KIRA6 abrogates the generation of myeloid-derived suppressor cells and overcomes resistance to anti-PD-1 therapy

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Therefore, it is unmet need to target MDSCs to achieve better outcome of ICB therapy. Inositol-requiring enzyme 1α (IRE1α) is identified as a key regulator for generation of MDSC. Here, we evaluated the potential of KIRA6, an inhibitor for IREα kinase activity and RNase activity, to abrogate MDSC mediated immune suppression. KIRA6 significantly suppressed 4T1 tumor growth, decreased MDSC population and enhanced T cell infiltration. Two dosages of KIRA6 treatment directly inhibited extramedullary myelopoiesis and MDSC generation in vivo. KIRA6 abrogated the induction of MDSCs from bone marrow cells and abolished the immunosuppressive capability of MDSCs in vitro. Meanwhile, KIRA6 not only attenuated G-CSF production from tumor cells thereby blocking the induction of MDSCs, but also caused apoptosis of tumor cells. Moreover, KIRA6 treatment diminished MDSC generation, restored T cell proportion in both local and systemic immune landscapes and eventually overcame resistance to anti-PD-1 therapy. Our work establishes the evidence for KIRA6 as an impressive agent for abrogating MDSC mediated immune suppression, killing tumor, and overcoming ICB resistance. Health sciences/Diseases/Cancer/Cancer microenvironment Health sciences/Medical research/Preclinical research Myeloid-derived suppressor cell Immune Checkpoint Inhibitor Immunotherapy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Immune checkpoint blockade (ICB) therapy has improved survival for cancer patients through harnessing the immune system, making it one of the cornerstones of cancer treatment regimens in a broad range of cancers nowadays 1 – 6 . However, the overall response rates remain low across many types of cancer, which limits the benefit of ICB therapy for majority of cancer patients 7 – 9 . Many immunosuppressive factors in the tumor microenvironment (TME) have been found to collaboratively restrain the activation and effector function of T cells despite the PD-1 blockade 10 . The generation of regulatory immune cells, such as myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs) or regulatory T cells, is the major tumor-driven mechanisms of immune suppression, which alternatively impair antitumor T cell activity within the TME 10 – 14 . Therefore, it is unmet need to target the suppressive immune cells in the TME to achieve better outcome of ICB therapy. Myeloid-derived suppressor cells (MDSCs) constitute a major component of the TME, which are key orchestrators of immunosuppression in TME and could inhibit T cell function through multiple mechanisms, such as arginase-1 (ARG1) and reactive oxygen species (ROS) 15 , 16 . The potent immune modulation capability of MDSCs enables them to drive tumor progression, metastasis, and resistance to cancer therapies, therefore MDSC infiltration is found to correlate with failure of ICB treatment and poor patient survival 16 – 20 . Considering their established role as therapeutic barriers, great efforts have been made to develop MDSC-targeting strategies for cancer therapy, including eliminating MDSC, blocking MDSC recruitment, and abrogating the suppressive function of MDSCs 15 , 21 – 24 . Although targeting MDSCs in preclinical models get improved outcomes, there are still no drugs approved for clinical usage, urging for novel pharmacologic agents to dismantle this suppressive axis. MDSCs consist of a group of heterogeneous myeloid cells, including polymorphonuclear MDSCs (PMN-MDSCs) and monocytic MDSCs (M-MDSCs) 16 . It is generally believed that MDSCs originate from hematopoietic progenitor cells in the bone marrow and spleen 16 , 25 – 27 . This aberrant myelopoiesis process is driven by multiple cytokines and growth factors, such as granulocyte colony-stimulating factor (G‑CSF), promoting their differentiation into immunosuppressive effectors rather than functional counterparts 28 – 31 . Endoplasmic reticulum (ER) stress is observed in MDSCs because of high secretory and metabolic activity during the immunosuppressive myelopoiesis in tumor milieu 32 – 35 . Inositol-requiring enzyme 1α (IRE1α), an important kinase and RNase of ER stress response, is identified as a key regulator for generation of MDSC 33 . On the one hand, IRE1α-mediated XBP1 cleavage promotes acquisition of suppressive activity of MDSC in cancer 33 . On the other hand, the kinase activity of IRE1α potentiates the expression of G-CSF and granulocyte/macrophage colony-stimulating factor (GM-CSF) from tumor cells through activation of JNK pathway, which further facilitates mobilization of hematopoietic progenitor cells and pathological myelopoiesis 36 . The dual effects of IRE1α make it an ideal target for abrogating MDSCs generation. KIRA6 (kinase-inhibiting RNase-attenuators), is developed for diabetes treatment by inhibiting IRE1α kinase activity and RNase activity simultaneously 37 . However, its potential for cancer treatment, especially for targeting MDSC generation and immunotherapy, remains unexplored. Here, we evaluate the antitumor activity of KIRA6 for breast cancer in both animal model and in vitro model. KIRA6 significantly suppresses 4T1 tumor progression and reprograms the tumor immune landscape by enhancing T cell infiltration and decreasing MDSC population. Short-term KIRA6 treatment and cell culture confirm that KIRA6 directly inhibits MDSC generation and function. Meanwhile, KIRA6 not only attenuates G-CSF production therefore blocks the induction of MDSCs, but also causes apoptosis in tumor cells. Moreover, KIRA6 treatment diminishes MDSC generation, restores T cell proportion in both local and systemic immune landscapes and eventually overcomes resistance to anti-PD-1 therapy. Our work establishes KIRA6 as an MDSC-targeting agent for overcoming ICB resistance and provides a promising combination strategy for achieving better immunotherapy. Materials and Methods Reagents Reagents used in this study are summarized in Supplementary Material, Table S1 . Tumor cell culture 4T1 cells (ATCC, CRL-2539) were cultured with RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/mL), streptomycin (100 mg/mL) at 37°C in 5% CO 2 -humidified atmosphere. 4T1 cells were plated overnight in complete RPMI 1640 medium before KIRA6 treatment. 4T1 cells were cultured with fresh culture medium with or without KIRA6 at indicated concentrations. For cell viability assay and flow cytometry analysis, cells were treated with vehicle or KIRA6 for 24 hours. For the preparation of RNA and protein sample, cells were treated with KIRA6 for 4 hours before sample collection. For tumor condition medium, cells were treated with KIRA6 for 4 hours and then washed and culture with fresh medium for 24 hours. Cell culture medium was collected and centrifuged to obtain tumor condition medium. Mouse model All animal experiments were performed according to institutional guidelines and approved by the ethical board of Zhuhai People’s Hospital. Female BALB/c mice (6–8 weeks of age) were purchased from Bestest Biotechnology Company (Zhuhai, China). 2×10 5 4T1 cells were injected subcutaneously into the flank of BALB/c mice. Mice were intraperitonially injected with vehicles, KIRA6 (10 mg/kg) every other day or anti-PD-1 antibody (200 µg) every three days in designated groups. Tumor sizes were measured with caliper when tumors were palpable. Tumor volumes were calculated by (length 2 × width)/2. Isolation of leukocytes from bone marrow cells and splenocytes Bone marrow cells were harvested by flushing the femurs and tibias of mice with PBS supplemented with 1% FBS using syringe. Splenocytes were obtained by homogenizing the spleen using nylon mesh and gently aspirating through a 21-gauge needle 26 , 36 . Red blood cells were removed by ACK lysis buffer. Isolated cells were then washed and resuspended for cell culture, or flow cytometry analysis. Isolation of tumor infiltrating immune cells Mouse tumors were cut into small pieces and digested with 0.05% collagenase type IV, 0.002% DNase I in RPMI 1640 supplemented with 10% FBS. The dissociated cells were filtered through 100 µm mesh before washed and resuspended for FACS analysis or cell culture 31 . Flow cytometry Cells cultured in vitro , leukocytes from peripheral blood, spleen and tumor samples were prepared and suspended in PBS buffer supplemented with 1% heat-inactivated FBS, then stained with desired antibodies. Cell apoptosis was stained with APC Annexin V Apoptosis Detection Kit with 7-AAD. Cell cycle was detected with Cell Cycle and Apoptosis Analysis Kit. Data was acquired on DxP Athena (Cytek) and analyzed with FlowJo software. Generation of MDSC from bone marrow cells Bone marrow cells were plated in 24-well plates in complete RPMI medium (with 10% FBS) supplemented with tumor condition medium in the presence of KIRA6. Cells were cultured at 37°C in 5% CO 2 -humidified atmosphere for 3 days 36 . Co-culture of MDSC and splenocytes Splenocytes were isolated from homogenized spleen of naïve mice and stained with 1.5 µM CFSE for 10min at 37°C according to the manufacturer's instructions. Then, splenocytes were co-cultured with induced MDSCs at 2:1 ratio in the presence of 1 µg/ml anti-CD3 antibody, 1 µg/ml anti-CD28 antibody and 20 U/mL recombinant IL-2. Cells were cultured for 3–5 days and then collected, stained with surface markers, and analyzed by flow cytometry 27 , 36 . Immunoblotting The proteins were prepared with RIPA Lysis and Extraction Buffer and then quantified with BCA Protein Assay Kit. Proteins were separated by 10% SDS-PAGE, immunoblotted with anti p-ERK, ERK, p-Myc, Myc, ARG1 and β-Actin antibody. Antibody binding was detected using horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG antibody and visualized with Western Chemiluminescent kit. RNA-seq and analysis Cells were lysis with TRIzol for RNA purification. The library was constructed and then sequenced by Azenta on an Illumina instrument using a 2×150 paired-end (PE) configuration according to the manufacturer’s instructions. All data are publicly available in the GEO database. Differentially expressed genes were identified using DESeq2 (v1.34.0). Functional enrichment analyses were performed by Gene Set Enrichment Analysis (GSEA) or Gene Ontology (GO). G-CSF ELISA analysis For analysis of G-CSF, tumor condition medium collected from vehicle or KIRA6 pretreated 4T1 cells were assessed for G-CSF concentration using the mouse G-CSF ELISA kits (MultiSciences) according to the manufacturer’s instructions. Statistical analysis IBM SPSS Software (IBM Corporation) and GraphPad Prism (GraphPad Software) were used for statistical analysis. The significance of differences between groups was examined by the Student’s t-test or Mann–Whitney test, as appropriate. The overall survival curves were generated by the Kaplan–Meier method and analyzed using the log-rank test. P < 0.05 was considered significant. Results KIRA6 Inhibits Tumor Growth and Modulates Intratumoral Immune Cell Infiltration To evaluate the antitumor efficacy of KIRA6, tumor-bearing mice were treated with KIRA6 in 4T1 tumor model (Fig. 1a). KIRA6 induced a significant reduction in tumor growth compared to vehicle-treated controls (Fig. 1b-c). Meanwhile, no significant changes in body weight were observed in between KIRA6 treatment mice and control mice (Fig. 1d), indicating favorable tolerability and absence of overt systemic toxicity at this dosage. Moreover, we further characterized the tumor immune microenvironment in KIRA6-treated mice via flow cytometry (Fig. 1e). KIRA6 significantly enhanced T cell infiltration in tumor tissues, with a marked increase in CD3⁺ T lymphocytes (Fig. 1f), CD4⁺ T helper cells (Fig. 1g), and CD8⁺ cytotoxic T cells (Fig. 1h) relative to vehicle-treated tumors. Conversely, KIRA6 injection markedly suppressed CD11b⁺ myeloid cells (Fig. 1i), with specific reductions in both monocytic MDSCs (M-MDSCs, Fig. 1j) and polymorphonuclear MDSCs (PMN-MDSCs, Fig. 1k). Collectively, our data suggests that KIRA6 not only suppresses 4T1 tumor progression but also reprograms the tumor immune landscape by enhancing T cell infiltration and attenuating immunosuppressive myeloid populations. Short-term KIRA6 Treatment Reprograms Systemic Immunity To examine the direct immunological effects of KIRA6, tumor-bearing mice received short-term daily treatment for two consecutive days before sacrifice at day 14 (Fig. 2a). This brief intervention did not significantly reduce tumor weight compared to vehicle controls (Fig. 2b). Analysis of peripheral blood revealed that KIRA6 significantly increased the percentages of CD3⁺, CD4⁺, and CD8⁺ T cells within CD45⁺ leukocytes, while reducing CD11b⁺ cells, M-MDSCs, and PMN-MDSCs (Fig. 2c-d). In addition to circulating immune cells, we also examined the alteration in the spleen. KIRA6-treated mice exhibited marked reduction in spleen weight (Fig. 2e-f), suggesting potential effects on extramedullary hematopoiesis. In line with our observation in the peripheral blood, KIRA6 increased the proportions of CD3⁺, CD4⁺, and CD8⁺ T cells in CD45⁺ splenocytes, while decreasing CD11b⁺ cells, M-MDSCs, and PMN-MDSCs (Fig. 2g-h). These systemic immunomodulatory effects precede measurable tumor reduction, highlighting the direct immune reprogramming effect of KIRA6. KIRA6 Directly Inhibits MDSC Differentiation and Function In Vitro To validate the direct effects of KIRA6 on MDSCs, bone marrow cells from naïve BALB/c mice were cultured in 4T1 tumor-conditioned medium (TCM) in the presence of KIRA6 for 3 days. TCM potently induced PMN-MDSC differentiation, while KIRA6 suppressed TCM-induced MDSC in a dose-dependent manner (Fig. 3a-b). Meanwhile, KIRA6 treatment significantly induced PMN-MDSC apoptosis (Fig. 3a and 3c). Moreover, western blot analysis revealed KIRA6 markedly downregulated expression of the immunosuppressive enzyme arginase-1 (ARG1) in TCM-induced MDSCs (Fig. 3d). To further assess functional consequences, TCM-induced MDSCs were co-cultured with CFSE-labeled splenocytes. While TCM-MDSCs profoundly suppressed T cell proliferation, KIRA6 pretreatment completely abolished their inhibitory capability on both CD4⁺ T cells and CD8⁺ T cells (Fig. 3e-g). In summary, our data suggests that KIRA6 directly inhibits MDSC generation and suppressive function in vitro . KIRA6 Attenuates G-CSF Production from Tumor Cells to Decrease MDSC Generation To investigate direct effects on tumor cells, 4T1 cells were treated with KIRA6 for 4 hours followed by RNA sequencing. Transcriptomic profiling revealed significant alterations in cytokine/chemokine expression (Fig. 4a-b). Specifically, the mRNA level of granulocyte colony-stimulating factor (CSF3/G-CSF), a critical factor for generation and mobilization of MDSC, was significantly reduced after being treated with KIRA6 (Fig. 4c). ELISA analysis confirmed the decreased G-CSF secretion (Fig. 4d). To explore the impact on MDSC biology, bone marrow cells were then cultured in conditioned medium from KIRA6-treated 4T1 cells. KIRA6-pretreated TCM exhibited significantly reduced PMN-MDSC induction (Fig. 4e-f). When co-cultured with CFSE-labeled splenocytes, MDSCs induced with KIRA6-pretreated TCM failed to suppress proliferation of either CD4⁺ or CD8⁺ T cells (Fig. 4g-i). Thus, KIRA6 remodels the tumor cytokine production to impair the generation and immunosuppressive function of MDSC. KIRA6 Inhibits Tumor Cell Proliferation and Induces Apoptosis We also assessed the direct antitumor effects on 4T1 cells with escalating doses of KIRA6 (1–10,000 nM). KIRA6 significantly suppressed cellular proliferation in a dose-dependent manner as measured by CCK-8 assay (Fig. 5a). In consistence, KIRA6 treatment induced G0/G1 cell cycle arrest, demonstrated by a significant increase in G0/G1 phase cells, concurrent with decreased S phase and G2/M phase populations (Fig. 5b–e). Furthermore, KIRA6 treatment significantly increased the sub-G1 apoptotic population (Fig. 5f). Annexin V/7-AAD staining confirmed KIRA6 treatment significantly induced both early-stage apoptosis and late-stage apoptosis of 4T1 cells (Fig. 5g-i). Collectively, these findings demonstrate that KIRA6 exerts direct antitumor effects through coordinated induction of G0/G1 cell cycle arrest and apoptosis at pharmacologically relevant concentrations. KIRA6 Suppresses Oncogenic Signaling Pathways To reveal the key pathways how KIRA6 influences 4T1 cells, gene set enrichment analysis (GSEA) was performed with RNA-seq data from KIRA6-treated 4T1 cells. Hallmark pathways including Myc Targets V1 (NES = -1.34), Myc Targets V2 (NES = -1.16), and Kras Signaling Up (NES = -1.19) were significantly downregulated in KIRA6-treated cells (Fig. 6a-c). Conversely, KIRA6-treated cells exhibited enrichment of tumor-suppressive pathways, including Kras Signaling Down (NES = 1.24, Fig. 6d). These bio-informative analysis results indicated that KIRA6 significantly downregulated the Kras/ERK/Myc pathway, which is responsible for both G-CSF expression and cancer cell proliferation. Western blot further confirmed downregulation of phosphorylated ERK1/2 (p-ERK1/2) and phosphorylated Myc (p-Myc) proteins following KIRA6 treatment (Fig. 6e), indicating suppression of ERK/Myc signaling axes. Moreover, Interferon-α Response pathway (NES = 1.21) was significantly enriched in KIRA6-treated cells (Fig. 6f), suggesting that KIRA6 treatment induced an intrinsic antitumor immune response. A core KIRA6-targeting gene signature (FPKM ≥ 5 in controls, log 2 FC <-1, p < 0.05) was identified from the RNA-seq data. Functional annotation (GO Molecular Function) revealed significant enrichment for snoRNA binding, RNA methyltransferase activity, et al., in the KIRA6-targeting gene signature (Fig. 6g). Notably, high expression of this signature in breast cancer patients correlated with significantly shorter overall survival (HR = 1.509, P = 0.0162, Fig. 6h), suggesting that KIRA6 targets a group of genes related to poor prognosis. KIRA6 Enhances Antitumor Immunity and Overcomes Anti-PD-1 Resistance Considering the potent modulation of KIRA6 on both immune microenvironment and tumor cells, we evaluated the therapeutic potential of combining KIRA6 with immune checkpoint blockade. 4T1 tumor-bearing mice were treated with anti-PD-1 (200 µg, i.p. every 3 days), KIRA6 (10 mg/kg, i.p. every other day), or both until sacrifice at day 22 (Fig. 7a). PD-1 blockade failed to inhibit tumor growth in this resistant model. KIRA6 treatment not only significantly suppressed tumor progression alone, but also overcame ICB resistance when combined with anti-PD1 therapy, achieving further tumor suppression (Fig. 7b-d). While anti-PD-1 monotherapy exhibited negligible effect on splenomegaly, the combination of KIRA6 significantly decreased spleen weight (Fig. 7e-f), indicating resolution of tumor-driven extramedullary myelopoiesis. Moreover, comprehensive immunophenotyping was performed to further analyze immune cell populations in the tumor and spleen. In line with the therapeutic outcome, PD-1 antibody treatment could not significantly modulate the proportion of tumor-infiltrating immune cells. KIRA6 monotherapy increased intratumoral CD3⁺ and CD8⁺ T cell infiltration while decreasing immunosuppressive CD11b⁺ myeloid cells and PMN-MDSCs, which were amplified in the combination group (Fig. 7g). In similar, enhanced antitumor immunity, with increased T cell proportions and reduced immunosuppressive myeloid cells, was observed in the spleens from KIRA6 and combo-treated mice (Fig. 7h). These findings suggest that KIRA6 remodels both local and systemic immune landscapes and improves the efficiency of anti-PD-1 therapy. Discussion Immune checkpoint blockade (ICB) therapy is successfully used for cancer treatment in many types of cancers, however only a small portion of patients benefit from PD-1/PD-L1 blockade therapy because of immune suppression mediated by alternative suppressive immune cells, such as MDSCs 8 , 10 . IRE1α is a key regulator for generation of MDSC, whose kinase activity and RNase activity could be inhibited by KIRA6 simultaneously 37 . However, its potential for targeting MDSC remains unexplored. Here, we evaluated the antitumor activity of KIRA6 for breast cancer, we examined the role of KIRA6 in decreasing MDSC generation, we revealed the effect of KIRA6 on myeloid-promoting cytokine production and survival of tumor cells, and we explored the potential of KIRA6 to improve the benefit from anti-PD-1 therapy. Our work establishes important evidence that KIRA6 potently inhibits systemic and local MDSC in preclinical tumor model and provides a promising agent for overcoming ICB resistance. MDSCs are one of the major immunosuppressive cells that restrain antitumor T cell responses in the TME, therefore great efforts have been made to develop MDSC-targeting strategies for cancer therapy 15 , 16 . Previous studies aimed to abolish MDSC mediated immune suppression through eliminating MDSCs, decreasing MDSC infiltration or abrogating their suppressive functions, such as the use of chemotherapies, chemokine inhibitors, or neutralizing antibodies 15 , 21 . These agents effectively abrogate immune suppression of MDSC on T cells and improve antitumor response. However, MDSCs rapidly expanded from aberrant myelopoiesis in bone marrow and spleen. We and others have revealed that myeloid progenitor cells significantly increase in the peripheral blood and spleen of cancer patients and serve as an important source of functional MDSCs 26 , 31 , 36 . These observations suggest that tumors systemically regulate myelopoiesis and continuously replenish MDSCs through chronic secretion of cytokines, such as G-CSF and GM-CSF. Thus, targeting the source of MDSCs is promising for a more sustainable benefit 38 . In this study, KIRA6 treatment significantly suppressed 4T1 tumor growth with decreased MDSC population and enhanced T cell infiltration. Two dosages of KIRA6 treatment potently inhibited MDSC generation and extramedullary hematopoiesis, as indicated by systemically decreased MDSC population and smaller spleen size. Moreover, KIRA6 inhibited MDSC biogenesis and eventually achieved in overcoming resistance to anti-PD-1 therapy. Our work identified KIRA6 as an impressive agent for targeting MDSC generation and overcoming ICB resistance. The expansion and acquisition of immune suppression capability are orchestrated by several key pathways, offering promising strategies to reprogram MDSCs 16 . As a result of high secretory and metabolic activity during the immunosuppressive myelopoiesis in tumor milieu, MDSCs are faced with ER stress 32 , 33 . When challenged with ER stress, several ER transmembrane sensors mediated signaling cascade determine cell fate, such as IRE1α and PERK 32 , 37 . These pathways act as key regulators for generation of suppressive MDSCs. IRE1α possesses RNase activities, is activated by trans-autophosphorylation through its own kinase activity 37 . The RNase activity of IRE1α enables processing unspliced XBP1 to its mature production, spliced XBP1 (XBP1s), which encodes the transcriptionally active XBP1 protein 39 . Genetic deletion of IRE1α completely abrogate suppressive activity of PMN-MDSCs in tumor model 33 . On the other hand, cancer cell intrinsic XBP1s favors the synthesis and secretion of cholesterol, which is absorbed by MDSCs and drives its immunosuppressive reprogramming 39 . The kinase activity of IRE1α mediates activation of JNK pathway and markedly potentiates the expression of G-CSF and GM-CSF in breast cancer cells 36 . The multiple effects of IRE1α on supporting MDSC generation highlights the potential of inhibiting the kinase and RNase activity simultaneously for cancer immunotherapy. Therefore, KIRA6, other than solo RNase activity inhibitor, such as 4µ8C, MKC8866, or STF-083010 40, 41 , was evaluated for antitumor activity in this study. KIRA6 not only directly decreased MDSC differentiation from bone marrow cells, but also attenuated G-CSF production from tumor cells and therefore blocked the sustainably induction of MDSCs. Our data revealed that the dual effect of KIRA6 on blocking MDSC generation. KIRA6 exhibited direct killing on tumor cells in addition to its immune modulation ability. In support with our findings, previous studies have demonstrated IRE1α promotes the survival, growth, and drug resistance of tumor cells, which underline the importance of IRE 1α in tumor biology 42 – 45 . For example, IRE1α pathway activates c-MYC signaling and promotes prostate cancer 41 ; reactivation of IRE1α caused acquired resistance to KRAS inhibitor, and inhibition of IRE1α overcame resistance to KRAS inhibitor 46 ; IRE1α RNase silences taxane-induced dsRNA through preventing NLRP3 inflammasome-dependent pyroptosis 47 . We found KIRA6 suppressed cellular proliferation in a dose-dependent manner, and induced cell cycle arrest and apoptosis at sub-micromolar concentration. KIRA6 treatment downregulated the phosphorylation of ERK1/2 and Myc proteins, indicating potent suppression on oncogenic signaling pathways. KIRA6 is also reported to inhibit KIT as well as its downstream signaling modules phosphorylated ERK1/2 at nanomolar concentrations and are sufficient to induce cell death in a KIT signaling-dependent leukemia cell line 48 . This study confirmed the direct cancer killing effect of KIRA6 on breast cancer at pharmacologically concentrations. KIRA6 treatment achieved more potent antitumor response and resulted in overcoming ICB resistance when combined with anti-PD1 therapy. The improved T cell response could benefit from various aspects of KIRA6 treatment. On the one hand, KIRA6 systemically reprograms myeloid population, with decreased MDSC in peripheral blood, spleen, and tumor tissue. Spleen is an important source for MDSC generation, while short-term KIRA6 treatment significantly decreased spleen weight, suggesting resolution of extramedullary hematopoiesis. KIRA6 abrogated myeloid cells mediated immune suppression, which is fundamental for overcoming ICB blockade. On the other hand, targeting IRE1α-XBP1 signaling have been found to restore anti-tumor capacity of T cells in cancer hosts 49 , 50 . IRE1α-XBP1 activation is found in T cell from patients with ovarian cancer, and suppresses mitochondrial activity and IFN-γ production. Blocking IRE1α-XBP1 pathway helps to restore the metabolic fitness and anti-tumor capacity of T cells in cancer hosts 50 . Previous studies suggest that inhibiting IRE1α rejuvenates T cells rather than interfere their antitumor functions. Therefore, KIRA6 abrogated MDSC mediated immune suppression without impeding T cell functions, making it a promising candidate of small molecule drug for cancer immunotherapy. Our study evaluated the antitumor activity of KIRA6 for breast cancer and dissected the underlying mechanism from both MDSC and cancer cell aspect. Our data revealed that KIRA6 potently inhibits MDSC generation and tumor progression in mouse model and uncovered its impressive role in overcoming ICB resistance. However, our study is conducted in animal and cell level. Future preclinical investigations in human models and clinical trials are required for a clearer translational impact. Declarations Authors’ contributions C. C., J.C., and X. L. contributed equally to this work. C. C., J. C., and X. L. designed and conducted experiments, analyzed data and wrote the manuscript; J. H., D. L., X. O., J. L., W. L., and S. X., conducted experiments; Y. Z., performed bioinformatic analysis; J.C., and Y. M. revised the manuscript; Z.M., Y. P. and H.-W. S. designed and supervised the research and revised the manuscript. Conflict of Interest The authors declare no potential conflicts of interest. Availability of Data and Materials All data relevant to the study are included in the article or uploaded as supplementary information. The data generated in this study are available within the article and its Supplementary Data files. Any data used in this study that are not included in the paper or supplementary files can be made available upon request from the corresponding author. Acknowledgments This work was supported by the National Natural Science Foundation of China (82471769, 82103306, 82203518, and 82272103), the Guangdong Provincial Key Laboratory of Tumor Interventional Diagnosis and Treatment (2021B1212040004), the Natural Science Foundation of Guangdong Province of China (2022B1515020010), Guizhou Provincial Basic Research Program (Natural Science) (No. MS [2025] 419), and the Xiangshan Talent Project of Zhuhai People's Hospital (2023XSYC-02). Funders were not involved in the research and publication. Ethics approval All animal experiments were performed in accordance with the guidance and approved by the Experimental Animal Ethics Committee of Zhuhai People’s Hospital (No. 20250222-01). Patient consent statement Not applicable. 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14:11:48","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7263160/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7263160/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41419-025-08401-6","type":"published","date":"2025-12-27T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":88862433,"identity":"ce3f1191-aa09-4854-a62f-0ebfae6d9ec2","added_by":"auto","created_at":"2025-08-12 07:55:23","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":830850,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKIRA6 inhibits 4T1 tumor growth and modulates the tumor immune microenvironment.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003e Experimental treatment schedule. Female BALB/c mice with implanted 4T1 tumors were intraperitoneal (\u003cem\u003ei.p.\u003c/em\u003e) injected with vehicle or KIRA6 every other day for two weeks. \u003cstrong\u003e(b)\u003c/strong\u003e Representative image of tumors from indicated groups at sacrifice. Scale bar,1 cm. \u003cstrong\u003e(c)\u003c/strong\u003e Tumor growth curves. Mean tumor volume of 4T1 tumors from vehicle or KIRA6 treated mice at the indicated time points. Results are shown as mean ± SEM. n = 5/group; *, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05. \u003cstrong\u003e(d)\u003c/strong\u003e Body weight of vehicle or KIRA6 treated mice over the treatment period. Data is presented as mean ± SEM. n = 5/group; ns, not significant. \u003cstrong\u003e(e)\u003c/strong\u003e Flow cytometry analysis of CD3\u003csup\u003e+\u003c/sup\u003e T cells, CD11b\u003csup\u003e+\u003c/sup\u003e myeloid cells, CD4\u003csup\u003e+\u003c/sup\u003e T cells, CD8\u003csup\u003e+\u003c/sup\u003e T cells, M-MDSCs (CD11b\u003csup\u003e+\u003c/sup\u003eLy6C\u003csup\u003e+\u003c/sup\u003eLy6G\u003csup\u003e-\u003c/sup\u003e) and PMN-MDSCs (CD11b\u003csup\u003e+\u003c/sup\u003eLy6C\u003csup\u003elow\u003c/sup\u003eLy6G\u003csup\u003e+\u003c/sup\u003e) in 7-AAD\u003csup\u003e-\u003c/sup\u003eCD45\u003csup\u003e+\u003c/sup\u003e cells from 4T1 tumors treated with vehicle or KIRA6. The percentage of indicated cell populations in CD45\u003csup\u003e+\u003c/sup\u003e cells is shown. \u003cstrong\u003e(f-k)\u003c/strong\u003e The frequency of the indicated immune cell subsets in total CD45\u003csup\u003e+\u003c/sup\u003e cells from tumor tissues (n = 5 /group). The results are shown as mean ± SEM. *, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ***, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u0026nbsp;\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7263160/v1/00b91841898343a3976a042f.png"},{"id":88862432,"identity":"83127241-d200-49bb-9a1c-f79f2ab89ce0","added_by":"auto","created_at":"2025-08-12 07:55:23","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1070110,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eShort-term KIRA6 treatment reprograms systemic immunity.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003e Treatment schedule. 4T1 tumor-bearing mice received \u003cem\u003ei.p.\u003c/em\u003e injections of KIRA6 or vehicle daily for two consecutive days and were sacrificed at day 14. \u003cstrong\u003e(b)\u003c/strong\u003e Tumor weight at sacrifice. Results are shown as mean ± SEM. n = 5/group; ns, not significant. \u003cstrong\u003e(c)\u003c/strong\u003e Flow cytometry analysis of immune cell subsets in 7-AAD\u003csup\u003e-\u003c/sup\u003eCD45\u003csup\u003e+\u003c/sup\u003e cells from peripheral blood of 4T1 bearing mice treated with vehicle and KIRA6. The percentage of gated cell populations in CD45\u003csup\u003e+\u003c/sup\u003e cells is shown. \u003cstrong\u003e(d)\u003c/strong\u003e The proportion of T cell subsets and myeloid populations in CD45\u003csup\u003e+\u003c/sup\u003e cells from peripheral blood. The data is summarized as mean ± SEM. n = 5/group; *, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ****, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001. \u003cstrong\u003e(e)\u003c/strong\u003e Image of spleens from indicated groups at sacrifice. Scale bar,1 cm. \u003cstrong\u003e(f)\u003c/strong\u003e Spleen weight at sacrifice. Results are shown as mean ± SEM. n = 5/group; ***, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001. \u003cstrong\u003e(g)\u003c/strong\u003e Flow cytometry analysis of splenic immune cell subsets in 7-AAD\u003csup\u003e-\u003c/sup\u003eCD45\u003csup\u003e+\u003c/sup\u003e cells from indicated groups. The percentage of gated cell subsets in total CD45\u003csup\u003e+\u003c/sup\u003e cells is shown.\u003cstrong\u003e (h)\u003c/strong\u003e The proportion of splenic immune cell populations in CD45\u003csup\u003e+\u003c/sup\u003e cells. The data is summarized as mean ± SEM. n = 5/group; *, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ***, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7263160/v1/f9de18b9fd0e597163e2d599.png"},{"id":88862437,"identity":"0e8787c9-67c1-4c35-8587-16e02e1b22ff","added_by":"auto","created_at":"2025-08-12 07:55:23","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1629591,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKIRA6 directly impairs MDSC generation and immunosuppressive function.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003e Bone marrow cells were cultured in control medium, 4T1 tumor-conditioned medium (TCM), or TCM with KIRA6 at indicated concentrations for 3 days. The quantification of PMN-MDSC and apoptosis of cultured cells were examined by flow cytometry. \u003cstrong\u003e(b)\u003c/strong\u003e The number of PMN-MDSCs (CD11b\u003csup\u003e+\u003c/sup\u003e Ly6G\u003csup\u003e+\u003c/sup\u003e) from indicated groups are summarized as mean ± SEM. n = 4/group; *, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ****, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001. \u003cstrong\u003e(c)\u003c/strong\u003e The proportion of apoptotic cells are shown as mean ± SEM. n = 4/group; *, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; ****, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001. \u003cstrong\u003e(d)\u003c/strong\u003e Western blot analysis of ARG1 expression in MDSCs cultured in (a). \u003cstrong\u003e(e) \u003c/strong\u003eCFSE stained splenocytes were cultured alone or co-cultured with MDSCs induced with control medium, 4T1 tumor-conditioned medium (TCM), or TCM with KIRA6 (1μM) for 3 days (MDSC:T cell = 1:2). The proliferation of T cells was analyzed by flow cytometry.\u003cstrong\u003e (f-g) \u003c/strong\u003eThe proportion of non-proliferated CD4⁺ T cells (f) and CD8⁺ T cells (g) are shown as mean ± SEM (n = 4).\u003cstrong\u003e \u003c/strong\u003e**, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ***, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7263160/v1/9da8abd27a606a8dd3882ee4.png"},{"id":88862442,"identity":"f40a5cac-339c-4a5f-b1c7-019cf7818f81","added_by":"auto","created_at":"2025-08-12 07:55:24","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1014556,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKIRA6 inhibits G-CSF secretion from tumor cells to decrease MDSC generation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003e Volcano plot of RNA-seq data from 4T1 cells treated with vehicle or KIRA6 (1μM). \u003cstrong\u003e(b)\u003c/strong\u003eHeatmap of differentially expressed cytokines/chemokines. \u003cstrong\u003e(c)\u003c/strong\u003e The mRNA expression of Csf3 (G-CSF).\u003cstrong\u003e \u003c/strong\u003eThe data is shown as mean ± SEM (n = 3). **, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01. \u003cstrong\u003e(d)\u003c/strong\u003e The secretion of G-CSF in conditioned medium from vehicle or KIRA6 (1μM) treated 4T1 tumor cells. The results are shown as mean ± SEM (n = 4). **, \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01. \u003cstrong\u003e(e)\u003c/strong\u003e Bone marrow cells were cultured in control medium, TCM from vehicle or KIRA6 (1μM) pre-treated 4T1 tumor cells for 3 days. The number of PMN-MDSC in cultured cells were examined by flow cytometry. \u003cstrong\u003e(f)\u003c/strong\u003e The number of PMN-MDSCs in (e) are summarized as mean ± SEM. n = 4/group; ****, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001.\u003cstrong\u003e (g) \u003c/strong\u003eSplenocytes were cultured alone or co-cultured with MDSCs induced with control medium, TCM from vehicle or KIRA6 (1μM) pre-treated 4T1 tumor cells for 3 days (MDSC:T cell = 1:2). The proliferation of T cells was monitored by flow cytometry.\u003cstrong\u003e (h-i) \u003c/strong\u003eThe percentage of non-proliferated CD4⁺ T cells (h) and CD8⁺ T cells (i) are summarized as mean ± SEM (n = 4).\u003cstrong\u003e \u003c/strong\u003e**, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7263160/v1/0861ddaaf230a493e9ce43d1.png"},{"id":88863660,"identity":"242d0d0e-b486-44dd-90e2-4030d49c2262","added_by":"auto","created_at":"2025-08-12 08:03:24","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":837102,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKIRA6 directly inhibits 4T1 tumor cell proliferation by inducing cell cycle arrest and apoptosis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003e Cell viability of 4T1 tumors cultured for 24h in control conditions or with KIRA6 treatment at indicated concentrations were measured by CCK-8 assay. n = 5; **, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01. \u003cstrong\u003e(b)\u003c/strong\u003e Representative flow cytometry analysis for cell cycle distribution of 4T1 cells cultured in indicated conditions. \u003cstrong\u003e(c-f)\u003c/strong\u003e The percentages of G0/G1 phase (c), S phase (d), G2/M phase (e), sub-G1 apoptotic population (f) in (b) are summarized as mean ± SEM (n = 3).\u003cstrong\u003e \u003c/strong\u003e*, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ***, \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001. \u003cstrong\u003e(g)\u003c/strong\u003e Flow cytometry analysis for apoptosis of 4T1 cells cultured in indicated conditions. \u003cstrong\u003e(h-i)\u003c/strong\u003e The percentages of early-stage apoptosis (h) and late-stage apoptosis (i) are presented as mean ± SEM (n = 3). *, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7263160/v1/365a825a804563bcdc1920c7.png"},{"id":88862448,"identity":"c41d808c-9529-46ff-8a7b-7d95a4927411","added_by":"auto","created_at":"2025-08-12 07:55:24","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1329411,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKIRA6 suppresses oncogenic signaling pathways.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a-d)\u003c/strong\u003e GSEA enrichment plot for Hallmark pathways enriched in vehicle- or KIRA6-treated 4T1 cells. NES, normalized enrichment score. \u003cstrong\u003e(e)\u003c/strong\u003eWestern blot analysis of p-ERK1/2, total ERK1/2, p-Myc, and total Myc in vehicle and KIRA6-treated cells. β-actin was used as loading control. \u003cstrong\u003e(f)\u003c/strong\u003eGSEA enrichment plot for \u003cem\u003eInterferon-α Response\u003c/em\u003e in vehicle and KIRA6-treated 4T1 cells. \u003cstrong\u003e(g)\u003c/strong\u003e Functional enrichment (GO Molecular Function) of KIRA6-downregulated genes. The top ten terms are shown. \u003cstrong\u003e(h)\u003c/strong\u003eKaplan-Meier survival analysis for overall survival in TCGA breast cancer cohort (n = 1069). KIRA6-targeting gene signature was scored by Gene Set Variation Analysis (GSVA). Patients were stratified into high score group (n = 380) or low score group (n = 689) by the cutoff value (score = -0.4141) determined by receiver operating characteristic (ROC) curve. HR, Hazard ratios.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7263160/v1/c2339561984a663b60a4599c.png"},{"id":88862446,"identity":"d33cf5b6-587d-4581-b894-830951de6e8c","added_by":"auto","created_at":"2025-08-12 07:55:24","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1039817,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKIRA6 overcomes anti-PD-1 resistance and enhances antitumor immunity.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003e Tumor treatment regimen. 4T1 tumor bearing mice were treated with vehicle, anti-PD-1 antibody (200 µg \u003cem\u003ei.p.\u003c/em\u003e every three days, red arrows), KIRA6 (10 mg/kg \u003cem\u003ei.p.\u003c/em\u003e every other days, blue arrows), or combined treatment from day 9 to 21. \u003cstrong\u003e(b)\u003c/strong\u003e Tumor growth curves. Mean tumor volume of 4T1 tumors from indicated groups are shown as mean ± SEM. n = 5/group; *, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ***, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003cstrong\u003e (c)\u003c/strong\u003e Image of tumors from indicated groups at sacrifice. Scale bar, 1 cm. \u003cstrong\u003e(d)\u003c/strong\u003e Tumor weight at sacrifice. Results are shown as mean ± SEM. n = 5/group; ns, not significant; *, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ***, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003cstrong\u003e (e)\u003c/strong\u003e Image of spleens from indicated groups at sacrifice. Scale bar, 1 cm. \u003cstrong\u003e(f)\u003c/strong\u003e Spleen weight at sacrifice. Results are shown as mean ± SEM. n = 5/group; *, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; ***, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001. \u003cstrong\u003e(g) \u003c/strong\u003eThe percentage of CD3\u003csup\u003e+\u003c/sup\u003e T cells, CD8\u003csup\u003e+\u003c/sup\u003e T cells, CD11b\u003csup\u003e+\u003c/sup\u003e myeloid cells, PMN-MDSCs in CD45\u003csup\u003e+\u003c/sup\u003e cells from 4T1 tumors treated with indicated drugs were analyzed by flow cytometry. The results are shown as mean ± SEM. *, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01. \u003cstrong\u003e(h) \u003c/strong\u003eThe percentage of splenic CD3\u003csup\u003e+\u003c/sup\u003e T cells, CD8\u003csup\u003e+\u003c/sup\u003e T cells, CD11b\u003csup\u003e+\u003c/sup\u003e myeloid cells, PMN-MDSCs in CD45\u003csup\u003e+\u003c/sup\u003e cells from indicated groups were quantified by flow cytometry. The data is shown as mean ± SEM. *, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ***, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; ****, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7263160/v1/b021420bd9f347ad52303302.png"},{"id":101552419,"identity":"b2d15ad3-8676-498f-b0ed-e7c6a3636cbc","added_by":"auto","created_at":"2026-01-31 08:12:15","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8161298,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7263160/v1/5c4f5195-6b66-4634-b3da-842a0647e3af.pdf"},{"id":88862430,"identity":"8dd6c56f-e6bd-487f-8224-f59f8e9de2fd","added_by":"auto","created_at":"2025-08-12 07:55:23","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":122385,"visible":true,"origin":"","legend":"Supplementary Materials-Table S1","description":"","filename":"SupplementaryMaterials.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7263160/v1/2e79ee2cade54a3e74299753.pdf"},{"id":88863659,"identity":"0db05829-dac5-43e2-9bc5-7091fd57ab1a","added_by":"auto","created_at":"2025-08-12 08:03:23","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":479837,"visible":true,"origin":"","legend":"Supplementary Materials-Original Western Blot","description":"","filename":"SupplementaryMaterialsOriginalWesternBlot.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7263160/v1/2961c86923393c8af7c460e6.pdf"}],"financialInterests":"(Not answered)","formattedTitle":"KIRA6 abrogates the generation of myeloid-derived suppressor cells and overcomes resistance to anti-PD-1 therapy","fulltext":[{"header":"Introduction","content":"\u003cp\u003eImmune checkpoint blockade (ICB) therapy has improved survival for cancer patients through harnessing the immune system, making it one of the cornerstones of cancer treatment regimens in a broad range of cancers nowadays\u003csup\u003e\u003cspan additionalcitationids=\"CR2 CR3 CR4 CR5\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e–\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. However, the overall response rates remain low across many types of cancer, which limits the benefit of ICB therapy for majority of cancer patients\u003csup\u003e\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e–\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Many immunosuppressive factors in the tumor microenvironment (TME) have been found to collaboratively restrain the activation and effector function of T cells despite the PD-1 blockade\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. The generation of regulatory immune cells, such as myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs) or regulatory T cells, is the major tumor-driven mechanisms of immune suppression, which alternatively impair antitumor T cell activity within the TME\u003csup\u003e\u003cspan additionalcitationids=\"CR11 CR12 CR13\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e–\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Therefore, it is unmet need to target the suppressive immune cells in the TME to achieve better outcome of ICB therapy.\u003c/p\u003e\u003cp\u003eMyeloid-derived suppressor cells (MDSCs) constitute a major component of the TME, which are key orchestrators of immunosuppression in TME and could inhibit T cell function through multiple mechanisms, such as arginase-1 (ARG1) and reactive oxygen species (ROS) \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. The potent immune modulation capability of MDSCs enables them to drive tumor progression, metastasis, and resistance to cancer therapies, therefore MDSC infiltration is found to correlate with failure of ICB treatment and poor patient survival\u003csup\u003e\u003cspan additionalcitationids=\"CR17 CR18 CR19\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e–\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Considering their established role as therapeutic barriers, great efforts have been made to develop MDSC-targeting strategies for cancer therapy, including eliminating MDSC, blocking MDSC recruitment, and abrogating the suppressive function of MDSCs\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan additionalcitationids=\"CR22 CR23\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e–\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Although targeting MDSCs in preclinical models get improved outcomes, there are still no drugs approved for clinical usage, urging for novel pharmacologic agents to dismantle this suppressive axis.\u003c/p\u003e\u003cp\u003eMDSCs consist of a group of heterogeneous myeloid cells, including polymorphonuclear MDSCs (PMN-MDSCs) and monocytic MDSCs (M-MDSCs)\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. It is generally believed that MDSCs originate from hematopoietic progenitor cells in the bone marrow and spleen\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e–\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. This aberrant myelopoiesis process is driven by multiple cytokines and growth factors, such as granulocyte colony-stimulating factor (G‑CSF), promoting their differentiation into immunosuppressive effectors rather than functional counterparts\u003csup\u003e\u003cspan additionalcitationids=\"CR29 CR30\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e–\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Endoplasmic reticulum (ER) stress is observed in MDSCs because of high secretory and metabolic activity during the immunosuppressive myelopoiesis in tumor milieu\u003csup\u003e\u003cspan additionalcitationids=\"CR33 CR34\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e–\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Inositol-requiring enzyme 1α (IRE1α), an important kinase and RNase of ER stress response, is identified as a key regulator for generation of MDSC\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. On the one hand, IRE1α-mediated XBP1 cleavage promotes acquisition of suppressive activity of MDSC in cancer\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. On the other hand, the kinase activity of IRE1α potentiates the expression of G-CSF and granulocyte/macrophage colony-stimulating factor (GM-CSF) from tumor cells through activation of JNK pathway, which further facilitates mobilization of hematopoietic progenitor cells and pathological myelopoiesis\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. The dual effects of IRE1α make it an ideal target for abrogating MDSCs generation.\u003c/p\u003e\u003cp\u003eKIRA6 (kinase-inhibiting RNase-attenuators), is developed for diabetes treatment by inhibiting IRE1α kinase activity and RNase activity simultaneously\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. However, its potential for cancer treatment, especially for targeting MDSC generation and immunotherapy, remains unexplored. Here, we evaluate the antitumor activity of KIRA6 for breast cancer in both animal model and \u003cem\u003ein vitro\u003c/em\u003e model. KIRA6 significantly suppresses 4T1 tumor progression and reprograms the tumor immune landscape by enhancing T cell infiltration and decreasing MDSC population. Short-term KIRA6 treatment and cell culture confirm that KIRA6 directly inhibits MDSC generation and function. Meanwhile, KIRA6 not only attenuates G-CSF production therefore blocks the induction of MDSCs, but also causes apoptosis in tumor cells. Moreover, KIRA6 treatment diminishes MDSC generation, restores T cell proportion in both local and systemic immune landscapes and eventually overcomes resistance to anti-PD-1 therapy. Our work establishes KIRA6 as an MDSC-targeting agent for overcoming ICB resistance and provides a promising combination strategy for achieving better immunotherapy.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eReagents\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eReagents used in this study are summarized in Supplementary Material, Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eTumor cell culture\u003c/strong\u003e\u003c/p\u003e\u003cp\u003e4T1 cells (ATCC, CRL-2539) were cultured with RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/mL), streptomycin (100 mg/mL) at 37°C in 5% CO\u003csub\u003e2\u003c/sub\u003e-humidified atmosphere.\u003c/p\u003e\u003cp\u003e4T1 cells were plated overnight in complete RPMI 1640 medium before KIRA6 treatment. 4T1 cells were cultured with fresh culture medium with or without KIRA6 at indicated concentrations. For cell viability assay and flow cytometry analysis, cells were treated with vehicle or KIRA6 for 24 hours. For the preparation of RNA and protein sample, cells were treated with KIRA6 for 4 hours before sample collection. For tumor condition medium, cells were treated with KIRA6 for 4 hours and then washed and culture with fresh medium for 24 hours. Cell culture medium was collected and centrifuged to obtain tumor condition medium.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eMouse model\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eAll animal experiments were performed according to institutional guidelines and approved by the ethical board of Zhuhai People’s Hospital. Female BALB/c mice (6–8 weeks of age) were purchased from Bestest Biotechnology Company (Zhuhai, China). 2×10\u003csup\u003e5\u003c/sup\u003e 4T1 cells were injected subcutaneously into the flank of BALB/c mice. Mice were intraperitonially injected with vehicles, KIRA6 (10 mg/kg) every other day or anti-PD-1 antibody (200 µg) every three days in designated groups. Tumor sizes were measured with caliper when tumors were palpable. Tumor volumes were calculated by (length\u003csup\u003e2\u003c/sup\u003e × width)/2.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eIsolation of leukocytes from bone marrow cells and splenocytes\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eBone marrow cells were harvested by flushing the femurs and tibias of mice with PBS supplemented with 1% FBS using syringe. Splenocytes were obtained by homogenizing the spleen using nylon mesh and gently aspirating through a 21-gauge needle\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Red blood cells were removed by ACK lysis buffer. Isolated cells were then washed and resuspended for cell culture, or flow cytometry analysis.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eIsolation of tumor infiltrating immune cells\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eMouse tumors were cut into small pieces and digested with 0.05% collagenase type IV, 0.002% DNase I in RPMI 1640 supplemented with 10% FBS. The dissociated cells were filtered through 100 µm mesh before washed and resuspended for FACS analysis or cell culture\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eFlow cytometry\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eCells cultured \u003cem\u003ein vitro\u003c/em\u003e, leukocytes from peripheral blood, spleen and tumor samples were prepared and suspended in PBS buffer supplemented with 1% heat-inactivated FBS, then stained with desired antibodies. Cell apoptosis was stained with APC Annexin V Apoptosis Detection Kit with 7-AAD. Cell cycle was detected with Cell Cycle and Apoptosis Analysis Kit. Data was acquired on DxP Athena (Cytek) and analyzed with FlowJo software.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eGeneration of MDSC from bone marrow cells\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eBone marrow cells were plated in 24-well plates in complete RPMI medium (with 10% FBS) supplemented with tumor condition medium in the presence of KIRA6. Cells were cultured at 37°C in 5% CO\u003csub\u003e2\u003c/sub\u003e-humidified atmosphere for 3 days\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eCo-culture of MDSC and splenocytes\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eSplenocytes were isolated from homogenized spleen of naïve mice and stained with 1.5 µM CFSE for 10min at 37°C according to the manufacturer's instructions. Then, splenocytes were co-cultured with induced MDSCs at 2:1 ratio in the presence of 1 µg/ml anti-CD3 antibody, 1 µg/ml anti-CD28 antibody and 20 U/mL recombinant IL-2. Cells were cultured for 3–5 days and then collected, stained with surface markers, and analyzed by flow cytometry\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eImmunoblotting\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eThe proteins were prepared with RIPA Lysis and Extraction Buffer and then quantified with BCA Protein Assay Kit. Proteins were separated by 10% SDS-PAGE, immunoblotted with anti p-ERK, ERK, p-Myc, Myc, ARG1 and β-Actin antibody. Antibody binding was detected using horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG antibody and visualized with Western Chemiluminescent kit.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eRNA-seq and analysis\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eCells were lysis with TRIzol for RNA purification. The library was constructed and then sequenced by Azenta on an Illumina instrument using a 2×150 paired-end (PE) configuration according to the manufacturer’s instructions. All data are publicly available in the GEO database. Differentially expressed genes were identified using DESeq2 (v1.34.0). Functional enrichment analyses were performed by Gene Set Enrichment Analysis (GSEA) or Gene Ontology (GO).\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eG-CSF ELISA analysis\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eFor analysis of G-CSF, tumor condition medium collected from vehicle or KIRA6 pretreated 4T1 cells were assessed for G-CSF concentration using the mouse G-CSF ELISA kits (MultiSciences) according to the manufacturer’s instructions.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eIBM SPSS Software (IBM Corporation) and GraphPad Prism (GraphPad Software) were used for statistical analysis. The significance of differences between groups was examined by the Student’s t-test or Mann–Whitney test, as appropriate. The overall survival curves were generated by the Kaplan–Meier method and analyzed using the log-rank test. \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 was considered significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eKIRA6 Inhibits Tumor Growth and Modulates Intratumoral Immune Cell Infiltration\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eTo evaluate the antitumor efficacy of KIRA6, tumor-bearing mice were treated with KIRA6 in 4T1 tumor model (Fig.\u0026nbsp;1a). KIRA6 induced a significant reduction in tumor growth compared to vehicle-treated controls (Fig.\u0026nbsp;1b-c). Meanwhile, no significant changes in body weight were observed in between KIRA6 treatment mice and control mice (Fig.\u0026nbsp;1d), indicating favorable tolerability and absence of overt systemic toxicity at this dosage. Moreover, we further characterized the tumor immune microenvironment in KIRA6-treated mice via flow cytometry (Fig.\u0026nbsp;1e). KIRA6 significantly enhanced T cell infiltration in tumor tissues, with a marked increase in CD3⁺ T lymphocytes (Fig.\u0026nbsp;1f), CD4⁺ T helper cells (Fig.\u0026nbsp;1g), and CD8⁺ cytotoxic T cells (Fig.\u0026nbsp;1h) relative to vehicle-treated tumors. Conversely, KIRA6 injection markedly suppressed CD11b⁺ myeloid cells (Fig.\u0026nbsp;1i), with specific reductions in both monocytic MDSCs (M-MDSCs, Fig.\u0026nbsp;1j) and polymorphonuclear MDSCs (PMN-MDSCs, Fig.\u0026nbsp;1k). Collectively, our data suggests that KIRA6 not only suppresses 4T1 tumor progression but also reprograms the tumor immune landscape by enhancing T cell infiltration and attenuating immunosuppressive myeloid populations.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eShort-term KIRA6 Treatment Reprograms Systemic Immunity\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eTo examine the direct immunological effects of KIRA6, tumor-bearing mice received short-term daily treatment for two consecutive days before sacrifice at day 14 (Fig.\u0026nbsp;2a). This brief intervention did not significantly reduce tumor weight compared to vehicle controls (Fig.\u0026nbsp;2b). Analysis of peripheral blood revealed that KIRA6 significantly increased the percentages of CD3⁺, CD4⁺, and CD8⁺ T cells within CD45⁺ leukocytes, while reducing CD11b⁺ cells, M-MDSCs, and PMN-MDSCs (Fig.\u0026nbsp;2c-d). In addition to circulating immune cells, we also examined the alteration in the spleen. KIRA6-treated mice exhibited marked reduction in spleen weight (Fig.\u0026nbsp;2e-f), suggesting potential effects on extramedullary hematopoiesis. In line with our observation in the peripheral blood, KIRA6 increased the proportions of CD3⁺, CD4⁺, and CD8⁺ T cells in CD45⁺ splenocytes, while decreasing CD11b⁺ cells, M-MDSCs, and PMN-MDSCs (Fig.\u0026nbsp;2g-h). These systemic immunomodulatory effects precede measurable tumor reduction, highlighting the direct immune reprogramming effect of KIRA6.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eKIRA6 Directly Inhibits MDSC Differentiation and Function In Vitro\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eTo validate the direct effects of KIRA6 on MDSCs, bone marrow cells from naïve BALB/c mice were cultured in 4T1 tumor-conditioned medium (TCM) in the presence of KIRA6 for 3 days. TCM potently induced PMN-MDSC differentiation, while KIRA6 suppressed TCM-induced MDSC in a dose-dependent manner (Fig.\u0026nbsp;3a-b). Meanwhile, KIRA6 treatment significantly induced PMN-MDSC apoptosis (Fig.\u0026nbsp;3a and 3c). Moreover, western blot analysis revealed KIRA6 markedly downregulated expression of the immunosuppressive enzyme arginase-1 (ARG1) in TCM-induced MDSCs (Fig.\u0026nbsp;3d). To further assess functional consequences, TCM-induced MDSCs were co-cultured with CFSE-labeled splenocytes. While TCM-MDSCs profoundly suppressed T cell proliferation, KIRA6 pretreatment completely abolished their inhibitory capability on both CD4⁺ T cells and CD8⁺ T cells (Fig.\u0026nbsp;3e-g). In summary, our data suggests that KIRA6 directly inhibits MDSC generation and suppressive function \u003cem\u003ein vitro\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eKIRA6 Attenuates G-CSF Production from Tumor Cells to Decrease MDSC Generation\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eTo investigate direct effects on tumor cells, 4T1 cells were treated with KIRA6 for 4 hours followed by RNA sequencing. Transcriptomic profiling revealed significant alterations in cytokine/chemokine expression (Fig.\u0026nbsp;4a-b). Specifically, the mRNA level of granulocyte colony-stimulating factor (CSF3/G-CSF), a critical factor for generation and mobilization of MDSC, was significantly reduced after being treated with KIRA6 (Fig.\u0026nbsp;4c). ELISA analysis confirmed the decreased G-CSF secretion (Fig.\u0026nbsp;4d). To explore the impact on MDSC biology, bone marrow cells were then cultured in conditioned medium from KIRA6-treated 4T1 cells. KIRA6-pretreated TCM exhibited significantly reduced PMN-MDSC induction (Fig.\u0026nbsp;4e-f). When co-cultured with CFSE-labeled splenocytes, MDSCs induced with KIRA6-pretreated TCM failed to suppress proliferation of either CD4⁺ or CD8⁺ T cells (Fig.\u0026nbsp;4g-i). Thus, KIRA6 remodels the tumor cytokine production to impair the generation and immunosuppressive function of MDSC.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eKIRA6 Inhibits Tumor Cell Proliferation and Induces Apoptosis\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eWe also assessed the direct antitumor effects on 4T1 cells with escalating doses of KIRA6 (1–10,000 nM). KIRA6 significantly suppressed cellular proliferation in a dose-dependent manner as measured by CCK-8 assay (Fig.\u0026nbsp;5a). In consistence, KIRA6 treatment induced G0/G1 cell cycle arrest, demonstrated by a significant increase in G0/G1 phase cells, concurrent with decreased S phase and G2/M phase populations (Fig.\u0026nbsp;5b–e). Furthermore, KIRA6 treatment significantly increased the sub-G1 apoptotic population (Fig.\u0026nbsp;5f). Annexin V/7-AAD staining confirmed KIRA6 treatment significantly induced both early-stage apoptosis and late-stage apoptosis of 4T1 cells (Fig.\u0026nbsp;5g-i). Collectively, these findings demonstrate that KIRA6 exerts direct antitumor effects through coordinated induction of G0/G1 cell cycle arrest and apoptosis at pharmacologically relevant concentrations.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eKIRA6 Suppresses Oncogenic Signaling Pathways\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eTo reveal the key pathways how KIRA6 influences 4T1 cells, gene set enrichment analysis (GSEA) was performed with RNA-seq data from KIRA6-treated 4T1 cells. Hallmark pathways including \u003cem\u003eMyc Targets V1\u003c/em\u003e (NES = -1.34), \u003cem\u003eMyc Targets V2\u003c/em\u003e (NES = -1.16), and \u003cem\u003eKras Signaling Up\u003c/em\u003e (NES = -1.19) were significantly downregulated in KIRA6-treated cells (Fig.\u0026nbsp;6a-c). Conversely, KIRA6-treated cells exhibited enrichment of tumor-suppressive pathways, including \u003cem\u003eKras Signaling Down\u003c/em\u003e (NES = 1.24, Fig.\u0026nbsp;6d). These bio-informative analysis results indicated that KIRA6 significantly downregulated the Kras/ERK/Myc pathway, which is responsible for both G-CSF expression and cancer cell proliferation. Western blot further confirmed downregulation of phosphorylated ERK1/2 (p-ERK1/2) and phosphorylated Myc (p-Myc) proteins following KIRA6 treatment (Fig.\u0026nbsp;6e), indicating suppression of ERK/Myc signaling axes. Moreover, \u003cem\u003eInterferon-α Response\u003c/em\u003e pathway (NES = 1.21) was significantly enriched in KIRA6-treated cells (Fig.\u0026nbsp;6f), suggesting that KIRA6 treatment induced an intrinsic antitumor immune response.\u003c/p\u003e\u003cp\u003eA core KIRA6-targeting gene signature (FPKM ≥ 5 in controls, log\u003csub\u003e2\u003c/sub\u003eFC \u0026lt;-1, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05) was identified from the RNA-seq data. Functional annotation (GO Molecular Function) revealed significant enrichment for snoRNA binding, RNA methyltransferase activity, et al., in the KIRA6-targeting gene signature (Fig.\u0026nbsp;6g). Notably, high expression of this signature in breast cancer patients correlated with significantly shorter overall survival (HR = 1.509, \u003cem\u003eP\u003c/em\u003e = 0.0162, Fig.\u0026nbsp;6h), suggesting that KIRA6 targets a group of genes related to poor prognosis.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eKIRA6 Enhances Antitumor Immunity and Overcomes Anti-PD-1 Resistance\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eConsidering the potent modulation of KIRA6 on both immune microenvironment and tumor cells, we evaluated the therapeutic potential of combining KIRA6 with immune checkpoint blockade. 4T1 tumor-bearing mice were treated with anti-PD-1 (200 µg, \u003cem\u003ei.p.\u003c/em\u003e every 3 days), KIRA6 (10 mg/kg, \u003cem\u003ei.p.\u003c/em\u003e every other day), or both until sacrifice at day 22 (Fig.\u0026nbsp;7a). PD-1 blockade failed to inhibit tumor growth in this resistant model. KIRA6 treatment not only significantly suppressed tumor progression alone, but also overcame ICB resistance when combined with anti-PD1 therapy, achieving further tumor suppression (Fig.\u0026nbsp;7b-d). While anti-PD-1 monotherapy exhibited negligible effect on splenomegaly, the combination of KIRA6 significantly decreased spleen weight (Fig.\u0026nbsp;7e-f), indicating resolution of tumor-driven extramedullary myelopoiesis.\u003c/p\u003e\u003cp\u003eMoreover, comprehensive immunophenotyping was performed to further analyze immune cell populations in the tumor and spleen. In line with the therapeutic outcome, PD-1 antibody treatment could not significantly modulate the proportion of tumor-infiltrating immune cells. KIRA6 monotherapy increased intratumoral CD3⁺ and CD8⁺ T cell infiltration while decreasing immunosuppressive CD11b⁺ myeloid cells and PMN-MDSCs, which were amplified in the combination group (Fig.\u0026nbsp;7g). In similar, enhanced antitumor immunity, with increased T cell proportions and reduced immunosuppressive myeloid cells, was observed in the spleens from KIRA6 and combo-treated mice (Fig.\u0026nbsp;7h). These findings suggest that KIRA6 remodels both local and systemic immune landscapes and improves the efficiency of anti-PD-1 therapy.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eImmune checkpoint blockade (ICB) therapy is successfully used for cancer treatment in many types of cancers, however only a small portion of patients benefit from PD-1/PD-L1 blockade therapy because of immune suppression mediated by alternative suppressive immune cells, such as MDSCs\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. IRE1α is a key regulator for generation of MDSC, whose kinase activity and RNase activity could be inhibited by KIRA6 simultaneously\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. However, its potential for targeting MDSC remains unexplored. Here, we evaluated the antitumor activity of KIRA6 for breast cancer, we examined the role of KIRA6 in decreasing MDSC generation, we revealed the effect of KIRA6 on myeloid-promoting cytokine production and survival of tumor cells, and we explored the potential of KIRA6 to improve the benefit from anti-PD-1 therapy. Our work establishes important evidence that KIRA6 potently inhibits systemic and local MDSC in preclinical tumor model and provides a promising agent for overcoming ICB resistance.\u003c/p\u003e\u003cp\u003eMDSCs are one of the major immunosuppressive cells that restrain antitumor T cell responses in the TME, therefore great efforts have been made to develop MDSC-targeting strategies for cancer therapy\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Previous studies aimed to abolish MDSC mediated immune suppression through eliminating MDSCs, decreasing MDSC infiltration or abrogating their suppressive functions, such as the use of chemotherapies, chemokine inhibitors, or neutralizing antibodies\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. These agents effectively abrogate immune suppression of MDSC on T cells and improve antitumor response. However, MDSCs rapidly expanded from aberrant myelopoiesis in bone marrow and spleen. We and others have revealed that myeloid progenitor cells significantly increase in the peripheral blood and spleen of cancer patients and serve as an important source of functional MDSCs\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. These observations suggest that tumors systemically regulate myelopoiesis and continuously replenish MDSCs through chronic secretion of cytokines, such as G-CSF and GM-CSF. Thus, targeting the source of MDSCs is promising for a more sustainable benefit\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. In this study, KIRA6 treatment significantly suppressed 4T1 tumor growth with decreased MDSC population and enhanced T cell infiltration. Two dosages of KIRA6 treatment potently inhibited MDSC generation and extramedullary hematopoiesis, as indicated by systemically decreased MDSC population and smaller spleen size. Moreover, KIRA6 inhibited MDSC biogenesis and eventually achieved in overcoming resistance to anti-PD-1 therapy. Our work identified KIRA6 as an impressive agent for targeting MDSC generation and overcoming ICB resistance.\u003c/p\u003e\u003cp\u003eThe expansion and acquisition of immune suppression capability are orchestrated by several key pathways, offering promising strategies to reprogram MDSCs\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. As a result of high secretory and metabolic activity during the immunosuppressive myelopoiesis in tumor milieu, MDSCs are faced with ER stress\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. When challenged with ER stress, several ER transmembrane sensors mediated signaling cascade determine cell fate, such as IRE1α and PERK\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. These pathways act as key regulators for generation of suppressive MDSCs. IRE1α possesses RNase activities, is activated by trans-autophosphorylation through its own kinase activity\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. The RNase activity of IRE1α enables processing unspliced XBP1 to its mature production, spliced XBP1 (XBP1s), which encodes the transcriptionally active XBP1 protein\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Genetic deletion of IRE1α completely abrogate suppressive activity of PMN-MDSCs in tumor model\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. On the other hand, cancer cell intrinsic XBP1s favors the synthesis and secretion of cholesterol, which is absorbed by MDSCs and drives its immunosuppressive reprogramming\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. The kinase activity of IRE1α mediates activation of JNK pathway and markedly potentiates the expression of G-CSF and GM-CSF in breast cancer cells\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. The multiple effects of IRE1α on supporting MDSC generation highlights the potential of inhibiting the kinase and RNase activity simultaneously for cancer immunotherapy. Therefore, KIRA6, other than solo RNase activity inhibitor, such as 4µ8C, MKC8866, or STF-083010\u003csup\u003e40, 41\u003c/sup\u003e, was evaluated for antitumor activity in this study. KIRA6 not only directly decreased MDSC differentiation from bone marrow cells, but also attenuated G-CSF production from tumor cells and therefore blocked the sustainably induction of MDSCs. Our data revealed that the dual effect of KIRA6 on blocking MDSC generation.\u003c/p\u003e\u003cp\u003eKIRA6 exhibited direct killing on tumor cells in addition to its immune modulation ability. In support with our findings, previous studies have demonstrated IRE1α promotes the survival, growth, and drug resistance of tumor cells, which underline the importance of IRE 1α in tumor biology\u003csup\u003e\u003cspan additionalcitationids=\"CR43 CR44\" citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e–\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. For example, IRE1α pathway activates c-MYC signaling and promotes prostate cancer\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e; reactivation of IRE1α caused acquired resistance to KRAS inhibitor, and inhibition of IRE1α overcame resistance to KRAS inhibitor\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e; IRE1α RNase silences taxane-induced dsRNA through preventing NLRP3 inflammasome-dependent pyroptosis\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. We found KIRA6 suppressed cellular proliferation in a dose-dependent manner, and induced cell cycle arrest and apoptosis at sub-micromolar concentration. KIRA6 treatment downregulated the phosphorylation of ERK1/2 and Myc proteins, indicating potent suppression on oncogenic signaling pathways. KIRA6 is also reported to inhibit KIT as well as its downstream signaling modules phosphorylated ERK1/2 at nanomolar concentrations and are sufficient to induce cell death in a KIT signaling-dependent leukemia cell line\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. This study confirmed the direct cancer killing effect of KIRA6 on breast cancer at pharmacologically concentrations.\u003c/p\u003e\u003cp\u003eKIRA6 treatment achieved more potent antitumor response and resulted in overcoming ICB resistance when combined with anti-PD1 therapy. The improved T cell response could benefit from various aspects of KIRA6 treatment. On the one hand, KIRA6 systemically reprograms myeloid population, with decreased MDSC in peripheral blood, spleen, and tumor tissue. Spleen is an important source for MDSC generation, while short-term KIRA6 treatment significantly decreased spleen weight, suggesting resolution of extramedullary hematopoiesis. KIRA6 abrogated myeloid cells mediated immune suppression, which is fundamental for overcoming ICB blockade. On the other hand, targeting IRE1α-XBP1 signaling have been found to restore anti-tumor capacity of T cells in cancer hosts\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. IRE1α-XBP1 activation is found in T cell from patients with ovarian cancer, and suppresses mitochondrial activity and IFN-γ production. Blocking IRE1α-XBP1 pathway helps to restore the metabolic fitness and anti-tumor capacity of T cells in cancer hosts\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. Previous studies suggest that inhibiting IRE1α rejuvenates T cells rather than interfere their antitumor functions. Therefore, KIRA6 abrogated MDSC mediated immune suppression without impeding T cell functions, making it a promising candidate of small molecule drug for cancer immunotherapy.\u003c/p\u003e\u003cp\u003eOur study evaluated the antitumor activity of KIRA6 for breast cancer and dissected the underlying mechanism from both MDSC and cancer cell aspect. Our data revealed that KIRA6 potently inhibits MDSC generation and tumor progression in mouse model and uncovered its impressive role in overcoming ICB resistance. However, our study is conducted in animal and cell level. Future preclinical investigations in human models and clinical trials are required for a clearer translational impact.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eC. C., J.C., and X. L. contributed equally to this work. C. C., J. C., and X. L. designed and conducted experiments, analyzed data and wrote the manuscript; J. H., D. L., X. O., J. L., W. L., and S. X., conducted experiments; Y. Z., performed bioinformatic analysis; J.C., and Y. M. revised the manuscript; Z.M., Y. P. and H.-W. S. designed and supervised the research and revised the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no potential conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of Data and Materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data relevant to the study are included in the article or uploaded as supplementary information. The data generated in this study are available within the article and its Supplementary Data files. Any data used in this study that are not included in the paper or supplementary files can be made available upon request from the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (82471769, 82103306, 82203518, and 82272103), the Guangdong Provincial Key Laboratory of Tumor Interventional Diagnosis and Treatment (2021B1212040004), the Natural Science Foundation of Guangdong Province of China (2022B1515020010), Guizhou Provincial Basic Research Program (Natural Science) (No. MS [2025] 419), and the Xiangshan Talent Project of Zhuhai People\u0026apos;s Hospital (2023XSYC-02). Funders were not involved in the research and publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments were performed in accordance with the guidance and approved by the Experimental Animal Ethics Committee of Zhuhai People\u0026rsquo;s Hospital (No. 20250222-01).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePatient consent statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSharma P, Goswami S, Raychaudhuri D, Siddiqui BA, Singh P, Nagarajan A\u003cem\u003e, et al.\u003c/em\u003e Immune checkpoint therapy-current perspectives and future directions. 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Nature\u003cem\u003e.\u003c/em\u003e 2018; 562: 423-428.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-disease","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddis","sideBox":"Learn more about [Cell Death \u0026 Disease](http://www.nature.com/cddis/)","snPcode":"41419","submissionUrl":"https://mts-cddis.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Myeloid-derived suppressor cell, Immune Checkpoint Inhibitor, Immunotherapy","lastPublishedDoi":"10.21203/rs.3.rs-7263160/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7263160/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Immune checkpoint blockade (ICB) therapy is one of the cornerstones of cancer treatment regimens, but the overall response rates remain low because of suppressive immune cells, such as myeloid-derived suppressor cells (MDSCs). Therefore, it is unmet need to target MDSCs to achieve better outcome of ICB therapy. Inositol-requiring enzyme 1α (IRE1α) is identified as a key regulator for generation of MDSC. Here, we evaluated the potential of KIRA6, an inhibitor for IREα kinase activity and RNase activity, to abrogate MDSC mediated immune suppression. KIRA6 significantly suppressed 4T1 tumor growth, decreased MDSC population and enhanced T cell infiltration. Two dosages of KIRA6 treatment directly inhibited extramedullary myelopoiesis and MDSC generation in vivo. KIRA6 abrogated the induction of MDSCs from bone marrow cells and abolished the immunosuppressive capability of MDSCs in vitro. Meanwhile, KIRA6 not only attenuated G-CSF production from tumor cells thereby blocking the induction of MDSCs, but also caused apoptosis of tumor cells. Moreover, KIRA6 treatment diminished MDSC generation, restored T cell proportion in both local and systemic immune landscapes and eventually overcame resistance to anti-PD-1 therapy. Our work establishes the evidence for KIRA6 as an impressive agent for abrogating MDSC mediated immune suppression, killing tumor, and overcoming ICB resistance.","manuscriptTitle":"KIRA6 abrogates the generation of myeloid-derived suppressor cells and overcomes resistance to anti-PD-1 therapy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-12 07:55:19","doi":"10.21203/rs.3.rs-7263160/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2025-09-25T16:16:07+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-09-17T07:26:07+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-09-13T07:33:27+00:00","index":3,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-09-08T05:57:07+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-08-31T15:26:43+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-08-12T14:38:14+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2025-08-12T13:26:38+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-01T09:24:53+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-31T14:08:38+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cell Death \u0026 Disease","date":"2025-07-31T14:08:37+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-disease","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddis","sideBox":"Learn more about [Cell Death \u0026 Disease](http://www.nature.com/cddis/)","snPcode":"41419","submissionUrl":"https://mts-cddis.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a22f148d-9c57-45e1-96fb-ab23bbc05561","owner":[],"postedDate":"August 12th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":52486131,"name":"Health sciences/Diseases/Cancer/Cancer microenvironment"},{"id":52486132,"name":"Health sciences/Medical research/Preclinical research"}],"tags":[],"updatedAt":"2026-01-31T08:12:06+00:00","versionOfRecord":{"articleIdentity":"rs-7263160","link":"https://doi.org/10.1038/s41419-025-08401-6","journal":{"identity":"cell-death-and-disease","isVorOnly":false,"title":"Cell Death \u0026 Disease"},"publishedOn":"2025-12-27 05:00:00","publishedOnDateReadable":"December 27th, 2025"},"versionCreatedAt":"2025-08-12 07:55:19","video":"","vorDoi":"10.1038/s41419-025-08401-6","vorDoiUrl":"https://doi.org/10.1038/s41419-025-08401-6","workflowStages":[]},"version":"v1","identity":"rs-7263160","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7263160","identity":"rs-7263160","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}