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High STAT5A activity promotes CD8+ T-cell neoplasia
Figure S7C). All three inhibitors reduced the viability of cS5Ahi-derived CTL cells already at low concentrations, with the half maximal inhibitory concentration (IC50) being ~4.1 μM for AC-3-19, ~0.005 μM for ruxolitinib and ~0.03 μM for tofacitinib (Online Supplementary Figure S7C). Sensitivity to STAT5 inhibition could be confirmed in human PTCL cell lines. AC-3-19 treatment reduced the viability of Mac2A, FePD, SU-DHL-1, Mac1 and SR786 (IC50 5-10 μM) (Online Supplementary Figure S7D). When we used ruxolitinib we found that Mac1- and Mac2A cells that harbor a JAK2 translocation were sensitive.54 Control cell lines were only affected at significantly high- er concentrations (AC-3-19: <20 μM) (Online Supplementary Figure S7D, summary on mutations in human cells in Online Supplementary Table S7). In addition, ruxolitinib blocked neoplastic cell growth in cS5Ahi mice, as exemplified by reductions of splenomegaly and lym- phadenopathy (Figure 7D-E). The specificity of the treat- ment was validated by reduced pYSTAT5 in lymph nodes and spleen (Figure 7F, Online Supplementary Figure S7E). Most impressively, the T-cell infiltration in peripheral organs was drastically reduced in treated mice (Figure 7G, Online Supplementary Figure S7F, G). Collectively, these data verify that malignant cS5Ahi-CTL and human PTCL cells critically depend on STAT5 signaling.
Discussion
PTCL patients face an unfavorable prognosis as chemotherapy results in a poor 5-year overall survival rate.2,55 Targeted treatment options only exist for ALK+ anaplastic large cell lymphoma and are urgently needed for the other 30 PTCL entities. The cohort of patients we investigated revealed increased expression and activity of both STAT5A and STAT5B proteins in mature T-cell lym- phomas. Using cS5Ahi transgenic mice we unambiguously associated STAT5A activity with PTCL phenotype. Importantly, mRNA expression changes of cS5Ahi mice closely matched gene expression profiles of human T-cell neoplasia and overlapped with the profile of the more aggressive hSTAT5BN642H mouse model.32 Importantly, clin- ical JAK1/2/3 inhibitors ruxolitinib and tofacitinib38 and selective STAT5 inhibitors39 reduced neoplastic PTCL cell growth.
Previous transgenic models addressing leukemogenesis downstream of STAT5 employed either wt or STAT5 gain- of-function variants in a lymphoid-restricted manner resulting in expansion of mature T cells, lymphoblastic or B-cell lymphoma.56-59 Broad hematopoietic expression resulted in enhanced granulopoiesis.60 Here, we engi- neered expression of the well-characterized hyperactive STAT5A variant cS5F using the vav-promoter starting expression from the hematopoietic stem cell stage.40 cS5Ahi mice developed effector/memory- CD8+ T-cell malignan- cies featuring pronounced organ infiltration. Transgene expression was observed in all major blood lineages test- ed, but neoplastic expansion of CD8+ T cells was most prominent. This argues for the susceptibly of the T-cell lin- eage to STAT5 hyperactivation and it emphasizes the role of JAK1/3/STAT3/5 signaling in the outgrowth of T-cell lymphomas. We suggest that CD8+ T cells outcompete cS5AF-mediated effects on other lineages due to faster cell cycle progression, higher survival, cytokine sensitivity and cell fate-specific expression differences. We speculate that
STAT5-mediated effects are more negatively controlled in myeloid over lymphoid cell types and that low pYSTAT5 levels, as mimicked in cS5Alo mice, do not promote neo- plasia. Gene-dosage and graded STAT5 activity levels in our study showed a clear positive correlation between STAT5 activation status and CD8+ T-cell numbers.34,35 Senescence, diminished oligomer formation or differenti- ation capacity might contribute to the differences between cS5Alo and cS5Ahi. Although the level of pYSTAT5 in cS5Alo mice was significant, it was neverthe- less lower than in cS5Ahi or hSTAT5BN642H mice, and malig- nant transformation did not occur in the cS5Alo animals. Thus, the real driver of PTCL disease might be the amount of pYSTAT5 resulting in enhanced and prolonged tran- scriptional activity.
In PTCL several factors account for STAT5 activation – such as mutations in STAT5 or in upstream signaling com- ponents (e.g. IL-2R, JAK1, JAK3). Surprisingly, recurrent mutations in epigenetic and chromatin remodeling factors parallel JAK/STAT activation and the epigenetic landscape is also shaped by STAT3/5 and its versatile interaction partners.52,61 However, until now a detailed understanding of these factors in PTCL is lacking. Increased pYSTAT5 levels have also been reported upon autocrine PDGFα sig- naling in PTCL, NOS.62 In CTCL, overexpression of onco- genic miR-155,49 downregulation of tumor-suppressive miR-22,50 enhanced progression by lymphotoxin-α-depen- dent lymphangiogenesis,63 STAT5-dependent CD80 expression,51 resistance to vorinostat48 and risk of disease progression64 were attributed to enhanced STAT5 signal- ing. The identification of recurrent and mutually exclusive gain-of-function mutations in STAT3 suggests that these proteins share redundant functions in PTCL.18,23,28 Thus, dual STAT3/5 inhibition is needed in future therapy, which could be approached by SH2-domain blockers. Certainly, mutations or translocations in the upstream JAK proteins boost STAT5 signaling,25,29,30,65 further emphasizing the potential benefit of JAK and STAT inhibitors.
The cS5Ahi model recapitulates clinical features of PTCL with high STAT5 activation. Among the few high-fidelity mouse models for other PTCL subsets,9,66-69 the cS5Ahi model closely phenocopies pathological hallmarks of cytotoxic CD8+ T-cell PTCL. Importantly, cS5Ahi-express- ing cells are hypersensitive to the Food and Drug Administration-approved JAK1/2 inhibitor ruxolitinib, which is currently being studied in phase II clinical trials for the treatment of relapsed PTCL and adult T-cell leukemia/lymphoma (NCT01431209, NCT02974647, NCT01712659). In line with this, JAK-inhibitor treatment of Sézary syndrome,29 T-cell prolymphocytic leukemia,54,70 and adult T-cell leukemia/lymphoma71 revealed reduced proliferation and immunomodulatory effects on the PTCL tumor microenvironment.3 Moreover, a small molecule inhibitor of STAT5 SH2 domain-phosphopeptide interac- tions39 resulted in strongly reduced viability in PTCL lines with high STAT5 activity and its successor compound showed significant effects in acute myeloid leukemia.53 Although these inhibitors are currently considered lead compounds, specific STAT5 inhibitors are also expected to enter clinical trials and combinations with kinase inhibitors could be tested.
In conclusion, our cS5Ahi transgenic mouse model indi- cates that STAT5 is a driver and STAT5 itself or its interac- tion partners are potential drug targets in PTCL. The STAT5-dependent CD8+ PTCL mouse model described
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