Targeting the HuR oncogenic role with a new class of cytoplasmic dimerization inhibitors

Abstract

The development of novel therapeutics that exploit alterations in the activation state of key cellular signaling pathways due to mutations in upstream regulators has generated the field of personalized medicine. These first-generation efforts have focused on actionable mutations identified by deep sequencing of large numbers of tumor samples. We propose that a second-generation opportunity exists by exploiting key downstream “nodes of control” that contribute to oncogenesis and are inappropriately activated due to loss of upstream regulation and microenvironmental influences. The RNA-binding protein HuR represents such a node. Since HuR functionality in cancer cells is dependent on HuR dimerization and its nuclear/cytoplasmic shuttling, we developed a new class of molecules targeting HuR protein dimerization. A structure-activity relationship algorithm enabled development of inhibitors of HuR multimer formation that were soluble, had micromolar activity, and penetrated the blood-brain-barrier (BBB). These inhibitors were evaluated for activity validation and specificity in a robust cell-based assay of HuR dimerization. SRI-42127, a molecule that met these criteria, inhibited HuR multimer formation across primary patient-derived glioblastoma (PDGx) xenolines, leading to arrest of proliferation, induction of apoptosis, and inhibition of colony formation. SRI-42127 had favorable attributes with central nervous system penetration and inhibited tumor growth in mouse models. RNA and protein analysis of SRI-42127-treated PDGx xenolines across glioblastoma molecular subtypes confirmed attenuation of targets upregulated by HuR. These results highlight how focusing on key attributes of HuR that contribute to cancer progression, namely cytoplasmic localization and multimerization, has led to the development of a novel, highly effective inhibitor.

Introduction

HuR belongs to the ELAV-family of mRNA-binding proteins, and since our seminal observation of its upregulation in tumors of the central nervous system (CNS) and colon cancer, it has become a clear chemotherapeutic target for many types of cancer (1,2). Increases in HuR expression, abnormal cytoplasmic localization, and molecule dimerization are reported for most cancer types and aberrantly regulate thousands of transcripts involved in tumor genomic instability, cell cycle progression, immune resistance, and the suppression of apoptosis (3–5). Several approaches have been taken in the past decade to inhibit HuR function in cancer cells. These approaches include inhibition of i) HuR/mRNA interaction, ii) HuR dimerization/multimerization, iii) HuR nuclear/cytoplasmic shuttling, and iv) HuR expression. To date, the following key compounds/scaffolds have been identified as potential inhibitors of HuR function: MS-444 as a blocker of HuR dimerization and nuclear/cytoplasmic shuttling (6); CMLD1, CMLD2, quercetin, azaphilone derivatives, DHTS, suramin, NSC#84126 as the modulators of HuR/mRNA interaction (7–13) and okicenone, trichostatin, 5-aza-2’-deoxycytidine (AZA) as the inhibitors of HuR shuttling (6,14). The IC₅₀s of these HuR inhibitors range from 300 nM to 10 uM in the direct biochemical assays and from 1 to 100 uM in in vitro assays. The inhibition of xenograft tumor growth in vivo has been reported for colorectal, lung, melanoma, glioma, and pancreatic cancer primarily using si- or miRNA constructs (15–21). Despite the plethora of HuR inhibitors that have been proposed, none target the specific oncogenic driven changes related to HuR of subcellular localization and dimerization in in vivo tumor models.

In a recent manuscript, we reported on a strategy to optimize searches for inhibitors of HuR cytoplasmic multimerization in transformed cells (22). In the current report, we utilized a combination of medicinal chemistry with a cell-based HuR-specific biochemical assay to identify a new class of molecules that disrupt HuR cytoplasmic dimerization. This approach of developing a detailed understanding of the biological role with the cell-based assays capable of allowing readout of cancer-promoting mechanism-of-action with concurrent medicinal chemistry modifications augments the chance of success in targeted therapy development. We confirmed that the discovered HuR inhibitors are cytotoxic and suppress glioma growth and progression using in vitro and in vivo models. A detailed analysis of cell signaling pathways affected by these inhibitors has been performed on patient-derived glioma (PDGx) cell lines revealing disruption of HuR-dependent cell signaling pathways essential for the tumor cell proliferation, survival, invasion, DNA damage repair, and anti-tumor immune resistance, thereby providing molecular biomarkers for patient outcomes and future drug development.

Materials and methods

Cell lines and cell culture

XD456 (ProNeural), JX6 (Classical), JX10 (Classical), JX12p (Classical), JX22p (Mesenchymal), X14p (Classical), and X1524 (ProNeural) primary patient-derived glioma xenolines (PDGx) with molecular subgroup were previously established (19); neurospheres were formed and maintained as previously described in the Neurobasal-A medium (Gibco, Carlsbad, CA) supplemented with a B-27 supplement without vitamin A (Gibco), N-2 supplement (Gibco), 2mM L-Glutamine (Mediatech, Manassas, VA), 100 U/ml Penicillin/Streptomycin (Mediatech), the basal growth factors EGF, 20 ng/ml and bFGF, 20 ng/ml (Thermo Fisher Scientific, Waltham, MA) (23). U251 (Sigma-Aldrich, St. Louis, MO), U87 (Sigma-Aldrich), and LN229 (ATCC, Manassas, VA) glioma cell lines were maintained in DMEM/F12 medium (Thermo Fisher Scientific) supplemented with 10% Fetal Bovine Serum (Gibco), 2mM L-Glutamine (Mediatech), and 100 U/ml Penicillin/Streptomycin (Mediatech). Primary human neurons with neuronal medium and poly-l-lysine were purchased from ScienCell Research Laboratories (Carlsbad, CA) and maintained according to the company’s protocol on the poly-l-lysine coated plates. Primary human astrocytes with astrocyte medium were purchased from ScienCell Research Laboratories and were retained according to the company’s protocol. Stable cell lines expressing the doxycycline-inducible Fluc, HuR-Nluc plus HuR-Cluc constructs were developed and maintained as previously described (22); the clone-stability was ensured by utilizing Hydromycin B and G418 Sulfate (Mediatech) cell-selection antibiotics in the DMEM/F12 medium (Thermo Fisher Scientific) supplemented with 10% Fetal Bovine Serum (Gibco), 2mM L-Glutamine (Mediatech), and 100 U/ml Penicillin/Streptomycin (Mediatech) (22).

Split firefly luciferase assays

The split firefly luciferase assays and data analysis were performed as previously described (22). Briefly, the assays were performed in 96 well plates with a clear bottom (Corning Inc., Corning, NY). 30,000–50,000 cells were plated per well; the constructs were induced by doxycycline (0.75 ug/ml) for 48 hours. Compounds (or vehicles as the control) were administered for 6 hours. The luminescence signals were detected by using InfinitiM200 plate-reader (TECAN, Morrisville, NC) in the presence of the luciferase substrate luciferin (D-luciferin, potassium salt) from GoldBio (St Louis, MO). A multichannel pipette (XL 3000ITM, Denville Scientific, South Plainfield, NJ) was used in all procedures to ensure the experiment quality. The established inhibitors of HuR dimerization (MS444 and DHTS) were utilized as a positive control (Fig. S1 A–C).

Drug synthesis and medicinal chemistry

All reactions were carried out in an oven- or flame-dried glassware under argon atmosphere using the standard gas-tight syringe, cannula, and septa. All the reactions were performed using anhydrous solvents (DMF, THF, CH2Cl2, 1,4-Dioxane, 1-Butanol, CHCl3, DME) purchased from Sigma-Aldrich. Microwave reactions were carried out using CEM discover Labmate System with Intelligent Technology for Focused Microwave Synthesizer (Explorer 48). The precise details of compound synthesis and compound characteristics were provided (Data file S1).

Computational docking study

Computational docking study was performed to investigate the binding mode of SRI-42127 compound at HuR (details were included in the Data file S1). SRI-42127 was docked to HuR via the induced-fit docking protocol (24).

Cell cycle analysis

Cell cycle distribution was evaluated using a standard staining procedure with propidium iodide (PI) (Sigma-Aldrich) followed by flow cytometry as described (25).

Cell viability assay

PrestoBlue cell viability reagent (ThermoFisher Scientific) was used for cell viability assays as previously described (22). The inhibitory dose-response data were normalized to the control (treatment with vehicles); the inhibitory dose-response curves, which were fitted by using the Boltzmann equation, and the corresponding IC₅₀s were generated by using OriginPro software (OriginLab Corporation, CA) (20).

Immunohistochemistry

Neurons and astrocytes were fixed in 3.7% paraformaldehyde (pH 7.4) for 13 min at room temperature. 0.3% Triton X-100 in PBST buffer (0.1% Tween 20) was used for cell permeabilization, 30 min at room temperature. Blocking buffer (3% BSA, 22.52 mg/ml glycine in PBS) was used to block unspecific antibody binding, 30 min at room temperature. Primary cleaved caspase-3 antibody (Cell Signaling Technology) was utilized at 1:200 dilution in PBS (1% BSA) buffer overnight at 4°C. The next day, cells were incubated with secondary antibody (Alexa Fluor 594 Goat anti-rabbit IgG, Invitrogen) in PBS (1% BSA) at 1:2000/5000 dilution for 1 hour at room temperature in the dark. DAPI was utilized for nuclear staining; images were obtained with EVOSFl (Life Technologies) imaging system.

The immunostaining on the brain tissue (fixed in paraformaldehyde, 4%) was performed as described previously (19). Briefly, tissue was permeabilized with 0.5% Triton X-100 in PBST buffer for 30 min, followed by three times wash with PBST and blocking with the Universal blocking buffer for 30 min. Then BEAT^tm Blocker kit from Zymed laboratories (Carlsbad, CA) was utilized according to the manufactory protocol to block unspecific antibody binding to endogenous mouse IgG. The primary antibodies anti-HuR from Santa Cruz Biotechnology (1:100), anti-Bcl2 from Santa Cruz Biotechnology (1:50), anti-Mcl1 (1:100) from Cell Signaling Technology were utilized overnight at 4°C in PBS (1% BSA) buffer. The corresponding secondary antibodies Alexa Fluor 594 Goat anti-rabbit IgG (1:1000) from Invitrogen or Alexa Fluor 594 Donkey anti-mouse IgG (1:1500) from Life Technologies (Eugene, OR, USA ) were utilized in PBS (3% BSA) buffer for 1 hour at room temperature. The fluorescence signal was calibrated based on the secondary-only and primary-only control staining.

Western blotting, antibodies, nuclear and cytoplasmic protein fractionations

Western blotting was performed as previously described (22). Nuclear and cytoplasmic protein fractionations were performed by using NE-PER Nuclear and Cytoplasmic extraction reagents (ThermoFisher Scientific) (22). Anti-lamin A/C and anti-Tubulin alpha antibodies were utilized to verify nuclear and cytoplasmic fractions, respectively. Antibodies against lamin A/C, cleaved PARP, cleaved caspase 3, SOX2, Mcl1 were purchased from Cell Signaling Technology (Danvers, MA). Anti-Tubulin alpha antibody was purchased from Sigma-Aldrich. Antibodies against HuR, actin, and Bcl2 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). A polyclonal anti-HuR antibody for HuR/mRNA co-immunoprecipitation was purchased from MBL-International (Woburn, MA).

HuR/mRNA co-immunoprecipitation assay and Taqman data

The HuR-mRNA co-immunoprecipitation (IP) was performed by utilizing RiboCluster Profiler RIP-assay Kit (MBL-International) (see Materials and Methods in Supplementary Material). The quantifications of Bcl2, Mcl1, and 18S transcripts were performed by using Taqman technique with the following gene-specific probes: Hs00153350_m1, Hs03003631_g1, and Hs00766187_m1 (ThermoFisher Scientific) for Bcl2, 18S, and Mcl1, respectively (19).

mRNA isolation from adherent cell culture

mRNA isolation and purification from adherent cells were performed as previously described by using the RNeasy Mini Kit and QIAshredder columns (Qiagen, Valencia, CA) (19).

Colony formation assays

The soft agar colony formation assay was performed by using 0.9% and 0.45% agarose for the bottom and the top layers, respectively; cells were incorporated in the top layer. Seaplaque low-melting temperature agarose was from Lonza (Rockland, ME). Five hundred cells per well were utilized in both assays, which have been performed in 6 well plates; cells were treated with vehicles or with the desired drugs, which were administered twice per week for three weeks. The Crystal violet solution (0.1%) was utilized for colony staining. Colonies were counted after three weeks of treatment and images were obtained by using Amersham Imager-600 reader (Piscataway, NJ).

Illumina global RNA-sequencing data

Illumina global RNA-sequencing data were generated by utilizing PDGx neurospheres, which have been treated with SRI-42127, 3 uM for 12 hours, or with vehicles as the control (Data file S2). RNA was isolated by using TRIzol reagent (Invitrogen, Carlsbad, CA). The RNA samples were processed in the UAB Sequencing Core facility (see Materials and Methods in Supplementary Material).

Proteomic data

The cell pellets from PDGx neurospheres, which have been treated with SRI-42127, 3 uM for 18 hours or with vehicles as the control, were processed in the UAB CCC Mass Spectrometry/Proteomics shared facility. The proteomic data were generated by using standard procedures (see Materials and Methods in Auxiliary Supplementary Materials and Data files S3 and S4).

Kinase profiling assays

The kinase inhibitory potentials of SRI-42127 and SRI-41664 compounds were evaluated by ThermoFisher Scientific’s SelectScreenTM Kinase Profiling Service (the detailed reports and the 10-point titration results for inhibition of NTRK1, AAK1, IRAK1, PIM1, and cMET kinases were presented in the Data file S5.

In vivo mouse glioma model

The athymic nude mice and PDGx XD456 cells transduced with the firefly luciferase and EGFP constructs were utilized for the in vivo mouse glioma model as previously described (19) (see Materials and Methods in Auxiliary Supplementary Materials).

Pharmacokinetic (PK) assessment

The in vivo PK study for SRI-42127 was performed by Pharmaron (Beijing, China); pharmacokinetics were evaluated in the C57Bl/6 mice; the detailed report is provided (Data file S6).

Statistical analysis

Statistical interpretations were achieved by using a Student’s t-test (when only two groups of data were analyzed) and one-way ANOVA with Turkey’s post hoc test (when multiple data groups were analyzed). Correlation analysis was performed using Pearson’s correlation. The results are shown as the mean±SD; P values less than 0.05 were considered to be statistically significant.

Data and materials availability

All data associated with this study are shown in the manuscript, in the Supplementary Figures, Supplementary Table, Auxiliary Supplementary Materials of the Cancer Research journal website, and GEO repository.

Results

HuR expression is associated with poor prognosis for glioma patients

We analyzed the clinical outcomes of glioma patients harboring low or high expression of ELAVL1 (HuR) by utilizing R2: Genomics Analysis and Visualization Platform (http://r2.amc.nl). Low expression levels of ELAVL1 were associated with favorable prognosis (REMBRANDT Madhavan - 550 MAS.5.0-u133p2 study) (Fig. 1A); the expression levels of the ELAVL1 and ACTB (actin) as a control in the glioma samples (REMBRANDT Madhavan - 550 MAS.5.0-u133p2 study) compared to the normal brain samples (N Brain 44 Harris study) are illustrated in the Fig. 1B. Note the significant ELAVL1 overexpression in the glioma group compared to the normal brain group. Next, we analyzed the influence of the ELAVL1 expression on the outcome of patients with different glioma grades. ELAVL1 expression significantly impacted the prognoses of patients harboring WHO grades I, II, and III tumors and did not significantly affect the outcome of patients with WHO grade IV (Fig. 1C). The ELAVL1 expression normalized to the ACTB expression in the tumors of different grades compared to the normal brain is shown in Fig. 1D. Note the increase in average ELAVL1 expression with higher tumor grade. In our next step, we performed a mini ontology analysis of gene sets, which exhibited significant positive or negative correlations with the ELAVL1 expression, for normal brain and for each tumor grade based on R2: platform data (Fig. 1D and Fig. S1). We found that the low grades (I and II) and the high grades (III and IV) tumors harbored different ELAVL1-dependent gene sets and exhibited a different degree of ELAVL1 dependence. The increase in tumor grade was associated with an enhancement in the number of ELAVL1- dependent cell signaling pathways (Fig. 1E). Note that the expression of the gene set, which determines cell cycle progression, was not significantly correlated with ELAVL1 expression in low-grade tumors; however, it exhibited a strong positive correlation with ELAVL1 expression in grades III and IV (Fig. S2). The gene set representing DNA repair cell signaling pathways was homogeneously negatively correlated with ELAVL1 expression in low-grade tumors; however, it harbored two distinguishing groups of genes with positive and negative correlations with ELAVL1 expression in grade IV tumors.

Figure. 1. Correlations of HuR (ELAVL1) expression in brain tumors with clinical outcomes.

(A) Kaplan-Meier overall survival curves (x-axis represents the time since brain tumor diagnosis) demonstrate statistically significant differences (P=1.9×10⁻¹⁶, long-rank test) between ”high ELAVL1 expression” versus “low ELAVL1 expression” (median cut-off modus) for grouping of all brain tumors. (B) Plots illustrate ELAVL1 and ACTB (as a control) mRNA expression in the brain tumor samples versus normal brain samples. (C) Kaplan-Meier overall survival curves according to the tracks ”high ELAVL1 expression” versus “low ELAVL1 expression” (median cut-off modus) are shown for the following brain tumor subsets: grades 1, 2; grade 3; and grade 4. Kaplan-Meier overall survival curves are exhibiting statistically significant differences according to the tracks ”high ELAVL1 expression” versus “low ELAVL1 expression” for grade 1, 2 subsets (P=1.2×10⁻², long-rank test) and grade 3 subset (P=5.4×10⁻⁴, long-rank test). Grades 1 and 2 consist of astrocytoma (n=65), glioblastoma multiforme (n=2), oligodendroglioma (n=30), mixed (n=4). Grade 3 consists of astrocytoma (n=58), oligodendroglioma (n=23), mixed (n=3), unknown (n=1). Grade 4 consists of glioblastoma multiforme (n=134). (D) The graph illustrates the enhancement of ELAVL1 /ACTB mRNA ratios with the increase of the brain tumor grade. Results are shown as mean±SD; statistical significance was determined by Student t-test. (E) The plot illustrates the gene sets, which exhibited significant positive or negative correlations with the ELAVL1 expression for normal brain and grades one through four brain tumors.

Therefore, extending results from previous publications (19, 22, 26, 27), our data analysis verifies that ELAVL1 overexpression favors glioma progression, and its action is grade-dependent. Consequently, ELAVL1 targeting in high-grade gliomas offers a favorable benefit/risk ratio with identifiable gene sets for monitoring its activity.

Discovery of a new inhibitor class targeting HuR cytoplasmic dimerization

In our previous report, we showed that HuR multimerization is essential for glioma progression and developed a cell-based high throughput split firefly luciferase - HuR assay for the search and optimization of HuR dimerization inhibitors (22). Screening compounds using the split luciferase - HuR assay and established inhibitors of HuR dimerization (as positive control) led us to identify the commercially available compound A-92 (an inhibitor of the general control nonderepressible-2 kinase) as a new inhibitor of the HuR dimerization. Fig. 2A illustrates an inhibitory dose-response for the A-92 compound in a split firefly luciferase - HuR assay. Specific and robust inhibition of the HuR dimerization (HuR-Cluc+HuR-Nluc) compared to the control (Fluc-full length luciferase) occurred following cell treatment for 6 hours with A-92, IC₅₀=4.5±0.5 uM (n=8). The maximum inhibition of HuR dimerization was 93±2% (n=8) after 6 hours of cell treatment. The luminescence signal from cells overexpressing the control Fluc construct decreased by 32±7% (n=8) following treatment with A-92; this value represents the cytotoxic effect of A-92 compound over a period of 6 hours.

Figure 2. A new class of inhibitors of HuR dimerization prevents HuR oligomerization in cell-based split firefly luciferase - HuR assay.

(A) The graph represents the dose-response of inhibition of HuR dimerization for A-92 compound, IC₅₀=4.5±0.5 uM (n=8); A-92 structure is shown beside the graph. Red-filled squares represent luminescence signal from HuR dimer reporter (U251 cells co-expressing HuR-Nluc and HuR-Cluc constructs); blue-filled circles represent luminescence signal from control reporter (U251 cells expressing Fluc construct). (B) The graph represents the dose-response of inhibition of HuR dimerization for SRI-41664 compound, IC₅₀=2.4±0.2 uM (n=6); SRI-41664 structure is shown beside the graph. Red-filled squares represent signal from HuR dimer reporter; blue circles represent signal from the control reporter. (C) The graph represents the dose-response of inhibition of HuR dimerization for SRI-42127 compound, IC₅₀=1.2±0.2 uM (n=6); modifications with resulting SRI-42124 and SRI-42127 compound structures are shown beside the graph. Red squares represent signal from HuR dimer reporter; blue circles represent signal from the control reporter. Results are shown as mean±SD in all graphs. All dose-responses were fitted by the Boltzmann function in OriginPro software. Note that the half inhibitions were not reached for A-92, SRI-41664, and SRI-42127 compounds in the control reporter assay. The IC₅₀s for A-92, SRI-41664, and SRI-42117 compounds (4.5±0.5 uM (n=8), 2.4±0.2 uM (n=6), and 1.2±0.2 uM (n=6), respectively, in the HuR dimerization reporter assay) were significantly different, P<0.05, Student t-test.

Next, optimization of A-92 was performed. First, we resynthesized the A-92 compound and renamed it SRI-41964 (Data file S1). SRI-41964 exhibited identical chemical and HuR inhibitory properties (IC₅₀=4.5 uM) as the original compound A-92. We employed medicinal chemistry and the cell-based split firefly luciferase - HuR assay for lead compound optimization resulting in the generation SRI-41664 (Fig. 2B, Data file S1). SRI-41664 exhibited improved HuR inhibition potency as well as improved aqueous solubility compared to the compound SRI-41964 due to the following structural modifications: i) the excision of a pyrazole ring from SRI-41964 yielding SRI-42125 with improved aqueous solubility but HuR inhibition potency similar to that of the original compound, ii) a scaffold hop of the triazolopyrimidine template of SRI-42125 to an equivalent 5-6-fused imidazopyridazine template. SRI-41664 inhibited HuR oligomerization with an IC₅₀=2.4±0.2 uM (n=6) in cell-based assay after 6 hours of treatment (Fig. 2B). The maximum inhibition of HuR dimerization was 93±1% (n=6) after 6 hours of cell treatment with the SRI-41664 compound. The luminescence signal from cells overexpressing the control Fluc construct decreased by 38±5% (n=6) after cell treatment with SRI-41664 for 6 hours.

In our next step of optimizing lead compounds (SRI-41964 and SRI-41664), a reduction of kinase inhibition potential was performed. This was achieved by the introduction of a methyl group on the pyrazolylamino and a pyranylamino NH resulting in compounds SRI-42124 and SRI-42127, respectively (Fig. 2C and Data file S1). Compound SRI-42124 had poor aqueous solubility that precluded compound evaluation in the cell-based assay. Compound SRI-42127 inhibited HuR dimerization in the cell-based assay with IC₅₀=1.2±0.2 uM (n=6), 6 hours of treatment, the maximum inhibition of the HuR dimerization was 90±1% (n=6) (Fig. 2C). The similar results were achieved in the four independent reporter cell clones overexpressing HuR-Cluc+HuR-Nluc constructs (Fig. S3). The luminescence signal from cells overexpressing the control Fluc construct decreased by 26±9% (n=6) after cell treatment with SRI-42127 for 6 hours. Compound SRI-42127 had reduced kinase inhibition potential compared to the parent SRI-41664 compound (Fig. S4 and Data file S5). Note that SRI-42127 retained its HuR inhibition potential in the presence of all evaluated kinase inhibitors (Fig. S5). Hence, the new lead compound SRI-42127 was established.

Structural modifications that we pursued in the optimization of the lead compounds and the HuR inhibition potencies of the compounds are presented in Data file S1. A schematic summary of the key structure-activity relationship (SAR) finding that emerged from our study is presented in Fig. 3A. Structural changes that led to compounds with HuR inhibition IC₅₀ values below 10 uM are presented in Fig 3B.

Figure 3. Schematic illustration of optimization of the lead HuR dimerization inhibitor scaffolds.

(A) Schematic illustration of key SAR findings. (B) A chart is listing structural changes that yielded compounds with HuR dimerization with IC₅₀s below 10 uM. The following accumulative information is presented in the chart for each compound: a molecular structure (column one), an identification number (column two), a molecular weight (column three), a maximum compound concentration utilized in the assay (column four), a maximum value of inhibition of HuR dimerization (column five), a maximum value of inhibition of the control reporter (column six), an IC₅₀ of inhibition of HuR dimerization (column seven). Columns five through seven represent the mean data of at least three experiments for each compound. Modifications for all compounds with resulting compound structures and HuR inhibition potentials are shown in Table S2.

Newly identified inhibitors of HuR dimerization suppressed glioma progression, in vitro

The glioma suppressive potentials of the new class of HuR multimerization inhibitors were evaluated by utilizing SRI-41664 and SRI-42127 compounds in several cell-based assays. First, we confirmed that the SRI-41664 and SRI-42127 compounds are cytotoxic for established and PDGx cell lines. Fig. 4A illustrates the inhibitory dose responses of SRI-41664 and SRI-42127 compounds in the established U251, LN229, and the PDGx XD456 glioma cell lines. The IC₅₀s for the SRI-41664 compound were 3.4±0.5 uM (n=5), 3.8±0.5 uM (n=5), and 4.8±1.1 uM (n=5); the IC₅₀s for compound SRI-42127 were 2.8±0.6 uM (n=5), 3.2±0.6 uM (n=5), and 4.0±0.6 uM (n=5) for U251, LN229, and XD456 cell lines, respectively, after 48 hours of treatment. We employed a HuR-overexpression approach to confirm SRI-42127 inhibitory potency against HuR dimerization. In the U251 glioma cells with dox-inducible HuR-dimerization reporters, an increase in HuR dimerization by two fold led to a significant increase in the SRI-42127 IC₅₀ for inhibition of HuR dimerization from 1.2±0.1 uM (n=4) to 1.9±0.1 uM (n=4), 6 hours of treatment (P<0.05, Student t-test); the IC₅₀ of SRI-42127 for inhibition of cell viability, 48 hours after cell treatment with SRI-42127, increased from 2.8±0.4 uM (n=4) to 8.0±0.8 uM (n=4) (P<0.05, Student t-test) after the enhancement of HuR-dimer formation by two fold (Fig. S6 A). The overexpression of the control reporter Fluc construct by two-fold did not significantly alter IC₅₀s of SRI-42127 (Fig. S6 B). Similar results have been achieved in XD456 cells; the enhancement of HuR-dimer formation approximately by two-fold, increased an IC₅₀ of SRI-42127 for inhibition of cell viability, after 48 hours of treatment, by 1.9±0.2 (n=4) fold (P<0.05, Student t-test). Fig. S7 A–E illustrates HuR expression in established and PDGx glioma cell lines and the IC₅₀s of SRI-42127 compound to inhibit cell viability after 48 hours of treatment (data include parental TMZ-resistant and stem cell lines).

Figure 4. Lead compounds suppress glioma progression in vitro.

(A) Graphs illustrate cell-viability dose-responses for lead compounds in established and PDGx cell lines (48 hours of treatment). Results are shown as mean±SD, n=5 for all cell lines. (B) Flow cytometry plots illustrate cell-cycle distribution in cell lines after SRI-42127 treatment (10 uM, 18 hours) versus control (vehicle treatment). G1, S, and G2 phase values are shown in percent (%), data were analyzed by FlowJo v10. The bar graph represents the average numbers of cells (mean±SD, %) in each phase of the cell cycle after treatment with SRI-42127 compound normalized to the corresponding control values (vehicle treatment). The alterations in cell numbers in G1 and S phases after treatment with SRI-42127 versus control were significant for all cell lines, P<0.05, Student t-test. The alterations in G2 phases after treatment with SRI-42127 versus control were significant only for U251 and LN221 cell lines, P<0.05, Student t-test. (C) Phase-contrast images illustrate primary human neurons after SRI-42127 treatment (48 hours) versus control (vehicle treatment). Scale bar, 100 um. (D) The graph illustrates that neuron numbers were not affected by the SRI-42127 treatment (48 hours) versus control (vehicle treatment). Results are shown as mean±SD (n=3). (E) The graph illustrates the metabolic alterations in primary human neurons after SRI-42127 treatment (48 hours) versus control. Results are shown as mean±SD (n=3). (F) The graph illustrates the percent of cells with cleaved caspase-3 immunostaining in primary human neurons and astrocytes after SRI-42127 treatment (up to 25 uM) for 48 hours. Results are shown as mean±SD of at least three experiments for each cell type. Representative images of cleaved caspase-3 immunostaining after SRI-42127 treatment versus control (vehicle) are shown for neurons; the scale bar is 200 um. DAPI marks the cell nucleus.

HuR is one of the key regulators of cell cycle progression in cancer cells, particularly high-grade glioma (28); therefore, the arrest of cell cycle progression by the newly identified compounds would be supportive of HuR-targeted inhibition. Fig. 4B illustrates representative histograms of cell cycle progression in the established U251, LN229, and the PDGx XD456 glioma cell lines in the control (treatment with vehicles) and after treatment with SRI-42127 compound. The bar graph represents the average numbers of cells in each phase of the cell cycle after treatment with the SRI-42127 compound normalized to the corresponding control values (vehicle treatment). Note a significant accumulation of cells in the G1 and the reduction in the S phases after treatment with SRI-42127, 10 uM for 18 hours. The average accumulations of cells in the G1 phase increased by 97±33% (n=3), 56±28% (n=3), and 210±40% (n=3) in U251, LN229, and XD456 cell lines, respectively, after treatment with SRI-42127 compared to the control. The average reductions of cells in the S phase were 65±4% (n=3), 60±3% (n=3), and 70±3% (n=3) in U251, LN229, and XD456 cell lines, respectively, after treatment with SRI-42127 compared to the control.

Primary human neuronal cells were evaluated as the control and exhibited no morphological changes after treatment with SRI-42127 at up to 15 uM for 48 hours compared to the control (vehicle treatment); the cell numbers and cell sprouting remained unchanged (Fig. 4C, D). The cellular metabolism declined by about 44% after treatment with SRI-42127, 25 uM for 48 hours (Fig. 4E). There were no significant alterations in patterns of cleaved caspase-3 immunostaining after 48 hours of treatment with SRI-42127 at concentrations up to 15 uM compared to the vehicle control (Fig. 4F). Similar results were obtained in primary human astrocytes; we did not observe significant alterations in cleaved caspase-3 activation after treatment with SRI-42127 up to 15 uM (Fig. 4F). Therefore, we conclude that compound SRI-42127 inhibited glioma cell growth and had low neuronal and astrocyte cytotoxicity at concentrations below 15 uM.

Novel HuR inhibitors induce apoptosis and reduce tumorigenicity in established and PDGx glioma cell lines

HuR protein plays a significant role in stabilizing and positively regulating mRNAs of the anti-apoptotic Bcl2-family (19,29) that contributes to the protection of genetically unstable glioma cells from apoptosis. Therefore, we evaluated the Bcl2-family in the established and PDGx cell lines after cell treatment with SRI-41664 and SRI-42127 compounds and observed a significant reduction in the expression of the Bcl2-family at the mRNA and protein levels (Fig. 5A, B). After 24 h of treatment with 10 uM of SRI-41664, there were a 94–96% reduction in Bcl2 mRNA and an 87–96% reduction in Mcl1 mRNA for U251, U87, LN229, and PDGx XD456 glioma cell lines compared to control. Likewise, for SRI-42127, there were an 86–96% reduction for Bcl2 mRNA and an 87–95% reduction for MCl1 mRNA versus control. The average decreases of Bcl2/18S and Mcl1/18S mRNA ratios were 96±2% and 95±3%, 95±2% and 86±7%, 95±2% and 87±8%, 96±2% and 90±9% based on three experiments in the established U251, U87, LN229, and PDGx XD456 cell lines, respectively, after cell treatment with SRI-42127, 10 uM for 24 hours compared to the control. A significant reduction in the interaction of the HuR protein with Bcl2 and Mcl1 mRNAs after cell treatment with SRI-42127 compound compared to the control was confirmed in the HuR/mRNA co-immunoprecipitation assay performed in U251 cells (Fig. S8). Consequently, protein levels of Bcl2 and Mcl1 were evaluated in U251 and PDGx XD456 glioma cells after vehicle (control) or inhibitor (SRI-41664 or SRI-42127) treatment for 48 hours. The representative western blots in Fig. 5B illustrate a dramatic reduction of Bcl2 and Mcl1 protein levels in the cytoplasmic fractions of both cell lines after treatment with SRI-41664 or SRI-42127 compounds compared to the control. The reduction was associated with a significant mitochondria clusterization and depolarization of the mitochondrial membrane potential evaluated by using JC-1 dye (Fig. S9 A,B) and was accompanied by the sign of apoptosis: cleaved caspase-3 and cleaved PARP (Fig. 5C). Similar results were observed in three independent experiments. SRI-41664 and SRI-42127 compounds exhibited stronger glioma inhibitory potency than the A-92 compound; Fig. S10 A–C represents A-92 compound glioma inhibitory potentials, in vitro, in comparison to lead compound SRI-41664.

Fig. 5. Induction of apoptosis and reduction of tumorigenicity of established and PDGx glioma cell lines after treatment with lead compounds.

(A) Graphs illustrate the percent reduction of Bcl2/18S and Mcl1/18S mRNAs following cell treatment with lead compounds versus control (vehicle treatment). Results are shown as mean±SD (n=3), differences statistically significant for all cell lines with P<0.005, Student t-test. (B) Representative western blots confirm the reduction of Bcl2 and Mcl1 proteins in cytoplasmic fractions of U251 and XD456 cell lines following treatment with lead compounds, 10 uM for 48 hours. SOX2 reduction in nuclear fractions indicates a decrease in tumorigenicity of cell lines after treatment with lead compounds. (C) Representative western blots illustrate appearances of cleaved PARP and cleaved caspase-3 in glioma cell lines after treatment with lead compounds, 10 uM for 48 hours. (D) Graphs illustrate a significant reduction of colony formations in U251 and XD456 cell lines after SRI-42127 (3 uM and 10 uM treatments) versus control (vehicle treatment). Results are shown as mean±SD, (n=3) and (n=4) for attached and anchorage-independent assays, respectively; P<0.005, Student t-test for all assays. Examples of colony formations are shown in the U251 cell line.

Note that short-term treatment with SRI-42127 (3 uM and 10 uM, 8–12 hours of treatment), utilized in the HuR-dimerization reporter assays, did not alter the total HuR protein levels (Fig. S11A). However, we observed a decrease in HuR cytoplasmic levels after prolonged treatment with A-92, SRI-41664, and SRI-42127 compounds versus control and an accumulation of nuclear HuR with the following HuR proteolysis and disintegration (Fig. 5B, Fig. S10B). The experiments in the presence of cycloheximide (an inhibitor of protein synthesis) alone or in combination with SRI-42127 (3 uM and 10 uM) suggested that that the disruption of the HuR dimerization by SRI-42127 might facilitate HuR degradation (Fig. S11B). Also, the activation of cleaved caspase-3 (Fig. 5C) associated with the prolonged treatment with lead compounds might contribute to HuR proteolysis and disintegration in agreement with previous reports (30,31).

Colony formation assays (attached and anchorage-independent) were performed in U251 and XD456 glioma cells and showed a significant reduction in glioma cells’ ability to form colonies after treatment with SRI-42127 (3 uM and 10 uM) for two weeks compared to vehicle treatment (Fig. 5D). The number of colonies significantly decreased from 337±31 (n=3) to 49±28 (n=3) and from 402±38 (n=3) to 34±12 (n=3) in U251 and XD456 cell lines, respectively, in the attached cell colony formation assay with SRI-42127, 3uM. The number of colonies significantly decreased from 436±28 (n=4) to 90±70 (n=4) and from 283±62 (n=4) to 39±6 (n=4) in U251 and XD456 cell lines, respectively, in the soft agar colony formation assay with SRI-42127, 3uM. No significant numbers of colonies were detected in the presence of SRI-42127, 10 uM in U251 and XD456 cell lines in both assays.

Hence, our data confirmed the impairment of the in vitro growth and reduction of tumorigenicity of both established and patient-derived glioma cell lines following treatment with the new class of HuR dimerization inhibitors.

Analysis of the cell-signaling pathways altered in patient derived glioma neurospheres by SRI-42127

The Illumina global RNA-Sequencing was performed on PDGx-derived glioma neurospheres representing the different molecular subtypes (classical, neuronal, and mesenchymal) after treatment with vehicle (control) or SRI-42127 (3 uM) for 12 h (Data file S2). First, analysis of direct HuR mRNA targets and their corresponding cell signaling pathways was performed using previously published gene sets of direct HuR targets (11). Table S1 illustrates the evaluated gene sets, representing known direct HuR mRNA targets, and their corresponding cell signaling pathways. The gene set average values, which exhibited significant alterations following treatment with the SRI-42127 compound as compared to the control, are highlighted in green. Note that the gene sets corresponding to cell cycle progression, MAPK signaling pathways, TP53- dependent transcriptional pathways, stress-response cell signaling pathways, RHO GTPase- dependent pathways, membrane trafficking pathways, cellular senescence and developmental pathways, cell signaling pathways associated with the Epstein-Barr virus infection are significantly down-regulated in at least four of five evaluated neurosphere cell lines.

Next, we utilized the Enrichr software for overall gene ontology enrichment for gene sets significantly altered in PDGx neurospheres by SRI-42127 compound compared to the control (Fig. 6). Gene sets that exhibited significant up-regulation or down-regulation of more than 50% across all the evaluated cell lines after treatment with SRI-42127 were selected for the enrichment analysis. Fig. 6A illustrates the enrichment for down-regulated cell signaling pathways and targeted subcellular structures. The detailed cluster plots of the enriched down-regulated genes and corresponding cell signaling pathways and targeted subcellular structures are shown in Fig. 6A and Data file S7. The networks of downregulated transcriptional factors and associated pathways are shown in Fig. S12. Significantly down-regulated pathways (associated with glioma progression) included: cell cycle progression, ii) DNA replication, iii) transport mature transcript to the cytoplasm, iv) DNA strand elongation, v) capped intron-containing pre-mRNA processing, vi) DNA repair, vii) the unwinding of DNA, viii) alpha-linolenic (omega 3) and linoleic (omega 6) metabolism, ix) chromosome maintenance, x) messenger RNA splicing. We and others have previously reported that HuR may control the centrosome and microtubule-organizing center in cancer cells (25,32,33). Therefore, without surprise, the enrichment analysis confirmed that the centrosome, mitotic spindle, pericentriolar materials, microtubule-organizing center, and telomeric region of chromosome are among of the subcellular structures affected by SRI-42127 treatment.

Figure 6. Gene ontology enrichment analysis for gene sets significantly altered in PDGx neurospheres by SRI-42127, 3 uM 12 hours of treatment compared to control (vehicle treatment).

(A) Enrichment of the significantly down-regulated cell-signaling pathways/processes (chart on the left) and targeted subcellular structures (chart on the right) for significantly down-regulated gene sets after SRI-42127 treatment compared to control (vehicle treatment). (B) Enrichment of the significantly up-regulated cell signaling pathways/processes (chart on the left) and targeted subcellular structures (chart on the right) for significantly up-regulated gene sets after SRI-42127 treatment compared to control (vehicle treatment). Genes that exhibited the significant up-regulation or down-regulation on more than 50% across all of the evaluated PDGx neurosphere cell lines (JX12p, X14P, XD456, X1524, and JX22p) after treatment with SRI-42127 versus control have been selected for the enrichment analysis; statistical significance was determined by Student t-test. The bar graphs illustrating the relative combined scores are attached to each chart on the right side. The results are generated based on RNA-Seq data.

Fig. 6B illustrates the enrichment of the significantly up-regulated cell signaling pathways and targeted subcellular structures. The detailed cluster plots of the enriched up-regulated genes and corresponding cell-signaling pathways and targeted subcellular structures are shown in Fig. 6B and Data file S7. There was significant up-regulation of the cellular pathways responsible for i) SRP-dependent co-translational protein targeting to the membrane and ER, ii) ribosomal biogenesis, iii) ncRNA processing, iv) nuclear-transcribed mRNA processing and decay components of the ribosome and polysome were the main targeted sub-cellular structures. The ribosomal reorganization is the common adaptive response to translational and environmental stress (34); therefore, the ribosome reorganizing pathways are considered the cell’s main compensatory response pathways. Also, importantly, the data show an up-regulation of the Tubb2A transcript by more than two-fold in four of five neurosphere cell lines (Data file S2). Since TUBB2a is associated with neuronal protection, this pattern is consistent with the absence of toxicity in neurons treated with SRI42127 (Fig. 4) (35).

Next, quantitative proteomics data was generated using glioma neurospheres of different molecular subtypes (classical, neuronal, and mesenchymal) in control (treatment with vehicles) and after treatment with SRI-42127, 3 uM for 18 hours (Data files S3 and S4). The enrichment of proteins, which exhibited a significant down-regulation by more than 50 % after cell treatment with the SRI-42127, revealed that all neurosphere cell lines uniformly had down-regulation of RNA binding function and sign of apoptosis (Fig. 6 and Fig S13A,B). Additionally, we found a significant down-regulation of: i) protein sets responsible for protein transporter activity, small protein activating enzyme activity, cadherin binding, double-stranded DNA binding, DNA dependent ATPase activity, nucleoside-triphosphatase activity, ADP binding in the neurospheres of the classical subtype; ii) the protein sets involved in transcriptional factor activity, transcription initiation factor activity, nuclear localization sequence binding in the neurospheres of the proneuronal subtype (Fig. S13 A and Data file S4).

Thus, the data analysis suggested that cell-signaling pathways, which require HuR direct mRNA targets, were mostly down-regulated in neurospheres after treatment with the SRI-42127 compound. However, some gene set modifications were specific to the glioblastoma molecular subtypes.

SRI-42127 crosses the blood brain barrier and suppresses glioma growth

The pharmacokinetic (PK) evaluation of SRI-42127 in a mouse model revealed a significant penetration into the CNS with a brain-to-plasma concentration ratio of 0.42±0.05, n=9 (Fig. S14 A–C and Data file S6). The half-life of compound SRI-42127 was short 0.16 h using a dose of 10 mg/kg by IP. Next, we evaluated the impact of SRI-42127 on intracranial glioma growth in a mouse model. Because the half-life of SRI-42127 was relatively short, we used twice a day dosing at 15mg/Kg, administered for three weeks (see Methods). There was a reduction of tumor progression in the mouse group treated with SRI-42127 compared to the control group (treated with vehicles) based on the detection of tumor reporter luminescence signal (Fig.7 A). Also, a reduction in tumor mass was confirmed in brain slices from the mouse group treated with compound SRI-42127 compared to the control group based on the detection of tumor reporter EGFP signal. Immunostaining for HuR, Bcl2, and MCl1 confirmed a reduction of HuR, Bcl2, and Mcl1 expression in the tumors from the mouse group treated with SRI-42127 versus the control group (Fig. 7 B,C,D).

Figure 7. Glioma growth inhibition by SRI-42127, in vivo, and computational modeling of SRI-42127 binding to HuR.

(A-D) SRI-42127 suppresses glioma progression in vivo. Mice were randomly divided into two groups after four and half days of the intracranial tumor establishment and treated with vehicle (control group) or SRI-42127 compound (SRI-42127 group), 15 mg/kg, twice a day for three weeks via intraperitoneal injection. Representative scans illustrate the tumor reporter images superimposed with mouse images; luminescence/color scales are beside the corresponding scans. Graphs illustrate luminescence signals from intracranial tumors on the second (top) and third (bottom) weeks of treatment; results are shown as mean±SD (radiance, p/sec/cm2/sr); the differences between groups were not statistically significant, P=0.07 (n=5) for the second week, P=0.069 (n=5) for the third week, Student t-test. The weight changes in group treated with SRI-42127 compound were not significant compared to the control group (P>0.05, Student t-test). Immunostaining for HuR (B), Bcl2 (C), and MCl1 (D) on tumor brain tissue from control and treated with SRI-42127 compound mouse groups. (E). Computational docking of SRI-42127 at HuR. SRI-42127 compound and key residues of HuR are represented with green and purple carbons, respectively. Hydrogen bonds, hydrophobic contacts, and pi-pi stacking (or pi-cation interactions) are indicated by cyan, orange, and green dash lines, respectively. Grey and blue ribbons represent RRM1 and RRM2 domains, respectively.

Hence, we conclude that the new class of inhibitors of HuR dimerization (particularly compound SRI-42127) exhibited encouraging, however not yet statistically significant, glioma-inhibitory potential in vivo.

Putative binding model of SRI-42127 at HuR.

A computational docking study was performed to investigate the binding mode of lead SRI-42127 compound at HuR (details are included in Data file S1). The apo structure of HuR was prepared from a crystal structure of RNA/HuR complex (PDB ID: 4ED5) using the Protein Preparation Wizard (24). Fig. 7 E illustrates the docked pose of SRI-42127 at HuR. According to the putative binding mode, SRI-42127 is able to interact with four functionally important residues Y26, I103, R97, and R153 of HuR. MM-GBSA, an in silico binding free energy scoring approach, was used to estimate the binding affinity of SRI-42127 at HuR. Specifically, MM-GBSA scoring was performed on the fixed conformation of the docked pose of SRI-42127 at HuR using VSGB solvation model and OPLS3e force field. The same scoring was performed on the UMP (U8 from HuR co-crystal structure), which resides in the same region as SRI-42127 to obtain a reference value. SRI-42127 showed a stronger MM-GBSA binding free energy score than the UMP (−57.6 kcal/mol versus −22.7 kcal/mol), indicating that SRI-42127 is indeed capable of forming energetically favorable interactions at the RM1-RM2 putative binding site on HuR.

Therefore, one of the possible modes of action could be that the binding of SRI-42127 to RM1-RM2 induces a conformational change of HuR monomer that disrupts the optimal interface needed for HuR dimerization and mRNA binding.

Discussion

Glioma is one of the deadly tumors of the CNS and accounts for about eighty percent of all malignant brain tumors (36). Standard therapy with surgery, radiotherapy, and chemotherapy with temozolomide (TMZ) represent the first line of treatment for glioblastoma, the most malignant, over the past decade (37,38). However, due to high tumor heterogeneity, genomic instability, their diffusely infiltrating nature, and the development of drug resistance, glioma remains incurable and basically poorly responsive to treatment (38, 39).

HuR plays a crucial role in glioma development and transition to the higher grade (1,19,22,33, 39–41). HuR’s role is unique for each tumor grade and depends on integrated transcriptome and proteome, including cellular kinome, which is involved in the HuR post-translational modifications and is grade-specific (42,43). Glioma HuR-dependence is progressive with tumor grade. HuR overexpression in low-grade glioma correlated with an overall decrease of transcriptional factor activity, down-regulation of cell-signaling pathways responsible for DNA repair, and, therefore, an accumulation of genomic instability. The overexpression of HuR in higher-grade tumors results in unleashed cell cycle progression, chemoresistance, immune resistance, aberrant cell signal transduction, suppression of differentiation, and apoptosis. In our work, we evaluated the impact of a new class of HuR inhibitors on the progression of high-grade primary and established glioma cell lines. Uniformly, we observed an arrest of cell cycle progression, the appearance of apoptosis, a reduction of tumor stemness, and a decrease of colony formation after glioma cell treatment. First, enrichment of cell signaling pathways indicated that centrosomes and cell division apparatuses were significantly impacted. These data are in agreement with prior reports from our laboratory and elsewhere showing that HuR colocalizes with centrosomes in a phosphorylation-dependent manner, controls protein synthesis in the near proximity to centrosomes, and affects the cytoskeleton architecture of the mitotic spindles, and influences the centrosome-dependent aspects of tumor cell division (25,32,33,44,45). Second, there was a significant decrease in the expression of HuR-dependent mRNAs of cyclins, which are essential for cell cycle progression (12, 28,33,46). Third, there was a marked down-regulation of Bcl2 family members (Fig. 5) consistent with our prior reports with HuR knockdown or inhibition (19,41). This family of proteins plays a critical role in the prevention of apoptosis in tumor cells (19,29,46), and the appearance of apoptotic markers (cleaved PARP and cleaved caspase 3) after SRI-42127 treatment (Fig. S13B) is consistent with their suppression. Taken together, the molecular and cellular effects of SRI-42127 on glioma cells are consistent with HuR-targeted inhibition.

Recently, it has been reported that a correlation exists between a patient’s gender and response to glioblastoma therapy as well as overall survival (specifically for GBM of mesenchymal, proneural, and neural subtypes) (47). Standard therapy is more effective in female patients compared to male, which exhibited up-regulation of the HuR-dependent transcriptome essential for cell cycle progression. In this regard, HuR inhibitors may serve as an adjuvant treatment modality for male patients with up-regulated cell cycle progression. The female group, which was associated with the activation of the integrin signaling components following standard therapy, might also benefit from the HuR inhibitors due to the fact that several members of the integrin signaling components (to include ITGB4, ITGA4, ITGB1, ITGA3, ICAM1, VCAM1, VIM) and chemokine family (to include CCL2, CCL8, CCL13, CXCL1, CXCL2, CXCL3, CXCL5, CCL20) are direct HuR mRNA targets. Therefore, sex differences in GBM biology may be useful to predict suppression of tumor invasion and angiogenesis in the female group and suppression of tumor proliferation in the male group by the HuR inhibitors. A detailed transcriptome analysis of the therapeutics targets combined with the profiling of patient transcriptome is essential for the development of personalized medicine. Our work profiled the transcriptomics and proteomic alterations of the different glioma subtypes after treatment with the new class of HuR inhibitors; we plan to extend our analysis using in vivo mouse glioma models to further understand sex-, age-, and subtype-specific benefits of HuR inhibitors.

Overall, the most unfavorable HuR-dependent transformations for patients harboring gliomas are i) cell-cycle progression and ii) development of the tumor heterogeneity with the consequent drug resistance (3,19, 25,26,28,41,48,49). HuR-dependent cell cycle progression is well documented (10,16,24,28,41); the mechanisms of drug resistance are still under investigation and consist of the HuR-dependent activation of the ABC drug transporters, the anti-apoptotic pathways related to the mitochondria function, and the HuR-dependent intercellular gene transfer (14,19,29,48). Similar to gliomas, colon cancer cell viability and mitochondria function exhibited strong dependence from the anti-apoptotic Bcl2-family (7), transcripts of Bcl2 and Mcl1 are established HuR-mRNA targets. Depolarization of mitochondria membrane potential with an induction of apoptosis has been observed in colon cancer cells after treatment with the HuR inhibitor, CMLD-2 (7). Therefore, we predict that colon cancer (as well as breast, lung, bladder, oral, ovarian, and prostate cancers) will likely be sensitive to HuR inhibitors; however, the detailed benefits of treatment and the most patient-sensitive group remain to be determined.

Twenty-six compounds, which exhibited a HuR-inhibitory potential with IC₅₀s less than 10 uM in the cell-based HuR dimerization assay, have been synthesized and evaluated in vitro. Six recently developed compounds have IC₅₀s below 1uM. With new HuR dimerization inhibitors, we confirmed the disruption of HuR-related oncogenic characteristics in glioma cell lines similar to disruptions induced by MS444 and DHTS, established inhibitors of HuR (6,10,11,22,48). All inhibitors of HuR dimerization (like A-92, SRI-41664, SRI-42127, MS444, DHTS) were cytotoxic for glioma cell lines, and the reductions of cell viability were associated with the appearance of apoptosis and a reduction in the Bcl2-family at the mRNA and protein levels (10,48). The arrest of cell cycle progression and uniform decrease in cell transition to the S phase of the cell cycle were observed for all the above HuR dimerization inhibitors. A reduction in cancer cell line stemness following treatment with MS444, DHTS, and SRI-42127 compounds was confirmed in our experiments and was reported by others as well (10,48). The comparison of RIP-chip profiling of HeLa cells treated with DHTS (11) and glioma cells treated with SRI-42127 revealed common alterations in HuR-dependent gene sets and cell signaling pathways. However, MS444 and DHTS compounds have unknow BBB permeability, require a high concentration for activity, and are challenging to create soluble solutions for administration. SRI-42127 compound exhibited superior BBB permeability, a stronger potency for inhibition of HuR dimerization compared to MS444 and is readily soluble. The in vivo compound assessment, in conjunction with future lead compound optimization, will focus on the improvement in the PK characteristics as our next steps. In conclusion, our work has identified a new class of HuR multimerization inhibitors and detailed the lead compound inhibitory potentials on glioma progression in vitro and in vivo.

Statement of Significance.

Findings utilize a cell-based mechanism of action assay with a structure-activity relationship compound development pathway to discover inhibitors that target HuR dimerization, a mechanism required for cancer promotion.