Romidepsin and Afatinib Abrogate Jak-Signal Transducer and Activator of Transcription Signaling and Elicit Synergistic Antitumor Effects in Cutaneous T-Cell Lymphoma.

Cutaneous T-cell lymphomas are mature lymphoid neoplasias resulting from the malignant transformation of skin-resident T-cells. A distinctive clinical feature of cutaneous T-cell lymphomas is their sensitivity to treatment with histone deacetylase inhibitors. However, responses to histone deacetylase inhibitor therapy are universally transient and noncurative, highlighting the need for effective and durable drug combinations. In this study, we demonstrate that the combination of romidepsin, a selective class I histone deacetylase inhibitor, with afatinib, an EGFR family inhibitor, induces strongly synergistic antitumor effects in cutaneous T-cell lymphoma models in vitro and in vivo through abrogation of Jak-signal transducer and activator of transcription signaling. These results support a previously unrecognized potential role for histone deacetylase inhibitor plus afatinib combination in the treatment of cutaneous T-cell lymphomas.

Oncogenic Vav1-Myo1f induces therapeutically targetable macrophage-rich tumor microenvironment in peripheral T cell lymphoma.

Peripheral T cell lymphoma not otherwise specified (PTCL-NOS) comprises heterogeneous lymphoid malignancies characterized by pleomorphic lymphocytes and variable inflammatory cell-rich tumor microenvironment. Genetic drivers in PTCL-NOS include genomic alterations affecting the VAV1 oncogene; however, their specific role and mechanisms in PTCL-NOS remain incompletely understood. Here we show that expression of Vav1-Myo1f, a recurrent PTCL-associated VAV1 fusion, induces oncogenic transformation of CD4+ T cells. Notably, mouse Vav1-Myo1f lymphomas show T helper type 2 features analogous to high-risk GATA3+ human PTCL. Single-cell transcriptome analysis reveals that Vav1-Myo1f alters T cell differentiation and leads to accumulation of tumor-associated macrophages (TAMs) in the tumor microenvironment, a feature linked with aggressiveness in human PTCL. Importantly, therapeutic targeting of TAMs induces strong anti-lymphoma effects, highlighting the lymphoma cells' dependency on the microenvironment. These results demonstrate an oncogenic role for Vav1-Myo1f in the pathogenesis of PTCL, involving deregulation in T cell polarization, and identify the lymphoma-associated macrophage-tumor microenvironment as a therapeutic target in PTCL.

Jak-STAT Inhibition Mediates Romidepsin and Mechlorethamine Synergism in Cutaneous T-Cell Lymphoma.

Sézary syndrome is an aggressive and disseminated form of cutaneous T-cell lymphoma associated with dismal prognosis in which the histone deacetylase inhibitor romidepsin has shown remarkable activity as a single agent. However, clinical responses to romidepsin are typically transient, highlighting the need for more effective therapies. In this study, we show synergistic antilymphoma effects of romidepsin in combination with mechlorethamine, an alkylating agent, in cutaneous T-cell lymphoma cell lines and primary samples with strong antitumor effects in an in vivo model of Sézary syndrome. Mechanistically, gene expression profiling points to abrogation of Jak/signal transducer and activator of transcription (STAT) signaling as an important mediator of this interaction. Consistently, the combination of mechlorethamine plus romidepsin resulted in downregulation of STAT5 phosphorylation in romidepsin-sensitive cell lines and primary Sézary syndrome samples, but not in romidepsin-resistant tumors. Moreover, in further support of Jak/STAT signaling as a modulator of romidepsin activity in cutaneous T-cell lymphoma, treatment with romidepsin in combination with Jak inhibitors resulted in markedly increased therapeutic responses. Overall, these results support a role for romidepsin plus mechlorethamine in combination in the treatment of cutaneous T-cell lymphoma and uncover a previously unrecognized role for Jak/STAT signaling in the response to romidepsin and romidepsin-based combination therapies in Sézary syndrome.

FYN-TRAF3IP2 induces NF-κB signaling-driven peripheral T cell lymphoma.

Angioimmunoblastic T cell lymphoma (AITL) and peripheral T cell lymphoma not-otherwise-specified (PTCL, NOS) have poor prognosis and lack driver actionable targets for directed therapies in most cases. Here we identify FYN-TRAF3IP2 as a recurrent oncogenic gene fusion in AITL and PTCL, NOS tumors. Mechanistically, we show that FYN-TRAF3IP2 leads to aberrant NF-κB signaling downstream of T cell receptor activation. Consistent with a driver oncogenic role, FYN-TRAF3IP2 expression in hematopoietic progenitors induces NF-κB-driven T cell transformation in mice and cooperates with loss of the Tet2 tumor suppressor in PTCL development. Moreover, abrogation of NF-κB signaling in FYN-TRAF3IP2-induced tumors with IκB kinase inhibitors delivers strong anti-lymphoma effects in vitro and in vivo. These results demonstrate an oncogenic and pharmacologically targetable role for FYN-TRAF3IP2 in PTCLs and call for the clinical testing of anti-NF-κB targeted therapies in these diseases.

Biology and Molecular Pathogenesis of Mature T-Cell Lymphomas.

Peripheral T-cell lymphomas (PTCLs) constitute a highly heterogeneous group of hematological diseases with complex clinical and molecular features consistent with the diversity of the T-cell type from which they originate. In the past several years, the systematic implementation of high-throughput genomic technologies for the analysis of T-cell malignancies has supported an exponential progress in our understanding of the genetic drivers of oncogenesis and unraveled the molecular complexity of these diseases. Recent findings have helped redefine the classification of T-cell malignancies and provided novel biomarkers to improve diagnosis accuracy and analyze the response to therapy. In addition, multiple novel targeted therapies including small-molecule inhibitors, antibody-based approaches, and immunotherapy have shown promising results in early clinical analysis and have the potential to completely change the way T-cell malignancies have been treated traditionally.

GATA3-Controlled Nucleosome Eviction Drives MYC Enhancer Activity in T-cell Development and Leukemia.

Long-range enhancers govern the temporal and spatial control of gene expression; however, the mechanisms that regulate enhancer activity during normal and malignant development remain poorly understood. Here, we demonstrate a role for aberrant chromatin accessibility in the regulation of MYC expression in T-cell lymphoblastic leukemia (T-ALL). Central to this process, the NOTCH1-MYC enhancer (N-Me), a long-range T cell-specific MYC enhancer, shows dynamic changes in chromatin accessibility during T-cell specification and maturation and an aberrant high degree of chromatin accessibility in mouse and human T-ALL cells. Mechanistically, we demonstrate that GATA3-driven nucleosome eviction dynamically modulates N-Me enhancer activity and is strictly required for NOTCH1-induced T-ALL initiation and maintenance. These results directly implicate aberrant regulation of chromatin accessibility at oncogenic enhancers as a mechanism of leukemic transformation. SIGNIFICANCE: MYC is a major effector of NOTCH1 oncogenic programs in T-ALL. Here, we show a major role for GATA3-mediated enhancer nucleosome eviction as a driver of MYC expression and leukemic transformation. These results support the role of aberrant chromatin accessibility and consequent oncogenic MYC enhancer activation in NOTCH1-induced T-ALL.This article is highlighted in the In This Issue feature, p. 1631.

RHOA G17V Induces T Follicular Helper Cell Specification and Promotes Lymphomagenesis.

Angioimmunoblastic T cell lymphoma (AITL) is an aggressive tumor derived from malignant transformation of T follicular helper (Tfh) cells. AITL is characterized by loss-of-function mutations in Ten-Eleven Translocation 2 (TET2) epigenetic tumor suppressor and a highly recurrent mutation (p.Gly17Val) in the RHOA small GTPase. Yet, the specific role of RHOA G17V in AITL remains unknown. Expression of Rhoa G17V in CD4+ T cells induces Tfh cell specification; increased proliferation associated with inducible co-stimulator (ICOS) upregulation and increased phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase signaling. Moreover, RHOA G17V expression together with Tet2 loss resulted in development of AITL in mice. Importantly, Tet2-/-RHOA G17V tumor proliferation in vivo can be inhibited by ICOS/PI3K-specific blockade, supporting a driving role for ICOS signaling in Tfh cell transformation.

First-in-class inhibitor of the T cell receptor for the treatment of autoimmune diseases.

Modulating T cell activation is critical for treating autoimmune diseases but requires avoiding concomitant opportunistic infections. Antigen binding to the T cell receptor (TCR) triggers the recruitment of the cytosolic adaptor protein Nck to a proline-rich sequence in the cytoplasmic tail of the TCR's CD3ε subunit. Through virtual screening and using combinatorial chemistry, we have generated an orally available, low-molecular weight inhibitor of the TCR-Nck interaction that selectively inhibits TCR-triggered T cell activation with an IC50 (median inhibitory concentration) ~1 nM. By modulating TCR signaling, the inhibitor prevented the development of psoriasis and asthma and, furthermore, exerted a long-lasting therapeutic effect in a model of autoimmune encephalomyelitis. However, it did not prevent the generation of a protective memory response against a mouse pathogen, suggesting that the compound might not exert its effects through immunosuppression. These results suggest that inhibiting an immediate TCR signal has promise for treating a broad spectrum of human T cell-mediated autoimmune and inflammatory diseases.

The curious origins of angioimmunoblastic T-cell lymphoma.

PURPOSE OF REVIEW: Once an obscure disease, recent studies have transformed our understanding of angioimmunoblastic T-cell lymphoma (AITL). In this review, we summarize new major advances in the genetics and biology of AITL. RECENT FINDINGS: Genome wide sequencing studies have dissected the repertoire of the genetic alterations driving AITL uncovering a highly recurrent Gly17Val somatic mutation in the small GTPase RHOA and major role for mutations in epigenetic regulators, such as TET2, DNMT3A and IDH2, and signaling factors (e.g., FYN and CD28). These findings support a multistep model of follicular T helper cell transformation in AITL and pinpoint novel candidates for the development of targeted therapies in this disease. SUMMARY: AITL originates from follicular T helper cells and is characterized by the presence of RHOA G17V mutation together with genetic alterations in TET2, DNMT3A, and IDH2. Research efforts now focus on the elucidation of the specific roles and interplay of these genetic alterations in the pathogenesis of AITL.

Maintenance of immune tolerance by Foxp3+ regulatory T cells requires CD69 expression.

Although FoxP3(+) regulatory T cells are key players in the maintenance of immune tolerance and autoimmunity, the lack of specific markers constitute an obstacle to their use for immunotherapy protocols. In this study, we have investigated the role of the C-type lectin receptor CD69 in the suppressor function of Tregs and maintenance of immune tolerance towards harmless inhaled antigens. We identified a novel FoxP3(+)CD69(+) Treg subset capable to maintain immune tolerance and protect to developing inflammation. Although CD69(+) and CD69(-)FoxP3(+) Tregs exist in homeostasis, only CD69-expressing Tregs express high levels of CTLA-4, ICOS, CD38 and GITR suppression-associated markers, secrete high amounts of TGFß and have potent suppressor activity. This activity is regulated by STAT5 and ERK signaling pathways and is impaired by antibody-mediated down-regulation of CD69 expression. Moreover, immunotherapy with FoxP3(+)CD69(+) Tregs restores the homeostasis in Cd69(-/-) mice, that fail to induce tolerance, and is also highly proficient in the prevention of inflammation. The identification of the FoxP3(+)CD69(+) Treg subset paves the way toward the development of new therapeutic strategies to control immune homeostasis and autoimmunity.

Is CD69 an effective brake to control inflammatory diseases?

Early studies described CD69 as a leukocyte activation marker, and suggested its involvement in the activation of different leukocyte subsets as well as in the pathogenesis of chronic inflammation. However, recent investigations have showed that CD69 knockout mice exhibit an enhanced or reduced susceptibility to different experimental models of inflammatory diseases, including those mediated by T helper 17 (Th17) lymphocytes. In this regard, the expression of CD69, both in Th17 lymphocytes and by a subset of regulatory T cells, has an important role in the control of the immune response and the inflammatory phenomenon. Therefore, different evidence indicates that CD69 exerts a complex immunoregulatory role in humans, and that it could be considered as a target molecule for the therapy of immune-mediated diseases.

Eukaryotic elongation factor 2 controls TNF-α translation in LPS-induced hepatitis.

Bacterial LPS (endotoxin) has been implicated in the pathogenesis of acute liver disease through its induction of the proinflammatory cytokine TNF-α. TNF-α is a key determinant of the outcome in a well-established mouse model of acute liver failure during septic shock. One possible mechanism for regulating TNF-α expression is through the control of protein elongation during translation, which would allow rapid cell adaptation to physiological changes. However, the regulation of translational elongation is poorly understood. We found that expression of p38γ/δ MAPK proteins is required for the elongation of nascent TNF-α protein in macrophages. The MKK3/6-p38γ/δ pathway mediated an inhibitory phosphorylation of eukaryotic elongation factor 2 (eEF2) kinase, which in turn promoted eEF2 activation (dephosphorylation) and subsequent TNF-α elongation. These results identify a new signaling pathway that regulates TNF-α production in LPS-induced liver damage and suggest potential cell-specific therapeutic targets for liver diseases in which TNF-α production is involved.

The leukocyte activation antigen CD69 limits allergic asthma and skin contact hypersensitivity.

BACKGROUND: Allergic diseases have a major health care impact in industrialized countries. The development of these diseases is influenced by exposure to allergen and to immunological and genetic factors. However, the molecular mechanisms underlying the inflammatory response that triggers allergy are not well defined. OBJECTIVE: We have investigated the role of the leukocyte activation antigen CD69 in the regulation of two allergic diseases, asthma and contact dermatitis. METHODS: Analysis of two models of allergic diseases in CD69 knockout and wild-type mice: ovalbumin-induced allergic airway inflammation (BALB/c genetic background) and contact hypersensitivity to oxazolone (C57BL/6J genetic background). RESULTS: CD69 deficiency dramatically enhanced the inflammatory response in the ovalbumin-induced asthma model of antigen-induced airway allergy. CD69 knockout mice showed exacerbated pulmonary eosinophil recruitment, high vascular cell adhesion molecule 1 expression levels in lung vasculature, and enhanced T(H)2 and T(H)17 cytokines in the bronchoalveolar space and lung tissue. In the hapten-induced cutaneous contact hypersensitivity model, both CD69 deficiency and treatment with anti-CD69 mAb increased inflammation. Treatment with contact allergens induced enhanced T(H)1 and T(H)17 responses in CD69 deficient mice, and neutralizing anti-IL-17 antibodies reduced skin inflammation. In both experimental systems, adoptive transfer of lymph node cells from CD69 knockout mice increased the inflammatory response in recipient mice. CONCLUSION: These results demonstrate that the early activation receptor CD69 is an intrinsic modulator of immune allergic processes through the negative regulation of allergen-induced T-cell effector responses.

Treatment of cells with n-alpha-tosyl-L-phenylalanine-chloromethyl ketone induces the proteolytic loss of STAT6 transcription factor.

The implication of the STAT6 transcription factor in several human diseases makes the regulation of its activity a topic of great biological interest. The activation of this transcription factor is tightly regulated by kinases, phosphatases, and proteases. The initial aim of this study was to investigate the utility of protease inhibitors in controlling STAT6 activation. Among all inhibitors analyzed, n-alpha-tosyl-L-phenylalanine-chloromethyl ketone (TPCK) was found to inhibit the IL-4-induced STAT6 activation. Unexpectedly, this inhibition was accompanied by a loss of STAT6 protein. Thus, TPCK promoted the loss of STAT6 by a mechanism sensitive to the serine-protease inhibitor 4-(2-aminoetyl)-benzenesulfonyl fluoride. However, the effects of TPCK seemed not to be mediated by its protease inhibitory activity since multiple protease inhibitors tested had no effect on STAT6 expression. The results found suggest that the effect of TPCK was mediated by its alkylating activity. Thus, cysteine reactive and thiol antioxidant compounds prevented the loss of STAT6 induced by TPCK. The reactivity of thiol groups on STAT6 was moreover demonstrated with biotinylated sulfhydryl-reactive compounds. Analysis of other signaling molecules indicated that STAT5, but not other STATs, Shc, or c-Rel, was also affected by TPCK, suggesting a common downregulatory mechanism for STAT6 and STAT5. These results reveal a novel mechanism of action of TPCK in inducing a selective loss of STAT proteins. These findings may have implications for diseases in which STAT proteins are involved.

Kaempferol inhibits IL-4-induced STAT6 activation by specifically targeting JAK3.

IL-4 is involved in several human diseases including allergies, autoimmunity, and cancer. Its effects are mainly mediated through the transcription factor STAT6. Therefore, investigation of compounds that regulate STAT6 activation is of great interest for these diseases. Natural polyphenols are compounds reported to have therapeutic properties in diseases involving IL-4 and STAT6. The aim of this study was to investigate the effect of these compounds in the activation of this transcription factor. We found that in hemopoietic cells from human and mouse origin, some flavonoids were able to inhibit the activation of STAT6 by IL-4. To identify molecular mechanisms, we focused on kaempferol, the compound that showed the greatest inhibitory effect with the lowest cell toxicity. Treatment of cells with kaempferol did not affect activation of Src kinase by IL-4 but did prevent the phosphorylation of JAK1 and JAK3. Further enzymatic analysis demonstrated that kaempferol blocked the in vitro phosphorylation activity of JAK3 without affecting JAK1, suggesting that it specifically targeted JAK3 activity. Accordingly, kaempferol had no effect on STAT6 activation in nonhemopoietic cell lines lacking JAK3, supporting its selective inhibition of IL-4 responses through type I receptors expressing JAK3 but not type II lacking this kinase. The inhibitory effect of kaempferol was also observed in IL-2 but not IL-3-mediated responses and correlated with the inhibition of MLC proliferation. These findings reveal the potential use of kaempferol as a tool for selectively controlling cell responses to IL-4 and, in general, JAK3-dependent responses.