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B. Maurer et al.
Introduction
Peripheral (mature) T-cell lymphomas (PTCL) are het- erogeneous neoplasms often accompanied by aggressive courses and extranodal organ infiltration. PTCL have vari- able histology, immunophenotype, and molecular fea- tures.1,2 The World Health Organization (WHO) classifi- cation of lymphoid neoplasms distinguishes more than 30 mature T- and natural killer (NK)-cell neoplasms. The most common subtype is PTCL, not otherwise specified (NOS), which collects together cases not attributable to other, better defined, entities. PTCL, NOS is a highly dynamic category with respect to consensus features, proposed cell of origin, and prognostic subsets based on molecular signatures.3 PTCL, NOS with a follicular T- helper (TFH) cell phenotype relates to angioimmunoblastic T-cell lymphoma (AITL), with which it is co-categorized in a provisional group.4 Another distinguishable PTCL, NOS subset is constituted by cases with cytotoxic fea- tures. High-throughput methodologies helped in such histogenetic assignments and in the prognostically rele- vant separation of non-TFH type PTCL, NOS from AITL and anaplastic large cell lymphoma.2,3,5-7 Eminent prob- lems, particularly in PTCL, NOS, are the inefficacy of the poly-chemotherapies historically designed for aggressive B-cell lymphomas and the lack of high-fidelity mouse models8-10 in which to investigate biological principles and to address preclinical questions.
The molecular landscape of PTCL, NOS reveals that altered T-cell receptor signaling, epigenetic modifiers, and immune evasion mechanisms are common.3,5-7,11-17 Activating mutations in the JAK-STAT pathway affecting mostly the interleukin-2 receptor (IL2R), JAK1, JAK3, STAT3, and STAT5B were found in many mature T- and NK-cell neoplasms.18,19 The entities with the highest inci- dence of STAT5B and STAT3 mutations are anaplastic large cell lymphoma, cutaneous T-cell lymphoma (CTCL; comprising mycosis fungoides and Sézary syndrome), enteropathy-associated T-cell lymphoma, hepatosplenic T-cell lymphoma, NK/T-cell lymphoma, T-cell prolym- phocytic leukemia, and the auto-aggressive CD8+ T-large granular lymphocyte leukemia.15,20-22 Furthermore, muta- tions in chromatin remodelers, GTPases, DNA repair machinery or co-repressors have been associated with JAK/STAT hyperactivation.19
STAT5BN642H is the most frequent recurrent gain-of-func- tion mutation in the closely related genes encoding for the transcription factors STAT5A and STAT5B. It is associ- ated with unfavorable disease progression in patients15,23-31 and leads to an aggressive CD8+ T-cell neoplasia in mice.32 JAK-STAT signaling is a central cancer pathway driving survival and cell cycle progression, but it also promotes differentiation and senescence as safety pathways. STAT5A and STAT5B play important roles in immune cells33 and absence of lymphoid STAT5 results in loss of CD8+, γδ, and regulatory T cells (Treg)34 Differentiation of CD8+ T cells is regulated by STAT5 in a dose-dependent manner35 and enhances effector and memory CD8+ T-cell survival and proliferation. High levels of tyrosine phos- phorylated STAT5 (pYSTAT5) are associated with a neg- ative prognosis in many myeloid neoplasms.36
Aggressive CD8+ T-cell neoplasia resulted in early death upon STAT5BN642H expression.32 Enhanced pYSTAT5 can also be mimicked by the hyperactive Stat5aS710F variant (cS5F).37 We generated and compared graded STAT5 activ-
ity mouse models within the hematopoietic system. Low activity models displayed only a modest CD8+ T-cell expansion, whereas those with high STAT5 activity developed aggressive CD8+ PTCL-like disease reminis- cent of human PTCL, NOS with cytotoxic features. Although STAT5A- and STAT5B-induced changes largely overlap, STAT5B hyperactivation was more aggressive than STAT5A hyperactivation. Comparative analyses revealed that STAT5A and STAT5B overexpression is common in human mature T-cell lymphomas. The clini- cal JAK1/2/3 inhibitors ruxolitinib and tofacitinib38 as well as a selective STAT5 inhibitor39 specifically reduced viabil- ity of PTCL cells. Ruxolitinib blocked PTCL disease in vivo. We conclude that STAT5 activation drives PTCL and that patients with PTCL can benefit from JAK/STAT inhibitors.
Methods
Animals and generation of transgenic mice
Mice were maintained on a C57BL/6N background, housed in a specific-pathogen-free facility under standardized conditions and monitored daily for signs of disease. All animal experiments were carried out according to the animal license protocols (BMWF-66.009/0281-I/3b/2012, BMWFW-68.205/0166- WF/V/3b/2015, BMWFW-68.205/0117-WF/V/3b/2016 and BMWFW-68.205/0103-WF/V/3b/2015) approved by the institu- tional Ethics Committee and the Austrian Ministry BMWF authorities. All transgenic mice were hemizygous. Non-trans- genic littermates served as controls. We used the vav-hematopoi- etic vector vav-hCD4 (HS21/45)40 to generate transgenic mice expressing cS5F in the hematopoietic system at different levels (called cS5Alo [B6N-Tg(Vav-cS5F)564Biat] and cS5Ahi [B6N- Tg(Vav-cS5F)565Biat]), as described in the Online Supplementary Methods. Details of hSTAT5B and hSTAT5BN642H mice have been published.32 All primers used are listed in Online Supplementary Table S1.
Patients’ samples
Retrospective immunohistological analysis, approved by the ethics committee of the Medical University of Vienna (1437/2016), was done on formalin-fixed, paraffin-embedded patients’ specimens of 35 PTCL, NOS, 14 AITL, 6 mycosis fun- goides, and 29 CTCL (from 23 patients) cases and 5 non-dis- eased lymph nodes, kindly provided by the Medical University of Vienna, Austria, the Karl Landsteiner University of Health Sciences, St. Poelten, Austria, Wilheminenspital (Wiener Krankenanstaltenverbund), Vienna, Austria, and the University Hospital Brno, Czech Republic. Samples were included in this study after patients had given informed consent in accordance with the Declaration of Helsinki. Diagnoses of samples were made according to the 2008 WHO criteria by experienced hematopathologists or expert dermatopathologists. Patients with CTCL were diagnosed according to the WHO-EORTC classification for cutaneous lymphomas as follows: mycosis fun- goides (stage IA: n=10, stage IB: n=1, stage IIA: n=1, stage IIB: n=6), Sézary syndrome (n=2), and lymphomatoid papulosis (n=3).
Histological analysis of murine and human sections
Formalin-fixed, paraffin-embedded 3 μm consecutive mouse organ sections were stained with hematoxylin (Merck, Darmstadt, Germany) and eosin G (Carl Roth, Karlsruhe, Germany). Immunohistochemistry was performed using anti-
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haematologica | 2020; 105(2)