Inhibition of CHK 1 (Checkpoint Kinase 1) Elicits Therapeutic Effects in Pulmonary Arterial Hypertension

Abstract

OBJECTIVE:

Pulmonary arterial hypertension (PAH) is a debilitating disease associated with progressive vascular remodeling of distal pulmonary arteries leading to elevation of pulmonary artery pressure, right ventricular hypertrophy, and death. Although presenting high levels of DNA damage that normally jeopardize their viability, pulmonary artery smooth muscle cells (PASMCs) from patients with PAH exhibit a cancer-like proproliferative and apoptosis-resistant phenotype accounting for vascular lumen obliteration. In cancer cells, overexpression of the serine/threonine-protein kinase CHK1 (checkpoint kinase 1) is exploited to counteract the excess of DNA damage insults they are exposed to. This study aimed to determine whether PAH-PASMCs have developed an orchestrated response mediated by CHK1 to overcome DNA damage, allowing cell survival and proliferation.

APPROACH AND RESULTS:

We demonstrated that CHK1 expression is markedly increased in isolated PASMCs and distal PAs from patients with PAH compared with controls, as well as in multiple complementary animal models recapitulating the disease, including monocrotaline rats and the simian immunodeficiency virus–infected macaques. Using a pharmacological and molecular loss of function approach, we showed that CHK1 promotes PAH-PASMCs proliferation and resistance to apoptosis. In addition, we found that inhibition of CHK1 induces downregulation of the DNA repair protein RAD 51 and severe DNA damage. In vivo, we provided evidence that pharmacological inhibition of CHK1 significantly reduces vascular remodeling and improves hemodynamic parameters in 2 experimental rat models of PAH.

CONCLUSIONS:

Our results show that CHK1 exerts a proproliferative function in PAH-PASMCs by mitigating DNA damage and suggest that CHK1 inhibition may, therefore, represent an attractive therapeutic option for patients with PAH.

Pulmonary arterial hypertension (PAH) is a vascular remodeling disease characterized by vasoconstriction and progressive obliteration of distal pulmonary arteries (PA) leading to elevation of PA pressure, right ventricular (RV) failure, and death.¹ Although extensive proliferation and resistance to apoptosis of PA smooth muscle cells (PASMCs) has become increasingly acknowledged to play an important role in PAH, current therapies are mainly dedicated to counteract vasoconstriction and do not reverse disease progression.² This underlines a pressing need to improve our understanding of the disease and identify new therapeutic targets that directly contribute to vascular remodeling.³

The accumulating evidence that PAH and cancer cells share a certain degree of similarity in terms of cellular and molecular mechanisms driving their survival and excessive proliferation may provide clues to treat the disease.^4,5 We previously demonstrated that to combat threats posed by DNA damage, sustained activation of DNA damage response associated with overexpression of the Mre11-Rad50-Nbs1 DNA damage sensor complex and members of the DNA repair machinery, such as PARP-1 (poly[ADP-ribose] polymerase 1) and OGG1 (8-oxoguanine DNA glycosylase), occurs in PAH-PASMCs allowing efficient DNA repair and thus cell survival and proliferation.^6–8 Although prevention of DNA damage–induced cell death seems to be an adaptive mechanism used by PAH-PASMCs to expand, the source of DNA damage, as well as the molecular players engaged to orchestrate t he repair of DNA lesion remain largely unknown. As observed in cancer cells, PAH-PASMCs experience numerous stresses, such as oxidative and shear stress, inflammation, and pseudo-hypoxia, which are major sources of DNA damage.⁴ In addition, active proliferation of PAH-PASMCs is expected to generate significant levels of replication stress (RS)-associated DNA damage, commonly defined as slowing or stalling in replication fork progression and characterized by the generation of single-strand DNA coated by RPA (replication protein A) proteins.^9,10 In the face of persistent DNA damage or RS, activation of the DNA damage checkpoints is necessary to ensure cell survival.¹¹

Although structurally unrelated, CHK1 (checkpoint kinase 1) and CHK2 are serine/threonine kinases that serve as nexus between DNA damage sensors and components of the cell cycle machinery.¹² Despite their quite overlapping functions, CHK1 plays a predominant role compared with CHK2 in cell survival and cell response to DNA damaging agents.^13,14 In response to DNA damage and DNA RS, CHK1 is phosphorylated on serines 317 and 345 by the ATR (ataxia telangiectasia and Rad3–related) kinase, an activation process facilitated by adaptor proteins DNA TopBP1 (topoisomerase 2-binding protein 1) and CLSPN (Claspin).^13,15 Once activated, CHK1, in turn, phosphorylates multiple proteins to temporarily halting the progression of cell replication and division, initiating DNA repair and triggering apoptosis in response to irreversible DNA damage.¹³ In this regard, overexpression/hyperactivation of CHK1 is a hallmark of many cancer types,^16,17 enhancing cancer cell survival by preventing the accumulation of DNA damage.¹⁸ In keeping with this, targeted CHK1 inhibition alone or combined with DNA damaging agents have been shown to exert antitumor activity in preclinical studies by exacerbating DNA breaks and inducing apoptosis.^16,17 Various CHK1 inhibitors have also been developed and tested in clinical trials¹⁹ as anticancer drugs either alone or in combination with chemotherapy.

In the present study, we documented that DNA damage checkpoint signaling is aberrantly and constitutively activated in PASMCs from patients with PAH. We demonstrated that PAH-PASMCs rely on efficient-CHK1 DNA damage stress response for survival and proliferation. Moreover, pharmacological inhibition of CHK1 provides therapeutic benefit in 2 experimental rat models of PAH.

MATERIALS AND METHODS

Human Tissue Samples and Pulmonary Vascular Smooth Muscle Cell Isolation

Experimental procedures using human cells conformed to the principles outlined in the Declaration of Helsinki and were performed with the approval of Laval University and the IUCPQ (Institut Universitaire de Cardiologie et de Pneumologie de Québec) Biosafety and Ethics Committees. Tissues were obtained from patients who had previously given written informed consent. Healthy lung tissues (controls) were obtained during lung resection for tumors. Lung samples were taken at distance from the tumor and demonstrated normal lung parenchyma. PAH diagnosis was previously confirmed by right heart catheterization. PAH and control tissues were obtained from Respiratory Health Network tissue bank (Major Resources Table in the online-only Data Supplement). PAH-PASMCs (n=10 cell lines) were isolated from small PAs (<1000 μm diameter) from patients with PAH. αSMA (α smooth muscle actin) staining was used to confirm the smooth muscle phenotype of the cells in culture. Controls PASMCs (n=10 cells lines) were either purchased from Cell Application or isolated from non-PAH patients as previously described.^6,8 Cells were used at passages 4 to 8 for experiments.

Animal Models

Experiments were performed according to the guidelines of the Canadian Council on Animal Care and approved by the institutional animal care and use committees of University Laval and University of Pittsburgh. Tissue samples from simian immunodeficiency virus–infected rhesus macaques (12 months postinfection) were collected from the same animals as in the previous report.²⁰ Because male rats develop more severe monocro talineinduced PAH than females,^21,22 the therapeutic potential of MK-8776 in the monocrotaline model was only investigated in males. Male Sprague-Dawley rats (250–300 g body weight) were purchased from Charles River. Monocrotaline-induced PAH was developed by a single subcutaneous injection of monocrotaline (60 mg/kg; Sigma, St Louis, MO), as previously described.⁸ Control rats received saline. Two weeks post monocrotaline injection or in 1-year-old male and female Fawn-Hooded Rats (FHR), rats were randomly divided into 2 groups to receive MK-8776 (1 mmol/L thrice a week) or its vehicle (4% DMSO in PBS) by intratracheal nebulization for 2 additional weeks. Briefly, rats were anesthetized with 3% isoflurane for 15 minutes and placed on an angled platform. The tongue was gently pulled out with forceps to visualize the oropharynx and vocal cords through transillumination. A 16-gauge intravenous catheter was inserted in the lumen of the trachea, and the position was confirmed by visualization of breath on a dental mirror placed at the end of the catheter. A nebulizer (Aeroneb, Aerogen) was plugged into the catheter, and a volume of 50 μL of vehicle or MK-8776 was delivered as a mist into trachea with O₂ 0.1 L/min for 7 to 10 seconds. At the end of protocol, all rats were anesthetized with 2% to 3% isoflurane and underwent closed-chest right heart catheterization. A polyethylene catheter connected to a pressure transducer (SciSence catheters) was inserted into the right external jugular vein and threaded into the RV and the PA to obtain RV systolic pressure, mean PA pressure (mPAP), and RV cardiac output, as previously described.^7,8 Total pulmonary resistance was calculated by dividing mPAP by cardiac output. All hemodynamic measurements and analyses were performed blinded to the condition.²³ Following the measurements, heart, lungs, and other major organs were harvested. The RV was dissected from the left ventricle and the septum and weighted to quantify the extent of hypertrophy by the Fulton index as follow: RV/(left ventricle+septum).

Morphometric Analysis of PA and In Situ Immunofluorescence

Lungs were collected, fixed with 4% paraformaldehyde for 24 hours, paraffin embedded, sectioned at 5 μm and processed for Elastica van Gieson or immunohistochemical stainings. The medial wall thickness of distal PA (<75 μm) was quantified and expressed as follows: ([medial thickness×2]/external diameter)×100. For immunofluorescence staining, tissue sections were deparaffinized in xylene and rehydrated in a graded ethanol-water series. Sections were subjected to antigen retrieval in citrate buffer (0.01 M, pH 6.0) in a microwaveable pressure cooker for 20 minutes. Sections were blocked with 5% goat serum for 2 hours and then incubated with indicated primary antibodies in a humidified chamber overnight at 4°C (Major Resources Table in the online-only Data Supplement). After washes, sections were further incubated for 1 hour at room temperature with appropriate fluorescent-dye conjugated secondary antibodies. Sections were mounted onto coverslips using DAPI (4′,6-diamidino-2-phenylindol) Fluoromount G mounting medium. Sections were examined by confocal microscopy using an Axio Observer microscope (Zeiss), and images were acquired using Zen system (Zeiss). At least 15 randomly selected arteries per animal were measured in a blinded fashion to assess vascular remodeling, proliferation, and apoptosis. Intensity of CHK1 staining was quantified via the integrated density function found in Image J software (National Institutes of Health).

Cell Culture and Treatments

PASMCs were grown in high-glucose DMEM supplemented with 10% FBS (Thermo Fisher Scientific) and 1% antibiotic/antimycotic (Thermo Fisher Scientific). MK-8776 was purchased from ApexBio and dissolved in DMSO. Small interfering RNA targeting CHK1, and its scrambled control small interfering RNA were synthesized by Life Technologies. PAH-PASMCs were transfected with either siCHK1 (small interfering RNA for CHK1) or control small interfering RNA using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA) at a final concentration of 20 nM according to the manufacturer’s instructions. To assess the effects of CHK1 overexpression in control PASMCs, cells were infected with adenoviruses expressing human CHK1 gene (Vector Biolabs) at the multiplicity of infection of 50 and used for experiments 16 hours postinfection. Cells receiving empty vector adenovirus and untreated cells were used as control groups. To supplement miR (microRNA)-424 activity in PAH-PASMCs, cells were transfected with hsa-miR-424–5p mimics or scramble mimics (Life Technologies) at a final concentration of 50 nM. Upregulation of miR-424 after transfection was validated by real-time polymerase chain reaction (PCR). To induce DNA damage, cells were treated with hydroxyurea (1 and 2 mmol/L) for 24 hours or Etoposide (10 and 100 μM) for 6 hours.

In Vitro Analysis of Cell Proliferation, Resistance to Apoptosis and DNA Damage

Cells were used at passages 4 to 9 for experiments. PASMCs were cultured for 48 hours in 10% FBS (a condition that is known to promote proliferation) or 0.1% FBS (a starvation condition that promotes apoptosis) in presence or absence of MK-8776, siCHK1 or their respective controls. Cell proliferation and resistance to apoptosis were quantified by Ki67 and transferase-mediated dUTP nick-end labeling (DeadEnd Fluorometric TUNEL (system, Promega), respectively. To assess DNA damage, cells, treated as indicated, were stained with γH2AX (H2A histone family member X and gamma-H2AX) and p(S4/S8)-RPA32. Nuclei were stained with DAPI. The percentages of Ki67, TUNEL, γH2AX, and pRPA 32 (phosphorylated replication protein A2)-positive cells were calculated. At least 300 cells were counted for each cell line. The sources and working dilutions of primary antibodies are listed in the Major Resources Table in the online-only Data Supplement.

Western Blotting Analyses

Total proteins were extracted from either PASMC or distal PAs using a 2% Chaps protein extraction buffer supplemented with a protease-inhibitor cocktail (Roche). Protein concentration was determined using the Bradford method. Equal amounts of protein were loaded on SDS PAGE, electrophoresed, and transferred onto polyvinylidene fluoride membranes using a liquid blotting system (Bio-Rad). After blocking with either 5% nonfat dry milk or 5% goat serum in TBS-T buffer for 1 hour, membranes were incubated overnight at 4°C with indicated primary antibodies. The sources and working dilutions of primary antibodies are listed in the Major Resources Table in the online-only Data Supplement. Membranes were then incubated with appropriate horseradish peroxidase–conjugated secondary antibodies for 2 hours. Antibodies were revealed using ECL reagents (Perkin–Elmer) and using the imaging Chemidoc MP system (Bio-Rad Laboratories). Protein expression was quantified using the Image lab software (Bio-Rad Laboratories) and normalized to Amido black, as previously reported.⁸

Quantitative Real-Time PCR

Total RNA was extracted from cells or lungs using TRIzol Reagent following the manufacturer’s instructions. RNA quality was checked, and RNA was quantified using a NanoDrop spectrophotometer. Reverse transcription and amplification of miR-424 was performed using the TaqMan MicroRNA Assay miR-424 (ThermoFisher Scientific). Real-time PCR was performed using the QuantStudio 7 Flex real-time PCR system (Applied Biosystems). The expression levels of miR-424 were normalized against U6 small nuclear RNA using the 2-ΔCT method. Each sample was analyzed in triplicate.

Statistical Analysis

One-way ANOVA was performed for data with comparisons among groups. Linear mixed model was performed for repeated measures form the same subjects. For 1-way ANOVA, statistical models were investigated for heterogeneous variances and tested whether models could be reduced to the same variance among groups. When effect that specifies heterogeneity in the covariance structure was significant (heteroscedasticity) compared with the same variance, the statistical analysis was performed using separate residual variance per group. The Satterthwaite degree of freedom statement was added for unequal variance structures. For linear mixed models, a heterogeneous covariance structure with the same correlation among conditions was used. The normality assumption was verified with the Shapiro-Wilk tests, after a Cholesky factorization on residuals from the statistical model. The Brown and Forsythe variation of Levene test statistic was used to verify the homogeneity of variances. Posteriori comparisons were performed using the Tukey comparison. The Nelson-Aalen estimate was used for the overall survival at follow-up, and the log-rank tests were performed to compare groups. The results were considered significant with P≤0.05. All analyses were conducted using the statistical package SAS, version 9.4 (SAS Institute Inc, Cary, NC) and R (R Core Team [2018], Foundation for Statistical Computing, Vienna, Austria).

RESULTS

PAH-PASMCs Display Elevated Levels of DNA Damage/RS and Exhibit Constitutive Activation of the ATR/CHK1 Pathway

Given that PAH and cancer cells display similarities^4,5 and CHK1 overexpression confers survival advantage in cancer cells experiencing DNA damage/RS,^13,14,16 we hypothesized that increased levels of CHK1 and markers of DNA damage/RS are also features of hyperproliferating PAH-PASMCs. We first compared levels of CHK1 expression and activity in isolated PASMCs from control and patients with PAH. We found that hyperproliferating PAH-PASMCs, but not PAH-PAECs, exhibit significantly increased CHK1 protein expression compared with their normal counterparts (Figure 1A and Figure I in the online-only Data Supplement). In addition, CHK1 auto-phosphorylation (S296), a marker of CHK1 kinase activity, was increased in isolated PAH-PASMCs (Figure 1A). To further demonstrate that PAH-PASMCs experience endogenous DNA damage/RS, phosphorylation levels of H2AX and RPA32, 2 markers of DNA damage and RS,²⁴ were measured. Consistent with above findings showing elevated CHK1 expression and activity in PAH-PASMCs compared with control cells, phospho-RPA32 (S4/S8) and H2AX (S139) were increased in PAH-PASMCs (Figure 1A). Since numerous studies implicate ATR as a major kinase mediating DNA damage/RS and a direct upstream activator of CHK1,^13,25 we next measured expression of total ATR, ATR autophosphorylation at Thr 1989 (a marker for ATR activation²⁶), CHK1 phosphorylation at S345 (an ATR phosphorylation site²⁵), as well as CLSPN (known to mediate ATR-CHK1 physical interaction²⁷) and BLM (Bloom’s syndrome protein helicase; a responder to RS regulated by CHK1²⁸). Whereas no significant difference was observed for ATR, PAH-PASMCs exhibited increased expression of phospho-ATR, phospho-CHK1, CLSPN, and BLM compared with control cells (Figure 1B and Figure IIA in the online-only Data Supplement). Additionally, along with CHK2, activity of DNA-PK (DNA-dependent protein kinase), required to maintain CHK1-CLSPN complex stability for optimal RS response,²⁹ was increased (Figure IIB in the online-only Data Supplement). Collectively, our results demonstrate that constitutive ATR-CHK1 signaling is a hallmark of PAH-PASMCs. To complement our analysis and confirm that overexpression of CHK1 is not simply an artifact of in vitro cell culture, CHK1 expression level was assessed by immunofluorescence in distal PAs from control and patients with PAH, as well as in 3 complementary animal models of PAH, namely the monocrotaline rat, the FHR, and the simian simian immunodeficiency virus–infected macaque models. In keeping with in vitro findings, fluorescence intensity of CHK1 was increased in distal PAs from patients with PAH and mainly detected in αSMA-positive cells (Figure 2A and Figure III in the online-only Data Supplement); a feature recapitulated in rats (monocrotaline and FHR), as well as simian immunodeficiency virus–infected macaques exhibiting hemodynamic and structural manifestations of PAH (Figure 2B and 2C and Figure III in the online-only Data Supplement).

Figure 1. Pulmonary arterial hypertension (PAH)-pulmonary artery smooth muscle cells (PASMCs) display elevated levels of DNA damage/replication stress and exhibit constitutive activation of the ATR (ataxia telangiectasia and Rad3–related)/CHK1 (checkpoint kinase 1) pathway.

A, Representative Western blots and corresponding densitometric analyses of CHK1, p(S296)-CHK1, p(S4/S8)-RPA32 and γH2AX (H2A histone family member X and gamma-H2AX) in isolated PASMCs from control (Ctrl; n=4–10) and PAH (n=5–10) patients. B, Representative Western blots and corresponding densitometric analyses of p(S345)-CHK1, p(Thr1989)-ATR and ATR in isolated PASMCs from Ctrl (n=10) and PAH (n=10) patients. Protein expression was normalized to Amido black (AB). Data are presented as mean±SEM. *P<0.05 and **P<0.01.

Figure 2. CHK1 (checkpoint kinase 1) is overexpressed in distal pulmonary arteries (PAs) from pulmonary arterial hypertension (PAH) patients and animal models.

A, Double immunofluorescence staining for αSMA (α smooth muscle actin; green) and CHK1 (red) in lungs from control donors (n=6) and patients with PAH (n=6) and corresponding quantification of CHK1 signal intensity, demonstrating overexpression of CHK1 in remodeled PAs. B, Double immunofluorescence staining for αSMA (green) and CHK1 (red) and corresponding quantification of integrated CHK1 signal intensity showing increased expression of CHK1 in remodeled distal PAs from Fawn-Hooded rats (FHR) and monocrotaline (MCT)-treated rats compared with control rats (n=5–7 rats per group). C, Double immunofluorescence staining for αSMA (white) and CHK1 (red) and corresponding quantification of integrated CHK1 signal intensity showing increased expression of CHK1 in PAs from simian immunodeficiency virus (SIV)-infected macaques suffering PAH (n=5 macaques per group). Pink dots symbolize females. Scale bars: 20 μm in A and B; 50 μm in C. **P<0.01. AU indicates arbitrary unit; DAPI, 4′,6-diamidino-2-phenylindol; and RVSP, right ventricular systolic pressure.

Decreased miR-424 Expression Accounts for Increased CHK1 Upregulation in PAH-PASMCs

Although numerous studies have located ATR as the main activator of CHK1,¹³ little is known about the mechanisms responsible for increased CHK1 abundance in diseased cells. Interestingly, CHK1 was identified as a direct target of miR-424,³⁰ which acts as a sensitizer of DNA damage inducers in cancer cells.³¹ In addition, miR-424 was identified as a master regulator of PAH progression, diminished in PAECs isolated from patients with PAH, and exerting antiproliferative effects.³² Based on these published results, we reasoned that diminished miR-424 expression might occur in PAH-PASMCs favoring CHK1 upregulation. As assessed by real-time quantitative PCR, we demonstrated that, as previously observed for PAH-PAECs,³² miR-424 was significantly decreased in PAH-PASMCs compared with control cells (Figure 3A). Decreased miR-424 expression was also detected in lungs from PAH rat models (Figure IV in the online-only Data Supplement). To determine whether miR-424 contributes to the regulation of CHK1, we transfected PAH-PASMCs with scrambled control oligo or miR-424 mimics. We found that miR-424 supplementation in PAH-PASMCs substantially reduces CHK1 expression (Figure 3B) indicating that downregulation of miR-424 accounts at least in part for the upregulation of CHK1 in PAH-PASMCs. Next, to demonstrate that CHK1 is activated in response to DNA damage, we tested the effects of well-known inducers of DNA damage, namely hydroxyurea (an antimetabolite that depletes nucleotide pools) and etoposide (an inhibitor of DNA topoisomerases required for releasing torsional stress accumulating during replication). Both treatments resulted in increased levels of markers for DNA damage, that is, pRPA32 and γH2AX, as well as increased activity of CHK1 (phospho S296; Figure 3 and Figure V in the online-only Data Supplement).

Figure 3. Regulation of CHK1 (checkpoint kinase 1) expression and activity by miR (microRNA)-424 and DNA damage inducers, respectively.

A, Expression of miR-424 in isolated pulmonary artery smooth muscle cells (PASMCs) from control (Ctrl; n=5) and pulmonary arterial hypertension (PAH; n=7) patients. B, Representative Western blot and corresponding densitometric analysis demonstrating reduced expression of CHK1 in PAH-PASMCs (n=3) transfected with miR-424 mimics (50 nM) for 48 h. C and D, Representative Western blots and corresponding quantifications showing increased activity of CHK1 in Ctrl PASMCs (n=4) after treatment or not with hydroxyurea (HU, C), Etoposide (Eto, D) or their corresponding Ctrls for 48 h. E and F, Representative immunofluorescence images and corresponding quantifications of p(S4/S8)-RPA32- and γH2AX (H2A histone family member X and gamma-H2AX)-positives PAH-PASMCs after treatment or not with HU (E) or Eto (F) for 48 h. mRNA and protein expressions were normalized to U6 and Amido black (AB), respectively. Data are presented as mean±SEM; *P<0.05; **P<0.01 and ***P<0.001. NT indicates non treated; and pRPA32, phosphorylated replication protein A2.

Inhibition of CHK1 Exacerbates DNA Damage and Reduces PAH-PASMC Proliferation and Resistance to Apoptosis

Because CHK1 activation is known to mitigate DNA damage and RS, we tested whether inhibition of CHK1 diminishes sustained PAH-PASMC proliferation and resistance to apoptosis. To this end, the selective CHK1 kinase inhibitor MK-8776 (also called SCH 900776) tested in clinical trials in patients with cancer^33,34 was used to treat PAH-PASMCs. As previously reported,^16,35 pharmacological inhibition of CHK1 resulted in diminished CHK1 protein levels and hyperphosphorylation of CHK1 on S317 and S345 (because of the loss of CHK1-mediated feedback inhibition of ATR), validating the inhibitory effects of MK-8776 on CHK1 (Figure VI in the online-only Data Supplement). Because MK-8776 produced a more robust response at the dose of 1 μM, subsequent experiments were performed at this dose. As assessed by Ki67 and TUNEL labeling, we found that MK-8776 significantly reduced PAH-PASMC proliferation and resistance to apoptosis (Figure 4A and 4B). To strengthen our results, we checked the expression levels of the cyclin-dependent kinase inhibitor p21(Cip1/Waf1) known to respond to CHK1 inhibition.³⁶ As expected, pharmacological inhibition of CHK1 resulted in increased expression of p21 (Figure VI in the online-only Data Supplement). To validate our findings and safeguard against potential MK-8776 off target effects, small interfering RNA–mediated CHK1 silencing was performed in PAH-PASMCs. Western blot analysis revealed that efficient knockdown of CHK1 in PAH-PASMCs (Figure VI in the online-only Data Supplement). Consistent with the effects seen with MK-8776, PAH-PASMC proliferation and survival were impeded after molecular CHK1 inhibition (Figure 4A and 4B and Figure VI in the online-only Data Supplement). As a corollary experiment, we wondered whether CHK1 overexpression using adenoviral infection was sufficient to establish a proproliferative and apoptosis-resistant phenotype in control PASMCs. We found that increased expression of CHK1 did not increase the proliferative ability of control PASMCs challenged or not with hydroxyurea. In contrast, overexpression of CHK1 led to a decrease in apoptosis in control cells exposed to hydroxyurea, thus reinforcing the notion that CHK1 is protective under stress conditions.

Figure 4. Inhibition of CHK1 (checkpoint kinase 1) reduces pulmonary arterial hypertension (PAH)-pulmonary artery smooth muscle cell (PASMC) proliferation and resistance to apoptosis and exacerbates DNA damage.

A, Proliferation (Ki67) and (B) apoptosis (TUNEL [terminal deoxynucleotidyl transferase dUTP nick-end labeling]) were measured after treatments of PAH-PASMCs with the CHK1 pharmacological inhibitor MK-8776 (1 μM) or vehicle (Veh, DMSO), as well as siCHK1 (small interfering RNA for CHK1) or siSCRM (scrambled small interfering RNA; 20 nM) for 48 h. Representative immunofluorescence images of Ki67 and TUNEL-positive cells as well as corresponding quantifications are shown. C, Representative immunofluorescence images and corresponding quantifications of p(S4/S8)-RPA32- and γH2AX (H2A histone family member X and gamma-H2AX)-positives PAH-PASMCs after treatment or not with MK-8776 (1 μM), siCHK1 (20 nM) or their corresponding controls for 48 h. D, Representative Western blot and corresponding densitometric analyses of RAD51 in PAH-PASMCs (n=4) exposed to MK-8776 for 48 h. Scale bar=50 μm. Data are expressed as fold change relative to untreated cells; protein expression was normalized to Amido black (AB). Experiments were performed in triplicate in at least 4 PAH-PASMC cell lines.*P<0.05 and ****P<0.0001. DAPI indicates 4′,6-diamidino-2-phenylindol; NT, non treated; and pRPA32, phosphorylated replication protein A2.

We next examined whether accumulation of DNA lesions occurs in PAH-PASMCs treated with CHK1 inhibitors. To this end, expression levels of 2 different markers for DNA damage, that is, phospho-RPA32 (S4/ S8) and γH2AX, were assessed by immunofluorescence (Figure 4C) in PAH-PASMCs exposed or not to MK-8776 or siCHK1. As expected, CHK1 inhibition resulted in an increased proportion of PAH-PASMCs positive for pRPA32 or with >10 γH2AX foci, a feature of RS,¹⁸ indicative of increased amounts of DNA damage.

These results prompted us to investigate the effects of CHK1 inhibition on DNA repair factors. Previous studies have demonstrated that CHK1 phosphorylates and inhibits E2F6,³⁷ a repressor of E2F-dependent transcription known to cope with high levels of DNA damage by activating genes involved in DNA damage repair and cell cycle progression.³⁸ Thus, we assessed the protein level of the E2F target gene RAD51 in PAH-PASMCs challenged or not with MK-8776 for 48 hours. We observed that RAD51 is upregulated in PAH-PASMCs compared with control cells (Figure VIII in the online-only Data Supplement) and that treatment with MK-8776 diminished its expression level (Figure 4D). Taken together, these findings indicate that CHK1 overexpression in PAH-PASMCs represents a mechanism in place to cope with RS/DNA damage and thus providing a proliferative advantage.

Pharmacological Inhibition of CHK1 Improves PAH in Rodent Models

As most patients with PAH are diagnosed at an advanced stage of the disease, we next investigated whether pharmacological inhibition of CHK1 improved established PAH in the FHR model that spontaneously develops PH.³⁹ We initially performed a pilot dose-response study of MK-8776 as a nebulized solution in FHR rats. Three doses of MK-8776 from 0.5 to 1 mmol/L and vehicle were given 2× a week (Figure 5A). In this dose-response study, hemodynamic improvement was greatest in the group receiving 1 mmol/L (data not shown). This correlates with increased pRPA32 and γH2AX expression, as well as enhanced phosphorylation of CHK1 on S345, as assessed by WB on dissected PAs (Figure 5B) indicative of accumulation of DNA damage. In view of these data, a new experiment on a larger sample size was conducted at a dose of 1 mmol/L. As measured by right heart catheterization, pharmacological inhibition of CHK1 in FHR significantly reduced RV systolic pressure (56±9 versus 40±4 mm Hg) and mPAP (41±7 versus 23±9 mm Hg) as compared to vehicle-treated FHR (Figure 5C and 5D). There were no differences in hemodynamic improvement according to sex. RV hypertrophy, assessed by the Fulton index, remained unchanged in MK-8776-treated rats (Figure 5E). Cardiac output was increased after treatment with MK-8776, although the difference did not reach statistical significance (Figure 5F), and total pulmonary resistance was significantly reduced after CHK1 inhibition (Figure 5G). To determine whether MK-8776 exerts beneficial effects on pulmonary vascular remodeling, medial wall thickness of small PAs was measured. As shown in Figure 5H, the percentage of medial wall thickness was significantly decreased in MK-8776-treated rats as compared to the FHR vehicle group. In agreement with this, PASMC proliferation within distal PAs tends to reduce in MK-8776-treated rats, as evaluated by Ki67 and αSMA double immunostaining on lung tissue, as well as P21 immunoblot (Figure 5I and Figure IX in the online-only Data Supplement).

Figure 5. MK-8776 improves pulmonary hypertension in the Fawn-Hooded rat (FHR) model.

A, Schematic representation of the experimental design. (B) Expression of p(S4/S8)-RPA32, γH2AX (H2A histone family member X and gamma-H2AX), p(S345)-CHK1 (checkpoint kinase 1) and CHK1 were assessed by Western blot in dissected distal pulmonary arteries (PAs) from FHR treated or not with increasing doses of MK-8776 for 2 wk. C–G, Right ventricular systolic pressure (RVSP, C), mean PA pressure (mPAP, D), cardiac output (CO, E), total pulmonary vascular resistance (TPR, F) and right ventricular hypertrophy (Fulton index, G) were measured in FHR+vehicle (Veh) and FHR+MK-8776 (1 mmol/L thrice a week for 2 wk). H, Representative images of distal PAs and corresponding quantification of vascular remodeling as determined by the measure of the medial wall thickness by using, Elastica van Gieson (EVG) staining. I, Representative images of distal PAs labeled with Ki67 in red. Vascular smooth muscle cells were labeled using αSMA staining (α smooth muscle actin, green). The graph on the right represents the percentage of pulmonary artery smooth muscle cells (PASMCs) positive for Ki67 in distal PAs. Scale bar=20μm; n=6–10 rats/group (mean of 15 vessels/rat). Pink dots correspond to females. AB indicates Amido black. *P<0.05 and **P<0.01.

Considering that no animal model fully mimics human PAH and in accordance with recent recommendations on optimal preclinical studies in PAH,⁴⁰ we thus decided to test the therapeutic potential of MK-8776 in a second animal model. To this end, we used the monocrotaline rat model, for which increased CHK1 expression was noticed (Figure 2B). Two weeks after monocrotaline injection, male rats were randomly allocated to receive nebulized MK-8776 or its vehicle for 2 weeks. A third group consisted of control rats without monocrotaline injection (Figure 6A). As expected, vehicle-treated monocrotaline rats exhibited elevated RV systolic pressure and mPAP compared with control rats. As observed in the FHR model, inhibition of CHK1 using MK-8776 was associated with increased expression of pRPA32, γH2AX, and phosphorylated CHK1 (S345; Figure 6B) and resulted in a significant reduction in RV systolic pressure, mPAP, and total pulmonary resistance (Figure 6). MK-8776-treated rats presented diminished RV hypertrophy (Figure 6E) and increased cardiac output as compared to vehicle-treated monocrotaline rats (Figure 6F). Regarding pulmonary vascular remodeling, vehicle-treated monocrotaline rats showed an increase in vessel wall thickness, which was significantly reduced by treatment with MK-8776 (Figure 6H). This histological improvement was accompanied by a decreased trend in PASMC proliferation within distal PAs (Figure 6H), as assessed by quantifying the proportion of Ki67-positive–PASMCs and p21 expression levels in PAs by immunofluorescence and immunoblot, respectively (Figure IX in the online-only Data Supplement). Despite this hemodynamic improvement, no significant difference in survival was observed between the groups (Figure IX in the online-only Data Supplement).

Figure 6. Pharmacological inhibition of CHK1 (checkpoint kinase 1) using MK-8776 improves hemodynamic parameters and vascular remodeling in the monocrotaline (MCT) rat model.

A, Schematic representation of the experimental design. B, Expression of p(S4/S8)-RPA32, γH2AX (H2A histone family member X and gamma-H2AX), p(S345)-CHK1 and CHK1 was assessed by Western blot in dissected distal pulmonary arteries (Pas) from MCT treated or not with MK-8776 1 mmol/L thrice a week for 2 wk. C–G, Right ventricular systolic pressure (RVSP), mean pulmonary artery pressure (mPAP), cardiac output (CO), total pulmonary resistance (TPR) and right ventricular hypertrophy were measured in control (Ctrl), MCT+vehicle (Veh) and MCT+MK-8776 (1 mmol/L thrice a week for 2 wk) rats. H, Representative images of distal PAs stained with Elastica van Gieson (EVG) or labeled with Ki67 (proliferation, red). Vascular smooth muscle cells were labeled using αSMA (α smooth muscle actin, green). Graphs on the right represent the degree of vascular remodeling (as determined by the measure of the medial wall thickness) and the percentage of pulmonary artery smooth muscle cells (PASMCs) positive for Ki67 in distal PAs. Scale bar=20μm; n=5–15 rats/group (mean of 15 vessels/rat). AB indicates Amido black. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001.

DISCUSSION

Primary activated by ATR in response to a broad spectrum of DNA insults interfering with DNA replication, CHK1 is essential for cancer cell viability by coordinating DNA repair, DNA replication, and subsequent cell cycle progression. Indeed, substantial works have shown that CHK1 levels could be used to identify tumors with high levels of RS and that CHK1 inhibitors selectively kill cancer cells exhibiting elevated levels of RS^16,41 whereas healthy tissues or tumors exhibiting low levels of RS are largely nonresponsive to ATR/CHK1 inhibitors. In the present study, we demonstrated that hyperproliferating PAH-PASMCs exhibit increased levels of γH2AX and pRPA32, 2 markers of DNA damage/RS. We demonstrated that PAH-PASMCs display enhanced expression and activation of CHK1 and that its pharmacological or molecular inhibition induced further accumulation of DNA damage. Our results are consistent with recent studies demonstrating that, as observed in cancer cells, PAH cells progressively develop coping mechanisms to chronically stressful conditions allowing their survival and proliferation.^4,7,8 More importantly, we showed that pharmacological inhibition of CHK1 improves established PAH in 2 clinically relevant PAH rat models.

In the present study, we provide evidence that decreased expression levels of miR-424 accounts for elevated CHK1 expression in PAH-PASMCs. Interestingly, although downregulation of miR-424 was originally documented in PAH-PAECs promoting their proliferation,³² CHK1 protein expression did not differ between control and PAH-PAECs, indicating cell-type-specific regulation of CHK1 expression in PAH. In addition, we cannot exclude the fact that other factors also contribute to increased CHK1 expression in PAH-PASMCs. Prior studies have demonstrated that treatment of cancer cells with the pan-HDAC (histone deacetylase) inhibitor vorinostat substantially reduced CHK1 mRNA expression,⁴² and a similar effect was observed following inhibition of mTOR (mammalian target of rapamycin) signaling.⁴³ Given that pulmonary vascular remodeling in PAH is associated with increased expression of HDAC and activation of mTOR signaling,^44,45 these factors may also be involved in the regulation of CHK1 in PAH-PASMCs.

In cancer, 2 approaches have been developed to kill unwanted cells.⁴⁶ The first approach, stress overload, aims to intensify existing stress to surpass the buffer capacities of the cells. The second approach, called stress sensitization, aims to inhibit stress coping mechanisms and thus reach an amount of stress incompatible with cell viability. Targeting stress support pathways and, specifically, the DNA damage response network was proven successful in combating a variety of cancers. Although we found that pharmacological or molecular inhibition of CHK1 led to a pronounced accumulation of DNA damage and an antiproliferative effect in all PAH-PASMC cell lines examined, effect on resistance to apoptosis was more heterogeneous. Because, persistent and massive DNA damage has been documented to trigger permanent cell cycle arrest; a state known as senescence,⁴⁷ it can be speculated that some PAH-PASMC cell lines may become senescent rather than apoptotic. The identification of the molecular mechanisms underlying the divergent sensitivity of MK-8776 in terms of cell death is an important subject for a future study and for potential clinical implication. In addition, although MK-8776 is considered as an inhibitor highly specific for CHK1, we cannot rule out that it may have little impact CHK2 activity, which could explain the greater antisurvival effect observed with this drug compared with treatment with siCHK1.

Inhibition of CHK1 alone has demonstrated beneficial effects in multiple preclinical models of cancer^48–50 and many studies have documented robust cytotoxicity in cells lacking p53⁵¹ as well as synergistic antitumor effects with different drugs, such as PARP-1, HDAC, and BRD4 (bromodomain-containing protein 4) inhibitors.^52–54 Interestingly, inhibition of PARP-1, HDACs, and BRD4 was reported to improve vascular remodeling in PAH animal models,^7,55 leading to the assumption that combined inhibition of DNA damage response pathways may achieve a more robust response. Consistent with our observation that activated CHK1 is a hallmark of isolated PAH-PASMCs, activation of ATR was also detected, supporting the view that ATR and CHK1 function as a kinase cascade. Besides ATR-CHK1 activation, augmented activation of CHK2 and DNA-PK was noted in PAH-PASMCs emphasizing a coordinated ATR- and ATM (ataxia-telangiectasia mutated)-dependent DNA damage response. Because CHK1 and CHK2 exert partly redundant function, it will be interesting to determine whether CHK1/CHK2 dual inhibitors may provide a therapeutic advantage over single CHK1 inhibitors. Given that PAH is a heterogeneous disorder and ATR/CHK1 inhibitors have been documented to achieve maximal efficacy in cancer studies based on the abundance of diverse factors, identification of potential predictive biomarker of CHK1 inhibitor sensitivity seems mandatory to define their potential use for PAH patient stratification and maximize their impact in the clinic.

Antineoplastic drugs are often associated with cardiotoxicity suggesting that inhibition of CHK1 may negatively impact the already weakened heart in PAH. To this end, intratracheal nebulization of MK-8776 was used in the present study to preferentially target its effect on the lungs with minimal cardiac exposure. We did not observe any macroscopic and functional difference between vehicle and MK-8776-treated rats confirming early clinical trials data.³³ Nevertheless, further studies are warranted to test putative toxicity effects.

In summary, our results demonstrate that the ATR-CHK1 signaling is activated in PAH-PASMCs and that inhibition of this axis provides significant therapeutic effects in 2 complementary animal models mimicking PAH. This indicates that inhibition of CHK1 may represent a new therapeutic avenue for patients with PAH by blocking or reversing pulmonary vascular remodeling, a key pathological feature of PAH for which current approved therapies have limited efficacy. Our findings set the ground for future studies deciphering the molecular mechanisms underlying MK-8776 action and exploring its ability to potentiate the effects of antiremodeling drugs in preclinical models of PAH. Finally, our data provide the rationale to investigate the implication of CHK1 in other proliferative cardiovascular diseases characterized by DNA damage, such as carotid artery restenosis.

Highlights.

• Hyperproliferative pulmonary artery smooth muscle cells from patients with pulmonary arterial hypertension and animal models display elevated levels of DNA damage and exhibit constitutive activation of the ATR (ataxia telangiectasia and Rad3–related)/CHK1 (checkpoint kinase 1) pathway.

• In vitro, pharmacological or molecular inhibition of CHK1 exacerbates DNA damage and reduces pulmonary arterial hypertension–pulmonary artery smooth muscle cell proliferation and resistance to apoptosis.

• Lung-specific targeting of CHK1 is associated with histological and hemodynamic improvement in 2 rat models of pulmonary arterial hypertension.

Nonstandard Abbreviations and Acronyms

αSMAα

smooth muscle actin

ATR

Ataxia telangiectasia and Rad3–related

CHK1

checkpoint kinase 1

CLSPN

Claspin

FHR

Fawn-hooded rat

mPAP

mean pulmonary artery pressure

mTOR

mammalian target of rapamycin

PA

pulmonary artery

PAH

pulmonary arterial hypertension

PASMC

pulmonary artery smooth muscle cells

RS

replication stress

RV

right ventricle

TopBP1

topoisomerase 2-binding protein 1