The Journal of Pathology, ORIGINAL PAPER
Anti-tumour effects of PIM kinase inhibition on progression and chemoresistance of hepatocellular carcinoma

Ming-Sum Leung1†, Kristy Kwan-Shuen Chan1†, Wen-Juan Dai1, Cheuk-Yan Wong1, Kwan-Yung Au1, Pik-Ying Wong1, Carmen Chak-Lui Wong1,2, Terence Kin-Wah Lee3, Irene Oi-Lin Ng1,2, Weiyuan John Kao4, Regina Cheuk-Lam Lo1,2
1Department of Pathology, The University of Hong Kong,
2State Key Laboratory of Liver Research (The University of Hong Kong),
3Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University,
4Department of Industrial and Manufacturing Systems Engineering, Biomedical Engineering Program of Faculty of Engineering and Faculty of Medicine, The University of Hong Kong
† These authors contributed equally
Correspondence to: R C Lo, Department of Pathology, The University of Hong Kong, Queen Mary Hospital, Pokfulam, Hong Kong SAR, PR China. E-mail: [email protected]

 
Conflict of interest: R.C. Lo receives research funding from Nan Fung Life Sciences not related to the submitted work. All other authors declare no conflicts of interest.
Running title: PIM inhibitors in HCC Word count (abstract): 249
Word count (main text): 3912

 

 
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as doi:10.1002/path.5492

ABSTRACT

Hepatocellular carcinoma (HCC) is a biologically aggressive cancer. Targeted therapy is in need to tackle challenges in the treatment perspective. A growing body of evidence suggests a promising role of pharmacological inhibition of PIM (proviral integration site for Moloney murine leukaemia virus) kinase in some human haematological and solid cancers. Yet to date the potential application of PIM inhibitors in HCC is still largely unexplored. In the present study we investigated the pre-clinical efficacy of PIM inhibition as a therapeutic approach in HCC. Effects of PIM inhibitors on cell proliferation, migration, invasion, chemosensitivity and self-renewal were examined in vitro. The effects of PIM inhibitors on tumour growth and chemoresistance in vivo were studied using xenograft mouse models. Potential downstream molecular mechanisms were elucidated by RNA- sequencing (RNA-seq) of tumour tissues harvested from animal models. Our findings demonstrate that PIM inhibitors SGI-1776 and PIM447 reduced HCC proliferation, metastatic potential, and self-renewal in vitro. Results from in vivo experiments supported the role of PIM inhibition in suppressing of tumour growth and increasing chemosensitivity of HCC toward cisplatin and doxorubicin, the two commonly used chemotherapeutic agents in trans-arterial chemoembolisation (TACE) for HCC. RNA-seq analysis revealed downregulation of the MAPK/ERK pathway upon PIM inhibition in HCC cells. In addition, LOXL2 and ICAM1 were identified as potential downstream effectors. Taken together, PIM inhibitors demonstrated remarkable anti-tumourigenic effects in HCC in vitro and in vivo. PIM kinase inhibition is a potential approach to be exploited in formulating adjuvant therapy for HCC patients of different disease stages.

 

Keywords: PIM kinase; inhibitor; liver cancer; therapeutics; TACE; hypoxia.

INTRODUCTION

Hepatocellular carcinoma (HCC) is a common and aggressive cancer. Surgical resection is the mainstay of treatment and trans-arterial chemoembolisation (TACE) is recommended in intermediate-stage HCC or as bridging therapy for liver transplantation. However, chemoresistance, tumour recurrence and metastasis remain key issues to be tackled in improving the survival outcome. Potent molecular therapies that augment the efficacy of surgical and local ablative therapeutic modalities could best serve to maximise survival benefit of HCC patients. To this end, our group previously analysed the expression and characterised the role of PIM1, a functional molecular target in human HCC [1]. PIM1 is a fundamental member in the PIM (proviral integration site for Moloney murine leukaemia virus) family of constitutively activated serine/threonine kinases. We observed PIM1 overexpression in primary HCC tissues and further enhanced expression in metastatic tumour tissues, suggesting a role of PIM1 in HCC tumourigenesis and metastasis. Using cell line models, PIM1 expression was upregulated under hypoxia and functionally, silencing of PIM1 by short hairpin RNA (shRNA) approach suppressed proliferation and invasion of HCC cells [1]. The oncogenic role of PIM1 was also revealed in other cancer types before and after our study [2–4].
Since PIM1 is a kinase, pharmacological inhibition is feasible. To date, PIM inhibitors are categorised into two main classes, ATP-mimetic inhibitors and ATP-competitive inhibitors, according to the mechanism of inhibition of ATP binding to PIM kinases. SGI- 1776 and AZD1208 are one of the first-tested PIM inhibitors in human cancers [5]. SGI-

1776 and AZD1208 induced apoptosis in leukaemia cells in vitro and suppressed tumour growth in vivo [6-8]. In solid cancers, SGI-1776 re-sensitised resistant cells to chemotherapeutic agent in prostate cancer [9]. Two recent studies demonstrated the inhibitory effect on in vivo tumour growth in breast cancer with SGI-1776 and AZD1208 [10,11]. While clinical trials with SGI-1776 were terminated due to cardiotoxicity (NCT00848601), emerging PIM inhibitors have been invented and tested. For instance, PIM447 was shown to be effective in myeloma using cell line models [12] with a recently reported clinical trial demonstrating that it was well tolerated and showed anti-tumour activity in multiple myeloma patients [13].
In HCC, PIM kinases were collectively shown to exert oncogenic functions [14,15]. In particular, PIM1 is an appealing therapeutic target in liver cancer. Intra-tumoural hypoxia is frequently observed in HCC with the rapid rate of tumour growth or following TACE. In this regard, PIM1 was recognised to be induced by hypoxia in human colon cancer, pancreatic cancer, cervical cancer and HCC cell lines [1,16]. With the critical role of hypoxia in regulating pro-tumourigenic phenotypes including metastasis, chemoresistance and stemness [17] which our group has also previously demonstrated [18,19], hypoxia-induced genetic events are deemed strategic in the therapeutic perspective. Moreover, it was demonstrated in our previous study that PIM1 is a fast responder to hypoxia which was upregulated prior to protein stabilisation of the master regulator hypoxia-inducible factor-1α (HIF-1α) [1]. This implies that PIM1 inhibition likely brings additional therapeutic merits than targeting HIFs alone.

 
In this study, we investigated and demonstrated the preclinical efficacy of pharmacological inhibition of PIM kinase with SGI-1776 and PIM447 in HCC using cell line models and patient-derived tumour xenograft (PDTX). Downstream mechanisms elicited by PIM inhibition were elucidated by RNA-sequencing (RNA-seq) of tumour tissues from the animal models.

 

 

MATERIALS AND METHODS Clinical samples
Human HCC samples were collected from liver resection specimens at Queen Mary Hospital, Hong Kong. Samples were fixed in 10% formalin for paraffin embedding (FFPE). Use of human samples was approved by the Institutional Review Board of The University of Hong Kong/Hospital Authority Hong Kong West Cluster.
Cell lines and tissue culture

Human HCC cell lines MHCC-97L and Huh7 were used. MHCC-97L was from Professor Z.Y. Tang, Fudan University (Shanghai, PR China). Huh7 was obtained from the Japanese Collection of Research Bioresources. Huh7 cells were maintained in Dulbecco’s Modified Eagle’s Minimal Essential Medium (DMEM) with high glucose (Gibco, Thermo Fisher Scientific, Waltham, MA, USA); MHCC-97L was cultured in DMEM high-glucose with sodium pyruvate. All media were supplemented with 10% foetal

bovine serum (FBS), penicillin at 100 units/ml and streptomycin at 100 μg/ml (Gibco). For normoxic condition, cell lines were cultured in a humidified incubator with 20% O2/5% CO2/balanced N2 at 37 °C. For hypoxic condition, cells were incubated in a modular incubator chamber with 1% O2/5% CO2/balanced N2. The HCC cell lines were authenticated by short-tandem repeat profiling.
IC50 determination

PIM inhibitors SGI-1776 and PIM447 were purchased from Selleck Chemicals (Houston, TX, USA). Both inhibitors were reconstituted in dimethyl sulfoxide (DMSO). An assay using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was used to estimate the effect of PIM inhibitors on MHCC-97L and Huh7 cell number. DMSO and MTT were purchased from Sigma-Aldrich (St. Louis, MO, USA). HCC cells were seeded at a density of 5 × 103 cells per well in a 96-well plate and incubated for 24 h. After incubation, cells were treated with a gradient of concentrations of SGI-1776 or PIM447 for 24 h. Control cells were treated with DMSO only. Fifty µg of MTT was added to each well and incubated at 37 °C for 2 h. The media were removed and the lysis buffer (0.2 N hydrochloric acid: isopropanol, 1:4) was added to each well. Absorbance was read at 570 nm using an Infinite F200 microplate reader (Tecan, Mannedorf, ZH, Switzerland).
Cell proliferation assay

Cell proliferation was estimated using MTT assays. Two thousand cells were seeded in each well of 96-well plate and incubated for 24 h. MHCC-97L cells and Huh7 cells were

treated with a gradient of concentrations of SGI-1776 or PIM447 for 1, 3 and 5 days. DMSO was used as mock control. Cells culture medium and DMSO or PIM447 were replenished every other day. Fifty µg of MTT was added to each well and incubated at 37 °C for 2 h. The culture medium was removed and the lysis buffer (0.2 N hydrochloric acid: isopropanol, 1:4) was added to each well. Absorbance was read at 570 nm. Data were collected at day 0, 1, 3 and 5.
Apoptosis assays

Apoptosis was measured using Annexin V staining, performed as described previously [20]. HCC cells (1X105) were seeded in a 12-well plate and incubated for 24 h. MHCC- 97L and Huh7 cells were treated with SGI-1776 or PIM447, cisplatin (Pharmachemie BV, Haarlem, Netherlands), doxorubicin (Pharmachemie BV), or combined agents. All treated cells were incubated in normoxic or hypoxic condition for 24 h. Media and cells were collected and stained with FITC-conjugated Annexin V (BD Biosciences, San Jose, CA, USA) and propidium iodide (PI) staining solution (BD Biosciences). Samples were analysed by flow cytometry using a BD LSR Fortessa Analyzer (BD Biosciences) or BD FACSCalibur flow cytometer with CellQuest software (BD Biosciences). The results were analysed using FlowJo software (v7.6.1, Tree Star Inc., Ashland, OR, USA).
In vivo studies

Male athymic nude mice (BALB/CAnN-nu) of 6-8-weeks old were used for in vivo experiments. The mice were provided and maintained by the Laboratory Animal Unit

(LAU), HKU. PIM447 (purchased from MedChemExpress, Monmouth Junction, NJ, USA) were dissolved in 15% DMSO and 5% dextrose (Gibco). All animal experiments were carried out according to the Animals (Control of Experiments) Ordinance (Cap. 340) and approved by the Committee on the Use of Live Animals in Teaching and Research (CULATR) of HKU.
Subcutaneous injection model

Two million MHCC-97L cells were resuspended in serum free medium and mixed with matrigel (1:1, vol/vol; Corning Inc, Corning, NY, USA) and injected subcutaneously into the flank of nude mice. Tumour size was measured using a digital calliper. Tumour volume (mm3) was calculated by the equation: 1/2 x length x width2. Treatment started on day 13 after tumour cell inoculation. The mice were randomised into vehicle or treatment groups and treated with vehicle (5% dextrose with 15% DMSO) or PIM447 (formulated in 5% dextrose with 15% DMSO) at 60 mg/kg or 100 mg/kg, 3 days per week for two weeks. Vehicle and PIM447 were administered to mice by oral gavage. The mice were sacrificed after the end of treatment on day 27. For combined treatment of PIM447 with cisplatin or doxorubicin, treatment started on day 12 after tumour cell inoculation. The mice were randomised to vehicle and treatment groups as follows: (i) vehicle (5% dextrose with 15% DMSO and 0.9% saline), (ii) PIM447 at 60 mg/kg and 0.9% saline, (iii) cisplatin at 1 mg/kg and 5% dextrose with 15% DMSO, (iv) doxorubicin at 1 mg/kg and 5% dextrose with 15% DMSO, (v) combined PIM447 at 60 mg/kg and cisplatin at 1 mg/kg and (vi) combined PIM447 at 60 mg/kg and doxorubicin at 1 mg/kg. Treatment was given

to the mice for two days per week for two weeks. Dextrose with DMSO and PIM447 were administered by oral gavage. Saline, cisplatin, and doxorubicin were administered by intraperitoneal (IP) injection. The mice were sacrificed after the end of treatment on day 26.
Patient-derived tumour xenografts (PDTX) were established with HCC resection specimens from Queen Mary Hospital, Hong Kong [21]. For propagation, tumours harvested from mice were washed in F12/DMEM medium (Gibco) supplemented with 1% P/S and minced into approximately 1 mm3 pieces using surgical blades. Tumour pieces were homogenised using a gentleMACS dissociator (Miltenyi Biotec, Bergisch Gladbach, Germany) in the presence of Liberase at 10 μg/ml (Roche, Basel, Switzerland) and DNase I at 250 μg/ml (Roche). A single cell suspension was collected by filtering the mixture through a 100 μm nylon cell strainer. The cell suspension was pelleted by centrifugation for 3 min at 700 rpm. Cell viability and counting of the dissociated cells was assessed by trypan blue staining (Gibco) and a haemocytometer. One million tumour cells were resuspended in 50 µl F12/DMEM medium and mixed with 50 µl matrigel and injected subcutaneously into the flank of nude mice. Tumour growth was monitored, and treatment was started when tumours reached 4 mm in maximal dimension. Mice were randomised to vehicle and treatment groups. Mice were treated with vehicle or PIM447 (60 mg/kg) for 3 days per week for two weeks by oral gavage. The first dose of treatment was given on day 24 after tumour cell inoculation. The mice were sacrificed after the end of treatment on day 38. Tumours and major organs from all

animal experiments were collected and processed to FFPE sections for histological examination; part of the tumour tissue was snapped frozen.
Experimental details for western blotting analysis, immunohistochemical staining, Transwell migration and invasion assays, the tumoursphere formation assay, RNA- sequencing (RNA-seq) and RT-qPCR of tumour tissue from mouse models, and Statistical and bioinformatics analyses are available in supplementary materials, Supplementary materials and methods.
RESULTS

Determination of IC50 of PIM inhibitors in HCC cell lines

MTT assays were performed to determine the IC50 for each inhibitor in MHCC-97L and Huh7 HCC cell lines (Figure 1A). The IC50 of SGI-1776 in Huh7 and MHCC-97L was 7.6 µM and 13.0 µM, respectively. The IC50 of PIM447 in Huh7 and MHCC-97L was 9.3 µM and 22.2 µM, respectively. Huh7 showed a lower IC50 than MHCC-97L for inhibitors, suggesting that Huh7 is more sensitive to PIM inhibition than MHCC-97L, a metastatic HCC cell line. With reference to the IC50 results we examined the alteration of pAKT (Ser473), a downstream target of PIM1 as demonstrated by shRNA approach in our previous study [1], at a gradient concentration of PIM inhibitors. By western blotting, expression of pAKT decreased upon administration of both SGI-1776 and PIM447 in both cell lines (supplementary material, Figure S1A–D). As an expected finding, expression of PIM1 itself was not altered upon administration of PIM inhibitors (supplementary material, Figure S1E–H).

 
PIM inhibitors suppress proliferation, migration and invasion of HCC in vitro

The in vitro effects of PIM inhibitors on HCC proliferation, migration and invasion were investigated with Huh7 and MHCC-97L cell lines. Both SGI-1776 and PIM447 could significantly inhibit proliferation of Huh7 and MHCC-97L cells in a dose-dependent manner (Figure 1B). Cell migratory (supplementary material, Figure S2A,B) and invasive abilities (supplementary material, Figure S2C,D) of HCC cells were also reduced upon PIM inhibition in a dose-dependent manner. To ensure that such effects were not attributed to cell proliferation, the assays were also performed with addition of mitomycin C which halted cell proliferation. Consistent results were obtained, that both inhibitors suppress migration and invasion of HCC cells (supplementary material, Figure S3).
PIM inhibitors attenuate in vivo tumour growth of HCC

With the encouraging results from the in vitro assays we proceeded to in vivo experiments. The effect on in vivo tumour growth was studied by a subcutaneous injection mouse model. MHCC-97L cells were injected to the flanks of nude mice which were then randomised into two groups receiving PIM447 or vehicle control, respectively. At experimental endpoint, reduced tumour size was consistently observed in the PIM447 treated groups, and a higher dose (100 mg/kg) led to a more pronounced reduction of tumour growth (Figure 2A). One mouse from the MHCC-97L-bearing group treated with PIM447 at 100 mg/kg was sacrificed before the end of experiment due to pronounced weight loss. The findings on in vivo tumour growth were further supported by adopting

PDTX (PDTX#5) model that we previously developed from HCC patients [21]. PIM1 expression could be detected in the tumour cells by immunohistochemical staining (Figure 2B). Consistently, with the PDTX model we could observe attenuation of in vivo tumour growth upon PIM447 administration (Figure 2B). Histological examination of major organs from the MHCC-97L-bearing mice and PDTX including the heart, lungs, liver, spleen, and kidneys did not reveal significant necrosis. Mild non-specific inflammation in the liver was detected in one MHCC-97L xenograft from the 60 mg/kg treatment group.
PIM inhibition hampers chemoresistance and stemness in HCC

Hypoxia is a known mechanism inducing chemoresistance of cancer cells. A previous report by Chen et al illustrated the role of PIM1 in contributing to chemoresistance of pancreatic cancer cells [22]. To understand whether PIM inhibitors modulate chemosensitivity of HCC cells, we first resorted to compare the effect on apoptosis towards conventional chemotherapies cisplatin and doxorubicin with or without co- treatment with SGI-1776. Cisplatin and doxorubicin are the two most commonly administered chemotherapy via TACE for HCC patients. From the results, we found that SGI-1776 as a single agent could significantly increase apoptosis of HCC cells compared with DMSO control (Figure 3). Secondly, when SGI-1776 was given in combination with cisplatin (Figure 3A and supplementary material, Figure S4A) or doxorubicin (Figure 3B and supplementary material, Figure S4B), the apoptotic fraction of HCC cells was significantly augmented when compared with single treatment with cisplatin or

doxorubicin alone. As mentioned in the introduction, TACE typically triggers intra- tumoural hypoxia. Being a hypoxia-responsive molecular target, PIM1 is possibly upregulated in the tumour microenvironment after TACE. In this connection, we next performed the chemosensitivity experiments with PIM447 under normoxic (Figure 3C,D) and hypoxic (supplementary material, Figure S4C,D) conditions, respectively. Similar results were obtained. Use of PIM447 significantly increased apoptosis of HCC cells. When PIM447 was administered together with cisplatin or doxorubicin, the pro-apoptotic effect on HCC cells was enhanced compared with cisplatin or doxorubicin alone. To enrich the clinical significance of our findings, we evaluated the immunohistochemical expression of PIM1 in clinical HCC samples showing histological evidence of TACE effect. PIM1 staining was observed in peri-necrotic tumour cells in 6 of 8 (75%) cases, suggesting enhanced PIM1 expression in the TACE clinical subset (supplementary material, Figure S5A). In addition, immunohistochemical expression for PIM2 and PIM3 was assessed in the clinical cohort of our previous study on PIM1 [1]. Among the 52 available cases, none expressed PIM2 while 15 cases (28.8%) showed focal PIM3 expression in tumour cells (supplementary material, Figure S5B,C). Co-expression of PIM1 and PIM3 was observed in 7 cases. By Western blotting with HCC cell lines, PIM3 was endogenously expressed but not responsive to hypoxia (supplementary material, Figure S5D).
After that, the combinatorial effects of PIM inhibitors and conventional chemotherapeutic agents in vivo were studied by a subcutaneous injection assay. After inoculation of

MHCC-97L cells, the mice were randomised to vehicle control group, PIM447 group, cisplatin group, doxorubicin group, PIM447 + cisplatin group, and PIM447 + doxorubicin group. Our findings showed that PIM447 as a single agent could inhibit tumour growth when compared with control. Combined PIM447 + cisplatin and PIM447 + doxorubicin showed significantly more pronounced suppression in tumour growth compared with cisplatin alone and doxorubicin alone, respectively (Figure 4A). The change of body weight across the groups at end of treatment was similar (supplementary material, Table S1). Histological examination of major organs including the heart, lungs, liver, spleen, and kidneys from the mice did not reveal significant necrosis (supplementary material, Figure S6A). One mouse each from the PIM447 only, cisplatin only, doxorubicin only and PIM447 + doxorubicin groups showed focal non-specific inflammation in the liver (supplementary material, Figure S6B). Chemoresistance is a pivotal characteristic feature for cancer stemness. After showing that PIM inhibitors functionally lower chemoresistance of HCC cells, we next asked whether PIM inhibitors could modulate other cancer stemness phenotypes. To gain further insights on this question, we further assessed how the tumoursphere formation ability of HCC cells could be modulated by PIM inhibition. Results from tumoursphere formation assay showed a decrease in both number and size of tumourspheres upon administration of PIM447, suggestive of reduced self-renewal ability of HCC cells (Figure 4B).
Deciphering the downstream molecular events upon PIM inhibition in HCC

To obtain a more comprehensive picture on the genetic alterations elicited by PIM inhibitors in HCC cells, tumours harvested from the control and inhibitor (60 mg/kg) treatment groups in subcutaneous injection assay in Figure 2A (n=2 from each group) were subjected to RNA-seq analysis (Figure 5A and supplementary material, Table S2). After filtering, a total of 16,163 protein-coding genes were identified for downstream analysis. Based on the threshold FDR<0.05 and fold-change ≥1.2, 213 genes (1.32%) were defined as differential expressed genes (DEGs) which 103 genes were upregulated and 110 genes were downregulated in the PIM447-treated samples (Figure 5B,C and supplementary material, Table S3). Gene targets were further shortlisted based on their known biological significance in human cancers. The alteration of lysyl oxidase homolog 2 (LOXL2) and intercellular adhesion molecule 1 (ICAM1) was subsequently validated by RT-qPCR in tumour lysates collected from PDTX (Figure 5D). ICAM1 downregulation was also observed in tumourspheres derived from PIM inhibitor-treated MHCC-97L cells (supplementary material, Figure S7A). Following that, all DEGs were subjected to KEGG pathway enrichment analysis by DAVID software. Sixteen pathways were identified with the MAPK signalling pathway showing the largest number of DEG being involved (Figure 5E). The alteration of MAPK/ERK pathway was validated by western blotting with tumour lysates from the animal model, as illustrated by a downregulation of pERK1/2 in the PIM447 treatment group (Figure 5F). Similarly, pERK1/2 expression was reduced upon treatment of PIM inhibitors to HCC cells in vitro (supplementary material, Figure S7B).

 
DISCUSSION

In the present study, we have demonstrated the in vitro and in vivo preclinical efficacy of PIM inhibitors in HCC. PIM inhibitors exerted anti-tumour effects on growth and metastatic potential of HCC. In addition, we extended our investigation and explored how PIM inhibitors modulate chemosensitivity and other properties of cancer stemness. Underpinning the clinical relevance of our findings, PIM inhibitors are potent adjuvant agents to suppress the aggressive tumour phenotypes of HCC cells. Considering the suppression of self-renewal ability of HCC cells as demonstrated with the tumoursphere formation assay, PIM kinase inhibition could potentially reduce tumour relapse after surgical resection or local ablative therapy. In particular, PIM inhibitors may be able to counteract the hypoxia-induced adverse effects elicited by TACE. Practically, PIM inhibitors could be applied to augment the therapeutic effects of TACE as single-agent therapy or in combination therapy with cisplatin and doxorubicin.
As a matter of fact, PIM inhibitors were reported to exert anti-tumourigenic effect in conjunction with other targeted therapies for human cancers. For instance, PIM inhibitors were found to sensitise tumour cells to Bcl-2 inhibitor-induced apoptosis [23] and augment therapeutic response of myeloma cells to lenalidomide [24]. Simultaneous inhibition of PIM and PI3K hampered growth of chronic lymphocytic leukaemia cells [25]. A very recent study reported the efficacy of combined PIM/PI3K/mTOR targeting in neuroblastoma [26]. Another recent study reported the role of PIM kinases on anti-

vascular endothelial growth factor (VEGF) resistance of colon and pancreatic cancers, justifying the utility of PIM inhibitors and anti-VEGF therapy to augment treatment response [27]. In one recent report, PIM inhibitors enhanced the efficacy of anti- programmed death-1 (anti-PD1) therapy through potentiating T cell response in melanoma model [28]. Among the above, anti-VEGF and anti-PD1 antibodies have been frequently reported in clinical trials for HCC [29,30]. In view of the practical issue of drug resistance, PIM inhibitors could be a potential candidate in combinatorial therapies with these agents to maximise therapeutic benefits in HCC patients suffering from different stages of disease. In vivo studies using an immunocompetent mouse model will facilitate a more precise and in-depth characterisation of the effectors of PIM inhibitors in the modulation of immune microenvironment and anti-PD1 response.
Considering downstream effectors, PIM inhibitors had been reported to act through c- myc, cell cycle progression, anti-apoptotic and pro-survival proteins in previous studies on human cancers [24,31–33]. Given the expanding spectrum of the effects of PIM inhibitors such as on chemoresistance and stemness as demonstrated in our present study, additional downstream molecular mechanisms are possible. Results from our RNA-seq data highlighted the HIF signalling pathway and the central carbon metabolism pathway, which are in line with the findings from our previous report [1]. In addition, we have here validated the downregulation of MAPK/ERK pathway upon PIM inhibition in HCC cells. The MAPK/ERK pathway is a known deregulated pathway in human cancers and plays a role in cancer progression and chemoresistance [34–38]. We noted a

relatively less pronounced suppression of pERK1/2 upon PIM inhibitor treatment in vitro (supplementary material, Figure S7B), which might hint that other cell types in the tumour microenvironment are involved in the process. Furthermore, gene targets identified from our RNA-seq data, LOXL2 and ICAM1, may suggest a role of PIM inhibitors in modulating the tumour microenvironment to facilitate metastasis. LOXL2 is a collagen-modifying enzyme and a key extracellular matrix component mediating metastasis across different cancer types [39–42]. Lines of evidence suggest that LOXL2 is tightly associated with epithelial-mesenchymal transition in the metastasis process. In our previous study we demonstrated the upregulation of LOXL2 in tumour and serum samples of HCC patients. LOXL2 is regulated by HIF-1α and functions to modify the extracellular matrix to favour the formation of metastatic niche [43]. ICAM1 is a transmembrane protein that mediates cell-cell interaction upon exogenous stimuli. Expression of ICAM1 in HCC tumour from clinical samples has been reported and a high ICAM1 serum level in HCC patients was an independent poor prognosticator [44]. Using cell line model, ICAM1-positive HCC cells demonstrated increased stemness features in vitro [45]. In this regard, we could detect ICAM1 downregulation in tumourspheres from the PIM447-treated group (supplementary material, Figure S7A), suggesting that ICAM1 is possibly mediating stemness properties in HCC upon PIM inhibition. These altogether contribute to enrich our understanding on the different mechanistic actions of PIM inhibitors in HCC.
In summary, findings from our present study portray PIM kinase inhibition as a potential therapeutic strategy for HCC. In vivo anti-tumour effects were illustrated by cell line-

derived xenograft model and substantiated by PDTX model, shedding light on the possibility of personalised medicine by employing PIM inhibition. Evaluation on pharmacokinetics and systemic adverse effects in clinical studies will be needed to translate the results into patient care. At the stage of pre-clinical investigation, further studies focusing on patient-derived tissue models, such as xenografts and organoids, would provide insights on the selection of predicted responders to this emerging anti- cancer agent for HCC patients.
Acknowledgments

This work was supported by Health and Medical Research Fund, Food and Health Bureau, Hong Kong SAR (05161056). The authors thank Dr Lo-Kong Chan, Dr Carmen Leung, Ms Aki Tse, Ms Joyce Lee, Dr Puiyan Lee, and Faculty Core Facility, LKS Faculty of Medicine, The University of Hong Kong for their technical support.
Author contributions statement

Conception and design: R-CL, ML and K-KC. Data acquisition: ML, K-KC, CW, PW, KA, WD, R-CL, T-KL, C-CW and I-ON. Writing of manuscript: R-CL, ML, K-KC and WJK. Supervision of study: R-CL and WJK.
Data availability statement

The RNA-seq data is deposited in NCBI’s Gene Expression Omnibus (GEO) and is accessible through accession number GSE142871 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE142871).

 
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FIGURE LEGENDS

Figure 1. PIM inhibitors reduce cell proliferation of HCC in vitro. (A) IC50 determination of SGI-1776 (left panel) and PIM447 (right panel) in MHCC-97L and Huh7 cells (n=3). (B) Cell proliferation of MHCC-97L cells and Huh7 cells treated with SGI- 1776 (left panel) and PIM447 (right panel) (n=3). Mean ± SD. *p<0.05, **p<0.01, ***p<0.001
Figure 2. In vivo tumour growth of HCC is attenuated upon PIM inhibition. (A) In vivo effects on tumour growth of PIM447 (at 60 mg/kg and 100 mg/kg) by subcutaneous injection assay in nude mice using MHCC-97L cell line. Upper panel – experimental scheme; middle panel – image of subcutaneous tumours at experimental endpoint (left) and growth curves of tumours (right) (control: n=5; PIM447 at 60 mg/kg: n=5; PIM447 at 100 mg/kg: n=4); lower panel – representative H&E images of tumour and major organs from PIM447-treated mice (magnification x200). (B) In vivo effects on tumour growth in PDTX treated with PIM447 (at 60 mg/kg) by subcutaneous injection assay. Upper panel – experimental scheme; middle panel – image of subcutaneous tumour at experimental endpoint (left) and growth curves of tumours (right) (control: n=5; PIM447 at 60 mg/kg: n=5); lower panel – immunohistochemical staining for PIM1 in tumour section from PDTX (left) and representative H&E images of tumour and major organs from PIM447-treated mice (right) (magnification x200). Mean ± SD. *p<0.05, **p<0.01, ***p<0.001.
Figure 3. PIM inhibitors enhance chemosensitivity of HCC cells toward cisplatin and doxorubicin in vitro. Results of apoptosis assay with Annexin V/PI staining: (A) MHCC-97L treated with SGI-1776 (5 µM), cisplatin (3 µg/ml) or combined SGI-1776 and cisplatin (n=7). (B) MHCC-97L treated with SGI-1776 (5 µM), doxorubicin (0.5 µg/ml) or combined SGI-1776 and doxorubicin. (C) MHCC-97L treated with PIM447 (10 µM), cisplatin (3 µg/ml) or combined PIM447 and cisplatin (n=3). (D) MHCC-97L treated with PIM447 (10 µM), doxorubicin (0.5 µg/ml) or combined PIM447 and doxorubicin (n=3). Mean ± SD. ns not significant, *p<0.05, **p<0.01, ***p<0.001.

 

Figure 4. PIM inhibition hampers chemoresistance of HCC in vivo and suppresses tumoursphere formation. (A) In vivo effects on tumour growth by subcutaneous injection assay in nude mice treated with control (5% dextrose and DMSO), PIM447 (60 mg/kg), cisplatin (1mg/kg), doxorubicin (1 mg/kg), combined PIM447 (60 mg/kg) and cisplatin (1 mg/kg), or combined PIM447 (60 mg/kg) and doxorubicin (1 mg/kg). Left panel – image of subcutaneous tumours at experimental endpoint; right upper panel – growth curves of tumours (control: n=5; PIM447: n=4; cisplatin: n=5; doxorubicin n=5; PIM447+cisplatin: n=5; PIM447+doxorubicin: n=4); right lower panel – body weight of mice during treatment period. (B) Number and size of tumourspheres of Huh7 and MHCC-97L cells treated with PIM447 (6 µM or 10 µM, respectively) versus DMSO. Number of tumourspheres for size measurement: Huh7 (DMSO and PIM447), n=50; MHCC-97L (DMSO), n=50; MHCC-97L (PIM447), n=22). The experiment was performed twice; result from a representative experiment performed in triplicates was shown. Scale bar = 200 µm. Mean ± SD. *p<0.05, ***p<0.001, ****p<0.0001
Figure 5. Elucidation of downstream molecular mechanisms upon PIM inhibition in HCC (A) Simplified presentation of workflow on tissue sample preparation for RNA- seq. (B) Upper panel – Proportion of the DEG in the RNA-seq result; lower panel – volcano plot showing the global distribution of transcriptional changes by comparing PIM447-treated group versus control. DEG was defined as FDR < 0.05 and fold-change (PIM447/Control) ≥ 1.2. (C) Heatmap of the DEGs obtained from RNA-seq data. (D) Relative mRNA expression of LOXL2 and ICAM1 in the mouse PDTX tumour tissues samples. Lines represent mean values. (E) KEGG pathway enrichment analysis of the DEGs. (F) Immunoblot for pERK1/2 and ERK1/2 in tumour lysates from the MHCC-97L xenografts; each lane represents sample from one mouse. Signal intensity for pERK relative to ERK was quantified. Mean ± SD. *p<0.05.

 

 

 

 

 

 

 

 
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