Deciphering the Immunomodulatory Role of Cyclin-Dependent Kinase 4/6 Inhibitors in the Tumor Microenvironment.

Cancer is characterized by persistent cell proliferation driven by aberrant cell cycle regulation and stimulation of cyclin-dependent kinases (CDKs). A very intriguing and potential approach for the development of antitumor medicines is the suppression of CDKs that lead to induction of apoptosis and cell cycle arrest. The shift of the cell cycle from the G0/G1 phase to the S phase, which is characterized by active transcription and synthesis, depends on the development of the cyclin D-CDK4/6 complex. A precise balance between anticancer activity and general toxicity is demonstrated by CDK inhibitors, which can specifically block CDK4/6 and control the cell cycle by reducing the G1 to S phase transition. CDK4/6 inhibitors have recently been reported to exhibit significant cell growth inhibition via modulating the tumour microenvironment in cancerous cells. One significant new understanding is that these inhibitors serve important functions in the interaction among tumour cells and the host immune system in addition to being cytostatic. Herein, we discuss the biological significance of CDK4/6 inhibitors in cancer therapeutics, as well as their biological impact on T cells and other important immune cells. Furthermore, we explore the integration of preclinical findings of these pharmaceuticals' ability to enhance antitumor immunity.

The Immunological Role of CDK4/6 and Potential Mechanism Exploration in Ovarian Cancer.

Background: Ovarian cancer (OC) is one of the most lethal gynecologic cancers. Growing evidence has proven that CDK4/6 plays a key role in tumor immunity and the prognosis of many cancers. However, the expression and function of CDK4/6 in OC remain unclear. Therefore, we aimed to explore the influence of CDK4/6 in OC, especially on immunity. Methods: We analyzed CDK4/6 expression and prognosis using The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO) and Genotype Tissue Expression (GTEx) data. Subsequently, we used the cytoHubba plug-in of Cytoscape software and starBase to identify the noncoding RNAs (ncRNAs) regulating CDK4/6. Finally, we verified the effect of CDK4/6 on immunity in OC cell lines and animal models. Results: CDK4/6 expression was higher in OC tissues than in normal ovarian tissues, and the high expression levels of CDK4/6 contributed to the immunosuppressive state of OC and were thus related to the poor prognosis of OC patients. This was also in general agreement with the results of OC cell line and animal experiments. Mechanistically, the CDK4/6 inhibitor palbociclib increased the secretion of interferon (IFN)-γ and the interferon-stimulated gene (ISG) response, thereby upregulating the expression of antigen-presenting molecules; this effect was partly dependent on the STING pathway and thus activated immunity in OC. Additionally, according to public data, the LRRC75A-AS1-hsa-miR-330-5p axis could inhibit the immune response of OC patients by upregulating CDK4/6, leading to a poor prognosis. Conclusion: CDK4/6 affects the immune microenvironment of OC and correlates with the prognosis of OC patients.

Combined BRAF, MEK, and CDK4/6 Inhibition Depletes Intratumoral Immune-Potentiating Myeloid Populations in Melanoma.

Combined inhibition of BRAF, MEK, and CDK4/6 is currently under evaluation in clinical trials for patients with melanoma harboring a BRAFV600 mutation. While this triple therapy has potent tumor-intrinsic effects, the impact of this combination on antitumor immunity remains unexplored. Here, using a syngeneic BrafV600ECdkn2a-/-Pten-/- melanoma model, we demonstrated that triple therapy promoted durable tumor control through tumor-intrinsic mechanisms and promoted immunogenic cell death and T-cell infiltration. Despite this, tumors treated with triple therapy were unresponsive to immune checkpoint blockade (ICB). Flow cytometric and single-cell RNA sequencing analyses of tumor-infiltrating immune populations revealed that triple therapy markedly depleted proinflammatory macrophages and cross-priming CD103+ dendritic cells, the absence of which correlated with poor overall survival and clinical responses to ICB in patients with melanoma. Indeed, immune populations isolated from tumors of mice treated with triple therapy failed to stimulate T-cell responses ex vivo While combined BRAF, MEK, and CDK4/6 inhibition demonstrates favorable tumor-intrinsic activity, these data suggest that collateral effects on tumor-infiltrating myeloid populations may impact antitumor immunity. These findings have important implications for the design of combination strategies and clinical trials that incorporate BRAF, MEK, and CDK4/6 inhibition with immunotherapy for the treatment of patients with melanoma.

A patient with melanoma that became sensitized to immunotherapy after treatment with a CDK4/6 inhibitor.

Background: Despite the profound effect that checkpoint inhibitors and BRAF/MEK inhibitors have had on survival in patients with metastatic melanoma, treatment options remain limited for those who demonstrate poor response or develop resistance to these modalities. The prospect of tumor sensitization to these treatments is therefore an attractive one. Results: We describe the case of a patient who developed a sustained response to trametinib and pembrolizumab, despite prior resistance to both these therapies, after receiving treatment with a CDK4/6 inhibitor. Discussion: We further outline the preclinical data supporting a possible role for the use of CDK4/6 inhibitors in tumor sensitization to immunotherapy.

Beyond the Cell Cycle: Enhancing the Immune Surveillance of Tumors Via CDK4/6 Inhibition.

Inhibitors of the cyclin-dependent kinases 4 and 6 (CDK4/6) were originally designed to block proliferation and cell cycle progression of cancer cells in which the activity of these kinases is dysregulated. CDK4/6 inhibitors have already been FDA approved for the treatment of estrogen receptor (ER)-positive breast cancer and are being tested in numerous other cancer types. However, several recent studies have identified novel effects of CDK4/6 inhibitors on tumor growth, most notably an indirect effect resulting from the activation of immune surveillance. This Perspective discusses these recent observations, including the effects that CDK4/6 inhibitors may have on immune cells themselves. It is likely that CDK4/6 inhibitors will have a broader impact than their expected induction of cell cycle arrest in the treatment of human cancers. Mol Cancer Res; 16(10); 1454-7. ©2018 AACR.

Inhibition of CDK4/6 protects against radiation-induced intestinal injury in mice.

Radiotherapy causes dose-limiting toxicity and long-term complications in rapidly renewing tissues, including the gastrointestinal tract. Currently, there is no FDA-approved agent for the prevention or treatment of radiation-induced intestinal injury. In this study, we have shown that PD 0332991 (PD), an FDA-approved selective inhibitor of cyclin-dependent kinase 4/6 (CDK4/6), prevents radiation-induced lethal intestinal injury in mice. Treating mice with PD or a structurally distinct CDK4/6 inhibitor prior to radiation blocked proliferation and crypt apoptosis and improved crypt regeneration. PD treatment also enhanced LGR5+ stem cell survival and regeneration after radiation. PD was an on-target inhibitor of RB phosphorylation and blocked G1/S transition in the intestinal crypts. PD treatment strongly but reversibly inhibited radiation-induced p53 activation, which blocked p53-upregulated modulator of apoptosis-dependent (PUMA-dependent) apoptosis without affecting p21-dependent suppression of DNA damage accumulation, with a repair bias toward nonhomologous end joining. Further, deletion of PUMA synergized with PD treatment for even greater intestinal radioprotection. Our results demonstrate that the cell cycle critically regulates the DNA damage response and survival of intestinal stem cells and support the concept that pharmacological quiescence is a potentially highly effective and selective strategy for intestinal radioprotection.

The G1/S Specific Cyclin D2 Is a Regulator of HIV-1 Restriction in Non-proliferating Cells.

Macrophages are a heterogeneous cell population strongly influenced by differentiation stimuli that become susceptible to HIV-1 infection after inactivation of the restriction factor SAMHD1 by cyclin-dependent kinases (CDK). Here, we have used primary human monocyte-derived macrophages differentiated through different stimuli to evaluate macrophage heterogeneity on cell activation and proliferation and susceptibility to HIV-1 infection. Stimulation of monocytes with GM-CSF induces a non-proliferating macrophage population highly restrictive to HIV-1 infection, characterized by the upregulation of the G1/S-specific cyclin D2, known to control early steps of cell cycle progression. Knockdown of cyclin D2, enhances HIV-1 replication in GM-CSF macrophages through inactivation of SAMHD1 restriction factor by phosphorylation. Co-immunoprecipitation experiments show that cyclin D2 forms a complex with CDK4 and p21, a factor known to restrict HIV-1 replication by affecting the function of the downstream cascade that leads to SAMHD1 deactivation. Thus, we demonstrate that cyclin D2 acts as regulator of cell cycle proteins affecting SAMHD1-mediated HIV-1 restriction in non-proliferating macrophages.

Targeting human melanoma neoantigens by T cell receptor gene therapy.

In successful cancer immunotherapy, T cell responses appear to be directed toward neoantigens created by somatic mutations; however, direct evidence that neoantigen-specific T cells cause regression of established cancer is lacking. Here, we generated T cells expressing a mutation-specific transgenic T cell receptor (TCR) to target different immunogenic mutations in cyclin-dependent kinase 4 (CDK4) that naturally occur in human melanoma. Two mutant CDK4 isoforms (R24C, R24L) similarly stimulated T cell responses in vitro and were analyzed as therapeutic targets for TCR gene therapy. In a syngeneic HLA-A2-transgenic mouse model of large established tumors, we found that both mutations differed dramatically as targets for TCR-modified T cells in vivo. While T cells expanded efficiently and produced IFN-γ in response to R24L, R24C failed to induce an effective antitumor response. Such differences in neoantigen quality might explain why cancer immunotherapy induces tumor regression in some individuals, while others do not respond, despite similar mutational load. We confirmed the validity of the in vivo model by showing that the melan-A-specific (MART-1-specific) TCR DMF5 induces rejection of tumors expressing analog, but not native, MART-1 epitopes. The described model allows identification of those neoantigens in human cancer that serve as suitable T cell targets and may help to predict clinical efficacy.

Role of Cdk4 in lymphocyte function and allergen response.

We recently described a new adhesion pathway in lymphocytes that is dependent on Cyclin-dependent kinase (Cdk) 4 activity and mediates lymphocyte interactions with endothelial matrix. We showed that Cdk4(-/-) mice had impaired recruitment of lymphocytes following bleomycin model of acute lung injury. In this study, we characterized the development and function of hematopoietic cells in Cdk4(-/-) mice and assessed the response of Cdk4(-/-) mice to allergen challenge. Cdk4(-/-) mice had hypoplastic thymuses with decreased total thymocyte cell numbers and increased CD4/CD8 double negative cells. Cdk4(-/-) bone marrow (BM) chimeric mice showed similar findings. Thymocytes from either Cdk4(-/-) or Cdk4(-/-) BM chimeric mice proliferated equally well as wild type controls in response to IL-2 activation. However Cdk4(-/-) thymocytes had decreased adhesion to both endothelial cell matrix and fibronectin compared to wildtype (WT) controls, whereas Cdk4(-/-) and WT splenocytes had similar adhesion. When Cdk4(-/-) BM chimeric mice and wild type BM chimeric mice were sensitized and challenged by intranasal administration of ovalbumin, we found no differences in allergic responses in the lung and airways between the two groups, as measured by inflammatory cell infiltrate, airway hyperreactivity, IgE levels and cytokine levels. In summary, we show that Cdk4 plays a previously unrecognized role in thymocyte maturation and adhesion, but is not required for thymocyte proliferation. In addition, Cdk4 is not required for lymphocyte trafficking to the lung following allergen sensitization and challenge.

Apurinic/apyrimidinic endonuclease 1 is a key modulator of keratinocyte inflammatory responses.

Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1) functions in both DNA repair and redox signaling, making it an attractive emerging therapeutic target. However, the role of APE1 in cutaneous inflammatory responses is largely unknown. In this study, we report that APE1 is a key upstream regulator in TLR2-dependent keratinocyte inflammatory responses. We found that nuclear expression of APE1 in epidermal layers was markedly up-regulated in psoriatic skin. APE1 was essential for the transcriptional activation and nuclear translocation of hypoxia-inducible factor-1alpha and NF-kappaB, both of which are crucial for inflammatory signaling in keratinocytes. Moreover, APE1 played a crucial role in the expression of TLR2-mediated inflammatory mediators, including TNF-alpha, CXCL8, and LL-37, in HaCaT cells and human primary keratinocytes. Silencing of APE1 attenuated cyclin D1/cyclin-dependent kinase 4 expression and phosphorylation of ERK1/2 and Akt, thereby affecting keratinocyte proliferation. Importantly, TLR2-induced generation of reactive oxygen species contributed to the nuclear translocation and expression of APE1, suggesting an autoregulatory circuit in which the subcellular localization of APE1 is associated with the production of APE1 per se through reactive oxygen species-dependent signaling. Taken together, these findings establish a role for APE1 as a master regulator of TLR2-dependent inflammatory responses in human keratinocytes.

Inhibitory effect of RNA pool complexity on stimulatory capacity of RNA-pulsed dendritic cells.

Tumor cells that show downregulation of their tumor-associated antigens (TAAs) may be able to escape immune-mediated elimination. Therefore, efficient vaccine strategies attempt to target multiple TAAs simultaneously. This is easily achieved in dendritic cell (DC)-based vaccines by introducing antigens in the form of RNA. Although insufficient message may hinder adequate expression of individual TAAs when using total-tumor RNA, high amounts of individual RNAs as pools yield DCs presenting high numbers of specific peptide-major histocompatibility complex ligands with epitopes derived from different TAAs. We used the transfer of RNAs encoding the well-defined melanoma TAAs tyrosinase, Melan-A, CDK4mut, gp100, SNRP116mut, and GPNMBmut to characterize DCs at the levels of transfected RNA, expressed protein and peptide-major histocompatibility complex ligand presentation. TAA-encoding RNA was rapidly degraded in the DCs, allowing only a single surge in protein expression shortly after transfection. We compared the functional capacity of DCs transfected with pools of 3 versus 6 RNAs. Whereas functional assays demonstrated a decrease in stimulatory capacity of DCs transfected with a pool of 3 RNAs by only 30% as compared with single RNAs, a 60% loss was seen with 6 RNAs. We conclude that larger RNA pools result in diminished presentation of individual epitopes and suggest that smaller pools of RNA be transfected into separate DC populations which are then pooled to create multiplex vaccines.

Cigarette tar phenols impede T cell cycle progression by inhibiting cyclin-dependent kinases.

Cigarette smoking causes profound suppression of pulmonary T cell responses, which is associated with increased susceptibility to respiratory tract infections and decreased tumor surveillance. We previously demonstrated that the phenolic compounds in cigarette tar inhibit blastogenesis and interfere with human T cell cycle progression. To identify the mechanism by which cell cycle arrest occurs, we examined the effects of these compounds on cyclin-dependent kinases (Cdk) that control the G0/G1 transition. We found that hydroquinone inhibited induction of Cdk4 and Cdk6 kinase activities by >80%, while catechol and phenol were markedly less potent. HQ did not affect mitogenic induction of the Cdk6 protein, but inhibited expression of cyclin D3 by >90% resulting in a dramatic reduction in proper Cdk6/Cyclin D3 complex formation.