TROP2 Is Associated with Primary Resistance to Immune Checkpoint Inhibition in Patients with Advanced Non-Small Cell Lung Cancer.

PURPOSE: Mechanisms of primary resistance to inhibitors of the programmed cell death-1 (PD-1)/programmed death-ligand 1 (PD-L1) signaling axis in non-small cell lung cancer (NSCLC) are still poorly understood. While some studies suggest the involvement of trophoblast cell surface antigen 2 (TROP2) in modulating tumor cell resistance to therapeutic drugs, its specific role in the context of PD-1/PD-L1 axis blockade is not definitively established. EXPERIMENTAL DESIGN: We performed high-throughput analysis of transcriptomic data from 891 NSCLC tumors from patients treated with either the PD-L1 inhibitor atezolizumab or chemotherapy in two large randomized clinical trials. To confirm our results at the protein level, we complemented this transcriptional approach by performing a multiplex immunofluorescence analysis of tumor tissue samples as well as a proteomic profiling of plasma. RESULTS: We observed a significant association of TROP2 overexpression with worse progression-free survival and overall survival on PD-L1 blockade, independent of other prognostic factors. Importantly, we found increased TROP2 expression to be predictive of survival in patients treated with atezolizumab but not chemotherapy. TROP2 overexpression was associated with decreased T-cell infiltration. We confirmed these results at the proteomic level both on tumor tissue and in plasma. CONCLUSIONS: Our results suggest an important contribution of TROP2 expression to the primary resistance to PD-L1 blockade in NSCLC. TROP2-biomarker-based strategy may be relevant in selecting patients with NSCLC who are more likely to benefit from a combination of immunotherapy and an anti-TROP2 agent.

Upregulation of Indoleamine 2,3-Dioxygenase 1 in Tumor Cells and Tertiary Lymphoid Structures is a Hallmark of Inflamed Non-Small Cell Lung Cancer.

PURPOSE: Overexpression of the tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase 1 (IDO1) has been reported in several tumor types, including non-small cell lung cancer (NSCLC), and has been shown to promote tumor-immune evasion and inhibit T-cell activation through increased tryptophan degradation and the production of several immunosuppressive metabolites collectively known as kynurenines. However, it remains unclear whether IDO1 expression by tumor cells is detrimental specifically in the context of programmed cell death protein 1 (PD-1)/programmed death ligand 1 (PD-L1) axis blockade. EXPERIMENTAL DESIGN: We analyzed the transcriptome of 891 NSCLC tumor samples from patients enrolled in two large randomized clinical trials investigating the safety and activity of atezolizumab, a humanized IgG1 mAb that targets PD-L1, versus docetaxel in patients with advanced NSCLC. We complemented these transcriptomics results at the protein level by using multiplex immunofluorescence and at the functional level with in vitro experiments. RESULTS: The increased expression of the tryptophan-catabolizing enzyme IDO1 was significantly associated with improved objective response, progression-free survival, and overall survival in patients treated with PD-L1 inhibitors, but not in those treated with chemotherapy. Strikingly, inflamed tumors had higher levels of IDO1, and IDO1 was also expressed in tertiary lymphoid structures (TLS) by mature follicular dendritic cells. L-kynurenine impaired the differentiation of antibody-producing B cells induced by follicular helper T (Tfh)/B-cell interactions, a hallmark process within TLS. CONCLUSIONS: IDO1 pathway in NSCLC is driven by the immune system rather than by tumor cells. Targeting IDO1 in combination with anti-PD-1/PD-L1 might be beneficial only in patients with inflamed tumors and particularly in those bearing TLS.

Fucoidan Independently Enhances Activity in Human Immune Cells and Has a Cytostatic Effect on Prostate Cancer Cells in the Presence of Nivolumab.

Fucoidan compounds may increase immune activity and are known to have cancer inhibitory effects in vitro and in vivo. In this study, we aimed to investigate the effect of fucoidan compounds on ex vivo human peripheral blood mononuclear cells (PBMCs), and to determine their cancer cell killing activity both solely, and in combination with an immune-checkpoint inhibitor drug, Nivolumab. Proliferation of PBMCs and interferon gamma (IFNγ) release were assessed in the presence of fucoidan compounds extracted from Fucus vesiculosus, Undaria pinnatifida and Macrocystis pyrifera. Total cell numbers and cell killing activity were assessed using a hormone resistant prostate cancer cell line, PC3. All fucoidan compounds activated PBMCs, and increased the effects of Nivolumab. All fucoidan compounds had significant direct cytostatic effects on PC3 cells, reducing cancer cell numbers, and PBMCs exhibited cell killing activity as measured by apoptosis. However, there was no fucoidan mediated increase in the cell killing activity. In conclusion, fucoidan compounds promoted proliferation and activity of PBMCs and added to the effects of Nivolumab. Fucoidan compounds all had a direct cytostatic effect on PC3 cells, as shown through their proliferation reduction, while their killing was not increased.

IDO Targeting in Sarcoma: Biological and Clinical Implications.

Sarcomas are heterogeneous malignant mesenchymal neoplasms with limited sensitivity to immunotherapy. We recently demonstrated an increase in Kynurenine Pathway (KP) activity in the plasma of sarcoma patients treated with pembrolizumab. While the KP has already been described to favor immune escape through the degradation of L-Tryptophan and production of metabolites including L-Kynurenine, Indoleamine 2,3 dioxygenase (IDO1), a first rate-limiting enzyme of the KP, still represents an attractive therapeutic target, and its blockade had not yet been investigated in sarcomas. Using immunohistochemistry, IDO1 and CD8, expression profiles were addressed within 203 cases of human sarcomas. At a preclinical level, we investigated the modulation of the KP upon PDL1 blockade in a syngeneic model of sarcoma through mRNA quantification of key KP enzymes within the tumor. Furthermore, in order to evaluate the possible anti-tumor effect of IDO blockade in combination with PDL1 blockade, an innovative IDO inhibitor (GDC-0919) was used. Its effect was first assessed on Kynurenine to Tryptophan ratio at plasmatic level and also within the tumor. Following GDC-0919 treatment, alone or in combination with anti-PDL1 antibody, tumor growth, immune cell infiltration, and gene expression profiling were measured. IDO1 expression was observed in 39.1% of human sarcoma cases and was significantly higher in tumors with high CD8 infiltration. In the pre-clinical setting, blockade of PDL1 led to a strong anti-tumor effect and was associated with an intratumoral inflammatory cytokines signature driven by Ifng but also with a modulation of the KP enzymes including Ido1 and Ido2. IDO1 inhibition using GDC-0919 resulted in (i) a significant decrease of plasmatic Kynurenine to Tryptophan ratio and in (ii) a decrease of tumoral Kynurenine. However, GDC-0919 used alone or combined with anti-PDL1, did not show anti-tumoral activity and did not affect the tumor immune cell infiltrate. In order to elucidate the mechanism(s) underlying the lack of effect of GDC-0919, we analyzed the gene expression profile of intratumoral biopsies. Interestingly, we have found that GDC-0919 induced a downregulation of the expression of pvr and granzymes, and an upregulation of inhba and Dtx4 suggesting a potential role of the IDO pathway in the control of NK function.

Progressive Parkinsonism by acute dysfunction of excitatory amino acid transporters in the rat substantia nigra.

Parkinson's disease (PD) is characterized by the progressive degeneration of substantia nigra (SN) dopamine neurons, involving a multifactorial cascade of pathogenic events. Here we explored the hypothesis that dysfunction of excitatory amino acid transporters (EAATs) might be involved. Acutely-induced dysfunction of EAATs in the rat SN, by single unilateral injection of their substrate inhibitor l-trans-pyrrolidine-2,4-dicarboxylate (PDC), triggers a neurodegenerative process mimicking several PD features. Dopamine neurons are selectively affected, consistent with their sustained excitation by PDC measured by slice electrophysiology. The anti-oxidant N-acetylcysteine and the NMDA receptor antagonists ifenprodil and memantine provide neuroprotection. Besides oxidative stress and NMDA receptor-mediated excitotoxicity, glutathione depletion and neuroinflammation characterize the primary insult. Most interestingly, the degeneration progresses overtime with unilateral to bilateral and caudo-rostral evolution. Transient adaptive changes in dopamine function markers in SN and striatum accompany cell loss and axonal dystrophy, respectively. Motor deficits appear when neuron loss exceeds 50% in the most affected SN and striatal dopamine tone is dramatically reduced. These findings outline a functional link between EAAT dysfunction and several PD pathogenic mechanisms/pathological hallmarks, and provide a novel acutely-triggered model of progressive Parkinsonism.

Preferential vulnerability of mesencephalic dopamine neurons to glutamate transporter dysfunction.

Nigral depletion of the main brain antioxidant GSH is the earliest biochemical event involved in Parkinson's disease pathogenesis. Its causes are completely unknown but increasing number of evidence suggests that glutamate transporters [excitatory amino acid transporters (EAATs)] are the main route by which GSH precursors may enter the cell. In this study, we report that dopamine (DA) neurons, which express the excitatory amino acid carrier 1, are preferentially affected by EAAT dysfunction when compared with non-DA neurons. In rat embryonic mesencephalic cultures, l-trans-pyrrolidine-2,4-dicarboxylate, a substrate inhibitor of EAATs, is directly and preferentially toxic for DA neurons by decreasing the availability of GSH precursors and lowering their resistance threshold to glutamate excitotoxicity through NMDA-receptors. In adult rat, acute intranigral injection of l-trans-pyrrolidine-2,4-dicarboxylate induces a large regionally selective and dose-dependent loss of DA neurons and alpha-synuclein aggregate formation. These data highlight for the first time the importance of excitatory amino acid carrier 1 function for the maintenance of antioxidant defense in DA neurons and suggest its dysfunction as a candidate mechanism for the selective death of DA neurons such as occurring in Parkinson's disease.

Glutamate leakage from a compartmentalized intracellular metabolic pool and activation of the lipoxygenase pathway mediate oxidative astrocyte death by reversed glutamate transport.

Astrocytes have essential roles for neuron survival and function, so that their demise in neurodegenerative insults, such as ischemia, deserves attention. A major event of the cell death cascade in ischemia is the reversed operation of excitatory amino acid transporters (EAAT), releasing glutamate. Cytotoxicity is conventionally attributed to extracellular glutamate accumulation. We previously reported that mimicking such dysfunction by EAAT substrate inhibitors, whose uptake induces glutamate release by heteroexchange, triggers glutathione (GSH) depletion and oxidative death of differentiated astrocytes in culture. Here we demonstrate that astrocyte death, although correlated with glutamate release, is not resulting from high extracellular glutamate-mediated toxicity. L-glutamate per se was gliotoxic only at concentrations much higher than the maximum reached with the potent EAAT substrate inhibitor L-trans-pyrrolidine-2,4-dicarboxylate (PDC), and toxicity was lower. Moreover, high glutamate concentrations offered protection against PDC. Protection was also provided by L-aspartate, which is both transported by EAAT and metabolized into glutamate, and by inhibiting glutamine synthetase, which uses transported glutamate to synthesize glutamine. Neither D-aspartate, a metabolically inert EAAT substrate, nor compounds that can provide glutamate intracellularly but are not EAAT substrates offered protection. Interestingly, only the compounds providing protection prevented PDC-induced GSH depletion. These data strongly suggest that reversed uptake-mediated astrocyte death results from the leakage of glutamate from a compartmentalized intracellular metabolic pool specifically fuelled by EAAT, crucial for preserving GSH contents. In addition, we provide evidence for a minor contribution of the cystine-glutamate antiporter x(c) (-) but a major role of the 5-lipoxygenase pathway in this death mechanism.