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Modulating the activity of tumor-infiltrating dendritic cells (TiDC) provides opportunities for novel cancer interventions. In this article, we report on our study of the uptake of mRNA by CD8α+ cross-presenting TiDCs upon its intratumoral (i.t.) delivery. We exploited this property to deliver mRNA encoding the costimulatory molecule CD70, the activation stimuli CD40 ligand, and constitutively active Toll-like receptor 4, referred to as TriMix mRNA. We show that TiDCs are reprogrammed to mature antigen-presenting cells that migrate to tumor-draining lymph nodes (TDLN). TriMix stimulated antitumor T-cell responses to spontaneously engulfed cancer antigens, including a neoepitope. We show in various mouse cancer models that i.t. delivery of TriMix mRNA results in systemic therapeutic antitumor immunity. Finally, we show that the induction of antitumor responses critically depends on TiDCs, whereas it only partially depends on TDLNs. As such, we provide a platform and a mechanistic rationale for the clinical testing of i.t. administration of TriMix mRNA. Cancer Immunol Res; 4(2); 146–56. ©2015 AACR.

Supplementary Fig. S1. Delivery of TriMix mRNA to the tumor reprograms TiDCs. Supplementary Fig. S2. Delivery of TriMix mRNA to tumors induces tumor-specific CTLs.

Supplemental Figure 1 shows the prognostic value of the classical RASSF proteins RASSF1, RASSF3, RASSF5 and RASSF6 in MM patients. Supplemental Figure S2 shows the cellular localization of RASSF4. Supplemental Figure S3 demonstrates the inducible doxycycline-dependent overexpression of human RASSF4 and provides additional evidence for the anti-myeloma activity of RASSF4 overexpression in human cell lines. Supplemental Figure S4 provides a comparison of the RASSF4 expression levels of the primary MM cells (n=10) and a selected panel of HMCL (n=9) obtained through microarray analysis. Supplemental Figure S5 shows additional data on the effect of RASSF4 overexpression on tumor development and the confirmation of RASSF4 overexpression on protein level. Supplemental Figure S6 provides an overview of the top 20 differentially expressed genes between all control and RASSF4 overexpressing human multiple myeloma cell lines and subsequent pathway enrichment analysis of the genes downregulated after RASSF4 overexpression. Supplemental Figure S7 provides additional evidence for the anti-myeloma activity of trametinib in combination with the HDACi quisinostat or panobinostat and shows the effect of the HDACi panobinostat and/or the MEK1/2 inhibitor trametinib on RASSF4 mRNA expression.

RAS mutations occur frequently in multiple myeloma (MM), but apart from driving progression, they can also stimulate antitumor effects by activating tumor-suppressive RASSF proteins. Although this family of death effector molecules are often silenced in cancers, functional data about RASSF proteins in MM are lacking. Here, we report that RASSF4 is downregulated during MM progression and correlates with a poor prognosis. Promoter methylation analysis in human cell lines revealed an inverse correlation between RASSF4 mRNA levels and methylation status. Epigenetic modulating agents restored RASSF4 expression. Enforced expression of RASSF4 induced G2-phase cell-cycle arrest and apoptosis in human cell lines, reduced primary MM cell viability, and blocked MM growth in vivo. Mechanistic investigations showed that RASSF4 linked RAS to several pro-death pathways, including those regulated by the kinases MST1, JNK, and p38. By activating MST1 and the JNK/c-Jun pathway, RASSF4 sensitized MM cells to bortezomib. Genetic or pharmacological elevation of RASSF4 levels increased the anti-MM effects of the clinical relevant MEK1/2 inhibitor trametinib. Kinome analysis revealed that this effect was mediated by concomitant activation of the JNK/c-Jun pathway along with inactivation of the MEK/ERK and PI3K/mTOR/Akt pathways. Overall, our findings establish RASSF4 as a tumor-suppressive hub in MM and provide a mechanistic rationale for combining trametinib with HDAC inhibitors or bortezomib to treat patients with tumors exhibiting low RASSF4 expression.Significance: These findings provide a mechanistic rationale for combining trametinib with HDAC inhibitors or bortezomib in patients with multiple myeloma whose tumors exhibit low RASSF4 expression. Cancer Res; 78(5); 1155–68. ©2017 AACR.

Information about material and methods, that further support the date described in this article. Additional references and legends to the supplmental figures are also provided.

Supplemental Table S1 summarizes the patient characteristics. Supplemental Table S4 provides an overview of all kinases differentially phosphorylated in RASSF4 overexpressing cells treated with trametinib compared to parental cells treated with trametinib and/or RASSF4 overexpressing cells. Supplemental Table S5 summarizes significance and combination index (CI) values calculated for the different drug concentrations used in the combination experiments.

List of differentially expressed genes between all control and RASSF4 overexpressing human multiple myeloma cell lines

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PDF file - 117K, Uptake of LV-tSMAC transduced dying human melanoma cells enables dendritic cells to activate tumor-infiltrating lymphocytes.

PDF file - 184K, The therapeutic effect of LV-tSMAC involves effector T-cells.