ORMDL3 restrains type-I interferon signaling and anti-tumor immunity by promoting RIG-I degradation

Reviewer #1 (Public review):

Summary:

The authors aim to investigate the role of ORMDL3 in regulating Type 1 interferon (IFN) responses and its effect on tumor growth inhibition. The study focuses on the mechanisms involving the RIG-I pathway and USP10-mediated degradation and attempts to establish a link between ORMDL3 expression and the effectiveness of cancer therapy. The authors also explore the broader implications of ORMDL3 in immune signaling, particularly within the context of Type 1 IFN signaling and its therapeutic potential.

Strengths:

• The manuscript explores a novel aspect of cancer immunology by examining the relationship between ORMDL3 and Type 1 IFN signaling, potentially offering new therapeutic avenues.
• A variety of experimental approaches are employed, including knockdown models, overexpression assays, and protein interaction analyses, to elucidate the role of ORMDL3 in modulating immune responses.
• The findings suggest a potential mechanism by which ORMDL3 affects the tumor microenvironment and immune responses, which could have significant implications for understanding cancer progression and therapy.

Weaknesses:

• The study does not clearly establish the relationship between Type 1 IFN and cancer therapy, and more robust data are needed to support the claim that tumor growth inhibition occurs via Type 1 IFN upregulation following ORMDL3 knockdown.
• There is ambiguity regarding whether ORMDL3 has a positive or negative role in the Type 1 IFN pathway, especially given conflicting findings in the literature that link higher ORMDL3 levels to increased Type 1 IFN expression.
• The use of certain experimental models, such as HEK293T cells (which are not typical Type 1 IFN producers), raises concerns about the validity and generalizability of the results. Further clarity is needed regarding the rationale for using the same tag in overexpression experiments.
• The manuscript contains several inconsistencies and lacks detailed explanations of critical areas, such as the mechanism by which ORMDL3 facilitates USP10 transfer to RIG-I despite no direct interaction between ORMDL3 and RIG-I.

Reviewer #2 (Public review):

Summary:

The authors identified ORMDL3 as a negative regulator of the RLR pathway and anti-tumor immunity. Mechanistically, ORMDL3 interacts with MAVS and further promotes RIG-I for proteasome degradation. In addition, the deubiquitinating enzyme USP10 stabilizes RIG-I and ORMDL3 disturbs this process. Moreover, in subcutaneous syngeneic tumor models in C57BL/6 mice, they showed that inhibition of ORMDL3 enhances anti-tumor efficacy by augmenting the proportion of cytotoxic CD8-positive T cells and IFN production in the tumor microenvironment (TME).

Strengths:

The paper has a clearly arranged structure and the English is easy to understand. It is well written. The results are clearly supporting the conclusion.

Author response:

• The study does not clearly establish the relationship between Type 1 IFN and cancer therapy, and more robust data are needed to support the claim that tumor growth inhibition occurs via Type 1 IFN upregulation following ORMDL3 knockdown.

We thank the reviewer’s concern. In Figure 6 we detected the expression of IFNB1 and ISGs in MC38 and LLC tumor upon ORMDL3 knockdown. At the mean time, we also used IHC to explore the abundance of RIG-I and ORMDL3 in these tumors. In addition, in figure S5 we performed western blots to detect the expression of RIG-I with or without ORMDL3 knockdown. All these results support our hypothesis that that ORMDL3 is a negative regulator of interferon via modulating RIG-I abundance.

• There is ambiguity regarding whether ORMDL3 has a positive or negative role in the Type 1 IFN pathway, especially given conflicting findings in the literature that link higher ORMDL3 levels to increased Type 1 IFN expression.

We appreciate the reviewer’s concern. In our system and experiments, we validated that ORMDL3 is a negative regulator of interferon, although there is also literature that links higher ORMDL3 levels to increased type-I IFN response. ORMDL3 has been reported associated with rhinovirus-induced childhood asthma (Nature. 2007;448(7152):470-473; N Engl J Med. 2013 Apr 11;368(15):1398-407), and ORMDL3 level is positively associated with rhinovirus abundance (N Engl J Med. 2013 Apr 11;368(15):1398-407). There are reports indicating that ORMDL3 supports the replication of rhinovirus (for example, Am J Respir Cell Mol Biol. 2020 Jun;62(6):783-792). This phenomenon is consistent with our findings that higher ORMDL3 expression leads to lower interferon production, which facilitates viral replication. We believe that the different experimental conclusions obtained in these experiments are due to different experiment condition and different stimulation. In our research, we provided comprehensive studies at the molecular, cellular, and animal levels to support the conclusion that ORMDL3 is a negative regulator of type-I interferon.

• The use of certain experimental models, such as HEK293T cells (which are not typical Type 1 IFN producers), raises concerns about the validity and generalizability of the results. Further clarity is needed regarding the rationale for using the same tag in overexpression experiments.

We thank the reviewer’s suggestion. Besides HEK293T, in Figure 1C and 1D we also used A549 and BMDM to overexpress ORMDL3 and stimulate them with polyI:C or polyG:C, Our results showed that ORMDL3 especially inhibits RLR signaling. Additionally, in Figure 3H we found that the endogenous RIG-I expression decreased when we overexpressed ORMDL3 in BMDM. Regarding the issue of using different protein tags, we plan to use different tags to validate our results.

• The manuscript contains several inconsistencies and lacks detailed explanations of critical areas, such as the mechanism by which ORMDL3 facilitates USP10 transfer to RIG-I despite no direct interaction between ORMDL3 and RIG-I.

There are some ERMC (ER-mitochondria contact) proteins that mediate the interaction between ER and mitochondria. ORMDL3 locates in ER, and it has been reported to be associated with calcium transportation. At the meantime, the calcium transfer between ER and mitochondria plays an important role in protein synthesis. It is possible that some ERMC proteins mediate the interaction between ORMDL3 and MAVS. In addition, we also validated that ORMDL3 interacts with USP10 (Figure 5B). Although ORMDL3 and RIG-I do not interact directly, we generated a mechanistic model that ORMDL3 and MAVS recruit USP10 and RIG-I to ERMCS respectively, thus USP10 could form a complex with RIG-I (Figure 5C) and regulate the stability of RIG-I upon RNA sensing.