankyrase inhibition reduced NuMA levels. Materials and Methods Eighteen CRC explants were treated with AZ1366 single agent or in combination for 28 days and treatment responses were assessed. Pharmacokinetic (AZ1366 drug concentrations) and pharmacodynamic effects (Axin2 levels) were investigated over 48 hours. Immunohistochemistry of nuclear β-catenin levels as well as western blot was employed to examine the treatment effects on the WNT pathway as well as NuMA. Conclusions Combination AZ1366 and irinotecan achieved greater anti-tumor effects compared to monotherapy. Activity was limited to CRC explants that displayed irinotecan resistance and increased protein levels of tankyrase and NuMA.