Kdm6a and Kdm6b: Altered expression in malignant pleural mesothelioma
Abstract
Malignant pleural mesothelioma (MPM) is an uncommon yet highly aggressive form of cancer originating from the lining of the lungs, known as the pleura. Its primary etiology is unequivocally linked to prior exposure to asbestos fibers. The current standard of care for patients diagnosed with MPM typically involves a chemotherapy regimen combining cisplatin with either pemetrexed or, alternatively, raltitrexed. Despite these therapeutic interventions, the prognosis for most patients remains grim, with the majority succumbing to the disease within 24 months of diagnosis. This stark reality underscores the critical and urgent need for the identification and development of novel, more effective therapeutic strategies for this devastating cancer.
Inflammation is increasingly recognized as a pivotal and integral element in the complex pathogenesis of MPM, playing a significant role in its initiation and progression. In line with this understanding, recent investigations have highlighted the Kdm6 family of histone demethylases, specifically Kdm6a and Kdm6b, as crucial regulators involved in various inflammatory processes. Given their established roles in inflammation, these genes represent intriguing potential novel candidate targets for therapeutic intervention in MPM. To explore this possibility, we employed reverse transcription-polymerase chain reaction (RT-PCR) to meticulously examine the expression profiles of Kdm6a and Kdm6b. This analysis was conducted across a panel of established MPM cell lines and, critically, in a cohort of snap-frozen patient tissue samples obtained during surgery. This patient cohort encompassed a range of histologies, including benign pleural lesions, as well as the epithelial, biphasic, and sarcomatoid subtypes of MPM, providing a comprehensive representation of the disease. Our findings revealed that both Kdm6a and Kdm6b messenger RNA (mRNA) levels were significantly overexpressed in MPM samples when compared to benign controls, suggesting a potential oncogenic role.
However, subsequent experimental investigations aimed at assessing the therapeutic utility of targeting Kdm6a/b using a specific small molecule inhibitor, GSK-J4, yielded unexpected and clinically challenging results. Our studies demonstrated that the anti-proliferative activity of GSK-J4 was unexpectedly higher at lower drug concentrations in cell lines derived from normal mesothelial cells compared to those originating from malignant mesothelioma cells. This differential sensitivity suggests that normal cells might be more susceptible to the inhibitor’s effects than cancer cells, raising concerns about potential off-target toxicity in a clinical setting. Furthermore, treatments with GSK-J4 were found to be paradoxically associated with the induction of apoptosis in a manner that appeared to also involve an increased expression of pro-inflammatory cytokines. This observation raises a significant clinical concern, as the induction of a robust pro-inflammatory cytokine response, often referred to as a “cytokine storm,” could exacerbate the inflammatory component of MPM and lead to severe systemic toxicity. As such, our results, despite the initial finding of Kdm6 family overexpression in MPM, collectively demonstrate that these genes may not be suitable candidates for targeted therapy with inhibitors like GSK-J4, primarily due to their potential to elicit an adverse cytokine storm and their undesirable differential toxicity profile.
Introduction
Malignant pleural mesothelioma (MPM) is an exceptionally aggressive and often fatal inflammatory cancer, profoundly associated with prior exposure to asbestos. This formidable malignancy originates from the mesothelial cells that line the pleural, peritoneal, and pericardial cavities. Characteristically, MPM is marked by an extended latency period between asbestos exposure and disease onset, often spanning decades. Consequently, the vast majority of patients present at an advanced stage of the disease, leading to a consistently poor prognosis with a median survival time typically ranging from 6 to 12 months. Current epidemiological estimates highlight the global burden of this disease, suggesting that approximately 43,000 individuals succumb to MPM each year, corresponding to a mortality rate of around 6.2 per million population. While numerous countries have progressively banned the use of asbestos, and preliminary data may suggest a potential decline in mesothelioma incidence in these regions, there remains some debate and contention surrounding the accuracy of these long-term projections. Furthermore, the long-term health risks posed to younger individuals who have been exposed to asbestos, still prevalent in many existing buildings, are not yet fully quantified but could be substantial. Nevertheless, it is broadly accepted that, despite concerted efforts to abate asbestos, MPM mortality rates are projected to continue their upward trajectory, increasing by 5-10% annually in most industrialized nations over the next two to three decades. This global challenge is compounded by the fact that asbestos consumption continues to escalate in emerging economies, such as Brazil, which forebodes a predicted new wave of MPM cases in these regions.
The current standard of care for MPM patients involves a combination chemotherapy regimen, typically comprising pemetrexed (or raltitrexed) in conjunction with cisplatin. Unfortunately, this conventional treatment approach yields a patient response rate that is disappointingly low, ranging only between 23% and 40%, and critically, it is not curative. This significant therapeutic gap underscores an urgent and critical imperative to identify and develop novel therapeutic avenues and strategies for this devastating disease, with the overarching aim of substantially improving patient outcomes and quality of life.
Dysregulation of epigenetic transcriptional control mechanisms, particularly alterations in promoter DNA methylation patterns and aberrant histone post-translational modifications, is a well-established and pervasive feature observed across a wide spectrum of human malignancies, including mesothelioma. Recognizing this fundamental aspect of cancer biology, the pharmaceutical industry has dedicated substantial resources and efforts towards developing pharmacological agents specifically designed to target the intricate components of the epigenetic machinery. The initial promise of targeting the epigenetic machinery in MPM emerged from early data from a phase I clinical trial investigating vorinostat, a histone deacetylase (HDAC) inhibitor. In this trial, a modest but encouraging observation was made: 4 out of 13 patients (30%) with MPM who received vorinostat achieved stable disease for a duration exceeding 4 months, with two unconfirmed partial responses. However, despite these initial glimmering hopes, when vorinostat progressed to a phase III trial (VANTAGE 014) as a second-line or third-line therapy in a large cohort of 660 pretreated advanced MPM patients, it unfortunately failed to demonstrate any improvement in overall survival. Consequently, the consensus recommendation was that vorinostat was unsuitable as a therapeutic option for MPM patients in this setting.
Despite the setback with vorinostat, evidence continues to accumulate, strongly suggesting that targeting the epigenetic machinery may indeed represent a viable and promising therapeutic option in MPM. For instance, our own recent investigations have demonstrated that KAT5 (lysine acetyltransferase 5), another critical epigenetic regulator, was significantly overexpressed in MPM, positioning it as a suitable and promising candidate for therapeutic intervention. Furthermore, the potential significance of targeting the epigenetic machinery in MPM has been underscored by compelling data emerging from studies focusing on MPM patients who harbor mutations in the BRCA1-associated protein 1 (BAP1) gene. BAP1 plays indispensable roles in chromatin remodeling, a process vital for gene regulation. Intriguingly, the loss of functional BAP1 in MPM cells has now been mechanistically linked to altered sensitivity to HDAC inhibitors, mediated through the specific regulation of HDAC2, an important member of the HDAC family.
In a recent comprehensive analysis of MPM, a distinct subset of genes was identified as being silenced through the epigenetic modification known as histone H3 lysine 27 trimethylation (H3K27me3). This specific chromatin mark is most frequently found at or in close proximity to the promoter regions of genes that are transcriptionally silent. The Polycomb repressive complex 2 (PRC2) is the enzymatic machinery responsible for catalyzing the trimethylation of Histone H3 at lysine 27 (H3K27me2/3). This complex contains the lysine methyltransferase EZH2 as a key catalytic subunit. Consequently, targeting this complex with small molecule inhibitors, such as the methyltransferase inhibitor DZNep, has been shown to represent a potential therapeutic option in MPM. Of particular note, sensitivity to DZNep has been specifically linked to a subset of MPM patients who carry mutations in the BAP1 gene. Building upon these promising preclinical observations, a phase II clinical trial investigating the EZH2 inhibitor tazmetostat (EPZ-6438) in mesothelioma is currently underway, highlighting the active pursuit of epigenetic therapies in this disease.
The enzymes responsible for demethylating H3K27me3 have been collectively identified as members of the Kdm6 family. This family notably includes Kdm6a (also known as Utx) and Kdm6b (also known as JMJD3), both of which have been rigorously demonstrated to catalyze the demethylation of H3K27me3. The precise roles of these histone demethylases in the complex landscape of cancer are, however, less uniformly defined. There are numerous instances in various cancers where a loss of expression of these demethylases is observed, often through genetic mutation, suggesting a tumor-suppressor role. Conversely, many other studies have reported the overexpression of these proteins in different cancer types, implying a potential oncogenic function. Given this dual nature, specific inhibitors for the Kdm6 family, such as GSK-J1 and GSK-J4, have now been successfully developed, offering tools to probe their therapeutic potential.
Considering the pronounced pro-inflammatory nature characteristic of MPM, coupled with the established therapeutic potential of targeting the PRC2 complex, it is plausible that the Kdm6 family of demethylases could also represent a promising candidate for therapeutic intervention. In the present study, we systematically assessed the expression levels of Kdm6 family members in MPM and rigorously investigated the effects of Kdm6 inhibition on MPM cellular health and viability. Our findings, however, provide a nuanced perspective, indicating that while the Kdm6 family is indeed overexpressed in MPM, selective inhibition of this family may not be a suitable therapeutic option for MPM patients. This conclusion is primarily driven by observations suggesting potential adverse effects, including the elicitation of a cytokine storm, which could exacerbate the disease.
Materials and Methods
Primary Tumor Samples
Surgical specimens were ethically obtained as discarded tumor samples from patients who had undergone extended pleuro-pneumonectomy at Glenfield Hospital in Leicester, UK. To provide crucial control comparisons, benign specimens were acquired from patients with no history or diagnosis of malignant pleural mesothelioma. Prior to sample collection, full informed consent was meticulously obtained from each participating patient, and the entire study protocol adhered to stringent ethical guidelines, receiving formal approval from the relevant Hospital Ethics Committee (Leicestershire REC references 6742 and 6948). The collected sample cohort encompassed a diverse range of histologies, including 5 benign lesions and 17 malignant pleural mesothelioma (MPM) samples. Within the MPM group, the breakdown of histologies was as follows: 7 epithelial subtypes, 4 sarcomatoid subtypes, and 6 biphasic subtypes, ensuring a comprehensive representation of MPM variants. Detailed demographic and pathological information for these samples is meticulously provided in Table I. All research activities involving these precious samples were conducted at St. James’s Hospital, under the explicit approval of the SJH/AMNCH research ethics committee (Approval number 041017/8704).
Cell Culture
All malignant pleural mesothelioma (MPM) cell lines utilized in this study were meticulously maintained under highly controlled environmental conditions: a humidified atmosphere containing 5% v/v carbon dioxide (CO2) in appropriate growth media, which was consistently supplemented with 10% v/v fetal bovine serum (FBS) and a standard antibiotic cocktail of penicillin-streptomycin (500 U/ml) to prevent bacterial contamination. All cell culture reagents were of high quality and procured from Lonza (Walkersville, MD, USA). The study employed a diverse panel of cell lines, comprising both normal mesothelial cell lines (LP9 and Met5A) and a comprehensive collection of malignant MPM cell lines. This extensive malignant panel included NCI-H2596, MMP, MMB, NCI-H2052, NCI-H28, Ju77, One58, RS-5, DM-3, ACC-MESO-1, ACC-MESO-4, Y-MESO-8D, Y-MESO-9, Y-MESO-12, Y-MESO-14, NCI-H226, and REN. Several of these specialized cell lines were generously provided by collaborating research institutions: ACC-MESO-1, ACC-MESO-4, Y-MESO-8D, Y-MESO-9, Y-MESO-12, and Y-MESO-14 were kindly supplied by Yoshitaka Sekido from the Aichi Cancer Center Research Institute, Nagoya, Japan. NCI-H2052, One-58, and JU77 cells were obtained from Duncan Stewart at the University of Leicester, UK. The REN and NCI-H226 cell lines were provided by Dean Fennell from Queen’s University, Belfast, Northern Ireland. NCI-H28 and the immortalized non-tumorigenic mesothelial cell line, Met-5A, were purchased from the ATCC (LGC Promochem, Teddington, UK). Lastly, DM-3 and RS-5 cell lines were acquired from the Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ). To ensure the authenticity and integrity of the cell lines used, the NCI-H226 cell line underwent rigorous authentication through STR-profiling (Short Tandem Repeat profiling) conducted by Source Bioscience, Nottingham, UK.
Total RNA Isolation and RT-PCR Amplification
Total RNA was meticulously extracted from both primary tumor samples and cell lines using TRI reagent, strictly adhering to the manufacturer’s prescribed instructions. This method ensures efficient and high-quality RNA isolation. For cDNA synthesis, a precise amount of total RNA, specifically 250 ng for primary tumors and 1000 ng (or 1 µg) for cell lines, was first pre-treated by enzymatic digestion with RQ1 DNase (Promega, Madison, WI, USA) to eliminate any contaminating genomic DNA. Following the successful inactivation of the RQ1 DNase, the treated messenger RNA (mRNA) was subsequently reverse-transcribed into complementary DNA (cDNA) using RevertAid reverse transcriptase (Thermo Fisher Scientific) in conjunction with random hexamers (Roche), strictly following the manufacturer’s guidelines. The synthesized cDNA was then stored at -20˚C to maintain its integrity until further use. The expression levels of Kdm6a, Kdm6b, and the housekeeping gene 18S rRNA were subsequently quantified using RT-PCR (Reverse Transcription Polymerase Chain Reaction). The specific primer sequences utilized for these amplifications are precisely outlined in Table II. The PCR cycling conditions were optimized as follows: an initial denaturation step at 95˚C for 5 minutes, followed by 35 cycles consisting of 1 minute at 95˚C (denaturation), 1 minute at 58˚C (annealing), and 1 minute at 72˚C (extension), concluding with a final extension step at 72˚C for 10 minutes. The resulting PCR products were then separated and visualized by electrophoresis on a 2% agarose gel. For quantitative analysis, product quantification was performed using TINA 2.09c densitometry software (Raytest, Isotopenmeßgeräte GmbH, Straubenhardt, Germany). The mRNA expression levels of the experimental genes were normalized to either 18S rRNA or β-actin controls, both of which serve as reliable internal loading controls, and were expressed as a ratio of experimental gene expression to control gene expression, allowing for comparative analysis.
Drug Treatment and Cellular Viability Assays
The specific small molecule inhibitor, GSK-J4, was procured from Selleck (St. Louis, MO, USA; catalog number O7753). Prior to use, GSK-J4 was precisely dissolved in dimethyl sulfoxide (DMSO) to achieve a stock concentration of 1 M. For all drug treatment experiments, cells were first subjected to a period of serum starvation (0.5% v/v FBS) for 24 hours. This starvation period was implemented to synchronize cells and reduce background proliferative signals. Following serum starvation, either the drug (GSK-J4) or an equivalent volume of vehicle (DMSO) was added to the cell cultures, and the cells were subsequently incubated for an additional 48 hours to allow for the inhibitor’s effects to manifest. Cellular viability, a critical endpoint for assessing drug efficacy, was then comprehensively assessed using two distinct methodologies: either a resazurin reduction assay, a colorimetric method previously described and validated, or a BrdU ELISA (Enzyme-Linked Immunosorbent Assay) supplied by Roche Diagnostics, Ltd., Sussex, UK, strictly adhering to the manufacturer’s instructions. These complementary assays provided robust and reliable measures of cell proliferation and metabolic activity.
Cellular Apoptosis (FACS)
To quantitatively assess the induction of cellular apoptosis in response to GSK-J4 treatment, NCI-H226 cells were initially seeded into 6-well plates at a density of 1×10^5 cells per well and allowed to adhere overnight to ensure stable cell attachment. The following day, the complete culture media was carefully removed, and the cells were washed with 100 ml of phosphate-buffered saline (PBS). Subsequently, serum-depleted media (0.5% FBS) was added, and the cells were incubated for an additional 24 hours to induce a quiescent state and sensitize them to apoptotic triggers. Following this pre-treatment, cells were then exposed to various concentrations of GSK-J4, meticulously diluted in serum-depleted media, and incubated in the presence of the drug for a further 48 hours. Where appropriate, control cells were treated with either vehicle (DMSO) or left untreated, receiving media only, to provide baseline measurements. After the treatment period, the culture media containing detached and apoptotic cells was carefully transferred to labeled FACS (Fluorescence-Activated Cell Sorting) tubes and immediately placed on ice to halt further cellular processes. Any remaining adherent cells were harvested by trypsinization and then transferred to their corresponding FACS tubes, ensuring that all cells, both floating and adherent, were collected for analysis. The cells were then centrifuged at 1300 rpm for 3 minutes, and all supernatants were discarded. The resulting cell pellet was gently resuspended and washed in 1 ml of 1X binding buffer (BB), diluted in ice-cold PBS, then centrifuged again, and finally resuspended in 100 µl of BB. A total of 2 µl of Annexin V (IQ Products BV, Groningen, the Netherlands), a fluorescent probe that binds to externalized phosphatidylserine on apoptotic cells, was added to each tube, with the exception of the negative control and media-only samples. The tubes were then incubated at 4˚C for 20 minutes, protected from light, to allow for Annexin V binding. After incubation, the cells were washed in 1 ml of 1X binding buffer, and the supernatant was removed. Prior to flow cytometric analysis, the cell pellet was resuspended in 400 µl of BB containing 0.5 µg/ml Propidium Iodide (PI) (Invitrogen, Paisley, UK), a nucleic acid stain that enters cells with compromised membranes, except for the negative control and FMO (fluorescence minus one) control for PI, for which only BB was used. Samples were then analyzed by flow cytometry to quantify early and late apoptotic populations.
Cellular Apoptosis (Caspase-3/-7 Activation)
To provide an additional and distinct measure of apoptosis, the activation of effector caspases-3 and -7 was quantified. NCI-H226 cells were seeded at a density of 4×10^3 cells per well into Corning® 96-Well Flat Clear Bottom Black Polystyrene Tissue Culture-treated plates and allowed to adhere overnight. The following day, the culture media was removed, and the cells were washed with 100 ml of PBS. Subsequently, the cells were incubated in serum-depleted media (0.5% FBS) for a further 24 hours to promote cellular quiescence. At this point, the cells were treated with GSK-J4 at various concentrations and incubated for an additional 48 hours. Following the treatment period, caspase-3/-7 activation was then precisely measured using a FluoroFire caspase-3/-7 fluorescent assay kit, strictly adhering to the manufacturer’s instructions (Molecutools, Dublin, Ireland). This assay quantifies the enzymatic activity of activated caspases, providing a direct biochemical readout of apoptosis.
In Silico Analysis
For broader contextualization and validation of our findings, data-mining of publicly available mesothelioma datasets was systematically conducted using established bioinformatics platforms. These platforms included Oncomine (accessible at www.oncomine.org), a comprehensive cancer microarray database and web-based data mining platform, and cBioPortal (accessible at www.cbioportal.org), a resource for exploring multidimensional cancer genomics data. All analyses on these platforms were performed using their default settings to ensure consistency and comparability with other published studies. It is important to acknowledge that the results presented here are, in whole or in part, based upon data originally generated by The Cancer Genome Atlas Research Network (accessible at cancergenome.nih.gov), a landmark program that has provided a foundational understanding of the molecular basis of cancer.
Statistical Analysis
All quantitative data generated from the experimental procedures are meticulously expressed as the mean ± standard error of the mean (SEM) derived from multiple independent experiments (n=3). To assess the statistical significance of observed differences, appropriate statistical tests were rigorously applied. Depending on the data distribution and experimental design, either the Mann-Whitney U test (for non-parametric data), the unpaired Student’s t-test (for comparing two independent groups with parametric data), or a one-way ANOVA (Analysis of Variance) followed by Dunnett’s post-test (for comparing multiple groups to a single control) were utilized. These statistical analyses were performed using Graphpad Prism 5.01 software. A p-value of less than 0.05 was predetermined as the threshold for statistical significance, ensuring that only robust and meaningful differences were interpreted as statistically relevant.
Results
Kdm6 Family Members are Ubiquitously Expressed in Mesothelioma Cell Lines
To comprehensively assess the baseline expression profiles of Kdm6 family members, we utilized RT-PCR to meticulously examine the messenger RNA (mRNA) levels of Kdm6a and Kdm6b across a diverse panel of cell lines. This panel included cell lines derived from normal pleura, specifically LP9 and Met5A, as well as a range of established mesothelioma cell lines. Our findings consistently demonstrated that both Kdm6a and Kdm6b were readily detectable and ubiquitously expressed in all cell lines tested. This widespread presence suggests a fundamental role for these genes in both normal mesothelial cell biology and in the context of mesothelioma.
Kdm6 Family Members are Overexpressed in Malignant Pleural Mesothelioma
To translate our in vitro observations to a clinically relevant context, we proceeded to assess the expression of Kdm6 family members in primary patient material. RT-PCR was performed on a carefully curated panel of snap-frozen benign pleura and malignant pleural mesothelioma (MPM) tumor samples, all isolated during surgical procedures from affected patients. Densitometric analysis of the electrophoresis gels, which provided a semi-quantitative measure of mRNA abundance, revealed a statistically significant increase in the expression of both Kdm6a mRNA (P=0.0036) and Kdm6b mRNA (P=0.0122) in the MPM tumor samples when directly compared to the normal benign pleura samples. This robust overexpression in malignant tissue suggests a potential role for these genes in MPM pathogenesis. When further stratified by histological subtype, a significant overexpression of Kdm6a mRNA was consistently observed across all histological subtypes of MPM (epithelial, biphasic, and sarcomatoid). In contrast, significant alteration in Kdm6b mRNA expression was predominantly observed only within the biphasic subtype, indicating a potentially subtype-specific role for Kdm6b.
To corroborate and extend these findings through independent data sources, we performed in silico analyses. Using the Oncomine database, we queried the expression of both Kdm6 family members in the Gordon et al. dataset, which is an independent large-scale gene expression dataset for mesothelioma. In this particular dataset, only Kdm6a was shown to be significantly overexpressed in the MPM specimens when compared to either benign pleura or lung tissue (P=0.007), while Kdm6b levels did not exhibit significant alteration. Furthermore, utilizing cBioPortal, we examined an available mesothelioma TCGA (The Cancer Genome Atlas) next-generation sequencing (NGS) dataset comprising 87 samples. This analysis indicated that overexpression of both Kdm6a and Kdm6b was present, albeit in a relatively small subset of patients within this cohort. Taken together, despite some variations across different datasets and analytical approaches, the overall results from both our experimental work and in silico analyses collectively suggest that the Kdm6 family could indeed represent a plausible candidate for therapeutic targeting in MPM.
Inhibition of the Kdm6 Family Results in Decreased Malignant Pleural Mesothelioma Proliferation and Altered Expression of Pro-Inflammatory Cytokines
Currently, the only known selective inhibitor targeting both Kdm6a and Kdm6b is GSK-J1, which has a corresponding cell-active pro-drug form known as GSK-J4. We initiated our investigation into the functional impact of Kdm6 inhibition by testing the effects of GSK-J1 on the proliferative capacity of NCI-H226 cells, a representative MPM cell line. The results unequivocally indicated that GSK-J1 was capable of effectively inhibiting MPM cellular proliferation, suggesting its therapeutic potential. Subsequently, we extended our evaluation to the pro-drug form, GSK-J4, and confirmed that it similarly inhibited MPM cellular proliferation in two distinct MPM cell lines tested.
Given the established role of Kdm6a as a crucial regulator in inflammatory processes, the ability of GSK-J4 to potentially block pro-inflammatory cytokine expression, and the well-recognized pro-inflammatory nature of MPM itself, we then rigorously examined the effect of GSK-J4 on the expression of a panel of key pro-inflammatory cytokines in MPM cells. Surprisingly, and contrary to initial expectations for an anti-inflammatory effect, treatment of MPM cells with GSK-J4 resulted in a significant upregulation of several pro-inflammatory cytokines. Specifically, we observed a statistically significant increase in the expression of CXCL1 (P=0.0072), CXCL2 (P=0.004), and CXCL8/IL-8 (P=0.0038). This unexpected induction of pro-inflammatory mediators upon Kdm6 inhibition raises important questions regarding the therapeutic suitability of such inhibitors in a disease with a strong inflammatory component.
Non-Malignant Pleural Cells Are More Sensitive to GSK-J4 than Malignant Pleural Mesothelioma Cells
While GSK-J4 demonstrated significant anti-proliferative effects on malignant pleural mesothelioma cell lines, and given that the expression of Kdm6a and Kdm6b was found to be almost ubiquitous across our entire panel of cell lines (including normal and malignant), we proceeded to critically examine the sensitivity of normal mesothelial cells (represented by the LP-9 cell line) in parallel with two malignant MPM cell lines (NCI-H226 and REN). Using two distinct and complementary methods for assessing cellular proliferation—a resazurin-based assay and a BrdU incorporation assay—our findings consistently demonstrated that the normal pleural cell line (LP-9) exhibited greater sensitivity to GSK-J4 compared to the malignant MPM cell lines. To confirm that this differential sensitivity was not due to variations in baseline expression, we re-screened all cell lines for Kdm6a and Kdm6b mRNA expression and confirmed that both genes were indeed expressed in all cell lines. This increased sensitivity of the normal pleural cell line to Kdm6 inhibition was further corroborated by an observed increase in the levels of apoptosis in these normal cells when compared to the MPM cell lines, suggesting a less favorable therapeutic window.
Discussion
Malignant pleural mesothelioma (MPM) is an exceptionally aggressive and therapeutically challenging cancer, characterized by very limited treatment options. Currently, the established first-line therapy, a combination of cisplatin and an anti-folate agent, unfortunately yields suboptimal outcomes. Most MPM patients exhibit a poor response to this or other available therapies, and even among responders, the duration of response is typically short, with rapid development of treatment resistance. There is presently no universally defined second-line therapy, which consequently underscores an urgent and critical need to identify novel therapeutic avenues to improve patient outcomes in this devastating disease. Recent ground-breaking demonstrations have revealed that MPMs are, in fact, polyclonal tumors, formed by the coalescence of different independent subclones. This inherent intra-tumoral heterogeneity implies that each subclone may harbor its own distinct set of molecular alterations. Such heterogeneity can significantly contribute to the emergence of drug-resistant subpopulations, necessitating multi-targeted therapeutic approaches to effectively overcome the complex issue of clonality and achieve durable responses.
The histone methyltransferase EZH2 is intimately associated with the H3K27me3 histone post-translational modification mark, which is commonly found in regions of silenced chromatin or at bivalent “poised” promoters, indicating a state of readiness for gene expression or repression. The lysine demethylases that precisely catalyze the removal of this H3K27me3 mark have been unequivocally identified as Kdm6a (also known as Utx) and Kdm6b (also known as Jmjd3). Given the potentially crucial importance of this specific histone mark in BAP1-mutated MPM, a subset of the disease with distinct molecular characteristics, our study sought to systematically examine the expression patterns of these lysine demethylases in MPM. An initial comprehensive screen revealed that the messenger RNA (mRNA) for both Kdm6a and Kdm6b was ubiquitously and commonly expressed across all cell lines tested, encompassing those derived from both normal mesothelial cells and malignant MPM cells.
However, when their expression was examined in primary patient tumor samples, our findings demonstrated a statistically significant elevation of mRNA levels for both Kdm6a and Kdm6b in the malignant tumors compared to benign pleura. This overexpression in patient tumors reinforces their potential involvement in MPM pathogenesis. When these patient samples were further stratified according to histological subtype, we observed no consistent or significant overexpression of Kdm6a across all histological subtypes. For Kdm6b, elevated mRNA levels were found to be statistically significant only in the biphasic subtype. In this particular regard, it is imperative to acknowledge that the current sample size of our patient cohort is clearly insufficient to draw definitive and robust comparisons across all histological subtypes. A substantially larger sample size would be required in future investigations to comprehensively address this aspect of Kdm6 expression. Our in silico analyses, using independent public datasets, further confirmed that altered expression of these Kdm family members indeed occurs in MPM. However, these analyses also suggested that significant overexpression might be predominantly restricted to Kdm6a, with less consistent or pronounced changes for Kdm6b in certain datasets.
It could also be argued that utilizing normal pleural tissues obtained from patients undergoing resection for lung cancer would represent a more suitable and less inflammatory control group than the benign inflammatory pleural samples employed in the current study. We concur with this perspective and believe that, moving forward, incorporating such controls would provide a valuable additional comparative basis, allowing for a distinction between non-inflammatory pleura and inflammatory pleura. Nevertheless, it is critical to consider that MPM is inherently a pro-inflammatory cancer, and members of the Kdm6 family have been robustly demonstrated to play pivotal roles in the regulation of pro-inflammatory responses. Furthermore, Kdm6b has been implicated as a key element in “inflammaging,” a process that can contribute to tumor progression. In this context, our data definitively demonstrate that overexpression of these demethylases occurs in malignant tissue when compared to the benign pleural plaques, which themselves exhibit a significant inflammatory profile. This observation helps to disentangle a potential confounding issue: our data suggest that the observed upregulation of Kdm6a and Kdm6b in malignant tissue is not merely a consequence of general inflammation, but rather may be specifically linked to the malignant state itself.
Given the established pro-inflammatory nature of MPM and the observed elevated expression of Kdm6a and Kdm6b in our patient samples, we logically proceeded to examine the functional effects of inhibitors specific to members of the Kdm6 family, namely GSK-J1 and its cell-active pro-drug GSK-J4, on MPM cellular health and pro-inflammatory cytokine expression. Both drugs demonstrated significant anti-proliferative activity on MPM cell lines, with the cell-active ethyl ester pro-drug GSK-J4 exhibiting greater anti-proliferative effects at lower concentrations than GSK-J1. While GSK-J4 was originally shown to prevent LPS-mediated induction of pro-inflammatory cytokines in human primary macrophages, our investigation revealed a surprising and clinically concerning outcome: treatment of an MPM cell line with GSK-J4 resulted in a significant upregulation of several pro-inflammatory cytokines, including IL-8. This paradoxical induction of IL-8 by anticancer regimens has been previously observed in experimental models of mesothelioma. Furthermore, elevated levels of IL-8 have also been found in mesothelioma patients undergoing therapy following pleurectomy or extrapleural pneumonectomy (EPP), or in MPM patients treated with tumor necrosis factor-alpha (TNF-α). It has also been noted that elevated levels of IL-8 may not consistently serve as a reliable marker for predicting response to therapy. The critical observation that GSK-J4 is capable of elevating the expression of several pro-inflammatory cytokines raises a serious concern that inhibition of Kdm6 family members in MPM patients could potentially induce cytokine response syndrome (CRS), a severe systemic inflammatory response sometimes observed in MPM.
The increased production of pro-inflammatory cytokines in MPM cells upon GSK-J4 treatment was paradoxically accompanied by decreased cellular proliferation and an associated induction of apoptosis, as well as evidence of necrosis. Furthermore, a crucial finding was our ability to demonstrate that cells derived from normal pleura were significantly more sensitive to GSK-J4 compared to those derived from malignant MPM cells. These results suggest that while the expression of Kdm6 family members is indeed significantly elevated in MPM, therapeutically targeting these enzymes with inhibitors like GSK-J4 may not spare the patients’ normal pleural tissue, posing a considerable toxicity challenge. However, it is important to reconcile this with our observation that in primary tissues, the levels of the Kdm6 family are generally much higher in patient tumors than in normal pleura. This discrepancy between the in vitro cell line sensitivity and the in vivo tissue expression levels remains an important area for future investigation and resolution.
In conclusion, our collective results indicate that the Kdm6 family of histone demethylases may indeed represent a novel therapeutic target for the treatment of MPM, given their overexpression in malignant tissue. However, the unexpected findings of potential damage to normal pleura and, more critically, the possibility of inducing a severe cytokine storm upon Kdm6 GSK467 inhibition with agents like GSK-J4, necessitate significant caution and further rigorous studies. We firmly believe that extensive additional research will be required to comprehensively determine whether targeting the Kdm6 family is a feasible therapeutic strategy in MPM, or perhaps only applicable to a specific subset of MPM patients. Furthermore, future investigations should explore the potential for multi-targeting approaches, combining epigenetic therapies, including (or alongside) agents like GSK-J4, to overcome the complexities and challenges presented by this aggressive and heterogeneous cancer.
Acknowledgements
The authors express profound gratitude to Dr. Warren Thomas, Dr. Yoshitaka Sekido, Dr. Dean Fennell, and Dr. Hannu Norppa for their invaluable generosity in providing access to various mesothelioma cell lines, which were indispensable for this research. The present study received partial financial support for consumables from the Masters in Translational Oncology program at Trinity College Dublin (TCD), specifically for Sian Cregan’s research efforts.