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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

Cyclin-dependent kinases (CDK) control the cell cycle and play a crucial role in oncogenesis. Pharmacologic inhibition of CDK has contributed to the recent clinical approval of dual CDK4/6 inhibitors for the treatment of breast and small cell lung cancer. While the anticancer cell effects of CDK inhibitors are well-established, preclinical and early clinical studies describe additional mechanisms of action such as chemo- and radiosensitization or immune stimulation. The latter offers great potential to incorporate CDK inhibitors in immune-based treatments. However, dosing schedules and accurate timing of each combination partner need to be respected to prevent immune escape and resistance. In this review, we provide a detailed summary of CDK inhibitors in the two solid cancer types head and neck cancer and glioblastoma multiforme; it describes the molecular mechanisms of response vs. resistance and covers strategies to avoid resistance by the combination of immunotherapy or targeted therapy. Supplementary Information The online version contains supplementary material available at 10.1007/s10555-020-09940-4.

This study aimed to refine combined targeted approaches on well-characterized, low-passage tumor models. Upon in vivo xenografting in immunodeficient mice, three cell lines from locally advanced or metastatic HNSCC were established. Following quality control and basic characterization, drug response was examined after therapy with 5-FU, Cisplatin, and cyclin-dependent kinase inhibitors (abemaciclib, THZ1). Our cell lines showed different in vitro growth kinetics, morphology, invasive potential, and radiosensitivity. All cell lines were sensitive to 5-FU, Cisplatin, and THZ1. One cell line (HNSCC48 P0 M1) was sensitive to abemaciclib. Here, Cyto-FISH revealed a partial CDKN2a deletion, which resulted from a R58* mutation. Moreover, this cell line demonstrated chromosome 12 polysomy, accompanied by an increase in CDK4-specific copy numbers. In HNSCC16 P1 M1, we likewise identified polysomy-associated CDK4-gains. Although not sensitive to abemaciclib per se, the cell line showed a G1-arrest, an increased number of acidic organelles, and a swollen structure. Notably, intrinsic resistance was conquered by Cisplatin because of cMYC and IDO-1 downregulation. Additionally, this Cisplatin-CDKI combination induced HLA-ABC and PD-L1 upregulation, which may enhance immunogenicity. Performing functional and molecular analysis on patient-individual HNSCC-models, we identified CDK4-gains as a biomarker for abemaciclib response prediction and describe an approach to conquer intrinsic CDKI resistance.

Simple Summary This study examined the therapeutic potential of a combined therapy approach, based on clinical approved drugs (5-FU, Cisplatin, cetuximab) and cyclin-dependent kinase inhibitors (CDKi, dinaciclib, palbociclib, THZ1). We identified individual effects on head and neck squamous cell carcinoma cells, including induction of apoptosis/necrosis, and senescence as well as reduced invasiveness. Besides, we describe the relevance of the sequential timing of each combination partner to achieve synergistic effects. Another interesting finding of our study is the upregulation of immunologically relevant molecules on the tumor cell surface under certain CDKi-drug combinations. Here, dinaciclib and palboclicb had highest impact on immunogenicity, which even exceeded effects of the standard drugs. Finally, a therapeutic in vivo approach partially confirmed cell line-based results. Here, effective tumor growth control was seen when cisplatin was combined with dinaciclib. However, antitumoral effects were highly individual and nicely confirm the heterogeneity of this tumor entity. Abstract Cyclin-dependent kinase inhibitors (CDKi´s) display cytotoxic activity against different malignancies, including head and neck squamous cell carcinomas (HNSCC). By coordinating the DNA damage response, these substances may be combined with cytostatics to enhance cytotoxicity. Here, we investigated the influence of different CDKi´s (palbociclib, dinaciclib, THZ1) on two HNSCC cell lines in monotherapy and combination therapy with clinically-approved drugs (5-FU, Cisplatin, cetuximab). Apoptosis/necrosis, cell cycle, invasiveness, senescence, radiation-induced γ-H2AX DNA double-strand breaks, and effects on the actin filament were studied. Furthermore, the potential to increase tumor immunogenicity was assessed by analyzing Calreticulin translocation and immune relevant surface markers. Finally, an in vivo mouse model was used to analyze the effect of dinaciclib and Cisplatin combination therapy. Dinaciclib, palbociclib, and THZ1 displayed anti-neoplastic activity after low-dose treatment, while the two latter substances slightly enhanced radiosensitivity. Dinaciclib decelerated wound healing, decreased invasiveness, and induced MHC-I, accompanied by high amounts of surface-bound Calreticulin. Numbers of early and late apoptotic cells increased initially (24 h), while necrosis dominated afterward. Antitumoral effects of the selective CDKi palbociclib were weaker, but combinations with 5-FU potentiated effects of the monotherapy. Additionally, CDKi and CDKi/chemotherapy combinations induced MHC I, indicative of enhanced immunogenicity. The in vivo studies revealed a cell line-specific response with best tumor growth control in the combination approach. Global acting CDKi’s should be further investigated as targeting agents for HNSCC, either individually or in combination with selected drugs. The ability of dinaciclib to increase the immunogenicity of tumor cells renders this substance a particularly interesting candidate for immune-based oncological treatment regimens.

Background Mlh1-knock-out-driven mismatch-repair-deficient (dMMR) tumors can be targeted immunologically. By applying therapeutic tumor vaccination, tumor growth is delayed but escape mechanisms evolve, including upregulation of immune-checkpoint molecules (LAG-3, PD-L1). To counteract immune escape, we investigated the therapeutic activity of a combined tumor vaccine-immune-checkpoint inhibitor therapy using α-PD-L1. Design In this trial, Mlh1-knock-out mice with established gastrointestinal tumors received single or thrice injections of α-PD-L1 monoclonal antibody clone 6E11 (2.5 mg/kg bw, q2w, i.v.) either alone or in combination with the vaccine. Longitudinal flow cytometry and PET/CT imaging studies were followed by ex vivo functional immunological and gene expression assays. Results 6E11 monotherapy slightly increased median overall survival (mOS: 6.0 weeks vs. control 4.0 weeks). Increasing the number of injections (n = 3) improved therapy outcome (mOS: 9.2 weeks) and was significantly boosted by combining 6E11 with the vaccine (mOS: 19.4 weeks vs. 10.2 weeks vaccine monotherapy). Accompanying PET/CT imaging confirmed treatment-induced tumor growth control, with the strongest inhibition in the combination group. Three mice (30%) achieved a complete remission and showed long-term survival. Decreased levels of circulating splenic and intratumoral myeloid-derived suppressor cells (MDSC) and decreased numbers of immune-checkpoint-expressing splenic T cells (LAG-3, CTLA-4) accompanied therapeutic effects. Gene expression and protein analysis of residual tumors revealed downregulation of PI3K/Akt/Wnt-and TGF-signaling, leading to T cell infiltration, reduced numbers of macrophages, neutrophils and MDSC. Conclusions By successful uncoupling of the PD-1/PD-L1 axis, we provide further evidence for the safe and successful application of immunotherapies to combat dMMR-driven malignancies that warrants further investigation.