Mer Receptor Tyrosine Kinase Signaling: PREVENTION OF APOPTOSIS AND ALTERATION OF CYTOSKELETAL ARCHITECTURE WITHOUT STIMULATION OR PROLIFERATION

Mer is a member of the Axl/Mer/Tyro3 receptor tyrosine kinase family, a family whose physiological function is not well defined. We constructed a Mer chimera using the epidermal growth factor receptor (EGFR) extracellular and transmembrane domains and the Mer cytoplasmic domain. Stable transfection of the Mer chimera into interleukin 3 (IL-3)-dependent murine 32D cells resulted in ligand-activable surface receptor that tyrosine autophosphorylated, stimulated intracellular signaling, and dramatically reduced apoptosis initiated by IL-3 withdrawal. However, unlike multiple other ectopically expressed receptor tyrosine kinases including full-length EGFR or an EGFR/Axl chimera, the Mer chimera did not stimulate proliferation. Moreover, and in contrast to EGFR, Mer chimera activation induced adherence and cell flattening in the normally suspension-growing 32D cells. The Mer chimera signal also blocked IL-3-dependent proliferation leading to G(1)/S arrest, dephosphorylation of retinoblastoma protein, and elongation of cellular processes. Unlike other agonists that lead to a slow (4-8 days) ligand-dependent differentiation of 32D cells, the combined Mer and IL-3 signal resulted in differentiated morphology and growth cessation in the first 24 h. Thus the Mer chimera blocks apoptosis without stimulating growth and produces cytoskeletal alterations; this outcome is clearly separable from the proliferative signal produced by most receptor tyrosine kinases

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Interplay of IKK/NF-kappaB signaling in macrophages and myofibers promotes muscle degeneration in Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a lethal X-linked disorder associated with dystrophin deficiency that results in chronic inflammation and severe skeletal muscle degeneration. In DMD mouse models and patients, we find that IkappaB kinase/NF-kappaB (IKK/NF-kappaB) signaling is persistently elevated in immune cells and regenerative muscle fibers. Ablation of 1 allele of the p65 subunit of NF-kappaB was sufficient to improve pathology in mdx mice, a model of DMD. In addition, conditional deletion of IKKbeta in mdx mice elucidated that NF-kappaB functions in activated macrophages to promote inflammation and muscle necrosis and in skeletal muscle fibers to limit regeneration through the inhibition of muscle progenitor cells. Furthermore, specific pharmacological inhibition of IKK resulted in improved pathology and muscle function in mdx mice. Collectively, these results underscore the critical role of NF-kappaB in the progression of muscular dystrophy and suggest the IKK/NF-kappaB signaling pathway as a potential therapeutic target for DMD

Interplay of IKK/NF-κB signaling in macrophages and myofibers promotes muscle degeneration in Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a lethal X-linked disorder associated with dystrophin deficiency that results in chronic inflammation and severe skeletal muscle degeneration. In DMD mouse models and patients, we find that IκB kinase/NF-κB (IKK/NF-κB) signaling is persistently elevated in immune cells and regenerative muscle fibers. Ablation of 1 allele of the p65 subunit of NF-κB was sufficient to improve pathology in mdx mice, a model of DMD. In addition, conditional deletion of IKKβ in mdx mice elucidated that NF-κB functions in activated macrophages to promote inflammation and muscle necrosis and in skeletal muscle fibers to limit regeneration through the inhibition of muscle progenitor cells. Furthermore, specific pharmacological inhibition of IKK resulted in improved pathology and muscle function in mdx mice. Collectively, these results underscore the critical role of NF-κB in the progression of muscular dystrophy and suggest the IKK/NF-κB signaling pathway as a potential therapeutic target for DMD