Steven M. Chirieleison

Cdk5 disruption attenuates tumor PD-L1 expression and promotes antitumor immunity

Cyclin suppresses antitumor immunity Despite the dramatic success of cancer immunotherapy, many types of cancer do not respond. Understanding why could help us to find ways to enhance the overall responsiveness of tumors to immunotherapies. Dorand et al. report that cyclin-dependent kinase 5 (Cdk5), an enzyme that is highly expressed by neurons in many brain cancers, may dampen the ability of T cells to reject tumors. In a mouse model of medulloblastoma, if tumors were Cdk5 deficient, T cells were able to remove them. This heightened antitumor immunity correlated with reduced expression of the inhibitory molecule programmed cell death ligand 1 (PD-L1), a target of current cancer immunotherapies. Science, this issue p. 399 Cyclin-dependent kinase 5 expression by tumors inhibits antitumor immunity in human and mouse medulloblastoma. Cancers often evade immune surveillance by adopting peripheral tissue– tolerance mechanisms, such as the expression of programmed cell death ligand 1 (PD-L1), the inhibition of which results in potent antitumor immunity. Here, we show that cyclin-dependent kinase 5 (Cdk5), a serine-threonine kinase that is highly active in postmitotic neurons and in many cancers, allows medulloblastoma (MB) to evade immune elimination. Interferon-γ (IFN-γ)–induced PD-L1 up-regulation on MB requires Cdk5, and disruption of Cdk5 expression in a mouse model of MB results in potent CD4+ T cell–mediated tumor rejection. Loss of Cdk5 results in persistent expression of the PD-L1 transcriptional repressors, the interferon regulatory factors IRF2 and IRF2BP2, which likely leads to reduced PD-L1 expression on tumors. Our finding highlights a central role for Cdk5 in immune checkpoint regulation by tumor cells.

Synthetic Biology Reveals the Uniqueness of the RIP Kinase Domain

The RIP kinases (RIPKs) play an essential role in inflammatory signaling and inflammatory cell death. However, the function of their kinase activity has been enigmatic, and only recently has kinase domain activity been shown to be crucial for their signal transduction capacity. Despite this uncertainty, the RIPKs have been the subject of intense pharmaceutical development with a number of compounds currently in preclinical testing. In this work, we seek to determine the functional redundancy between the kinase domains of the four major RIPK family members. We find that although RIPK1, RIPK2, and RIPK4 are similar in that they can all activate NF-κB and induce NF-κB essential modulator ubiquitination, only RIPK2 is a dual-specificity kinase. Domain swapping experiments showed that the RIPK4 kinase domain could be converted to a dual-specificity kinase and is essentially indistinct from RIPK2 in biochemical and molecular activity. Surprisingly, however, replacement of RIPK2’s kinase domain with RIPK4’s did not complement a nucleotide-binding oligomerization domain 2 signaling or gene expression induction defect in RIPK2−/− macrophages. These findings suggest that RIPK2’s kinase domain is functionally unique compared with other RIPK family members and that pharmacologic targeting of RIPK2 can be separated from the other RIPKs.

Live-cell visualization of gasdermin D-driven pyroptotic cell death

Pyroptosis is a form of cell death important in defenses against pathogens that can also result in a potent and sometimes pathological inflammatory response. During pyroptosis, GSDMD (gasdermin D), the pore-forming effector protein, is cleaved, forms oligomers, and inserts into the membranes of the cell, resulting in rapid cell death. However, the potent cell death induction caused by GSDMD has complicated our ability to understand the biology of this protein. Studies aimed at visualizing GSDMD have relied on expression of GSDMD fragments in epithelial cell lines that naturally lack GSDMD expression and also lack the proteases necessary to cleave GSDMD. In this work, we performed mutagenesis and molecular modeling to strategically place tags and fluorescent proteins within GSDMD that support native pyroptosis and facilitate live-cell imaging of pyroptotic cell death. Here, we demonstrate that these fusion proteins are cleaved by caspases-1 and -11 at Asp-276. Mutations that disrupted the predicted p30-p20 autoinhibitory interface resulted in GSDMD aggregation, supporting the oligomerizing activity of these mutations. Furthermore, we show that these novel GSDMD fusions execute inflammasome-dependent pyroptotic cell death in response to multiple stimuli and allow for visualization of the morphological changes associated with pyroptotic cell death in real time. This work therefore provides new tools that not only expand the molecular understanding of pyroptosis but also enable its direct visualization.

An IκB Kinase-Regulated Feedforward Circuit Prolongs Inflammation

Loss of NF-κB signaling causes immunodeficiency, whereas inhibition of NF-κB can be efficacious in treating chronic inflammatory disease. Inflammatory NF-κB signaling must therefore be tightly regulated, and although many mechanisms to downregulate NF-κB have been elucidated, there have only been limited studies demonstrating positive feedforward regulation of NF-κB signaling. In this work, we use a bioinformatic and proteomic approach to discover that the IKK family of proteins can phosphorylate the E3 ubiquitin ligase ITCH, a critical downregulator of TNF-mediated NF-κB activation. Phosphorylation of ITCH by IKKs leads to impaired ITCH E3 ubiquitin ligase activity and prolongs NF-κB signaling and pro-inflammatory cytokine release. Since genetic loss of ITCH mirrors IKK-induced ITCH phosphorylation, we further show that the ITCH(-/-) mouses spontaneous lung inflammation and subsequent death can be delayed when TNF signaling is genetically deleted. This work identifies a new positive feedforward regulation of NF-κB activation that drives inflammatory disease.

Nucleotide-binding oligomerization domain (NOD) signaling defects and cell death susceptibility cannot be uncoupled in X-linked inhibitor of apoptosis (XIAP)-driven inflammatory disease

The X-linked inhibitor of apoptosis (XIAP) protein has been identified as a key genetic driver of two distinct inflammatory disorders, X-linked lymphoproliferative syndrome 2 (XLP-2) and very-early-onset inflammatory bowel disease (VEO-IBD). Molecularly, the role of XIAP mutations in the pathogenesis of these disorders is unclear. Recent work has consistently shown XIAP to be critical for signaling downstream of the Crohns disease susceptibility protein nucleotide-binding oligomerization domain-containing 2 (NOD2); however, the reported effects of XLP-2 and VEO-IBD XIAP mutations on cell death have been inconsistent. In this manuscript, we describe a CRISPR-mediated genetic system for cells of the myeloid lineage in which XIAP alleles can be replaced with disease-associated XIAP variants expressed at endogenous levels to simultaneously study inflammation-related cell death and NOD2 signaling. We show that, consistent with previous studies, NOD2 signaling is critically dependent on the BIR2 domain of XIAP. We further used this system to reconcile the aforementioned inconsistent XIAP cell death data to show that XLP-2 and VEO-IBD XIAP mutations that exhibit a loss-of-function NOD2 phenotype also lower the threshold for inflammatory cell death. Last, we identified and studied three novel patient XIAP mutations and used this system to characterize NOD2 and cell death phenotypes driven by XIAP. The results of this work support the role of XIAP in mediating NOD2 signaling while reconciling the role of XLP-2 and VEO-IBD XIAP mutations in inflammatory cell death and provide a set of tools and framework to rapidly test newly discovered XIAP variants.

Chemical disruption of the pyroptotic pore-forming protein gasdermin D inhibits inflammatory cell death and sepsis

Necrosulfonamide inhibits pyroptosis through direct inhibition of gasdermin D. Targeting gasdermin D Gasdermin D (GSDMD) is a key downstream effector in inflammasome-driven, caspase-1–dependent and lipopolysaccharide-driven, caspase-11–dependent pyroptosis. Upon activation and caspase-dependent proteolytic cleavage, GSDMD oligomerizes to form pores that facilitate pyroptotic cell death. Here, Rathkey et al. report necrosulfonamide (NSA) to be an inhibitor of GSDMD and GSDMD-mediated pyroptosis. They propose that NSA binds to cysteine 191 and inhibits the oligomerization of GSDMD dimers. NSA could be used as a template to develop more potent inhibitors of GSDMD, an important goal for the treatment of septic shock. In the same issue, Sollberger et al. independently report a pyrazolo-oxazepine scaffold–based molecule to be an inhibitor of GSDMD. Dysregulation of inflammatory cell death is a key driver of many inflammatory diseases. Pyroptosis, a highly inflammatory form of cell death, uses intracellularly generated pores to disrupt electrolyte homeostasis and execute cell death. Gasdermin D, the pore-forming effector protein of pyroptosis, coordinates membrane lysis and the release of highly inflammatory molecules, such as interleukin-1β, which potentiate the overactivation of the innate immune response. However, to date, there is no pharmacologic mechanism to disrupt pyroptosis. Here, we identify necrosulfonamide as a direct chemical inhibitor of gasdermin D, the pyroptotic pore-forming protein, which binds directly to gasdermin D to inhibit pyroptosis. Pharmacologic inhibition of pyroptotic cell death by necrosulfonamide is efficacious in sepsis models and suggests that gasdermin D inhibitors may be efficacious clinically in inflammatory diseases.

Unique BIR domain sets determine inhibitor of apoptosis protein–driven cell death and NOD2 complex signal specificity

The unique positioning of similar domains determines the signaling specificity of IAP family proteins. Structure-function specificity of IAP proteins Inhibitor of apoptosis proteins (IAPs) are involved in regulating cell death and inflammation. As such, both gain and loss of IAP function are associated with various diseases, from cancer to immune disorders. Chirieleison et al. examined the functional redundancy and selectivity of IAP family members as conferred by their structural motifs. The authors found that at least one IAP protein was critical for cell survival and that the catalytic domain of each protein, which provides E3 ubiquitin ligase activity, is redundant among the proteins but that its positioning within a triplet of protein-protein interaction domains confers distinct signaling functions among the proteins. These findings have implications for drug development as well as our understanding of the pathogenesis of immune disorders caused by IAP loss. The mammalian IAPs, X-linked inhibitor of apoptosis protein (XIAP) and cellular inhibitor of apoptosis protein 1 and 2 (cIAP1 and cIAP2), play pivotal roles in innate immune signaling and inflammatory homeostasis, often working in parallel or in conjunction at a signaling complex. IAPs direct both nucleotide-binding oligomerization domain-containing 2 (NOD2) signaling complexes and cell death mechanisms to appropriately regulate inflammation. Although it is known that XIAP is critical for NOD2 signaling and that the loss of cIAP1 and cIAP2 blunts NOD2 activity, it is unclear whether these three highly related proteins can compensate for one another in NOD2 signaling or in mechanisms governing apoptosis or necroptosis. This potential redundancy is critically important, given that genetic loss of XIAP causes both very early onset inflammatory bowel disease and X-linked lymphoproliferative syndrome 2 (XLP-2) and that the overexpression of cIAP1 and cIAP2 is linked to both carcinogenesis and chemotherapeutic resistance. Given the therapeutic interest in IAP inhibition and the potential toxicities associated with disruption of inflammatory homeostasis, we used synthetic biology techniques to examine the functional redundancies of key domains in the IAPs. From this analysis, we defined the features of the IAPs that enable them to function at overlapping signaling complexes but remain independent and functionally exclusive in their roles as E3 ubiquitin ligases in innate immune and inflammatory signaling.

Abstract A050: Cdk5 disruption attenuates tumor PD-L1 expression via regulation of IFN-γ signaling components and promotes antitumor immunity

In order to escape normal immune surveillance, tumors mimic peripheral tissue tolerance mechanisms such as the expression of programmed cell death-ligand 1 (PD-L1), the inhibition of which can lead to potent anti-tumor responses. Here we show the contribution of cyclin dependent kinase 5 (Cdk5) to immune evasion. Cdk5 is a proline-directed serine/threonine kinase, which is highly expressed in post-mitotic neurons and directs a variety of homeostatic functions from cytoskeletal rearrangement to neurotransmitter signaling. Studies in xenograft models have linked Cdk5 activity to the generation and metabolic activity of primary tumors within the central nervous system (CNS). Here we show that Cdk5, expressed in both murine and human medulloblastoma (MB) cell lines, plays a major role in the ability of MB to avoid immune detection. First, we observed that decreased Cdk5 expression was associated with an increase in the number of infiltrating CD3+ cells in the tumor mass of clinical MB samples. Additionally, using publicly available datasets, we found that decreased Cdk5 expression is correlated with better overall survival or fewer distant metastasis in melanoma, breast cancer, glioma, and lung adenocarcinoma. Next, using a CRISPR/Cas-9 approach, we silenced Cdk5 in a murine model of Sonic Hedgehog (SHH) pathway MB. Rather than causing intrinsic growth defects in MB, interference of Cdk5 activity sensitizes tumors to killing by the normal host immune system in a CD4+ T cell-dependent manner. This rejection is associated with increased IFN-γ expression in the tumor microenvironment, as well as increased PD-L1 expression by myeloid populations in both subcutaneous and intracranial tumors. Mechanistically, we observed an attenuated response to IFN-γ stimulated expression of programmed death ligand 1 (PD-L1) on MB cells. This blunted response was recapitulated when MB cells were treated with Roscovitine, a non-selective Cdk5 inhibitor. Furthermore, using a quantitative global phosphoproteomics approach, we found that Cdk5 knockdown also increased phosphorylation of S-440 and S-443 on interferon regulatory factor binding protein 2 (IRF2BP2), an upstream co-repressor of interferon regulatory factor 2 (IRF-2). Increased phosphorylation of IRF2BP2 corresponded with stable expression of IRF-2 with a concomitant decrease in surface expression of PD-L1. These observations highlight a critical role for Cdk5 in the immune escape mechanisms of primary CNS tumors and provides new therapeutic targets for PD-1/PD-L1 directed immunotherapy. Citation Format: Rodney D. Dorand, Jr., Joseph Nthale, Jay T. Myers, Deborah S. Barkauskas, Stefanie Avril, Steven M. Chirieleison, Tej K. Pareek, Derek W. Abbott, Duncan S. Stearns, John J. Letterio, Alex Y. Huang, Agne Petrosiute. Cdk5 disruption attenuates tumor PD-L1 expression via regulation of IFN-γ signaling components and promotes antitumor immunity [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A050.