MST1/2 inhibitor XMU‐MP‐1 alleviates the injury induced by ionizing radiation in haematopoietic and intestinal system

Abstract The Hippo signalling pathway has been considered as potential therapeutic target in self‐renewal and differentiation of stem and progenitor cells. Thus, mammalian Ste20‐like kinase 1/2 (MST1/2) as the core serine‐threonine kinases in the Hippo signalling pathway has been investigated for its role in immunological disease. However, little information of MST1/2 function in bone marrow suppression induced by ionizing radiation was fully investigated. Here, we reported that MST1/2 inhibitor XMU‐MP‐1 could rescue the impaired haematopoietic stem cells (HSCs) and progenitor cells (HPCs) function under oxidative stress condition. Also, XMU‐MP‐1 pretreatment markedly alleviated the small intestinal system injury caused by the total body irradiation 9 Gy and extended the average survival days of the mice exposed to the lethal dose radiation. Therefore, irradiation exposure causes the serious pathological changes of haematopoietic and intestinal system, and XMU‐MP‐1 could prevent the ROS production, the haematopoietic cells impairment and the intestinal injury. These detrimental effects may be associated with regulating NOX/ROS/P38MARK pathway by MST1/2.


| INTRODUC TI ON
There are constant research, ongoing innovation and technical and scientific advancements in order to tackle different forms of cancer.
Various molecules have been considered as the therapeutic targets for cancer prevention. [1][2][3][4][5][6][7] Excluding chemotherapy, radiation therapy is another cancer treatment that uses high doses of radiation to kill cancer cells and shrink tumours. However, ionizing radiation can induce irreversible damage in all radiosensitive organs. The level of damage depends on the radiation dose absorbed, radiation type and organ sensitivity. The haematopoietic system is most sensitive to radiation, especially the HSCs undergo senescence in vitro and in vivo following irradiation exposure. 8,9 Therefore, the bone marrow suppression would be observed by encompassing all cell lines causing anaemia, leucopenia, thrombocytopenia and neutropenia. 10 The ROS production is one crucial reason for radiation damage; furthermore, the persistent oxidative stress is observed in haematopoietic stem/progenitor cells (HSPCs). Previous researches revealed the persistent ROS elevation induced oxidative stress associated with the activation of NOX4 signalling. 11,12 NOX4 mediates ROS production in radiation-induced senescent cells and contributes to normal tissue damage after IR via the recruitment of inflammatory cells and the exacerbation of tissue inflammation. In addition, radiation-induced ROS production was diminished by genetic or pharmacological inhibition of NOX4. 13 High-dose irradiation (>10 Gy) can also cause severe intestinal toxicity, known as gastrointestinal syndrome. Under homeostatic conditions, rapid turnover of enterocytes is driven by ISCs located at the base of the crypts. XMU-MP-1 as an inhibitor of mammalian Ste20-like kinase 1/2 (MST1/2), displayed excellent in vivo pharmacokinetics and was able to promote mouse intestinal repair. The Hippo pathway regulates the self-renewal and differentiation of stem and progenitor cells and plays key roles in controlling organ size and regeneration. 14,15 MST1/2, mammalian homologs of Hippo, are a core pair of serine-threonine kinase in the Hippo signalling pathway that regulates the cell cycle and apoptosis. 16 A recent study showed that MST1/2 are also crucial for CD8α+ dendritic cell-mediated antigen presentation to CD8+ T cells, which requires a balance of metabolic activity and cytokine signalling depending on MST1/2 activity. 17 And it is reported that MST1/2 functioned to control ROS production by regulating mitochondrial trafficking and mitochondrion-phagosome juxtaposition. 18 Studies of MST1-deficient mice have demonstrated that MST1 regulates the production of reactive oxygen species (ROS) through FOXO3A (forkhead box O3A), thereby preventing apoptosis of peripheral T cells. 19 Meanwhile, the roles of MST1/2 among HSPCs in vivo did not reach an agreement. We hypothesized that MST1/2 could associate with the ROS production in the mitochondria of HSPCs, and XMU-MP-1 as a putative MST1/2 inhibitor was utilized as a novel radioprotectant to ameliorate TBI-induced residual BM injury. The effect of XMU-MP-1 on the intestinal system injury induced by radiation was assessed as well.  For assessing the radiation injury in the haematopoietic system, animals (n = 5 per group) were exposed to total body irradiation (TBI) at a single sublethal dose of 4.0 Gy and then monitored for 9 days. Regarding the radiation challenge to the small intestine, mice (n = 5 per group) were exposed to 9 Gy TBI, and small intestine was harvested for the histol-

| Peripheral blood cells and bone marrow nucleated cells (BMNCs) counts
Blood samples isolated by orbital sinus were kept in the tube with ethylenediaminetetraacetic acid (EDTA) and tested by Celltac E hemocytometer (Nihon Kohden). The BMNCs were collected by flushing the femur with cold PBS and analysed with the hemocytometer.

| ROS level analysis on HSCs, HPCs and BMNCs with irradiation exposure.
Reactive oxygen species level of HSCs, HPCs and BMNCs was tested by the flow cytometry with 2,7-dichlordihydrofluorescein diacetate

| The immune cells proportion analysis of the peripheral blood
For analysis of multilineage blood cells, FITC-Anti-GR-1 (BD), FITC-Anti-CD45R/B220 and FITC-Anti-CD11b antibodies were used for the test of the peripheral blood. And the proportion of positive marked cells was measured on a flow cytometer (BD Accuri™ C6 Plus).

| Histological analysis
Mice were sacrificed, and jejunum was isolated after TBI 3.5 days, fixed overnight in 10% neutral formalin and embedded in paraffin.
5μm sections were prepared and stained with haematoxylin and eosin (H&E) according to standard protocols and observed under a light microscope (Olympus Corp. Japan).

| Organ weight index detection
Animals were sacrificed organs including the thymus, spleen, heart, kidney, lung and liver were isolated. Organ index was calculated using the following equation: Organ index = organ weight/body weight.

| Statistical analysis
The results were expressed as the mean ± SD. An unpaired t-test (twotails) was used for the majority of comparisons. Significant differences between experimental groups were evaluated using one and two-way analysis of variance (ANOVA) with repeated measures followed by post hoc comparisons with Tukey's multiple paired comparison test.

| XMU-MP-1 rescues the damage of the haematopoietic system
Haematopoietic system is vulnerable to ionizing radiation, especially, WBC as a sensitive marker of irradiation effect is associated with the irradiation dose and time. The results ( Figure 1) show that the WBC of the mice exposed to 4 Gy TBI decreased to 24.7% of that in the control group (p < 0.001). XMU-MP-1 could significantly rescue the WBC impairment after IR by increasing the WBC to 56.4% of pre-irradiation (p < 0.05). The effect of radiation exposure on RBCs, HCT, PLTs and BMNCs showed the same pattern with the WBC, while the HGB level was not obviously influenced at 9 days after IR.

| XMU-MP-1 reduces the ROS level of haematopoietic system
Radiation generates ROS that can interact with a various cellular separately compared with irradiation alone. While ROS level in HPCs of the irradiated mice shows the similar pattern, but the difference is not significant. NOX4, as the phagocyte-type oxidase, is responsible for the production of ROS. As Figure 2C shown that the NOX4 expression of the mice exposed to 4 Gy TBI was induced obviously to 1.5-fold (BMNCs, p < 0.05), 1.3-fold (HPCs, p < 0.05) and 2.2fold (HSCs, p < 0.001) compared with the control groups. And NOX4 expression associated with ROS induction could be significantly decreased by XUM-MP-1 pretreatment, which recovered to 72% (BMNCs, p < 0.05), 74% (HPCs, p < 0.05) and 52% (HSCs, p < 0.001) compared with 4.0 Gy TBI. Additionally, the HSCs protein expression of γ-H2AX, Nrf2 and PP38 was measured in the mice exposed to 4 Gy TBI, the result shows the same pattern with the ROS induction in the HSCs.

| XMU-MP-1 improves the immune cells impairment induced by TBI 4 Gy
The radiation exposure can suppress or weaken the immune system. CD11b was used to label myeloid lineage cells including monocytes, neutrophils and eosinophils. As Figure 3 shown that the proportion of CD11b+ cells depleted obviously (control,

| XMU-MP-1 relieves the intestinal injury induced by 9 Gy TBI
To determine the effect of XMU-MP-1 on the intestinal system exposed to irradiation, a higher dose of 9 Gy TBI was applied to mice.
The radiation experiment was set up as the diagram Figure 4A shown. The results of haematoxylin and eosin staining ( Figure 4B

| XMU-MP-1 extends the survival days exposed to 7.5 Gy TBI
To evaluate the radioprotection of XMU-MP-1 to the acute radiation challenge, the lethal dose radiation of 7.5 Gy was applied to the mice with XMU-MP-1 treatment (shown in Figure 5A). And the results ( Figure 5B) show that there existed a turning point of the body weight of irradiated mice at the 6th day after radiation.
As Figure 5C shown, the average survival days of mice treated with

F I G U R E 3 XMU-MP-1 relieves the immune cells impair induced by 4 Gy TBI.
Mice were administrated with XMU-MP-1 for 7 days before being exposed to TBI 4 Gy. Bone marrow cells were harvested 9 days after IR and tested the cells proportion by flow cytometry. Results are expressed as Mean ± SD, n = 5 per group. (Significance: *p < 0.05, **p < 0.01 and ***p < 0.001) irradiated mice suffered from the weight loss starting at the 6th day.
Although suffering with extreme weight loss, there were more mice survived in the XMU-MP-1 pretreated group. Therefore, XMU-MP-1 could provide a tolerance of the body weight loss challenge.

| DISCUSS ION
Ionizing radiation exposure on body can activate the boost of ROS, consequently induce a various reaction on DNA, protein and lipids. Double-strand breaks (DSBs), although rare, are probably the most lethal mechanism and are commonly produced by ionizing radiation. 25 Regarding the radiosensitivity, the quiescent and slowly

ACK N OWLED G EM ENTS
This work was supported by the National Natural Science Foundation of China (81972975, 81972847); the key technologies R&D project of Tianjin (17YFZCSY00670).

CO N FLI C T O F I NTE R E S T
The authors declare no conflicts of interest.