1126 Molecular mechanisms of DC activation by melanoma cells responding to LTX-315

Background

Oncolytic peptides are emerging as attractive candidates for the development of novel anticancer regimens¹, reflecting broad cytolytic activities against a variety of malignant (but not normal) cells and a pronounced potential for immunostimulation.² LTX-315 is a synthetic nonameric cationic peptide derived from bovine lactotransferrin³ that has been associated with a pronounced capacity to elicit tumor-targeting immune responses in various preclinical models of cancer.² Specifically, LTX-315 has been shown to (1) cause immunogenic cell death (ICD) ⁴ coupled to the release of immunostimulatory cytokines and damage-associated molecular patterns (DAMPs)^5,6, (2) deplete the tumor microenvironment (TME) of immunosuppressive cells such as CD4⁺CD25⁺FOXP3⁺ regulatory T (T_REG) cells and myeloid-derived suppressor cells (MDSCs)⁷, and (3) synergize with immunogenic chemotherapy⁸ or immune checkpoint inhibitors (ICIs)⁷ in the control of syngeneic mouse tumor models. However, the effects of tumor cells responding to LTX-315 on dendritic cells (DCs) remain to be precisely elucidated.

Methods

Human A375 and mouse B16F10 cells were used as models of melanoma, in vitro (A375, B16F10) and in vivo (B16F10) upon subcutaneous injection in immunocompetent C57BL/6 mice. Flow cytometry, ELISA, RT-PCR and immunoblotting were used to assess DC maturation in vitro and in vivo, immunoblotting was employed to investigate the molecular mechanisms underlying DC activation, knockout and knockdown systems were harnessed to demonstrate mechanistic involvement. Therapeutic assays with B16F10 melanoma cells growing in immunocompetent vs Myd88^-/- mice coupled to tumor rechallenge were employed to confirm activation of tumor-targeting immunity coupled to the establishment of immunological memory.

Results

LTX-315 mediated the release of immunostimulatory DAMPs and nucleic acids (NAs) from melanoma cells that drove robust DC activation as monitored by surface immunophenotyping for CD80, CD83, CD86 and MHC class II and cytokine (TNF, IL-1β) secretion. This immunostimulatory effect mechanistically depended on TLR9 (detecting LTX-315-DNA complexes), TLR8 (detecting LTX315-RNA complexes) and TLR7 (which was activated by LTX-315 per se), as well as on the common TLR signal transducer MYD88. Accordingly, the in vivo anticancer activity of LTX-315 against B16F10 melanoma cells was significantly reduced in Myd88^-/- mice, and Myd88^-/- mice eradicating B16F10 tumors upon LTX-315 treatment remained susceptible to a rechallenge with B16F10 cells.

Conclusions

In addition to induce immunogenic cell death, LTX-315 also activates DC via MYD88-dependent pathways that mediate optimal immunostimulatory effects coupled to the establishment of cancer-specific immunological memory in preclinical melanoma models.

References

Camilio KA, Berge G, Ravuri CS, Rekdal O, Sveinbjørnsson B. Complete regression and systemic protective immune responses obtained in B16 melanomas after treatment with LTX-315. Cancer Immunol Immunother 2014;63(6):601–613. Camilio KA, Wang MY, Mauseth B, et al. Combining the oncolytic peptide LTX-315 with doxorubicin demonstrates therapeutic potential in a triple-negative breast cancer model. Breast Cancer Res. 2019;21(1):9. Eike LM, Yang N, Rekdal Ø, Sveinbjørnsson B. The oncolytic peptide LTX-315 induces cell death and DAMP release by mitochondria distortion in human melanoma cells. Oncotarget 2015;6(33):34910–34923. Kepp O, Marabelle A, Zitvogel L, Kroemer G. Oncolysis without viruses – inducing systemic anticancer immune responses with local therapies. Nat Rev Clin Oncol 2020;17(1):49–64. Kroemer G, Galassi C, Zitvogel L, Galluzzi L. Immunogenic cell stress and death. Nat Immunol 2022;23(4):487–500. Vitale I, Yamazaki T, Wennerberg E, et al. Targeting cancer heterogeneity with immune responses driven by oncolytic peptides. Trends Cancer 2021;7(6):557–572. Yamazaki T, Pitt JM, Vétizou M, et al. The oncolytic peptide LTX-315 overcomes resistance of cancers to immunotherapy with CTLA4 checkpoint blockade. Cell Death Differ. 2016;23(6):1004–1015. Zhou H, Forveille S, Sauvat A, et al. The oncolytic peptide LTX-315 triggers immunogenic cell death. Cell Death Dis 2016;7(3):e2134.

Ethics Approval

This study was approved by Weill Cornell Medicine IACUC.