David Foulad

Effects of MAPK and PI3K Pathways on PD-L1 Expression in Melanoma

Purpose: PD-L1 is the main ligand for the immune inhibitory receptor PD-1. This ligand is frequently expressed by melanoma cells. In this study, we investigated whether PD-L1 expression is controlled by melanoma driver mutations and modified by oncogenic signaling inhibition. Experimental Design: Expression of PD-L1 was investigated in a panel of 51 melanoma cell lines containing different oncogenic mutations, including cell lines with innate and acquired resistance to BRAF inhibitors (BRAFi). The effects of targeted therapy drugs on expression of PD-L1 by melanoma cells were investigated. Results: No association was found between the level of PD-L1 expression and mutations in BRAF, NRAS, PTEN, or amplification of AKT. Resistance to vemurafenib due to the activation of alternative signaling pathways was accompanied with the induction of PD-L1 expression, whereas the resistance due to the reactivation of the MAPK pathway had no effect on PD-L1 expression. In melanoma cell lines, the effects of BRAF, MEK, and PI3K inhibitors on expression of PD-L1 were variable from reduction to induction, particularly in the presence of INFγ. In PD-L1–exposed lymphocytes, vemurafenib paradoxically restored activity of the MAPK pathway and increased the secretion of cytokines. Conclusions: In melanoma cell lines, including BRAFi-resistant cells, PD-L1 expression is variably regulated by oncogenic signaling pathways. PD-L1–exposed lymphocytes decrease MAPK signaling, which is corrected by exposure to vemurafenib, providing potential benefits of combining this drug with immunotherapies. Clin Cancer Res; 20(13); 3446–57. ©2014 AACR.

Release of HMGB1 in Response to Proapoptotic Glioma Killing Strategies: Efficacy and Neurotoxicity

Purpose: In preparation for a phase I clinical trial using a combined cytotoxic/immunotherapeutic strategy with adenoviruses (Ad) expressing Flt3L (Ad-Flt3L) and thymidine kinase (Ad-TK) to treat glioblastoma (GBM), we tested the hypothesis that Ad-TK+GCV would be the optimal tumor-killing agent in relation to efficacy and safety when compared with other proapoptotic approaches. Experimental Design: The efficacy and neurotoxicity of Ad-TK+GCV was compared with Ads encoding the proapoptotic cytokines [tumor necrosis factor-α, tumor necrosis factor–related apoptosis-inducing factor (TRAIL), and Fas ligand (FasL)], alone or in combination with Ad-Flt3L. In rats bearing small GBMs (day 4), only Ad-TK+GCV or Ad-FasL improved survival. Results: In rats bearing large GBMs (day 9), the combination of Ad-Flt3L with Ad-FasL did not improve survival over FasL alone, whereas Ad-Flt3L combined with Ad-TK+GCV led to 70% long-term survival. Expression of FasL and TRAIL caused severe neuropathology, which was not encountered when we used Ad-TK+/−Ad-Flt3L. In vitro, all treatments elicited release of high mobility group box 1 protein (HMGB1) from dying tumor cells. In vivo, the highest levels of circulating HMGB1 were observed after treatment with Ad-TK+GCV+Ad-Flt3L; HMGB1 was necessary for the therapeutic efficacy of AdTK+GCV+Ad-Flt3L because its blockade with glycyrrhizin completely blocked tumor regression. We also showed the killing efficacy of Ad-TK+GCV in human GBM cell lines and GBM primary cultures, which also elicited release of HMGB1. Conclusions: Our results indicate that Ad-TK+GCV+Ad-Flt3L exhibit the highest efficacy and safety profile among the several proapoptotic approaches tested. The results reported further support the implementation of this combined approach in a phase I clinical trial for GBM.

Gene therapy-mediated delivery of targeted cytotoxins for glioma therapeutics

Restricting the cytotoxicity of anticancer agents by targeting receptors exclusively expressed on tumor cells is critical when treating infiltrative brain tumors such as glioblastoma multiforme (GBM). GBMs express an IL-13 receptor (IL13Rα2) that differs from the physiological IL4R/IL13R receptor. We developed a regulatable adenoviral vector (Ad.mhIL-4.TRE.mhIL-13-PE) encoding a mutated human IL-13 fused to Pseudomonas exotoxin (mhIL-13-PE) that specifically binds to IL13Rα2 to provide sustained expression, effective anti-GBM cytotoxicity, and minimal neurotoxicity. The therapeutic Ad also encodes mutated human IL-4 that binds to the physiological IL4R/IL13R without interacting with IL13Rα2, thus inhibiting potential binding of mhIL-13-PE to normal brain cells. Using intracranial GBM xenografts and syngeneic mouse models, we tested the Ad.mhIL-4.TRE.mhIL-13-PE and two protein formulations, hIL-13-PE used in clinical trials (Cintredekin Besudotox) and a second-generation mhIL-13-PE. Cintredekin Besudotox doubled median survival without eliciting long-term survival and caused severe neurotoxicity; mhIL-13-PE led to ∼40% long-term survival, eliciting severe neurological toxicity at the high dose tested. In contrast, Ad-mediated delivery of mhIL-13-PE led to tumor regression and long-term survival in over 70% of the animals, without causing apparent neurotoxicity. Although Cintredekin Besudotox was originally developed to target GBM, when tested in a phase III trial it failed to achieve clinical endpoints and revealed neurotoxicity. Limitations of Cintredekin Besudotox include its short half-life, which demanded frequent or continued administration, and binding to IL4R/IL13R, present in normal brain cells. These shortcomings were overcome by our therapeutic Ad, thus representing a significant advance in the development of targeted therapeutics for GBM.

Antiglioma Immunological Memory in Response to Conditional Cytotoxic/Immune-Stimulatory Gene Therapy: Humoral and Cellular Immunity Lead to Tumor Regression

Purpose: Glioblastoma multiforme is a deadly primary brain cancer. Because the tumor kills due to recurrences, we tested the hypothesis that a new treatment would lead to immunological memory in a rat model of recurrent glioblastoma multiforme. Experimental Design: We developed a combined treatment using an adenovirus (Ad) expressing fms-like tyrosine kinase-3 ligand (Flt3L), which induces the infiltration of immune cells into the tumor microenvironment, and an Ad expressing herpes simplex virus-1–thymidine kinase (TK), which kills proliferating tumor cells in the presence of ganciclovir. Results: This treatment induced immunological memory that led to rejection of a second glioblastoma multiforme implanted in the contralateral hemisphere and of an extracranial glioblastoma multiforme implanted intradermally. Rechallenged long-term survivors exhibited anti-glioblastoma multiforme–specific T cells and displayed specific delayed-type hypersensitivity. Using depleting antibodies, we showed that rejection of the second tumor was dependent on CD8+ T cells. Circulating anti‐glioma antibodies were observed when glioblastoma multiforme cells were implanted intradermally in naïve rats or in long-term survivors. However, rats bearing intracranial glioblastoma multiforme only exhibited circulating antitumoral antibodies upon treatment with Ad-Flt3L + Ad-TK. This combined treatment induced tumor regression and release of the chromatin-binding protein high mobility group box 1 in two further intracranial glioblastoma multiforme models, that is, Fisher rats bearing intracranial 9L and F98 glioblastoma multiforme cells. Conclusions: Treatment with Ad-Flt3L + Ad-TK triggered systemic anti–glioblastoma multiforme cellular and humoral immune responses, and anti–glioblastoma multiforme immunological memory. Release of the chromatin-binding protein high mobility group box 1 could be used as a noninvasive biomarker of therapeutic efficacy for glioblastoma multiforme. The robust treatment efficacy lends further support to its implementation in a phase I clinical trial. (Clin Cancer Res 2009;15(19):6113–27)

Study of the Efficacy, Biodistribution, and Safety Profile of Therapeutic Gutless Adenovirus Vectors as a Prelude to a Phase I Clinical Trial for Glioblastoma

Glioblastoma multiforme (GBM) is the most common and most aggressive primary brain tumor in humans. Systemic immunity against gene therapy vectors has been shown to hamper therapeutic efficacy; however, helper‐dependent high‐capacity adenovirus (HC‐Ad) vectors elicit sustained transgene expression, even in the presence of systemic anti‐adenoviral immunity. We engineered HC‐Ads encoding the conditional cytotoxic herpes simplex type 1 thymidine kinase (TK) and the immunostimulatory cytokine fms‐like tyrosine kinase ligand 3 (Flt3L). Flt3L expression is under the control of the regulatable Tet‐ON system. In anticipation of a phase I clinical trial for GBM, we assessed the therapeutic efficacy, biodistribution, and clinical and neurotoxicity with escalating doses of HC‐Ad‐TetOn‐Flt3L + HC‐Ad‐TK in rats. Intratumoral administration of these therapeutic HC‐Ads in rats bearing large intracranial GBMs led to long‐term survival in _70% of the animals and development of antiglioma immunological memory without signs of neuropathology or systemic toxicity. Systemic anti‐adenoviral immunity did not affect therapeutic efficacy. These data support the idea that it would be useful to develop HC‐Ad vectors further as a therapeutic gene‐delivery platform to implement GBM phase I clinical trials.

Combination of pan-RAF and MEK inhibitors in NRAS mutant melanoma

BackgroundApproximately 20% of melanomas contain a mutation in NRAS. However no direct inhibitor of NRAS is available. One of the main signaling pathways downstream of NRAS is the MAPK pathway. In this study we investigated the possibility of blocking oncogenic signaling of NRAS by inhibiting two signaling points in the MAPK pathway.MethodsFourteen NRAS mutated human melanoma cell lines were treated with a pan-RAF inhibitor (PRi, Amgen Compd A), a MEK inhibitor (MEKi, trametinib) or their combination and the effects on proliferation, cell cycle progression, apoptosis, transcription profile and signaling of the cells were investigated.ResultsThe majority of the cell lines showed a significant growth inhibition, with high levels of synergism of the PRi and MEKi combination. Sensitive cell lines showed induction of apoptosis by the combination treatment and there was a correlation between p-MEK levels and synergistic effect of the combination treatment. Proliferation of sensitive cell lines was blocked by the inhibition of the MAPK pathway, which also blocked expression of cyclin D1. However, in resistant cell lines, proliferation was blocked by combined inhibition of the MAPK pathway and cyclin D3, which is not regulated by the MAPK pathway. Resistant cell lines also showed higher levels of p-GSK3β and less perturbation of the apoptotic profile upon the treatment in comparison with the sensitive cell lines.ConclusionsThe combination of PRi + MEKi can be an effective regimen for blocking proliferation of NRAS mutant melanomas when there is higher activity of the MAPK pathway and dependence of proliferation and survival on this pathway.

Targeted Toxins for Glioblastoma Multiforme: pre-clinical studies and clinical implementation

Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults. GBM is very aggressive due to its poor cellular differentiation and invasiveness, which makes complete surgical resection virtually impossible. Therefore, GBMs invasive nature as well as its intrinsic resistance to current treatment modalities makes it a unique therapeutic challenge. Extensive examination of human GBM specimens has uncovered that these tumors overexpress a variety of receptors that are virtually absent in the surrounding non-neoplastic brain. Human GBMs overexpress receptors for cytokines, growth factors, ephrins, urokinase-type plasminogen activator (uPA), and transferrin, which can be targeted with high specificity by linking their ligands with highly cytotoxic molecules, such as Diptheria toxin and Pseudomonas exotoxin A. We review the preclinical development and clinical translation of targeted toxins for GBM. In view of the clinical experience, we conclude that although these are very promising therapeutic modalities for GBM patients, efforts should be focused on improving the delivery systems utilized in order to achieve better distribution of the immuno-toxins in the tumor/resection cavity. Delivery of targeted toxins using viral vectors would also benefit enormously from improved strategies for local delivery.

Erratum to: Antitumor activity of the ERK inhibitor SCH722984 against BRAF mutant, NRAS mutant and wild-type melanoma

Deborah JL Wong and Lidia Robert contributed equally to this work. The online version of the original article can be found under doi:10.1186/1476-4598-13-194.

Abstract 915: Melanomas with rare BRAF mutations and their responses to MAPK pathway blocking drugs.

The BRAF V600E driver mutation is found in more than 50% of melanomas. Less common BRAF mutations of V600K, V600R, L597S, and G466K have also been identified. In these rare BRAF mutants, the effects of vemurafenib (Vem) and other MAPK pathway inhibitors on signaling, growth rate and clinical course are not well understood. In cell lines with rare BRAF mutations, we investigated the effect of Vem, inhibitors of Pan-RAF (Amgen 2112819), MEK (trametinib), ERK (MERK SCH772984), Pan-RAFi+ MEKi and ERKi+Vem on growth and signaling. These results were compared to the clinical responses to BRAF and MEK inhibitors in patients with the original tumors. Regardless of the mutation, no paradoxical activation of MAPK pathway by Vem was observed and the Pan-Rafi decreased levels of pMEK in all the cell lines. A heterozygous V600K mutated cell line was sensitive to Vem and more sensitive to the other drugs. Clinically, the duration of response to Vem in the patient the cell line was derived from was 7 months, and an additional 3 months to the combination of Vem and MEKi upon progression on Vem. In contrast, a homozygous V600K mutated cell line was resistant to Vem but sensitive to other MAPK inhibitors. The patient the cell line was derived from had a 5 month response to Vem followed by a 2 month response to the addition of a MEKi. BRAF L597S cell line was only sensitive to ERKi and G466K and V600R mutated cell lines were resistant to all the drugs. In general, Vem was the least effective followed by Pan-RAFi with ERKi stronger than both in most cell lines. The combination of Vem and ERKi showed similar or slightly higher growth inhibition than ERKi alone. In conclusion, MAPK inhibitors are not as effective in melanoma cell lines with rare BRAF mutations. However, more growth inhibition can be achieved by MEKi-Pan-RAFi combination. These in vitro data seem to be correlated with the clinical outcomes. *Charles Ng, equal contribution Citation Format: Mohammad S. Atefi, Charles P. Ng, Deborah J. Wong, Lidia Robert, Helena Escuin-Ordinas, Amanda Lassen, David Foulad, Begonya Comin-Anduix, Ahmad A. Samatar, Antoni Ribas. Melanomas with rare BRAF mutations and their responses to MAPK pathway blocking drugs. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 915. doi:10.1158/1538-7445.AM2013-915

Abstract 917: High antitumor activity of the ERK inhibitor SCH722984 against BRAF-mutant, NRAS-mutant and wild-type melanoma cell lines.

MAPK pathway dysregulation via mutations in proteins such as BRAFV600E and NRASQ61 is a key feature in melanomas, leading to constitutive ERK signaling. Currently, there are no FDA-approved targeted therapies effective in vemurafenib-resistant BRAFV600E mutant, NRAS mutant, or wild-type melanoma. The effect of SCH722984, an allosteric inhibitor of ERK 1/2, was determined in a panel of 53 melanoma cell lines, including wild-type, NRAS-mutant and vemurafenib-sensitive and resistant BRAF-mutant melanoma. 54% (13 of 24) of BRAF mutants, including 4 with innate vemurafenib resistance, were sensitive to SCH722984 with an IC50 Citation Format: Deborah J. Wong, Lidia Robert, Mohammad S. Atefi, David Foulad, Earl V. Avramis, Begonya Comin-Anduix, Ahmed A. Samatar, Antoni Ribas. High antitumor activity of the ERK inhibitor SCH722984 against BRAF-mutant, NRAS-mutant and wild-type melanoma cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 917. doi:10.1158/1538-7445.AM2013-917