August 22, 2016
Here at Oncology Discovery, we’ve elaborated on strategies to improve response rates to checkpoint inhibitors. Instead of penning another post on the I/O competitive landscape, we now will attempt to sharpen our focus around a specific target. To the best of our knowledge, this protein has been overlooked by industry for any therapeutic application.
The target in question is IRAK3 (also known as IRAKM). IRAK3 has a well-established role in regulating innate immune responsiveness, mediating immune tolerance, and is a potent negative regulator of TLR/IL1R signaling. This post will attempt to elaborate on why we believe targeting IRAK3 is a differentiated, tractable, and worthwhile I/O strategy.
The interleukin-1 receptor-associated kinases (IRAKs) are mediators of toll-like receptor (TLR) and interleukin-1 receptor (IL1R) pro-inflammatory signaling. (For a detailed look at IRAK family kinases in cancer, the interested reader is referred to an open-access review manuscript published in the British Journal of Cancer.) Four IRAK family members exist, and although there are certain functional redundancies, each family member plays a unique role related to innate immune signaling. As shown in Figure 1, IRAK proteins share a conserved protein domain structure: an N-terminal domain, important for IRAK dimerization and interaction with MyD88, a proline/serine/threonine-rich (ProST) domain, and a kinase or pseudokinase domain. IRAK1, IRAK2, and IRAK3 also contain a C-terminal domain, which is required for interaction with TRAF6, an E3 ubiquitin ligase. IRAK4, IRAK2 and IRAK1 relay signaling to TRAF6, activating the NFκB and MAPK pathways, ultimately resulting in pro-inflammatory signaling. Importantly, IRAK3 acts as a brake to the TLR/IL1R pathway: IRAK3 inhibits downstream activation by gumming up (forming non-productive IRAK heterodimers) the Myddosome. More on the Myddosome complex below.
At the cell surface, TLR and IL1R activation is mediated through engagement with pathogen-associated molecular patterns (PAMPs) and pro-inflammatory cytokines (i.e. IL1). TLRs and IL1R share a common Toll/IL1R homology (TIR) domain, which functions in PAMP/cytokine binding. TLR engagement induces a hierarchical assembly of the MyD88-IRAK4-IRAK2 signaling complex, known as the Myddosome – hardcore protein biochemists can marvel at the Myddosome crystal structure in a 2010 Nature manuscript by Lin, et al. Simply put, IRAK3 forms heterodimers with, and inactivates, IRAK1 and IRAK2, thus preventing downstream TRAF6 and NFκB activation and subsequent inflammatory signaling. At the transcriptional level, IRAK3 expression is regulated by NFκB; IRAK3 activity serves as a critical negative feedback loop for the TLR/IL1R pathway.
IRAK3 expression is generally confined to monocytes and macrophages, as well as some epithelial tissues. Indeed, RNA-sequencing data derived from human tissues, as shown in Figure 3, reveals a high level of IRAK3 expression in bone marrow. In our view, the relatively tissue-restricted expression pattern of IRAK3 could enable a precision medicine strategy by potentially limiting collateral inflammation-mediated adverse events against non-target tissues.
IRAK3 was first described as a negative regulator of host immune defense. Consistent with this, mice deficient in IRAK3 exhibit improved host defense against pathogenic infections, such as Klebsiella pneumoniae. As shown in Figure 4, IRAK3 dampens inflammatory signaling through inhibition of TLR-mediated activation of NFκB, inhibition of AP-1 activation, and direct binding to and sequestration of the CD80 co-stimulatory molecule. More recently, IRAK3 has been shown to suppress inflammation in response to glucocorticoids during Haemophilus influenzae infection.
Evidence to Support Targeting IRAK3 as an Effective I/O Strategy
Tumors can circumvent immunosurveillance through TGF-β-mediated induction of IRAK3 on tumor associated macrophages (TAMs). TAMs are a major component of the immune cell infiltrate within the tumor microenvironment (TME). Factors within the TME are thought to convert infiltrating macrophages into alternatively activated, or M2 phenotype. Unlike their cytotoxic counterparts, TAMs exhibit poor antigen presenting capability, and produce factors that suppress T-cell activity. As shown in Figure 5, IRAK3 (IRAKM) expression is is significantly higher on TAMs versus their Peritoneal Macrophage (PEM) counterparts. TAMs isolated from the syngeneic Lewis Lung Carcinoma tumor model exhibit defective inflammatory cytokine production. Furthermore, the growth of the Lewis Lung Carcinoma model is significantly impaired in IRAK3-/- mice. TAMs isolated from IRAK3-/- animals exhibit increased IL12, IFN-γ and TNF-α levels — cytokines important for promoting type 1 immunity (Figure 6).
TGF-β production has long been known to be a mechanism of cancer immune evasion. In their 2011 Oncogene manuscript, Standiford, et al. defined the relationship between IRAK3 and TGF-β levels using human PBMCs and mouse macrophages (Figure 6). In both cell contexts, addition of exogenous TGF-β results in increased IRAK3 levels in a time-dependent manner. They also demonstrate an upregulation in IRAK3 levels in PBMCs co-cultured with lung cancer cell lines, implicating the activity of a soluble factor, such as TGF-β. They refined their hypothesis, and closed the loop, by using a TGF-β neutralizing antibody, which prevented IRAK3 upregulation. Thus, the upregulation of IRAK3 via TGF-β is a mechanism by which tumors evade the immune system.
Tumor cells deactivate the innate immune response through CD44/TLR-mediated upregulation of IRAK3. Prior to the Standiford Oncogene manuscript, Fresno and colleagues investigated the mechanisms of tumor-induced monocyte inactivation in a 2005 Journal of Immunology manuscript. They used a co-culture system to examine the effects of tumor cells on human monocytes. As shown in Figure 7, monocytes exhibit decreased levels of TNF-α, and IL12 in response to tumor cell line co-culture. IRAK3 (IRAKM) accumulates rapidly over time in human monocytes after exposure to various tumor cell lines, with kinetics similar to the TLR4 ligand LPS. Their investigation indicated that both membrane-bound and soluble molecule(s) from tumor cells result in increased IRAK3 transcription in human monocytes. They demonstrated that hemagglutinin (HA) – a factor secreted by and overexpressed on the cell surface of cancer cells – is responsible for inducing IRAK3 expression (Figure 7). Interestingly, both CD44 and TLR4 are capable of binding to HA. Furthermore, both CD44 and TLR4 are capable in mediating HA-induced IRAK3 expression, as demonstrated in experiments using neutralizing anti-CD44 and/or anti-TLR4 antibodies. The immune tolerance phenotype induced by cancer cell/monocyte co-culture system can be prevented through blocking IRAK3 via siRNA. Thus, IRAK3 is a critical mediator of cancer cell-mediated tolerance of the innate immune system. Supporting evidence for the role of IRAK3 in promoting immune tolerance in human cancer is depicted in Figure 8. Chronic myeloid leukemia patient circulating CD14+ cells exhibit increased levels of IRAK3 as compared to healthy controls. These cells are in direct contact with the tumor microenvironment, so they provide an ideal disease setting to test this hypothesis.
IRAK3 depletion enhances dendritic cell vaccine activity. Dendritic cells are potent, professional antigen presenting cells. Thus far, their use in vaccine-based cancer therapy has been met with mixed success. As a therapeutic class, dendritic cell (DC) vaccines must fulfill three criteria in order for optimal functionality. First, dendritic cells must be capable of migration to lymphoid tissues to present immunizing antigen (Ag). Next, the DCs must acquire and maintain a mature stimulatory phenotype, typified by the expression of the chemokine receptor, CCR7, which plays a chemosensing role (in response to CCL19 and CCL21) to direct dendritic cells to T cells within lymphoid organs. Finally, the dendritic cells must persist over time. Clinical studies of DC vaccines have demonstrated that typically <5% of DCs reach the lymph node, even if injected in nearby tissues; most cells die and fail to persist. Additionally, the maturation signals that are imparted on DC vaccines ex vivo are often rapidly attenuated, due to the presence of endogenous inhibitors within the host immune system.
To investigate immune-regulatory role of IRAK3 as it relates to dendritic cell vaccines, Turnis et al. generated IRAK3-/- DCs. As shown in Figure 9, IRAK3-/- DCs produce more type 1, pro-inflammatory cytokines (IL12, TNFα, and IL6), and less type 2 skewing (MCP1, CCL2) cytokines, than their wildtype counterparts following LPS stimulation. These data demonstrate that depletion of IRAK3 results in enhanced DC maturation and polarization towards a type 1 phenotype. In addition, removal of IRAK3 from DCs results in enhanced in vitro and in vivo migratory activity as compared to wildtype counterparts (Figure 10). Not surprisingly, IRAK3-/- DCs also exhibit enhanced longevity in vitro, relative to wildtype counterparts (Figure 10). Thus, targeting IRAK3 in the context of DCs yields improvements on the three criteria required for DC vaccine efficacy – better migration, acquisition, and maintenance of a mature phenotype, and enhanced longevity. Does improvement in these three critical dimensions improve the overall activity of DC vaccines? The answer is yes. As shown in Figure 11, vaccination of syngeneic tumor models with IRAK3-/- DCs results in enhanced tumor clearance and increased survival. Animals vaccinated with a wildtype DC vaccine exhibit no significant benefit in anti-tumor activity or survival as compared to vehicle control. In contrast, the IRAK3-/- DC vaccine provides robust anti-tumor activity, exhibiting tumor stasis over the 35 day study period. Targeting IRAK3 in the context of a I/O strategy may therefore result in enhanced DC survival, migration, and antigen presentation, leading to an enhanced anti-tumor immune response.
IRAK3 expression levels provide diagnostic and prognostic value. As shown in Figure 12, Saenger et al. describe a whole-blood based 4-gene model (including IRAK3) that predicts overall survival of melanoma patients that are treated with the anti-CTLA4 immune checkpoint antibody tremelimumab. Their model performed better than clinical variables, including tumor staging parameters. In these patients, elevation of IRAK3 expression is a predictor of shortened survival following anti-CTLA4 therapy, which is consistent with the negative immune regulatory role of IRAK3. Additionally, in support of the cancer-specific role of elevated IRAK3 expression in peripheral myeloid cells, Caba et al. published a four-gene diagnostic predictor set based on peripheral blood profiling of Pancreatic Ductal Adenocarcinoma (PDAC) patients (Figure 15). Thus, in addition to predicting poor clinical outcomes, elevated peripheral blood IRAK3 expression is also diagnostic of cancer.
Feasibility of Targeting IRAK3 Using a Small-Molecule Approach
Although IRAK family members have been successfully targeted using ATP-competitive small-molecule inhibitors, targeting IRAK3 with small-molecule approaches presents a unique challenge. Unlike IRAK1 and IRAK4, IRAK3 is a pseudokinase. Pseudokinases are characterized by the lack of conserved motifs involved in nucleotide binding or catalytic kinase activity. Interestingly, approximately 10% of the human kinome can be classified as pseudokinases. Figure 16 gives a schematic depiction of conserved motifs and residues that contribute to nucleotide binding and catalytic activity in kinases. Approximately 40% of pseudokinases retain nucleotide binding activity, but most are deficient in catalyzing phosphoryl transfer, the key chemical reaction, which is central to kinase function.
A resource for all things pseudokinase was published recently by Murphy, et al. This manuscript categorized 31 pseudokinases, dividing them into four classes on the basis of ligand-binding properties. IRAK3 is defined as a Class I pseudokinase; IRAK3 does not exhibit nucleotide or cation binding. As shown in Figure 17, IRAK3 lacks a key HRD motif, which contributes the catalytic aspartic acid residue. However, IRAK3 is still capable of binding the ATP-competitive small-molecule inhibitors DAP and VI16832, potentially indicating the presence of an intact ATP binding cleft (Figure 18). Given these data, it’s possible that IRAK3 does bind nucleotides and cations, but the binding affinities are just below the limit of detection for the assay used. Further biochemical investigation of the ATP-binding cleft region of IRAK3 could help determine the feasibility of an ATP-competitive small molecule inhibitor targeting approach.
But how does one successfully target a kinase devoid of any catalytic activity using small molecules? Any approach that seeks to target IRAK3 should ultimately aim to prevent IRAK3 heterodimerization with members of the Myddosome. This would halt dampening of pro-inflammatory TLR/IL1R signaling, and potentially translate into an improved innate immune anti-tumor response. Luckily, pharmacological targeting of pseuodokinases is not necessarily a new concept. The Gray laboratory are leaders in this field, and have pioneered chemical approaches to target the HER3 pseudokinase using covalent inhibitors. A medicinal chemistry strategy aimed at targeting IRAK3 could similarly exploit nucleophilic residues nearby the IRAK3 ATP-binding pocket, which would kill the protein by marking it for degradation, or potentially preventing heterodimerization with Myddosome members through an allosteric mechanism. Another approach could seek to entirely eliminate the IRAK3 protein, through exploiting the E3 ubiqutin ligase and proteasomal machinery, a topic we’ve opined about previously. For example, linking a covalent warhead to a thalidomide analogue would allow for the targeting of IRAK3 and recruitment of Cereblon, resulting in subsequent proteasomal degradation of IRAK3. Nonetheless, the medicinal chemistry efforts required for either of these complementary strategies should not be underestimated; entire biotechnology companies exist with the sole purpose of harnessing the E3 ubiquitin ligase machinery. Nonetheless, suitable chemical probes that are able to bind to and disrupt IRAK3 heterodimerization are required to explore the feasibility of the small-molecule targeting approach.
Other Approaches to Target IRAK3
Beyond the small-molecule approaches we’ve outlined above, one could envision using several additional modalities, perhaps for slightly different therapeutic applications. For example, knockdown of IRAK3 in TAMs could be achieved through either systemic or local delivery of an RNA-based modality (e.g. siRNA). Alternatively, gene editing approaches (e.g. CRISPR) could allow for complete knockout of IRAK3. Gene-editing could be of particular use in ex-vivo applications, such as generating an autologous IRAK3 knockout DC vaccine.
Applications of IRAK3-Targeting Technology
Immuno-oncology combinations would be the the most obvious application of IRAK3-targeting technology. Even though remarkable progress has been made with the advent of checkpoint inhibitors, most patients still fail to respond. The presence of a non-inflamed tumor microenvironment represents a significant barrier for checkpoint inhibition. Indeed, an inflamed microenvironment may be necessary for tumors to respond effectively to checkpoint inhibitors. As we’ve seen from the data presented above, inhibition of IRAK3 could potentially address this challenge by converting immunosuppresive type 2 (MDSC) myeloid cells into an activated type 1 phenotype. Based on the available preclinical data, IRAK3 inhibition could potentially result in increased antigen presentation and a heightened level of pro-inflammatory cytokines, such as IFNγ, TNFα, and IL6 within the tumor microenvironment. On the whole, these effects could result in conversion of non-inflamed tumors to a inflamed phenotype, and thus potentially improve response rates to checkpoint inhibition.
Questions or comments? Email Garrett at firstname.lastname@example.org
Disclaimer: All opinions expressed on Oncology Discovery are my own and do not necessarily represent the position of my employer. The information presented within this article is not a solicitation for investment.
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