Immune checkpoint blockade offers an exciting, new adjuvant therapy to both solid and liquid tumors. However, the underlying mechanisms by which tumors alter their expression of immune checkpoint blockade ligands, such as programmed death ligand 1 (PD-L1), to achieve adaptive immune resistance needs to be further studied. We sought to investigate the mechanisms by which PD-L1 expression is regulated in response to IFNγ stimulation in medulloblastoma, a primary CNS neoplasm. MB is the most common pediatric CNS malignancy that is capable of metastasis to distant sites. MB is classified into four different molecular subgroups. Patched (PTCH), a signaling molecule of the Sonic Hedgehog (SHH) pathway, represents one subgroup and accounts for 25% of all sporadic human MB. Because MB develops from primitive cerebellar granule neuron precursor cells, it is thought that errors in development or migration of these neurons contribute to tumorigenesis. Cyclin Dependent Kinase 5 (CDK5) is a non-canonical member of the CDK family that does not require cyclin for activity but relies on binding to its obligate activators, p35 and p39 which are mostly expressed in post mitotic neurons. CDK5 is required for normal Central Nervous System (CNS) development.
Utilizing both short hairpin RNA (shRNA) approach and the bacterial CRISPR-Cas9 (Cr) system we silenced Cdk5 expression in a C57BL/6 (B6) syngeneic MB line derived from p53-/- PTCH+/- (MM1). In vitro proliferation experiments revealed that MM1-WT, MM1 cells transduced with shRNA targeting CDK5 (MM1-shCDK5), and MM1 cells transduced with non-silencing, scrambled RNA sequences (MM1-shNS) had similar growth kinetics. Since there were no intrinsic differences in in vitro growth kinetics, we subcutaneously injected immunocompetent C57BL/B6 mice with 5x104 tumor cells and measured tumor growth every three days. In our first experiment, 19 days after injection 7.7% (N=13) of mice injected with MM1-WT and 0% (N=7) injected with MM1-shNS were tumor free compared with 76.5% (N=13) of animals injected with MM1-shCDK5 Comparison of tumor free survival revealed statistically significant differences between the MM1-WT with MM1-shCDK5 groups (p <0.001) and MM1-shNS with MM1-shCDK5 (p <0.001). There was no significant difference between MM1-WT and MM1-shNS (p= 0.206). Furthermore, when injected into athymic Nu/Nu mice, MM1-WT and MM1-shCDK5 had similar subcutaneous growth kinetics. Therefore, we sought to interrogate the role of the immune system in enhanced killing of MM1-shCDK5 tumors. When exposed to IFNγ stimulation in vitro, genetic profiling of MM1-shCDK5 revealed a decrease in PD-L1 expression, a known immune checkpoint surface molecule, compared to MM1-WT. Assessing the downstream molecules that could account for differential IFNγ signaling, we found that upon stimulation of MM1-WT cells in vitro with 100ng/mL of recombinant murine IFNγ, interferon regulatory factor-1 (IRF-1) had a time dependent upregulation that corresponded with augmented cell surface expression of PD-L1. In IFNγ stimulated MM1-shCDK5 cells and MM1 transduced with Cr constructs targeting CDK5 (MM1-crCDK5), IRF-1 protein did not increase and corresponded with an absence of PD-L1 cell surface expression.
In summary, we have shown that Cdk5 is expressed in Type 2 MB and that silencing of its expression with shRNA results in an altered growth phenotype in vivo but not in vitro. Furthermore, eliminating the ability of MM1 tumors to express PD-L1 might lead to effective immune rejection of MM1-shCDK5. Mechanistically, we showed that this enhanced tumor rejection may be due in part to the failure of Cdk5 deficient MB cells to alter IRF-1 expression which serves as a transcription factor for PD-L1. These novel findings point to CDK5 as a potential immunotherapeutic target in MB.
Citation Format: Rodney Dixon Dorand, Jr., Agne Petrosiute, Joseph Nthale, Tej Pareek, Francesca Scrimieri, Jay Myers, Deborah Barkauskas, John J. Letterio, Duncan S. Stearns, Alex Y. Huang. Cyclin dependent kinase-5 regulates IFNγ induced PD-L1 expression via IRF-1 in medulloblastoma. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B158.
- ©2016 American Association for Cancer Research.