Carfilzomib modulates tumor microenvironment to potentiate immune checkpoint therapy for cancer

Abstract Impressive clinical benefit is seen in clinic with PD‐1 inhibitors on portion of cancer patients. Yet, there remains an urgent need to develop effective synergizers to expand their clinical application. Tumor‐associated macrophage (TAM), a type of M2‐polarized macrophage, eliminates or suppresses T‐cell‐mediated anti‐tumor responses. Transforming TAMs into M1 macrophages is an attractive strategy of anti‐tumor therapy. Here, we conducted a high‐throughput screening and found that Carfilzomib potently drove M2 macrophages to express M1 cytokines, phagocytose tumor cells, and present antigens to T cells. Mechanistically, Carfilzomib elicited unfolded protein response (UPR), activated IRE1α to recruit TRAF2, and activated NF‐κB to transcribe genes encoding M1 markers in M2 macrophages. In vivo, Carfilzomib effectively rewired tumor microenvironment through reprogramming TAMs into M1‐like macrophages and shrank autochthonous lung cancers in transgenic mouse model. More importantly, Carfilzomib synergized with PD‐1 antibody to almost completely regress autochthonous lung cancers. Given the safety profiles of Carfilzomib in clinic, our work suggested a potentially immediate application of combinational treatment with Carfilzomib and PD‐1 inhibitors for patients with solid tumors.

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EMBO Molecular Medicine has a "scooping protection" policy, whereby similar findings that are published by others during review or revision are not a criterion for rejection. Should you decide to submit a revised version, I do ask that you get in touch after three months if you have not completed it, to update us on the status. ***** Reviewer's comments ***** Referee #1 (Comments on Novelty/Model System for Author): The comments and advice are listed in the remarks to the authors.
Referee #1 (Remarks for Author): The authors have screened FDA-approved drugs and found that Carfilzomib drove IL-4 induced macrophages to express M1 cytokines, enhanced phagocytosis ability and promoted T cell proliferation. In vivo, Carfilzomib reprogrammed TAMs into M1-like macrophages, and promoted CD8+ T cell proliferation or activation to inhibit autochthonous lung cancers in a mouse model. And Carfilzomib synergized with PD-1 antibody to better control autochthonous lung cancers. They further identified that Carfilzomib activated IRE1α to recruit TRAF2, resulting in the activated NF-κB to induce expression of the proinflammatory cytokines. In general, this paper have interesting and important findings, which has proposed that the FDA-approved Carfilzomib can be combined together with anti-PD-1 antibody to improve immune therapy against EGFR mutant-lung cancer. This manuscript can be considered for publication and a revision is encouraged after the authors address the below concerns. My questions are listed below. 1. Fig. 1: In the mock sample, can Carfilzomib alone affect macrophage survival or expression of these proinflammatory cytokines? 2. Fig. 2: Can Carfilzomib alone affect T cell proliferation driven by anti-CD3/CD28 stimulation? Can Carfilzomib affect the expression levels of MHC-I and MHC-II and CD80 in macrophages, which are critical for antigen presentation? In Fig.2, CD86 levels were checked, while in the in vivo study shown in Fig. 6, CD80 expression was measured. The authors should include both CD80 and CD86 in Fig. 2 and Fig. 6. The effector on phagocytosis is enhanced by Carfilzomib treatment from 1% to 3%. This might be due to the limited sensitivity of the assay. Nevertheless, this change is not substantial. The authors are suggested to delete the strong description such as significantly. Similarly, words like "drastic" in the manuscript should be modified. 3. Fig. 4J: Carfilzomib treatment can increase TRAF2 binding to IRE1α. But in this figure, the reduced amount of IRE1α is shown after Carfilzomib treatment. IS this correct? Fig. 4A The Kira6 effect is shown differently in RAW264.7 (completely) vs BMDM (partially). This should be discussed. 4. Fig. S5C: The in vivo study did not observe difference of CD4+ T cells. However, the ex vivo data in Fig. 2 indeed show that Carfilzomib treatment enhances both CD8+ T cell and CD4+ T cell proliferation. This should be discussed. Referee #2 (Remarks for Author): The manuscript by Zhou et al presents a novel approach to increase the efficiency of PD-1 ICB in resistant cancer. They utilize a variety of experimental procedures and convincingly prove their points. The conclusions are both new and of high medical impact.
Qian Zhou and her colleagues use a smart screening system to identify approved drugs, that may transform M2 to M1 macrophages. They identified Carfilzomib, together with two other proteasome inhibitors and then meticulously work their way through its biological effects, its ability to render TAM inflammatory, the mode of action and the intracellular signaling involved. Then they address the effects of Carfilzomib in vivo in an autochthonous murine lung cancer model and show impressive results, especially in combination with a PD-1 inhibitor. Finally, they show similar effects on human ex vivo differentiated macrophages, which strongly argues for transferability to human application. Hence the combination of proteasome inhibitors my represent a new combination treatment to help all those cancer patients who are unresponsive to PD-1 checkpoint blockade. The only prerequisite would be a sufficient TAM infiltration of their tumor. The authors performed their experiments well examined their assumptions often with different methods. Hence I consider this a high quality paper from the scientific point of view. Unfortunately, the English language is, in many parts, not sufficient for publication. Minor points: 1) I really advise the authors to have the manuscript corrected by a native speaker or a commercial service for language and grammar.
2) In some parts the manuscript is also quite demanding for readers, who are not specialist to the field, so a bit more explanation on experimental systems in the results section would be helpful for the general readership.
3) Especially the model system of tumor-SN-treated Raw264.7 needs a (short) introduction.

Referee #3 (Comments on Novelty/Model System for Author):
Overall, this manuscript by Zhou et al is interesting and timely. The authors identified a way to reprogram immunosuppressive M2 macrophages toward antitumor M1 cells, which may help to develop a novel immunotherapeutic strategy. The experiments are in general informative and reasonably well designed. However, several concerns on the analysis, presentation and interpretation of data are raised (see below).

Referee #3 (Remarks for Author):
In this manuscript, Zhou et al found that carfilzomib, a proteasome inhibitor (PI), could drive murine and human M2 macrophages to partially exhibit M1-like phenotype and function. By screening FDA-approved drugs, the authors identified three PIs namely carfilzomib, bortezomib and MLN9708 that effectively elicited IL-1β expression in murine bone marrow-derived macrophages (BMDMs). Further examination revealed that carfilzomib-induced M1-like cells (Ci-M1). Importantly, Ci-M1 exhibited enhanced phagocytotic and antigen-presenting activity, suggesting that carfilzomib treatment at least partially endowed M2/TAM with phenotype and functions similar to M1 macrophages. Investigation into the underlying mechanism of carfilzomib-induced M2 transformation into M1-like cells indicated that this process was dependent on an IRE1α-TRAF2-NF-κB pathway. Therapeutically, systemic carfilzomib treatment could inhibit tumor growth and synergize with PD-1 inhibitors in vivo, an effect that was attenuated in the absence of macrophages. Overall, reprogramming immunosuppressive/protumor macrophages toward cells with antitumor function is emerging as an attractive notion. The present study is interesting and timely. The experiments are in general informative and reasonably well designed. I have some minor concerns about the analysis, presentation and interpretation of data. 1) Figure 1B: Three red dots are shown but they are not individually and specifically annotated. In addition, what does the x axis stand for? It is also unclear whether carfilzomib, bortezomib and MLN9708 are the only drugs in the library that were capable of activating IL-1β expression in IL4-induced M2 macrophages. Is there other PIs in the library that were not able to induce IL-1β expression? These issues should be clarified. 2) Figure 2A: Although carfilzomib could decrease CD206 and arginase 1 expression, this reduction was marginal. Are the levels of IL-6 and iNOS expression post carfilzomib treatment comparable to that in conventional M1 macrophages? A positive control is recommended for supporting the authors' claim that carfilzomib could reprogram M2 macrophage into M1-like population.
3) Figure 2C: The gating strategy is unclear. How was the CD86+ and CD206+ population defined? Was it based on isotypematched antibody staining or fluorescence minus one (FMO) control? This point is also applicable for other flow cytometric plots shown in the manuscript (e.g., Figure S2E and S2L). 4) Figure 2F and 2H: Both assays showed that carfilzomib could enhance macrophage phagocytotic ability, but the phagocytosis efficiencies exhibit a ten-fold difference. This should be clarified. 5) Figure 2L, y axis: Is it CD8+ % or CD4+ %? Figure 3D: Extra asterisks are shown on IL-6. 6) Figure 4: It is very interesting that the kinase activity of IRE1α, instead of the endoribonuclease activity and the activation of XBP1, mediated the carfilzomib induced reprograming of M2 macrophages. The authors are encouraged to discuss the impact of PIs and ER stress inducers on XBP1-deficienct cells.
***** Reviewer's comments ***** Referee #1 (Comments on Novelty/Model System for Author): The comments and advice are listed in the remarks to the authors.
Referee #1 (Remarks for Author): The authors have screened FDA-approved drugs and found that Carfilzomib drove IL-4 induced macrophages to express M1 cytokines, enhanced phagocytosis ability and promoted T cell proliferation. In vivo, Carfilzomib reprogrammed TAMs into M1-like macrophages, and promoted CD8+ T cell proliferation or activation to inhibit autochthonous lung cancers in a mouse model. And Carfilzomib synergized with PD-1 antibody to better control autochthonous lung cancers. They further identified that Carfilzomib activated IRE1α to recruit TRAF2, resulting in the activated NF-κB to induce expression of the proinflammatory cytokines. In general, this paper have interesting and important findings, which has proposed that the FDA-approved Carfilzomib can be combined together with anti-PD-1 antibody to improve immune therapy against EGFR mutant-lung cancer. This manuscript can be considered for publication and a revision is encouraged after the authors address the below concerns.

Reply:
We thank Reviewer for finding our work interesting and important.  The effector on phagocytosis is enhanced by Carfilzomib treatment from 1% to 3%.
This might be due to the limited sensitivity of the assay. Nevertheless, this change is not substantial. The authors are suggested to delete the strong description such as significantly. Similarly, words like "drastic" in the manuscript should be modified. (partially). This should be discussed.

Reply:
We are sorry for not presenting our result clearly enough. Figure 4A showed that IRE1a knockout severely inhibited expression of IL-1B and IL-6 in BMDM. Reply: We thank Reviewer for finding our study novel and compelling.
Minor points: 1) I really advise the authors to have the manuscript corrected by a native speaker or a commercial service for language and grammar.
Reply: Thanks so much for your suggestions. We have a native English-speaking colleague edited the language of our manuscript. I hope that the current version is ready for publishing.
2) In some parts the manuscript is also quite demanding for readers, who are not specialist to the field, so a bit more explanation on experimental systems in the results section would be helpful for the general readership.

Reply:
We thank Reviewer for pointing this out to us. We have explained our rationale for designing our experiments before going into results. We hope that our current version is more friendly to broader readership.
3) Especially the model system of tumor-SN-treated Raw264.7 needs a (short) introduction. Reply: Following Reviewer's suggestion, we have now annotated these three dots separately (New Figure 1B). We are sorry to make Reviewer confused. The X axis stands for the code of the drugs. We have now added the information in figure legend (Page 34, Line 950).
We checked our library carefully again. We confirmed that there are only three PIs in our library. During our screening, we found only Carfilzomib, Bortezomib and MLN9708 could activate IL-1β expression in IL4-induced M2 macrophages. We have now clarified this issue in the text (page 9, line 212).
2) Figure  3) Figure 2C: The gating strategy is unclear. How was the CD86+ and CD206+ population defined? Was it based on isotype-matched antibody staining or fluorescence minus one (FMO) control? This point is also applicable for other flow cytometric plots shown in the manuscript (e.g., Figure S2E and S2L).  Figure 2F and 2H: Both assays showed that carfilzomib could enhance macrophage phagocytotic ability, but the phagocytosis efficiencies exhibit a ten-fold difference.

4)
This should be clarified.

Reply:
We thank Reviewer for pointing this out to us. Figure 2F showed that the number of L1210-GFP cells were phagocytosed per 100 macrophages. In some cases, one macrophage may phagocytose multiple L1210 cells. Figure 2H indicated that the percentage of phagocytosing macrophage in total macrophage. We are using these different methods for describing phagocytosing efficiency induced by Carfilzomib. Figure 2L, y axis: Is it CD8+ % or CD4+ %? Figure 3D: Extra asterisks are shown on IL-6.

5)
Reply: Reviewer is correct. The Y axis is CD4 + % in Figure 2L (new Figure 2L). Thank you for the submission of your revised manuscript to EMBO Molecular Medicine. We have now received the enclosed report from the two referees who were asked to re-assess it. As you will see the referees are now supportive, and I am pleased to inform you that we will be able to accept your manuscript pending the following amendments: 1. In the main manuscript file, please remove the blue color font.
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8. For more information: There is space at the end of each article to list relevant web links for further consultation by our readers. Could you identify some relevant ones and provide such information as well? Some examples are patient associations, relevant databases, OMIM/proteins/genes links, author's websites, etc... 9. As part of the EMBO Publications transparent editorial process initiative (see our Editorial at http://embomolmed.embopress.org/content/2/9/329), EMBO Molecular Medicine will publish online a Review Process File (RPF) to accompany accepted manuscripts. a. In the event of acceptance, this file will be published in conjunction with your paper and will include the anonymous referee reports, your point-by-point response and all pertinent correspondence relating to the manuscript. Let us know if you do NOT agree with this. b. Please note that the Authors checklist will be published at the end of the RPF.
I look forward to seeing a revised version of your manuscript as soon as possible.

Kind regards Jingyi
Jingyi Hou Editor EMBO Molecular Medicine *** Instructions to submit your revised manuscript *** *** PLEASE NOTE *** As part of the EMBO Publications transparent editorial process initiative (see our Editorial at https://www.embopress.org/doi/pdf/10.1002/emmm.201000094), EMBO Molecular Medicine will publish online a Review Process File to accompany accepted manuscripts.
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Is there an estimate of variation within each group of data?
Statistical analysis was done on all experiment that we used mice. we have indicated the mouse numbers in Materials and Methods and in Figure legends.
In experiment when we used Carfilzomib or other treatments (as indicatated in the manuscript) to treat transgenic mice, computed tomography was conducted to confirm lung cancer in mice. Mice bearing no noticable lung cancers before treatment were excluded from analysis.
In our treatment studies, we chose a cohort of mice bearing similar burden of lung cancer. These mice were then randomized to different treatments.

Not applicable
No, there was no estimate of variation within each group of data.
The mice were randomized for treatment. We have indicated this in main text.
ImageJ software was used to analyze the CT images for determining tumor burdens.
Statement was included in material and methods.

Data
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