Prophylaxis with abemaciclib delays tumorigenesis in dMMR mice by altering immune responses and reducing immunosuppressive extracellular vesicle secretion.

BACKGROUND: The CDK4/6 inhibitor abemaciclib is an FDA-approved agent and induces T-cell-mediated immunity. Previously, we confirmed the therapeutic potential of abemaciclib on mismatch repair-deficient (dMMR) tumors in mice. Here, we applied a prophylactic administration/dosage setting using two preclinical mouse models of dMMR-driven cancer. METHODS: Mlh1-/- and Msh2loxP/loxP mice received repeated prophylactic applications of abemaciclib mesylate (75 mg/kg bw, per oral) as monotherapy or were left untreated. Blood phenotyping and multiplex cytokine measurements were performed regularly. The tumor microenvironment was evaluated by immunofluorescence and Nanostring-based gene expression profiling. Numbers, size and immune composition and activity of extracellular vesicles (EVs) were studied at the endpoint. FINDINGS: Prophylactic abemaciclib-administration delayed tumor development and significantly prolonged overall survival in both mouse strains (Mlh1-/-: 50.0 wks vs. control: 33.9 wks; Msh2loxP/loxP;TgTg(Vil1-cre: 58.4 wks vs. control 44.4 wks). In Mlh1-/- mice, pro-inflammatory cytokines (IL-2, IL-6) significantly increased, whereas IL-10 and IL-17A decreased. Circulating and splenic exhausted and regulatory T cell numbers were significantly lower in the abemaciclib groups. Deeper analysis of late-onset tumors revealed activation of the Hedgehog and Notch signaling in Mlh1-/- mice, and activation of the MAPK pathway in Msh2loxP/loxP;TgTg(Vil1-cre mice. Still, arising tumors had fewer infiltrating myeloid-derived suppressor cells (vs. control). Notably, prophylactic abemaciclib-administration prevented secretion of procoagulant EVs but triggered release of immunomodulatory EVs in Mlh1-/- mice. INTERPRETATION: Prophylactic abemaciclib prolongs survival via global immunomodulation. Prophylactic use of abemaciclib should be considered further for individuals with inherited dMMR. FUNDING: This work was supported by grants from the German research foundation [DFG grant number: MA5799/2-2] and the Brigitte und Dr. Konstanze Wegener-Stiftung to CM.

Endothelin-1 influences mechanical properties and contractility of hiPSC derived cardiomyocytes resulting in diastolic dysfunction.

A better understanding of the underlying pathomechanisms of diastolic dysfunction is crucial for the development of targeted therapeutic options with the aim to increase the patients' quality of life. In order to shed light on the processes involved, suitable models are required. Here, effects of endothelin-1 (ET-1) treatment on cardiomyocytes derived from human induced pluripotent stem cells (hiPSCs) were investigated. While it is well established, that ET-1 treatment induces hypertrophy in cardiomyocytes, resulting changes in cell mechanics and contractile behavior with focus on relaxation have not been examined before. Cardiomyocytes were treated with 10 nM of ET-1 for 24 h and 48 h, respectively. Hypertrophy was confirmed by real-time deformability cytometry (RT-DC) which was also used to assess the mechanical properties of cardiomyocytes. For investigation of the contractile behavior, 24 h phase contrast video microscopy was applied. To get a deeper insight into changes on the molecular biological level, gene expression analysis was performed using the NanoString nCounter® cardiovascular disease panel. Besides an increased cell size, ET-1 treated cardiomyocytes are stiffer and show an impaired relaxation. Gene expression patterns in ET-1 treated hiPSC derived cardiomyocytes showed that pathways associated with cardiovascular diseases, cardiac hypertrophy and extracellular matrix were upregulated while those associated with fatty acid metabolism were downregulated. We conclude that alterations in cardiomyocytes after ET-1 treatment go far beyond hypertrophy and represent a useful model for diastolic dysfunction.

Identifying Predictive Biomarkers for Head and Neck Squamous Cell Carcinoma Response.

The 5-year survival rate for head and neck squamous cell carcinoma (HNSCC) is approximately 65%. In addition to radio-chemotherapy, immunotherapy is an approach in the treatment of advanced HNSCC. A better understanding of the immune context would allow personalized treatment by identifying patients who are best suited for different treatment options. In our discovery cohort, we evaluated the expression profiles of CMTM6, PD-L1, CTLA-4, and FOXP3 in 177 HNSCCs from Caucasian patients of all tumor stages and different treatment regimens, correlating marker expression in tumor and immune cells with outcomes. Patients with CMTM6high-expressing tumors had a longer overall survival regardless of treatment. This prognostic benefit of CMTM6 in HNSCC was validated in an independent cohort. Focusing on the in the discovery cohort (n = 177), a good predictive effect of CMTM6high expression was seen in patients receiving radiotherapy (p = 0.07; log rank), but not in others. CMTM6 correlated with PD-L1, CTLA-4 and FOXP3 positivity, with patients possessing CMTM6high/FOXP3high tumors showing the longest survival regardless of treatment. In chemotherapy-treated patients, PD-L1 positivity was associated with longer progression-free survival (p < 0.05). In the 27 patients who received immunotherapy, gene expression analysis revealed lower levels of CTLA-4 and FOXP3 with either partial or complete response to this treatment, while no effect was observed for CMTM6 or PD-L1. The combination of these immunomodulatory markers seems to be an interesting prognostic and predictive signature for HNSCC patients with the ability to optimize individualized treatments.

CDK4/6 blockade provides an alternative approach for treatment of mismatch-repair deficient tumors.

Mismatch repair-deficient (dMMR) tumors show a good response toward immune checkpoint inhibitors (ICI), but developing resistance impairs patients' outcomes. Here, we compared the therapeutic potential of an α-PD-L1 antibody with the CDK4/6 inhibitor abemaciclib in two preclinical mouse models of dMMR cancer, focusing on immune-modulatory effects of either treatment. Abemaciclib monotherapy significantly prolonged overall survival of Mlh1-/- and Msh2loxP/loxP;TgTg(Vil1- cre) mice (Mlh1-/-: 14.5 wks vs. 9.0 wks (α-PD-L1), and 3.5 wks (control); Msh2loxP/loxP;TgTg(Vil1- cre): 11.7 wks vs. 9.6 wks (α-PD-L1), and 2.0 wks (control)). The combination was not superior to either monotherapy. PET/CT imaging revealed individual response profiles, with best clinical responses seen with abemaciclib mono- and combination therapy. Therapeutic effects were accompanied by increasing numbers of tumor-infiltrating CD4+/CD8+ T-cells and lower numbers of M2-macrophages. Levels of T cell exhaustion markers and regulatory T cell counts declined. Expression analysis identified higher numbers of dendritic cells and neutrophils within tumors together with high expression of DNA damage repair genes as part of the global stress response. In Mlh1-/- tumors, abemaciclib suppressed the PI3K/Akt pathway and led to induction of Mxd4/Myc. The immune-modulatory potential of abemaciclib renders this compound ideal for dMMR patients not eligible for ICI treatment.