Dual‐Epigenetically Relieving the MYC‐Correlated Immunosuppression via an Advanced Nano‐Radiosensitizer Potentiates Cancer Immuno‐Radiotherapy

Cancer cells can upregulate the MYC expression to repair the radiotherapy‐triggered DNA damage, aggravating therapeutic resistance and tumor immunosuppression. Epigenetic treatment targeting the MYC‐transcriptional abnormality may intensively solve this clinical problem. Herein, 5‐Aza (a DNA methyltransferase inhibitor) and ITF‐2357 (a histone deacetylase inhibitor) are engineered into a tungsten‐based nano‐radiosensitizer (PWAI), to suppress MYC rising and awaken robust radiotherapeutic antitumor immunity. Individual 5‐Aza depletes MYC expression but cannot efficiently awaken radiotherapeutic immunity. This drawback can be overcome by the addition of ITF‐2357, which triggers cancer cellular type I interferon (IFN‐I) signaling. Coupling 5‐Aza with ITF‐2357 ensures that PWAI does not evoke the treated model with high MYC‐related immune resistance while amplifying the radiotherapeutic tumor killing, and more importantly promotes the generation of IFN‐I signal‐related proteins involving IFN‐α and IFN‐β. Unlike the radiation treatment alone, PWAI‐triggered immuno‐radiotherapy remarkably enhances antitumor immune responses involving the tumor antigen presentation by dendritic cells, and improves intratumoral recruitment of cytotoxic T lymphocytes and their memory‐phenotype formation in 4T1 tumor‐bearing mice. Downgrading the radiotherapy‐induced MYC overexpression via the dual‐epigenetic reprogramming strategy may elicit a robust immuno‐radiotherapy.


Introduction
Radiotherapy, although clinically effective in cancer treatment, causes patients severe immune tolerance and immunosuppression. [1]One main reason is that cancer cells trickily increase the expression of MYC protein per se after receiving the radiation. [2]This protein possesses the capacity to repair DNA damage caused by radiotherapy, [3] continuing the proliferation of cancer cells and inhibiting the type I interferon (IFN-I) signaling pathway. [4]mpaired IFN signaling further impedes antigen presentation, disturbs the activation of cytotoxic T lymphocytes (CTLs), and triggers the expansion of tumorassociated regulatory T (Treg) cells. [5]These cascade effects that MYC overexpression brings in highly inhibit the radiotherapeutic outcomes. [6]Downregulating the MYC level of tumor cells may overcome the critical immunosuppressive barrier and elicit a robust immuno-radiotherapy.Abnormal epigenetic changes of gene expression including the DNA methylation Scheme 1. PWAI-relevant preparation and therapeutic mechanism.a) The synthetic routes of PWAI.b) Schematic illustration of PWAI(+)-mediated antitumor immunity.Acid-responsive PWAI NPs disassembled after cellular internalization, followed by the release of 5-Aza, ITF-2357, and W 6+ .Afterward, W 6+ sensitized cancer cells to X-ray irradiation and assisted in breaking the DNA.Meanwhile, combining 5-Aza with ITF-2357 suppressed MYC signaling to reverse the DNA damage repair and type I IFN signaling inhibition, imparting a robust antitumor immunity.
and chromatin deacetylation can maintain cancer cellular subpopulations that are highly resistant to cytotoxic therapies, promoting the tumorigenesis and cancer progression. [7]In the light of this, introducing DNA methyltransferase inhibitors (DNMTi) or histone deacetylase inhibitors (HDACi) may epigenetically remodulate gene-expression level. [8]Representatively, 5-Aza, a typical DNMTi, has been reported to downregulate MYC across a few cell lines. [9]However, bare MYC depletion without anti-tumor immune awakening impedes its application in immunotherapy.Given that the HDACi promotes cancer cellular IFN-I production, [10] epigenetic drug combination of DNMTi and HDACi may greatly eliminate radiotherapy-induced MYC protein and evoke a strong IFN-I signaling, which however is unknown.
In this study, we designed an advanced nano-radiosensitizer (PWAI) to launch a dual-epigenetic reprogramming in radiotherapy.The PWAI can coordinate high-Z tungsten metal ions (W 6+ ) with PEG-polyphenols by utilizing a metal-phenolic network and load 5-Aza and ITF-2357 into the hydrophobic core (Scheme 1).Under the stimulation of exotic radiation, X-ray-sensitive W 6+ efficiently destroyed the DNA strands of tumor cells by generating plentiful reactive oxygen species (ROS), [11] and meanwhile two types of released epigenetic inhibitors can inhibit the repairing performance of MYC protein on broken DNA strands.
During the dual-epigenetic programming process, we identified that although downregulating MYC protein, 5-Aza did not obviously trigger IFN-/IFN- generation.When used in combination with ITF-2357, apparent MYC inhibitory effect was recognized, and more importantly, levels of IFN-I signal-related proteins involving IFN-, IFN-, and the major histocompatibility complex I (MHC-I) were significantly enhanced.To treat 4T1 tumor-bearing mice with PWAI nano-radiosensitizers, we identified the awakened antitumor immune response after radiotherapy, including the maturation of dendritic cells (DCs), CTLs recruitment and their memory-phenotype formation, as well as the immune polarization of tumor-associated macrophages from immunosuppressive state to immunosupportive condition.Bringing the dual-epigenetic reprogramming in radiotherapy may provide a potential strategy for an advanced immuno-radiotherapy.

Synthesis and Characterization of PWAI NPs
11a,12] It reacted from a monomer with polyphenol groups and could decompose in an acidic environment.All products were verified by 1 H NMR spectroscopy (Figures S1-S6, Supporting Information).The polyphenol groups on the PEG chelated with W 6+ , self-assembling as PW NPs.Loading two inhibitors (5-Aza and ITF-2357) into hydrophobic core of PW NPs generated the PWAI nano-radiosensitizer.As shown by the transmission electron microscopy (TEM), the synthesized PWAI exhibited sphere morphology (Figure 1a).Elemental mapping and energy-dispersive X-ray spectroscopy (EDS) analyses revealed the topography of PWAI and the inclusion of tungsten metal (Figure 1b,c).PWAI maintained an average size of 102.3 ± 4.9 nm up to one week (Figure 1d,e).To prove the acid-decomposition nature of PWAI, the size variation at the indicated pH values was examined (Figure S7, Supporting Information).As pH value decreased from 7.4 to 5.5, PWAI dissembled and showed significant size reduction, and W 6+ thus released from PWAI (Figure 1f,g).Quantitatively, the release rate of W 6+ reached up to 76.6 ± 5.8% within 16 h (pH = 5.5).The pH-responsive release of 5-Aza and ITF-2357 was also confirmed in Figures S8 and S9 (Supporting Information).Furthermore, we experimentally verified that ITF-2357 was encapsulated into PWAI almost entirely based on the hydrophobic interaction; yet 5-Aza loading relied on both hydrophobicity and hydrogen bonding, primarily the hydrophobicity (Figure S10, Supporting Information).The participation of radiosensitizer W 6+ apparently enhanced the ROS production (sin-glet oxygen, hydroxyl radical, and superoxide anion) that X-ray triggered (Figure 1h and Figure S11, Supporting Information).Next, by labeling PWAI nanoparticles with IR780 fluorescent probe (shorted as PWAI@PEG-IR780), we confirmed their effective uptake by 4T1 cancer cells after 6-h co-culturing (Figure 1i).To evaluate the in vivo biodistribution of PWAI in 4T1 breast tumor-bearing mice, PWAI enriched in the tumor tissue (marked by the red circle) on 12-h postinjection, and the accumulation went up to a plateau on 24-h postinjection (Figure 1j,k and Figure S12, Supporting Information).To continue the monitoring for one more day, the fluorescence signals of main organs including the tumor decreased.Quantitatively, 13.34% of W was identified in tumor tissue at 24 h post-injection (Figure S13, Supporting Information).These data revealed a highly intratumoral enrichment of PWAI agents and their biodegradability in vivo.

PWAI Dual-Epigenetically Inhibits the MYC Expression and Halts Post-Radiotherapy DNA Repair
DNA demethylation has been reported as an effective approach to modulate the MYC gene transcription.To examine the demethylated product (5-hmc), cells treated by 5-Aza, PW(+) (PW plus X-ray irradiation) + 5-Aza, or PWA(+) (5-Aza-loaded PW NPs plus X-ray irradiation) exhibited high fluorescent signal of 5-hmc (Figure 2a).We further examined the MYC expression regulated by 5-Aza and identified a dramatic MYC depletion (Figure 2b).The inhibition of 5-Aza on DNA methylation of tumor cells correlates with its function on the MYC downregulation.Next, ITF-2357 working as an HDACi initiated histone 3 acetylation (Figure 2c).To examine the combined function of 5-Aza and ITF-2357 on MYC inhibition, we identified an obvious improvement (Figure 2d).Loading these two hydrophobic inhibitors with our coordinated polymer further amplified the MYC inhibition efficiency.Combined epigenetic therapy that simultaneously inhibits DNA methylation and histone deacetylation was more effective relative to 5-Aza alone in terms of the induction of MYC depletion.
In addition, MYC translational variation may affect the radiotherapeutic killing.It is well known that radiotherapy breaks down the cancer cellular DNA strands by releasing high level of ROS. [13]However, the breakage can be reversed by cancer cells through upregulating the MYC protein per se and protecting themselves from apoptosis. [14]As exhibited in Figure 2e-j and Figures S14 and S15 (Supporting Information), both PW and PWAI activated strong ROS fluorescent signal in 4T1 cancer cells after X-ray irradiation (labeled as PW(+) and PWAI(+)), PW(+) however did not remarkably affect cell viability and proliferation as PWAI(+) did.In the cloning test (Figure 2g), we demonstrated that PW(+) barely reduced the formation of cell clusters.In contrast, cell clusters were hardly identified in the group of PWAI(+) functionalizing with 5-Aza and ITF-2357.To test MYC level per group, unlike the barely visible result in PWAI(+) group, robust MYC expression was identified in PW(+) group.PW(+) treatment upregulated the production of MYC protein, which was successfully inhibited by the dual-epigenetic inhibitors loaded in PWAI.Taken together, as shown in the Figure 2k, W 6+ from the PWAI worked as an X-ray sensitizer to promote the generation of ROS and damage DNA.To fight against the radiotherapeutic destruction, tumor cells upregulated intracellular MYC proteins, which however could be halted by the combined use of 5-Aza and ITF-2357, stopping the DNA repair and the unrestricted growth and proliferation of tumor cells.Cancer cellular MYC level thereby negatively correlates with the radiotherapeutic killing efficacy.

PWAI Boosts Radiotherapeutic IFN Signaling for Efficient Immunomodulation
Highly expressed MYC of tumor cells causes antagonism of interferon-stimulated gene induction and epigenetic treatmentinduced antigen presentation, which ultimately inhibits the IFN-I signaling and the CTL function. [15]As investigated in Figure 3a,b and Figure S16 (Supporting Information), single radiotherapy (PW(+)) did not obviously trigger the expression of IFN- and IFN-.To introduce 5-Aza and ITF-2357 into the radiotherapy, PWAI(+) multiplied the IFN- and IFN- levels by 7.1 and 6.4 times, respectively.IFN-I signaling impacts the MHC-I level of cancer cells.From the Figure 3c, we observed that the surface expression of MHC-I on 4T1 cell membrane in groups of PWAI(+) and 5-Aza + ITF-2357 was much higher than that in PBS group, which may greatly contribute to the presentation of tumor-associated antigens.Here, abnormal MYC impacted by the radiotherapy (Figure 2d) goes against interferon signaling, which nevertheless can be remedied.
IFNs and MHC-I play pivotal roles in antigen presentation and immune regulation. [16]To examine the maturation of bone marrow-derived DCs (BMDCs) from different treatment groups, we identified that mature BMDCs co-culturing with PWAI(+)treated tumor cells increased by 2.5 times compared to the number in PBS group (Figure 3d,e and Figure S17, Supporting Information), while radiotherapy alone did not exhibit obvious promotion in the maturation of BMDCs.Next, same coculturing approach was adopted to educate bone marrow-derived macrophages (BMDMs).And we found that BMDMs growing with PW(+)-treated tumor cells showed highest level of CD206 expression, which surprisingly dropped to the lowest in PWAI(+) treatment group (Figure 3f,g and Figure S18, Supporting Information).The addition of 5-Aza and ITF-2357 inhibited the polarization of BMDMs to M2 immunosuppressive phenotype.Taken together, epigenetic modulation in radiotherapy not only promoted the IFN-I and MHC-I signaling, and also matured DCs and reversed the immunosuppressive response during the radiotherapy (Figure 3h).

PWAI Reactivates Radiotherapeutic Immunosuppression In Vivo
Then we constructed a 4T1 orthotopic tumor-bearing mouse model to study the effects of PWAI on intratumoral MYC expression and IFN-I pathway (Figure 4a).One million of 4T1 cells were surgically injected into the fourth left mammary pad of the mouse.When the tumor grew up to 100 mm 3 , all mice were randomly divided into six groups, and the therapeutic reagents were injected through the tail vein, respectively.For mice receiving X-ray irradiation, 6 Gy of X-ray irradiation was given to the tumor site on 24 h after drug injection.On the third day after three consecutive treatments, tumor tissues were collected for comprehensive analysis.We firstly analyzed the epigenetic changes in tumor tissue by Western blot, and found that they drove consistent trends as the previous results from in vitro experiments (Figure 4b,c).PWAI upregulated the acetylation levels inhibited by the radiotherapy but downregulated the expression of MYC in tumor tissue.Next, PWAI(+) enhanced the intratumoral levels of IFN- and IFN- by 2.6-folds and 2.3-folds, respectively, compared with the group receiving PBS (Figure 4d,e).Differently, PW-based radiotherapy barely affected the levels of these two immune factors.The MHC-I fluorescent signal correlated with the IFN-I level in a positive mode and appeared highest intensity in tumor tissue of mice treated with PWAI(+) (Figure 4f).
To further examine cytokines in tumors and sera, except for single radiotherapy, all other treatments increased the concentrations of four proinflammatory cytokines (interferon- (IFN-), tumor necrosis factor- (TNF-), interleukin-6 (IL-6) and interleukin-12p70 (IL-12p70)) (Figure 4g and Figures S19 and S20, Supporting Information) to higher levels.In contrast, the radiotherapy was the only one to upregulate the anti-inflammatory cytokine IL-10 (Figure 4h).IFN cytokines stimulate the DC maturation.After three repeating treatments, the frequency of     CD8 + T cells in tumor tissue after staining with DAPI (blue), CD3 + antibody (red), and CD8 + antibody (green), respectively.Scale bar: 20 μm.f,g) Flow analysis of Foxp3 + T regulatory cells in tumors.h) Quantification of IFN- + T lymphocytes in total CD45 + CD8 + cells in TDLN.i) The percentage of granzyme B + (GzmB + ) cells in CD8 + T cells in the TDLN.j) Flow cytometric examination of Ki67 + cells in CD8 + T cells in the TDLN.

PWAI Potentiates Systemic Antitumor Immune Responses
IFNs recruit T cells and enhance their tumoral infiltration. [17]ere a bilateral orthotopic breast tumor-bearing mouse model was constructed to test the IFN signaling triggered by PWAI on T cell immunity (Figure 5a).On 24 h after tail vein injection of different agents, X-ray irradiation (6 Gy) was optionally applied to the primary tumor of the mice, and the treatment cycle was continuously implemented for three times.On the third day after the treatment, the distant tumors and tumor-draining lymph nodes (TDLNs) of the mice per group were collected for analyses.

PWAI Exhibits Robust Tumor Eliminating Performance In Vivo
We finally evaluated the antitumor efficacy of PWAI on an orthotopic, bilateral 4T1 breast tumor model.On day 9, NPs were intravenously administrated, followed by X-ray irradiation on day 10 (Figure 6a).Three-cycle treatment distinguished the prominent therapeutic effect of PWAI.As depicted in Figure 6b-f, PWAI(+) curved the growth of both primary and distant tumor (Figure 5).We collected the distant tumor for hematoxylin and eosin (H&E) staining and observed that treatment with PWAI(+) resulted in extensive tumor necrosis with few nuclear staining (Figure S28, Supporting Information).This result indicated that the PWAI(+) treatment significantly caused systemic tumor elimination, rendering a systemic immune awakening.To analyze all other therapeutic groups, no equivalent influence was observed on their distant tumors.Given that, PWAI(+) treatment elongated the survival rate of mice (Figure 6g).Since that PWAI(+) awakened antitumor immune surveillance while eradicating tumor cells, immunological memory after therapy might be strongly stimulated as well.As demonstrated in Figure 6h,i and Figure S29 (Supporting Information), different from quiet PW(+), PWAI(+) increased the memory CD8 + T cells (CD44 high CD62L low ) by 1.8-folds based on the control group.
In summary, the designed PWAI NPs worked as an immunoregulating radiosensitizer, aiming to solve the radiotherapeutic issues in the clinic and achieve systemic tumor regression.In retrospect, traditional radiotherapy, which requires high-dose irradiation (60 Gy or higher), easily cause patients with severe systemic damage. [18]The occurrence of high-Z metallic radiosensitizers can minimize the above side effects by virtue of their capability to deposit low-dose X-ray energy on tumor sites and sensitize tumor cells to radiotherapy. [19]Representatively, NBTXR3 (hafnium oxide nanoparticles) and AGuIX (gadolinium-based nanoparticles) have entered clinic trails to locally amplify the tumor-killing efficacy and proposed a new standard in radiation oncology. [20]However, radiosensitizer-type treatment still focuses on local tumor control and barely inhibits the progress of remote or non-irradiated ones. [21]rein, inspired by the fact that cancer cells trickily increase the expression of MYC protein per se after receiving the radiation and cause patients' severe immune tolerance, we proposed to functionalize radiosensitizers with immunostimulatory agents.Specifically, two MYC inhibitors were encapsulated into tungsten-based nano-radiosensitizer, forming into PWAI NPs and eventually awakening robust systemic radiotherapeutic outcomes.The abscopal effects induced by PWAI(+) were proved by the extensive tumor cell necrosis (Figure S28, Supporting Information) and elevated infiltration of activated immune cells (Figure 5b-d) within distant tumors.
Inevitably, the unclear toxicities and nano-bio interactions of nanotherapeutic agents may limit their clinical application. [22]umerous biomedical engineering methods have therefore been adopted to optimize the biosafety of nanocarriers as much as possible.In our case, the amphiphilic PEG-polyphenols were utilized.Polyphenols, mainly coming from natural plants, guarantee PWAI excellent biodegradability and biosafety. [23]A strong coordination between polyphenols and metal ions endows PWAI physiological stability and pH-dependent responsiveness.Moreover, PEG decoration of PWAI increases the systemic circulation time of the nanoagents and reduces their immunogenicity. [24]herapeutic biosafety and biocompatibility can be reflected from the normal histological structure of major organs (Figure S30, Supporting Information) and the faint level variations of the serum biochemical markers (Figure S31, Supporting Information).

Conclusion
In this work, advanced nanoradiosensitizers of PWAI NPs were reported to achieve robust radiotherapeutic influence and simultaneously relieve the immunosuppression caused by MYC upregulation.MYC as a key immunosuppressive protein of diverse solid tumors is easily overexpressed under the stimulation of radiation therapy.We thus introduced MYC inhibitors (5-Aza and ITF-2357) to the coordination system of polyphenol molecules and high-Z W 6+ ions, forming PWAI.Combined 5-Aza and ITF-2357 obviously depleted MYC generation, and more importantly activated the IFN-I signaling pathway.To explore from in vitro and in vivo models, we identified that PWAI not just sensitized radiotherapeutic effect on tumor cell elimination, but enhanced CD8 + T cell cytotoxicity, proliferation and the corresponding immune memory.It sheds a light on the epigenetic potential in radiotherapeutic immune activation.

Statistical Analysis
Data were presented as mean ± standard deviation (SD).Two-way analysis of variance (ANOVA) with Tukey's test was performed for the analysis of tumor growth profiles.For other analyses, statistical significance was calculated via one-way ANOVA with Tukey's test (three or more groups) or unpaired two-tailed t-test (two groups).Log-rank (Mantel-Cox) test was used for the statistical analysis of survival rates.In all cases, differences were defined statistically significant if p < 0.05 (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).All statistical analyses were performed using GraphPad Prism 8.0 software.

Figure 4 .
Figure 4. PWAI(+) induces high intratumoural IFN-/ secretion and triggers robust immune response.a) Scheme of in vivo experimental design for (b)-(l).Mice were treated with (+) or without (−) X-ray irradiation one day after the injection of PBS, 5-Aza, ITF-2357, 5-Aza + ITF-2357, PW or PWAI one day.(+) and (−) refer to with and without 6 Gy X-ray irradiation, respectively.Tumors were collected on day 17 for further analysis (n = 5).b) Evaluation of pharmacologic targets of ITF-2357 and 5-Aza: histone acetylation by immunoblotting in tumor tissue after treatment.Histone 3 was used as loading controls.c) Immunoblot showing MYC expression in tumor tissue of mice treated with different agents.GAPDH used as loading control.d,e) Intratumoural IFN- and IFN- levels after treatments.f) Immunofluorescence images of tumor sections after various treatments.The MHC-I (green) and nucleus (blue) were stained with FITC and DAPI, respectively.Scale bar = 10 μm.g,h) Intratumoural IFN- and TNF- levels after the indicated treatments.i,j) Flow analysis of CD86 + activated DCs in tumors.k,l) Flow cytometry analysis of M2-like macrophages (CD45 + CD11b + F4/80 + CD206 + ) in tumor tissue.