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Published in final edited form as: Radiother Oncol. 2025 Feb 18;206:110794. doi: 10.1016/j.radonc.2025.110794

PIM kinase inhibition counters resistance to radiotherapy and chemotherapy in human prostate cancer

Anne Rajkumar-Calkins a,b,*, Vinay Sagar c,*, Jian Wang a, Shania Bailey d, Philip Anderson d, Sarki A Abdulkadir e, Austin N Kirschner a
PMCID: PMC12851849  NIHMSID: NIHMS2138831  PMID: 39978680

Abstract

Purpose:

PIM kinases are associated with treatment resistance and poor prognosis in prostate cancer through roles in DNA damage response, cellular metabolism, proliferation, and survival. We hypothesized PIM inhibition addresses treatment resistance to radiotherapy and docetaxel in prostate cancer.

Methods:

PIM inhibition in prostate cancer cell lines was examined by phosphorylated H2AX and colony formations assays. In normal and castrated mice with prostate tumor xenografts, tumor growth was monitored with daily oral PIM inhibition +/− fractionated radiotherapy (RT) or docetaxel. Radiotherapy was given 30 Gy in 15 treatments, mimicking clinical conventional daily treatment over 3 weeks in a translational murine model system.

Results:

PIM inhibition decreased radiotherapy-induced DNA-damage repair and decreased cell proliferation and survival. In mice, PIM inhibition increased the efficacy of both radiation and docetaxel to reduce tumor size in hormone-dependent and -independent xenografts. Xenografts showed altered gene expression changes, including downregulation of ribosomal pathways and upregulation of cardiomyocyte signaling pathways, due to PIM inhibition as analyzed by RNA-Seq. Immunostaining of multiple proteins, including COX-2 and MDM2, was altered by PIM inhibition.

Conclusions:

PIM inhibition addresses treatment resistance to docetaxel and radiotherapy in multiple prostate cancer models. Our data provide a strong rationale for testing PIM inhibitors in combination with standard therapies for treatment-resistant high-risk localized or metastatic prostate cancer in clinical trials.

Keywords: PIM kinase, prostate cancer, radiation, docetaxel, radiosensitization, model organisms/animal models of cancer

1. Introduction

Prostate cancer is a leading cause of cancer-related death in men with over 30,000 deaths per year in the United States, despite initially presenting with localized disease in 96% of cases (1). While over 90% of low-risk disease cases are cured by surgery or radiation, the best control rates in high-risk localized disease only approach 60–80%, depending on various risk factors and treatment modalities (2). Higher radiation doses improve outcomes (3–6), especially with modern image-guided techniques and simultaneous-integrated focal microboost design, but further escalation of whole gland dose is limited by toxicity (7–9). Androgen deprivation therapy (ADT) radiosensitizes prostate cells and is used in combination with radiation therapy for patients with high-risk localized disease (10–12). However, once cells metastasize and lose sensitivity to androgen deprivation (i.e., develop castration-resistance), chemotherapy is the primary treatment. Novel therapies to increase the efficacy of radiation and chemotherapy are needed to improve patient survival. We propose the inhibition of Proviral Integration site for Moloney murine leukemia virus (PIM) kinases (PIM1, PIM2, and PIM3) as a targeted therapy to enhance and address resistance to radiotherapy or chemotherapy for the treatment of prostate cancer.

PIM1 is highly expressed in high-risk prostate cancer and associated with aggressive behavior and poor prognosis (high Gleason grade), with tissue microarray staining highly positive in 64% of human prostate cancer specimens (13–15). PIM1 also synergizes with MYC to cause tumorigenesis in prostate cancer models, indicating its central role in aggressive tumor behavior (16, 17). The Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway upregulates the expression of PIM kinases, although a prostate cancer clinical trial for a JAK inhibitor lacked efficacy (NCT00638378). Instead, inhibiting PIM kinases in various malignancies is being investigated in several early phase clinical trials. Once expressed, PIM kinases are constitutively active serine/threonine kinases that phosphorylate numerous targets that activate the cell cycle, cellular metabolism, proliferation, survival, angiogenesis, and DNA damage response (18–21). Regarding the last, overexpression of PIM2 accelerated the removal of UV-induced DNA damage and correlated with reduced accumulation of phosphorylated H2AX (γH2AX) and increased Ataxia Telangiectasia Mutant protein kinase (ATM) activity (22). Furthermore, silencing of PIM3 increased γH2AX, reduced activation of ATM, and had radiosensitizing effects in pancreatic cancer cells (23).

In prostate cancer, PIM1 knockdown or inhibition reduced non-homologous end joining (NHEJ) by reducing ATM and DNA-PKcs activities, downregulating Ku70/86 expression, and reducing DNA-end binding activity of Ku70/86 (24). Recent studies show pan-PIM inhibitor AZD1208 has radiosensitizing properties with an androgen-sensitive, Myc-driven mouse prostate cancer cell line, Myc-CaP, both in vitro and in mouse xenografts (25). Here, we tested whether PIM inhibition via pan-PIM inhibitors PIM447 or AZD1208 increased the efficacy of radiotherapy in human-derived prostate cancer cell lines. We also tested preclinical models of human prostate cancer cell lines to investigate the combination of PIM inhibition with fractionated radiotherapy and docetaxel, a commonly used chemotherapy in prostate cancer.

2. Materials and Methods

Cell culture

LNCaP, 22Rv1, DU145, and PC3 cell lines were purchased from ATCC, authenticated, and confirmed Mycoplasma-free (IDEXX BioAnalytics) upon receipt and grown in RPMI (Corning) supplemented with 10% fetal bovine serum (Corning) and 1% penicillin-streptomycin (Gibco) at 37°C in the presence of 5% CO2.

Clonogenic Assay

This was performed as previously described (26), with radiation treatment delivered to tissue culture dishes via a Cs-137 chamber irradiator (Shepherd).

Tumor xenografts

All animal experiments were performed under protocol M-14–182 and M1700134 approved by the Institutional Animal Care and Use Committee to insure appropriate animal welfare. 6-week-old male nude mice (Foxn1nu/nu, Jackson Laboratory) were used for xenograft experiments. One million LNCaP or 22Rv1 cells were implanted subcutaneously into flanks for non-radiation experiments or into each hindlimb (just above the knee) for radiation experiments. Cells were implanted before castration for hormone-dependent LNCaP xenografts, but after castration for AR-v7-expressing hormone-independent 22Rv1 xenografts. When pre-specified graft sizes of 200–300 mm3 were reached, mice were randomized and treated once daily with either (1) 50mM acetate pH3 containing PIM447 25 mg/kg by oral gavage, with or without daily radiation treatments to the hindlimbs or (2) vehicle (0.5% Methocel E4M (Gallipot, St. Paul, MN, USA)/0.1% Tween 80), or AZD1208 30–45 mg/kg by oral gavage, with or without the addition of once-weekly docetaxel 6 mg/kg by intraperitoneal injection. Radiation was delivered via an orthovoltage 300 kVp/10 mA X-ray Pantak irradiator with custom lead blocks to irradiate the hindlimbs containing the xenografts while sparing the remaining mouse body. Mice were anesthetized by inhaled isoflurane during oral gavage and radiation treatments.

Immunoblotting and quantification of bands

Harvested tumor tissue was mechanically minced and lysed in RIPA buffer containing protease (Complete Mini, Sigma-Aldrich) and phosphatase inhibitors (PhosStop, Roche). Loading buffer containing beta-mercaptoethanol was added to the protein samples, which were heated to 95°C for 5 minutes, then separated by SDS-PAGE and transferred to membranes for immunoblotting, as described (27, 28). The following primary antibodies were used: rabbit anti-Androgen Receptor (AR) (PG-21 Millipore #06–680), rabbit anti-β-Actin (CST #5125), and mouse anti-phosphorylated H2AX (γH2AX) (Millipore #05–636). Following incubation with horseradish peroxidase-conjugated goat anti-rabbit/mouse (1:5000, Biorad) or goat anti-rat (1:5000, Santa Cruz) for 1 hour, signals were detected with Supersignal West Femto Maximum Sensitivity Substrate system (Pierce) followed by visualization with BioRad ChemiDoc Imager. Quantification analyses were performed by BioRad Image Lab software and ImageJ.

Immunohistochemistry / immunofluorescence

Tumors harvested within 3 hrs of final treatments were fixed in 10% zinc-formalin, paraffin embedded, sectioned, and processed for immunostaining. The following primary antibodies were used: rat anti-Ki67 (eBioscience #14-5698-80), rabbit anti-phosphorylated γH2AX (CST#9718), mouse anti-MDM2 (Santa Cruz #SC-965) and mouse anti-COX-2 (Cayman Chemical #160112). Slides were incubated with secondary antibodies labelled with Alexa Fluor 488 anti-rabbit (Thermo Scientific # A11008) and Alexa 594 anti-mouse (Molecular probes #A11005). Slides were counterstained with DAPI (Sigma #D-9542) and mounted with Prolong Gold Antifade reagent (Invitrogen/Molecular Probes #P36961). Immunofluorescence images were visualized using a fluorescent microscope or Leica A1R spectral confocal microscope. Quantification was performed by Nikon NIS-Elements software, counting γH2AX foci per cell nucleus. The γH2AX foci analysis is restricted to DAPI-positive nuclei.

RNA sequencing and analysis

Total RNA extraction from harvested 22Rv1 human castration-resistant prostate cancer xenografts was performed using the Qiagen RNeasy Purification kit, according to the manufacturer’s instructions.

RNA sequencing (RNA-Seq) was performed at HudsonAlpha Discovery (Huntsville, AL). Quality control was completed using FastQC version 0.11.5 (FastQC: a quality control tool for high throughput sequence data. Available online at: http://www.bioinformatics.babraham.ac.uk/projects/fastqc)(29), and trimming reads was performed using Trimm-o-Matic version 0.36 (30). 100 bp paired-end reads were mapped to the human transcriptome (Gencode version 29) (31) using Salmon version 0.14.1 (32). Differential expression analysis for AZD1208-treated and control 22Rv1 cells was performed using DESeq2 version 1.20 (33). Significantly upregulated and downregulated genes were subject to over-representation analysis using WebGestsalt (34, 35); individual proteins were queried in UniProtKB (36). Our full RNA analysis protocol and raw data is available upon request. Euler diagrams were generated using DeepVenn (37). Protein interaction web was produced by Lens for Enrichment and Network Studies (38) and is available for further exploration at https://hagrid.dbmi.pitt.edu/LENS/index.php/results/view/609071b7c0383.

Statistical analysis

For xenograft-based experiments, we used Power and Sample Size Program (39, 40) to calculate a sample size of at least 5 mice for 80% power to detect a change in tumor growth of at least 50 mm3 (25% of initial tumor volume) between different conditions. This calculation assumes a normal distribution with a standard deviation up to 25 mm3, which our experience with this tumor model is an appropriate assumption. Due to application of two independent treatments (radiation and PIM inhibition or docetaxel and PIM inhibition) in each experiment, immunohistochemical staining and tumor growth curves were analyzed by two-way ANOVA with Tukey’s test for multiple comparisons. p-values less than 0.05 were considered statistically significant (GraphPad Prism version 9.1.0 for Windows, GraphPad Software, www.graphpad.com).

3. Results

We investigated the effect of PIM inhibition on DNA damage response in castration-sensitive versus castration-resistant prostate cancer cells via analysis of γH2AX, a marker of DNA double-stranded breaks following radiation. Treatment with pan-PIM inhibitor PIM447 or DMSO control prior to radiation more than doubled expression of γH2AX 3 hours after radiation in castration-sensitive LNCaP cells (Figure 1A–B). By 6 hours after RT, γH2AX was roughly equivalent in both treatment conditions. Given prior results showing depletion of key actors of NHEJ following PIM inhibition (24), we also performed immunoblots of Ku70/86, but showed no alteration in total protein (Figure 1A). We found similar results with pan-PIM inhibitor AZD1208 (Supplementary Figure 1).

Figure 1. Immunoblots of prostate cancer cell lines treated with pan-PIM inhibitor PIM447.

Figure 1.

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(A-B) LNCap, (C-D) DU145, and (E-F) 22Rv1 cells were treated with DMSO or PIM447 (300 nM) for 1 hr, radiated, and incubated for indicated times. Total proteins were analyzed by Western blots. Bands are quantified using ImageJ software with correction for Actin loading control and normalization to DMSO control.

We tested whether castration-resistant prostate cancer cell lines DU145 and 22Rv1 also altered DNA damage response in response to PIM inhibition. If pretreated with PIM447, DU145 cells showed persistent γH2AX even 6 hours after radiation, with levels >10 times higher than irradiated cells without PIM447 (Figure 1C–D). In contrast, 22Rv1 cells showed less persistence of γH2AX, with similar reduction in maximum levels by 3 hours after treatment (Figure 1E–F). However, at both 1 and 3 hours after treatment, the total γH2AX was 39% higher in cells pretreated with PIM447. We found similar results with AZD1208 (Supplementary Figure 1). These results suggest that PIM inhibition acts as a radiosensitizer via DNA damage response in castration-sensitive and castration-resistant prostate cancer.

We next investigated whether PIM inhibition would affect cell proliferation in castration-resistant cell lines. At 2 Gy, following pre-treatment with PIM447, there was no significant reduction in colony survival in vitro for 22Rv1, DU145, and PC3 (Figure 2A; Supplementary Figure 2). Nevertheless, the numeric decrease in colony formation for 22Rv1 cells was selected as the most promising for evaluation of radiosensitization in vivo in a bifactorial trial design (Figure 2B). Starting at an average mouse tumor-graft volume of 461 mm3, PIM447 in combination with fractionated radiation resulted in no palpable tumor, compared to PIM447 alone or radiation treatment alone resulting in an average of 78% and 51% of initial volume, respectively.

Figure 2. Colony formation assay, mouse model, and tumor proliferation for prostate cancer cell lines treated with pan-PIM inhibitor PIM447.

Figure 2.

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(A) DU145, PC3, and 22Rv1 cells were treated with DMSO or PIM447 for 1 hr prior to radiation at indicated doses, with plating efficiency 43%, 68%, 83%, respectively. Colonies were counted 14 days after plating. (B) Treatment schematic for mice engrafted with 22Rv1 cells with subsequent treatment with PIM447 and/or radiation therapy. V = vehicle, P = PIM447, RT = 2 Gy radiation treatment each day. (C) Relative tumor size as measured by digital calipers twice weekly during experiment outlined in (B). Error bars represent SEM. Unpaired T-test, ** = p<0.01. (D) Immunofluorescence staining for Ki67 in harvested tumors from experiment outlined in B with (E) quantification of mean Ki67 staining by field-of-view (FoV). Two-way ANOVA, * = p<0.05, ** = p<0.01, *** = p<0.001, **** = p<0.0001.

We investigated biochemical markers of tumor response following treatment. Single treatment with radiation or PIM447 significantly lowered the average cell proliferation marker Ki67 by 52% and 21%, respectively (Figure 2D and 2E). Combination therapy with PIM447 and radiation drastically lowered Ki67 by 97%. Together, these results suggest that PIM inhibition enhances the effects of radiation to decrease cell proliferation and inhibit tumor growth.

We evaluated whether PIM inhibition alters the DNA damage response in vivo to corroborate altered γH2AX following radiation with PIM inhibition in cell lines (Figure 1). γH2AX expression was highest in 22Rv1 tumors treated with both PIM447 and radiation. In contrast to our cell line data, xenografts treated with PIM447 alone showed significant effects on γH2AX with increase in both number of cells with any γH2AX and number of γH2AX foci. Treatment with radiation alone resulted in the largest number of cells with >5 γH2AX foci. However, combination treatment with radiation and PIM447 resulted in the largest proportion of cells expressing >1 γH2AX focus (Figure 3A and 3B).

Figure 3. 22Rv1 Prostate tumor xenografts representative immunofluorescence staining and quantification.

Figure 3.

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(A-B) yH2AX, (C-D) COX-2, (E-F) MDM2 and (G) Western blotting of full length androgen receptor (AR-FL) and truncated, constitutively active androgen receptor (AR-v7) with actin loading control with (H) quantification by Image J with correction for actin loading control in harvested tumors from experiment outlined in Figure 2B. RFU = Relative fluorescence units. Two-way ANOVA, * = p<0.05, ** = p<0.01, *** = p<0.001, **** = p<0.0001.

PIM has multiple activities across cellular functions, including hypoxic and inflammatory response, survival, and transcription.(18) We studied proteins relevant to each of these processes in prostate cancer: COX-2, MDM2, and androgen receptor (AR). Treatment with PIM447, radiation alone, or combination therapy reduced COX-2 staining in the harvested xenografts. The greatest change from baseline expression was seen with PIM447 alone (Figure 3C and 3D).

PIM phosphorylates MDM2, preventing both degradation of MDM2 and p53.6 In 22Rv1 xenografts, treatment with PIM447 alone or radiation alone reduced MDM2 expression. With combination PIM447 and radiation, MDM2 expression profile more closely mimicked baseline, with overall increased MDM2 expression compared to monotherapies (Figure 3E and 3F).

In contrast to COX-2 and MDM2, expression of AR and constitutively active AR (AR-v7) was not altered by treatment with PIM447 alone. Radiation alone reduced expression of AR and AR-v7 by 71% and 59%. Adding PIM447 to radiation reduced AR and AR-v7 by 93% and 89% (Figure 3G and 3H). These results suggest an interaction between PIM inhibition and radiation influences AR expression.

Altogether, our data suggest PIM inhibition has effects on multiple different pathways that are relevant to inflammatory response and cell survival.

PIM inhibition downregulated expression and activity of NHEJ factors in PC3 cells and resulted in excess γH2AX after paclitaxel treatment in a prior study.(24) As prostate cancer can develop resistance to chemotherapy, we next tested whether PIM inhibition would synergize in vivo with docetaxel, a chemotherapeutic in the same class as paclitaxel.

LNCaP xenografts were treated with vehicle, docetaxel, AZD1208, or the combined drugs daily for 3 weeks following castration (Figure 4A). At the end of 3 weeks, mice who only underwent castration had 37% tumor size reduction, while combined therapy showed greatest effect with 85% size reduction. Treatment with AZD1208 alone was roughly equivalent to treatment with docetaxel alone, with tumor size reduction of roughly 72% and 70%, respectively (Figure 4B).

Figure 4. Prostate cancer mouse models treated by pan-PIM inhibitor AZD1208.

Figure 4.

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Treatment schematics for mice engrafted with androgen-sensitive LNCap cells (A) or 22Rv1 cells (C) with subsequent treatment with docetaxel 6 mg/kg once-weekly by intraperitoneal injection and/or AZD1208 30–45 mg/kg by daily oral gavage for 3 weeks. (B) Relative tumor size as measured by digital calipers twice weekly during experiment outlined in (A). Error bars are SEM. (D) Relative tumor size as measured by digital calipers twice weekly during experiment outlined in (A). Error bars are SEM. Two-way ANOVA, * = p<0.05, ** = p<0.01, *** = p<0.001, **** = p<0.0001.

We also queried if similar synergy occurred with castration-resistant prostate cancer. Castrated mice bearing 22Rv1 xenografts were randomized to the same treatments as above (Figure 4C). Like androgen-sensitive tumors, 22Rv1 tumors were smallest (60% reduction) with combination docetaxel/AZD1208 (Figure 4D). PIM inhibition or docetaxel alone stabilized tumor growth but did not cause significant regression. These results together demonstrate synergy of PIM inhibition with docetaxel in vivo in both castration-sensitive and castration-resistant models.

Our data showed dramatic differences in γH2AX between in vitro versus in vivo models, possibly due to cumulative effects of daily radiation and PIM inhibition over several weeks in the mouse model. We investigated mRNA expression after mice were treated with AZD1208 daily for 3 weeks. Fifteen genes were statistically significantly downregulated, of which 13 are translated into proteins (Supplementary Table 1). Nine genes were statistically significantly upregulated, of which 7 are translated into proteins (Supplementary Table 2). We summarize the functional significance of these proteins in Supplementary Tables 1 and 2 and grouped them by involvement in pathways as specified by Gene Ontology (41, 42) (Figure 5). We produced a protein interaction web for further exploration (Supplementary Figure 3; available at https://hagrid.dbmi.pitt.edu/LENS/index.php/results/view/609071b7c0383). The protein web verified that candidate proteins were more closely connected to PIM1 or PIM2 than to random proteins, with average 4.8 connections from altered protein to PIM1 or PIM2, versus 14.6 connections between random proteins to PIM1 or PIM2. This provides further evidence that the protein alterations were due to PIM inhibition rather than random chance.

Figure 5. Protein pathways from RNA-seq analysis influenced by pan-PIM kinase inhibitor AZD1208.

Figure 5.

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RNA-seq analysis for 22Rv1 human prostate tumor cells harvested following treatment with AZD1208 daily for 3 weeks normalized to treatment with vehicle, showing the individual proteins which are statistically significantly upregulated or downregulated in response to AZD1208. Proteins are grouped via pathways in which they are involved as classified by Gene Ontology.

Gene Ontology functional analysis (34) of the mRNA data showed cardiomyocyte signaling, vascular smooth muscle contraction, and apelin signaling among the top upregulated pathways, while the ribosome pathway was downregulated (Supplementary Figure 4). In contrast to individual protein analysis that were significantly impacted by PIM inhibition, no entire pathways met a threshold for significance.

4. Discussion

We demonstrated that PIM inhibition addresses resistance to radiotherapy and chemotherapy for prostate cancer treatment through radiosensitization and synergistic activity with docetaxel. Data indicates that PIM inhibition affects multiple factors leading to these endpoints, including fundamental DNA damage response/repair (altered γH2AX expression) and inflammatory pathways involving COX-2 and MDM2.

First, tumors cotreated with PIM447 and radiation had the highest γH2AX expression. This correlates with other studies investigating the interactions between PIM and elements of DNA damage and repair (13, 14, 22–24, 43). In our experiments, the interaction between PIM inhibition and radiation was stronger in vivo than in vitro. This may be due to additive DNA damage via fractionated radiotherapy with daily PIM inhibition over three weeks (mimicking clinical treatment with fractionated radiotherapy), as well as other biological factors, such as changes in the tumor microenvironment. This is encouraging for translation of PIM inhibition into clinical use as a radiosensitizer, since our fractionated radiation scheme was specifically chosen to closely approximate clinical treatments for prostate cancer, traditionally featuring 2 Gy per fraction. PIM inhibition enhances the effectiveness of radiation treatment and results in radiosensitization in prostate cancer cells.

We evaluated the potential anti-inflammatory role of PIM. COX-2 produces prostaglandin, an inflammatory protein. Other work has demonstrated that PIM inhibition reduces expression of proinflammatory cytokines and chemokines (44). When tumors were treated with both PIM inhibition and radiation, the COX-2 expression profile resembled tumors treated with radiation alone. The damage caused by radiation appears to override the anti-inflammatory effect of PIM inhibition, although the tumor-immune microenvironment was not studied to detail possible changes in immune cell infiltration or other inflammatory markers.

We also evaluated the expression of MDM2 following treatment with PIM447 and radiation. Overexpression of MDM2 is associated with increased distant metastases and cancer-specific mortality (45, 46). MDM2 ubiquitylates p53, resulting in proteasomal degradation. Meanwhile, p53 increases transcription of MDM2, forming a negative feedback loop between MDM2 and p53. PIM phosphorylates MDM2, resulting in its degradation (47). Prior work demonstrated that inhibition of MDM2 had radiosensitization effects in vitro and in vivo in a p53-dependent manner (48). In our study, three-week treatment with radiation or PIM447 monotherapies reduced MDM2 expression. However, MDM2 typically increases in surviving cells following radiation (49). More similarly to our results, another study with cells exposed to fractionated radiation also showed reduction in MDM2, with concomitant p53 accumulation (50). Despite having higher levels of p53, the cells with low MDM2 were radioresistant after repeated exposure to radiation. In our study, combination treatment rendered the MDM2 expression more similar to baseline. We suspect that re-establishing the oscillating MDM2-p53 negative feedback loop by preventing initial overaccumulation of MDM2 and p53 is another factor leading to the synergistic effect of the combination treatment. Therefore, PIM inhibition modulates response to radiation therapy via multiple pathways.

While our results did not show substantial change in AR with PIM inhibition, PIM inhibition did improve tumor control in combination with androgen deprivation for hormone-sensitive tumors. For patients with metastatic, androgen-sensitive disease at diagnosis, PIM inhibition could be combined with ADT to yield superior results, as demonstrated in our preclinical model. We also showed that PIM inhibition improved tumor control in castration-resistant prostate cancer cell line 22Rv1 and synergizes with docetaxel treatment in prostate cancer mouse models, which could be translated to clinical use in combination with either radiation for local control or docetaxel for metastatic disease.

Regarding gene expression analysis following in vivo treatment with PIM inhibitor, our RNA-seq analysis showed statistically significant up- and downregulation of multiple proteins (Supplementary Tables 1–2, Figure 5, Supplementary Figure 4). On broader whole-pathway analysis, no statistically significant relationships were found. Despite a lack of significance, several important connections between the pathway analysis data and other research exist. First, a relationship between PIM and ribosome regulation was previously established. Following ribosomal stress, PIM1 was destabilized and degraded, leading to cell cycle arrest (19, 51). Second, a relationship between PIM inhibition and cardiomyocyte signaling was implicated in the RNA-seq data. A trial of PIM inhibitor SGI-1776 was terminated prematurely due to unanticipated cardiac toxicity (NCT00848601) though no trials have noted cardiac toxicity with either PIM447 or AZD1208. Third, pathways in retinol metabolism and glucoronate interconversions were downregulated following PIM inhibition. This may be related to induction of cytochrome P450 enzymes (52, 53), which was also seen clinically with AZD1208 and resulted in limited ability to reach steady state in a phase 1 clinical trial due to increased drug clearance (54).

Our data adds to growing knowledge surrounding PIM kinases’ roles in treatment-resistant cancer. Within prostate cancer, PIM expression was previously correlated with aggressiveness, tumorigenicity in combination with MYC, and resistance to ADT and docetaxel (17). Overall, our results significantly add to the understanding of PIM inhibition in prostate cancer as a radiosensitizer and chemosensitizer. The observed interactions between PIM inhibition and radiation or docetaxel are multifaceted and complex. This work highlights the influence of PIM inhibition on DNA damage response and inflammatory pathways. Seven clinical trials with PIM inhibitors, PIM447 and AZD1208, have been registered in ClinicalTrials.gov, including two studies directed at solid tumors (Supplementary Table 3). Trials using PIM447 have been completed in AML, MDS, myelofibrosis, and multiple myeloma. None of the trials involving PIM447 showed unusual toxicities. With the promising data shown herein that PIM inhibition addresses treatment resistance in prostate cancer, PIM447 should move forward into clinical trials for patients with prostate cancer with aggressive local disease or metastatic disease, receiving radiotherapy and/or docetaxel.

Supplementary Material

Supplementary Materials

Supplementary Figure 1. Immunoblots of prostate cancer cell lines treated with pan-PIM inhibitor AZD1208. (A) LNCap, (B) DU145, and (C) PC3 cells were treated with DMSO or AZD1208 (1 μM, or where indicated at 5 μM) for 1 hr, irradiated with 4 Gy, and incubated for 6 hr (or 24 hr where indicated). Total proteins were analyzed by Western blots. Fold-change is noted based on bands quantification using ImageJ software with correction for Actin loading control and normalization to DMSO control. There is relatively more γH2AX expression in the AZD1208 or combination AZD1208+4Gy treatment groups compared to radiation alone or DMSO for all 3 cell lines, suggesting that AZD1208 reduces the efficiency of DNA damage repair allowing increased amounts and more persistence of γH2AX.

Supplementary Figure 2. Colony formation assay for prostate cancer cell lines treated by pan-PIM kinase inhibitor PIM447. DU145, PC3, and 22Rv1 cells were plated at the indicated cell densities and treated with DMSO or PIM447 (300 nM) for 1 hour prior to radiation at indicated doses. Colonies were stained and counted 14 days after plating.

Supplementary Figure 3. Protein interaction web for RNA-seq analysis. RNA-seq analysis of 22Rv1 tumor cells harvested following treatment with AZD1208 (dose) daily for 3 weeks normalized to treatment with vehicle, showing the individual proteins with altered expression (labeled circles) in response to AZD1208 dosing, with protein interaction network as defined and depicted by Lens for Enrichment and Network Studies (PMC4682415), with PIM1 and PIM2 as target proteins. Average path length from altered protein to PIM1 or PIM2 = 4.8 connections; altered protein to random protein = 11.1 connections; random protein to PIM1 or PIM2 = 14.6 connections; random protein to random protein = 14.4 connections.

Supplementary Figure 4. Upregulated and downregulated gene pathways in prostate cancer cells treated by pan-PIM kinase inhibitor AZD1208. RNA-Seq analysis of 22Rv1 tumor cells harvested following treatment with AZD1208 (dose) daily for 3 weeks normalized to treatment with vehicle, showing the 10 pathways with either highest or lowest enrichment scores.

Supplementary Figure 5. PIM kinase interaction network showing direct effects on multiple key cellular pathways, including cell cycle, cell survival, and gene expression. This project illustrates the influence of PIM kinase on radiosensitivity as shown by clonogenic assays and in vivo tumor grafts in mice, likely through reduced DNA damage repair shown by persistence of γH2AX, as well as numerous other pathways potentially altered.

[Acknowledgements]

We appreciate support from the Vanderbilt Clinical Oncology Research Career Development Program via NCI/NIH award 5K12CA090625-18. Although AstraZeneca and Novartis had no direct guidance on the research project and did not contribute to this manuscript, we appreciate supply of drug material for this project.

[Funding Statement]

This work was supported by the Radiological Society of North America (RSNA) Research Scholar Grant and the National Institutes of Health / National Cancer Institute grant 5K12CA090625-18 from the Vanderbilt Clinical Oncology Research Career Development Program to A.N. Kirschner.

Footnotes

Conflict of Interest:

A.N. Kirschner and S. A. Abdulkadir report Materials Transfer Agreements with AstraZeneca and Novartis. The other authors declare no potential conflicts of interest.

[Data Availability Statement for this Work]

Any data not already included in this article and associated supplementary materials will be shared on request to the corresponding author.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Materials

Supplementary Figure 1. Immunoblots of prostate cancer cell lines treated with pan-PIM inhibitor AZD1208. (A) LNCap, (B) DU145, and (C) PC3 cells were treated with DMSO or AZD1208 (1 μM, or where indicated at 5 μM) for 1 hr, irradiated with 4 Gy, and incubated for 6 hr (or 24 hr where indicated). Total proteins were analyzed by Western blots. Fold-change is noted based on bands quantification using ImageJ software with correction for Actin loading control and normalization to DMSO control. There is relatively more γH2AX expression in the AZD1208 or combination AZD1208+4Gy treatment groups compared to radiation alone or DMSO for all 3 cell lines, suggesting that AZD1208 reduces the efficiency of DNA damage repair allowing increased amounts and more persistence of γH2AX.

Supplementary Figure 2. Colony formation assay for prostate cancer cell lines treated by pan-PIM kinase inhibitor PIM447. DU145, PC3, and 22Rv1 cells were plated at the indicated cell densities and treated with DMSO or PIM447 (300 nM) for 1 hour prior to radiation at indicated doses. Colonies were stained and counted 14 days after plating.

Supplementary Figure 3. Protein interaction web for RNA-seq analysis. RNA-seq analysis of 22Rv1 tumor cells harvested following treatment with AZD1208 (dose) daily for 3 weeks normalized to treatment with vehicle, showing the individual proteins with altered expression (labeled circles) in response to AZD1208 dosing, with protein interaction network as defined and depicted by Lens for Enrichment and Network Studies (PMC4682415), with PIM1 and PIM2 as target proteins. Average path length from altered protein to PIM1 or PIM2 = 4.8 connections; altered protein to random protein = 11.1 connections; random protein to PIM1 or PIM2 = 14.6 connections; random protein to random protein = 14.4 connections.

Supplementary Figure 4. Upregulated and downregulated gene pathways in prostate cancer cells treated by pan-PIM kinase inhibitor AZD1208. RNA-Seq analysis of 22Rv1 tumor cells harvested following treatment with AZD1208 (dose) daily for 3 weeks normalized to treatment with vehicle, showing the 10 pathways with either highest or lowest enrichment scores.

Supplementary Figure 5. PIM kinase interaction network showing direct effects on multiple key cellular pathways, including cell cycle, cell survival, and gene expression. This project illustrates the influence of PIM kinase on radiosensitivity as shown by clonogenic assays and in vivo tumor grafts in mice, likely through reduced DNA damage repair shown by persistence of γH2AX, as well as numerous other pathways potentially altered.

NIHMS2138831-supplement-Supplementary_Materials.pdf (1.1MB, pdf)

Data Availability Statement

Any data not already included in this article and associated supplementary materials will be shared on request to the corresponding author.

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