Table1_Tumor PD-L1 expression and molecular profiling are not associated with immune checkpoint inhibitor-induced thyroid dysfunction in advanced NSCLC patients.docx

Background: Immune-checkpoint inhibitors (ICIs) have revolutionized the treatment of advanced non-small cell lung cancer (NSCLC), however are frequently associated with thyroid immune-related adverse events (IRAEs). We investigated the association between patient characteristics, tumor PD-L1 expression and molecular profile with the development of thyroid IRAEs in NSCLC patients.Methods: Single center, retrospective study including 107 NSCLC patients treated with PD-1/PD-L1 inhibitors from April 2016 to July 2020. All patients were euthyroid at baseline with at least two TSH measurements post-treatment initiation. The primary outcome was the difference in tumor PD-L1 expression in patients who developed any thyroid IRAEs versus those who remained euthyroid. Additional outcomes included development of overt thyroid dysfunction, the association of specific molecular alterations with thyroid IRAEs, and onset of thyroid IRAEs as a function of tumor PD-L1 expression.Results: Overall, 37 (34.6%) patients developed any thyroid dysfunction and 18 (16.8%) developed overt thyroid dysfunction. Tumor PD-L1 staining intensity was not associated with thyroid IRAEs. TP53 mutation was less likely to be associated with any thyroid dysfunction (p Conclusion: PD-L1 expression is not associated with the development of thyroid dysfunction in advanced NSCLC patients treated with ICIs, suggesting that thyroid IRAEs are unrelated to tumor PD-L1 expression.</p

The Sirt1 Activators SRT2183 and SRT3025 Inhibit RANKL-Induced Osteoclastogenesis in Bone Marrow-Derived Macrophages and Down-Regulate Sirt3 in Sirt1 Null Cells.

Increased osteoclast-mediated bone resorption is characteristic of osteoporosis, malignant bone disease and inflammatory arthritis. Targeted deletion of Sirtuin1 (Sirt1), a key player in aging and metabolism, in osteoclasts results in increased osteoclast-mediated bone resorption in vivo, making it a potential novel therapeutic target to block bone resorption. Sirt1 activating compounds (STACs) were generated and were investigated in animal disease models and in humans however their mechanism of action was a source of controversy. We studied the effect of SRT2183 and SRT3025 on osteoclastogenesis in bone-marrow derived macrophages (BMMs) in vitro, and discovered that these STACs inhibit RANKL-induced osteoclast differentiation, fusion and resorptive capacity without affecting osteoclast survival. SRT2183 and SRT3025 activated AMPK, increased Sirt1 expression and decreased RelA/p65 lysine310 acetylation, critical for NF-κB activation, and an established Sirt1 target. However, inhibition of osteoclastogenesis by these STACs was also observed in BMMs derived from sirt1 knock out (sirt1-/-) mice lacking the Sirt1 protein, in which neither AMPK nor RelA/p65 lysine 310 acetylation was affected, confirming that these effects require Sirt1, but suggesting that Sirt1 is not essential for inhibition of osteoclastogenesis by these STACs under these conditions. In sirt1 null osteoclasts treated with SRT2183 or SRT3025 Sirt3 was found to be down-regulated. Our findings suggest that SRT2183 and SRT3025 activate Sirt1 and inhibit RANKL-induced osteoclastogenesis in vitro however under conditions of Sirt1 deficiency can affect Sirt3. As aging is associated with reduced Sirt1 level and activity, the influence of STACs on Sirt3 needs to be investigated in vivo in animal and human disease models of aging and osteoporosis

Sirt1 Promotes a Thermogenic Gene Program in Bone Marrow Adipocytes: From Mice to (Wo)Men

Bone marrow adipose tissue (MAT) is influenced by nutritional cues, and participates in whole body energy metabolism. To investigate the role of Sirtuin1 (Sirt1), a key player in metabolism, in MAT, marrow adiposity was evaluated in inbred 5-month-old 129/Sv Sirt1 haplo-insufficient (Sirt1Δ/+) and wild type (WT) mice. Decreased expression of the thermogenic genes: Prdm16, Pgc1α, Foxc2, Dio2, and β3AR was detected in whole tibiae derived from Sirt1Δ/+ compared to WT female mice. Similarly, decreased expression of Prdm16 and Pgc1α was observed in primary bone marrow mesenchymal stem cell (BM-MSC) cultures obtained from Sirt1Δ/+ compared to WT female mice, suggesting a cell autonomous effect of Sirt1 in BM-MSCs. In vitro, Sirt1 over-expression in the mesenchymal embryonic fibroblast stem cell line C3HT101/2 increased Pgc1α and Prdm16 protein level. Similarly, pharmacologic activation of Sirt1 by SRT3025 increased Foxc2, Pgc1α, Dio2, Tfam, and Cyc1 expression while inhibition of Sirt1 by EX527 down-regulated UCP1 in C3HT101/2 cells. Importantly, in human femoral BM-MSCs obtained from female patients undergoing hip operations for fracture or osteoarthritis, Sirt1 activation by SRT3025 increased PGC1α mRNA and protein level. Blocking sclerostin, an inhibitor of the WNT pathway and a Sirt1 target, by the monoclonal humanized antibody (Sc-AbII), stimulated β3AR, PRDM16, and UCP1 gene expression, and increased PGC1α protein level. These results show that Sirt1 stimulates a thermogenic gene program in marrow adipocytes in mice and humans via PGC1α activation and sclerostin inhibition. The implications of these findings to bone health, hematopoiesis and whole body energy metabolism remain to be investigated.ISSN:1664-239

SRT2183 inhibits RANKL-induced osteoclastogenesis and pit formation in s<i>irt1</i>^<i>-/-</i> BMMs.

<p>(A) Sirt1 expression in WT- and in <i>Sirt1</i>^<i>-/-</i>-derived osteoclasts. PCR amplification of exons 1–9 of the s<i>irt1</i> gene (left panel) and Western blot analysis with Sirt1 antibody (right panel) demonstrates complete loss of Sirt1 protein in osteoclasts obtained from <i>Sirt1</i>^<i>Δ/Δ</i> (<i>Sirt1</i>^<i>-/-</i>) mice. (B) The effect of SRT2183 on osteoclast differentiation in <i>Sirt1</i>^<i>-/-</i>-derived BMMs. BMMs were inducted to osteoclastogenesis with RANKL in the presence or absence of SRT2183. TRAP staining performed 4 days post induction. (C) The effect of SRT2183 on pit formation in <i>Sirt1</i>^<i>-/-</i>-derived BMMs stimulated with RANKL. An eroded area (left panel) and pit formation assay (right) are shown. (D) The effect of SRT2183 on p65 acetylation (Lys310). Western blot analysis of p65K310 ac and p65 in SRT2183- and vehicle-treated osteoclasts 4 days post RANKL stimulation. (E) The effect of SRT2183 on AMPKα phosphorylation (Thr172). Western blot analysis of pAMPKα and AMPKα in SRT2183- and vehicle-treated osteoclasts 4 days post RANKL stimulation. (F) The effect of SRT2183 on IκBα protein level. Western blot analysis of IκBα and GAPDH in SRT2183- and vehicle-treated BMMs 24 hours post RANKL stimulation. (G-H) The effect of SRT2183 on Sirt3 protein (G) and gene expression (H). Western blot analysis of Sirt3 and GAPDH in SRT2183- and vehicle-treated osteoclasts 4 days post RANKL stimulation (G). Gene expression analysis by quantitative Real-Time PCR 4 days post RANKL stimulation is shown. Results are relative to <i>Polr2a</i> (H). (I) The effect of SRT2183 on superoxide dismutase 2 (Sod2) Lys68 acetylation. Western blot analysis of acetylated (ac) Sod2K68 and Sod2 in SRT2183- and vehicle-treated osteoclasts 4 days post RANKL stimulation. Data are Mean ± SEM (n = 3 independent experiments), analyzed by paired Student's <i>t</i>-test paired (C) or one-sample Student's <i>t</i>-test (H-I); ***<i>P</i><0.001, ****<i>P</i><0.0001, versus vehicle-treated BMMs. Magnification X40; scale bar 1mm.</p

SRT2183 activates AMPK and deacetylates RelA/p65 K310 in RANKL-induced BMMs.

<p>(A) The effect of SRT2183 on AMPKα phosphorylation (Thr172). Western blot analysis of pAMPKα and AMPKα in SRT2183- and vehicle-treated osteoclasts 4 days post RANKL stimulation. p- phosphorylated; AMPKα- AMP-activated protein kinase alpha. (B) The effect of SRT2183 on ACC phosphorylation. Western blot analysis of pACC and ACC in SRT2183- and vehicle-treated osteoclasts 4 days post RANKL stimulation. p- phosphorylated; ACC-Acetyl CoA Carboxylase. (C) The effect of SRT2183 on Sirt1 protein level in RANKL-stimulated osteoclasts. Western blot analysis of Sirt1 and HSP90 in SRT2183- and vehicle-treated osteoclasts 4 days post RANKL stimulation. (D) The effect of SRT2183 on IκBα protein level. Western blot analysis of IκBα and GAPDH in SRT2183- and vehicle-treated BMMs 24 hours post RANKL stimulation. (E) The effect of SRT2183 on p65 acetylation (Lys310). Western blot analysis of p65K310 ac and p65 in SRT2183- and vehicle-treated osteoclasts 4 days post RANKL stimulation. Data are Mean ±SEM (n = 3 independent experiments), analyzed by one-sample Student's <i>t</i>-test; *<i>P</i><0.05 versus vehicle-treated BMMs.</p

SRT2183 inhibits RANKL-induced osteoclastogenesis in bone marrow-derived macrophages (BMMs).

<p>(A) Chemical structure of SRT2183. (B-C) TRAP staining of SRT2183 or vehicle-treated BMMs inducted to osteoclastogenesis 4 days post RANKL stimulation (B). Total number of osteoclasts (left panel) and number of multinuclear cells (right) are shown (C). (D) The effect of SRT2183 on pit formation by RANKL-induced BMMs. A pit formation assay (left panel) and eroded area (right) are shown. (E) Time course of the effect of SRT2183 or vehicle (DMSO) administration on osteoclast differentiation. SRT2183 or a vehicle was added in the proliferation (a), differentiation (b, c) or maturation (c, d) phase. Arrows indicate periods of treatments with SRT2183. TRAP staining is shown. (F) The effect of SRT2183 on cell proliferation during the proliferation phase. SRT2183 or a vehicle were co-administrated with M-CSF for 72 hours on day of plating. (G-H) The effect of SRT2183 on cell viability and apoptosis during the proliferation phase (G) and the differentiation and maturation phase (H). SRT2183 or vehicle were co-administrated with M-CSF for 72 hours on day of plating (G) or with RANKL 3 days post plating (H). The graphs illustrate fold change in Caspase 3 activity and the percent change in living cells with time. Data are Mean ±SEM (n = 3 independent experiments), analyzed by 2 way ANOVA with nuclei number and treatment as the independent variables followed by Sidak's post-hoc correction (C), paired Student's <i>t</i>-test (D), one-sample Student's <i>t</i>-test (F-H), *<i>P</i><0.05; **<i>P</i><0.01 compared to vehicle-treated BMMs. Magnification X40; Scale bar 1mm.</p

SRT2183 inhibits RANKL-induced NFATc1 activation in bone marrow-derived macrophages (BMMs).

<p>(A) The effect of SRT2183 on NFATc1 protein level. Western blot analysis of NFATc1 and HSP90 in SRT2183- and vehicle-treated osteoclasts 4 days post RANKL stimulation. NFATc1- nuclear factor of activated T-cell cytoplasmic 1. (B) The effect of SRT2183 on DC-STAMP protein level. Western blot analysis of DC-STAMP and GAPDH in SRT2183- and vehicle-treated osteoclasts 4 days post RANKL stimulation. DC-STAMP- dendritic cell-specific transmembrane protein. (C) The effect of SRT2183 on mRNA expression of osteoclast markers and fusion-related genes. SRT2183 or vehicle were co-administrated with RANKL. Gene expression analysis by quantitative Real-Time PCR 4 days post RANKL stimulation is shown. Results are relative to <i>GAPDH</i>. Data are Mean ±SEM (n = 3 independent experiments), analyzed by one-sample Student's <i>t</i>-test, *<i>P</i><0.05; **<i>P</i><0.01; ***<i>P</i><0.001 compared to vehicle-treated BMMs.</p