Author response:
We particularly appreciate the reviewers’ detailed consideration of both the strengths and limitations of the study. The comments have helped us identify areas where the mechanistic interpretation can be further strengthened and where the conclusions should be more precisely defined.
We have carefully considered all of the points raised and provide below our provisional responses and planned revisions. We intend to address these comments comprehensively in the revised manuscript.
Reviewer #1 (Public review):
An expanded description of the histological types of ovarian cancer used to generate survival curves and demonstration of the expression of total IRAK4 would strengthen the manuscript.
If the information is available, it could be useful to indicate the percentages of different ovarian cancer histotypes analyzed in Figure 1.
Providing panels demonstrating the expression of total IRAK4 in Figure 4 would be informative.
We will expand the analysis/description of the ovarian cancer histo-types represented in the survival analyses and, where available, provide the percentages of the different histo-types analyzed in Figure 1. We will also provide additional panels demonstrating total IRAK4 expression in Figure 4. Indeed, these additions will provide greater clarity regarding the histological and molecular characteristics of the models used.
Reviewer #2 (Public review):
(1) UR241-2 decreases tumors at the injury site but apparently does not significantly decrease omental tumor burden in the syngeneic MiM model. It argues against simple nonspecific antitumor activity and supports a possible role for IRAK4 specifically within an inflammation/injury-dependent metastatic niche. The authors should make much more of this distinction-but also mechanistically prove it.
We agree that the differential response to UR241-2 at the injury site versus the omentum is an important finding and will make this distinction substantially more prominent in the revised manuscript. We appreciate that reviewer considers this pattern arguing against interpreting UR241-2 simply as a nonspecific inhibitor of ovarian tumor growth. Rather, the preferential reduction of tumor formation at the injury site is consistent with a role for IRAK4 in the establishment of tumors within an injury-associated inflammatory niche.
This interpretation is also consistent with our data that genetic IRAK4 depletion significantly prolonged survival than null control. This newly generated data will be added to the revised manuscript.
We will also clarify an important limitation our study pertaining to the omental comparison. Because the omentum is thin and multilayered, direct needle implantation does not provide the same spatial control as implantation at an injury site in the peritoneum; needle penetration may result in tumor-cell deposition beyond the intended omental compartment and complicate quantitative assessment of omental tumor burden. Thus, while we agree that the differential response may be informative, we will avoid interpreting the absence of a significant omental response as definitive evidence that IRAK4 is not involved in omental disease. This is stated so because, importantly, clinical observations have been reported indicating that surgically injured or partially resected omental tissue can support tumor-cell adhesion (Dong X et al. https://doi.org/10.1186/s13048-024-01401-8). This provides an additional rationale for considering injury/inflammation, rather than anatomical site alone, as the relevant biological feature needed for inflammation-mediated seeding. We will incorporate this evidence and provide the published literature citations to show that residual injured omentum, when present, could constitute an inflammatory niche where seeding can occur. Nevertheless, omentum is generally resected in advanced EOC (omentectomy), so it is not available for seeding during the recurrence and hence is largely irrelevant to our study theme.
(2) The manuscript does not yet establish that UR241-2's antitumor effects are mediated primarily through IRAK4. The compound has measurable activity against additional kinases, including MAP4K2 and LRRK2, and activity against other kinases is reported at higher concentrations. More importantly, there appears to be a substantial concentration disconnect across assays. IRAK4 phosphorylation is inhibited at nanomolar concentrations in some experiments, whereas colony formation/viability phenotypes occur largely in the micromolar range. For example, colony effects are reported at 5-20 µM and viability experiments at 20-60 µM. Thus, are the antiproliferative effects observed at 10-60 µM actually caused by IRAK4 inhibition? The manuscript needs a stronger pharmacological/genetic causality experiment. Ideally, the authors should test UR241-2 in IRAK4-knockdown/knockout cells. If UR241-2 retains essentially identical cytotoxic activity after IRAK4 loss, the mechanistic interpretation would need substantial revision. A rescue experiment with WT versus inhibitor-resistant IRAK4 would be even stronger.
We agree that establishing pharmacological causality is central to interpreting UR241-2’ s mechanism of action and designating it as a drug-like candidate. In particular, the reviewer appropriately points out that biochemical inhibition of IRAK4 occurs at lower concentrations than some of the antiproliferative and colony-formation phenotypes and that UR241-2 has measurable activity against additional kinases. However, nanoBret assay (Figure-2M) identifies UR241-1 as a very selective IRAK4 kinase inhibitor. The homologue IRAK1 is inhibited at ~1092-fold higher doses than IRAK4. Similarly, FLT3, MAP4K1, MAP4K2, MAP4K3, MAP4K5 and LRRK2 are inhibited at over 18-800 folds. We agree that doses of UR241-2’s at which kinase inhibition and anti-proliferative actions occur do not match. We will attempt to carefully distinguish the concentrations required for direct IRAK4 pathway inhibition from those producing broader cellular phenotypes and will strengthen the pharmacological-genetic evidence linking UR241-2 activity to IRAK4. We will specifically address whether the cellular effects of UR241-2 are diminished when IRAK4 is genetically depleted or otherwise functionally absent. These analyses will help determine the extent to which the higher-concentration antiproliferative effects can be attributed to IRAK4 versus potential off-target activities.
We will also revise the manuscript so that conclusions regarding UR241-2 mechanism are proportional to the available evidence and do not imply that all cellular effects at higher concentrations necessarily reflect IRAK4 inhibition.
(3) The distinction between host IRAK4 and tumor-cell IRAK4 is insufficiently resolved. The Il1r1 experiments manipulate the host, whereas IRAK4 knockdown manipulates the tumor cell. UR241-2, meanwhile, presumably inhibits IRAK4 in both compartments. Consequently, the current experiments combine at least two mechanistically distinct possibilities: tumor-intrinsic IRAK4 versus host IRAK4. The manuscript would be considerably stronger if these compartments were experimentally separated. For example, IRAK4-deficient tumor cells implanted into WT versus pathway-deficient hosts, or pharmacological treatment of mice bearing IRAK4-deficient tumor cells, could determine how much of UR241-2 efficacy is tumor-intrinsic versus microenvironment-mediated.
We agree that this is an important mechanistic distinction. The current experiments interrogate different compartments: IL1R1 manipulation primarily addresses the host inflammatory environment, whereas IRAK4 depletion in tumor cells addresses tumor-intrinsic signaling. UR241-2, in contrast, can potentially affect IRAK4 in both compartments.
We will therefore more explicitly separate these mechanisms in the revised manuscript. In particular, we will examine the available genetic and pharmacological data in the context of a model in which host IL1R1/inflammatory signaling establishes a permissive injury-associated environment, while tumor-cell IRAK4 contributes to tumor-cell responses within that environment. Where feasible, we will incorporate additional experiments to better distinguish tumor-intrinsic from host-mediated contributions to UR241-2 activity.
(4) The immune conclusions are presently associative. The increase in MHC-II-positive macrophages and neutrophils is interesting, but describing these populations as demonstrating an "antitumor immune response" is stronger than the evidence warrants. MHC-II expression does not itself demonstrate antitumor function. Likewise, neutrophils in ovarian cancer can be either tumor-promoting or tumor-suppressive depending on context. The authors show that UR241-2 changes immune composition/phenotype. They do not yet demonstrate that these cells mediate the therapeutic effect. This could be addressed by macrophage or neutrophil depletion, functional assays, cytokine profiling, T-cell activation measurements, or potentially single-cell profiling
We agree that the changes in macrophage and neutrophil populations are currently associative, and do not by themselves establish that these cells mediate the therapeutic response of UR241-2. MHC-II expression, for example, indicates a change in macrophage phenotype but is not sufficient evidence of antitumor activity, and neutrophil function can be context-dependent.
We will therefore revise the terminology to distinguish changes in immune-cell composition or phenotype from demonstrated immune-mediated tumor suppression. We will integrate these findings more appropriately into the inflammatory-niche model and will limit causal conclusions regarding macrophages and neutrophils unless supported by additional functional evidence.
(5) The drug-development claims are somewhat premature
The ADMET package is useful, but several features deserve more cautious interpretation. The compound shows: very high plasma protein binding, rapid mouse microsomal turnover, evidence of efflux, relatively rapid IV clearance, and measurable off-target kinase activity. The authors report mouse microsomal half-life of only ~8.7 min compared with ~209 min in human microsomes and an efflux ratio of ~4.14. These are not fatal problems for a proof-of-concept molecule, but they make language implying a near-clinical candidate premature. UR241-2 currently looks more convincing as a lead/tool compound demonstrating therapeutic tractability of IRAK4 than as an advanced drug candidate.
We agree that, although the ADMET characterization of UR241-2 is comprehensive and addresses key parameters relevant to drug development, its potential as a drug candidate should be interpreted cautiously.
Importantly, the available data supports appropriate cellular selectivity for IRAK4. Although the initial HotSpot kinase panel identified additional kinase interactions, most of these activities were substantially weaker than IRAK4 inhibition and were effectively dialed out in the cell-based NanoBRET assay, with target engagement generally requiring approximately 50–1000-fold higher concentrations than those required for IRAK4. Thus, the NanoBRET data provides a more physiologically relevant assessment of cellular target selectivity and supports preferential engagement of IRAK4 by UR241-2 at pharmacologically relevant concentrations.
We also interpret the high plasma protein binding of UR241-2 in the context of its intended pharmacological profile. Rather than necessarily representing a liability, extensive plasma binding may be compatible with development of a formulation or dosing strategy designed to provide sustained, controlled exposure. This could be advantageous for IRAK4, an important component of innate immune signaling, because prolonged and complete suppression may increase susceptibility to infectious complications, particularly in patients with advanced or treatment-exposed EOC who may already have compromised host defenses. Accordingly, a sustained but incomplete degree of IRAK4 inhibition may be preferable to continuous, complete target blockade. This hypothesis will require direct validation during further drug-development studies.
The relatively short microsomal half-life observed in mouse microsomes is recognized as a limitation for preclinical development and may complicate exposure and toxicity studies in rodents. However, UR241-2 demonstrated a substantially longer microsomal half-life in human microsomes (209 min) (the intended species), which supports further consideration of the scaffold for human drug development. Collectively, these findings support UR241-2 as a pharmacological proof-of-concept and a tractable lead scaffold, while acknowledging that additional medicinal chemistry and pharmacokinetic optimization will be necessary to establish a suitable development candidate
However, we will revise the manuscript to position UR241-2 as an investigational lead/tool compound demonstrating the therapeutic tractability of IRAK4 while clearly acknowledging its current development limitations. We will also distinguish proof-of-concept efficacy from the properties required for progression toward a clinical candidate.
(6) Exposure-response relationships need considerably more attention. Analysis of IRAK4 and/or NF-κB pathway in the treated tumors should be examined
We agree that linking systemic exposure to tumor pharmacodynamics would strengthen interpretation of the in vivo findings. We will further examine the available tumor samples and pharmacodynamic data for evidence of IRAK4 and/or NF-κβ pathway modulation following UR241-2 treatment and relate these findings, where possible, to compound exposure and tumor response.
This analysis should help distinguish target engagement and pathway modulation from nonspecific effects occurring at higher systemic or tissue concentrations.
(7) Some mechanistic observations need deeper validation. The connections among IRAK4, adhesion, E-cadherin, WNT4 and ECM remodeling are intriguing but currently somewhat descriptive. At present, several pieces of this pathway appear adjacent rather than causally connected.
We agree that these observations currently provide a mechanistic framework but that some relationships are not yet established as directly causal. We will therefore more clearly distinguish findings that demonstrate pathway activity from those that represent downstream associations.
In particular, we will strengthen the discussion of how IRAK4 signaling may influence tumor-cell adhesion and establishment following tissue injury and will avoid presenting changes in E-cadherin, WNT4, or ECM-associated factors as independently proven causal steps unless directly supported by the data. Where appropriate, additional analyses will be used to clarify the relationship among these processes.
We appreciate the reviewers' careful evaluation of the manuscript. The comments have helped us sharpen the central model: that tissue injury creates an inflammatory environment that promotes tumor establishment and that IRAK4 represents an important signaling node within this process. The revised manuscript will strengthen the mechanistic evidence for this model, clarify the distinction between injury-associated metastatic establishment and generalized tumor growth, and more appropriately define the translational potential and limitations of UR241-2.
