A Role of JAK/STAT Pathway in Cutaneous T-Cell Lymphomas: Exploring Its Effects for Targeted Therapy

INTRODUCTION

Development of targeted therapy in Cutaneous T-Cell Lymphoma (CTCL) patients still requires actionable mutated genes and deregulated pathways to be identified. We have recently published the mutational status of a number of human CTCL lesions, and found JAK/STAT signaling pathway to frequently harbor somatic mutations (Vaqué et al 2014). In this line of evidence, activating mutations in JAK kinases have been reported in human hematological malignancies (Kameda T1 2010) and may serve as indicators for targeted therapy. Therefore, we decided to analyze the mutational status of JAK/STAT pathway in a greater cohort of human CTCL patient samples and cell lines, by using massive parallel sequencing techniques, to shed light on its possible role in the pathogenesis and to uncover its potential as a new therapeutic target for this disease. To this end, we have studied the biological and molecular effects that targeted inhibition of the JAK/STAT signaling pathway, exerts in human CTCL cells, and explore its potential use as a targeted therapy.

MATERIAL AND METHODS

NGS: We searched for mutations in the catalytic domain of JAK and STAT genes in 39 CTCL patients using targeted NGS techniques: HaloPlex (Agilent) and sequencing in IonTorrent (LifeTech) and Illumina sequencers. Proliferation, apoptosis, cell cycle and DNA synthesis assays: CTCL cell lines were: My-La (MF), HH (MF) and HUT-78 (SS) (ECACC,Salisbury, UK). Effects in proliferation was assessed using CellTiter-Glo® Luminescent Cell Viability Assay (Promega, USA). To analyze the effects on cell survival, we evaluated early and delayed cell death provoked by FACS using FlowCellect Annexin Red Kit (EMD Millipore Corporation, USA). The distribution of cells among different phases of the cell cycle was evaluated by FACS using propidium iodide (PI, Sigma-Aldrich, USA) and Click-iT® EdU Alexa Fluor® 488 Flow Cytometry Assay Kit (Technologies-Thermo Fisher Scientific, USA) according to manufacturer’s instruction. Gene expression (GEP) studies: mRNA was extracted with Trizol and mRNA Array-based expression analysis was perfomed using a Whole Human Genome Agilent 4 × 44K v1 Oligonucleotide Microarray.

RESULTS:

Using our approach we found JAK/STAT pathway mutated in up to 24% of the CTCL samples. 3 mutations were found in JAK1, 5 in JAK3 and 1 in STAT5A genes. Interestingly, most of these mutations affected the tyrosine kinase domain of JAK kinases, a hotspot for activating mutations described in multiple types of human cancer.

Thereafter, we decided to explore the biological effects of targeted JAK/STAT inhibition, using specific JAK inhibitors (JAKi) in preclinical CTCL models. To this end, CTCL cell lines were incubated at different time points with increasing concentrations of JAKi. These treatments inhibited JAK/STAT signaling in CTCL cells, as assessed by western-blot using P-STAT-1,-3 and -5 antibodies. Biologically, JAKi provoked a marked inhibition of cell proliferation by a mechanism impinging the control of DNA replication. This was accompanied with a modest increase in cell death. To better understand the molecular mechanisms controlled by aberrant JAK/STAT signaling, we also performed an array-based mRNA expression analysis (GEP) in HUT78 cell line (JAK1Y654F) treated at different time points (0, 0.5 and 3h) with JAKi (at a concentration of IC₅₀). Our results showed a number of genes regulated by JAK/STAT signaling which activity has been described as T-cell activation, differentiation and proliferation (i.e. PAG1,TNF or EGR1).These genes may serve as potential indicators for therapy using JAK/STAT inhibitors or as surrogate markers for drug response.

CONCLUSIONS

Our results show frequent mutations affecting JAK/STAT downstream signaling in samples from patients with CTCL. Targeted inhibition of this pathway reveals an aberrant CTCL growing controlled by this signaling. Thus, these results provide evidence for the use of JAK/STAT inhibitors on the basis of a targeted massive sequencing analysis in specific cases of CTCL.