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Published in final edited form as: J Invest Dermatol. 2024 Jan 14;144(7):1579–1589.e8. doi: 10.1016/j.jid.2023.12.010

Romidepsin and afatinib abrogate JAK-STAT signaling and elicit synergistic antitumor effects in cutaneous T-cell lymphoma

Bobby B Shih 1, Cindy Ma 1, Jose R Cortes 1,#, Clara Reglero 1, Hannah Miller 1, S Aidan Quinn 1, Robert Albero 1,§, Anouchka P Laurent 1, Adam Mackey 1, Adolfo A Ferrando 1,2,3,4,#, Larisa Geskin 5, Teresa Palomero 1,2
PMCID: PMC11193653  NIHMSID: NIHMS1960155  PMID: 38219917

Abstract

Cutaneous T-cell lymphomas (CTCL) are mature lymphoid neoplasias resulting from the malignant transformation of skin-resident T cells. A distinctive clinical feature of CTCL is their sensitivity to treatment with histone deacetylase (HDAC) inhibitors. However, responses to HDAC inhibitor therapy are universally transient and non-curative, highlighting the need for effective and durable drug combinations. Here we demonstrate that the combination of romidepsin, a selective class I HDAC inhibitor, with afatinib, an epidermal growth factor receptor (EGFR) family inhibitor, induces strongly synergistic antitumor effects in CTCL models in vitro and in vivo via abrogation of JAK-STAT signalling. These results support a previously unrecognized potential role for HDAC inhibitor plus afatinib combination in the treatment of CTCL.

Keywords: Cutaneous T-cell lymphoma, romidepsin, combination therapy, preclinical models

Introduction

Cutaneous T-cell lymphomas (CTCL) are a heterogeneous group of mature T-cell malignancies that originate from skin-homing neoplastic CD4+ T-cells (Willemze et al., 2005). Mycosis Fungoides (MF) is the most common form of CTCL, constituting ~60% of cutaneous T-cell lymphomas (Willemze et al., 2019, Willemze et al., 2005). Clinically, MF is an indolent neoplasia typically manifesting with erythematous patches and plaques on the skin and with a median survival of 11.4 years (Kim et al., 2003). In contrast, Sezary syndrome (SS), a disseminated form of CTCL with erythroderma, lymphadenopathy and circulating clonal malignant T cells in the peripheral blood (Willemze et al., 2019, Willemze et al., 2005) has an aggressive clinical course and a 5-year survival rate of ~36% (Willemze et al., 2005). Genomic profiling studies of indolent and aggressive CTCL reveal a heterogeneous genomic landscape but also recurrent mechanisms, including loss of TP53; alterations in genes encoding epigenetic regulators controlling DNA methylation (TET2), reading and writing of epigenetic histone tail modifications (CREBBP, KMT2D and KMT2C) and nucleosome positioning (SMARCA4 and CHD3); and alterations in signaling factors driving increased MAPK, NF-κB and NFAT activity upon T cell receptor stimulation (Chang et al., 2018, Choi et al., 2015, da Silva Almeida et al., 2015, McGirt et al., 2015, Prasad et al., 2016, Ungewickell et al., 2015, Wang et al., 2015, Woollard et al., 2016).

In patients with early-stage disease, skin-directed therapies, such as topical glucocorticoids, phototherapy, local radiotherapy, and topical mechlorethamine, are commonly used (Lessin et al., 2013, Talpur et al., 2014, Wang and Bagot, 2019, Willemze et al., 2018), while systemic therapies including bexarotene, histone deacetylase inhibitors, methotrexate and the brentuximab vedotin antibody-drug conjugate are reserved for patients with more advanced and aggressive disease (Duvic et al., 2001, Olsen et al., 2007, Prince et al., 2017, Whittaker et al., 2010). The relatively high activity of romidepsin, a selective class I HDAC inhibitor, with an overall response rate of ~38% as a single agent, and its favorable toxicity profile (Whittaker et al., 2010) has prompted the investigation of combinations with chemotherapy (methotrexate, mechlorethamine), hypomethylating agents (5-azacitidine) and kinase inhibitors (ruxolitinib) (Cieza-Diaz et al., 2021, Cortes et al., 2021, Falchi et al., 2021). Yet, the clinical superiority of these drug combinations remains to be established (Cieza-Diaz et al., 2021, Cortes et al., 2021, Falchi et al., 2021, Makena et al., 2017, Quaglino et al., 2017). Here we present the results of a systematic screen of romidepsin in combination with a small-molecule-inhibitor library of bioactive and FDA-approved drugs and describe a synergistic pro-apoptotic antitumor effect of romidepsin in combination with the ErbB tyrosine kinase inhibitors afatinib/afatinib dimaleate in CTCL.

Results

High Throughput Drug Screening Identifies Romidepsin and Afatinib Synergism

To systematically explore the landscape of clinically relevant drugs capable of inducing increased antitumor effects with romidepsin in CTCL we treated MYLA, a model CTCL cell line established from a plaque biopsy of an 82-year-old Caucasian patient with mycosis fungoides (Supplementary Table 1), with the Prestwick Chemical Library, a collection of bioactive compounds that contains 1,014 FDA-approved drugs, alone and in combination with romidepsin (Figure 1a). As expected, HDAC inhibitors entinostat, mocetinostat and vorinostat enhanced the activity of romidepsin, used in our screen at sensitizing concentrations (IC20) (Figure 1b, Supplementary Figure 1). In agreement with previous reports from our group (Cortes et al., 2021), our screen recovered an interaction between ruxolitinib, a JAK-STAT inhibitor, and romidepsin (Excess over Bliss (EOB) = 6.9) (Supplementary Table 2) and extended this interaction to additional JAK-STAT inhibitors including pacritinib, cerdulatinib, and gandotinib (Supplementary Table 2). In addition, and most notably, we observed a marked synergistic antitumor effect of romidepsin in combination with afatinib and afatinib dimaleate with EOB scores of 11.4 and 10, respectively (Supplementary Table 2). Of note, afatinib dimaleate is chemically identical to afatinib, except for the addition of two molar equivalents of maleic acid, a commonly used method to increase compound solubility for oral delivery (2023, Gupta et al., 2018).

Figure 1. High throughput drug screening to identify drug synergies with romidepsin and effect of romidepsin, afatinib/afatinib dimaleate and their combination in CTCL in vitro and in vivo.

Figure 1.

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(a) Experimental design for high throughput screen and synergistic candidate evaluation. Cells are plated in 384 well plates in triplicate and treated for 72 hours with: (1) Prestwick Chemical Library at 1 μM, (2) romidepsin alone at IC20, and (3) romidepsin at IC20 plus the Prestwick library compoundsThe expected effect of combination is calculated using the Bliss independence model and synergy is quantified by calculating the difference between the observed and expected effect of combination. Top hits are then validated using in vitro and in vivo models of CTCL. (b) Volcano plot summary of high throughput synergy drug screening. Excess over bliss (EOB) is the difference between the observed and expected effect of drug using the bliss independence model. Blue lines represent −10 and +10 EOB. Red line represents p-value = 0.05. (c) Representative dose response map representation of the combination of romidepsin and afatinib/afatinib dimaleate in MYLA (MF) and HUT78 (SS) cell lines at 72 hours. (d) Synergy quantification using four independent models of synergy scoring for the combination of romidepsin and afatinib/afatinib dimaleate in MYLA (MF) and HUT78 (SS) cell lines. (e) Evaluation of apoptosis in MYLA (MF) and HUT78 (SS) cell lines after 24, 48 and 72 hours of treatment with vehicle (DMSO), romidepsin, afatinib dimaleate, or the combination using flow cytometry analysis after annexin-V/7-AAD staining. (f) Evaluation of apoptosis in romidepsin-sensitive and -resistant Sezary Syndrome primary samples after 24, 48 and 72 hours of treatment with vehicle (DMSO), romidepsin, afatinib dimaleate, or the combination using flow cytometry analysis after annexin-V/7-AAD staining. P-values were calculated using a two-tailed Student’s t-test. Error bars represent mean +/− s.d. n=3. (g) Tumor burden measured by IVIS bioluminescent imaging of mice treated with either DMSO, romidepsin, afatinib dimaleate, or the combination after 12 days of treatment. P-values were calculated using the Mann-Whitney test. *p ≤0.05, **p ≤0.01, ***p ≤0.001. Error bars represent mean +/− s.d. Panel (a) created with BioRender.com.

Following on this result, we further evaluated the interaction between romidepsin and afatinib using a non-constant ratio dose response experimental design for synergy scoring in MYLA cells and also in HUT78, a second CTCL cell line derived from a 53-year-old Caucasian male patient with Sezary Syndrome (Supplementary Table 1). In cell viability assays, the combination of romidepsin and afatinib/afatinib dimaleate induced a greater than additive effect in cell viability assays compared to either single agent alone (Figure 1c), with average synergy scores greater than 10 in both cell lines (Figure 1d). In this context, treatment with romidepsin and afatinib in combination resulted in marked antitumor effects at subtherapeutic single agent concentrations at which either single agent alone has no or negligible antitumor activity (Figure 1c). Flow cytometry analysis of cell cycle progression following 5-ethynyl-2´-deoxyuridine (EdU) incorporation and DNA content staining revealed G1 arrest and substantial loss of cells in S-phase in MYLA and HUT78 CTCL cells treated with romidepsin plus afatinib in combination for 24 and 48 hours, respectively (Supplementary Figures 2-3). In addition, we observed a synergistic time-dependent increase in sub-G0 apoptotic cells following treatment with romidepsin plus afatinib as compared to cells treated with either agent as monotherapy (Supplementary Figures 2-3). Similarly, we observed a significantly greater proportion of apoptotic cells in combination treatment compared to either single agent (P<0.05) in a time-response analysis using Annexin-V staining apoptosis assays (Figure 1e).

Next, and to directly evaluate the relevance of these results in primary patient samples from aggressive forms of CTCL, we tested the effectiveness of romidepsin and afatinib in malignant CD4+ T cells from patients with SS. Briefly, CD4+ T-cells enriched from peripheral blood of SS patients were activated with CD3/CD28 for 24h and treated with an IC50 concentration of romidepsin, afatinib dimaleate and their combination for 72 hours. In these assays, we observed significant antitumor responses in single agent conditions (Figure 1f). Moreover, treatment with the romidepsin plus afatinib dimaleate combination resulted in significant decrease in cell viability (P<0.05) when compared to either single agent in 4/5 patient samples tested (Figure 1f), including one romidepsin-resistant patient.

Following these observations, we evaluated the efficacy and therapeutic activity of romidepsin and afatinib dimaleate as single agents and in combination in vivo using a SS xenograft model. In these experiments, we injected luciferized HUT78 cells orthotopically in NRG immunodeficient mice. After verifying tumor engraftment by in vivo bioimaging, we segregated animals in therapeutic groups ensuring equal representation of bioluminescent signal, for treatment with vehicle, romidepsin, afatinib dimaleate, or romidepsin plus afatinib dimaleate in combination. Following 12 days of treatment, animals treated with romidepsin showed markedly attenuated tumor growth compared with vehicle treated controls, while afatinib dimaleate treatment elicited only minor antitumor effects in tumor growth (Figure 1g). In contrast, mice treated with romidepsin plus afatinib dimaleate in combination showed a marked reduction in tumor burden compared with vehicle only and single drug treatment arms (Figure 1g, Supplementary Figure 4).

Combination with afatinib dimaleate overcomes acquired romidepsin resistance in CTCL

Secondary resistance to HDAC inhibitors at the time of disease progression and relapse is a common clinical occurrence in CTCL (Foss et al., 2015, Olsen et al., 2007, Whittaker et al., 2010). To test if the combination of afatinib and romidepsin could overcome therapy resistance, we established a HDAC inhibitor-resistant cellular model by treating HUT78 cells with low-dose romidepsin over an extended period of time. Following selection, HUT78 cells showed striking resistance to up 14 nM romidepsin at 72 hours whereas treatment with 5 nM romidepsin of non-selected cells resulted in complete cell death (Figure 2b). Interestingly, HDACi-resistant HUT78 cells seem to display diminished response to afatinib or afatinib dimaleate when compared to unselected cells (Figure 1c). However, and most notably, the combination of romidepsin plus afatinib and romidepsin plus afatinib dimaleate remained highly synergistic (SynergyFinder Score>30) and induced overt antitumor effects at concentrations in which single agent treatment resulted in negligible cell death (Figures 2a and 2c). Taken together, these results support a potential role for the combination of afatinib plus romidepsin in the treatment of romidepsin-resistant relapsed CTCL.

Figure 2. Effect of the combination treatment with romidepsin and afatinib/afatinib in a romidepsin resistant cell line model of CTCL.

Figure 2.

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(a) Dose response map representation of the combination of romidepsin and afatinib/afatinib dimaleate at 72 hours in romidepsin resistant HUT78 (SS). (b) Dose response cell viability curves for HUT78 and romidepsin resistant HUT78 cell lines (SS) at 72 hours with treatment of romidepsin. (c) Synergy quantification using four independent models of synergy scoring for the combination of romidepsin and afatinib/afatinib dimaleate in romidepsin resistant HUT78 cell lines (SS).

Romidepsin and afatinib dimaleate induce down-regulation of JAK-STAT signaling in CTCL.

The markedly synergistic antitumor effects of romidepsin and afatinib in CTCL support a mechanistic interaction by which epigenetic perturbation following HDAC inhibition and inhibition of EGFR signaling suppress oncogenic programs strictly required for CTCL cell growth, proliferation and survival (Chambers et al., 2003, Glaser et al., 2003, Peart et al., 2005). To examine possible molecular mechanisms underlying the synergistic anti-neoplastic effects of these drugs we examined the transcriptional impact of treatment with romidepsin, afatinib dimaleate and both drugs in combination at an early timepoint (24 hours) before overt induction of apoptosis. RNAseq profiling of MYLA cells treated with romidepsin revealed broad changes in gene expression with 817 up-regulated and 541 down-regulated genes compared with vehicle-only treatment controls. Treatment with afatinib dimaleate treatment resulted in 412 up-regulated and 431 down-regulated genes (log2FC > 1.2; P < 0.05) (Figure 3a-b). In addition, and consistent with perturbation of cellular homeostasis through complementary non-overlapping mechanisms, treatment with romidepsin plus afatinib dimaleate in combination induced substantially broader gene expression changes (1,013 up-regulated and 1,025 down-regulated genes) (Figure 3a-b). Across these treatments the gene expression signatures resulting from romidepsin and afatinib treatment were largely non-overlapping, in agreement with their distinct mechanisms of action. In contrast, 402 genes were convergently upregulated following treatment with romidepsin and romidepsin plus afatinib dimaleate. However, across the board, the combination treatment induced a largely distinct transcriptional downregulation program non-overlapping with those of romidepsin or afatinib dimaleate (Figure 3a-b). Transcripts downregulated following treatment with romidepsin and afatinib dimaleate in combination included genes involved in cell cycle regulation (CCAR2, CCNB2, CCND3, CCNY, CDNK1A), cytokine (IL15RA, IL2RB, IL6R), JAK/STAT (JAK1, STAT5A), TGF-β (TAB2) and WNT signaling (TCF7L2, WNT10B) (Figure 3c). Moreover, Gene Set Enrichment Analysis (GSEA) annotation of the combination treatment-associated transcriptional programs revealed negative enrichment of MAPK (Normalized Enrichment Score (NES) = 1.714, False Discovery Rate (FDR) = 0.179), PI3K-AKT-mTOR (NES = 1.519, FDR = 0.208), IL2-STAT5 (NES = 1.433, FDR = 0.232), ERBB (NES = 1.351, FDR = 0.258) and JAK-STAT (NES = 1.321, FDR = 0.267) signaling-associated genesets (Figure 3d, Supplementary Figure 5). Among these pathways, suppression of JAK/STAT signaling has been linked with enhanced antitumor responses to romidepsin in CTCL (Cortes et al., 2021) suggesting a potential effector role in mediating the drug synergism between this HDAC inhibitor and afatinib. In support of this hypothesis, western blot analysis of JAK-STAT signaling in MYLA and HUT78 cells revealed marked downregulation of phospho-STAT1 (p-STAT1), phospho-STAT3 (p-STAT3) and phospho-STAT5 (p-STAT5) following treatment with afatinib dimaleate and afatinib dimaleate plus romidepsin compared with vehicle only and romidepsin-only treated cells (Figure 4). Of note, decreased signaling was coupled with reduced levels of total STAT1 and STAT5 in both MYLA and HUT78 cells (Figure 4a-b). Additionally, and in agreement with EGFR inhibition by afatinib, treatment with romidepsin plus afatinib in combination resulted in decreased HER2 and HER3 phosphorylation, and protein levels (Supplementary Figure 6). These results support that inhibition of EGFR/HER2/HER3 signaling by afatinib, can abrogate JAK-STAT signaling by downregulation of STAT1, STAT3 and STAT5 activation.

Figure 3. Analysis of differential gene expression induced by the treatment of romidepsin, afatinib dimaleate, or the combination in CTCL cell lines by RNA-seq.

Figure 3.

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(a-b) Circos plots representing the relative proportion of significantly up- (a) and down-regulated (b) genes in each treatment condition normalized to vehicle condition (DMSO). Circos plot node width and bar plots represent the number of genes that are either common, shared, or unique between each treatment condition. (c) Heatmap representation of the normalized expression of all significantly dysregulated genes for cells treated with the combination of romidepsin and afatinib dimaleate compared to vehicle (DMSO). Genes are clustered using unsupervised complete-linkage hierarchical clustering. (d) Gene Set Enrichment Analysis enrichment plot representing the KEGG JAK-STAT signaling pathway (NES = 1.321, FDR = 0.267) with (e) genes comprising the leading edge represented as a heatmap of normalized expression values. R: romidepsin, A: afatinib dimaleate, RA: romidepsin plus afatinib dimaleate.

Figure 4. Analysis of signaling pathways regulated in CTCL cell lines treated with romidepsin, afatinib dimaleate and their combination.

Figure 4.

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(a) Western blot analysis of phosphorylated and total STAT1, STAT3 and STAT5 in protein lysates prepared from MYLA (MF) and HUT78 (SS) cell lines treated with vehicle (DMSO), romidepsin, afatinib dimaleate, and their combination for 24 hours. GAPDH is used as loading control (b) Western blot quantification of total signal normalized to GAPDH.

Discussion

Genome-wide mutation profiling studies have markedly improved our understanding of the genetics and mechanisms of CTCL, however, the highly heterogeneous mutational landscape of this disease offers little opportunity for the development of targeted therapies (da Silva Almeida et al., 2015, Kiel et al., 2015, McGirt et al., 2015, Prasad et al., 2016, Ungewickell et al., 2015).

Histone deacetylases (HDACs) are a family of epigenetic enzymes that remove acetyl groups from both histone and non-histone targets. Clinically, HDAC inhibitors have shown significant activity in hematologic malignancies, in particular the treatment of peripheral T cell lymphomas (Bates et al., 2015). In CTCL, romidepsin has been approved since 2009 for patients with relapsed and refractory disease based on the results of two phase II open-label, multicenter clinical trials, which showed an overall response rate of 34% with 6% complete responses (Piekarz et al., 2009). Despite these well-documented clinical benefits, the efficacy of romidepsin is limited to a fraction of patients and is further curtailed by the emergence of progressive resistant disease (Martinez-Escala et al., 2016, Piekarz et al., 2009). In this setting, the identification of new therapeutic agents active and synergistic with HDAC inhibitor therapy in CTCL is urgently needed.

Here we identify afatinib, a clinically approved EGFR family inhibitor, as an active antitumor drug in CTCL. In addition, and most notably, we report a remarkable synergistic activity of afatinib in combination with romidepsin in preclinical models. Specifically, we document that the combination of afatinib and romidepsin is highly synergistic in MF and SS CTCL cell lines. Activity of this combination is also demonstrated in aggressive CTCL primary patient samples in vitro and in an in vivo disseminated mouse model of SS. In addition, the combination of romidepsin plus afatinib remained highly active in SS cells from a romidepsin resistant patient at relapse and in SS HUT78 cells selected for romidepsin resistance, both findings of potential direct clinical impact for the treatment of relapsed and refractory disease.

Oncogenic EGFR signaling contributes to malignant transformation through activation of downstream JAK/STAT, MAPK, and mTOR pathways. Gain of function mutations and amplifications affecting member of the EGFR family are common drivers in lung and breast cancer (Tu et al.) Interestingly, point mutation and copy number alterations in members of the EGFR signaling pathway have been identified in CTCL (Supplementary Figure 7). Moreover, amplifications in chromosome 17q, where ERBB2 is located is a highly recurrent event in CTCL (Chang et al., 2018).

Mechanistically, signaling and transcriptomic profiling of CTCL cells lines treated with romidepsin plus afatinib revealed downregulation of JAK-STAT signaling. JAK-STAT activation in CTCL results from cell extrinsic (up-regulation of cytokine signaling) and cell intrinsic (gain-of-function genetic alterations) mechanisms, which convergently drive constitutive JAK1, JAK3, STAT3, and STAT5B activation (Damsky and Choi, 2016). In early stage CTCL, upregulation of STAT5 has been reported to drive tumor growth by increasing expression of anti-apoptotic proteins and cell cycle genes (Kopp et al., 2013, Litvinov et al., 2014, Qin et al., 2001, Tracey et al., 2003). In advanced stages of CTCL, constitutive expression of STAT3 further drives proliferation and resistance to apoptosis through increased expression of BCL2 and miR-21 oncogenic microRNA (Eriksen et al., 2001, Nielsen et al., 1999, Nielsen et al., 1997, Qin et al., 2001, van der Fits et al., 2012). Activation of JAK-STAT signaling has been linked to resistance to HDAC inhibitors in B-cell lymphoma cell lines (Iqbal et al., 2010). Conversely, class I HDACs have been shown to interact with and regulate STAT1, STAT2 and STAT3 (Gupta et al., 2012, Nusinzon and Horvath, 2003). In addition, the inhibition of class I HDACs can suppress STAT3 phosphorylation and upregulates STAT3 acetylation, resulting in decreased activity via increased nuclear export (Gupta et al., 2012, Liu et al., 2013). Furthermore, the synergistic interaction between romidepsin and mechlorethamine in CTCL has been linked with downregulation of JAK/STAT signaling (Cortes et al., 2021). However, it should be noted that the combination of romidepsin with mechloretamine induces downregulation of JAK1 protein levels, while suppression of JAK-STAT signaling following treatment with romidepsin plus afatinib is accompanied by decreased phosphorylation and total levels of STAT1 and STAT5 pointing to a convergent but molecularly distinct mechanism. In all, these results demonstrate a previously unreported synergistic interaction between romidepsin and afatinib via suppression of JAK-STAT signaling with a distinct mechanism of action. Finally, given the clinical efficacy of JAK-STAT inhibition in CTCL (Moskowitz et al., Moskowitz et al.) as well as studies suggesting JAK-STAT inhibition potentiates HDACi mediated cytotoxicity (Yumeen et al.), our results strongly support a central role for JAK-STAT inhibition in combination therapeutics in CTCL.

Importantly, we have demonstrated increased effect of treatment using the combination of romidepsin and afatinib dimaleate in romidepsin-resistant primary Sezary Syndrome samples. The relevance of these findings is highlighted by the common occurrence of resistance to therapy following treatment with HDAC inhibitors. In this context, synergistic drug combination therapies capable of eliciting more profound and durable responses are urgently needed to improve therapeutic efficacy and reduce the incidence of HDACi resistance. HDACi combinations with tyrosine kinase, proteasome, and immune checkpoint inhibitors, as well as other epigenetic modifiers (Cortes et al., 2021, Falchi et al., 2021) are all being tested in clinical trials. The strong synergism of afatinib plus romidepsin documented here in aggressive and HDAC inhibitor resistant models of CTCL support the investigation of this drug combination for the treatment of relapsed and refractory disease.

Materials and Methods

Cell Lines

We obtained the HUT78 (TIB-161) CTCL cell lines from the ATCC and purchased the mycosis fungoides MYLA cell line (95051032) from Millipore-Sigma (St. Louis, MO, USA) (Supplementary Table 1)

Cell Culture

We cultured all CTCL cell lines in RPMI 1640 medium with 2.05 mM L-Glutamine (Gibco) supplemented with penicillin (100 μg mL−1), streptomycin (50 μg mL−1), 20% fetal bovine serum (FBS) and 20 ng mL−1 human recombinant IL-2 (Peprotech, Rocky Hill, NJ, USA) at 37°C in a humidified atmosphere containing 5% CO2. We checked periodically for mycoplasma contamination using the Universal Mycoplasma Detection Kit (ATCC). We cultured primary Sezary Syndrome lymphoma cells in RPMI 1640 medium with 2.05 mM L-Glutamine supplemented with penicillin (100 μg mL−1), streptomycin (50 μg mL−1), 20% human serum (Sigma-Aldrich, St. Louis, MO, USA), 10 μg mL−1 IL-2 and 10 μg mL−1 IL-7 (PeproTech, Rocky Hill, NJ, USA). Prior to drug treatments, Sezary cells were stimulated for 24 hours with 10 μg mL−1 plated anti-CD3 (clone HIT3a) and 2 μg mL−1 soluble anti-CD28 (clone CD28.2) both from Thermo Fisher (Waltham, MA, USA).

Primary CTCL samples

We obtained de-identified peripheral blood mononuclear cells (PBMCs) isolated from patients who met WHO-EORTC criteria for Sezary Syndrome. Patient samples were collected with written informed consent and analysis was conducted under the supervision of the CUIMC Institutional Review Board (AAAQ8751). We isolated CD4+ T cells via negative selection using the Human CD4+ T cell isolation kit (Miltenyi, Auburn, CA, USA).

High throughput drug screening

High throughput drug screening was performed by the High-Throughput Screening Center at the JP Sulzberger Columbia Genome Center. MYLA CTCL cells were seeded in 384 well plates and treated with romidepsin IC20 concentration, 1 μM of each compound from the Prestwick Chemical Library (GreenPharma, Orléans, France) alone, or in combination with romidepsin in triplicate. We measured cell viability at 72 hours using the CellTiter-Glo luminescent assay (Promega, Madison, WI, USA).We calculated the treatment effects on cell viability by normalizing to DMSO and thimerosol controls.

The expected cell viability of the combination treatment, assuming both drugs acts independently, is calculated using the Bliss model (Bliss, 1939). The synergy score for each drug combination is defined as the difference between the observed cell viability and the predicted cell viability using the Bliss model, termed excess over bliss (EOB). A drug combination was considered synergistic if it demonstrated an EOB > 10 and p-value < 0.05.

Cell Cycle Analyses

For the analysis of cell proliferation and cell cycle we utilized the Click-iT EdU Pacific Blue Flow Cytometry Assay Kit (Invitrogen, Waltham, MA, USA). We treated CTCL cell lines with vehicle, single drugs and drug combinations for 24, 48 and 72 hours, in triplicate. At each time point, we counted and re-suspended the cells in fresh media at a concentration of 5*105 cells mL−1 with 10 mM EdU (5-ethynyl-2´-deoxyuridine) for 2 hours. After incubation, we collected, fixed and permeabilized the cells by re-suspending cells them in ice-cold 70% ethanol and stored them at −20°C until the time of analysis. For EdU labelling, we incubated the cells with the Click-iT reaction cocktail, following the manufacturer’s protocol. Cells were stained for DNA content by incubating them in PI (propidium iodide)/RNase Staining Solution (Cell Signaling Technology, Danvers, MA, USA) for 15 minutes prior to FACS analysis.

Drugs

We purchased the HDAC inhibitor drugs romidepsin (S3020), entinostat (S1053) and mocetinostat (S1122); the EGFR family inhibitors afatinib (S1011) and afatinib dimaleate (S7810); and thimerosol (S3646) from Selleckchem (Houston, TX, USA). For in vivo drug formulation, romidepsin was prepared at a final concentration of 0.12 mg mL−1 in 1.2% DMSO and 30% PEG-400. Afatinib dimaleate was prepared at a final concentration of 1.2 mg mL−1 (5% DMSO, 30% PEG-300).

Cell proliferation and drug response analyses

We plated cells at a density of 5x105 cells mL−1 in 96-well plates and treated them in triplicate with single drug or drug combination using a HP D300e Digital Dispenser (HP, Palo Alto, CA, USA). For each drug or drug combination tested, treatment wells are normalized to the highest volume of DMSO and a DMSO and thimerosol (500 μM) controls are included in triplicate. We measured cell growth and viability after 72 hours using the Cell Counting Kit 8 (ab228554) (Cambridge, UK), a WST-8 / CCK8 tetrazolium salt based colorimetric assay. Response to treatment was measured by normalizing signal to DMSO and thimerosol controls. We used GraphPad Prism 7.0 to generate non-linear fit dose-response curves and calculate the mean inhibitory concentration (IC20, IC50) for each drug at 72 hours of treatment.

To validate the screen results, we evaluated the combination of romidepsin and the top hits from the screen, mocetinostat and entinostat, using a WST-8 tetrazolium salt-based cell viability assay. To verify that effects of drug combinations were synergistic, we utilized a non-constant ratio dose response design using the IC20 concentration for each drug as the maximum drug dose for both drugs tested in the MYLA and HUT78 cell lines. Synergy was then evaluated using SynergyFinder 3.0, a R package that calculates synergy scores using the bliss, loewe, highest single agent (HSA), and zero interaction potency (ZIP) models (Berenbaum, 1989, Bliss, 1939, Loewe, 1953, Yadav et al., 2015). A drug combination was defined as synergistic if the average synergy score across all combination doses tested was greater than 10 for any single scoring model. A score between −10 and 10 is considered additive and a score below −10 is considered antagonistic.

Evaluating synergy using non-constant ratio dose response

To evaluate synergy, we utilized a non-constant ratio design when performing drug response assays for combination treatment. We plated cells at a density of density of 5x105 cells mL−1 in 96-well plates and treated them romidepsin at 0.2, 0.4, 0.6, 0.8 and 1x IC20 or the drug of interest at 0.25, 0.5, 0.75 and 1x IC20 in triplicate. Cells were also treated at every possible combination of the doses used for romidepsin and the drug of interest (total 20 combination treatments) in triplicate. As controls, cells are treated with DMSO or thimerosol in triplicate. We then measured cell viability using the Cell Counting Kit 8 (ab228554) at 72 hours of treatment. Synergy was evaluated using SynergyFinder 3.0 (v3.4.2) (Zheng et al., 2022), as before.

Apoptosis Analyses

For analysis of apoptosis, we treated CTCL cell lines and primary patient PBMC’s with vehicle, single drugs and drug combinations for 24, 48, and 72 hours and analyzed them by flow cytometry after staining with PE-conjugated Annexin V and 7-AAD (PE Annexin V Apoptosis Detection Kit I) (BD Bioscience, Franklin Lakes, NJ, USA), following the manufacturer’s protocol.

RNA isolation

Total RNA was extracted from CTCL cell lines after treatment with single drugs and combination using the RNeasy kit (Qiagen, Hilden, Germany) following manufacturer’s protocol. RNA quality and concentration was determined using a Nanodrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA)

RNAseq and gene expression profiling

RNAseq libraries were prepared from total RNA isolated from treated CTCL cell lines and sequenced by Azenta Life Sciences (South Plainsfield, New Jersey, USA). Paired-end sequencing (2x150 bp) was performed on a HiSeq series sequencing instrument (Illumina, San Diego, CA, USA). We obtained between 28.9 and 41.0 million paired-end reads per sample, with an average of 36.4 million reads. We mapped reads to the GRCh38.p12 reference genome using STAR v2.7.9 (Dobin et al., 2013). Count matrices were generated with featureCounts v2.0.0 (Liao et al., 2014) with gencode human transcript reference annotation v31.

Generation of luciferase expression CTCL cell lines

We transfected the FUW-mCherry-Puro-Luc lentiviral vector carrying the mCherry and luciferase genes together with the plasmid vectors encoding the Gag-Pol (pCMV ΔR8.91) and V-SVG (pMD.G VSVG) viral proteins into HEK293T cells using the JetPEI transfection reagent (Polyplus Transfection, Illkirch-Graffenstaden, France). We used viral-particle containing supernatants for infection of HUT78 cells by spinoculation using standard procedures. We assessed the efficiency of infection by detection of mCherry expressing cells by flow cytometry.

Mouse husbandry and procedures

We maintained all animals in specific pathogen-free facilities at the Irving Cancer Research Center at the Columbia University Medical Center campus. All animal procedures are approved by the Institutional Animal Care and Use Committee (IACUC) at Columbia University Medical Center.

For tumor transplantation assays, we injected 300,000 luciferized HUT78 T-cells subcutaneously into Rag1/Il2rg double knockout recipient mice (NOD.Cg-Rag1tm1Mom Il2rgtm1Wjl/SzJ, Stock No: 007799, Jackson Laboratory). We monitored tumor development by luciferase bioimaging with the In Vivo Imaging System (IVIS, Xenogen, Alameda, CA). Tumors were given 7 days after initial injection to engraft and were evaluated by IVIS imaging. Mice were then assigned to one of four treatment groups: DMSO, romidepsin, afatinib dimaleate, or combination. Romidepsin was administered at 1.2 mg kg−1 via intraperitoneal injection every four days (Cortes et al., 2021). Afatinib dimaleate was administered at 6 mg kg−1 via oral gavage daily for five days with two days of rest. Afatinib dimaleate dosing was determined after performing dose escalation experiment to determine maximum tolerated dose.

Western blot analysis

We prepared whole cell protein lysates using RIPA buffer supplemented with cOmplete, mini, EDTA-free Protease Inihibitor Cocktail (Roche, Basel, Switzerland). Protein quatification was performed using the Pierce BCA Protein Assay Kit following manufacturer’s protocol (Thermo Scientific, Waltham, MA, USA). Total cell lysate was then reduced and denatured using NuPAGE Sample Reducing Agent and NuPAGE LDS Sample Buffer (Thermo Scientific, Waltham, MA). We loaded 30 ug of denatured whole cell lysate onto a 4% to 12% Bis-Tris gel (Thermo Scientific, Waltham, MA), separated protein by gel electrophoresis, and transferred the proteins to a nitrocellulose membrane for western blot analysis. The following antibodies were used: phospho-HER3 (Tyr1289) (Cell Signaling #4791), HER3 (Cell Signaling #12708), phospho-HER2 (Tyr1221/1222) (Cell Signaling #2243), HER2 (Cell Signaling #2165), phospho-STAT1 (Tyr701) (Cell Signaling #7649), STAT1 (Cell Signaling #9172), phospho-STAT3 (Tyr705) (Cell Signaling #9145), STAT3 (Cell Signaling #9139), phospho-STAT5 (Tyr694) (Cell Signaling #9314), STAT5 (Cell Signaling #94205).

Statistical analyses

We evaluated significance in apoptosis assays using two-tailed Student’s t-tests and in in vivo bioluminescence quantification of tumor growth using a Mann-Whitney U test.

Supplementary Material

1

Acknowledgements

B.B.S. is supported by the Ruth L. Kirschstein NRSA administered by the National Cancer Institute (1F31CA261153). C.R. and R.A.G were supported by Career Development Program Awards from the Leukemia and Lymphoma Society. A.P.L. is supported by a Postdoctoral Fellowship by the Lymphoma Research Foundation. T.P. is supported by National Institutes of Health U01 5U01CA243073-04 and R01CA256341-01 awards and Department of Defense RA210160 award. T.P. was a recipient of a Velocity Award sponsored by the Herbert Irving Comprehensive Cancer Center (HICCC) at Columbia University. This research was funded in part through the NIH/NCI Cancer Center Support Grant P30CA013696 and used the Genomics and High Throughput Screening and the Oncology Precision Therapeutics and Imaging Cores at the HICCC at Columbia University.

Footnotes

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Conflict of Interest

Dr. Geskin serves on the Advisory Board for Therakos, Inc., Merck & Co., and Celgene Corporation and is an investigator for Actelion Pharmaceuticals US, Inc., Merck & Co., Inc., Kyowa Kirin Pharmaceutical Development, Inc. and Celgene Corporation. Dr. Palomero is the recipient of a research grant from Kura Technologies.

Data Availability Statement

RNAseq datasets related to this article are available in the Gene Expression Omnibus data base (accession# GSE237167, https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE237167).

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Associated Data

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

Supplementary Materials

1
NIHMS1960155-supplement-1.pdf (11.1MB, pdf)

Data Availability Statement

RNAseq datasets related to this article are available in the Gene Expression Omnibus data base (accession# GSE237167, https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE237167).

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