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

<i>ZNF367</i> Inhibits Cancer Progression and Is Targeted by miR-195

<div><p>Background</p><p>Several members of the zinc finger protein family have been recently shown to have a role in cancer initiation and progression. Zinc finger protein 367 (<i>ZNF367</i>) is a member of the zinc finger protein family and is expressed in embryonic or fetal erythroid tissue but is absent in normal adult tissue.</p><p>Methodology/Principal Findings</p><p>We show that <i>ZNF367</i> is overexpressed in adrenocortical carcinoma, malignant pheochromocytoma/paraganglioma and thyroid cancer as compared to normal tissue and benign tumors. Using both functional knockdown and ectopic overexpression in multiple cell lines, we show that <i>ZNF367</i> inhibits cellular proliferation, invasion, migration, and adhesion to extracellular proteins <i>in vitro</i> and <i>in vivo</i>. Integrated gene and microRNA expression analyses showed an inverse correlation between <i>ZNF367</i> and miR-195 expression. Luciferase assays demonstrated that miR-195 directly regulates <i>ZNF367</i> expression and that miR-195 regulates cellular invasion. Moreover, integrin alpha 3 (<i>ITGA3</i>) expression was regulated by <i>ZNF367</i>.</p><p>Conclusions/Significance</p><p>Our findings taken together suggest that <i>ZNF367</i> regulates cancer progression.</p></div

<i>ZNF367</i> regulates <i>ITGA3</i> expression.

<p>(<b>A</b>) <i>ZNF367</i> knockdown upregulates <i>ITGA3</i> expression in SW13 cells. Error bars represent ± SEM. (<b>B</b>) The correlation between <i>ITGA3</i> and <i>ZNF367</i> mRNA expression in adrenocortical tumor samples. X and Y axes represent log 2–transformed values. (<b>C</b>) Western blot quantification of ITGA3 protein expression with <i>ZNF367</i> knockdown. (<b>D</b>–<b>E</b>) ITGA3 expression with ZNF367 overexpression. Error bars represent ± SEM.</p

<i>ZNF367</i> mRNA and protein levels in adrenocortical carcinoma, papillary thyroid cancer, and pheochromocytoma/paraganglioma compared to benign and normal tissue samples for each cancer type.

<p>(A and B) Expression level in the normal adrenal cortex, benign adrenocortical adenomas, and adrenocortical carcinomas; (C and D) normal adrenal medulla, benign and malignant pheochromocytoma/paraganglioma tissue samples; and (E and F) normal thyroid and papillary thyroid cancer tissue samples. The Y axis on each graph represents the percentage of mRNA expression using the 2^∧-ΔCt*100% method ± SEM. *p<0.05, **p<0.001, ***p<0.001 (Kruskal-Wallis test). Representative immunohistochemistry images are from normal, benign, and malignant tumor samples at 20X magnification.</p

The effect of <i>ZNF367</i> knockdown on growth in SW13 cells in vitro and in vivo.

<p>(A) <i>ZNF367</i> knockdown increases cellular proliferation. The Y axis represents relative fluorescent units (RFU), and the X axis indicates days post-transfection. *p<0.05 relative to the negative control. Error bars represent ± SD. (B) <i>ZNF367</i> knockdown enhances tumor growth in vivo. SW13 cells were transfected with the negative control (n = 4) and siRNA (n = 4) into the right and left flank of each mouse. After 48 hours of transfection, 3×10⁶ cells were injected in athymic nude mice, and tumor growth was measured weekly. The Y axis represents the tumor volume and X axis the weeks of tumor measurement after flank injection. *p<0.05 and error bars represent ± SD.</p

MiR-195 targets ZNF367.

<p>(<b>A</b>) The correlation between miR-195 and <i>ZNF367</i> expression in an adrenocortical tumor. The Pearson correlation coefficient is indicated by r with its p value. (<b>B</b>) Ectopic overexpression of pre-miR-195 results in downregulation of <i>ZNF367</i>. <i>ZNF367</i> mRNA expression in SW13 cells transfected with pre-miR-195 and the pre-negative control at 5 nM for 24 hours (presented as a fold-change relative to the pre-negative control). Error bars represent ± SEM. The right panel shows the Western blot of the ZNF367 protein from SW13 cells transfected with pre-miR-195 and the negative control. (<b>C</b>) Pre-miR-195 overexpression increases invasion in SW13 cells compared to the negative control. The right panel shows the mean number of invaded cells on the Y axis. (<b>D</b>) <i>ITGA3</i> mRNA expression is increased after transfection of miR-195 and the negative control in SW13 cells. Error bars represent ± SEM. (<b>E</b>) The binding site of miR-195 in the <i>ZNF367</i> 3′UTR, along with the mutant construct in the predicted seed region. In the right panel, the luciferase assay demonstrates decreased luminescence in SW13 cells co-transfected with miR-195 and the negative control at 5 nM, with the empty vector, wild-type <i>ZNF367</i> 3′UTR, or MUT-<i>ZNF367</i> 3′UTR vector (mutated in the first three nucleotides of the seed sequence). The luminescence was read after 24 hours of transfection. The Y axis represents the ratio of <i>ZNF367</i> 3′UTR to the empty vector. *p value < 0.05 compared to the negative control. Error bars represent ± SEM.</p