Ruxolitinib with resminostat exert synergistic antitumor effects in Cutaneous T-cell Lymphoma

Fani Karagianni; Christina Piperi; Vassiliki Mpakou; Aris Spathis; Periklis G Foukas; Maria Dalamaga; Vasiliki Pappa; Evangelia Papadavid

doi:10.1371/journal.pone.0248298

. 2021 Mar 11;16(3):e0248298. doi: 10.1371/journal.pone.0248298

Ruxolitinib with resminostat exert synergistic antitumor effects in Cutaneous T-cell Lymphoma

Fani Karagianni ^1,^*,^#, Christina Piperi ^2,^#, Vassiliki Mpakou ³, Aris Spathis ⁴, Periklis G Foukas ⁴, Maria Dalamaga ^1,², Vasiliki Pappa ³, Evangelia Papadavid ¹

Editor: Salvatore Papa⁵

PMCID: PMC7951910 PMID: 33705488

Abstract

Background

The combination of JAK/STAT and HDAC inhibitors exerted beneficial effects in haematological malignancies, presenting promising therapeutic CTCL targets. We aim to investigate the efficacy of JAK1/2i ruxolitinib in combination with HDACi resminostat in CTCL in vitro.

Material & methods

Non-toxic concentrations of ruxolitinib and/or resminostat were administered to MyLa (MF) and SeAx (SS) cells for 24h. Cytotoxicity, cell proliferation and apoptosis were estimated through MTT, BrdU/7AAD and Annexin V/PI assay. Multi-pathway analysis was performed to investigate the effect of JAK1/2i and/or HDACi on JAK/STAT, Akt/mTOR and MAPK signalling pathways.

Results

Both drugs and their combination were cytotoxic in MyLa (p<0.05) and in SeAx cell line (p<0.001), inhibited proliferation of MyLa (p<0.001) and SeAx (p<0.001) at 24h, compared to untreated cells. Moreover, combined drug treatment induced apoptosis after 24h (p<0.001) in MyLa, and SeAx (p<0.001). The combination of drugs had a strong synergistic effect with a CI<1. Importantly, the drugs’ combination inhibited phosphorylation of STAT3 (p<0.001), Akt (p<0.05), ERK1/2 (p<0.001) and JNK (p<0.001) in MyLa, while it reduced activation of Akt (p<0.05) and JNK (p<0.001) in SeAx.

Conclusion

The JAKi/HDACi combination exhibited substantial anti-tumor effects in CTCL cell lines, and may represent a promising novel therapeutic modality for CTCL patients.

1. Introduction

Cutaneous T-cell lymphomas (CTCLs) are rare skin malignancies, forming a heterogeneous group of non-Hodgkin lymphomas derived from skin-homing mature T-cells [1]. Mycosis Fungoides (MF) is considered as the commonest type of CTCL, characterized by patches and infiltrated plaques on the skin, which eventually evolve into tumors [2, 3], whereas, Sezary syndrome (SS), the leukemic variant of MF, characterized by erythroderma, lymphadenopathy and the presence of a malignant T-cell clone in the peripheral blood and the skin [3, 4]. In advanced stages, the survival rate is poor (a median survival < 4 years, and only 13 months for those with nodal involvement) and patients often die due to an incompetent immune system [5–8].

MF and SS are considered as different diseases since they arise from distinct T-cell subsets: MF, from skin-resident effector memory T-cells (TRm) and SS, from the central memory T-cells (TCm) [9, 10]. The fundamental difference in the putative cell of origin between SS (TCM-derived) and MF (TRM-derived) is consistent with their distinct clinical behaviors, since TCm may be found in all three compartments, i.e. in peripheral blood, lymph node and skin, while resident TRm-cells remain localized at the skin. Detection of these malignant T-cell clones is critical for the diagnosis of CTCL, with TCm expressing CCR7⁺/L-selectin⁺ and TRm expressing CCR7⁺/L-selectin^- [11]. Recently, the genomic profiling of CTCL has been determined using high-throughput technologies, Next generation sequencing (NGS) and whole genome sequencing (WGS), to identify genetic alterations or abnormal gene expression between healthy controls and patients suffering from CTCL [12–14]. Therefore, many studies sought to explore the genetic landscape of MF/SS and have identified several mutated genes affecting cell cycle regulation, chromatin remodeling and major signal transduction pathways, including the Janus kinase (JAK)-signal transducer and activator of transcription protein (STAT), the T-cell receptor (TCR)/nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and the mitogen-activated protein kinase (MAPK) [14–16].

Dysregulation of the JAK/STAT pathway is implicated in the pathogenesis of solid tumors [17], as well as in some hematologic malignancies where abnormal activation of JAK2 signaling has been reported [18]. Of interest, activation of the JAK-STAT pathway has also been implicated in CTCL progression, leading to constitutive expression of STAT3 and STAT5, upregulation of cell survival (Bcl-2 and Bcl-xL) and cell cycle genes (Cyclin D, c-Myc), Th2 cytokines (IL-4) and mir-155 [19].

Several drugs inhibiting the aforementioned pathway have been shown to exhibit a therapeutic potential, including ruxolitinib (initially FDA-approved as Jakinib^®), a potent JAK1/2 approved for the treatment of primary myelofibrosis, and also studied in psoriasis and rheumatoid arthritis [10]. Ruxolitinib has been shown to cause a dose-dependent inhibition of cell proliferation, with concurrent activation of apoptosis, a marked and rapid inhibition of STAT activation and inhibition in DNA synthesis in CTCL cells [20].

Another class of drugs with potential modulatory effects in MF/SS pathogenesis is the family of Histone Deacetylase (HDACs) inhibitors. HDACs are implicated in a wide range of cellular processes [21–23] including JAK2/STAT3 signaling pathway inhibition through regulation of suppressor of cytokine signaling proteins (SOCS), a family of genes involved in JAK2-STAT3 signaling pathway inhibition [24, 25], while they are also highly upregulated in CTCL [26] and in B- and T- cell lymphoma patients [27].

HDAC inhibitors have been shown to interfere with cell proliferation, apoptosis and migration of neoplastic cells via the down-regulation of several pathways implicated in the activation of cytokines, chemokines, growth factors and protein kinases [28]. Two HDAC inhibitors (HDACi), vorinostat^® and romidepsin^® (FK228) have achieved good clinical efficacy in CTCL with objective responses of 25–30%, leading to the FDA approval of both drugs for the treatment of CTCL in 2006 and 2009, respectively [29]. Currently, resminostat, an HDAC inhibitor targeting HDAC class I, IIb and IV is tested in a phase II clinical trial in Europe and Japan as a maintenance therapy in advanced CTCL (RESMAIN, NCT02953301).

Resminostat was reported to up-regulate a gene expression signature representative of the Th1 cell type, and down-regulate genes of the Th2 cell type, thus favouring the beneficial Th1 cell phenotype. Furthermore, resminostat exhibited a reduction at the mRNA and protein secretion of the Th2 and itch-mediating cytokine IL-31, suggesting that it might improve pruritus [30].

It can be concluded from the above studies that these monotherapies are partially effective in haematological malignancies.

In the present work, we investigated for the first time the antitumor activity of JAK2 ruxolitinib and HDAC inhibitor resminostat in the cytotoxicity, proliferation and apoptosis of CTCL cell lines, along with their impact in major cellular signaling pathways. The combined effect of these drugs was shown to target different upstream and downstream molecules in JAK/STAT pathway.

2. Material and methods

2.1 Cell lines and drugs

The human CTCL cell lines, MyLa and SeAx, were kindly provided by Dr Jan P. Nikolay (Klinik für Dermatologie, Venerologie und Allergologie, Universitätsmedizin Mannheim, Ruprecht-Karls-Universität Heidelberg, Mannheim, Deutschland), already tested and authenticated. Both cell lines were cultured in RPMI 1640, supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin, and treated with/without JAK1/2 inhibitor (15μM ruxolitinib) and/or HDAC inhibitor (1 μM resminostat) at 37°C in a humidified atmosphere with 5% CO₂, for 24h. The HDAC inhibitor was a generous gift from 4SC AG (Germany) and the JAK inhibitor was a gift from Novartis Incyte (USA). Both inhibitors were dissolved in DMSO according to the manufacturer’s instruction. Therefore, vehicle controls or untreated cells were treated with 0.1% DMSO for all the experiments. T cell stimulation was achieved through PMA (50ng/ml) and ionomycin (1μg/ml) treatment prior to the multi-pathway analysis.

2.2 Cytotoxicity assay

The 3-(4,5-dimethylthiazol-2-yl)-2,5-dimethyltetrazolium bromide (MTT) assay (ThermoFisher Scientific) was used to measure the cytotoxicity in untreated and drug-treated CTCL cell lines. Briefly, 10⁴ cells were seeded in 96-well plates with the addition of the appropriate drugs. After 24h, the culture medium was removed and substituted with fresh one, along with MTT solution, for 4 hours and DMSO at 37°C. The absorbance was measured at 570 nm wavelength in a microplate reader (Tecan Infinite F200, Austria).

2.3 BrdU assay

In this method, BrdU (an analog of the DNA precursor thymidine) was incorporated into newly synthesized DNA by cells entering and progressing through the S (DNA synthesis) phase of the cell cycle. The incorporated BrdU (BD Pharmingen) was stained with specific anti-BrdU fluorescent antibody. The levels of cell-associated BrdU were then measured by flow cytometry. Briefly, 10⁵ cells were seeded in 24-well plate for 24h with the presence/absence of specific drugs at the determined concentrations according to the manufacturer’s instruction.

2.4 Apoptosis assay

FACS analysis using the Annexin V/PI staining method was used to determine the apoptotic rate in drug-treated and control cells. Briefly, 10⁵ cells were seeded and cultured with or without the addition of ruxolitinib and/or resminostat at the indicated concentrations for 24h. Cells were afterwards washed with ice-cold PBS and centrifuged at 500×g for 5 min. Cell pellet was re-suspended in ice-cold annexin binding buffer, and annexin-V-FITC antibody, along with propidium iodide (AssayDesigns, U.S.A.) were added and cells were incubated for 10 min in the dark. Samples were run on a Cytomics FC500 Flow Cytometry Analyzer (Beckman Coulter, USA) and cell viability and apoptosis were calculated using a quadrant on the Annexin-V-FITC/PI histogram. Living cells were negatively stained for both assays, early apoptotic cells were only Annexin-V-FITC positive, necrotic cells were only PI positive and late apoptotic cells were positively stained for both assays. The synergistic effect between resminostat and ruxolitinib was determined by the combination index (CI) based on the Chou-Talalay method [31], as previously described [32]. The CI value was determined by the following equation: CI = sum of apoptosis of single agent treatment/apoptosis upon combined treatment. The combination effect was defined as “synergistic”, “additive” or “antagonistic” when CI was <1, 1 and >1, respectively. A combination index (CI) of > 1 indicates antagonism, a CI of 1 denotes additivity, and a CI of < 1 indicates synergism. More specifically, CI values ranging from 0.1–0.3 are considered to indicate strong synergism, 0.3–0.7 synergism, and 0.7–0.85 moderate synergism.

2.5 Multipathway panel analysis

Pellets from stimulated cells with PMA (50ng/ml) and ionomycin (1μg/ml) were collected at 24h and 48h post drug treatment, lysed and further subjected to multipathway analysis (Millipore, MILLIPLEX MAP Multi-Pathway Magnetic Bead) of JAK/STAT [p-STAT3 (ser427), p-STAT5(Tyr694/699)], Akt/mTOR [p-Akt (Ser473), p-p70S6K (Thr412)] and MAP kinase [p-ERK1/2 (Thr185/Tyr187), p-p38 (Thr180/Tyr182), p-JNK (Thr183/Tyr185) and p-NF-κB (Ser536 p65 subunit)] signalling pathways, after the aforementioned drug treatments for 24h and 48h using Luminex 200TM (Luminex Corporation Austin, TX, USA) technology, according to the manufacturer’s instructions.

2.6 Statistical analysis

Statistical analyses were performed using the SPSS software (version 16.0; SPSS, Inc., Chicago, IL., USA). All experiments were performed in triplicates, in 3 independent repeats, and the results were presented as the mean ± standard deviation. One-way Anova and Bonferroni methods were used to compare the means among the different groups. A two-sided p value <0.05 was considered statistically significant.

3. Results

3.1 Resminostat and/or ruxolitinib induce cytotoxicity in CTCL cell lines

Different concentrations (0.3μΜ, 1μΜ and 3μΜ) of resminostat were initially tested in both CTCL cell lines used (S1 Fig), in order to determine cytotoxicity. Among the three concentrations used, only 1μΜ and 3μΜ resminostat significantly reduced the cell viability of MyLa and SeAx cell lines at 24h compared to cells treated with 0.1% DMSO (S1A Fig). Between the two concentrations of 1μΜ and 3μΜ resminostat, no significant differences were found. Therefore, the concentration of 1μΜ was selected for further combination experiments.

In accordance to resminostat, different concentrations (1μM, 15μM and 30μM) of ruxolitinib were also tested in CTCL cell lines (S1B Fig) Among the three concentrations used, only the concentration of 15μΜ and 30μΜ ruxolitinib significantly reduced the cell viability of MyLa and SeAx cell lines at 24h compared to cells treated with 0.1% DMSO. Between the two concentrations of 15μΜ and 30μΜ ruxolitinib, no significant differences were found. Therefore the concentration of 15μM was selected for further combination experiments.

The combination of 1μM resminostat/15 μΜ ruxolitinib was shown to significantly reduce the cell growth of MyLa cells (Fig 1A) after 24h (p<0.05) of treatment compared to control, whereas in SeAx cell line, a statistically significant reduction was obtained also at 24h (p<0.001) (Fig 1B).

3.2 Combined treatment of resminostat and ruxolitinib induces cell cycle arrest

To further investigate the inhibitory effects of resminostat and/or ruxolitinib on the proliferation of CTCL cell lines, we examined the cell cycle distribution after a 24h treatment with resminostat and/or ruxolitinib (Fig 1C and 1D). The percentages of treated MyLa and SeAx cells, with either resminostat and/or ruxolitinib was higher in G0/G1 phase compared to control cells at significant level (p<0.001 for MyLa and p<0.05 for SeAx). There was a further increase in the percentage of the treated cells with both drugs, and this increase was even higher in the combination therapy (p<0.001). No significant changes were observed in G2/M phase. On the contrary, as expected, there was a significant decrease in S phase of both cell lines in monotherapies and combination therapy compared to untreated cells indicating that these drugs inhibit cell proliferation of MyLa and SeAx cell line at 24h of treatment.

3.3 Combined treatment of resminostat and ruxolitinib induces apoptosis in CTCL cell lines

Following the effect of resminostat and ruxolitinib in cell cycle proliferation, we proceeded to investigate their impact on apoptosis’ induction (Fig 2).

Treatment of MyLa cells with either resminostat or ruxolitinib did not exhibit any significant effects on apoptosis after 24h (Fig 2A and 2B). However, their combination significantly increased early apoptotic (22.1%, p<0.001) and late apoptotic cell numbers (22%, p<0.001) at the 24h time point, compared to control cells (Fig 2A and 2B).

Similarly, single treatment of SeAx cells with resminostat or ruxolitinib did not seem to affect apoptotic cell numbers after 24h (Fig 2C and 2D). However, their combination significantly increased early apoptotic (19.1%, p<0.001) and late apoptotic cells (14.5%, p<0.001) at the 24h post-treatment time point, compared to control cells (Fig 2C and 2D).

The combination treatment has synergistic effects in both cell lines (Fig 3). In MyLa cells CI was 0.062 at 24h post treatment, whereas in SeAx cells, the CI was 0.141 at 24h. These values indicate strong synergism for both cell lines.

3.4 Inhibition of key signalling pathways after exposure to ruxolitinib, resminostat and their combination

In order to further investigate the signal transduction pathways that are activated by single or combinational resminostat and ruxolitinib treatment, we performed a multi-pathway analysis in both cell lines at two different time points.

Resminostat treatment of MyLa cells resulted in decreased Akt (p<0.001) phosphorylation levels both at 24h and 48h, compared to control cells (Fig 4B), while ruxolitinib significantly reduced p-Akt (p<0.001, Fig 4B), p-p70S6K (p<0.05, Fig 4F) and p-p38 (p<0.05, Fig 4G) levels at 24h compared to controls. Ruxolitinib treatment significantly reduced p-STAT3 (p<0.001, Fig 4A) and p-JNK (p<0.001, Fig 5D) levels at 48h post-treatment.

Fig 5 — Effect of resminostat and/or ruxolitinib in the activation of key signalling pathways in SeAx cells. Values are the mean ± standard deviation of two experiments run in triplicates. * p<0.05, **p<0.001 statistically significant differences versus untreated and single drugs or combination.

Interestingly, in MyLa cells the combination of the two drugs was found to significantly decrease p-STAT3 (p<0.001, Fig 4A), p-AKT (p<0.001, Fig 4B), p-ERK1/2 (p<0.045) and p-JNK (p<0.001, Fig 4D) at 24h, compared to controls. Furthermore, a significant reduction was observed at p-STAT3 (p<0.001, Fig 4A), p-AKT (p<0.001, Fig 4B), p-JNK (p<0.001, Fig 4D) and p-p38 (p<0.001, Fig 4G) at 48h post-treatment, compared to control cells.

In the SeAx cell line, resminostat treatment did not show any significant effects in the activated signaling pathways compared to control cells neither at 24h nor 48h after treatment. However, ruxolitinib treatment was shown to reduce p-JNK levels (p<0.001, Fig 5D) at 24h compared to control cells. Of note, the combination of the two drugs was found to significantly decrease p-AKT (p<0.05, Fig 5B) at 24h and at 48h and p-JNK (p<0.001, Fig 5D) only at 24h compared to controls (Fig 5).

4. Discussion

Current treatment approaches for MF/SS are not curative and present high rates of relapse. Patients with relapsed or refractory MF/SS display a poor prognosis, and a need for more efficient treatment options. The only curative option in MF/SS is stem cell transplantation [33]. Complete responses with existing monotherapies are still rare and data on survival under approved therapies are yet to be collected. The development of standardized approaches for the treatment of advanced disease, resistant and recurrent disease and especially standardized strategies for maintenance therapy of these patients in remission are at present a particularly interesting area of research [32]. Therefore, there is a need to develop new drugs or use of repurposed drugs that can provide more durable responses, and improve the poor outcomes of patients with advanced stage MF and SS.

Our results demonstrate for the first time that the combination of ruxolitinib, a JAK1/2i with resminostat, a HDACi demonstrates efficacy in an in vitro model of CTCL, highlighting its significance as a promising novel therapeutic modality for CTCL patients that fail to benefit from monotherapy.

More specifically, we found that ruxolitinib and resminostat, as monotherapies, manage to inhibit cell viability of both CTCL cell lines tested. Moreover, their combination significantly reduced the cell viability of MyLa and SeAx cells after both 24h of treatment. In line with this observation, previous studies have already shown that resminostat treatment inhibits proliferation of multiple myeloma cells [34]. Moreover, Civallero et al. have demonstrated the antitumor activity of ruxolitinib and vorinostat^®, another HDAC inhibitor, in cell lines of hematologic malignancies, including Hodgkin’s lymphoma and selected subtypes of non-Hodgkin’s lymphoma, multiple myeloma and CTCL [35]. These cell lines exhibited sensitivity to ruxolitinib and vorinostat^® mono-therapy, while their combination was shown to synergistically increase their inhibitory effects and induce a cessation of growth in tumor cells. This finding agrees with our results which highlight the beneficial effect of combinational ruxolitinib and resminostat therapy in inhibiting CTCL cell survival. Most importantly, resminostat which exhibits a particular specificity towards HDAC6, in combination with ruxolitinib may present a therapeutic advantage in overcoming resistance.

Furthermore, the drugs’ combinational treatment appeared most effective in inhibiting cell proliferation, as well as inducing apoptosis while increasing late apoptosis at 24h in both CTCL cell lines tested, compared to monotherapies. Our findings are in line with previous published data from Civallero et al. demonstrating that the combination of ruxolitinib and vorinostat^® induced apoptosis in 12 cell lines of hematological malignancies, including a CTCL cell line [36]. In addition, another study showed that ruxolitinib alone enhanced apoptosis in MyLa cells, with an increase of almost 20% [20]. Our data also revealed a differential response of MyLa and SeAx cells to ruxolitinib/resminostat combinational treatment, in terms of apoptosis. Indeed, the percentage of late apoptotic MyLa cells was found to be 22% after 24h of treatment, whereas late apoptotic SeAx cells reached only 14.5%. Taken together, these data indicate a potential debulking activity for the drug combination.

Combined treatment of resminostat with ruxolitinib was shown to have a strong synergistic effect at both CTCL cell lines with a CI = 0.062 for MyLa cells and CI = 0.141 for SeAx cells. Moreover, our data revealed that the two drugs exhibit differential profiles of inhibition in terms of key signaling molecules activation in the CTCL cell lines tested, thus further confirming that MF and SS should be considered as different diseases, arising from distinct T-cell subsets [9, 34, 37]. Malignant T cells present variations in the activation of cellular signaling due to extensive crosstalk between different signal transduction pathways.

It is evident from our data that the resminostat/ruxolitinib drug combination affects the activation of Akt and JNK in both cell lines, whereas it also inhibits JAK/STAT and MAPK activation in MF cell line, a finding that further indicates the differential genetic and epigenetic mechanisms implicated in MF and SS.

In the MyLa cell line, we have shown that ruxolitinib alone or in combination with resminostat led to the inhibition of STAT3 phosphorylation, 48h post-treatment. These observations are in accordance with previous results demonstrating that STAT3 and STAT5 are constitutively expressed in CTCL [19]. We found that the combination of ruxolitinib and resminostat inhibited the phosphorylation of STAT3 and blocked JAK/STAT pathway. This could be mainly attributed to ruxolitinib’s inhibitory effect on JAK2, and may also be the additive result of resminostat targeting on the JAK pathway. On the contrary, Perez et al. observed a marked and rapid inhibition of STAT activation with ruxolitinib that was abolished after 3h of treatment [20].

Furthermore, we showed that Akt phosphorylation was decreased after 24h of treatment with either ruxolitinib or resminostat in MyLa cell line, whereas their combination was more effective than monotherapies at 48h. The observed inhibition of Akt phosphorylation might have also contributed to the inhibition of cell proliferation and the induction of apoptosis in both MF and SS cell lines tested an issue that requires further studies, mostly due to the involvement of PI3K/Akt/mTOR pathway in autophagy and also the implication of HDAC6 in the same process. Similarly, the combination of jakitinib^® and vorinostat^® has been previously shown to affect autophagy in cell lines of hematological malignancies [38]. We have previously shown that activation of AKT/mTOR pathway in MF is correlated with NOTCH1, p-ERK, and p-STAT3 and is implicated in the acquisition of a more aggressive phenotype. Moreover, the combination of p-AKT, p-p70S6K, and p-4E-BP1 emerged, from our observations, as a significant potential prognostic marker in patients with advanced disease stage [35, 38]. In the CTCL cell lines tested herein, a small decrease in the phosphorylation of the downstream p-70S6K protein was observed upon treatment, indicating a possible involvement of PI3K signaling pathway, instead of mTOR.

Regarding the phosphorylation of MAP kinases in MyLa cell line, marked changes in p-ERK1/2 and p-p38 were observed only after treatment with the drugs’ combination for 24h and 48h, respectively. In myeloproliferative neoplasms (MPN), p-ERK was shown to be blocked by ruxolitinib, whereas p-p38 seems to remain unaffected [37]. On the other hand, Klemke et al. had shown that all CTCL cell lines exhibit ERK phosphorylation 5min after TCR stimulation, but this is not detectable after prolonged (45min) TCR stimulation [39].

Furthermore, we found that the JNK phosphorylation was decreased upon drug combination at 24h and 48h of treatment, indicating the importance of JNK signaling in MyLa cells [40] and its targeting potential in MF treatment that needs to be further investigated. A previous study reported the production of high levels of VEGF through JAK3- and JNK-dependent signal pathways from malignant T cells in CTCL, thus revealing potential new therapeutic targets in CTCL [39]. On the other hand, Cardoso et al. have demonstrated that the incubation of MPN cells with BM-derived stroma impairs the cytotoxic action of vorinostat^® and ruxolitinib combination, an effect possibly attributed to the activation of relevant survival pathways, including JNK and PI3K [40]. Therefore, inhibition of these pathways in the same system may abrogate the protective effects exerted by the stroma. These results point towards a novel therapeutic approach for the treatment of MPN patients, which relies on the dual targeting of both neoplastic clones and the microenvironmental cues.

In the SeAx cell line, the inhibition of signaling pathways upon single or combined ruxolitinib/resminostat drug treatment was less prominent. No significant inhibition of STAT3 was achieved upon drug treatment at any time point, possibly because STAT3 phosphorylation may be restored by alternative kinase families, such as MAPK and c-Jun [41]. However, ruxolitinib alone or in combination with resminostat was shown very effective in reducing p-JNK levels after 24h of treatment.

Most important, the ruxolitinib/resminostat drug combination significantly decreased p-AKT levels at 24h and 48h, in SeAx cells, indicating an important target for future treatments that needs to be further investigated. Previous studies from our group and others have also detected high levels of phosphorylated Akt in CTCL patients [35, 42–44], highlighting the importance of using AKT/mTOR signaling pathway-related targets in CTCL treatment [44, 45].

Conclusion

Collectively, our data demonstrate for the first time the combinational inhibitory effect of ruxolitinib and resminostat in the proliferation and apoptosis of CTCL cell lines and further reveal the importance of AKT, ERK1/2 and JNK signalling as potential targets for CTCL disease management. These encouraging results are based on in vitro observations; therefore, they need to be further confirmed in primary patient-derived cells (cultured isolated T lymphocytes from peripheral blood and skin biopsies) in preclinical and clinical studies, validating the efficacy of this therapeutic strategy in vitro. Moreover, the synergistic impact of the proposed drug combination on oncogenic transformation could enable the effective use of lower doses of these agents, leading to better tolerability, and hopefully, avoidance or delay of the development of drug resistance.

Supporting information

S1 Fig. Testing various concentration of ruxolitinib and resminostat for cell cytotoxicity in CTCL at various concentrations at 24h.

Values are the mean ± standard deviation of three experiments. *p <0.05, statistically significant differences versus untreated and single drugs.

(TIF)

Click here for additional data file.^{(470.1KB, tif)}

Data Availability

All relevant data are within the manuscript.

Funding Statement

The author(s) received no specific funding for this work.

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PLoS One. doi: 10.1371/journal.pone.0248298.r001

Decision Letter 0

Salvatore Papa

11 Aug 2020

PONE-D-20-21841

Ruxolitinib with resminostat exert synergistic antitumor effects in Cutaneous T-cell Lymphoma

PLOS ONE

Dear Dr. Karagianni,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please revise the use of MTT assay as a cell proliferation assay. As correctly stated by the reviewer this assay is a cytotoxicity assay rather than a proliferative assay. To determine the effect on cell proliferation the authors are encouraged to preform cell cycle analyses with PI and BrdU staining and analyses of cyclin expression by immunoblots WB analyses.

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Reviewer #1: Yes

Reviewer #2: Partly

**********

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #2: No

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Reviewer #2: Yes

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Reviewer #1: The author here shows an interesting finding of synergistic effect of ruxolitinib and resminostat to inhibit proliferation and induce apoptosis on Cutaneous T-cell lymphomas. Although there are certain concerns I would like the author to address:

1. Figure 1B shows cell survival of SeAx cells around 60% at 24h whereas 80% at 48h for combination treatment with 1uM resm/15 uM rux This is surprising as mostly in case for any anticancer drug, cell death should be increasing with increase in time point, or we may say cell survival should be decreasing. Also, its contradicting to the result obtained from Apoptosis study in Figure 3, where percentage of apoptotic cells (early + late) are more in 48h (almost 60%) compared to 24 h (35%). The author should explain this discrepancy.

2. Although the author have written a detailed discussion of their finding, but it would be better for the manuscript to have a

short (conclusion section to summarize the manuscript for ease of an reader) separately following the discussion and before author contribution. Not just as a part of abstract section.

3. I would prefer the author to mention in full before using abbreviations like WGS for Whole genome sequencing or NGS for Next-Generation Sequencing, if they mean so. (Introduction section, page 11 line 68).

4. The figures representations could be improved (eg. In fig 3B and fig 3D the asterisk marks does not have consistent font size within or in comparison as well).

5. Each figure caption could start with a title in bold (to explain what this data/figure is about in short) then followed by the legend. This helps the reader to quick glance through. (eg., Fig 1. Cell viability assay of resminostat and/or ruxolitinib on CTCL. Effect of resminostat and/or ruxolitinib in cell proliferation in Myla (A) and SeAx (B) cell lines at 24h and 48h. Values are the mean � standard deviation of three experiments. *p <0.05, **p<0.001 statistically significant differences versus untreated and single drugs or combination.

Reviewer #2: This manuscript describes an interesting study, reporting the induction of apoptosis in 2 lymphoma cell lines following treatment with specific concentrations of resminostat and ruxolitinib alone and in combination. However, the manuscript is lacking 3 independent repeats of all data and despite a clear increase in efficacy when resminostat and ruxolitinib are employed in combination as opposed to their use as individual compounds, to describe the result as synergistic is incorrect as this has not been investigated in the study. Describing the MTT as a proliferation assay is also incorrect. However, the assessment of cell cycle using annexin v/PI staining and the status of proteins in a variety of cell signalling pathways, using the MILLIPLEX MAP Multi-Pathway Magnetic Bead is sound.

The authors refer to a poor survival rate (line 57) for those with lymphoma (MF). Please can the authors include some survival statistics so it is clear to the reader the current outcome for these patients. Furthermore, since the study describes putative new therapies for these patients, please can the authors include details of the current treatment strategies, why these are inadequate and therefore why new treatments are necessary. The authors report previous work revealing the importance of a variety of signalling pathways, including the JAK-STAT pathway in MF/SS, and importantly suggest the use of inhibitors targeting this pathway as a possible therapeutic strategy. However, it is unclear as to why the authors have chosen to examine the combined effect of two compounds targeting the same pathway and proteins. It would be useful to have this explained more clearly in the introduction. Since it is unlikely these compounds would be employed as a monotherapy, it would be important to establish if any synergy exists with current therapies. This should be addressed in the discussion.

Although the authors described decreased “proliferation” of the cells in response to combination treatment, in this study the MTT assay has been employed, which does not measure the proliferation rate of cells. The MTT assay is a colorimetric assay which merely reflects the number of cells present based on the activity of the mitochondria. It is a major error to refer to this data as measuring proliferation.

Although in line with previous data from the literature, the authors describe data from 1 cell line from a MF and a SS lymphoma. They report the data as an “in vitro model of CTCL”, which is misleading. It should be acknowledged that these findings need to be validated in a wider panel of cells, preferably primary patient derived cells, to confirm the efficacy of this therapeutic strategy in vitro. i.e. expand comment on line 338.

It is unclear as to the vehicle solution in which the drugs have been dissolved. Furthermore, it is not clear as to whether vehicle control treated cells have been used as the “control” cells (line 175) or are these really completely “untreated” cells (line 174). If a vehicle treated cell control has not been employed, this seriously limits the impact of the findings described throughout the study as it is wholly incorrect.

The statistics in this manuscript have been performed correctly, however, data from multiple independent repeats must be presented with standard error of the mean as opposed to standard deviation (line 163). Furthermore, some conclusions have been formed from only 2 independent repeats of experiments (line 162 “at least twice”). It is most appropriate to run 3 independent repeats of all experiments.

The authors describe a decrease in the number of cells and increased apoptosis following treatment with both resminostat and ruxolitinib, showing this drug combination represents an interesting strategy for investigation. However, in places throughout the manuscript, including the title, the authors refer to synergy between the two compounds (line 245, 339). Synergy between the inhibitors has not been shown or investigated in this manuscript. To examine synergistic or additive effects between 2 compounds, a range of concentrations of both drugs must be investigated alone and in combination using the CalcuSyn analyser of combined drug effects. It is wrong to suggest synergy has been observed in this study.

The authors refer to testing the efficacy of resminostat and ruxolitinib across a range of concentrations. Please can this data be included to explain why the concentrations of 1um and 15um, respectively, were chosen for future experiments. What was the reason for this choice of concentration?

The manuscript is written in standard English. There are some small errors for correction:

Line 197: “same wise” – please find an alternative.

Line 307: typo “\\”

Line 265: extra space

Line 267: extra comma

Throughout: et al. must be in italics

**********

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Reviewer #1: Yes: Dr. Arindam Pramanik

Reviewer #2: No

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PLoS One. 2021 Mar 11;16(3):e0248298. doi: 10.1371/journal.pone.0248298.r002

Author response to Decision Letter 0

6 Feb 2021

Dear Editor-in-Chief,

We would like to thank you and the reviewers for your thoughtful evaluation of our manuscript and for your most welcome comments/suggestions. Accordingly, we have now revised our manuscript to reflect these comments.

In the revised Text all changes/additions/modifications made in response to the Reviewers’ points are marked with track changes.

Please find below a point-by-point response to the issues raised by the Reviewers:

Academic Editor:

Please revise the use of MTT assay as a cell proliferation assay. As correctly stated by the reviewer this assay is a cytotoxicity assay rather than a proliferative assay. To determine the effect on cell proliferation the authors are encouraged to perform cell cycle analyses with PI and BrdU staining and analyses of cyclin expression by immunoblots WB analyses.

RESPONSE: We would like to thank the academic editor for this comment. We have revised the use of MTT as a proliferation assay to cytotoxicity assay and we have also performed cell cycle analyses with 7AAD and BrdU staining. Data are shown in Figure 1.

Due to time restriction, we have not performed cyclin expression, but we believe that 7AAD/BrdU analysis clearly demonstrated the effect of the two agents in cell proliferation.

Reviewer #1:

The author here shows an interesting finding of synergistic effect of ruxolitinib and resminostat to inhibit proliferation and induce apoptosis on Cutaneous T-cell lymphomas. Although there are certain concerns, I would like the author to address:

1. Figure 1B shows cell survival of SeAx cells around 60% at 24h whereas 80% at 48h for combination treatment with 1uM resm/15 uM rux. This is surprising as mostly in case for any anticancer drug, cell death should be increasing with increase in time point, or we may say cell survival should be decreasing. Also, its contradicting to the result obtained from Apoptosis study in Figure 3, where percentage of apoptotic cells (early + late) are more in 48h (almost 60%) compared to 24 h (35%). The author should explain this discrepancy.

Response: We have re-evaluated the effect of ruxolitinib and resminostat in cell proliferation using the 7AAD/BrdU staining and provide the new data in the revised manuscript. Based on these experiments, cell viability, cell proliferation and apoptotic data are in agreement (Figures 1 and 2).

2. Although the author have written a detailed discussion of their finding, but it would be better for the manuscript to have a short (conclusion section to summarize the manuscript for ease of an reader) separately following the discussion and before author contribution. Not just as a part of abstract section.

Response: We have included a conclusion section following the discussion.

3. I would prefer the author to mention in full before using abbreviations like WGS for Whole genome sequencing or NGS for Next-Generation Sequencing if they mean so. (Introduction section, page 11 line 68).

Response: The full descriptions of abbreviations are provided (line 76-77).

4. The figures representations could be improved (eg. In fig 3B and fig 3D the asterisk marks does not have consistent font size within or in comparison as well).

Response: The figure representations were improved and the asterisks in Figure 3 (B and D) were corrected with consistent font size.

Response: All figure captures have been corrected as proposed by the reviewer (figure legend file).

Reviewer #2:

This manuscript describes an interesting study, reporting the induction of apoptosis in 2 lymphoma cell lines following treatment with specific concentrations of resminostat and ruxolitinib alone and in combination.

1. However, the manuscript is lacking 3 independent repeats of all data and despite a clear increase in efficacy when resminostat and ruxolitinib are employed in combination as opposed to their use as individual compounds, to describe the result as synergistic is incorrect as this has not been investigated in the study.

Response: All experiments performed in triplicates and three independent repeats were performed. The authors corrected this statement at line 198-199.

We have also determined the synergistic activity of resminostat and ruxolitinib by calculating the combination index (CI). The synergistic activity of the two drugs (CI<1 for all combinations) is described in the revised manuscript [see sections Materials and Methods (line 174-183), Results (line 270-272), Discussion (line 353-355) and Figure 3.

Dr Maria Dalamaga has performed the synergistic experiments and she has been added as a co-author to this manuscript.

2. Describing the MTT as a proliferation assay is also incorrect. However, the assessment of cell cycle using annexin v/PI staining and the status of proteins in a variety of cell signalling pathways, using the MILLIPLEX MAP Multi-Pathway Magnetic Bead is sound.

Response: ΜΤΤ is a colorimetric assay for assessing cell metabolic activity. Tetrazolium dye assays can also be used to measure cytotoxicity (loss of viable cells) or cytostatic activity (shift from proliferation to quiescence) of potential medicinal agents and toxic materials. However, the authors agreed to assess the proliferation (rapidly dividing cells) by performing cell cycle analysis with 7AAD and BrdU. The corresponding method is described in Material and Methods section (line 153-160), the data are described in Results Section (line 237-249) and in Discussion section (line 340-341). These experiments were performed by Aris Spathis and Periklis G. Foukas, and have been added as co-authors in the revised manuscript.

3. The authors refer to a poor survival rate (line 57) for those with lymphoma (MF). Please can the authors include some survival statistics, so it is clear to the reader the current outcome for these patients.

Response: On line 64-65, statistics of the poor survival for the advanced stage disease have been added.

4. Furthermore, since the study describes putative new therapies for these patients, please can the authors include details of the current treatment strategies, why these are inadequate and therefore why new treatments are necessary. The authors report previous work revealing the importance of a variety of signalling pathways, including the JAK-STAT pathway in MF/SS, and importantly suggest the use of inhibitors targeting this pathway as a possible therapeutic strategy. However, it is unclear as to why the authors have chosen to examine the combined effect of two compounds targeting the same pathway and proteins. It would be useful to have this explained more clearly in the introduction. Since it is unlikely these compounds would be employed as a monotherapy, it would be important to establish if any synergy exists with current therapies. This should be addressed in the discussion.

Response: The authors have added a more descriptive paragraph at the beginning of the Discussion (line 303-315) and at the end of Introduction (line 125-126).

5. Although the authors described decreased “proliferation” of the cells in response to combination treatment, in this study the MTT assay has been employed, which does not measure the proliferation rate of cells. The MTT assay is a colorimetric assay which merely reflects the number of cells present based on the activity of the mitochondria. It is a major error to refer to this data as measuring proliferation.

Response: As it has been mentioned above, the authors agreed and assessed the proliferation assay by performing cell cycle analysis with 7AAD and BrdU.

6. Although in line with previous data from the literature, the authors describe data from 1 cell line from a MF and a SS lymphoma. They report the data as an “in vitro model of CTCL”, which is misleading. It should be acknowledged that these findings need to be validated in a wider panel of cells, preferably primary patient derived cells, to confirm the efficacy of this therapeutic strategy in vitro. i.e. expand comment on line 338.

Response: In current line 432-434 (previous line 338) the phrase ‘…. in primary patient derived cells (cultured isolated T lymphocytes from peripheral blood and skin biopsies)…. validating the efficacy of this therapeutic strategy in vitro….’ was added.

7. It is unclear as to the vehicle solution in which the drugs have been dissolved. Furthermore, it is not clear as to whether vehicle control treated cells have been used as the “control” cells (line 175) or are these really completely “untreated” cells (line 174). If a vehicle treated cell control has not been employed, this seriously limits the impact of the findings described throughout the study as it is wholly incorrect.

Response: On line 139-142, a description was added where it is stated where the drugs had been dissolved. All cells that were characterized as ‘untreated’, where cells that were treated with 0.1% DMSO (vehicle control).

8. The statistics in this manuscript have been performed correctly, however, data from multiple independent repeats must be presented with standard error of the mean as opposed to standard deviation (line 163). Furthermore, some conclusions have been formed from only 2 independent repeats of experiments (line 162 “at least twice”). It is most appropriate to run 3 independent repeats of all experiments.

Response: On line 199, the authors changed the phrase ‘at least twice’ by the phrase ‘in 3 independent repeats’.

9. The authors describe a decrease in the number of cells and increased apoptosis following treatment with both resminostat and ruxolitinib, showing this drug combination represents an interesting strategy for investigation. However, in places throughout the manuscript, including the title, the authors refer to synergy between the two compounds (line 245, 339). Synergy between the inhibitors has not been shown or investigated in this manuscript. To examine synergistic or additive effects between 2 compounds, a range of concentrations of both drugs must be investigated alone and in combination using the CalcuSyn analyser of combined drug effects. It is wrong to suggest synergy has been observed in this study.

Response: The authors added the following sentence: ‘The synergistic effect between resminostat and ruxolitinib was determined by the combination index (CI) based on the Chou-Talalay method [31], as previously described [32]. The CI value was determined by the following equation: CI=sum of apoptosis of single agent treatment/apoptosis upon combined treatment. The combination effect was defined as “synergistic”, “additive” or “antagonistic” when CI was <1, 1 and >1, respectively’. A combination index (CI) of > 1 indicates antagonism, a CI of 1 denotes additivity, and a CI of < 1 indicates synergism. More specifically, CI values ranging from 0.1–0.3 are considered to indicate strong synergism, 0.3–0.7 synergism, and 0.7–0.85 moderate synergism. (line 174-183).

10. The authors refer to testing the efficacy of resminostat and ruxolitinib across a range of concentrations. Please can this data be included to explain why the concentrations of 1um and 15um, respectively, were chosen for future experiments. What was the reason for this choice of concentration?

Response: The authors have added the MTT assay with the various concentrations of both drugs to choose the concentrations for the combined experiments. Therefore, supplementary figure 1 has been added.

11. The manuscript is written in standard English. There are some small errors for correction:

Line 197: “same wise” – please find an alternative.

Line 307: typo “\\”

Line 265: extra space

Line 267: extra comma

Throughout: et al. must be in italics

Response: We have performed a grammar and spell check throughout the manuscript.

In more detail:

Previous line 197: line 262 changed to ‘similarly’

Previous line 307: line 399, the typo ‘/’ was deleted

Previous line 265: extra space on line 256 was deleted

Previous line 267: extra comma on line 359 was deleted

Et al. was changed to italics throughout the manuscript.

Trusting that we have adequately addressed the Reviewers’ concerns, we would like to thank you for your help in significantly improving our work.

With kind regards,

Fani Karagianni

Corresponding author

Attachment

Submitted filename: PONE-D-20-21841R1 -ANSWERS TO REVIEWERS COMMENTS.doc

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PLoS One. doi: 10.1371/journal.pone.0248298.r003

Decision Letter 1

Salvatore Papa

24 Feb 2021

Ruxolitinib with resminostat exert synergistic antitumor effects in Cutaneous T-cell Lymphoma

PONE-D-20-21841R1

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PLoS One. doi: 10.1371/journal.pone.0248298.r004

Acceptance letter

Salvatore Papa

2 Mar 2021

PONE-D-20-21841R1

Ruxolitinib with resminostat exert synergistic antitumor effects in Cutaneous T-cell Lymphoma

Dear Dr. Karagianni:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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on behalf of

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Associated Data

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

Supplementary Materials

S1 Fig. Testing various concentration of ruxolitinib and resminostat for cell cytotoxicity in CTCL at various concentrations at 24h.

Values are the mean ± standard deviation of three experiments. *p <0.05, statistically significant differences versus untreated and single drugs.

(TIF)

Click here for additional data file.^{(470.1KB, tif)}

Attachment

Submitted filename: PONE-D-20-21841R1 -ANSWERS TO REVIEWERS COMMENTS.doc

Click here for additional data file.^{(63KB, doc)}

Data Availability Statement

All relevant data are within the manuscript.

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PERMALINK

Ruxolitinib with resminostat exert synergistic antitumor effects in Cutaneous T-cell Lymphoma

Fani Karagianni

Christina Piperi

Vassiliki Mpakou

Aris Spathis

Periklis G Foukas

Maria Dalamaga

Vasiliki Pappa

Evangelia Papadavid

Roles

Abstract

Background

Material & methods

Results

Conclusion

1. Introduction

2. Material and methods

2.1 Cell lines and drugs

2.2 Cytotoxicity assay

2.3 BrdU assay

2.4 Apoptosis assay

2.5 Multipathway panel analysis

2.6 Statistical analysis

3. Results

3.1 Resminostat and/or ruxolitinib induce cytotoxicity in CTCL cell lines

Fig 1. Cell viability of resminostat and/or ruxolitinib in CTCL.

3.2 Combined treatment of resminostat and ruxolitinib induces cell cycle arrest

3.3 Combined treatment of resminostat and ruxolitinib induces apoptosis in CTCL cell lines

Fig 2. Apoptotic effect of resminostat and/or ruxolitinib in CTCL cell line.

Fig 3. Synergistic effect of resminostat and ruxolitinib in CTCL cell lines.

3.4 Inhibition of key signalling pathways after exposure to ruxolitinib, resminostat and their combination

Fig 4. Activation of key pathways in a Mycosis Fungoides cell line.

Fig 5. Activation of key pathways in a Mycosis Fungoides cell line.

4. Discussion

Conclusion

Supporting information

Data Availability

Funding Statement

References

Decision Letter 0

Salvatore Papa

Roles

Author response to Decision Letter 0

Decision Letter 1

Salvatore Papa

Roles

Acceptance letter

Salvatore Papa

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

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