Joanna Majkut

Identification of an acetylation-dependant Ku70/FLIP complex that regulates FLIP expression and HDAC inhibitor-induced apoptosis.

FLIP is a potential anti-cancer therapeutic target that inhibits apoptosis by blocking caspase 8 activation by death receptors. We report a novel interaction between FLIP and the DNA repair protein Ku70 that regulates FLIP protein stability by inhibiting its polyubiquitination. Furthermore, we found that the histone deacetylase (HDAC) inhibitor Vorinostat (SAHA) enhances the acetylation of Ku70, thereby disrupting the FLIP/Ku70 complex and triggering FLIP polyubiquitination and degradation by the proteasome. Using in vitro and in vivo colorectal cancer models, we further demonstrated that SAHA-induced apoptosis is dependant on FLIP downregulation and caspase 8 activation. In addition, an HDAC6-specific inhibitor Tubacin recapitulated the effects of SAHA, suggesting that HDAC6 is a key regulator of Ku70 acetylation and FLIP protein stability. Thus, HDAC inhibitors with anti-HDAC6 activity act as efficient post-transcriptional suppressors of FLIP expression and may, therefore, effectively act as ‘FLIP inhibitors’.

Differential affinity of FLIP and procaspase 8 for FADD's DED binding surfaces regulates DISC assembly

Death receptor activation triggers recruitment of FADD, which via its death effector domain (DED) engages DEDs in procaspase 8 and its inhibitor FLIP to form death-inducing signalling complexes (DISCs). The DEDs of FADD, FLIP and procaspase 8 interact with one another using two binding surfaces defined by α1/α4 and α2/α5 helices respectively. Here we report that FLIP has preferential affinity for the α1/α4 surface of FADD, whereas procaspase 8 has preferential affinity for FADD’s α2/α5 surface. These relative affinities contribute to FLIP being recruited to the DISC at comparable levels to procaspase 8 despite lower cellular expression. Additional studies, including assessment of DISC stoichiometry and functional assays, suggest that following death receptor recruitment, the FADD DED preferentially engages FLIP using its α1/α4 surface and procaspase 8 using its α2/α5 surface; these tripartite intermediates then interact via the α1/α4 surface of FLIP DED1 and the α2/α5 surface of procaspase 8 DED2.

SAHA overcomes FLIP-mediated inhibition of SMAC mimetic-induced apoptosis in mesothelioma

Malignant pleural mesothelioma (MPM) is a highly pro-inflammatory malignancy that is rapidly fatal and increasing in incidence. Cytokine signaling within the pro-inflammatory tumor microenvironment makes a critical contribution to the development of MPM and its resistance to conventional chemotherapy approaches. SMAC mimetic compounds (SMCs) are a promising class of anticancer drug that are dependent on tumor necrosis factor alpha (TNFα) signaling for their activity. As circulating TNFα expression is significantly elevated in MPM patients, we examined the sensitivity of MPM cell line models to SMCs. Surprisingly, all MPM cell lines assessed were highly resistant to SMCs either alone or when incubated in the presence of clinically relevant levels of TNFα. Further analyses revealed that MPM cells were sensitized to SMC-induced apoptosis by siRNA-mediated downregulation of the caspase 8 inhibitor FLIP, an antiapoptotic protein overexpressed in several cancer types including MPM. We have previously reported that FLIP expression is potently downregulated in MPM cells in response to the histone deacetylase inhibitor (HDACi) Vorinostat (SAHA). In this study, we demonstrate that SAHA sensitizes MPM cells to SMCs in a manner dependent on its ability to downregulate FLIP. Although treatment with SMC in the presence of TNFα promoted interaction between caspase 8 and the necrosis-promoting RIPK1, the cell death induced by combined treatment with SAHA and SMC was apoptotic and mediated by caspase 8. These results indicate that FLIP is a major inhibitor of SMC-mediated apoptosis in MPM, but that this inhibition can be overcome by the HDACi SAHA.

Nanoencapsulation of ABT-737 and camptothecin enhances their clinical potential through synergistic antitumor effects and reduction of systemic toxicity

The simultaneous delivery of multiple cancer drugs in combination therapies to achieve optimal therapeutic effects in patients can be challenging. This study investigated whether co-encapsulation of the BH3-mimetic ABT-737 and the topoisomerase I inhibitor camptothecin (CPT) in PEGylated polymeric nanoparticles (NPs) was a viable strategy for overcoming their clinical limitations and to deliver both compounds at optimal ratios. We found that thrombocytopenia induced by exposure to ABT-737 was diminished through its encapsulation in NPs. Similarly, CPT-associated leukopenia and gastrointestinal toxicity were reduced compared with the administration of free CPT. In addition to the reduction of dose-limiting side effects, the co-encapsulation of both anticancer compounds in a single NP produced synergistic induction of apoptosis in both in vitro and in vivo colorectal cancer models. This strategy may widen the therapeutic window of these and other drugs and may enhance the clinical efficacy of synergistic drug combinations.

FLIP: a targetable mediator of resistance to radiation in non-small cell lung cancer

Resistance to radiotherapy due to insufficient cancer cell death is a significant cause of treatment failure in non–small cell lung cancer (NSCLC). The endogenous caspase-8 inhibitor FLIP is a critical regulator of cell death that is frequently overexpressed in NSCLC and is an established inhibitor of apoptotic cell death induced via the extrinsic death receptor pathway. Apoptosis induced by ionizing radiation (IR) has been considered to be mediated predominantly via the intrinsic apoptotic pathway; however, we found that IR-induced apoptosis was significantly attenuated in NSCLC cells when caspase-8 was depleted using RNA interference (RNAi), suggesting involvement of the extrinsic apoptosis pathway. Moreover, overexpression of wild-type FLIP, but not a mutant form that cannot bind the critical death receptor adaptor protein FADD, also attenuated IR-induced apoptosis, confirming the importance of the extrinsic apoptotic pathway as a determinant of response to IR in NSCLC. Importantly, when FLIP protein levels were downregulated by RNAi, IR-induced cell death was significantly enhanced. The clinically relevant histone deacetylase (HDAC) inhibitors vorinostat and entinostat were subsequently found to sensitize a subset of NSCLC cell lines to IR in a manner that was dependent on their ability to suppress FLIP expression and promote activation of caspase-8. Entinostat also enhanced the antitumor activity of IR in vivo. Therefore, FLIP downregulation induced by HDAC inhibitors is a potential clinical strategy to radiosensitize NSCLC and thereby improve response to radiotherapy. Overall, this study provides the first evidence that pharmacological inhibition of FLIP may improve response of NCSLC to IR. Mol Cancer Ther; 15(10); 2432–41. ©2016 AACR.

Cytoplasmic FLIP(S) and nuclear FLIP(L) mediate resistance of castrate-resistant prostate cancer to apoptosis induced by IAP antagonists

Expression of tumor necrosis factor-α (TNFα) in the serum of prostate cancer patients is associated with poorer outcome and progression to castrate-resistant (CRPC) disease. TNFα promotes the activity of NFκB, which regulates a number of anti-apoptotic and proinflammatory genes, including those encoding the inhibitor of apoptosis proteins (IAPs); however, in the presence of IAP antagonists, TNFα can induce cell death. In the presence of recombinant or macrophage-derived TNFα, we found that IAP antagonists triggered degradation of cIAP1 and induced formation of Complex-IIb, consisting of caspase-8, FADD and RIPK1 in CRPC models; however, no, or modest levels of apoptosis were induced. This resistance was found to be mediated by both the long (L) and short (S) splice forms of the caspase-8 inhibitor, FLIP, another NFκB-regulated protein frequently overexpressed in CRPC. By decreasing FLIP expression at the post-transcriptional level in PC3 and DU145 cells (but not VCaP), the Class-I histone deacetylase (HDAC) inhibitor Entinostat promoted IAP antagonist-induced cell death in these models in a manner dependent on RIPK1, FADD and Caspase-8. Of note, Entinostat primarily targeted the nuclear rather than cytoplasmic pool of FLIP(L). While the cytoplasmic pool of FLIP(L) was highly stable, the nuclear pool was more labile and regulated by the Class-I HDAC target Ku70, which we have previously shown regulates FLIP stability. The efficacy of IAP antagonist (TL32711) and Entinostat combination and their effects on cIAP1 and FLIP respectively were confirmed in vivo, highlighting the therapeutic potential for targeting IAPs and FLIP in proinflammatory CRPC.

Abstract LB-305: Development and pre-clinical assessment of a first-in-class small molecule inhibitor of FLIP

Background Evasion of cell death is a major cause of resistance to anti-cancer therapies, making proteins that regulate cell death clinically relevant therapeutic targets. The anti-apoptotic protein FLIP is frequently overexpressed in a number of cancers, including non-small cell lung cancer (NSCLC) and colorectal cancer (CRC), and has been shown by us and others to be a major mediator of drug resistance. FLIP and procaspase-8 form complexes with the adaptor protein FADD in response to a variety of clinically relevant stimuli, including ligation of death receptors, such as TRAIL-R1 and R2, and treatment with chemotherapeutic agents. In these complexes, FLIP modulates the activation of procaspase-8, and thereby apoptosis and necroptosis - two major cell death mechanisms. We have found that there are important differences between FLIP and procaspase-8 in terms of their preferred modes of interaction with FADD that are potentially therapeutically exploitable [1]. Herein, we report the development and pre-clinical characterization of first-in-class inhibitors of FLIP. Methods Molecular modelling of the FLIP-FADD interaction; cell-free screening assays; cell-based activity assays; biophysical binding assays; on-target mechanism-of-action studies, in vivo studies. Results Molecular modelling of the FLIP-FADD interaction identified a putative drug-binding pocket on FLIP against which a virtual small-molecule screen was carried out. Subsequent biochemical screening of selected compounds using a FLIP-FADD protein-protein interaction assay identified hits with on-target activity. Medicinal chemistry optimization of these hits identified a series of compounds that are able to disrupt FLIP’s interaction with the DISC and display nanomolar activity in NSCLC and CRC cell-based assays (i.e. caspase activation, cell death and cell survival), which is in line with their binding affinity in an orthogonal biophysical assay (isothermal calorimetry). The pro-apoptotic effects of these FLIP inhibitors were enhanced upon addition of death ligands, such as TRAIL, and lead-molecules have been shown to potentiate the effects of the standard-of-care chemotherapeutics such as e.g. cisplatin (NSCLC) or 5FU (CRC). FLIP overexpression and CRISPR-mediated procaspase-8 deletion abrogated the effects of these novel inhibitors consistent with the expected mechanism-of-action. In addition, using peripheral blood mononuclear cells (PBMCs), we demonstrate that FLIP inhibitors have selectivity against cancer cells. Finally, we identified lead molecules with ADME profiles suitable for in vivo evaluation and using these compounds, single-agent anti-tumor effects have been demonstrated in xenograft models. Conclusions The first-in-class inhibitors of FLIP developed in this study have the potential for broad application in treatment of cancer, either as monotherapy or in combination with other agents. Acknowledgements This work was supported by a Seeding Drug Discovery award from the Wellcome Trust. 1. Majkut, J., et al., Differential affinity of FLIP and procaspase 8 for FADD9s DED binding surfaces regulates DISC assembly. Nat Commun, 2014. 5: p. 3350. Citation Format: Joanna Majkut, Catherine Higgins, Jennifer Fox, Greti E. Fiedler, Timothy Harrison, Daniel B. Longley. Development and pre-clinical assessment of a first-in-class small molecule inhibitor of FLIP [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-305. doi:10.1158/1538-7445.AM2017-LB-305

Abstract C4: Development and pre-clinical assessment of a first-in-class small molecule inhibitor of FLIP for treatment of NSCLC

Background Evasion of cell death is a major cause of resistance to cancer therapy, making proteins that regulate cell death clinically relevant therapeutic targets. The anti-apoptotic protein FLIP is frequently overexpressed in a number of cancers, including non-small cell lung cancer (NSCLC), and has been shown by us and others to be a major mediator of drug resistance. FLIP and procaspase-8 form complexes with the adaptor protein FADD in response to a variety of clinically relevant stimuli, including ligation of death receptors, such as TRAIL-R1 and R2, and treatment with chemotherapeutic agents. In these complexes, FLIP modulates the activation of procaspase-8, and thereby apoptosis and necroptosis - two major cell death mechanisms. We recently reported that there are important differences between FLIP and procaspase-8 in terms of both their binding affinities and preferred modes of interaction with FADD that are potentially therapeutically exploitable [1]. We now report our subsequent work leading to the development and pre-clinical characterisation of first-in-class inhibitors of FLIP. Methods Molecular modelling of the FLIP-FADD complex; virtual small molecule library screening; cell-free screening assays; cell-based activity assays; biophysical binding assays; in vivo anti-tumor studies. Results Molecular modelling of the FLIP-FADD complex identified a putative drug-binding pocket on FLIP against which a virtual small-molecule screen was carried out. Subsequent biochemical screening of selected compounds using a FLIP-FADD protein-protein interaction assay identified hits with on-target activity. Medicinal chemistry optimisation of these hits afforded lead and back-up series with nanomolar activity in cell-based assays (i.e. caspase activation, cell death and cell survival), which is in line with their binding affinity in an orthogonal biophysical assay (isothermal calorimetry). The pro-apoptotic effects of these FLIP inhibitors were enhanced upon addition of death ligands, such as TRAIL, and lead-molecules have been shown to potentiate the effects of the standard-of-care chemotherapeutic cisplatin. FLIP overexpression and procaspase-8 depletion abrogated the effects of these novel inhibitors consistent with the expected mechanism-of-action. Lead molecules have been identified with ADME profiles suitable for in vivo evaluation. Using these compounds, single-agent anti-tumor effects have been demonstrated in NSCLC xenograft models. Conclusions The novel first-in-class inhibitors of FLIP developed in this study have the potential for broad application in treatment of NSCLC, either as monotherapy or in combination with other agents. Acknowledgements This work was supported by a Seeding Drug Discovery award from the Wellcome Trust (reference: 099470). 1. Majkut, J., et al., Differential affinity of FLIP and procaspase 8 for FADD9s DED binding surfaces regulates DISC assembly. Nat Commun, 2014. 5: p. 3350. Citation Format: Joanna Majkut, Catherine Higgins, Adnan Malik, Zsusannah Nemeth, Peter Blurton, Ray J. Boffey, Trevor R. Perrior, David Haigh, Timothy Harrison, Daniel B. Longley. Development and pre-clinical assessment of a first-in-class small molecule inhibitor of FLIP for treatment of NSCLC. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C4.

Abstract C5: FLIP protein-protein interaction inhibitors enhance sensitivity of colorectal cancer cells to chemotherapy and TRAIL

Background Colorectal Cancer (CRC) is the second most common cause of cancer death, with 40% of patients with this disease obtaining no benefit from current chemotherapy. Novel therapeutic strategies are needed to improve CRC patient response rates and survival. FLIP is an inhibitor of the extrinsic apoptotic pathway that binds to FADD at death-inducing signalling complexes (DISCs), such as those formed by the TNF-α-related apoptosis inducing ligand (TRAIL) receptors TRAIL-R1 and TRAIL-R2, thereby blocking homodimerization and activation of procaspase-8 and inhibiting apoptosis induction. We previously reported that FLIP blocks apoptosis induced by TRAIL and standard-of-care chemotherapeutics (5-Fluorouracil, oxaliplatin and SN38) in CRC models. Moreover, FLIP is frequently overexpressed in CRC and its overexpression correlates with poor prognosis. Subsequently, we have developed novel small molecule inhibitors that target FLIP9s critical protein-protein interactions, preventing its interaction with FADD and therefore promoting activation of caspase-8 and apoptosis induction. Methods A DISC recruitment assay was used to assess levels of FLIP at the TRAIL-R2 DISC. Caspase activity, cell viability and apoptosis induction were assessed in CRC models treated with FLIP inhibitors alone and in combination with TRAIL or standard-of-care chemotherapeutics. Mechanism-of-action was assessed using caspase-8-targeted siRNA and FLIP overexpressing models. Results Using CRC cell line models, it was demonstrated that FLIP recruitment to the DISC is inhibited by FLIP inhibitors from the lead series. As a result, TRAIL-induced caspase-8 and caspase-3/7 activity were enhanced and increased levels of apoptosis cells were observed. Decreased cell viability was observed that was proportionate to the levels of apoptosis induced. Cell death triggered by FLIP inhibitors was shown to be caspase-8-dependent consistent with the expected mechanism-of-action. Importantly, inhibitors of FLIP also enhanced apoptosis induction in response to 5-Fluorouracil, oxaliplatin and SN38. Conclusion We have developed inhibitors of FLIP that decrease its recruitment to the TRAIL-R2 DISC and increase TRAIL-induced caspase activation and apoptosis. Moreover, these inhibitors synergise with 5-Fluorouracil, oxaliplatin and SN38, suggesting that this novel class of agents has therapeutic potential in CRC when used in conjunction with standard-of-care chemotherapeutic agents. Acknowledgements This work was supported by a Seeding Drug Discovery award from the Wellcome Trust (reference: 099470). Citation Format: Jennifer P. Fox, Joanna Majkut, Catherine Higgins, Zsuzsanna Nemeth, Adnan Malik, Christopher J. Scott, Peter Blurton, Ray J. Boffey, Trevor R. Perrior, Timothy Harrison, Daniel B. Longley. FLIP protein-protein interaction inhibitors enhance sensitivity of colorectal cancer cells to chemotherapy and TRAIL. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C5.

Abstract LB-037: Development and preclinical assessment of a first-in-class small molecule inhibitor of the major cell death regulator protein FLIP

Background Evasion of cell death is a major cause of resistance to cancer therapy, making proteins that regulate cell death clinically-relevant therapeutic targets. The anti-apoptotic protein FLIP is frequently overexpressed in a number of cancers and has been shown by us and others to be a major mediator of drug resistance. FLIP and procaspase-8 form complexes with the adaptor protein FADD in response to a variety of clinically-relevant stimuli, including: ligation of death receptors, such as TRAIL-R1 and R2; and cytotoxic chemotherapeutics. In these complexes, FLIP modulates the activation of procaspase-8, and thereby apoptosis and necroptosis - two major cell death mechanisms. We recently reported that there are important differences between FLIP and procaspase-8 in terms of both their binding affinities and preferred modes of interaction with FADD that are potentially therapeutically exploitable [1]. We now report our subsequent work leading to the development and pre-clinical characterisation of first-in-class inhibitors of FLIP. Methods Molecular modelling of the FLIP-FADD complex; virtual small molecule library screening; cell-free screening assays; cell-based activity assays; biophysical binding assays; in vivo anti-tumor studies. Results Molecular modelling of the FLIP-FADD complex identified a putative drug-binding pocket on FLIP against which a virtual small-molecule screen was carried out. Subsequent biochemical screening of selected compounds using a FLIP-FADD protein-protein interaction assay identified hits with on-target activity. Medicinal chemistry optimisation of these hits afforded lead and back-up series with nanomolar activity in cell-based assays (i.e. caspase activation, cell death and cell survival), which is in line with their binding affinity in an orthogonal biophysical assay (isothermal calorimetry). Lead compounds have been shown to block recruitment of FLIP to the TRAIL-R2 death-inducing signalling complex (DISC), confirming their on-target activity. Moreover, the pro-apoptotic effects of these FLIP inhibitors were enhanced upon addition of death ligands, such as TRAIL; and lead-molecules have been shown to potentiate the effects of standard-of-care chemotherapeutics and radiotherapy. To further confirm the mechanism of action, FLIP overexpression and procaspase-8 depletion abrogated the effects of these novel inhibitors. Lead molecules have been identified with ADME profiles suitable for in vivo evaluation. Using these compounds, single-agent anti-tumor effects have been demonstrated in xenograft models Conclusions The novel, first-in-class inhibitors of FLIP developed in this study have the potential for broad application in a range of cancers, either as monotherapy or in combination with other agents. Acknowledgements This work was supported by a grant from the Wellcome Trust9s Seeding Drug Discovery Initiative (reference: 099470). Reference 1. Majkut, J., et al., Differential affinity of FLIP and procaspase 8 for FADD9s DED binding surfaces regulates DISC assembly. Nat Commun, 2014. 5: p. 3350. Citation Format: Joanna Majkut, Catherine Higgins, Adnan Malik, Zsusannah Nemeth, Peter Blurton, Ray Boffey, Trevor R. Perrior, Patrick G. Johnston, David Haigh, Timothy Harrison, Daniel B. Longley. Development and preclinical assessment of a first-in-class small molecule inhibitor of the major cell death regulator protein FLIP. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-037. doi:10.1158/1538-7445.AM2015-LB-037