Callum M. Sloss

Selective BRAFV600E Inhibition Enhances T-Cell Recognition of Melanoma without Affecting Lymphocyte Function

Targeted therapy against the BRAF/mitogen-activated protein kinase (MAPK) pathway is a promising new therapeutic approach for the treatment of melanoma. Treatment with selective BRAF inhibitors results in a high initial response rate but limited duration of response. To counter this, investigators propose combining this therapy with other targeted agents, addressing the issue of redundancy and signaling through different oncogenic pathways. An alternative approach is combining BRAF/MAPK-targeted agents with immunotherapy. Preliminary evidence suggests that oncogenic BRAF (BRAF(V600E)) contributes to immune escape and that blocking its activity via MAPK pathway inhibition leads to increased expression of melanocyte differentiation antigens (MDA). Recognition of MDAs is a critical component of the immunologic response to melanoma, and several forms of immunotherapy capitalize on this recognition. Among the various approaches to inhibiting BRAF/MAPK, broad MAPK pathway inhibition may have deleterious effects on T lymphocyte function. Here, we corroborate the role of oncogenic BRAF in immune evasion by melanoma cells through suppression of MDAs. We show that inhibition of the MAPK pathway with MAPK/extracellular signal-regulated kinase kinase (MEK) inhibitors or a specific inhibitor of BRAF(V600E) in melanoma cell lines and tumor digests results in increased levels of MDAs, which is associated with improved recognition by antigen-specific T lymphocytes. However, treatment with MEK inhibitors impairs T lymphocyte function, whereas T-cell function is preserved after treatment with a specific inhibitor of BRAF(V600E). These findings suggest that immune evasion of melanomas mediated by oncogenic BRAF may be reversed by targeted BRAF inhibition without compromising T-cell function. These findings have important implications for combined kinase-targeted therapy plus immunotherapy for melanoma.

The role of intracellular Ca2+ in the regulation of proteinase- activated receptor-2 mediated nuclear factor kappa B signalling in keratinocytes

1 In this study, we examined the role of Ca2+ in linking proteinase‐activated receptor‐2 (PAR2) to the nuclear factor kappa B (NFκB) pathway in a skin epithelial cell line NCTC2544 stably expressing PAR2 (clone G). 2 In clone G, PAR2‐mediated NFκB luciferase reporter activity and NFκB DNA‐binding activity was reduced by preincubation with BAPTA‐AM but not BAPTA. Trypsin stimulation of inhibitory kappa B kinases, IKKα and IKKβ, was also inhibited following pretreatment with BAPTA‐AM. 3 BAPTA/AM also prevented PAR2‐mediated IKKα activation in cultured primary human keratinocytes. 4 The effect of BAPTA‐AM was also selective for the IKK/NFκB signalling axis; PAR2 coupling to ERK, or p38 MAP kinase was unaffected. 5 Pharmacological inhibition of the Ca2+‐dependent regulatory protein calcineurin did not inhibit trypsin‐stimulated IKK activity or NFκB‐DNA binding; however, inhibition of Ca2+‐dependent protein kinase C isoforms or InsP3 formation using GF109203X or the phospholipase C inhibitor U73122, respectively, reduced both IKK activity and NFκB‐DNA binding. 6 Mutation of PAR2 within the C‐terminal to produce a mutant receptor, which does not couple to Ca2+ signalling, but is able to activate ERK, abrogated NFκB‐DNA binding and IKK activity stimulated by trypsin. 7 These results suggest a predominant role for the InsP3/Ca2+ axis in the regulation of IKK signalling and NFκB transcriptional activation.

MAP kinase phosphatase-2 plays a critical role in response to infection by Leishmania mexicana

In this study we generated a novel dual specific phosphatase 4 (DUSP4) deletion mouse using a targeted deletion strategy in order to examine the role of MAP kinase phosphatase-2 (MKP-2) in immune responses. Lipopolysaccharide (LPS) induced a rapid, time and concentration-dependent increase in MKP-2 protein expression in bone marrow-derived macrophages from MKP-2+/+ but not from MKP-2−/− mice. LPS-induced JNK and p38 MAP kinase phosphorylation was significantly increased and prolonged in MKP-2−/− macrophages whilst ERK phosphorylation was unaffected. MKP-2 deletion also potentiated LPS-stimulated induction of the inflammatory cytokines, IL-6, IL-12p40, TNF-α, and also COX-2 derived PGE2 production. However surprisingly, in MKP-2−/− macrophages, there was a marked reduction in LPS or IFNγ-induced iNOS and nitric oxide release and enhanced basal expression of arginase-1, suggesting that MKP-2 may have an additional regulatory function significant in pathogen-mediated immunity. Indeed, following infection with the intracellular parasite Leishmania mexicana, MKP-2−/− mice displayed increased lesion size and parasite burden, and a significantly modified Th1/Th2 bias compared with wild-type counterparts. However, there was no intrinsic defect in MKP-2−/− T cell function as measured by anti-CD3 induced IFN-γ production. Rather, MKP-2−/− bone marrow-derived macrophages were found to be inherently more susceptible to infection with Leishmania mexicana, an effect reversed following treatment with the arginase inhibitor nor-NOHA. These findings show for the first time a role for MKP-2 in vivo and demonstrate that MKP-2 may be essential in orchestrating protection against intracellular infection at the level of the macrophage.

Inactivation of JNK activity by mitogen-activated protein kinase phosphatase-2 in EAhy926 endothelial cells is dependent upon agonist-specific JNK translocation to the nucleus

We have investigated the termination of agonist-stimulated mitogen-activated protein (MAP) kinase activity in EAhy926 cells by MAP kinase phosphatase-2 (MKP-2). In cells expressing either wild-type (WT) or catalytically inactive (CI)-MKP-2, there was no significant differences in TNFalpha-stimulated JNK or p38 MAP kinase activity, however hydrogen peroxide (H2O2)-stimulated JNK activity was substantially reduced in WT-MKP-2 expressing clones and enhanced in cells expressing CI-MKP-2. Consistent with these findings, we observed substantial nuclear translocation of JNK occurred in response to H2O2 but not TNFalpha. Using a phosphospecific anti-JNK antibody, we found that TNFalpha-stimulated JNK activity was associated principally with the cytosol while in response to H2O2, JNK activity was found within the nucleus. These results show that the role of MKP-2 in terminating JNK activity is determined by the translocation of JNK to the nucleus, which is under agonist-specific regulation and not a universal cellular response to stimulation.

Enhanced Targeting of the EGFR Network with MM-151, an Oligoclonal Anti-EGFR Antibody Therapeutic

Although EGFR is a validated therapeutic target across multiple cancer indications, the often modest clinical responses to current anti-EGFR agents suggest the need for improved therapeutics. Here, we demonstrate that signal amplification driven by high-affinity EGFR ligands limits the capacity of monoclonal anti-EGFR antibodies to block pathway signaling and cell proliferation and that these ligands are commonly coexpressed with low-affinity EGFR ligands in epithelial tumors. To develop an improved antibody therapeutic capable of overcoming high-affinity ligand-mediated signal amplification, we used a network biology approach comprised of signaling studies and computational modeling of receptor–antagonist interactions. Model simulations suggested that an oligoclonal antibody combination may overcome signal amplification within the EGFR:ERK pathway driven by all EGFR ligands. Based on this, we designed MM-151, a combination of three fully human IgG1 monoclonal antibodies that can simultaneously engage distinct, nonoverlapping epitopes on EGFR with subnanomolar affinities. In signaling studies, MM-151 antagonized high-affinity EGFR ligands more effectively than cetuximab, leading to an approximately 65-fold greater decrease in signal amplification to ERK. In cell viability studies, MM-151 demonstrated antiproliferative activity against high-affinity EGFR ligands, either singly or in combination, while cetuximab activity was largely abrogated under these conditions. We confirmed this finding both in vitro and in vivo in a cell line model of autocrine high-affinity ligand expression. Together, these preclinical studies provide rationale for the clinical study of MM-151 and suggest that high-affinity EGFR ligand expression may be a predictive response marker that distinguishes MM-151 from other anti-EGFR therapeutics. Mol Cancer Ther; 14(7); 1625–36. ©2015 AACR.

G-protein-dependent and -independent pathways regulate proteinase-activated receptor-2 mediated p65 nf kappa b serine 536 phosphorylation in human keratinocytes

The mechanisms underpinning the coupling of GPCRs, such as PAR-2, to the phosphorylation of p65 NFkappaB have not been investigated. In the current study we found that trypsin and the selective PAR-2 activating peptide, 2f-LIGKV-OH, stimulated large and sustained increases in the serine 536 phosphorylation of p65/RelA in a transfected skin epithelial cell line and primary keratinocytes. Parallel experiments showed that in both cell types, p65 NFkappaB phosphorylation is mediated through the selective activation of IKK2. Treatment with PKC inhibitor GF109203X or PKCalpha siRNA reduced phosphorylation at 15 min but not 30 min, whilst rottlerin, a selective PKCdelta inhibitor and PKCdelta siRNA reduced the response at both time points. Pre-treatment of cells with the novel Gq/11 inhibitor YM-254890 and Gq/11 siRNA caused a similar pattern of inhibition and also reduced PAR-2-mediated NFkappaB transcriptional activity. Furthermore, stimulation of cells through a novel PAR-2 mutant PAR-2(34-43), delayed p65 phosphorylation but was without effect on the kinetics of ERK activation. Inhibition of Gi or G12/13 pathways by pertussis toxin pre-treatment or over-expression of the RGS mutant Lsc, also did not effect NFkappaB phosphorylation. Taken together these data indicate dependency for Gq/11 in early phosphorylation of p65 NFkappaB and this subsequently affects initial NFkappaB-dependent gene transcriptional activity, however later regulation of p65 is unaffected. Overall these novel data demonstrate an IKK2-dependent, predominantly G-protein-independent pathway involved in PAR-2 regulation of NFkappaB phosphorylation in keratinocytes.

Differential regulation of MAP kinase activation by a novel splice variant of human MAP kinase phosphatase-2

MAP kinase phosphatase-2 (MKP-2) is a member of the family of dual specificity phosphatases that functions to inactivate the ERK and JNK MAP kinase signalling pathways. Here, we identify a novel human MKP-2 variant (MKP-2-S) lacking the MAP kinase binding site but retaining the phosphatase catalytic domain. Endogenous MKP-2-S transcripts and proteins were found in PC3 prostate and MDA-MB-231 breast cancer cells and also human prostate biopsies. Cellular transfection of MKP-2-S gave rise to a nuclear protein of 33kDa which displayed phosphatase activity comparable to the formerly described long form of MKP-2 (MKP-2-L). Due to its lack of a kinase interacting motif (KIM), MKP-2-S did not bind to JNK or ERK; MKP-2-L bound ERK and to a lesser extent JNK. Protein turnover of adenoviral expressed MKP-2-S was accelerated relative to MKP-2-L, with a greater susceptibility to proteosomal-mediated degradation. MKP-2-S retained its ability to deactivate JNK in a similar manner as MKP-2-L and was an effective inhibitor of LPS-stimulated COX-2 induction. However, unlike MKP-2-L, MKP-2-S was unable to reverse serum-induced ERK activation or significantly inhibit endothelial cell proliferation. These findings reveal the occurrence of a novel splice variant of MKP-2 which is unable to bind ERK and may be significant in the dysregulation of MAP kinase activity in certain disease states, particularly in breast and prostate cancers.

Abstract A210: Mechanism of action of MM-151, a potent mixture of three human antibody antagonists targeting EGFR.

Epidermal growth factor receptor (EGFR) is a key regulator of cancer cell proliferation, apoptosis, invasion, and metastasis. However, the clinical benefits of EGFR targeted agents such as erlotinib and cetuximab have been modest. Using Merrimack9s Network Biology platform, a computational systems analysis was performed to identify how best to inhibit signaling through the EGFR-ERK network. These simulations predicted that enhanced inhibition is required to overcome the robust signal amplification inherent to this network and is best achieved through treatment with a combination of at least two noncompetitive ligand antagonists. The MM-151 therapeutic was developed to achieve the simulated design criteria and consists of a mixture of three fully human monoclonal antibodies directed against distinct non-overlapping epitopes in EGFR. Detailed in vitro and on-cell binding experiments, supported by computational simulations, with single component antibodies and their monovalent Fab variants, display a rich complexity of antibody affinity and avidity. All three antibodies are shown to have subnanomolar monovalent affinity to EGFR but to vary in the degree to which they functionally crosslink receptor (two antibodies display high avidity while the third has weak avidity). Ligand competition experiments with saturating antibody concentrations (EC90 of on-cell binding) demonstrate that two of the antibodies are each complete ligand antagonists while the third antibody is a partial (approx. 25%) antagonist. When combined into the MM-151 mixture, these antibodies simultaneously engage and robustly crosslink receptor to form a highly antagonistic therapeutic. Treatment of human tumor-derived cell lines with MM-151 elicits complete inhibition of ligand-mediated ERK signaling over a wide range of both EGF receptor density (as high as 2×10∘6 receptors per cell in the A431 cell line) and ligand burden (8 and 80nM EGF). In contrast, monoclonal EGFR targeted therapeutics, such as cetuximab and panitumumab, as well as oligoclonal inhibitors similar to MM-151, show weaker or even no effect on ERK signaling under similar conditions. Furthermore, the potency of monovalent MM-151 (Fab variants) is diminished in EGF mediated EGFR and ERK signaling, highlighting the importance of receptor crosslinking through antibody avidity as a determinant of efficacy. Together, these data demonstrate that MM-151 is a potent inhibitor of the EGFR-ERK signaling axis with the potential of improved efficacy over current EGFR targeted therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A210.

Abstract A144: Therapeutically targeting high-affinity ligand activation of EGFR with MM-151, an oligoclonal therapeutic.

The Epidermal Growth Factor Receptor (EGFR/ErbB1) is a receptor tyrosine kinase whose activation has been shown to play a key role in tumor growth and development. The EGFR ligand family is comprised of seven transmembrane precursor proteins whose expression and processing is highly regulated. These ligands can be classified based upon their affinity to EGFR; epidermal growth factor (EGF), betacellulin (BTC), heparin-binding epidermal growth factor (HB-EGF), and transforming growth factor alpha (TGFα) are considered high-affinity ligands, whereas amphiregulin (AREG), epiregulin (EREG), and epigen (EPI) are considered low-affinity ligands. EGFR overexpression in tumors is associated with higher risk of recurrence, metastasis, poorer survival and resistance to chemotherapy. Therefore, this pathway is a compelling target for the development of anti-EGFR therapeutics. Using Merrimack9s Network Biology approach, we performed a computational systems analysis to identify an optimal strategy to inhibit the EGFR-ERK signaling network, which is characterized by robust signal amplification from the receptor to downstream effectors. As a result, we developed MM-151, an oligoclonal therapeutic composed of three fully human monoclonal antibodies targeted to distinct EGFR epitopes. MM-151 inhibits EGFR pathway activation by the dual mechanism of EGFR ligand-blocking and enhancement of receptor downregulation. Our preclinical in vitro studies revealed greater potency for MM-151 versus existing monoclonal antibodies (e.g. cetuximab, panitumumab and nimotuzumab). Importantly, MM-151 was shown to inhibit in vitro ERK signaling and cell proliferation induced by both high- and low-affinity EGFR ligands, unlike existing monoclonal therapeutics, which only block low-affinity ligand-induced signaling and cell proliferation. In in vitro cell proliferation assays, several cell line models that were responsive to anti-EGFR monoclonals in the presence of the low-affinity ligand AREG became increasingly unresponsive to treatment upon titrating in increasing amounts of the high-affinity ligand EGF. Conversely, cells remained responsive to MM-151 even in the presence of high-affinity EGF ligand burden. In preclinical non-small cell lung cancer (NSCLC) and head and neck squamous cell carcinoma (HNSCC) EGFR-wildtype cell line models, overexpression of autocrine AREG is positively correlated with increased sensitivity to the anti-EGFR monoclonal cetuximab or tyrosine kinase inhibitor gefitinib (K. Yonesaka et al. , Clin. Cancer Res., 2008, 14: 6963–6973). In the clinic, metastatic colon cancer patients with wild type K-ras tumors highly expressing the low-affinity ligands AREG and EREG are more likely to exhibit disease control on cetuximab treatment (J. B. Baker et al. , Br. J. Cancer, 2011, 104: 488–495). Our preclinical data suggest that elevated high-affinity ligand expression would likely correlate with decreased patient response to anti-EGFR monoclonals, and that patients whose tumors are driven by high-affinity EGFR ligands might instead benefit from MM-151 treatment. Together, these data suggest that MM-151, capable of blocking both high and low affinity ligand-driven EGFR signaling, may have the potential to more broadly benefit lung and colon cancer patients as compared to existing EGFR-directed therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A144.

Abstract 2129: HB-EGF expression in gastric cancer promotes chemotherapy resistance and represents a promising target for molecular therapy and a novel biomarker of 5-FU resistance

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL The incidence of gastric cancer (GC) has been on the decline in the U.S. over the last 40 years, but remains high in Asia and Latin America. The primary treatment for early-stage GC is surgery. Many patients will also benefit from adjuvant 5-fluorouracil (5-FU) or platin-based chemotherapy. When it presents at an advanced stage, GC uniformly carries a poor prognosis, due to chemotherapy resistance. To improve the response of GC to chemotherapy, molecular-targeted therapies are needed. One of the EGFR family ligands called heparin-binding epidermal growth factor (HB-EGF) was recently found to be up-regulated in several epithelial-based malignancies, including GC. Our objective in this study was to evaluate the role of HB-EGF in GC chemotherapy resistance. We hypothesize that constitutive expression of HB-EGF is associated with chemotherapy resistance. Furthermore we propose that inhibition of HB-EGF enhances drug sensitivity and augments the apoptotic response to chemotherapy in GC. To test our hypothesis, we isolated RNA and protein from a panel of 6 GC cell lines and measured the basal HB-EGF expression compared to that of normal gastric epithelia. We similarly measured HB-EGF expression in response to 5-FU or cisplatin treatment, and then determined the response when cells were co-treated with HB-EGF or an inhibitor of HB-EGF (CRM197). We found that HB-EGF expression at both the protein and RNA levels was highly variable across the panel of GC cell lines. In addition, HB-EGF expression increased in response to drug treatment at 24 and 48 hours post-treatment. A positive correlation was observed between HB-EGF mRNA levels and chemoresistance to 5-FU. GC cell lines that are high-expressers of HB-EGF showed decreased cell growth when treated with CRM197, whereas low-expressers increased when treated with recombinant HB-EGF. The combination of CRM197 with anticancer agents increased apoptosis in high-expressers, but made no significant difference in low-expressers. Likewise, HB-EGF gene silencing using a lentiviral short-hairpin RNA significantly decreased high-expressers survival and proliferation. Similar to our findings using GC cell lines, we found that human gastric cancer tumor samples showed highly variable HB-EGF expression compared to their matching normal gastric epithelia. Our findings suggest that HB-EGF plays an important role in the proliferation and chemotherapy resistance of a subset of gastric cancers. HB-EGF may represent a promising molecular target in those select patients, in whom the gastric cancer expresses high levels of HB-EGF. Furthermore, HB-EGF expression may represent a novel biomarker for 5-FU chemoresistance in patients with gastric cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2129. doi:10.1158/1538-7445.AM2011-2129