Translating Science into Survival: Report on the Seventh International Cancer Immunotherapy Conference.

From September 20 to 23, 2023, the Seventh International Cancer Immunotherapy Conference was hosted jointly by the Cancer Research Institute, the European Network for Cancer Immunotherapy (ENCI), and the American Association for Cancer Research (AACR) in Milan, Italy. The four-day event covered the latest advances in cancer immunology and immunotherapy.

An adverse tumor-protective effect of IDO1 inhibition.

By restoring tryptophan, indoleamine 2,3-dioxygenase 1 (IDO1) inhibitors aim to reactivate anti-tumor T cells. However, a phase III trial assessing their clinical benefit failed, prompting us to revisit the role of IDO1 in tumor cells under T cell attack. We show here that IDO1 inhibition leads to an adverse protection of melanoma cells to T cell-derived interferon-gamma (IFNγ). RNA sequencing and ribosome profiling shows that IFNγ shuts down general protein translation, which is reversed by IDO1 inhibition. Impaired translation is accompanied by an amino acid deprivation-dependent stress response driving activating transcription factor-4 (ATF4)high/microphtalmia-associated transcription factor (MITF)low transcriptomic signatures, also in patient melanomas. Single-cell sequencing analysis reveals that MITF downregulation upon immune checkpoint blockade treatment predicts improved patient outcome. Conversely, MITF restoration in cultured melanoma cells causes T cell resistance. These results highlight the critical role of tryptophan and MITF in the melanoma response to T cell-derived IFNγ and uncover an unexpected negative consequence of IDO1 inhibition.

Tryptophan depletion results in tryptophan-to-phenylalanine substitutants.

Activated T cells secrete interferon-γ, which triggers intracellular tryptophan shortage by upregulating the indoleamine 2,3-dioxygenase 1 (IDO1) enzyme1-4. Here we show that despite tryptophan depletion, in-frame protein synthesis continues across tryptophan codons. We identified tryptophan-to-phenylalanine codon reassignment (W>F) as the major event facilitating this process, and pinpointed tryptophanyl-tRNA synthetase (WARS1) as its source. We call these W>F peptides 'substitutants' to distinguish them from genetically encoded mutants. Using large-scale proteomics analyses, we demonstrate W>F substitutants to be highly abundant in multiple cancer types. W>F substitutants were enriched in tumours relative to matching adjacent normal tissues, and were associated with increased IDO1 expression, oncogenic signalling and the tumour-immune microenvironment. Functionally, W>F substitutants can impair protein activity, but also expand the landscape of antigens presented at the cell surface to activate T cell responses. Thus, substitutants are generated by an alternative decoding mechanism with potential effects on gene function and tumour immunoreactivity.

Oncogene-dependent sloppiness in mRNA translation.

mRNA translation is a highly conserved and tightly controlled mechanism for protein synthesis. Despite protein quality control mechanisms, amino acid shortage in melanoma induces aberrant proteins by ribosomal frameshifting. The extent and the underlying mechanisms related to this phenomenon are yet unknown. Here, we show that tryptophan depletion-induced ribosomal frameshifting is a widespread phenomenon in cancer. We termed this event sloppiness and strikingly observed its association with MAPK pathway hyperactivation. Sloppiness is stimulated by RAS activation in primary cells, suppressed by pharmacological inhibition of the oncogenic MAPK pathway in sloppy cells, and restored in cells with acquired resistance to MAPK pathway inhibition. Interestingly, sloppiness causes aberrant peptide presentation at the cell surface, allowing recognition and specific killing of drug-resistant cancer cells by T lymphocytes. Thus, while oncogenes empower cancer progression and aggressiveness, they also expose a vulnerability by provoking the production of aberrant peptides through sloppiness.

Anti-tumour immunity induces aberrant peptide presentation in melanoma.

Extensive tumour inflammation, which is reflected by high levels of infiltrating T cells and interferon-γ (IFNγ) signalling, improves the response of patients with melanoma to checkpoint immunotherapy1,2. Many tumours, however, escape by activating cellular pathways that lead to immunosuppression. One such mechanism is the production of tryptophan metabolites along the kynurenine pathway by the enzyme indoleamine 2,3-dioxygenase 1 (IDO1), which is induced by IFNγ3-5. However, clinical trials using inhibition of IDO1 in combination with blockade of the PD1 pathway in patients with melanoma did not improve the efficacy of treatment compared to PD1 pathway blockade alone6,7, pointing to an incomplete understanding of the role of IDO1 and the consequent degradation of tryptophan in mRNA translation and cancer progression. Here we used ribosome profiling in melanoma cells to investigate the effects of prolonged IFNγ treatment on mRNA translation. Notably, we observed accumulations of ribosomes downstream of tryptophan codons, along with their expected stalling at the tryptophan codon. This suggested that ribosomes bypass tryptophan codons in the absence of tryptophan. A detailed examination of these tryptophan-associated accumulations of ribosomes-which we term 'W-bumps'-showed that they were characterized by ribosomal frameshifting events. Consistently, reporter assays combined with proteomic and immunopeptidomic analyses demonstrated the induction of ribosomal frameshifting, and the generation and presentation of aberrant trans-frame peptides at the cell surface after treatment with IFNγ. Priming of naive T cells from healthy donors with aberrant peptides induced peptide-specific T cells. Together, our results suggest that IDO1-mediated depletion of tryptophan, which is induced by IFNγ, has a role in the immune recognition of melanoma cells by contributing to diversification of the peptidome landscape.

Reversal of pre-existing NGFR-driven tumor and immune therapy resistance.

Melanomas can switch to a dedifferentiated cell state upon exposure to cytotoxic T cells. However, it is unclear whether such tumor cells pre-exist in patients and whether they can be resensitized to immunotherapy. Here, we chronically expose (patient-derived) melanoma cell lines to differentiation antigen-specific cytotoxic T cells and observe strong enrichment of a pre-existing NGFRhi population. These fractions are refractory also to T cells recognizing non-differentiation antigens, as well as to BRAF + MEK inhibitors. NGFRhi cells induce the neurotrophic factor BDNF, which contributes to T cell resistance, as does NGFR. In melanoma patients, a tumor-intrinsic NGFR signature predicts anti-PD-1 therapy resistance, and NGFRhi tumor fractions are associated with immune exclusion. Lastly, pharmacologic NGFR inhibition restores tumor sensitivity to T cell attack in vitro and in melanoma xenografts. These findings demonstrate the existence of a stable and pre-existing NGFRhi multitherapy-refractory melanoma subpopulation, which ought to be eliminated to revert intrinsic resistance to immunotherapeutic intervention.

Augmenting Immunotherapy Impact by Lowering Tumor TNF Cytotoxicity Threshold.

New opportunities are needed to increase immune checkpoint blockade (ICB) benefit. Whereas the interferon (IFN)γ pathway harbors both ICB resistance factors and therapeutic opportunities, this has not been systematically investigated for IFNγ-independent signaling routes. A genome-wide CRISPR/Cas9 screen to sensitize IFNγ receptor-deficient tumor cells to CD8 T cell elimination uncovered several hits mapping to the tumor necrosis factor (TNF) pathway. Clinically, we show that TNF antitumor activity is only limited in tumors at baseline and in ICB non-responders, correlating with its low abundance. Taking advantage of the genetic screen, we demonstrate that ablation of the top hit, TRAF2, lowers the TNF cytotoxicity threshold in tumors by redirecting TNF signaling to favor RIPK1-dependent apoptosis. TRAF2 loss greatly enhanced the therapeutic potential of pharmacologic inhibition of its interaction partner cIAP, another screen hit, thereby cooperating with ICB. Our results suggest that selective reduction of the TNF cytotoxicity threshold increases the susceptibility of tumors to immunotherapy.

Sulfated heterorhamnans from the green seaweed Gayralia oxysperma: partial depolymerization, chemical structure and antitumor activity.

Sulfated heterorhamnans produced by Gayralia oxysperma were utilized for the preparation of two homogeneous and highly sulfated Smith-degraded products (M(w) of 109 and 251 kDa), which were constituted principally by 3-linked α-L-rhamnosyl units 2- or 4-sulfate and 2-linked α-L-rhamnosyl units 4- or 3,4-sulfate, in different percentages. The homogeneous products and the crude extracts containing the sulfated heterorhamnans showed cytotoxic effect against U87MG cells. These sulfated polysaccharides induced an increase in the number of cells in G1 phase with concomitant increase of the mRNA levels of p53 and p21. The presence of 2-linked disulfated rhamnose residues together with the molecular weight could be important factors to be correlated with the inhibitory effect on human glioblastoma cells.

Flavone induces cell death in human hepatoma HepG2 cells.

Flavones have received considerable attention because of their antiproliferative properties and selective effects on cancer cells, making them good candidates for use in cancer therapy. In contrast to other flavones, little is known about the effects of the flavone core structure (2-phenyl-4H-1-benzopyran-4one) on cancer cells. Here, we report that flavone induces cell death in human hepatoma HepG2 cells. Furthermore, annexin-V+/PI- and SubG1 populations of HepG2 cells increased after flavone treatment. Exposure of HepG2 to flavone did not result in either cytochrome c release into the cytosol or changes in the mitochondrial membrane potential. Treatment of HepG2 cells with flavone for 24 h reduced the accumulation of intracellular ROS, which correlated with upregulation of Gred, CuZnSOD and MnSOD mRNA levels. Taken together, our results provided useful insights into the mechanism of cell death caused by flavones, in order to evaluate their future application in hepatocarcinoma therapy.

Parallel in vivo and in vitro melanoma RNAi dropout screens reveal synthetic lethality between hypoxia and DNA damage response inhibition.

To identify factors preferentially necessary for driving tumor expansion, we performed parallel in vitro and in vivo negative-selection short hairpin RNA (shRNA) screens. Melanoma cells harboring shRNAs targeting several DNA damage response (DDR) kinases had a greater selective disadvantage in vivo than in vitro, indicating an essential contribution of these factors during tumor expansion. In growing tumors, DDR kinases were activated following hypoxia. Correspondingly, depletion or pharmacologic inhibition of DDR kinases was toxic to melanoma cells, including those that were resistant to BRAF inhibitor, and this could be enhanced by angiogenesis blockade. These results reveal that hypoxia sensitizes melanomas to targeted inhibition of the DDR and illustrate the utility of in vivo shRNA dropout screens for the identification of pharmacologically tractable targets.