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.

Selective but not pan-CDK inhibition abrogates 5-FU-driven tissue factor upregulation in colon cancer.

Thromboembolic events are complications in cancer patients and hypercoagulability has been linked to the tissue factor (TF) pathway, making this an attractive target. Here, we investigated the effects of chemotherapeutics and CDK inhibitors (CDKI) abemaciclib/palbociclib (CDK4/6), THZ-1 (CDK7/12/13), and dinaciclib (CDK1/2/5/9) alone and in combination regimens on TF abundance and coagulation. The human colorectal cancer (CRC) cell line HROC173 was treated with 5-FU or gemcitabine to stimulate TF expression. TF+ cells were sorted, recultured, and re-analyzed. The effect of treatment alone or in combination was assessed by functional assays. Low-dose chemotherapy induced a hypercoagulable state and significantly upregulated TF, even after reculture without treatment. Cells exhibited characteristics of epithelial-mesenchymal transition, including high expression of vimentin and mucin. Dinaciclib and THZ-1 also upregulated TF, while abemaciclib and palbociclib downregulated it. Similar results were observed in coagulation assays. The same anticoagulant activity of abemaciclib was seen after incubation with peripheral immune cells from healthy donors and CRC patients. Abemaciclib reversed 5-FU-induced TF upregulation and prolonged clotting times in second-line treatment. Effects were independent of cytotoxicity, senescence, and p27kip1 induction. TF-antibody blocking experiments confirmed the importance of TF in plasma coagulation, with Factor XII playing a minor role. Short-term abemaciclib counteracts 5-FU-induced hypercoagulation and eventually even prevents thromboembolic events.

Short-term immune-checkpoint inhibition partially rescues perturbed bone marrow hematopoiesis in mismatch-repair deficient tumors.

Wide-spread cancer-related immunosuppression often curtails immune-mediated antitumoral responses. Immune-checkpoint inhibitors (ICIs) have become a state-of-the-art treatment modality for mismatch repair-deficient (dMMR) tumors. Still, the impact of ICI-treatment on bone marrow perturbations is largely unknown. Using anti-PD1 and anti-LAG-3 ICI treatments, we here investigated the effect of bone marrow hematopoiesis in tumor-bearing Msh2loxP/loxP;TgTg(Vil1-cre) mice. The OS under anti-PD1 antibody treatment was 7.0 weeks (vs. 3.3 weeks and 5.0 weeks, control and isotype, respectively). In the anti-LAG-3 antibody group, OS was 13.3 weeks and thus even longer than in the anti-PD1 group (p = 0.13). Both ICIs induced a stable disease and reduced circulating and splenic regulatory T cells. In the bone marrow, a perturbed hematopoiesis was identified in tumor-bearing control mice, which was partially rescued by ICI treatment. In particular, B cell precursors and innate lymphoid progenitors were significantly increased upon anti-LAG-3 therapy to levels seen in tumor-free control mice. Additional normalizing effects of ICI treatment were observed for lin-c-Kit+IRF8+ hematopoietic stem cells, which function as a "master" negative regulator of the formation of polymorphonuclear-myeloid-derived suppressor cell generation. Accompanying immunofluorescence on the TME revealed significantly reduced numbers of CD206+F4/80+ and CD163+ tumor-associated M2 macrophages and CD11b+Gr1+ myeloid-derived suppressor cells especially upon anti-LAG-3 treatment. This study confirms the perturbed hematopoiesis in solid cancer. Anti-LAG-3 treatment partially restores normal hematopoiesis. The interference of anti-LAG-3 with suppressor cell populations in otherwise inaccessible niches renders this ICI very promising for subsequent clinical application.