Granzyme M Research Articles

Abstract 131: Concordance of mRNA expression (nCounter) and protein expression (IHC) for the detection of PD-L1 in patients with advanced non-small cell lung cancer (NSCLC)

Abstract Background: PD-L1 immunohistochemistry (IHC) staining is currently accepted as the gold-standard biomarker for immune therapy in advanced non-small cell lung cancer (NSCLC). However, the use of various antibodies and cut-offs as well as certain degree of subjectivity in pathological evaluation has overshadowed the clear-cut predictive performance of PD-L1 expression. Multiplexed technologies can be of help in this setting providing an objective measurement of PD-L1 levels. On the other hand, gene expression signatures incorporating not only PD-L1 but also other components of the stroma might better capture the immune-context of the molecular heterogeneity of NSCLC tumors. nCounter gene expression technology is an alternative method to measure PD-L1 gene expression by digital counting proving a direct measurement of mRNA levels. Methods: A 7-gene ‘immune signature’ comprising CD4, CD8, programmed cell death-1 (PD-1), programmed death-ligand 1 (PD-L1), interferon gamma (IFNG), granzyme M (GZMM) and forkhead box P3 (FOXP3) were included in a customized nCounter panel (NanoString Technologies), used in our institution on a routine basis to simultaneously screen for relevant oncogenic-drivers (ALK, ROS1, RET, NTRK1 gene fusions and METΔ14 mutation). Total RNA obtained from formalin-fixed paraffin embedded (FFPE) samples was used for PD-L1 digital counting (nCounter) which was normalised with six housekeeping genes (ACTB, MRPL19, PSMC4, RPLP0, SF3A1, GAPDH) and compared with PD-L1 protein IHC evaluation using whole tissue section with 22C3 monoclonal mouse anti-PD-L1 antibody measured on tumor cells. Results: A total of 425 FFPE samples from advanced NSCLC were analyzed with the nCounter panel. Among them, 25 samples were not evaluable (5.9%). PD-L1 IHC was available for 163 FFPE samples and were compared with nCounter PD-L1 expression results. By IHC, 63/163 samples (38.65%) were scored as negative for PD-L1 protein expression, whereas 100/163 (61.35%) were evaluated as positive. Among positive, 62 (38.04%) and 38 (23.31%) presented a moderate (≥ 1-49%) and high PD-L1 staining (≥50%) respectively. Using an appropriate cut-off value (IHC≥1%), PD-L1 mRNA expression levels correlated with PD-L1 IHC evaluation with a 76% of concordance and a 0.755 Cohen’s kappa (confidence interval 95% 0.651- 0.858). Unsupervised clustering across of mRNA expression data from 395 samples using the seven-immune-related genes and correlations between each immune gene were performed and a high correlation was found between PD-1 and FOXP3 (r=0.9) and PD-1 with GZMM (r=0.8). Conclusions: PD-L1 mRNA gene expression shows promising in predicting PD-L1 protein expression in NSCLC. Further clinical validation is ongoing to confirm if PD-L1 gene expression by nCounter can be an alternative to IHC to select patients’ candidates for immune check-point inhibitors. Citation Format: Cristina Teixido, Elba Marin, Cristina Aguado, Laia Pare, Ana Gimenez-Capitan, Sandra Lopez-Prades, Andres Felipe Cardona, Carlos Cabrera, Elena Gonzalvo, Laura Lopez, Ruth Roman, Daniel Martinez, Ivana Sullivan, Pedro Jares, Aleix Prat, Miguel Angel Molina-Vila, Noemi Reguart. Concordance of mRNA expression (nCounter) and protein expression (IHC) for the detection of PD-L1 in patients with advanced non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 131.

Granzyme M and K release in human experimental endotoxemia

Granzymes are serine proteases involved in killing of tumor cells and virally infected cells. However, granzymes are also upregulated in blood under inflammatory conditions and contribute to cytokine release and processing. Here, we show that granzyme M (GrM) and to a lesser extent GrK are transiently elevated in the circulation following LPS administration in humans. GrM is released upon stimulation of whole blood with LPS or the gram-negative bacteria Escherichia coli BL21, Pseudomonas aeruginosa, and Neisseria meningitidis. GrK is only released upon stimulation with P. aeruginosa. Thus, GrM and GrK are differentially released in response to LPS and gram-negative bacteria.

Granzyme M: behind enemy lines

The granule-exocytosis pathway is the major mechanism via which cytotoxic lymphocytes eliminate virus-infected and tumor cells. In this pathway, cytotoxic lymphocytes release granules containing the pore-forming protein perforin and a family of serine proteases known as granzymes into the immunological synapse. Pore-formation by perforin facilitates entry of granzymes into the target cell, where they can activate various (death) pathways. Humans express five different granzymes, of which granzymes A and B have been most extensively characterized. However, much less is known about granzyme M (GrM). Recently, structural analysis and advanced proteomics approaches have determined the primary and extended specificity of GrM. GrM functions have expanded over the past few years: not only can GrM efficiently induce cell death in tumor cells, it can also inhibit cytomegalovirus replication in a noncytotoxic manner. Finally, a role for GrM in lipopolysaccharide-induced inflammatory responses has been proposed. In this review, we recapitulate the current status of GrM expression, substrate specificity, functions, and inhibitors.

Elevated granzyme M-expressing lymphocytes during cytomegalovirus latency and reactivation after allogeneic stem cell transplantation

Human cytomegalovirus (HCMV) reactivation can cause serious complications in allogeneic stem cell transplantation (SCT) patients. HCMV is controlled by cytotoxic lymphocytes that release antiviral granzymes. Recently, we have demonstrated that granzyme M (GrM) inhibits HCMV replication in vitro, however the physiological role of GrM and its cellular distribution during HCMV infection remains unknown. Here, we examined GrM expression in lymphocyte populations during HCMV infection. The percentage of GrM-expressing effector-memory CD4(+) T-cells was higher in HCMV latently-infected healthy individuals compared to that of uninfected individuals. SCT recipients had higher percentages of GrM-expressing CD4(+) T, CD8(+) T, γδT, and NKT cells. Despite lower total T-cell numbers, HCMV reactivation in SCT patients specifically associated with higher percentages of GrM-expressing CD4(+) (total and central-memory) T-cells. GrM was elevated in plasma during HCMV reactivation, pointing to extracellular perforin-independent functions of GrM. We conclude that GrM may be important in regulating HCMV latency and reactivation in SCT patients.

Granzyme M targets topoisomerase II alpha to trigger cell cycle arrest and caspase-dependent apoptosis

Cytotoxic lymphocyte protease granzyme M (GrM) is a potent inducer of tumor cell death. The apoptotic phenotype and mechanism by which it induces cell death, however, remain poorly understood and controversial. Here, we show that GrM-induced cell death was largely caspase-dependent with various hallmarks of classical apoptosis, coinciding with caspase-independent G2/M cell cycle arrest. Using positional proteomics in human tumor cells, we identified the nuclear enzyme topoisomerase II alpha (topoIIα) as a physiological substrate of GrM. Cleavage of topoIIα by GrM at Leu(1280) separated topoIIα functional domains from the nuclear localization signals, leading to nuclear exit of topoIIα catalytic activity, thereby rendering it nonfunctional. Similar to the apoptotic phenotype of GrM, topoIIα depletion in tumor cells led to cell cycle arrest in G2/M, mitochondrial perturbations, caspase activation, and apoptosis. We conclude that cytotoxic lymphocyte protease GrM targets topoIIα to trigger cell cycle arrest and caspase-dependent apoptosis.

Granzyme M targets host cell hnRNP K that is essential for human cytomegalovirus replication.

Human cytomegalovirus (HCMV) is the most frequent viral cause of congenital defects and HCMV infection in immunocompromised patients may trigger devastating disease. Cytotoxic lymphocytes control HCMV by releasing granzymes towards virus-infected cells. In mice, granzyme M (GrM) has a physiological role in controlling murine CMV infection. However, the underlying mechanism remains poorly understood. In this study, we showed that human GrM was expressed by HCMV-specific CD8(+) T cells both in latently infected healthy individuals and in transplant patients during primary HCMV infection. We identified host cell heterogeneous nuclear ribonucleoprotein K (hnRNP K) as a physiological GrM substrate. GrM most efficiently cleaved hnRNP K in the presence of RNA at multiple sites, thereby likely destroying hnRNP K function. Host cell hnRNP K was essential for HCMV replication not only by promoting viability of HCMV-infected cells but predominantly by regulating viral immediate-early 2 (IE2) protein levels. Furthermore, hnRNP K interacted with IE2 mRNA. Finally, GrM decreased IE2 protein expression in HCMV-infected cells. Our data suggest that targeting of hnRNP K by GrM contributes to the mechanism by which cytotoxic lymphocytes inhibit HCMV replication. This is the first evidence that cytotoxic lymphocytes target host cell proteins to control HCMV infections.

Cleavage of von Willebrand Factor by Granzyme M Destroys Its Factor VIII Binding Capacity

Von Willebrand factor (VWF) is a pro-hemostatic multimeric plasma protein that promotes platelet aggregation and stabilizes coagulation factor VIII (FVIII) in plasma. The metalloproteinase ADAMTS13 regulates the platelet aggregation function of VWF via proteolysis. Severe deficiency of ADAMTS13 is associated with thrombotic thrombocytopenic purpura, but does not always correlate with its clinical course. Therefore, other proteases could also be important in regulating VWF activity. In the present study, we demonstrate that VWF is cleaved by the cytotoxic lymphocyte granule component granzyme M (GrM). GrM cleaved both denaturated and soluble plasma-derived VWF after Leu at position 276 in the D3 domain. GrM is unique in that it did not affect the multimeric size and pro-hemostatic platelet aggregation ability of VWF, but instead destroyed the binding of VWF to FVIII in vitro. In meningococcal sepsis patients, we found increased plasma GrM levels that positively correlated with an increased plasma VWF/FVIII ratio in vivo. We conclude that, next to its intracellular role in triggering apoptosis, GrM also exists extracellularly in plasma where it could play a physiological role in controlling blood coagulation by determining plasma FVIII levels via proteolytic processing of its carrier VWF.

Human and mouse granzyme M display divergent and species-specific substrate specificities

Cytotoxic lymphocyte protease GrM (granzyme M) is a potent inducer of tumour cell death and a key regulator of inflammation. Although hGrM (human GrM) and mGrM (mouse GrM) display extensive sequence homology, the substrate specificity of mGrM remains unknown. In the present study, we show that hGrM and mGrM have diverged during evolution. Positional scanning libraries of tetrapeptide substrates revealed that mGrM is preferred to cleave after a methionine residue, whereas hGrM clearly favours a leucine residue at the P1 position. The kinetic optimal non-prime subsites of both granzymes were also distinct. Gel-based and complementary positional proteomics showed that hGrM and mGrM have a partially overlapping set of natural substrates and a diverged prime and non-prime consensus cleavage motif with leucine and methionine residues being major P1 determinants. Consistent with positional scanning libraries of tetrapeptide substrates, P1 methionine was more frequently used by mGrM as compared with hGrM. Both hGrM and mGrM cleaved α-tubulin with similar kinetics. Strikingly, neither hGrM nor mGrM hydrolysed mouse NPM (nucleophosmin), whereas human NPM was hydrolysed efficiently by GrM from both species. Replacement of the putative P1'-P2' residues in mouse NPM with the corresponding residues of human NPM restored cleavage of mouse NPM by both granzymes. This further demonstrates the importance of prime sites as structural determinants for GrM substrate specificity. GrM from both species efficiently triggered apoptosis in human but not in mouse tumour cells. These results indicate that hGrM and mGrM not only exhibit divergent specificities but also trigger species-specific functions.

The cytotoxic protease granzyme M is expressed by lymphocytes of both the innate and adaptive immune system

The cytotoxic serine protease granzyme M (GrM) is one of the five human granzymes, which are mainly expressed by cytotoxic T lymphocytes and/or NK cells. Upon perforin-dependent entry into a target cell, GrM cleaves specific substrates resulting in the onset of a unique cell death mechanism. However, the role of GrM in pathophysiological conditions is not clear yet. Knowledge of the expression and regulation of GrM by lymphocyte populations is instrumental for a better understanding of the contribution of this unique granzyme in health and disease. Two previous studies demonstrated GrM protein expression by lymphocytes of the innate immune system, i.e., NK cells, NKT cells, and γδ T cells, whereas its expression by CD8 + T cells remained controversial. In the present study, we have investigated the expression and regulation of GrM in lymphocyte subsets in more detail. Flow cytometry analysis with a novel specific antibody against human GrM confirmed high expression of this protease by NK cells, NKT cells, and γδ T cells. CD8 + T cells also expressed GrM and comparing the naive to early effector-memory, to late effector-memory, to effector subset, this expression gradually increased during differentiation. In contrast, CD4 + T cells hardly expressed GrM. Quantitative PCR analysis for GrM mRNA levels in the diverse lymphocyte sub-populations confirmed the FACS results. GrM protein expression by lymphocyte populations was not significantly affected by a panel of GrB-inducing cytokines, indicating that GrM expression is differentially regulated as compared to GrB. In conclusion, the human cytotoxic protease GrM is, besides by innate immune cells, also expressed by CD8 + effector T cells, in particular by the differentiated effector CD27 − CD45RO − subset. Our current findings support not only a role for GrM in the innate but also in the adaptive immune response.

NK Cell Protease Granzyme M Targets α-Tubulin and Disorganizes the Microtubule Network

Serine protease granzyme M (GrM) is highly expressed in the cytolytic granules of NK cells, which eliminate virus-infected cells and tumor cells. The molecular mechanisms by which GrM induces cell death, however, remain poorly understood. In this study we used a proteomic approach to scan the native proteome of human tumor cells for intracellular substrates of GrM. Among other findings, this approach revealed several components of the cytoskeleton. GrM directly and efficiently cleaved the actin-plasma membrane linker ezrin and the microtubule component alpha-tubulin by using purified proteins, tumor cell lysates, and tumor cells undergoing cell death induced by perforin and GrM. These cleavage events occurred independently of caspases or other cysteine proteases. Kinetically, alpha-tubulin was more efficiently cleaved by GrM as compared with ezrin. Direct alpha-tubulin proteolysis by GrM is complex and occurs at multiple cleavage sites, one of them being Leu at position 269. GrM disturbed tubulin polymerization dynamics in vitro and induced microtubule network disorganization in tumor cells in vivo. We conclude that GrM targets major components of the cytoskeleton that likely contribute to NK cell-induced cell death.

The expression of the novel cytotoxic protein granzyme M by large granular lymphocytic leukaemias of both T‐cell and NK‐cell lineage: an unexpected finding with implications regarding the pathobiology of these disorders

Granzyme M (GrM) is a novel cytotoxic protein normally exclusively expressed by natural killer (NK)-cells and cytotoxic T-cells with innate immune function. As most T-cell granular lymphocytic leukaemias (T-LGL) are thought to be derived from the adaptive immune system it was predicted that T-LGL would be GrM negative. Contrary to this hypothesis, bone marrow biopsy immunohistochemistry revealed that GrM was frequently expressed in both T-LGL (16 / 18) and NK-LGL (6 / 9). These unexpected results suggest commonality between T- and NK-LGL, providing further support to the notion that T-LGL is a disorder of dysregulated, chronically stimulated, adaptive cytotoxic T-cells.

Serpins prevent granzyme‐induced death in a species‐specific manner

Expression of serine protease inhibitors (serpins) is one of the mechanisms used by tumour cells to escape immune surveillance. Previously, we have shown that expression of serpins SPI-6 and SPI-CI, respectively, renders tumour cells resistant to granzyme B (GrB)-mediated death and granzyme M (GrM)-mediated death. To obtain better insight into the interaction between serpins and their target proteases, we investigated the roles of protease inhibitor (PI)-9 and SPI-6 in the resistance to GrB-mediated and CD95-mediated death in further detail. Neither human PI-9 nor its murine orthologue SPI-6 was capable of preventing CD95-induced apoptosis in murine or human cells, indicating that these serpins do not inhibit the activation of apical caspases in this pathway. High expression of PI-9 or SPI-6 did prevent apoptosis induced by human GrB. Strikingly, only SPI-6, and not PI-9, was capable of inhibiting murine GrB, suggesting that a difference in enzymatic specificity exists between the mouse and the human granzymes. In agreement with this suggestion, murine GrB was clearly less effective in inducing apoptosis in human cells. Similar species specificity was also observed for SPI-CI and GrM when either their capacity to associate or the effectiveness of GrM-induced cytotoxicity was analysed. Our findings therefore indicate a species diversity that has a clear effect on mixed in vitro effector target settings.