Contribution of STAT3 and RAD23B in Primary Sézary Cells to Histone Deacetylase Inhibitor FK228 Resistance

ORIGINAL ARTICLE

Contribution of STAT3 and RAD23B in Primary Se´zary Cells to Histone Deacetylase Inhibitor FK228 Resistance Rosie M. Butler1, Robert C. McKenzie1, Christine L. Jones1, Charlotte E. Flanagan1, Wesley J. Woollard1, Maria Demontis1, Silvia Ferreira1, Isabella Tosi1, Susan John2, Sean J. Whittaker1 and Tracey J. Mitchell1 FK228 (romidepsin) and suberoylanilide hydroxamic acid (vorinostat) are histone deacetylase inhibitors (HDACi) approved by the US Food and Drug Administration for cutaneous T-cell lymphoma (CTCL), including the leukemic subtype Se´zary syndrome. This study investigates RAD23B and STAT3 gene perturbations in a large cohort of primary Se´zary cells and the effect of FK228 treatment on tyrosine phosphorylation of STAT3 (pYSTAT3) and RAD23B expression. We report RAD23B copy number variation in 10% (12/119, P
0.01) of SS patients, associated with reduced mRNA expression (P ¼ 0.04). RAD23B knockdown in a CTCL cell line led to a reduction in FK228-induced apoptosis. Histone deacetylase inhibitor treatment significantly reduced pYSTAT3 in primary Se´zary cells and was partially mediated by RAD23B. A distinct pattern of RAD23B-pYSTAT3 coexpression in primary Se´zary cells was detected. Critically, Se´zary cells harboring the common STAT3 Y640F variant were less sensitive to FK228-induced apoptosis and exogenous expression of STAT3 Y640F, and D661Y conferred partial resistance to STAT3 transcriptional inhibition by FK228 (P
0.0024). These findings suggest that RAD23B and STAT3 gene perturbations could reduce sensitivity to histone deacetylase inhibitors in SS patients. Journal of Investigative Dermatology (2019) 139, 1975e1984; doi:10.1016/j.jid.2019.03.1130

INTRODUCTION Se´zary syndrome (SS) is the leukemic variant of cutaneous Tcell lymphoma (CTCL), is associated with poor prognosis (Kim et al., 2005), and is often refractory to treatment. The histone deacetylase (HDAC) inhibitors (HDACis) FK228 (romidepsin) and suberoylanilide hydroxamic acid (SAHA) (vorinostat) are approved by the US Food and Drug Administration for relapsed or refractory CTCL, and a response is seen in 30% of patients (Whittaker et al., 2010). The biological pathways by which HDACis exert their clinical efficacy are incompletely understood, and there is a clinical need to predict response. Increased expression of RAD23B (also known as HR23B) has been reported as a sensitivity determinant for SAHA response and proposed as a biomarker for tumor sensitivity to HDACi therapy (Fotheringham et al., 2009; Khan et al., 2010). RAD23B was first purified in a nucleotide excision repair complex and was named due to its homology to the

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St. John’s Institute of Dermatology, King’s College London, Guy’s Hospital, London, UK; and 2Department of Immunology, Infection and Inflammatory Disease, King’s College London, Guy’s Hospital, London, UK Correspondence: Tracey Mitchell, St. John’s Institute of Dermatology, School of Basic and Medical Biosciences, King’s College London, Guy’s Hospital, London, SE1 9RT, UK. E-mail: [email protected] Abbreviations: CN, copy number; CNV, copy number variation; CTCL, cutaneous T-cell lymphoma; HDAC, histone deacetylase; HDACi, histone deacetylase inhibitor; pYSTAT3, tyrosine-phosphorylated STAT3; SAHA, suberoylanilide hydroxamic acid; SS, Se´zary syndrome; TSA, trichostatin A; WT, wild type Received 4 January 2019; revised 23 February 2019; accepted 6 March 2019; accepted manuscript published online 22 March 2019; corrected proof published online 14 May 2019

Saccharomyces cerevisiae RAD23 gene (Masutani et al., 1994). Another RAD23 homologue, RAD23A, also exists in humans. However, RAD23A and RAD23B have been proposed to adopt distinct roles during HDACi response based on apoptosis studies (Fotheringham et al., 2009). RAD23B is a dual-function ubiquitin receptor thought to play a role in proteasomal targeting and transcriptional control (Chen and Madura, 2002; Kim et al., 2004; Wade and Auble, 2010; Wilkinson et al., 2001), along with its role in nucleotide excision repair (Masutani et al., 1994; Mueller and Smerdon, 1996; Watkins et al., 1993). RAD23B mediates tumor sensitivity to HDACi via its proteasomal role (Khan et al., 2010). RAD23B single nucleotide variant or copy number variation (CNV) have not yet been reported in CTCL. Increased levels of active, tyrosine-phosphorylated STAT3 (pYSTAT3) in tumors has been associated with resistance to SAHA (Fantin et al., 2008; Lu et al., 2014). STAT3 is a transcription factor that, upon cytokine-dependent phosphorylation and activation by JAK kinases, mediates T-cell proliferation, differentiation, and growth (Rawlings et al., 2015). STAT3 is vital for healthy immune function but is commonly dysregulated in cancer and is frequently perturbed in CTCL (da Silva Almeida et al., 2015; Kiel et al., 2015; Park et al., 2017; Ungewickell et al., 2015; Woollard et al., 2016). Somatic STAT3 SH2-domain Y640F and D661Y mutations have been shown to confer gain-of-function signaling in multiple cancer types (Couronne´ et al., 2013; Koskela et al., 2012; Pilati et al., 2011; Walker et al., 2016). Activated STAT3 is regulated by protein phosphatase activity (Kim et al., 2018) and by ubiquitin-dependent proteasomal degradation, suggesting a possible link between STAT3 and RAD23B (Thurman et al., 2012; Wei et al., 2012).

ª 2019 The Authors. Published by Elsevier, Inc. on behalf of the Society for Investigative Dermatology.

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Figure 1. RAD23B CNV in Se´zary cells is associated with reduced RAD23B mRNA expression. (a) A heat map indicating SNP array-derived CN data across chromosome 9 for P1eP16. (b) TaqMan quantitative PCR to validate RAD23B CN by using the standard curve method for P1eP11 and P13eP16 relative to 92 healthy control individuals. RAD23B CN was not tested in P12 due to lack of sample availability. (c) TaqMan qPCR to calculate RAD23B CN in peripheral blood mononuclear cell DNA from 119 SS patients and 92 healthy control individuals; solid and dashed lines represent the mean  three standard deviations, indicating criteria for calling CNV (P
0.01). (d) Gene expression TaqMan quantitative PCR for a subset of the patients from c. Statistical significance was tested using a one-way analysis of variance with multiple comparisons. *P < 0.05. CN, copy number; CNV, copy number variation; P, patient; SNP, single nucleotide polymorphism; SS, Se´zary syndrome.

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Here, we show that RAD23B CNV in primary Se´zary cells is associated with reduced RAD23B mRNA expression. Furthermore, in vitro, RAD23B knockdown partially mediates the HDACi-induced reduction of pYSTAT3 and confers partial resistance to FK228-induced apoptosis. We also show that FK228 significantly reduces pYSTAT3 expression in primary Se´zary cells and show a distinct profile of RAD23BSTAT3 coexpression. Our study shows that the common gain-of-function STAT3 Y640F mutation confers partial resistance to FK228.

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High-frequency RAD23B CNV and reduced RAD23B mRNA expression in primary Se´zary cells

FK228 down-regulates pYSTAT3 and RAD23B expression

Next, we investigated the effect of HDACi treatment on pYSTAT3, STAT3, and RAD23B protein coexpression and apoptosis by intracellular flow cytometry. Trichostatin A (TSA) and FK228 treatment of HUT-78 cells significantly reduced RAD23B, STAT3, and pYSTAT3 protein expression (P ¼ 0.0001) (Figure 2a) and induced apoptosis (Figure 2b). A distinct pattern of RAD23B-STAT3 coexpression was observed during HDACi treatment, whereby reduced STAT3 activity preceded loss of RAD23B expression (Figure 3a). Treatment with TSA or FK228 led to a substantial reduction in pYSTAT3; however, the majority of cells retained expression of RAD23B (Figure 3b). In apoptotic cells, the loss of pYSTAT3 was more pronounced, and an increased proportion of dual-negative cells was observed (Figure 3c). The RAD23BnegpYSTAT3þ population remained negligible under all conditions. Down-regulation of pYSTAT3 by FK228 and TSA is partially dependent on RAD23B

To investigate the relationship between RAD23B and pYSTAT3 during HDACi response, small interfering RNAmediated RAD23B knockdown was established in HUT-78 cells (Figure 4a and b), and apoptosis and pYSTAT3 expression were analyzed by flow cytometry (Figure 4c). In control

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Analysis of chromosome 9 SNP array data identified large-scale copy number (CN) loss in 3 of 16 SS patients and large-scale CN gain in one patient (Figure 1a). Specifically, CN loss of the region on the 9q31.2 band harboring the RAD23B gene was identified in 2 of 16 patients. TaqMan (Thermo Fisher Scientific, Waltham, MA) quantitative PCR confirmed the loss of RAD23B in all samples in which a CNV was identified and additionally identified a sample with RAD23B CN gain, where large-scale gain adjacent to this region was identified by SNP array (Figure 1b). Overall, Se´zary cells from 20% (3/15) of patients harbored RAD23B CNV (P
0.001) relative to none of 92 healthy control samples. A further 104 SS samples in addition to 15 of the original cohort (n ¼ 119) (Figure 1c) were analyzed, and RAD23B CNV was called in 10% (12/119) of patients (n ¼ 6 CN loss, 6 CN gain; P
0.001). Gene expression quantitative PCR showed that the RAD23B mRNA level did not differ significantly in Se´zary cells relative to healthy control samples (Figure 1d). However, Se´zary cells harboring RAD23B CNV showed significantly reduced RAD23B mRNA expression compared with those with normal RAD23B CN (P ¼ 0.04).

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Figure 2. HDACi treatment reduces pYSTAT3 expression and induces apoptosis. HUT-78 cells were treated with vehicle control, 1 mmol/L TSA, or 1 mmol/L FK228 and stained with FVS, annexin V-Pacific Blue, a-pYSTAT3-AF 647, a-STAT3-PE, and a-RAD23B-AF 488, before flow cytometry analysis. (a) Change in MFI plotted with standard error of the mean. Representative data from two biological repeats (b) HDACi-induced apoptosis. Alive indicates annexin Vneg FVSneg; Early Apoptotic indicates Annexin Vþ FVSneg; and Late Apoptotic indicates AnVþ FVSþ. Pooled data from two biological repeats. Bars represent the percentage of total cells. A total of 10,000 events were collected for each experiment. One-way analysis of variance with multiple comparisons was used to test the statistical significance relative to vehicle treated. ***P
0.0001, *P < 0.05. AF, Alexa Fluor; FVS, fixable viability stain. FVS, fixable viability stain; HDACi, histone deacetylase inhibitor; MFI, median fluorescence intensity; pYSTAT3, tyrosinephosphorylated STAT3; TSA, trichostatin A.

(scrambled small interfering RNA)-transfected cells, TSA and FK228 induced apoptosis in 60% and 45% of cells, respectively. After RAD23B knockdown, the proportion of apoptotic cells was reduced compared with control cells, highlighting the role for RAD23B in HDACi-induced apoptosis. In the control cells, TSA and FK228 reduced pYSTAT3 expression by 80% and 50%, respectively, compared with untreated cells. However, after RAD23B knockdown, pYSTAT3 was reduced by only 30% by TSA and 20% by FK228. These findings suggest a novel RAD23B-STAT3 relationship, whereby the absence of RAD23B reduces dephosphorylation of STAT3 in response to HDACi treatment. Immunoprecipitation of STAT3 from HUT-78 cells indicates that a direct interaction between RAD23B and STAT3 may be responsible for this relationship (see Supplementary Figure S1). www.jidonline.org

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Figure 3. A distinct pattern of pYSTAT3 and RAD23B co-expression in HUT-78 cells after HDACi treatment. HUT-78 cells were treated with vehicle control, 1 mmol/L TSA or 1 mmol/L FK228 and stained with FVS 780, Annexin V-Pacific Blue, a-pYSTAT3-AF 647, a-STAT3-PE and a-RAD23B-AF 488 prior to flow cytometry analysis. (a) Representative dot plots of RAD23B versus pYSTAT3 expression in live cells. (b) Dot plots were used to quantify the percentage of total cells with different pYSTAT3-RAD23B staining combinations in live cells or (c) early-apoptotic cells. 10,000 events were collected in each experiment. (b-c) represents pooled data from two biological repeats plotted with SEM. AF, Alexa Fluor; FVS, fixable viability stain; h, hour; M, mol/L; TSA, trichostatin A.

Primary Se´zary cells show a distinct pattern of RAD23B-STAT3 coexpression and variable apoptotic responses after HDACi treatment

In ex vivo primary Se´zary cells from six SS patients, FK228 significantly reduced total STAT3 expression in all patient samples tested (P
0.040) (Figure 5a) compared with CD4þ T cells from healthy control individuals. Increased pYSTAT3 expression was observed in three untreated primary Se´zary samples compared with healthy control individuals (p ¼ 0.001), but, consistent with data in HUT-78 cells, FK228 treatment significantly reduced pYSTAT3 in all Se´zary and control cells (p
0.0001) (Figure 5b). RAD23B and pYSTAT3 coexpression was investigated in live primary Se´zary cells. Although high levels of pYSTAT3 were detected in untreated Se´zary cells from three patients, pYSTAT3 was detected in up to 38% cells in these patients (Figure 5c). Untreated live Se´zary cells were predominantly RAD23BhighpYSTAT3e (62.4%), whereas 25.8% of cells were RAD23BhighpYSTAT3þ compared with 8.8% of healthy control cells (Figure 5d). Consistent with findings in HUT-78 cells, FK228 treatment led to an 18% reduction in the RAD23BhighpYSTAT3þ population, whereas there was a modest increase in the RAD23BhighpYSTAT3neg and RAD23BlowpYSTAT3neg populations. Furthermore, primary Se´zary cells did not express pYSTAT3 in the absence of high RAD23B expression, and apoptotic Se´zary cells expressed negligible levels of pYSTAT3 (Figure 5e). 1978

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The effect of FK228 on the induction of apoptosis was further investigated in primary Se´zary cells. Two samples were excluded because of low cell viability in untreated cells. One sample (P18) exhibited similar sensitivity to FK228 compared with the healthy control samples, and Se´zary cells from two patients (P3 and P19) showed increased sensitivity. Se´zary cells from one patient (P17) showed reduced sensitivity to FK228 compared with the other tumor and healthy samples (Figure 5f).

The STAT3 Y640F mutation persists in multiple SS tumor compartments

Genomic data for these patients were available to investigate the presence of RAD23B CNV and STAT3 gene perturbation. In these six samples, no RAD23B CNV was detected, and 5 of 6 samples were JAK/STAT3 wild type (WT). Se´zary cells derived from P17 harbored the STAT3 Y640F variant, had high levels of pYSTAT3 (Figure 5b), and were partially resistant to FK228-induced apoptosis (Figure 5f). The STAT3 Y640F variant was identified in 3 of 101 SS patients, including P17, previously reported by our group (Woollard et al., 2016). In this study, we showed the persistence of this A>T variant in serial clinical samples from multiple tumor compartments over time in P17. Furthermore, transcription of the variant allele was confirmed (Figure 6a). STAT3 Y640F was also detected in multiple tumor

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Figure 4. RAD23B knockdown decreases HDACi-induced apoptosis and reduces the effect of FK228 on pYSTAT3. HUT-78 cells were treated with RAD23B siRNA and appropriate controls for 48 hours as indicated. (a) RNA expression analyzed by real-time PCR for RAD23B and the cyclophilin control. (b) Whole cell lysates were subject to immunoblot and were probed with antibodies to detect RAD23B and the b-actin loading control. (c) Quantitative flow cytometry analysis of pYSTAT3 expression (left y-axis) and apoptosis (right y-axis) plotted with standard deviation. In a and b, n ¼ 1; in c, pooled data from three biological repeats for which 10,000 events were collected. HDACi, histone deacetylase inhibitor; pYSTAT3, tyrosine-phosphorylated STAT3; siRNA, small interfering RNA.

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compartments in two further patients (see Supplementary Figure S2 online). A comprehensive analysis of publicly available wholeexome sequencing data from mature T-cell malignancies identified STAT3 single nucleotide variants in 8.6% (40/466) of tumors (da Silva Almeida et al., 2015; Jiang et al., 2015; Kataoka et al., 2015; Kiel et al., 2015; McKinney et al., 2017; Moffitt et al., 2017). The STAT3 Y640F SH2-domain mutant, detected in 2.4% (11/466) of tumors, was the most frequently reported, and the STAT3 SH2-domain D661Y variant was detected in 1.9% (9/466) of tumors (see Supplementary Table S1 online). The STAT3 Y640F and D661Y variants confer partial resistance to FK228-mediated STAT3 transcriptional inhibition

We performed functional analysis using an in vitro model to assess the impact of mutant STAT3 on STAT3 activity after FK228 treatment. By immunoblot, we showed that the STAT3 Y640F and D661Y variants were constitutively phosphorylated in HEK293T cells (see Supplementary Figure S3 online). Reduction of STAT3 transcriptional activity after FK228 treatment has been reported (Tiffon et al., 2011); therefore, we tested the effect of FK228 treatment on STAT3 luciferase reporter activity (Figure 6b). In untreated cells, the STAT3 Y640F and D661Y variants conferred increased reporter activity compared with WT protein (P
0.0024). FK228 treatment reduced WT STAT3 reporter activity by 94% compared with untreated cells. In contrast, FK228 reduced STAT3 Y640F and D661Y reporter activity by only 40% or 55%, respectively, and therefore, the SH2-domain mutants had significantly increased activity compared with the WT after

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FK228 treatment (P
0.0001), suggesting partial resistance to HDACi-induced transcriptional inhibition. Thus, these in vitro data are consistent with the reduction in FK228induced apoptosis observed in primary Se´zary cells harboring the STAT3 Y640F variant. DISCUSSION This study has shown that STAT3 tyrosine phosphorylation in primary Se´zary cells is significantly reduced after FK228 treatment and that STAT3 gene mutations confer partial resistance to FK228. Furthermore, our data suggest that RAD23B and STAT3 may interact to modulate the response to FK228 in SS. We also identified a specific RAD23B CNV at the 9q31.2 locus in 10% of primary Se´zary tumors. Chromosome 9 structural abnormalities have been reported in SS (Espinet et al., 2004; Salgado et al., 2010); however, to our knowledge, there are no studies examining CNV at the RAD23B locus. We show that RAD23B CNV is associated with reduced RAD23B mRNA expression in primary Se´zary cells and that RAD23B knockdown downregulates FK228- and TSA-induced apoptosis in HUT-78 cells. This is consistent with previous findings that RAD23B knockdown in cell lines led to decreased apoptosis after SAHA treatment (Fotheringham et al., 2009; Khan et al., 2010). These data suggest that Se´zary cells with RAD23B CNV and reduced RAD23B expression may be less sensitive to FK228 therapy, although this could not be investigated because of a limited number of primary Se´zary cells derived from patients with known RAD23B CNV. We and others have previously shown constitutive activation of the STAT3 oncogene (Bromberg et al., 1999) in SS www.jidonline.org

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Figure 5. A distinct pattern of pYSTAT3 and RAD23B expression after FK228 treatment in primary Se´zary cells. Peripheral blood CD4þ T cells from two healthy volunteers (H1 and H2) and six SS patients (P3 and P17eP21) treated ex vivo with vehicle control or 1 mmol/L FK228 and stained with FVS, annexin V-Pacific Blue, a-pYSTAT3-AF 647, a-STAT3-PE, and a-RAD23B-AF 488, prior to analysis by flow cytometry. DMFI in untreated and treated cells plotted with SEM for (a) STAT3 or (b) pYSTAT3. (c) Representative dot plot from Se´zary cells from one patient showing RAD23B and pYSTAT3. (d, e) Percentage of (d) total live cells or (e) early-apoptotic cells in pooled Se´zary cells was quantified, shown with standard error of the mean. (f) Percentage increase in earlyand late-stage apoptosis relative to untreated cells. AF, Alexa Fluor; FVS, fixable viability stain; H, healthy volunteer; MFI, median fluorescence intensity; P, patient; pYSTAT3, tyrosine-phosphorylated STAT3; SS, Se´zary syndrome.

(McKenzie et al., 2012; Sommer et al., 2004), which has been linked to neoplastic transformation of CTCL (Sommer et al., 2004). Our data now show that FK228 treatment significantly reduces STAT3 transcriptional activity in vitro and confirm that STAT3 tyrosine phosphorylation in primary 1980

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Se´zary cells is significantly reduced after FK228 treatment. Our findings are consistent with studies showing that SAHA and FK228 reduce STAT3 activity in cell lines (Ierano et al., 2013; Tiffon et al., 2011). HDACi treatment has been shown to affect STAT3 cellular localization (Duvic et al.,

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Figure 6. The STAT3 Y640F mutant persists in multiple tumor compartments over time and confers partial resistance to STAT3 transcriptional inhibition by FK228. (a) Sanger sequencing of peripheral blood mononuclear cell DNA from serial blood samples (Blood I and II), lesional skin, and CD4þ-enriched Se´zary cell cDNA from patient 17. (b) Dual luciferase reporter assays were conducted in HEK293T cells transfected to express a STAT3 Firefly luciferase reporter, the control Renilla reporter, WT or mutant STAT3 protein, or the pCi empty vector. Bars represent Firefly/Renilla luminescence normalized to the empty vector, plotted with standard error of the mean. Pooled data from three biological repeats each conducted in triplicate. Statistical significance was tested for the mutants relative to WT within each condition using a two-way analysis of variance with multiple comparisons, ***P
0.001. WT, wild type.

2007) and acetylation, which in turn mediates STAT3 activity (Ray et al., 2005; Wang et al., 2005). FK228-dependent STAT3 inhibition is likely to contribute to its therapeutic effectiveness because STAT3 is an antiapoptotic factor (Lu et al., 2014). This is supported by our observation of pYSTAT3 expression in live, but not apoptotic, primary Se´zary cells after HDACi treatment. We have shown that the STAT3 Y640F mutation, reported in different mature T-cell malignancies (da Silva Almeida et al., 2015; Jiang et al., 2015; Kataoka et al., 2015; Kiel et al., 2015; McKinney et al., 2017; Moffitt et al., 2017), persists in multiple tissue compartments from three SS patients. We also observed in ex vivo primary Se´zary cells that the STAT3 Y640F mutant was associated with high levels of constitutive pYSTAT3 and was resistant to FK228-induced apoptosis compared with other tumor samples that were the STAT3 WT. This finding may explain the previous reported association of increased pYSTAT3 expression with reduced response to SAHA in CTCL (Fantin et al., 2008), although the role of the STAT3 genotype in HDACi resistance was not investigated. Our in vitro data showed that

STAT3 Y640F and D661Y, another common SH2 domain variant, both confer constitutive STAT3 phosphorylation and increased transcriptional activity, which is consistent with studies in other malignancies (Couronne´ et al., 2013; Koskela et al., 2012; Pilati et al., 2011). Critically, we have also shown that STAT3 gain-of-function mutations mediate partial resistance to FK228 inhibition of STAT3 transcriptional activity and induction of apoptosis. A recent report has identified the STAT3 Y640 residue as a site of TYK2-dependent phosphorylation, which suppresses STAT3 transcriptional activity (Mori et al., 2017). This was validated by showing that the Y640F mutation led to inactivation of the normal inhibitory regulation of this residue, enabling constitutive activation of the mutant STAT3 protein. The demonstration that STAT3 genotype influences primary tumor HDACi sensitivity could have significant clinical relevance in SS. Our findings show that pYSTAT3 response to HDACi is partially dependent on RAD23B. Specifically, we identified a distinct pattern of RAD23B and STAT3 coexpression after HDACi treatment, whereby pYSTAT3 expression is not detected in cells with low RAD23B expression. These findings suggest a direct STAT3-RAD23B protein-protein interaction in CTCL cells, which is also supported by the detection of RAD23B-STAT3 protein complexes by coimmunoprecipitation. Both STAT3 and RAD23B have been shown to interact with HDAC6 (Cheng et al., 2014; New et al., 2013). RAD23B mediates the degradation of ubiquitinylated proteins (Kim et al., 2004) and has been shown to contribute to SAHA responses (Khan et al., 2010). STAT3 is also regulated by ubiquitin-dependent protein degradation in T cells (Qu et al., 2012; Tanaka et al., 2011) and through phosphatase-dependent degradation of pYSTAT3 (Kim et al., 2018). Therefore, a potential explanation for the partial FK228 resistance of the STAT3 Y640F variant could be that this negative TYK2-phosphorylation site is also linked to RAD23B-mediated proteasomal degradation. Further work is now required to investigate the functional mechanism of the novel RAD23B-STAT3 interaction and to determine if these findings relate to STAT3 tumor genotype. Quantitative analysis of pYSTAT3 expression in primary Se´zary cells after FK228 treatment has, to our knowledge, not been previously reported. We have found frequent RAD23B and STAT3 gene perturbations in SS patients and have also identified a link between RAD23B expression, STAT3 genotype, and HDACi response in Se´zary cells. A proposed model summarizing the functional effect of HDACi on RAD23B and STAT3 in Se´zary cells is shown in Supplementary Figure S4 online. These findings suggest that STAT3 and RAD23B tumor genotype may influence the sensitivity to HDACi in SS patients. MATERIALS AND METHODS Patients and cell lines All samples used in this study were derived from patients who fulfilled the World Health Organization-European Organisation for Research and Treatment of Cancer diagnostic criteria for SS and were classified at blood stage B2 (Swerdlow et al., 2017) with an identical clonal T-cell receptor rearrangement identified in lesional skin and peripheral blood. All patients and healthy control www.jidonline.org

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STAT3, RAD23B, and HDACi Response in CTCL individuals provided written informed consent, and all samples were obtained from our ethically approved research tissue bank (Integrated Research Application System Project identification: 238203). The rationale for patient sample selection is included in the Supplementary Materials and Methods online. HUT-78 and HEK293T cells were purchased from the European Collection of Authenticated Cell Cultures.

Isolation of Se´zary cells

Peripheral blood CD4þ-enriched Se´zary cells were isolated using Lymphoprep (07851; STEMCELL Technologies, Vancouver, BC, Canada) after incubation with RosetteSep Human CD4þ T Cell Enrichment Cocktail (15022; STEMCELL Technologies).

Cell culture The HUT-78 cell line is a human T-lymphotropic virusenegative SSderived line (Bunn and Foss, 1996; Netchiporouk et al., 2017). HUT78 cells (maintained between passages 2 and 20) and primary CD4þ T cells were cultured in RPMI medium (11875085; Gibco, Waltham, MA) supplemented with 10% fetal bovine serum (10500064; Thermo Fisher Scientific) 100 U/ml penicillin, and 100 mg/ml streptomycin (P4333; MilliporeSigma, Burlington, MA). HEK293T cells (maintained between passages 2 and 25) were cultured in DMEM (61965026; Gibco) supplemented with 10% fetal bovine serum, penicillin, and streptomycin. Cells were cultured at 37  C in the presence of 5% CO2 and 95% humidity and were routinely tested for Mycoplasma species contamination using PCR (van Kuppeveld et al., 1994). Cells were treated with DMSO vehicle control, 1 mmol/L trichostatin A (T1952; MilliporeSigma), or 1 mmol/ L FK228 (romidepsin; HY-15149; Generon, Houston, TX) for 20e24 hours in complete medium.

Single nucleotide polymorphism array HumanOmni5Exome array (Illumina, San Diego, CA) of Se´zary cell DNA from 16 SS patients was analyzed with OncoSNP, version 1.4, after extraction of raw data (B-allele frequencies and log R ratios) with Illumina Genome Studio software (Woollard et al., 2016).

CN analysis Real-time PCR was performed on the ABI prism 7000 sequence detection system (Applied Biosystems, Foster City, CA). CN analysis was conducted with the Hs02323598_cn RAD23B probe/primer set and the TERT Ref CN and RNaseP Ref CN TaqMan probe/primer sets (Thermo Fisher Scientific). Each DNA sample was analyzed in quadruplet with the TaqMan Universal Mastermix II (4440040; Thermo Fisher Scientific) using the standard curve method. Patient samples were compared with a healthy control panel (HRC-1, 06041301; Sigma).

Gene expression analysis

Total RNA was extracted from CD4þ-enriched tumor samples with the RNeasy Plus Mini Kit (74136; Qiagen, Hilden, Germany). cDNA was synthesized with the High-Capacity RNA-to-cDNA kit (4387406; Thermo Fisher Scientific). Gene expression was determined by Hs00234102_m1 RAD23B and Hs01565700_g1 PPIA probe/primer sets by using the TaqMan Fast Advanced Mastermix (Thermo Fisher Scientific). Assays were performed in triplicate and analyzed by the DCt method.

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After fixation and permeabilization with 1% paraformaldehyde and 90% methanol, cells were stained with Alexa Fluor 647 anti-STAT3 Phospo (Tyr705) antibody (13A3-1, BioLegend), PE-STAT3 antibody (15H2B45, BioLegend) and RAD23B polyclonal Alexa Fluor 488 antibody (bs-15482R-A488; Bioss Antibodies, Woburn, MA). Flow acquisition of 10,000 events was performed with a BD FACSCanto II. Thresholds were determined with fluorescence-minus-one staining. Standardized machine settings were retained throughout the study. Data were analyzed with Flowing Software 2 (Perttu Terho, http://www.flowingsoftware.com). Change in median fluorescence intensity was calculated by subtracting the median fluorescence intensity of the fluorescence-minus-one from the sample. Standard error of the mean was calculated from the percentage coefficient of variance and event number. For single intracellular staining of pYSTAT3, cells were incubated with anti-Tyr705 pYSTAT3 antibody (no. 9145; Cell Signaling Technology, Danvers, MA) before incubation with the fluorescently labelled Alexa Fluor 555-conjugated goat anti-mouse antibody, as described (McKenzie et al., 2012). The Annexin V-PE apoptosis detection kit I (559763, BD Pharmingen) was used for independent assessment of apoptosis.

Immunoblotting For analysis of pYSTAT3 in HUT-78 cells, pYSTAT3 was stabilized by treatment with 100 mmol/L pervanadate (P0758; New England Biolabs, Ipswich, MA) for 2 minutes before harvesting. For immunoblots, 15 mg of whole cell lysate was probed with primary antibodies: rabbit monoclonal pYSTAT3 (1:1,000; Tyr705, D3A7, no. 9145, Cell Signaling Technologies), mouse monoclonal STAT3 (1:1,000; 124H6, no. 9139, Cell Signaling Technologies), rabbit polyclonal RAD23B (1:1,000; A302-306A; Bethyl Laboratories, Montgomery, TX), mouse monoclonal RAD23B (1:1,000; sc-136052; Santa Cruz Biotechnology, Dallas, TX), and rabbit monoclonal GAPDH (1:2,000; no. 14C10, Cell Signaling Technology). Duplicate blots were probed in parallel with pYSTAT3 and STAT3 antibodies before probing each blot with a-GAPDH. Signals were detected with horseradish peroxidase-conjugated anti-rabbit (P0448; Dako, Santa Clara, CA) or anti-mouse (ab6789; Abcam, Cambridge, UK) antibodies.

Small interfering RNA knockdown HUT-78 cells were electroporated with the Gene Pulser II Electroporation System (Bio-Rad, Hercules, CA) at 240 V and 975 mF with 100 nmol/L of RAD23B-specific (D-011759-04; Dharmacon, Lafayette, CO) or scrambled small interfering RNA (D-001206-1305; Dharmacon) and cultured for 48 hours, as described (Verma et al., 2010). RAD23B depletion was confirmed by immunoblot and by PCR of cDNA with RAD23B-specific primers (50 TGACCCCGAGGAGACGGTGA-0 3; reverse: 50 -GCAGGTGTGGAAGTGGGGGC-0 3) and cyclophilin-specific primers, as described (McKenzie et al., 2012).

PCR and Sanger sequencing Regions harboring single nucleotide variants in tumor-derived DNA or cDNA were amplified by PCR with AmpliTaq Gold DNA Polymerase (N8080241, Thermo Fisher Scientific) before Sanger sequencing (Source Bioscience, Cambridge, UK).

Detection of apoptosis, RAD23B, pYSTAT3, and STAT3

Cell transfection and luciferase reporter assays

Cells were stained with Fixable Viability Stain (FVS 780, 565388; BD, Franklin Lakes, NJ) and Annexin V-Pacific Blue (640918; BioLegend, San Diego, CA), following the manufacturer’s instructions.

HEK293T cells were transiently transfected with WT or mutant pCiSTAT3 constructs or pCi empty-vector control with poly(ethylenimine) (9002-98-6; MilliporeSigma), as described (Longo

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RM Butler et al.

STAT3, RAD23B, and HDACi Response in CTCL et al., 2013). HEK293T cells were cotransfected with 1 mg of the STAT3-luciferase reporter plasmid (kindly provided by Uwe Vinkemeier, University of Nottingham, Nottingham, UK) and 0.5 ng Renilla pRLTK control plasmid (John et al., 1999). Dual Luciferase Reporter Assays (E1980; Promega, Madison, WI) were performed in triplicate with an Orion II Microplate Luminometer (Berthold Detection Systems, Pforzheim, Germany). Firefly luciferase activity was standardized to Renilla.

Cheng F, Lienlaf M, Wang H-W, Perez-Villarroel P, Lee C, Woan K, et al. A novel role for histone deacetylase 6 in the regulation of the tolerogenic STAT3/IL-10 pathway in APCs. J Immunol 2014;193:2850e62.

Statistical analysis

Duvic M, Talpur R, Ni X, Zhang C, Hazarika P, Kelly C, et al. Phase 2 trial of oral vorinostat (suberoylanilide hydroxamic acid, SAHA) for refractory cutaneous T-cell lymphoma (CTCL). Blood 2007;109:31e9.

Statistical significance was assessed using a one-way analysis of variance with GraphPad Prism, version 7 (GraphPad Software, San Diego, CA).

Data availability statement Data sets related to this article can be found at https://www.ncbi. nlm.nih.gov/geo/query/acc.cgi?acc¼ GSE80650, hosted at the National Center for Biotechnology Information’s Gene Expression Omnibus Series, accession number GSE80650. ORCIDs Rosie M. Butler: https://orcid.org/0000-0003-1063-9097 Robert C. McKenzie: https://orcid.org/0000-0001-8885-733X Christine L. Jones: https://orcid.org/0000-0001-9539-7458 Charlotte E. Flanagan: https://orcid.org/0000-0002-5913-9192 Wesley J. Woollard: https://orcid.org/0000-0001-5567-8765 Maria Demontis: https://orcid.org/0000-0003-1927-4987 Silvia Ferreira: https://orcid.org/0000-0002-6865-5607 Isabella Tosi: https://orcid.org/0000-0002-2638-8539 Susan John: https://orcid.org/0000-0002-5620-1982 Sean J. Whittaker: https://orcid.org/0000-0003-0972-2600 Tracey J. Mitchell: https://orcid.org/0000-0001-8892-0642

CONFLICT OF INTEREST The authors state no conflict of interest.

ACKNOWLEDGMENTS This article represents independent research funded by the Guy’s and St Thomas’ Charity Prize PhD Programme in Biomedical and Translation Science and the King’s Bioscience Institute at King’s College London. The views expressed are those of the authors and not necessarily those of the Guy’s and St Thomas’ Charity or King’s College London. The authors acknowledge financial support from the Department of Health via the National Institute for Health Research comprehensive Biomedical Research Centre award to Guy’s & St Thomas’ National Health Service Foundation Trust in partnership with King’s College London and King’s College Hospital National Health Service Foundation Trust.

AUTHOR CONTRIBUTIONS Conceptualization: RMB, SJ, SJW, TJM; Data Curation: RMB, CLJ, WJW; Formal Analysis: RMB, CLJ, WJW, SJW, TJM; Funding Acquisition: SJW, TJM; Investigation: RMB, RCM, CEF, WJW, MD, SF, IT; Methodology: RMB, RCM, SJ, SJW, TJM; Project Administration: SJW, TJM; Resources: SJW, TJM; Software: RMB, CLJ, WJW, SJW, TJM; Supervision: SJ, SJW, TJM; Validation: RMB, RCM, WJW, CLJ; Visualization: RMB, RCM, TJM; Writing - Original Draft Preparation: RMB, SJW, TJM; Writing - Review and Editing: RMB, TJM

SUPPLEMENTARY MATERIAL

Couronne´ L, Scourzic L, Pilati C, Della Valle V, Duffourd Y, Solary E, et al. STAT3 mutations identified in human hematologic neoplasms induce myeloid malignancies in a mouse bone marrow transplantation model. Haematologica 2013;98:1748e52. da Silva Almeida AC, Abate F, Khiabanian H, Martinez-Escala E, Guitart J, Tensen CP, et al. The mutational landscape of cutaneous T cell lymphoma and Se´zary syndrome. Nat Genet 2015;47:1465e70.

Espinet B, Salido M, Pujol RM, Florensa L, Gallardo F, Domingo A, et al. Genetic characterization of Se´zary’s syndrome by conventional cytogenetics and cross-species color banding fluorescent in situ hybridization. Haematologica 2004;89:165e73. Fantin VR, Loboda A, Paweletz CP, Hendrickson RC, Pierce JW, Roth JA, et al. Constitutive activation of signal transducers and activators of transcription predicts vorinostat resistance in cutaneous T-cell lymphoma. Cancer Res 2008;68:3785e94. Fotheringham S, Epping MT, Stimson L, Khan O, Wood V, Pezzella F, et al. Genome-wide loss-of-function screen reveals an important role for the proteasome in HDAC inhibitor-induced apoptosis. Cancer Cell 2009;15: 57e66. Ierano C, Basseville A, To KKW, Zhan Z, Robey RW, Wilkerson J, et al. Histone deacetylase inhibitors induce CXCR4 mRNA but antagonize CXCR4 migration. Cancer Biol Ther 2013;14:175e83. Jiang L, Gu Z-H, Yan Z-X, Zhao X, Xie Y-Y, Zhang Z-G, et al. Exome sequencing identifies somatic mutations of DDX3X in natural killer/T-cell lymphoma. Nat Genet 2015;47:1061e6. John S, Vinkemeier U, Soldaini E, Darnell JE, Leonard WJ, Leonard WJ. The significance of tetramerization in promoter recruitment by Stat5. Mol Cell Biol 1999;19:1910e8. Kataoka K, Nagata Y, Kitanaka A, Shiraishi Y, Shimamura T, Yasunaga J, et al. Integrated molecular analysis of adult T cell leukemia/lymphoma. Nat Genet 2015;47:1304e15. Khan O, Fotheringham S, Wood V, Stimson L, Zhang C, Pezzella F, et al. HR23B is a biomarker for tumor sensitivity to HDAC inhibitor-based therapy. Proc Natl Acad Sci USA 2010;107:6532e7. Kiel MJ, Sahasrabuddhe AA, Rolland DCM, Velusamy T, Chung F, Schaller M, et al. Genomic analyses reveal recurrent mutations in epigenetic modifiers and the JAK-STAT pathway in Se´zary syndrome. Nat Commun 2015;6:8470. Kim EJ, Hess S, Richardson SK, Newton S, Showe LC, Benoit BM, et al. Immunopathogenesis and therapy of cutaneous T cell lymphoma. J Clin Invest 2005;115:798e812. Kim I, Mi K, Rao H. Multiple interactions of Rad23 suggest a mechanism for ubiquitylated substrate delivery important in proteolysis. Mol Biol Cell 2004;15:3357e65. Kim M, Morales LD, Jang I-S, Cho Y-Y, Kim DJ. Protein tyrosine phosphatases as potential regulators of STAT3 signaling. Int J Mol Sci 2018;19(9):2708. Koskela HLM, Eldfors S, Ellonen P, van Adrichem AJ, Kuusanma¨ki H, Andersson EI, et al. Somatic STAT3 mutations in large granular lymphocytic leukemia. N Engl J Med 2012;366:1905e13. Longo PA, Kavran JM, Kim M-S, Leahy DJ. Transient mammalian cell transfection with polyethylenimine (PEI). Methods Enzymol 2013;529:227e40.

Supplementary material is linked to the online version of the paper at www. jidonline org, and at https://doi.org/10.1016/j.jid.2019.03.1130.

Lu K, Wang X, Fang X, Feng L, Chen N, Li P, et al. Inhibition of STAT3 reverses the resistance to histone deacetylase inhibitors induced by interleukin-6 in chronic lymphocytic leukemia cells. Blood 2014;124:3632.

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SUPPLEMENTARY MATERIALS AND METHODS Patient selection

Samples from the 16 patients analyzed by SNP array (Figure 1) were from a cohort of untreated SS patients that we previously analyzed by next-generation sequencing, and therefore, comprehensive genotype data were available (Woollard et al., 2016). The additional cohort of 104 patients included for validation of RAD23B CNV by quantitative PCR (Figure 1c) were selected based on a diagnosis of SS (overall stage 1VA12) and the availability of genomic DNA isolated from peripheral blood mononuclear cells in which an identical clonal T-cell receptor gene rearrangement had been confirmed in skin and blood. The samples from the six patients selected for functional analyses (Figure 5) were based on the availability of

CD4þ-enriched tumor cells and were also from our nextgeneration sequencing study and, therefore, had STAT3/ RAD23B genotype data available. Coimmunoprecipitation

HUT-78 cell lysates were incubated with rabbit IgG negative control (NC-100-P1ABX, Thermo Fisher Scientific), rabbit polyclonal a-RAD23B antibody (A302-305A, Bethyl Laboratories) or rabbit monoclonal a-STAT3 antibody (no. 12640, Cell Signaling Technologies). Antibody-protein complexes were immunoprecipitated with protein A/G agarose beads (20421, Thermo Fisher Scientific) as per the manufacturer’s instructions. Immunoprecipitated proteins were analyzed by immunoblot.

a Patient 22 STAT3 A>T (Y640F)

Chromatogram

Blood

Involved node

b Supplementary Figure S1. RAD23B-STAT3 protein complexes detected by co-immunoprecipitation. Co-immunoprecipitation (IP) in untreated HUT-78 cells with IgG negative control or STAT3 or RAD23B antibodies, as described in the Supplementary Materials and Methods. Immunoblots of the input (whole cell lysate used for IP) or precipitated protein were probed with antibodies for STAT3 or RAD23B. Representative blot of three biological repeats. RAD23B-STAT3 complexes were not detected after immunoprecipitation with the a-RAD23B antibody, possibly due to steric hindrance. IB, immunoblot; IP, co-immunoprecipitation.

Patient 23 STAT3 A>T (Y640F)

Chromatogram

Blood

Lesional Skin

Supplementary Figure S2. The STAT3 Y640F mutant persists in multiple tumor compartments in SS patients. Sanger sequencing of tumor-derived DNA from multiple clinical samples from (a) patient 22 and (b) patient 23, analyzed with FinchTV software (Geospiza, Inc, Seattle, WA). SS, Se´zary syndrome.

Supplementary Figure S3. STAT3 Y640F and D661Y mutants are constitutively phosphorylated. Immunoblot in HEK293T cells expressing the pCi negative control, WT, or mutant STAT3 and probed with antibodies to detect pYSTAT3, STAT3, or the GAPDH loading control. Representative blot of three biological repeats. pYSTAT3, tyrosine-phosphorylated STAT3; WT, wild type.

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STAT3, RAD23B, and HDACi Response in CTCL Supplementary Figure S4. Model showing the effect of histone deacetylase inhibitors on STAT3 and RAD23B in Se´zary cells. (a) HDACi directly down-regulates STAT3, pYSTAT3, and RAD23B which, in association with proteasomal degradation of the RAD23B-pYSTAT3 protein complex, increases Se´zary cell apoptosis. (b) In contrast, the STAT3 Y640F gain-of-function mutation confers reduced HDACi sensitivity by blocking the STAT3-RAD23B interaction, which consequently reduces apoptosis. HDACi, histone deacetylase inhibitor; pYSTAT3, pYSTAT3, tyrosine-phosphorylated STAT3.

Supplementary Table S1. Comprehensive analysis of publicly available STAT3 single nucleotide variant data in whole-exomeesequenced mature T-cell lymphomas1 Total Disease patients, n AITL ATLL EATL HSTL MF NKTCL PTCL SS TPLL Total

11 81 71 63 14 27 9 154 36 466

Tumors with STAT3 single nucleotide variant, n

Frequency, %

0 19 10 6 1 3 0 1 0 40

0 23.5 14.1 9.5 7.1 11.1 0 0.6 0 8.6

Abbreviations: AITL, angioimmunoblastic T-cell lymphomas; ATLL, adult T-cell leukemia/lymphomas; EATL, enteropathy-associated T-cell lymphoma; HSTL, hepatosplenic T-cell lymphomas; MF, mycosis fungoides; NKTCL, natural killer/T-cell lymphoma; PTCL, peripheral T-cell lymphomas not otherwise specified; SS, Se´zary syndrome; TPLL, T-cell prolymphocytic leukemias. 1 N ¼ 466. Studies were analyzed when a full, publicly available single nucleotide variant list was available from whole-exomeesequenced tumors.

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