ICB、SIRT1与维生素D
一、SIRT1与促炎性T细胞趋化因子CXCL9、CXCL10和IFN-γ正相关，激活SIRT1促进T细胞浸润《Spatial proteomics reveals sirtuin 1 to be a determinant of T-cell infiltration in human melanoma》
Methods: We mapped the spatial distribution of proteins in TiL-enriched vs. TiL-low compartments in melanoma by combining microscopy, matrix-assisted laser desorption mass spectrometry imaging and liquid chromatography-mass spectrometry, as well as computational data mining. Pharmacological modulation of sirtuin 1 (SIRT1) activity in syngeneic mouse models was used to evaluate the efficacy of pharmacological SIRT1 activation in two syngeneic melanoma mouse models, one known to be α-programmed cell death protein 1 (PD-1) sensitive and the other α-PD-1 resistant.
Results: Spatial proteomics and gene ontology-based enrichment analysis identified > 145 proteins enriched in CD8high tumour compartments, including negative regulators of mammalian target of rapamycin signalling such as SIRT1. Multiplexed immunohistochemistry confirmed that SIRT1 protein was expressed more in CD8high than in CD8low compartments. Further analysis of bulk and single-cell RNA sequencing data from melanoma tissue samples suggested the expression of SIRT1 by different lymphocyte subpopulations (CD8+ T cells, CD4+ T cells and B cells). Furthermore, we showed in vivo that pharmacological SIRT1 activation increased the immunological effect of α-PD-1 ICI against melanoma cells in mice, which was accompanied by an increase in T-cell infiltration and T-cell-related cytokines, including interferon (IFN)-γ, CCL4, CXCL9, CXCL10 and tumour necrosis factor-α. In silico analysis of large transcriptional data cohorts showed that SIRT1 was positively associated with the proinflammatory T-cell chemokines CXCL9, CXCL10 and IFN-γ, and prolonged overall survival of patients with melanoma.
二、SIRT1介导β-catenin脱乙酰化；ICB超进展的发生机制之一就是IFNγ降低SIRT1活性从而造成β-catenin乙酰化的积聚，强化β-catenin信号传导。
《Intersection of immune and oncometabolic pathways drives cancer hyperprogression during immunotherapy》
To dissect how IFNγ activates β-catenin, we investigated the expression and posttranslational modification of β-catenin. IFNγ treatment did not alter the protein levels of total and phosphorylated β-catenin (Figures S4C and S4D) but increased β-catenin acetylation in A375 cells (Figure 4K). Acetylation of β-catenin increases its activity to stimulate gene transcription. P300 and Sirtuins are the main enzymes to catalyze acetylation and deacetylation of β-catenin, respectively 47–49. We treated A375 cells with L002, an inhibitor of P300 50, in the presence of IFNγ. As expected, L002 treatment reduced MYC expression. However, IFNγ was still able to increase MYC expression in L002 pretreated A375 cells, suggesting that P300 may not be involved in IFNγ-induced β-catenin signaling (Figure S4E). Then, we treated A375 cells with Salermide, a Sirtuin inhibitor 51. We observed that Salermide treatment induced β-catenin acetylation (Figure 4L) and target gene expression (Figure 4M). In addition, IFNγ failed to increase the expression of β-catenin target genes in cells co-treated with Salermide (Figure S4F–S4I). In line with this, Sirtinol, another Sirtuin inhibitor, also induced β-catenin signaling gene expression (Figure 4M). Notably, Salermide and Sirtinol failed to trigger the signaling gene expression of Hippo, NOTCH, and Hedgehog (Figure 4M). The Wnt-β-catenin signaling inhibitors, DKK1 and Wnt-C59, abolished the effect of Salermide on the expression of MYC, CCND1, VEGFA, and MMP14 (Figure S4J). The data suggest that IFNγ may induce the β-catenin signaling via reducing Sirtuin-mediated β-catenin deacetylation. Sirtuins are a class of NAD+ dependent deacetylases 52. IFNγ stimulated guanylate binding protein (GBP)-1 expression (positive control) 53, had no effect on SIRT1 expression (Figure 4N), but reduced the intracellular level of NAD+ (Figure 4O). To restore NAD+ and Sirtuin activity, we treated A375 cells with nicotinamide riboside (NR), an NAD+ precursor. Interestingly, NR treatment diminished IFNγ altered β-catenin acetylation (Figure 4P), TOP-FLASH reporter activity (Figure 4Q), and β-catenin target gene expression (Figure 4R and 4S), as compared with vehicle controls. Similarly, treatment with nicotinamide mononucleotide (NMN), also diminished the effect of IFNγ on the expression of β-catenin signaling genes, while expression of other oncogenic pathways, such as Hippo, NOTCH, and Hedgehog signaling, were not affected by NR or NMN (Figure 4S). Altogether, these data suggest that IFNγ enhances β-catenin acetylation via reducing NAD+ levels (Figure 4T). β-catenin can be acetylated at K49 and K345. We found that IFNγ did not affect the expression of K49-acetylated β-catenin (Figure S4K). Sirtuin may catalyze the deacetylation of β-catenin at K345 47. We established stable cells carrying K345R β-catenin mutation. Upon IFNγ (Figures 4U and 4V) and Salermide (Figures 4W and 4X) treatment, β-catenin acetylation (Figures 4U and 4W) and downstream gene activation (Figures 4V and 4X) were largely abrogated in K345R mutant cells. The data suggest that IFNγ may affect β-catenin acetylation at K345 via reducing Sirtuin activity. As an additional control, we treated non-HPD-prone mouse YUMM5.2 cells with IFNγ. IFNγ had no effect on the β-catenin signaling activity (Figure S4L) and intracellular NAD+ levels (Figure S4M). Thus, that IFNγ selectively alters the NAD+-β-catenin signaling activities in HPD-prone tumor models.
三、维生素D活化形式1,25(OH)2D3增强SIRT1的活性和脱乙酰化能力，从而增强β-catenin的脱乙酰化，抑制β-catenin
《SIRT1 Mediates the Antagonism of Wnt/β-Catenin Pathway by Vitamin D in Colon Carcinoma Cells》
Cancer initiation and progression result from genetic and epigenetic alterations caused by interactions between environmental and endogenous factors leading to aberrant cell signalling. Colorectal cancers (CRC) are linked to abnormal activation of the Wnt/β-catenin pathway, whose key feature is the nuclear accumulation of acetylated β-catenin in colon epithelial cells. Nuclear β-catenin acts as a transcriptional co-activator, targeting genes involved in cell proliferation and invasion. 1α,25-Dihydroxyvitamin D3 (1,25(OH)2D3 or calcitriol), the active form of vitamin D, antagonizes Wnt/β-catenin over-activation by engaging its high affinity receptor, VDR. Here we unveil that 1,25(OH)2D3-bound VDR activates Silent Information Regulator of Transcription, sirtuin 1 (SIRT1), leading to β-catenin deacetylation and nuclear exclusion, downregulation of its pro-tumourigenic target genes and inhibition of human colon carcinoma cell proliferation. Notably, orthogonal SIRT1 activation mimics nuclear exclusion of β-catenin while SIRT1 inhibition blocks the effects of 1,25(OH)2D3. Thus, SIRT1 emerges as a crucial mediator in the protective action of vitamin D against CRC. The mutual negative feedback loop unveiled here between Wnt and SIRT1 represents an important surrogate target in CRC. Since nuclear localisation of β-catenin is a critical driver of CRC that requires its acetylation, we provide a mechanistic foundation for the epidemiological evidence linking vitamin D deficiency and increased CRC risk and mortality.
四、临床试验的表明，ICB联合维生素D疗效好，风险小
1、《Vitamin D supplementation increases objective response rate and prolongs progression-free time in patients with advanced melanoma undergoing anti–PD-1 therapy》
Methods
A total of 200 patients with advanced melanoma were included in the study. All patients received anti–PD-1 immunotherapy (nivolumab or pembrolizumab) as first-line treatment. Serum vitamin D levels were measured in patients both before and every 12 weeks during treatment. Part of the group had vitamin D measured retrospectively from the preserved serum. The other part of the supplementation group was tested prospectively.
Results
The response rate in the group with low vitamin D levels and not supplemented was 36.2%, whereas in the group with normal baseline levels or a normal level obtained with supplementation was 56.0% (p = .01). Moreover, progression-free survival in these groups was 5.75 and 11.25 months, respectively (p = .03). In terms of overall survival, there was also a difference in favor of the group with normal vitamin D levels (27 vs. 31.5 months, respectively; p = .39).
Conclusions
In our opinion, maintaining the vitamin D level within the normal range during anti-PD-1 immunotherapy in advanced melanoma patients should be a standard procedure allowing the improvement of treatment outcomes.
2、《Vitamin D Status Is Associated With Immune Checkpoint Inhibitor Efficacy and Immune-related Adverse Event Severity in Lung Cancer Patients: A Prospective Cohort Study》
Vitamin D (VitD) is potentially immunomodulatory, so here we aimed to explore the relationships between serum VitD levels, immune checkpoint inhibitor (ICI) efficacy, and immune-related adverse events (irAEs). Serum 25-hydroxyvitamin D levels were quantified before and after ICI treatment in prospectively enrolled patients with advanced lung cancers. Of 77 enrolled patients, 29 developed 42 irAEs. Baseline 25(OH)D levels of partial response (PRs) patients were significantly higher than non-PR patients (19.39±7.16 vs. 16.28±5.99 ng/mL, P =0.04). The area under the curve of 25(OH)D >15.73 ng/mL to identify PR was 0.63 (95% CI, 0.51-0.76, P =0.047), and baseline 25(OH)D levels >15.73 ng/mL (odds ratio: 2.93, 95% CI, 1.10-7.79, P =0.03) and prior targeted therapy (odds ratio: 0.30, 95% CI, 0.10-0.92, P =0.04) were independent predictors of PR as best efficacy by multivariable logistic regression. With respect to irAEs, baseline 25(OH)D levels were higher in grade 1 irAE patients than in grade 2/3/4 irAE patients (20.07±8.64 vs. 15.22±2.30 ng/mL, P =0.02). However, the area under the curve was only 0.56 (95% CI, 0.42-0.70, P =0.39) for a baseline 25(OH)D of 20.99 ng/mL for predicting irAE occurrence. There was a direct monotonic relationship and U-shaped relationship between baseline 25(OH)D levels and ICI efficacy and irAE occurrence, respectively. Overall survival was significantly different between VitD sufficient, insufficient, and deficient patients (log-rank P =0.01), which remained after adjustment in Cox proportional hazards regression models. Baseline 25(OH)D levels seem to be associated with ICI efficacy and prognosis, it might be helpful to assess the baseline VitD status, and supplementation with VitD might bring some benefit to enhance ICI efficacy and reduce moderate-severe irAEs.
3、《Low Vitamin D Status Predicts Poor Clinical Outcome in Advanced Melanoma Treated With Immune Checkpoint or BRAF/MEK Inhibitors: A Prospective Non-Interventional Side-by-Side Analysis》
In melanoma and other malignancies, low vitamin D status is associated with increased risk and poor prognosis. However, there are limited data of the impact of 25(OH)D serum concentration (s.c.) on clinical outcome in advanced melanoma. We tested the hypothesis that vitamin D status is predictive of efficacy and safety in patients treated for metastasized melanoma with B-rapidly accelerated fibrosarcoma (BRAF), mitogen-activated protein kinase kinase (MEK), cytotoxic T lymphocyte-associated protein-4 (CTLA-4), and/or programmed cell death protein-1 (PD-1) inhibitors. Severe vitamin D deficiency was associated with markedly reduced overall (OS) and progress-free (PFS) survival, with increased tumor load , and with a trend for higher frequency of adverse events (AEs). An increase in average 25(OH)D s.c. of 1 ng/ml was associated with a 3.9% reduced risk for progressive disease , with a reduction of LDH s.c. of 3.86 U/l (p = 0.034, indicating a reduction of TL), and with a trend for reduced frequency of AEs (AE ratio -0.005; p = 0.295). Patients with average 25(OH)D s.c. ≥10 ng/ml and BRAF-mutant melanoma showed a trend for a higher frequency of AEs as compared to individuals with BRAF wild-type melanomas. Our data indicate that vitamin D deficiency is associated with poor clinical outcome in patients treated for metastasized melanoma with BRAF/MEK inhibitors or immunotherapy. Although it needs to be proven in future interventional trials whether optimizing serum 25(OH)D improves clinical outcome in these patients, we recommend that 25(OH)D s.c. should be analyzed and vitamin D deficiency treated in all patients with advanced melanoma.
五、维生素D可以用到很大的剂量
《High-dose vitamin D3 in adults with pulmonary tuberculosis: a double-blind randomized controlled trial》
Subjects were randomly assigned to receive oral vitamin D3 or a placebo concomitant with standard first-line antituberculosis drugs.
可以参考这个剂量，甚至在耐受的基础上探索更高剂量。
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