Deletion of IP6K1 in mice accelerates tumor growth by dysregulating the tumor-immune microenvironment

ABSTRACT A family of inositol hexakisphosphate kinases (IP6Ks) catalyzes the production of inositol pyrophosphate IP7 (5-diphosphoinositolpentakisphosphate) which is known to modulate various biological events such as cell growth. While targeting IP6K1 in various cancer cells has been well reported to control cancer cell motility and invasiveness, the role of host IP6K1 in tumor progression remains unknown. By using a syngeneic MC38 murine mouse colon carcinoma model, here we examined how host IP6K1 in the tumor microenvironment influences tumor growth. In IP6K1 knockout (KO) mice, the growth of MC38 tumor cells was markedly accelerated and host survival was significantly shortened compared with wild-type (WT). Our flow cytometric analysis revealed that tumors grown in IP6K1 KO mice had lower immune suppressive myeloid cells and M1 polarized macrophages. Notably, infiltration of both antigen-presenting dendritic cells and CD8+ cytotoxic T lymphocytes into the tumor tissues was remarkably abrogated in IP6K1 KO condition. These studies suggest that enhanced tumor growth in IP6K1 KO mice resulted from reduced anti-tumor immunity due to disturbed immune cell actions in the tumor microenvironment. In conclusion, we demonstrate that host IP6K1 acts as a tumor suppressor, most likely by fine-tuning diverse tumor-immune cell interactions, which might have implications for improving the host response against cancer progression.


Introduction
Inositol pyrophosphates are a class of high-energy signaling molecules characterized by the presence of pyrophosphate and monophosphate substitutions on the inositol ring Chakraborty 2018;Park et al. 2018). They participate in many cellular functions by binding specific proteins or by transferring their β phosphate to pre-phosphorylated serine residues to bring about serine pyrophosphorylation (S. . The most abundant inositol pyrophosphate in mammals, 5-diphosphoinositol pentakisphosphate (5-PP-IP 5 or 5-IP 7 ) is synthesized from inositol hexakisphosphate (IP 6 ) by inositol hexakisphosphate kinases (IP6Ks) (Saiardi et al. 1999;Saiardi et al. 2001;Chakraborty et al. 2011). IP6Ks are found in all eukaryotes, from lower organisms such as yeast to mammals possessing three IP6 K isoforms, IP6K1, 2, and 3. Pleiotropic signaling actions of IP6K1 include promotion of insulin release in pancreatic β cells (Bhandari et al. 2008;Landrier et al. 2019), maintenance of genome integrity (Rao et al. 2014), platelet coagulation (Ghosh et al. 2013), Akt signaling (Chakraborty et al. 2010), as well as presynaptic vesicle cycling Park et al. 2020).
Recent studies revealed key actions of IP6K1 in the control of tumorigenesis and cancer progression. When IP6K1 is depleted in mouse embryonic fibroblasts and cancer cells, they undergo cellular changes associated with decreased cell migration and dysregulated focal adhesion kinase (FAK) activity (Jadav et al. 2016;Fu et al. 2017). IP6K1-depleted HCT116 colorectal cancer cell xenografts showed reduced invasion in immunocompromised mice when compared with wildtype mice (Xu et al. 2020). Moreover, IP6K1 knockout (KO) in mice protects against 4-nitroquinoline-1-oxide (4-NQO)-induced carcinogenesis and diminishes the progression of invasive carcinoma (Jadav et al. 2016), suggesting IP6K1's pro-tumorigenic action in cancer cells. However, regarding tumor formation and progression, the role of host IP6K1 in this pathology remains poorly understood. Given that that complex cellular interactions in the tumor microenvironment have been shown to play a major role in tumor growth and progression, we hypothesized that IP6 K in the tumor microenvironment could contribute to a host protective response against cancer.
In the present study, we investigated whether host IP6K1 KO could control tumor growth using a syngeneic MC38 model which is a murine colon adenocarcinoma tumor model on the C57BL/6 background. Using the global IP6K1 KO mice, we show that the absence of the host IP6K1 results in significantly shortened survival compared to normal mice due to more rapidly growing tumors. By flow cytometric analysis we showed that tumors in IP6K1 KO mice contain high levels of CD11b + Gr1 + IL10 + myeloid cells (e.g., neutrophils, monocytes) and lower infiltration of M1-polarized tumor-associated macrophage (TAM), dendritic cells, and CD8 + killer T cells into tumors. Our data indicate that host IP6K1 acts as a tumor suppressor to regulate anti-tumor immune activities and control the recruitment and activation of cytotoxic T cells to the primary tumor through reprogramming tumor-immune control, and loss of this pathway increases tumor progression.

Ethics statement
All mice were bred and housed under specific pathogenfree, temperature-and humidity-controlled conditions in a 12-hour light-dark cycle at KAIST. They received a standard laboratory chow diet and water ad libitum. All experiments involving animals were conducted according to the ethical policies and procedures approved by the Committee for Animal Care at KAIST.

Mouse tumorigenesis and survival
For the tumor growth experiments, 6-7 weeks old male and female mice were inoculated MC38 colorectal cancer cells subcutaneously at both flanks of each mouse (2.5 × 10 5 cells/side). After 7 days passed from tumor injection, mice's body weight and tumor size were measured once every 2 to 3 days. Tumor volume was evaluated according to the general formula 0.5 × (width) 2 × (length) using a caliper, and Student's t-test was used to determine pvalues. For mouse survival experiments, 12-14 weeks old male mice were subjected to inject MC38 cells (5.0 × 10 5 cells/side) subcutaneously, and assessed survival rate and body weight daily. Mice were immediately euthanized when tumor volume exceeded 1000 mm 3 .

Immunohistochemistry
Tissues and tumors were embedded in the OCT compound, and 8 µm sections were prepared and stained with Hematoxylin and eosin Y solution (H&E) for histologic evaluation via light microscopy (KPNT, Korea). Sections were photomicrographed with a digital camera mounted on a light microscope (Olympus BX51, Japan), digitized, and analyzed. Analysis was performed on 10 fields of a section at 20x magnification.

Statistical analysis
Differences between averages were analyzed using a two-tailed Student's t-test. Data are expressed as means ± SEM.

MC38 tumor growth was promoted in IP6K1 KO mice
To determine whether host IP6K1 alters tumor growth in C57BL/6 mice, WT and IP6K1 KO mice were injected with syngeneic MC38 mouse colon carcinoma cells and observed for primary tumor growth and survival. The tumors between the two groups started showing significant differences on day 7 post-implantation ( Figure 1A). IP6K1 KO mice had markedly accelerated tumor growth by day 17, without notable changes in body weight ( Figures 1B−E). Accordingly, we found that mean survival time was significantly decreased in tumor-bearing IP6K1 KO animals, compared to WT mice ( Figure 1F). Bodyweight became significantly increased in IP6K1 KO mice after day 27 post-tumor inoculation ( Figure 1G), which reflects the higher tumor mass in IP6K1 KO mice compared to control.
Collectively, these results indicated the lack of IP6K1 in mice accelerated tumor growth and shortened host survival, suggesting the in vivo role of host IP6K1 as a tumor suppressor.
To explore the biology underlying the increased tumor burden in IP6K1 KO mice, we examined indicators of proliferation and angiogenesis. No differences were observed in protein levels of proliferating cell nuclear antigen (PCNA) and retinoblastoma protein (Rb) in tumor tissue from IP6K1 KO compared with WT mice (Supplementary Figure  S1A). Both Akt and Erk phosphorylation levels were also unchanged in tumor tissues from IP6K1 KO mice (Supplementary Figure S1B). Similarly, there was no change in the mRNA levels for angiogenic factors such as vascular endothelial growth factor (VEGF) and its receptor (VEGFR) between tumors grown in IP6K1 KO or WT hosts (Supplementary Figure S1C). These results collectively suggest no major role of host IP6K1 in supporting tumor-intrinsic growth properties.

IP6K1 KO mice show tumors with increased infiltration of immune cells with reduced M1 macrophages
The tumor microenvironment consists of multiple cell types, including various immune cells (Kenny et al. 2007;Hanahan and Coussens 2012). To examine if the deficiency of host IP6K1 in the tumor microenvironment leads to changes in lymphocytes or inflammatory cells during tumor progression, we harvested tumor-bearing tissues from IP6K1 KO and WT mice 2.5 weeks after the injection of MC38 cells and compared the type and quantity of infiltrating immune cells. Flow cytometry analysis revealed increased total viable CD45.2 + immune cells in tumors grown from IP6K1 KO mice (Supplementary Figure S2A), suggesting more tumor-infiltrating immune cells in the IP6K1 KO tumor microenvironment.
First, we measured CD11b + Gr1 + myeloid cells and found no apparent changes in tumor tissues grown from both WT and IP6K1 KO mice (Supplementary Figure S2B). However, tumors from IP6K1 KO mice contained increased levels of CD11b + Gr1 + IL10 + cells compared to control tissues (Supplementary Figure S2C). These results suggest that host IP6K1 deletion leads to more development of immunosuppressive tumorassociated neutrophils/monocytes, presumably establishing a highly immune-suppressive environment to promote tumor growth.
To further dissect changes in leukocyte populations, we next analyzed tumor-associated macrophages (TAMs) and identified significantly decreased CD80 + M1-polarized macrophages from tumor tissues in IP6K1 KO mice (Figure 2A). A marked reduction of the CD80 + IFN-γ M1 macrophages was further noted in the same tumor tissues from IP6K1 KO mice ( Figure 2B). These findings suggest that IP6K1 deficiency leads to more anti-tumorigenic, pro-inflammatory M1 polarization in tumor tissues. Since M1 macrophages can suppress tumor growth by directly killing tumor cells or indirectly controlling Th1 cell activities, diminished M1 TAMs found in tumors from IP6K1 KO conditions could contribute to the accelerated tumor growth. We further attempted in vitro macrophage polarization by using primary culture of bone marrow-derived macrophages (BMDMs). Levels of M1 markers (e.g. Il-6, iNOS) were rather increased in IP6K1 KO BMDMs, compared to control cells (Supplementary Figure S3A). Furthermore, major activities of Akt and other inflammation signaling events were similar between in vitro polarized IP6K1 KO and WT BMDMs (Supplementary Figure S3B), which further indicates that IP6K1 deletion may elicit its impact on TAMs throughout the in vivo tumor microenvironment but not in vitro culture conditions.

IP6K1 KO mice show tumors with decreased infiltration of dendritic cells
In addition to M1 TAMs, dendritic cells (DCs) are another major myeloid cell that contributes to antitumor immunity associated with a favorable outcome (Wylie et al. 2019;Wculek et al. 2020). The key role of conventional DCs in anti-tumor immunity depends on their ability to present tumor antigens and to secrete various cytokines to regulate T cell survival and their effector functions (Garris and Luke 2020). The infiltration of total CD11c + and CD11c + MHCII + DCs was markedly decreased in tumors formed in IP6K1 KO mice than in the tumors grown in WT control mice (Figures 3A and B). There is no error bar on day 32 in the IP6K1 KO group because only one IP6K1 KO mouse survived. Data are expressed as means ± SEM (*P < 0.05; **P < 0.01; ***P < 0.001, Student's t-test).
We further analyzed the effect of IP6K1 on dendritic cell homeostasis. We examined percentages of dendritic cells within the bone marrow (BM) and peripheral lymphoid organs. IP6K1 deletion did not alter the percentages of CD11c + cells within the thymus, BM, spleen, or peripheral lymph nodes ( Figure 3C and Supplementary Figure S4). Furthermore, the expression of activation markers such as CD80 and MHCII was comparable to control CD11c + cells (Figures 3D and E). To further examine whether the depletion of IP6K1 affects the dendritic cell maturation in vitro, we cultured WT and IP6K1 KO bone marrow dendritic cells (BMDCs) in vitro with GM-CSF and IL-4 for 9 days. BMDC maturation induced by lipopolysaccharide led to a robust elevation of CD80 with no difference between WT and IP6K1 KO BMDCs (Supplementary Figure S5), indicating that in vitro DC maturation is not affected by the loss of IP6K1. We also investigated whether loss of IP6K1 likewise affects dendritic cell migration. Under the stimulation with CCL5, the number of both WT and IP6K1 KO BMDCs similarly migrated towards the CCL5 (Supplementary Figure  S6), suggesting that loss of IP6K1 does not affect in vitro migration of dendritic cells.
CD8 + T cells in tumors grown in IP6K1 KO mice, relative to control tumor tissues ( Figures 4A and B). Collectively, these findings indicate that host IP6K1 deletion leads to failure of dendritic cell migration into the tumor microenvironment, thereby limiting activation and infiltration of anti-tumor CD8 + T cells, subsequently aiding tumor growth in IP6K1 KO mice.

Discussion
Here, we evaluated the role of host IP6K1 by using a syngeneic MC38 model, a commonly used preclinical model to investigate the complex actions of tumor cell-intrinsic and extrinsic factors in the control of tumorigenesis. We showed that the depletion of host IP6K1 markedly potentiated the growth of MC38 colorectal adenocarcinoma cells. Therefore, we describe the previously unidentified role of host IP6K1 in vivo as a tumor suppressor. FACS analyses revealed i) increased CD11b + Gr1 + IL10 + myeloid cells, ii) decreased antitumor, M1 TAMs, and iii) markedly reduced infiltration of dendritic cells into the tumor microenvironment in IP6K1 KO mice. Consequently, a significantly reduced amount of CD8 + killer T cells was observed in the tumor tissues, when host IP6K1 was deleted. Collectively, these data suggest a major contribution of host IP6K1 to the immune control of cancer cell growth in the tumor microenvironment.
Infiltration by inflammatory and immune cells is a hallmark of malignant tumors that can play a critical role in mounting an antitumor response (Labani-Motlagh et al. 2020;Murciano-Goroff et al. 2020). Most notably, we observed that tumor tissues of MC38 colon carcinoma in IP6K1 KO mice showed very little CD11c + dendritic cell infiltrate. When dendritic cells sense and present tumor antigens, naive antigen-specific CD4 + and CD8 + T cells are activated, thereby mediating anti-tumor activities (Gardner et al. 2020;Kim et al. 2021). Therefore, reduced detection of total and CD8 + killer T cell population in the tumor tissue from IP6K1 KO mice appears the consequence of defective IP6K1 KO dendritic cells. Thus, these findings suggest that an inefficient antitumor response due to the absence of dendritic cell actions might be the key event for the accelerated growth of MC38 cancer cells in IP6K1 KO mice. In addition, increased levels of IL10 + myeloid cells (e.g., neutrophils or myeloidderived suppressor cells), as well as lowered anti-tumor M1 TAMs from tumor tissues in IP6K1 KO mice, appear to be another major contributing factor to avoid immune surveillance. The next challenge is for us to dissect whether host IP6K1 deletion causes those cellular changes in a cell-autonomous manner or not. Conditional deletion of IP6K1 in different immune cells will be critical to fully understand mechanistic details underlying those cellular defects.
Previous studies also proposed IP6K1 and 5-IP 7 in the control of cellular migration from specific cell types such as neutrophils as well as cancer (Prasad et al. 2011;Jadav et al. 2016;Fu et al. 2017). When BMDCs were stimulated with CCL5, a major neoplastic tissue-derived chemokine, both WT and IP6K1 KO BMDCs were similarly responded, implying that in vitro dendritic cell migration potential may be independent of IP6K1. Marked reduction of IP6K1 KO dendritic cells in the tumor microenvironment may reflect complex molecular and cellular actions in vivo conditions but not in oversimplified in vitro culture settings. Further analyses will be needed to dissect the contribution of IP6K1 to the control of dendritic cell functions in the tumor tissues by using the conditional deletion of IP6K1 in dendritic cells. Examination of whether IP6K1 in T cells could directly influence their effector functions for antitumor activities is also required.
Since our approach using syngeneic tumor model demonstrate that targeting IP6K1 from the host promotes tumor growth, we believe that the host IP6K1 as a tumor suppressor may contribute to improving the host immune response against tumor progression. Future studies using various types of cancer cells as well as other IP kinase mouse models will be required to fully elucidate complex cell type-specific functions of host IP6K1 in modulating immune responses among tumor cells and their microenvironment. We further expect that therapeutics that modulate the levels of IP6Ks and their activities will be useful in the management of uncontrolled cancer progression.