The oncolytic peptide LTX-315 triggers immunogenic cell death

Abstract

LTX-315 is a cationic amphilytic peptide that preferentially permeabilizes mitochondrial membranes, thereby causing partially BAX/BAK1-regulated, caspase-independent necrosis. Based on the observation that intratumorally injected LTX-315 stimulates a strong T lymphocyte-mediated anticancer immune response, we investigated whether LTX-315 may elicit the hallmarks of immunogenic cell death (ICD), namely (i) exposure of calreticulin on the plasma membrane surface, (ii) release of ATP into the extracellular space, (iii) exodus of HMGB1 from the nucleus, and (iv) induction of a type-1 interferon response. Using a panel of biosensor cell lines and robotized fluorescence microscopy coupled to automatic image analysis, we observed that LTX-315 induces all known ICD characteristics. This conclusion was validated by several independent methods including immunofluorescence stainings (for calreticulin), bioluminescence assays (for ATP), immunoassays (for HMGB1), and RT-PCRs (for type-1 interferon induction). When injected into established cancers, LTX-315 caused a transiently hemorrhagic focal necrosis that was accompanied by massive release of HMGB1 (from close-to-all cancer cells), as well as caspase-3 activation in a fraction of the cells. LTX-315 was at least as efficient as the positive control, the anthracycline mitoxantrone (MTX), in inducing local inflammation with infiltration by myeloid cells and T lymphocytes. Collectively, these results support the idea that LTX-315 can induce ICD, hence explaining its capacity to mediate immune-dependent therapeutic effects.

Combination with SGT-53 overcomes tumor resistance to a checkpoint inhibitor

“…In cultured 4T1 mouse breast cancer cells, we observed changes indicative of ICD after SGT-53 treatment including increased surface expression of the ER protein CRT and release of cell death associated molecular patterns (HMGB1 and ATP). 34 Several additional alterations in immunogenicity were also observed with SGT-53 treatment including type I IFN responses and increased tumor expression of CD80, CD86, FAS, TAP1/2, and MHC I molecules in vivo . We have previously shown that SGT-53 treatment of MOC1 head and neck cancer cells could upregulate the expression of genes involved in antigen processing and presentation including CRT, calnexin, endoplasmic reticulum amino peptidase 1, and TAP1/2 involving stimulator of interferon genes (STING) pathway.…”

The oncolytic compound LTX-401 targets the Golgi apparatus

“…Downstream of mitochondria, then a variety of lethal signaling events (that can be partially inhibited by a combination of caspase inhibition by Z-VAD-fmk and RIP1 inhibition by necrostatin) come into action, as this is often observed, 23 in line with the increasing awareness that the clear-cut distinction between apoptotic and necroptotic degradation events is a didactic oversimplification. 24 Similar to LTX-315, 5 LTX-401 triggers all biochemical hallmarks of ICD including ATP release, calreticulin exposure, nuclear HMGB1 exodus and the induction of a type-1 interferon response. These findings, which have been obtained in vitro, suggest that LTX-401 can stimulate the full spectrum of events required for (ATP-dependent) attraction of DC precursors into the tumor bed, the (calreticulin-dependent) transfer of tumor antigens from cancer cells to DC precursors, the (HMGB1-dependent) maturation of DC for optimal antigen presentation of tumor-associated antigens, and (the type-1 interferon-dependent) recruitment of T cells into the tumor bed.…”

“…3,4 LTX-315 is being developed for the local treatment of superficial human cancers based on the observations that it causes potent cytotoxic effects and that it might stimulate a local immune response that may prolong its antineoplastic action over time. [5][6][7] This latter property makes LTX-315 an interesting lead compound, especially in view of its capacity to reverse the resistance of cancers to immune checkpoint blockade with anti-CTLA4 antibodies in preclinical models. 6 LTX-315 has the capacity of causing a necrotic cell death (i.e., cell death without caspase activation and without morphological features of apoptosis such as nuclear fragmentation) that is partially relying on mitochondrial membrane permeabilization by this oncolytic peptide, 4 which actually redistributes specifically towards the mitochondrial compartment.…”

“…9 Accordingly, LTX-315 triggers the exposure or release of several danger-associated molecular patterns (DAMPs) associated with ICD, namely the translocation of calreticulin (CALR) from the lumen of the endoplasmic reticulum to the cell surface; the release of ATP from cytoplasmic storage sites into the extracellular space; the exodus of high mobility group box 1 (HMGB1) from the nucleus to the extracellular milieu; and a type-1 interferon response. 5,10 Through the combination of these DAMPs, LTX-315-induced cell death can alert innate immune effectors including dendritic cells, hence starting a cascade of molecular events that culminates in a strong cellular immune response against tumor-associated antigens. 5,7 LTX-315 is a peptide encompassing nine amino acids residues, spurring interest in alternative compounds like LTX-401 that might share similar pharmacological properties.…”

“…5,10 Through the combination of these DAMPs, LTX-315-induced cell death can alert innate immune effectors including dendritic cells, hence starting a cascade of molecular events that culminates in a strong cellular immune response against tumor-associated antigens. 5,7 LTX-315 is a peptide encompassing nine amino acids residues, spurring interest in alternative compounds like LTX-401 that might share similar pharmacological properties. Indeed, LTX-401 has been shown to exert strong anticancer effects in a mouse model of melanoma, in which it stimulates infiltration of the tumor by T lymphocytes.…”