Serpins form a family of structurally related proteins, many of which function in plasma as inhibitors of serine proteases involved in inflammation, blood coagulation, fibrinolysis, and complement activation. To further characterize the mechanism by which serpins inhibit their target enzymes, we have studied the effect of temperature on the reaction of C1 inhibitor and the serine protease plasma kallikrein. At both 38 and 4 degrees C, C1 inhibitor (Mr 105,000) is cleaved by alpha-kallikrein (Mr 85,000 and 88,000) at position P1 (Arg444) of the reactive center, a reaction that leads to the formation of a covalent bimolecular enzyme-serpin complex (Mr 195,000) and cleaved but uncomplexed serpin (Mr 95,000). Between 38 and 4 degrees C, the product distribution is temperature-dependent, with more cleaved C1 inhibitor (Mr 95,000) formed at lower temperatures and correspondingly less Mr 195,000 complex. Studies employing intrinsic tryptophan fluorescence and 1H NMR spectroscopy show that this behavior is not caused by temperature-dependent conformational changes of kallikrein or C1 inhibitor. C1 inhibitor also behaves in this manner with the light chain of kallikrein and, to a lesser extent, with plasmin and C1s. These data are best explained by a branched reaction pathway, identical with the scheme describing the mechanism of action of suicide substrates. This scheme involves the formation of an enzyme-inhibitor intermediate, which can be stabilized into a covalent complex and/or dissociate into free enzyme and cleaved inhibitor, depending on the reaction conditions.

Introduction Renal Cell Carcinoma (RCC) is the deadliest urological malignancy. Profiling its complex microenvironment (TME) in situ is crucial to understand the mechanisms of progression and immune evasion that lead to metastasis and death. NanoString® GeoMx™ Digitial Spatial Profiling (DSP) platform facilitates these studies by enabling highly multiplexed, spatially resolved characterisation of proteins and RNA from FFPE tissue. DSP visualises and quantifies targets from areas of interest (AOI) using oligonucleotide-conjugated antibodies. Here, DSP is combined with automated image analysis (IA). When coupled with multiplexed immunofluorescence (IF), IA is able to automatically segment tumor from stroma and profile marker co-expression at single cell level. We present the advantages of using a combinatorial strategy, applied to clear cell RCC (ccRCC) tissue sections, in order to predict patient outcome. Methods 165 patients, grouped into 11 tumour microarray (TMA) slides were labelled with multiplex IF and scanned with a Zeiss Axioscan.z1. Scans were imported into Definiens Tissue Studio® IA software. Multiple TMA cores were sampled from matched non-cancerous kidney, primary, and venous thrombus (VTT) ccRCC. Tumor regions (labelled with Pan-cadherin and CA9) and stroma were segmented prior to automated immune quantification, where CD3, CD163, PD-1 and PD-L1 antibodies were used to profile the immune contexture. DSP was performed on the corresponding serial sections, where a 60-plex antibody panel was applied to each TMA core. Statistical analysis was performed on R Studio, where cox-proportional hazard ratios and Kaplan-Meier curves were used to correlate marker densities to risk of metastasis and cancer-related death. Results Both IA and DSP associated M2 macrophages (CD163) and T cells (CD3) to increased risk of metastasis and poor survival. IA demonstrated that tumor/stroma segmentation and single cell marker co-registration complements DSP analysis by allowing a more detailed profiling of the TME. In particular, a high density of PD-L1 positive tumor cells and PD-1 positive T-cells were correlated to poor survival in VTT and non-cancerous cores, respectively. DSP's high-plex ability is useful to investigate the relationship among the proteins of interest. It confirmed the T-cell exhaustion marker TIM-3 as a poor prognostic factor, thus demonstrating that quantifying only CD3 positive T cells may be insufficient to predict a precise prognosis. Conclusions This data demonstrates that both co-registration of cellular protein expression and highly plexed analysis can add value to the prediction of patient outcome and the risk of metastasis. We further report the prognostic significance of analysing the molecular signature of the immune contexture in both ccRCC tumorous and its adjacent non-cancerous tissue. Citation Format: Raffaele De Filippis, Sarah Warren, Youngmi Kim, Andrew White, Jason Reeves, Grant D. Stewart, David J. Harrison, Joe M. Beechem, Peter D. Caie. Combining automated image analysis and digital spatial profiling to investigate prognostic immune signatures in clear cell renal cell carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2670.

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New therapeutic modalities are required for an effective and recurrence-free lasting treatment of metastatic melanoma, which is one of the deadliest forms of cutaneous malignancy. 4'-Bromo-resveratrol (4BR), is a dual small molecule inhibitor of Sirtuin-1 and -3, both of which play tumor-promoting roles in melanoma. Earlier, we showed that 4BR imparted anti-proliferative effects against human melanoma cells in vitro. Here, we determined the in vivo efficacy and mechanism of 4BR against melanoma in a genetically engineered BRAF V600E / PTEN Null mouse model that recapitulates human disease with lymph nodes and lung metastases. Tumors were induced by topical application of 4-hydroxytamoxifen on shaved backs of 10-week-old mice, and the effects of 4-BR (30 mg/kg b.wt.; intraperitoneally; 3d/week for 5 weeks) treatment were assessed on pigmented melanoma tumors. 4BR treatment significantly reduced the size and volume of primary melanoma tumors, as well as the metastatic burden in lungs with no adverse effects. Further, mechanistic studies with skin/tumor samples demonstrated downregulation of cell proliferation markers (Ki67, PCNA, Survivin), melanoma promoting growth factor IGF1, as well as upregulation of the tumor suppressor protein IGFBP5. As Sirtuins-1 and -3 are linked to immunomodulation, to explore the mechanism of 4BR treatment, we performed differential gene expression analysis using the nCounter Pan-Cancer Immune panel (770 genes) and nSolver software. Results indicated that 4BR significantly downregulated (2-fold) expression of multiple genes promoting melanoma metastasis (Fn1, S1008a, Fap, Col3a1, Angpt2, Itga1, and Ptgs2), associated with chemokine/cytokines and their receptors (Ccr1, Ccl6, Il1b, Ccl24, Ccl9, Il1rl1 and Ccr5), and innate/adaptive immunity (Itgam, Nlrp3, Colec12 and Irf7). Overall, sirtuin inhibition by 4BR (or other means) appears to be a promising anti-cancer therapy with anti-metastatic and immunomodulatory effects, warranting further studies, including clinical investigations.

Imatinib mesylate (imatinib, Gleevec®, Novartis, Basel, Switzerland) inhibits T cells in vitro and in vivo (Dietz et al., Blood104: 1094–1099, 2004; Cwynarski et al., Leukemia18: 1332–1339, 2004). The drug blocks T cell cycle progression rather uniquely as it neither inhibits expression of CD69, an early marker of T cell activation, nor induces apoptosis. To characterize the molecular effects of imatinib leading to this mode of T-cell inhibition, we measured the changes in transcriptome (by Affymetrix U133 chips), proteome and phosphoproteome (by Western blotting, differential phosphoprotein expression and mass spectrometry). We found that phytohemagglutinin activated T cells pre-treated with imatinib had reduced expression of 983 transcripts and increased expression of 271 transcripts when compared to untreated PHA activated T cells by the factor of 1.5 or more (p<0.05). Among the prominently down-regulated transcripts were granzyme B, CTLA-4 and IL-2-receptor α-chain (CD25), all characteristic of activated T cells, as well as cyclins D2 and D3 and cyclin-dependent kinases 3, 4 and 7, the molecules regulating cell cycle progression. Among the up-regulated transcripts were Kruppel-like transcription factors 2 and 7, and p27, a finding compatible with the observed cell cycle inhibition. Furthermore, we selected and identified 30 proteins from 2-D gels that were up-regulated and/or hyperphosphorylated in imatinib treated activated T cells. Among these were four heterogeneous ribonucleoproteins, three lamins and γ-actin, all components of the nucleoskeleton at the interface of chromatin and inner nuclear membrane and involved in replication and transcription (Herrmann and Foisner, Cell. Mol. Life Sci.60: 1607–1612, 2003; Shumaker et al. Curr. Opinion Cell Biol.15: 358–366, 2003). Thus, imatinib-borne interference with T cell signal transduction affects the nuclear structure indicating for the first time that nucleoskeleton structural changes are associated with T cell activation status.