1Universitätsklinikum Heidelberg Klinik für Innere Med. III, Kardiologie, Angiologie u. Pneumologie Heidelberg, Deutschland
The broad utilization of immune checkpoint inhibitors (ICIs) dramatically improved cancer prognosis but recently leads to an increase in the severe adverse events of ICI-associated myocarditis (ICIM). So far, the underlying pathway is not fully understood.
To investigate the mechanism further, we aimed to characterize its transcriptional consequences in patients’ biopsies to explore unique, ICIM-defining alterations. Thus, we compared transcriptional changes of ICIM (n=9) with dilatative cardiomyopathy (DCM, n=11), virus-induced myocarditis (VIM, n=5) and patients receiving ICIs without confirmed evidence of myocarditis (n=4) in bulk-RNA-seq. We found 3784 upregulated genes in ICIM (FDR<0.05) compared to VIM or DCM. Within those genes, we identified an ICIM-defining gene pattern, including the genes of guanylate binding protein 5 and 6 (compared to VIM: GBP5 (log2 fc 3.21), GBP6 (log2 fc 5.37)). Both are prominent members of the interferon-gamma (IFNg) pathway and crucial for the assembly of the inflammasome. Histological assessments of ICIM-patients’ biopsies and an in vitro model of co-cultured neonatal rat cardiomyocytes (NRVM) with human mononuclear cells (hPBMC) revealed an upregulation of GBP5 as a cardiomyocyte-specific event (fc NRVM+hPBMC+PD1i/NRVM+PBMC=2.12, p=0.0286, Mann-Whitney Test).
To further investigate the biological relevance of GBP5 in ICIM in vivo we used CRISPR/Cas9 to generate an inducible, cardiomyocyte-specific Lox/Cre-driven knock-in truncation of GBP5 in C57BL6 mice (GBP5-icKI). The truncation, lacking the C-terminus of GBP5 results in a dysfunctional protein, which is no longer being able to form tetramers, which is essential for the inflammasome assembly. To additionally assess the interactions of tumors on these side effects and to create a tumor microenvironment, we used a tumor model (Ret-melanoma) in the GBP5-icKI mice by injecting melanoma cells subcutaneously. Age-matched wild-type (WT) C57BL/6N mice without tumor inoculation were included as controls. Both groups were subjected to combination anti–PD-1 and anti–CTLA-4 antibody treatment.
GBP5-icKI mice (n=3) showed no significant difference in the T-cell infiltration after antibody treatment in comparison control littermates (n=3). In contrast, antibody treatment leads to a higher CD8+-T-cell-infiltration in the wildtype animals (n=3) in comparison to the control animals. We also observed a significantly reduction of CD8+T-cells in the spleen of treated WT animals in comparison to untreated control group (CD8+ mean ± SEM: WT treated (n=3) 17.3% ± 1.65; WT control (n=3) 9.30 ± 0.56, p=0.0049, Mann-Whitney Test). In ICI-treated GBP5-icKI animals, a significantly upregulation of specific inflammation genes in the heart was blunted (e.g. IL6 and Caspase 1).
GBP5-icKI were protected from reduction in LVEF (left ventricular ejection fraction; mean ± SEM: GBP5-icKI + ICI (n=9) 52.5%±2.3%, GBP5-KI (n=6) 52.8%±2.8%) while wildtype animals showed a reduced LVEF upon ICI-therapy (mean ± SEM: WT treated (n=6) 50.3%±1.2%, WT untreated (n=6) 62.6%±2.5%, p=0.0087, Mann-Whitney Test).
In conclusion, we identified GBP5 in cardiomyocytes as a novel inflammatory regulators in cardiomyocytes, crucial for the pathogenesis of the ICIM. Therefore, pharmacological disruption of this molecular pathway may be a promising target for a specific treatment of ICIM.