IL-9 promotes migratory dissemination of malignant T-cells by activating the HIF-1α-Cofilin-1 axis in cutaneous T-cell lymphoma

Abstract

Cutaneous T-cell Lymphoma (CTCL) is a multistage disease characterized by rapid dissemination of malignant T lymphocytes from skin lesions to visceral organs and bone marrow. The cytokine IL-9 and its receptor (IL-9R) are aberrantly overexpressed in CTCL lesions and function to enhance tumor cell survival. Here, we uncovered a critical new role for IL-9 as a potent inducer of migration of malignant T-cells. Stimulation of IL-9R-expressing T-cell lymphoma cells with IL-9 induced a pseudohypoxic cellular state by elevating downstream levels of the pro-migratory and oxygen-sensing transcription factor, hypoxia inducible factor (HIF)-1α. High-throughput quantitative proteomics analyses of pseudohypoxic malignant T-cells identified the actin-modulating protein, Cofilin-1, as a pro-migratory CTCL-intrinsic target downstream of IL-9-HIF-1α signaling. Consistently, multicolor immunofluorescence staining revealed marked co-expression of Cofilin-1 with HIF-1α in both IL-9-treated human lymphoma cell lines and in patient CTCL skin biopsies compared to normal controls. Genetic knockdown of IL-9R or HIF-1α in human T-cell lymphoma lines by RNA interference significantly reduced both HIF-1α and Cofilin-1 co-expression and reversed IL-9-induced migration. Finally, pharmacological antagonism of HIF-1α activity using the FDA-designated orphan drug, echinomycin, significantly abrogated IL-9-triggered migration of both malignant T-cell lines as well as patient-derived T-cell lymphoma cells from CTCL biospecimens. Implications: Our results uncover a CTCL-intrinsic IL-9-HIF-1α-Cofilin-1 axis as a critical promoter of malignant T-cell migration. They further identify HIF-1α and Cofilin-1 as promising therapeutic targets to mitigate IL-9-induced CTCL dissemination.

INTRODUCTION

Cutaneous T cell lymphoma (CTCL) is a lymphoproliferative disorder of malignant clonal skin-resident or skin-homing T cells. Clinicopathologically, CTCL can broadly be classified into two types, mycosis fungoides (MF), which presents as erythematous skin lesions harboring epidermotropic lymphoid infiltrates, or the more aggressive Sezary syndrome (SZ) marked by lymph node and peripheral blood infiltration as well as cutaneous inflammation(1). While SZ and MF were previously believed to constitute two independent CTCL types, emerging data suggests they could in fact be linked such that SZ might be a more aggressive or migratory outgrowth of MF (2). Approximately 25% of MF patients present with secondary lesions of the lymph node, bone, lung, liver, or kidney (1). Once tumor cells disseminate from the primary site and colonize these vital internal organs, treatment of CTCL and its underlying symptoms becomes extremely difficult, reflecting its poor prognosis of 53% survival of patients with advanced disease (3). Clearly, tumor cell movement is a critical though poorly understood fate-determining process in CTCL.

Progression of CTCL is regulated by multiple cell types within the skin tumor microenvironment (TME), including immune cells, fibroblasts, and keratinocytes. Moreover, soluble cytokine, chemokine and growth factor mediators exert diverse activities on cellular functions and crosstalk within the TME. Because cytokines are indispensable orchestrators of tumor cell proliferation and metastastic dissemination, they have thus been a major focus of cancer research. For instance, significant association of IL-1β, IL-6, IL-8, IL-17, and transforming growth factor (TGF)-β with cancer metastasis and poor patient prognosis has been reported (4–6). In CTCL specifically, interleukin (IL)-2, IL-4, IL-7, IL-13, and IL-15 factor prominently in disease pathogenesis (7).

Among all cytokines, IL-9 is particularly critical in CTCL progression owing to its pleiotropic effects within the TME of the skin. Under normal physiologic conditions, IL-9 is secreted by skin resident T helper type 9 (Th9) cells and facilitates host defense against fungal pathogens (8). However, IL-9 is also profusely secreted by malignant cells in lesional skin of patients with MF. We (9) and others (10) have previously reported that IL-9 and its receptor, IL-9R, are both conspicuously enriched on malignant versus normal T-cells from CTCL patients and potently promote T-cell lymphoma survival. We further demonstrated that IL-9 not only reduced oxidative stress in patient CTCL T-cells (9) and keratinocytes (11) but also triggered a Warburg-like effect in both cell types (12). Such inflammatory settings coincided with IL-17-mediated induction of the oxygen-sensing transcription factor, HIF-1α (13). We thus hypothesized that pro-inflammatory IL-9 would similarly trigger HIF-1α upregulation as an essential adaptation to hypoxic stress conditions in highly proliferative CTCL lesions with increased oxygen demand (14). Indeed, HIF-1α fine-tunes gene expression of multiple critical pathways impacted by oxygen, such as glucose metabolism (15,16) and angiogenesis (17). Under low oxygen conditions, HIF-1α reduces reactive oxygen species (ROS) levels (18), thereby rendering cells more resilient to hypoxic stress. Strikingly, downstream effector targets of HIF-1α also include those with pivotal roles in cellular motility, migration, and tumor metastasis, including regulators of dynamic actin filament formation and depolymerization (19). Nonetheless, the precise roles of IL-9 and HIF-1α in CTCL dissemination, in particular their functional effects on the migratory machinery of malignant T-cells, have not been characterized.

Here, we identify IL-9 as a potent inducer of migration of IL-9R-expressing human malignant T-cell lymphoma lines and CTCL patient-derived malignant T-cells as examined in three-dimensional (3D) invasive motility assays by time-lapse video microscopy. Consistently, IL-9 treatment failed to stimulate migration of IL-9R-negative non-malignant T-cells or B-cell lymphoma cells, or IL-9R knockdown CTCL lines, thus highlighting the IL-9:IL-9R axis as a preferential driver of malignant T-cell migration. Western blot and multicolor immunofluorescence assays revealed significant elevation of HIF-1α protein expression in IL-9-stimulated CTCL cell lines as well as in skin lesions of patients with CTCL in comparison to healthy control specimens. Using an unbiased high-throughput proteomics platform, we further identified the actin-binding and filament turnover protein, Cofilin-1, as a pseudohypoxia-induced CTCL-intrinsic protein and subsequently validated it as a downstream target of IL-9-HIF-1α using several independent methodologies. Specifically, both HIF-1α and Cofilin-1 were concurrently elevated and co-expressed in IL-9-treated malignant T-cells as well as in patient CTCL skin biopsies. In agreement, inhibition of HIF-1α in multiple human CTCL lines, either by genetic knockdown via RNA interference (RNAi) or pharmacological antagonism with the FDA orphan drug, echinomycin (20), markedly suppressed both downstream Cofilin-1 protein level and resultant IL-9-induced migration. Our results thus uncover a pro-migratory IL-9-HIF-1α-Cofilin-1 migratory circuit intrinsic to malignant T-cells in CTCL, the therapeutic targeting of which markedly dampens IL-9-induced migration.

MATERIALS AND METHODS

Patient CTCL Biospecimens

CTCL patients (n = 12, Table S1) and healthy control volunteers (n = 12) were registered at Tata Memorial Hospital, Mumbai and B.Y.L. Nair Charitable Hospital, Mumbai, respectively. Patients with ages 18 or above, with known T cell lymphoma (cutaneous or peripheral) with blood involvement were included in the study. It was also ensured that patients were treatment naïve (with the exception of topical steroids). Samples were classified according to the Tumor, Node, Metastasis, Blood (TNMB) staging criteria. Patients excluded from the study included individuals with co-existing skin ailments such as psoriasis or autoimmune disorders or HIV/HCV/HBV infections, or those on immunosuppressive therapy. Eligible patients with ages ranging from 19 to 59 were recruited. Healthy individuals were recruited based on absence of any disease presentation, inflammatory skin disorders and infections. Punch skin biopsies from CTCL and healthy patients as well as blood samples from CTCL patients with known blood involvement (n = 9) were collected from Tata Memorial Hospital consistent with protocols approved by the Institutional Ethics Committee of Tata Memorial Centre and IIT Bombay, under document numbers IITB-IEC/2016/017. Written informed consent was obtained from all subjects, and all studies were conducted in accordance with both the Declaration of Helsinki and Institutional Ethics Committee guidelines at each respective hospital.

Cell Culture

Human T-cell lymphoma cell lines Jurkat E6.1(RRID:CVCL_0065), Hut-78 (RRID: CVCL_0337), and MJ (RRID:CVCL_1414), and human B-cell lymphoma cell lines, Raji (RRID:CVCL_0511) and NALM6 (RRID:CVCL_UJ05), were obtained from ATCC (Manassas, VA). Cell lines were cultured in RPMI-1640 (Jurkat, Hut78, NALM6, and Raji) or Iscove’s Modified Dulbecco Medium (MJ) containing 10% (Jurkat, NALM6 and Raji) or 20% (Hut78 and MJ) fetal bovine serum (FBS) and 1% penicillin-streptomycin (Gibco, Waltham, MA, USA) at 37°C, 5% CO₂. Peripheral blood mononuclear cells (PBMCs) were isolated from blood of either healthy individuals or patients with CTCL by Ficoll-Paque (Merck, Dermstadt, Germany) density gradient centrifugation and then washed. All the aforementioned cell lines were confirmed negative for mycoplasma contaminations using the Mycoplasma PCR Detection Kit (G238, Applied Biological Materials Inc.). T-cells from peripheral blood of healthy and CTCL patient samples were isolated from PBMCs by immunomagnetic negative selection using the EasySep^™ Direct Human T Cell Isolation Kit (STEMCELL Technologies, Cambridge, MA, USA, 19661). T-cells isolated from CTCL patient samples contained a mixture of both malignant and non-malignant T-cells. Activated human T-cells were generated by first collecting nonadherent plated PBMCs from healthy individuals, depleting monocytes, and activating T-cells by incubation with anti-CD3/CD28 Dynabeads Human T-cell Activator (1:1 ratio of beads:cells; Gibco, 11131D) for 48 hrs in the presence of IL-2 (20ng/ml, Gibco) in AIM-V Medium (Gibco) containing 5% heat-inactivated FBS. All primary T-cell cohorts were subsequently expanded and cultured in AIM-V Medium containing 5% heat-inactivated FBS and 1% penicillin-streptomycin.

Antibodies and Reagents

The following antibodies (abs) were used for flow cytometric analyses (all from BioLegend, San Diego, CA, USA): PE-conjugated anti-human IL-9R (CD129, clone AH9R7; 310404, RRID: AB_314817) and PE-conjugated mouse IgG2b isotype control (clone MPC-11; 400311, RRID: AB_2894969); PE-conjugated anti-human IL-2RG (CD132, clone TUGh4; 338605, RRID: AB_2233554) and PE-conjugated rat IgG2b isotype control (clone RTK4530; 400607, RRID: AB_326551). The following abs were used for Western blotting (all from Thermo Fisher Scientific, Waltham, MA, USA): Anti-HIF-1α (PA1–16601, RRID: AB_2117128), anti-Cofilin-1 (clone GT567; MA5–17275, RRID: AB_2538741), anti-β-actin (PA1–183, RRID: AB_2539914), horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (H+L, 31460, RRID: AB_228341), and HRP-conjugated goat anti-mouse IgG (H+L, 32430, RRID: AB_1965958). The following abs were used for immunofluorescence (all from Thermo Fisher Scientific): Anti-HIF-1α (PA1–16601) and Alexa Fluor 488-conjugated goat anti-rabbit IgG (H+L) highly cross adsorbed (A11008, RRID: AB_143165), anti-IL-2RG (PA5–119907, RRID: AB_2913479) and Alexa Fluor 594-conjugated goat anti-rabbit IgG (H+L) highly cross adsorbed (A11037, RRID: AB_2534095), anti-Cofilin-1 (clone GT567; MA5–17275), anti-IL-9R (clone IC2A6; MA5–48505, RRID: AB_3090895) and Alexa Fluor 488-conjugated goat anti-mouse IgG (H+L) cross adsorbed (A11001, RRID: AB_2534069), anti-CD3 clone CD3–12; MA5–16622, RRID: AB_2538118), Alexa Fluor 647-conjugated donkey anti-rat IgG (H+L) highly cross adsorbed (A78947, RRID: AB_2910635) and Alexa Fluor 594-conjugated donkey anti-rat IgG (H_L) Highly cross adsorbed (A21209, RRID: AB_2535795). Pseudohypoxia was induced with cobalt chloride (CoCl₂) (50μM, Sigma-Aldrich, St. Louis, MO, USA).

Flow Cytometry

Human T and B cell lymphoma lines, patient-derived malignant T-cells, and human primary activated and unactivated T-cells were stained with PE-conjugated anti-human IL-9R, anti-human IL-2RG, or respective isotype control abs (5μl ab per million cells). Cells were incubated 1 hr in the dark, washed twice in FACS buffer (2% FBS in PBS), data acquired on a BD FACSVerse (BD Biosciences, NJ, USA) and analyzed by BD FACSuite software.

RNA Interference

Endoribonuclease-prepared small interfering RNAs (esiRNAs) were used for genetic knockdown of human IL-9R gene (IL9R) and HIF-1α gene (HIF1A) expression. Specifically, MISSION^® esiRNA (MilliporeSigma, Burlington, MA, USA) targeting either HIF1A (EHU151981), IL9R (EHU079431) or negative control Firefly Luciferase (FLuc, EHUFLUC) were reconstituted in RNase-free Tris-EDTA (TE) buffer, pH 8.0 and transfected into either 0.5–1 × 10⁶ cells for Western blotting (30pmoles esiRNA) or 5 × 10⁴ cells for invasion assays (10pmoles esiRNA) in media devoid of serum using Lipofectamine RNAiMAX (Thermo Fisher Scientific) as per the manufacturer’s instructions. Transfected cells were centrifuged at 1000 rpm for 30 mins at 37°C, knockdown validated by Western blotting, and cell transfectants experimentally analyzed 18–48 hrs post-transfection in parallel with untransfected control cells.

IL-9 Receptor Overexpression

The full coding sequences of human IL-9R (NM_002186) and IL-2RG (NM_000206) genes were cloned into pCDNA3.1+P2A via HindIII/BamHI and EcoRI/XhoI restriction sites, respectively (GenScript, Piscataway, NJ, USA). Integration was confirmed by restriction digestion and DNA sequencing. The plasmid was then transfected into Jurkat cells using Lipofectamine LTX and Plus reagent (Thermo Fisher Scientific) according to the manufacturer’s protocol. Cell transfectants with high IL-9R surface protein expression were sorted 48 hrs post-transfection on a BD FACSAria (BD Biosciences) and maintained in complete RPMI media at 37°C with 5% CO₂.

Immunoblotting

Cancer cell lines as well as unactivated or activated human primary T lymphocytes were subjected to the following cytokine and/or drug treatments: IL-9 (50ng/ml, Peprotech, Cranbury, NJ, USA), CoCl₂ (50μM), HIF1A, IL9R or FLuc siRNAs (30pmoles), or echinomycin (5nM, Merck, SML0477) for 24 hrs at 37°C, 5% CO₂. Cells were lysed in RIPA buffer (Merck), lysates sonicated using Vibra-Cell Probe Sonicator (Sonics & Materials, Newtown, CT), and proteins pelleted by centrifugation at 16,000g. Protein extracts were quantified using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, 23225), resolved by SDS-PAGE, and then transferred onto PVDF membranes (BioRad, Hercules, CA, USA) using the Bio-Rad Semi-Dry Transfer Cell System (Bio-Rad). Membranes were blocked in 7.5% non-fat dry milk (HiMedia, Mumbai, India) in Tris-buffered saline, 0.1% Tween 20 (TBST, HiMedia) and incubated overnight at 4°C with anti-HIF-1α (1:1500) or anti-Cofilin-1 (1:500) abs in blocking solution. Blots were washed in TBST, incubated with HRP-conjugated secondary ab (1:10,000) for 2 hrs at RT, and imaged using the Excellent Chemiluminescent Substrate (ECL) Detection Kit (Elabscience, Houston, TX, USA) on the iBright 1500 Imaging System (Thermo Fisher Scientific).

Time-Lapse 3D Migration

Cells (5 × 10⁴) in either RPMI-1640, IMDM, or AIM-V media containing 10% FBS or 5% heat-inactivated FBS and 1% Pen-Strep were seeded in tissue culture-treated 24-well plates (Costar, Corning, NY, USA) and treated with either IL-9 (50ng/ml), CoCl₂ (50μM), HIF1A or IL-9R targeting siRNA (10pmoles) or echinomycin (5nM) and incubated under sterile conditions at 37°C and 5% CO₂. To separate 24-well plates, 500μl of 2% glutaraldehyde was added and incubated for 20 mins. The glutaraldehyde was thereafter aspirated and the plates were allowed to dry overnight under sterile conditions. Seeded cells above were collected and pelleted. Rat tail collagen type I (Gibco, 3mg/ml, pH 3.4) was mixed with 10x PBS, neutralized and diluted by addition of 1N NaOH to a final concentration of 1mg/ml collagen, pH 7.0 in 1x PBS, 0.01N NaOH and mixed with cells. The suspension was added to glutaraldehyde-coated wells for 45 mins at 37°C under sterile conditions until semi-solid collagen matrices containing embedded cells had formed. Complete media (RPMI-1640, IMDM, or AIM-V with 10% FBS or 5% heat-inactivated FBS and 1% Pen-Strep) either untreated or containing 50ng/ml IL-9, 5nM echinomycin, and/or 50μM CoCl₂ were layered atop the solidified gels. Cell migration and invasion were analyzed by time-lapse videomicroscopy over 24 hrs under 5% CO₂ and 37°C in a sterile environment using the Yokogawa CSU-X1 Spinning Disc Confocal Microscope (Electric Corporation, Yokogawa, Tokyo, Japan).

High Throughput Label-Free Quantitative Proteomics

CoCl₂ treated (50μM) versus untreated Jurkat cells (24 hrs, 37°C) were analyzed. Cells (3 × 10⁶) were lysed in Urea-Thiourea buffer (6M Urea, 2M Thiourea, pH 8.0, Merck) and sonicated using the Vibra-Cell Probe Sonicator (Sonics). Proteins were quantified by the Bradford assay (Himedia) and digested overnight at 37°C in Promega Trypsin Gold (Promega Corporation, Madison, WI, USA). Peptides were dried and de-salted on a C18 resin column (Merck). Purified peptides were quantified and approximately 600ng injected into the Thermo Scientific Q-Exactive Plus Mass Spectrometer via a liquid chromatography acetonitrile gradient of 120 min using the EasyNLC HPLC autosampler connected to a C18 PepMap analytical column (Thermo Fisher Scientific) for label-free quantitative proteomic analysis. The instrument was set to MS1 resolution of 70000 and MS2 resolution of 17500. MS spectra were analyzed using Proteome Discoverer 2.2 software (Thermo Fisher Scientific, RRID: SCR_014477). Discovery proteomics workflows were used with Mascot and SequestHT as search engines. This identified 3,220 protein hits with high confidence after false discovery rate (FDR)-based filtering that required at least two unique peptides per identified protein. Only unique peptides with abundance ratios of p < 0.05 were analyzed in order to identify significant differentially expressed proteins (DEPs).

Immunofluorescence

Skin biopsies of CTCL patients (n = 9) and healthy individuals (n = 9) were collected from Tata Memorial Hospital, Parel and B.Y.L. Nair Hospital, respectively (Mumbai, India) and stored in 40% formaldehyde, 13% methanol at RT until processed. Tissues were washed sequentially one time each in ethanol (Merck) solutions of 50%, 70%, 80%, and 95%, thrice in 100% ethanol, once each in 2:1, 1:1 and 1:2 ethanol:xylene solutions, three times in 100% xylene (Sisco Research Laboratories, Mumbai, India), once in 2:1, 1:1, 1:2 xylene:molten paraffin (Sisco Research Laboratories) solutions, once in 100% paraffin, and then mixed with molten paraffin overnight at 68°C. Tissues were then embedded in molten paraffin, solidified, paraffin blocks were removed from the mold, and tissues sectioned using a microtome. Sections of 5 micron thickness were cut and mounted on Silane coated glass slides (BioMarq, Hyderabad, India), dried, and stored at RT until staining.

For immunofluorescence staining of FFPE tissues, slides were heated at 60°C for 15–20 s to melt the paraffin, washed in 100% xylene, and then rehydrated by washing once sequentially in 100%, 95% and 85% ethanol. Antigen retrieval was performed by heating the slides in citrate buffer (10mM sodium citrate, 0.05% Tween 20, pH 6.0, Himedia and Merck, respectively) in a water bath at 95°C for 25 mins. Slides were cooled, dried, fixed in 4% paraformaldehyde (HiMedia) for 20 mins at 0°C, and blocked in 2% BSA (HiMedia) in PBS for 30 mins at RT. Anti-CD3 (1:200), HIF-1α (1:200) and Cofilin-1 (1:100) abs were diluted in blocking solution containing 0.1% Triton X-100 (Merck) and incubated with sectioned tissues overnight at 4°C. Excess ab was removed by washing, and sections were incubated with Alexa Fluor 488 or Alexa Fluor 594-conjugated abs (1:500) and DAPI (1:1000) solutions. Sections were mounted using ProLong Gold Antifade Mountant (Thermo Fisher Scientific) and imaged on a Zeiss LSM 780 Confocal Microscope (Zeiss, Oberkochen, Germany).

For immunofluorescence staining of cells, 0.5 × 10⁶ cells were seeded onto Poly-D-Lysine coated μ-Slide 8 Well Plates (ibidi GmbH, Gräfelfing, Germany) and treated with or without recombinant (r) human IL-9 (50ng/ml, Peprotech) for 24 hrs at 37°C in a tissue culture incubator. Cells were fixed with 4% ice cold paraformaldehyde for 10 mins at RT, washed thrice in PBS, permeabilized with 0.1% Triton X-100 in PBS for 20 mins at RT, washed thrice to remove excess detergent, and then blocked with 2% BSA in PBS for 2 hrs. Anti-HIF-1α (1:200), Cofilin-1 (1:100), IL-9R (1:100), IL-2RG (1:100) or CD3 (1:100) abs in 0.1% BSA were added to wells and incubated overnight at 4°C. Cells were washed twice with PBS followed by addition of Alexa Fluor 594, Alexa Fluor 488, or Alexa Fluor 647-conjugated secondary ab (all at 1:500) in 0.1% BSA along with DAPI (1:1000) and incubated in the dark for 2 hrs at RT. Cells were imaged as above and/or using an Super Resolution Confocal Eclipse Ti2 Microscope (Nikon, Melville, NY, USA).

MTT Assay

Jurkat and healthy primary activated T-cells (5 × 10⁴) were seeded in tissue culture-treated 96 well plates (CoStar, Corning, NY, USA) with echinomycin (0nM-400nM) for 24 hrs at 37°C, 5% CO₂ under sterile conditions. 10μl of MTT (0.25mg/ml final concentration, Sigma-Aldrich) was added to each well and incubated for 3 hrs at 37°C. Finally, 150μl of MTT stop solution (90μl DMSO and 60μl 30% SDS) was mixed into each well with gentle pipetting. Absorbances at 570 nm were measured after 15 min of shaking on a Multiskan FC Microplate Photometer (Thermo Fisher Scientific).

Cancer Dependency Map Analysis

Cancer Dependency Map (DepMap) analysis of RNA-seq data and CRISPR-based HIF1A and CFL1 gene knockout (KO) effects was performed using the publically available Broad Institute database (https://depmap.org/portal). The “Cell Line Selector” option was employed to generate a curated list of diverse malignant T cell lines with associated CRISPR-based KO gene screening data. Effects of HIF1A and CFL1 gene KO on cell viability were plotted as Gene Effect (Chronos) versus gene expression level for all respective cell lines. Density distribution plots were used to visualize CFL1 KO effects on T cell versus non-T cell lines. Data analyses and graphical depictions were rendered in R 3.4.1 using ggplot2 (RRID: SCR_014601), dplyr and ggrepel packages.

Cell Viability Assay

Cells (1 × 10⁵) were seeded in respective growth media, treated with IL-9 (50ng/ml), and incubated for 24 hours at 37°C, 5% CO₂. IL-9-treated or untreated cells were collected and subseqently stained with 0.4% trypan blue dye (Sigma-Aldrich). Cells were loaded onto a hemocytometer and live versus dead cells were quantified under a compound light microscope.

Apoptosis Assay

Transfected esiRNA-HIF1A or esiRNA-fluc control cells (1 × 10⁵) were collected and washed once with 1x Annexin V Binding Buffer (BioLegend, 40922). Cells were then stained concurrently with Annexin V and 7-AAD (5 μl each per 1 × 10⁶ cells) and incubated in the dark for 30 minutes at room temperature. After incubation, they were washed once with FACS staining buffer and analyzed using the Beckman Coulter CytoFLEX flow cytometer (Beckman Coulter, CA, USA).

Statistical Analysis

Unpaired, two-tailed Student t-test was used to compare data in Figs. 1–3 and Figs. 5–7. Background-based ANOVA from the Proteome Discoverer software (RRID: SCR_014477) was used to calculate significance of protein or peptide abundance changes relative to controls in Fig. 4. Specific statistical tests used are described in respective figure legends. Values of p < 0.05 were considered statistically significant. Asterisks denote p-values as follows: *, p < 0.05; **, p <0.01; ***, p < 0.001; ****, p < 0.0001. Prism 8.0.2 software (GraphPad, La Jolla, CA, USA, RRID: SCR_002798) and R version 3.4.1 were used to perform statistical analyses. Biological and technical replicate information is indicated in the figure legends.

Study Approval

All studies of human subjects and patient biospecimens were approved by the Ethics Committees of IIT Bombay and Tata Memorial Hospital, Mumbai under document number IITB-IEC/2016/017.

Data Availability

Data generated or analyzed for this study can be obtained from the corresponding authors upon reasonable request. Raw data for shotgun proteomics experiments of Jurkat cells can be found in the MassIVE Knowledge Base (RRID: SCR_013665), an open-source online public repository for mass spectrometric data under DOI:10.25345/C5ZG6GK16. The R scripts used to analyze and plot DepMap public data can be found at the following link https://github.com/Immunoengineering/CTCL.

RESULTS

The IL-9 receptor is enriched among malignant human T-cells

Functional expression of the IL-9 receptor complex requires heterodimeric pairing of the cytokine-specific IL-9 receptor α-chain (IL-9Rα) with the interleukin 2 receptor gamma chain (IL-2RG)(21). Flow cytometric analyses of both receptor chains revealed surface protein expression of both pairing subunits of the IL-9 cytokine receptor, IL-9R (Fig. 1A) and IL-2RG (Fig. 1B), by all patient CTCL T-cells examined (n = 3 patients). Mean expression levels of IL-9R and IL-2RG on patient CTCL T-cells were 194 ± 173 and 363 ± 64 (mean MFI ± SD), respectively. Immunofluorescence quadruple labeling of patient CTCL biospecimens for IL-9R and IL-2RG protein with the lymphocytic lineage marker, CD3, showed peripheral surface staining and strong colocalization of both receptor chains (r = 0.9665, Pearson correlation coefficient; n = 3 patient samples), thereby demonstrating IL-9 receptor complex expression by malignant patient T-cells (Fig. 1C). Notably, malignant T-cells from CTCL patients displayed increased levels of IL-9R and IL-2RG (390,852 ± 794,304 and 1,882 ± 2,765, respectively) in comparison to healthy, unactivated primary T-cells (256,420 ± 285,529 and 90 ± 43, respectively) (Supplementary Fig. S1A, B). Consistently, robust expression and colocalization of IL-9R and IL-2RG (r = 0.7, Pearson correlation coefficient) were also observed in the human CTCL T-cell line, MJ, as revealed by multicolor immunofluorescence (Fig. 1D). Indeed, flow cytometric analyses further confirmed high surface protein levels of both IL-9R (1,233 ± 12, mean MFI ± SD, Fig. 1E) and IL-2RG (2060 ± 16, Fig. 1F) on MJ cells as well as independently on another human CTCL T-cell line, Hut78 (860 ± 131, IL-9R; 3787 ± 33, IL-2RG, Figs. 1E–F). While IL-9R and IL-2RG were also present on the human malignant non-CTCL leukemia line, Jurkat and on activated human primary T-cells, respective levels were markedly lower in comparison to MJ or Hut78 CTCL lines (Figs. 1E–F). Strikingly, IL-9R and IL-2RG expression was either negligible and/or considerably reduced on unactivated human primary T-cells as well as on both B cell lymphoma lines in comparison to malignant T-cell lines (Figs. 1E–F). Together, these results demonstrate enriched protein expression of the IL-9 receptor complex on malignant T-cells of CTCL patients as well as on established human CTCL cell lines.

Figure 1. The IL-9 receptor is enriched on malignant T-lymphocytes.

A-B, Expression (MFI ± SD, left panels) and representative flow cytometric histograms (right panels) of A, IL-9R and B, IL-2RG surface protein level by CTCL patient-derived malignant T-cells. C-D, Representative quadruple immunofluorescence staining of C, a clinical CTCL skin biopsy and D, the established human CTCL T-cell line, MJ, for co-expression of IL-9R (green), IL-2RG (red), and CD3 (magenta). Nuclei were counterstained with DAPI (blue), with merged images shown. Size bars, 5–10 mm. E-F, Expression (MFI ± SD, left panels) and representative flow cytometric histograms (right panels) of E, IL-9R and F, IL-2RG surface protein expression by human T-cell lymphoma lines, Hut78 and MJ, T-cell leukemia line, Jurkat, activated and unactivated primary T-cells, and B-cell lymphoma lines, NALM6 and Raji. *p < 0.05. All results include n=3 biological replicates.

IL-9 induces migration of malignant T-lymphocytes

To examine whether the observed elevated expression of IL-9R on malignant CTCL T-cells functionally promotes migration, we tracked invasive movement of cells through 3D collagen matrices using real-time video microscopy. Indeed, when recombinant human IL-9 was layered atop the semi-solid collagen extracellular matrix (ECM) gel, migration distances over 24 hrs of embedded CTCL patient-derived T-cells, malignant T-lymphocytic lines (Hut78, MJ and Jurkat), and activated T-cells all significantly increased approximately two-fold versus untreated control conditions (Figs. 2A–E, respectively). On the other hand, cell types with negligible levels of the IL-9R complex, including unactivated primary T-cells as well as NALM6 and Raji B-cell lymphoma lines, showed no significantly increased invasive migration in response to IL-9 treatment (Figs. 2F–H, respectively). Consistently, genetic knockdown of IL-9Rα in Hut78, MJ, Jurkat, or primary activated T-cells attenuated IL-9-induced migration down to baseline untreated levels (Figs. 2I–L). These findings thus uncover an IL-9:IL-9 receptor pro-migratory axis as a critical driver of malignant human T-cell motility in CTCL.

Figure 2. IL-9 stimulates invasive migration of malignant T-cells.

A-L, Bar graphs (left panels) and representative migratory track plots (right panels) of cumulative distances traveled through collagen matrices over 24 hrs of embedded untreated (UT) versus IL-9-treated A-D, malignant T-lymphocytes (A, CTCL patient-derived T-cells, B, Hut78, C, MJ, D, Jurkat), E, activated T-cells, F, unactivated T-cells, G-H, B-cell lymphoma lines (G, NALM6, H, Raji), or I-L, IL-9R KD or fluc KD control variant I, Hut78, J, MJ, K, Jurkat, or L, activated T-cells, as determined by real-time videomicroscopy. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant. A-H, n=4 biological replicates; I-L, n=3 biological replicates.

IL-9 triggers downstream pro-migratory HIF-1α upregulation in malignant T-cells

Our group recently reported significant elevation of IL-9 in patient CTCL skin versus healthy controls as well as altered amount of reactive oxygen species (ROS) in CTCL patient T-cells (9). Given these results, we next investigated whether IL-9 in CTCL might thus directly control expression of HIF-1α, an established pro-migratory effector downstream of and highly responsive to ROS (22,23). Multiplex immunofluorescence labeling revealed a significant fold increase of cumulative HIF-1α intensity (9.5 ± 11.0, mean MFI fold change ± SD) in patient CTCL versus healthy control skin biospecimens (Figs. 3A–B). Consistently, HIF-1α showed strong colocalization with CD3-positive malignant T-cells (Figs. 3A–B). Biopsies from diverse stages and subtypes of T-cell lymphomas, including anaplastic lymphoma kinase (ALK)+ anaplastic large cell lymphoma (ALCL) and peripheral T-cell lymphoma (PTCL) were analyzed (Table S1), with no marked differences observed across specimens. Immunoblotting demonstrated a significant 2–3-fold induction of HIF-1α protein in IL-9-treated malignant T-cell lines, Hut78, MJ, and Jurkat as well as in activated primary T-cells versus respective untreated controls (Figs. 3C–F). In contrast, no such elevation of HIF-1α by IL-9 was found in cell types negative for the IL-9 receptor, including unactivated primary T-cells and B-cell lymphoma NALM6 and Raji cells (Figs. 3G–I). In agreement, incubation with positive control cobalt chloride (CoCl₂), an established stabilizer of HIF-1α protein and inducer of a hypoxia-like (e.g. pseudohypoxia) state (24), significantly stimulated HIF-1α expression in all cell types examined, including Hut78, MJ, and Jurkat malignant T-cells, activated and unactivated primary T-cells, and NALM6 and Raji malignant B-cells (Figs. 3C–I). Notably, CoCl₂ treatment also significantly stimulated cell migration of MJ (Fig. 3J) and Jurkat (Fig. 3K) malignant T-cells. Genetic silencing of HIF-1α or IL-9R expression in MJ or Jurkat via siRNA-mediated knockdown fully reversed IL-9-induced migration of both cell types down to or even exceeding baseline levels, while transfection with an siRNA control against Firefly Luciferase had no such inhibitory effect (Figs. 3J–K). Knockdown of IL-9R in malignant MJ, Hut78, or Jurkat T-cells fully abrogated IL-9 triggered HIF-1α upregulation (Supplementary Figs. S2A–C). Consistently, IL-9R overexpression (OE) in Jurkat cells resulted in even more pronounced induction of HIF1α by IL-9 treatment compared to untransfected control cells, further supporting IL-9R/IL-9 interaction in HIF1α upregulation (Supplementary Fig S2D). These results thus identify HIF-1α as a critical pro-migratory effector downstream of IL-9:IL-9 receptor signaling in CTCL malignant T-cells and validate CoCl₂ as a useful agonist for mimicking IL-9-based induction of both HIF-1α and cell migration. Moreover, they identify HIF-1α as a therapeutic target of IL-9-triggered migration, the inhibition of which abrogates unwanted migration of malignant T-cells.

Figure 3. IL-9 induces downstream pro-migratory HIF-1a in CTCL malignant T-cells.

A, Representative multiplex immunofluorescence staining of healthy versus CTCL patient skin biopsies for HIF-1a (green), CD3 (red), and DAPI (blue), with merged images shown. Size bars, 20 −50 μm. B, HIF-1a expression level (MFI ± SD) in patient CTCL versus healthy control skin biopsies as determined above by immunofluorescence staining, with bar graphs (left panel) and dot plots (right panel) of individual sample data points shown (n=4 biological replicates, n=8 technical replicates). C-I, Bar graphs (mean densitometric arbitrary units, AU ± SEM) and representative immunoblots of HIF-1a protein (94 kDa) levels in respective untreated (UT), IL-9-treated, or CoCl₂-stimulated cell cohorts, including C, Hut78, D, MJ, E, Jurkat, F, activated T-cells, G, unactivated T-cells, H, NALM6, and I, Raji. Data was normalized to respective actin (42 kDa) loading controls; n=4 biological replicates. J-K, Bar graphs showing cumulative distance traveled over 24 hrs by J, MJ and K, Jurkat cells. Representative track plots showing effect of IL-9, CoCl₂, siRNAHIF1A, or siRNAfluc on the migration potential of Jurkat cells over a period of 24 hrs (n=3 biological replicates). Mean ± SEM is plotted. *, p < 0.05; **, p < 0.01; ***p<0.001; ns, not significant.

Cofilin-1 actin modulator is co-induced with HIF-1α in malignant T-cells

To further dissect the IL-9-HIF-1α signaling pathway in malignant T-cell migration, we first leveraged CoCl₂ agonism as described above to chemically trigger a pseudohypoxic state (25) mirroring that of IL-9-induced HIF-1α upregulation. Cell lysates were then subjected to unbiased, high-throughput label-free quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify protein signaling clusters showing significant changes in expression between CoCl₂-induced pseudohypoxic versus untreated baseline conditions. Compared to untreated controls, incubation of malignant Jurkat T-cells with CoCl₂ (50μM) markedly induced expression of several HIF-1α pathway effector proteins, including Alpha-enolase (ENO1), Phosphoglycerate kinase (PGK1), L-lactate dehydrogenase B chain (LDHB), Triosephosphate isomerase (TP1) and Peroxiredixon-1 (PRDX1) (Table S2, Supplementary Fig. S1C). These results authenticated our CoCl₂-based approach for establishing a pseudohypoxic HIF-1α stabilized state resembling that of IL-9-triggered HIF-1α induction. The top 74 differentially expressed proteins between CoCl₂ treated versus untreated cohorts are depicted as both a heat map (Fig. 4A) and volcano plot (Fig. 4B). Additional functional annotation of top protein hits using the Database for Annotation, Visualization and Integrated Discovery (DAVID) as well as STRING pathway analysis identified significant enrichment of five major downstream clusters, including focal adhesion and actin-binding, glycolytic and metabolic, complement, lipid transport, as well as RNA binding, ribosomal, and heat shock proteins (Fig. 4C). Of these, the focal adhesion and actin-binding cluster showed the greatest enrichment score (22.32) and contained several established actin and motility-regulating cytoskeletal proteins, including Tropomyosin (TPM4), Tubulin (TUBB), and Moesin (MSN), among others (Table S3). A highly conspicuous top hit within this cluster was the pro-migratory, actin-severing protein, Cofilin-1 (26), owing to its significant and substantially increased abundance in CoCl₂-induced pseudohypoxic versus untreated non-hypoxic Jurkat cells (Fig. 4D). These results thus demonstrate upregulation of diverse actin-regulatory and migration effector proteins in HIF-1α induced, pseudohypoxic malignant T-cells. They further uncover Cofilin-1 as a potential candidate regulator of IL-9-HIF-1α triggered malignant T-cell migration.

Figure 4: Label-free quantitative proteomics reveals upregulation of actin cytoskeletal proteins in malignant T-cells by chemically induced hypoxia.

A, Heat map showing Top 74 differentially expressed proteins prepared using Metaboanalyst online tool (n=3 biological replicates, marked A-C). B, Volcano plot showing significantly increased versus decreased protein hits. Protein targets belonging to the focal adhesion cluster have been labeled. C, STRING network showing six clusters enriched in the LC-MS/MS data. D, Box plot of CFL-1 expression levels showing the distribution of data, with the horizontal line inside each box indicating the median value of expression levels in control versus CoCl₂ treated Jurkat cells; n=3 biological replicates.

The CTCL-intrinsic IL-9-HIF-1α axis promotes downstream Cofilin-1 expression in malignant T-cells

Cofilin-1 is an important cancer biomarker (27) given its overexpression in multiple tumor types and has been correlated with increased invasion, metastasis, and poor prognosis (28,29). Our unbiased proteomics results above identified co-induction of both Cofilin-1 and HIF-1α effectors in CoCl₂-stimulated pseudohypoxic malignant T-cells. We next chose to examine whether Cofilin-1 might be a direct downstream target of IL-9-HIF-1α in CTCL. Immunoblotting of malignant MJ (Fig. 5A) and Jurkat (Fig. 5B) lysates from either IL-9 or CoCl₂ treatment cohorts revealed significantly increased levels of Cofilin-1 (CFL-1) protein in comparison to untreated controls, thereby independently validating the LC-MS/MS data above and identifying CFL-1 as responsive not only to CoCl₂ but also directly to IL-9 stimulation. Consistently, multicolor immunofluorescence staining demonstrated a significant fold increase in CFL-1 protein level of 1.8 ± 0.4 (mean MFI fold change ± SD) in IL-9-rich CTCL versus IL-9-poor healthy skin biopsies, with CFL-1 positivity mostly confined to CD3-positive CTCL T-cells (Figs. 5C–D), suggestive of a CFL-1 role in disease pathogenesis. In agreement, immunofluorescence imaging of Jurkat cells incubated with IL-9 revealed significant co-induction of both CFL-1 and HIF-1α in comparison to untreated cells (Figs. 5E–F). Intriguingly, IL-9 treatment also altered cellular distribution of CFL-1 by triggering its localization from cytoplasmic to the cell membrane periphery of malignant T-cells (Figs. 5E–F), a region consistent with the known role of CFL-1 in actin regulation and motility. To directly assess if CFL-1 might be a downstream target under the control of IL-9-HIF-1α signaling, we transfected MJ and Hut78 malignant T-cells with endoribonuclease-prepared siRNAs targeting HIF-1α, which in the presence of IL-9 not only resulted in knockdown of HIF-1α protein as expected, but also significantly attenuated CFL-1 expression concurrently, as determined by immunoblotting (Figs. 6A–B). These results identified an IL-9-HIF-1α-Cofilin-1 pathway intrinsic to CTCL malignant T-cells and point to a potential role for the Cofilin-1 actin-severing protein in IL-9-induced migration.

Figure 5: Upregulation of Cofilin-1 and HIF-1a by IL-9 in CTCL.

Quantification of CFL-1 expression in control, IL-9 and CoCl₂ treated conditions measured by western blotting in A, MJ and B, Jurkat cells. Blots showing CFL-1 (21kDa) and actin (41kDa, loading control). CFL-1 expression levels have been plotted using bar graphs (n=4, biological replicates). C, CFL-1 expression was quantified in healthy and CTCL skin biopsies, with representative micrographs showing CFL1 (green), CD3 (red), DAPI-stained nuclei (blue) and merged images. D, Mean Fluorescence Intensity of CFL-1 for healthy and CTCL skin biopsies have been plotted. Dot plot showing data of individual samples (n=4 biological replicates). E, Co-expression of HIF-1α (green) and CFL-1 (red) in Jurkat cells under control vs. IL-9-treated conditions as determined by immunofluorescence (n=2 biological replicates). F, Bar graphs showing quantification of corrected fluorescence intensities (CFI) of HIF-1α and Cofilin-1 (Mean ± SEM). *p<0.05, **<0.01, ****p<0.0001.

Figure 6: Inhibition of HIF-1α suppresses Cofilin-1 expression in malignant T-cells.

A-B, Western blot quantitation of CFL-1 (21kDa) or HIF-1α (94kDa) normalized to b-actin (41kDa) in A, MJ or B, Hut78 cells in untreated control, IL-9-treated, siRNAHIF1A, siRNAfluc, or CoCl₂ sample or cell variant conditions (Mean ± SEM; n=3 biological replicates; *p<0.05, **<0.01).

Pharmacological inhibition of the HIF-1α-Cofilin-1 pathway reverses IL-9-stimulated migration of malignant T-cells

To further dissect both the translational and functional significance of therapeutically targeting the IL-9-HIF-1α-Cofilin-1 circuitry, we leveraged the FDA-approved orphan drug used in Acute Myeloid Leukemia (AML) and Graft-Versus-Host Disease (GVHD), echinomycin, which interferes with binding of HIF-1α to DNA (20). Treatment of MJ (Fig. 7A) and Jurkat (Fig. 7B) lines with echinomycin completely abrogated CFL-1 protein induction by IL-9 as determined by Western blotting, consistent with CFL-1 as a downstream target of the IL-9-HIF-1α pathway. In agreement, echinomycin also fully reversed IL-9 or CoCl₂-stimulated migration of CTCL patient-derived T-cells (Fig. 7C), MJ cells (Fig. 7D), Jurkat cells (Fig. 7E), and activated primary T-cells (Supplementary Fig. S3) down to untreated baseline levels. Given these promising results identifying echinomycin as a possible therapy in CTCL, we next investigated potential cytotoxic effects of echinomycin on both healthy patient and malignant T-cells. We first leveraged the publicly available Cancer Dependency Map (DepMap) portal to annotate diverse gene-dependent effects of HIF-1α disruption in several malignant T-cell leukemic and lymphoma cell lines. CRISPR-based HIF1A gene KO showed a nontoxic effect of >0.2, which fell well-outside the established toxicity range of <−1.0 (Supplementary Fig. S4A), indicating that HIF-1α inhibition via either genetic knockdown or echinomycin-based antagonism were unlikely to adversely affect T-cell viability. A similar DepMap nontoxic profile was obtained in the case of CFL-1 gene knockout, whether in malignant T-cell or non T-cell lines (Supplementary Fig. S4B) or cumulatively across T-cell versus other lines (Supplementary Fig. S4C). Likewise, IL-9 treatment also had no significant effect on cell viability over the short 24 hr duration (Supplementary Fig. S5A–H) of experiments, in contrast to its enhancement of cell viability reported over longer 72 hr timeframes of exposure (9). Consistently, annexin V and 7-AAD staining confirmed that HIF1A and IL-9R knockdown did not markedly affect apoptosis (Supplementary Fig. S5I–L). To strengthen these conclusions, we next performed functional MTT viability assays, the results of which showed half-maximal inhibitory concentrations (IC50) for echinomycin of 200nM for Jurkat cells (Supplementary Fig. S6A) and 100nM for primary T-cells (Supplementary Fig. S6B). Because these IC50 values were ≥ 20-fold greater than the echinomycin concentration (5nM) used to effectively inhibit IL-9-dependent migration, echinomycin should be well tolerated at this concentration. In total, these findings identify a pro-migratory IL-9:IL-9R dependent HIF-1α-Cofilin-1 circuit intrinsic to CTCL malignant T-cells. They further demonstrate that antagonism of HIF-1α-Cofilin-1 co-activity, either by HIF-1α genetic disruption or pharmacological antagonism using echinomycin, completely reverses IL-9-triggered migration of malignant T-cells in CTCL.

Figure 7: Echinomycin-based inhibition of HIF-1α attenuates IL-9-mediated CFL-1 expression and migration of malignant T lymphocytes.

Quantification of CFL-1 expression in untreated control versus IL-9, Echinomycin (E), or CoCl₂ treated conditions as measured by Western blotting of A, MJ or B, Jurkat cells. Blots show CFL-1 (21kDa) normalized to actin (41kDa) loading control. (n=3 biological replicates). C-E, Bar graphs showing cumulative distance traveled over 24 hrs by C, CTCL patient-derived malignant T-cells, D, MJ, or E, Jurkat cells. Data represents n=3 biological replicates. Representative track plots showing effects of IL-9, E, or CoCl₂ on migration over a period of 12 hrs. Mean ± SEM; *p<0.05, **<0.01, ***p<0.001, ****p<0.0001.

DISCUSSION

In this study, we identify critical roles for IL-9 in CTCL pathogenesis as a potent inducer of malignant T lymphocyte migration via downstream activation of HIF-1α and Cofilin-1 pathway effectors. Our investigation employed diverse human CTCL and non-CTCL control lines varying in IL-9R expression and consequent response to IL-9-induced migration as well as patient CTCL skin and blood biospecimens, time-lapse videomicroscopic analyses of cell migration, unbiased proteomics methodologies, multicolor immunofluorescence, flow cytometry, immunoblotting, viability assays, and genetic and pharmacological inhibitory approaches, including with the FDA-orphan drug, echinomycin. These findings thus add a new layer of mechanistic understanding to IL-9 triggered malignant T-cell migration in CTCL and uncover new opportunities for therapeutically targeting its HIF-1α and Cofilin-1 pro-migratory effectors to suppress IL-9 driven dissemination.

The IL-9 cytokine is conspicuously abundant within CTCL lesions (9) though its precise functional roles in CTCL progression, particularly with respect to invasive migration, have been largely undefined. Dissemination of malignant T-cells to internal organs is symptomatic of advanced-stage CTCL and coincides with discouragingly poor patient prognosis. Some cytokine/chemokine agonists, including members of the IL-22-CCL20-CCR6 axis, have been implicated as drivers of CTCL trafficking (30). CCL22 and CCL17 can also trigger CCR4-dependent transendothelial migration of CTCL cells (7). On the other hand, IL-9 may exhibit both pro- and anti-metastatic effects depending on context, which may differ according to the specific tumor type involved. For instance, in melanoma, breast, and cervical cancers, IL-9 reduces metastatic potential (31), while in pancreatic cancer models it augments cell migration (32). Hence, our findings of IL-9-triggered migratory enhancement of malignant T-cells is of great significance as it helps reconcile potentially disparate IL-9 functions by identifying it as pro-tumorigenic in the context of CTCL disease.

In support of IL-9 promotion of cancer cell migration in CTCL, expression level of the IL-9 receptor corresponded to migratory distances of malignant T-cells treated with IL-9. The IL-9 receptor is a heterodimer comprised of both IL9R in complex with IL2RG. Because IL-2RG is shared across diverse cytokine receptors (33), the IL-9R subunit represents the limiting determinant for IL-9 receptor complex expression and resultant response to IL-9. Notably, all IL-9R expressing cell types examined herein, including CTCL and non-CTCL cells, showed markedly enhanced migration under IL-9 treated versus untreated conditions. In contrast, IL-9 receptor negative non-CTCL cell types, including B-cell lymphoma lines, failed to respond to IL-9 stimulation. Our results are in agreement with an independent dataset available at the Human Protein Atlas (https://www.proteinatlas.org), confirming near absence of IL-9R across B-cell lines (34), thus underscoring the importance of IL-9:IL-9R interactions specifically in CTCL malignant T-cells, but not B-cell lymphoma. Interestingly, the IL-9 receptor was also expressed by activated, but not unactivated, primary T-cells, though at substantially reduced levels in comparison to malignant Hut78 and MJ T-cells. These results agree with a prior study identifying enriched IL-9R and IL-9 expression in CTCL patient T-cells and malignant Jurkat versus normal, unactivated T-cells (9).

Our group previously reported reduced oxidative stress in CTCL T-cells in response to IL-9 treatment (9), thereby leading us to hypothesize a potential downstream effect of IL-9:IL-9R on HIF-1α, an established suppressor of ROS (22). Apart from its prominent role as a key regulator of ROS and metabolic reprogramming, HIF-1α also has been previously shown to promote cancer metastasis via triggering of angiogenesis (35), proliferation (36), epithelial-mesenchymal transition (EMT)(37) and invasion (38). Hence, our findings of enhanced HIF-1α levels in IL-9 rich patient CTCL lesions versus healthy controls and in IL-9-stimulated malignant T-cell lines and patient cells support HIF-1α as a target of IL-9-induced migration. Consistently, IL-9 receptor negative cells did not show upregulation of HIF-1α by IL-9 incubation. Subsequent inhibition of HIF-1α, via genetic knockdown or small molecule-based antagonism with echinomycin, significantly suppressed IL-9-triggered migration, thus underscoring the essential role and targetability of HIF-1α in CTCL. It should however also be noted that because normal activated T-cells also expressed IL-9R commensurate with IL-9:HIF-1α-mediated migratory induction, adverse effects on T-cell migration and immunobiology should be considered when therapeutically inhibiting this pathway in CTCL. Nonetheless, our toxicity studies did not find adverse effects on T-cell viability at the effective echinomycin concentration used. Moreover, IL-9R and HIF-1α expression has also been reported in diverse cell types in the TME, including NK cells, macrophages, dendritic cells, myeloid-derived suppressor cells, and keratinocytes, the inhibition of which might impact cancer therapeutic effects targeting the IL-9-HIF-1α axis (11,39,40).

Migratory invasion of malignant T-cells through extracellular matrices critically depends on formation of invadopodia (41) and accompanying dynamic rearrangements and modulation of the actin cytoskeleton. Our unbiased quantitative proteomics analysis of pseudohypoxic malignant Jurkat cells identified the actin filament depolymerizer, Cofilin-1, as a major hit downstream of HIF-1α. Cofilin-1 plays significant functional roles in the formation of invadopodia and lamellipodia and has thus become an important biomarker of metastatic breast cancer and other solid tumor types (27,42). Additional support for the therapeutic value in targeting Cofilin-1 also arises from in-depth analysis of the DepMap database, which quantifies the impact of CRISPR-mediated CFL1 KO on cell viability. Fortuitously, malignant T cell lines, including Jurkat, Myla, and MJ, exhibited significantly higher sensitivity to Cofilin-1 KO as compared to other non-T cell lines. Intriguingly, a drug developed to reduce Cofilin-1 level was recently studied in hepatocellular carcinoma as an anti-metastatic therapy (43), thus underscoring the significance of our results herein linking Cofilin-1 to an upstream IL-9-HIF-1α pro-migratory circuit in CTCL. Indeed, expression of Cofilin-1 was significantly elevated not only in IL-9-rich CTCL skin lesions versus healthy controls, but also induced in tandem with HIF-1α upon direct IL-9 stimulation of patient-derived malignant T-cells or established lines. Strikingly, inhibition of HIF-1α activity, either by RNA interference or echinomycin-based pharmacological antagonism, significantly suppressed IL-9-triggered Cofilin-1 upregulation and cellular migration. These results thus identify Cofilin-1 as both a critical and targetable downstream member of the IL-9-HIF-1α pro-migratory pathway in CTCL malignant T-cells. While Cofilin-1 was previously implicated in hepatocellular carcinoma metastasis (44), this is the first report to our knowledge identifying it as an essential pro-migratory component of the IL-9-HIF-1α pathway in malignant T-cell lymphoma cells.

In summary, our work identifies an IL-9-triggered HIF-1α-Cofilin-1 pro-migratory circuit intrinsic to malignant T-cells. It further provides proof-of-concept that inhibition of this pathway, including with the FDA orphan drug for AML and GVHD, echinomycin, reverses IL-9-triggered migration. Our study thus invites continued evaluation of echinomycin as a potential therapy against migratory dissemination either alone, or possibly in combination with existing first line therapies for CTCL. It also rationalizes future testing and development of targeted therapies against additional IL-9-HIF-1α-CFL-1 pathway members in CTCL.

CONCLUSION

Our study identifies an IL-9-HIF-1α-Cofilin-1 pro-migratory circuit intrinsic to malignant T-cells in human CTCL. We further demonstrate that targeted disruption of IL-9-triggered downstream HIF-1α and Cofilin-1 activity, either via RNAi-based genetic knockdown or pharmacological antagonism with the clinical drug, echinomycin, attenuates tumor cell migration. These results thus uncover the IL-9:IL-9 receptor axis as a potential therapeutic target in CTCL.

ABBREVIATIONS:

CFL-1

Cofilin-1

CTCL

Cutaneous T-cell Lymphoma

HIF-1α

Hypoxia Inducible Factor-1 Alpha

IL

Interleukin

MF

Mycosis Fungoides

PBS

Phosphate Buffered Saline

RNAi

RNA Interference

ROS

Reactive Oxygen Species

siRNA

Small Interfering RNA

SZ

Sezary Syndrome

TME

Tumor Microenvironment