The generation of cortical projection neurons relies on the coordination of radial migration with branching. Here, we report that the multisubunit histone acetyltransferase Elongator complex, which contributes to transcript elongation, also regulates the maturation of projection neurons. Indeed, silencing of its scaffold (Elp1) or catalytic subunit (Elp3) cell-autonomously delays the migration and impairs the branching of projection neurons. Strikingly, neurons defective in Elongator show reduced levels of acetylated alpha-tubulin. Reduction of alpha-tubulin acetylation via expression of a nonacetylatable alpha-tubulin mutant leads to comparable defects in cortical neurons and suggests that alpha-tubulin is a target of Elp3. This is further supported by the demonstration that Elp3 promotes acetylation and counteracts HDAC6-mediated deacetylation of this substrate in vitro. Our results uncover alpha-tubulin as a target of the Elongator complex and suggest that a tight regulation of its acetylation underlies the maturation of cortical projection neurons.

Mutations in IKBKAP, encoding a subunit of Elongator, cause familial dysautonomia (FD), a severe neurodevelopmental disease with complex clinical characteristics. Elongator was previously linked not only with transcriptional elongation and histone acetylation but also with other cellular processes. Here, we used RNA interference (RNAi) and fibroblasts from FD patients to identify Elongator target genes and study the role of Elongator in transcription. Strikingly, whereas Elongator is recruited to both target and nontarget genes, only target genes display histone H3 hypoacetylation and progressively lower RNAPII density through the coding region in FD cells. Interestingly, several target genes encode proteins implicated in cell motility. Indeed, characterization of IKAP/hELP1 RNAi cells, FD fibroblasts, and neuronal cell-derived cells uncovered defects in this cellular function upon Elongator depletion. These results indicate that defects in Elongator function affect transcriptional elongation of several genes and that the ensuing cell motility deficiencies may underlie the neuropathology of FD patients.

Ruxolitinib is a small-molecule inhibitor of the JAK kinases, which has been approved for the treatment of myelofibrosis, a rare myeloproliferative neoplasm (MPN), but clinical trials are also being conducted in inflammatory-driven solid tumors. Increased infection rates have been reported in ruxolitinib-treated patients, and natural killer (NK) cells are immune effector cells known to eliminate both virus-infected and malignant cells. On this basis, we sought to compare the effects of JAK inhibition on human NK cells in a cohort of 28 MPN patients with or without ruxolitinib treatment and 24 healthy individuals. NK cell analyses included cell frequency, receptor expression, proliferation, immune synapse formation, and cytokine signaling. We found a reduction in NK cell numbers in ruxolitinib-treated patients that was linked to the appearance of clinically relevant infections. This reduction was likely due to impaired maturation of NK cells, as reflected by an increased ratio in immature to mature NK cells. Notably, the endogenous functional defect of NK cells in MPN was further aggravated by ruxolitinib treatment. In vitro data paralleled these in vivo results, showing a reduction in cytokine-induced NK cell activation. Further, reduced killing activity was associated with an impaired capacity to form lytic synapses with NK target cells. Taken together, our findings offer compelling evidence that ruxolitinib impairs NK cell function in MPN patients, offering an explanation for increased infection rates and possible longterm side effects associated with ruxolitinib treatment. Cancer Res; 75(11);

SummaryRuxolitinib (INCB018424) is the first JAK1/JAK2 inhibitor approved for treatment of myelofibrosis. JAK/STAT-signalling is known to be involved in the regulation of CD4 + T cells, which critically orchestrate inflammatory responses. To better understand how ruxolitinib modulates CD4 + T cell responses, we undertook an in-depth analysis of CD4 + T cell function upon ruxolitinib exposure. We observed a decrease in total CD3 + cells after 3 weeks of ruxolitinib treatment in patients with myeloproliferative neoplasms. Moreover, we found that the number of regulatory T cells (Tregs), pro-inflammatory T-helper cell types 1 (Th1) and Th17 were reduced, which were validated by in vitro studies. In line with our in vitro data, we found that inflammatory cytokines [tumour necrosis factor-a (TNF), interleukin (IL)5, IL6, IL1B] were also downregulated in T cells from patients (all P < 0Á05). Finally, we showed that ruxolitinib does not interfere with the T cell receptor signalling pathway, but impacts IL2-dependent STAT5 activation. These data provide a rationale for testing JAK inhibitors in diseases triggered by hyperactive CD4 + T cells, such as autoimmune diseases.In addition, they also provide a potential explanation for the increased infection rates (i.e. viral reactivation and urinary tract infection) seen in ruxolitinib-treated patients.

Type I protein kinase A (PKA) is targeted to the TCR-proximal signaling machinery by the A-kinase anchoring protein ezrin and negatively regulates T cell immune function through activation of the C-terminal Src kinase. RI anchoring disruptor (RIAD) is a high-affinity competitor peptide that specifically displaces type I PKA from A-kinase anchoring proteins. In this study, we disrupted type I PKA anchoring in peripheral T cells by expressing a soluble ezrin fragment with RIAD inserted in place of the endogenous A-kinase binding domain under the lck distal promoter in mice. Peripheral T cells from mice expressing the RIAD fusion protein (RIAD-transgenic mice) displayed augmented basal and TCR-activated signaling, enhanced T cell responsiveness assessed as IL-2 secretion, and reduced sensitivity to PGE2- and cAMP-mediated inhibition of T cell function. Hyperactivation of the cAMP–type I PKA pathway is involved in the T cell dysfunction of HIV infection, as well as murine AIDS, a disease model induced by infection of C57BL/6 mice with LP-BM5, a mixture of attenuated murine leukemia viruses. LP-BM5–infected RIAD-transgenic mice resist progression of murine AIDS and have improved viral control. This underscores the cAMP–type I PKA pathway in T cells as a putative target for therapeutic intervention in immunodeficiency diseases.