NOD2 is an intracellular sensor that contributes to immune defense and inflammation. Here we investigated whether NOD2 mediates its effects through control of microRNAs (miRNAs). miR-29 expression was upregulated in human dendritic cells (DCs) in response to NOD2 signals, and miR-29 regulated the expression of multiple immune mediators. In particular, miR-29 downregulated interleukin-23 (IL-23) by targeting IL-12p40 directly and IL-23p19 indirectly, likely via reduction of ATF2. DSS-induced colitis was worse in miR-29-deficient mice and was associated with elevated IL-23 and T helper 17 signature cytokines in the intestinal mucosa. Crohn's disease (CD) patient DCs expressing NOD2 polymorphisms failed to induce miR-29 upon pattern recognition receptor stimulation and showed enhanced release of IL-12p40 on exposure to adherent invasive E. coli. Therefore, we suggest that loss of miR-29-mediated immunoregulation in CD DCs might contribute to elevated IL-23 in this disease.

Leukemia is caused by the accumulation of multiple genomic lesions in hematopoietic precursor cells. However, how these events cooperate during oncogenic transformation remains poorly understood. We studied the cooperation between activated JAK3/STAT5 signaling and HOXA9 overexpression, two events identified as significantly co-occurring in T-cell acute lymphoblastic leukemia. Expression of mutant JAK3 and HOXA9 led to a rapid development of leukemia originating from multipotent or lymphoid-committed progenitors, with a significant decrease in disease latency compared with JAK3 or HOXA9 alone. Integrated RNA sequencing, chromatin immunoprecipitation sequencing, and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) revealed that STAT5 and HOXA9 have co-occupancy across the genome, resulting in enhanced STAT5 transcriptional activity and ectopic activation of FOS/JUN (AP1). Our data suggest that oncogenic transcription factors such as HOXA9 provide a fertile ground for specific signaling pathways to thrive, explaining why JAK/STAT pathway mutations accumulate in HOXA9-expressing cells. The mechanism of oncogene cooperation in cancer development remains poorly characterized. In this study, we model the cooperation between activated JAK/STAT signaling and ectopic HOXA9 expression during T-cell leukemia development. We identify a direct cooperation between STAT5 and HOXA9 at the transcriptional level and identify PIM1 kinase as a possible drug target in mutant JAK/STAT/HOXA9-positive leukemia cases. .

Mutations in the interleukin-7 receptor (IL7R) or the Janus kinase 3 (JAK3) kinase occur frequently in T-cell acute lymphoblastic leukemia (T-ALL) and both are able to drive cellular transformation and the development of T-ALL in mouse models. However, the signal transduction pathways downstream of JAK3 mutations remain poorly characterized. Here we describe the phosphoproteome downstream of the JAK3(L857Q)/(M511I) activating mutations in transformed Ba/F3 lymphocyte cells. Signaling pathways regulated by JAK3 mutants were assessed following acute inhibition of JAK1/JAK3 using the JAK kinase inhibitors ruxolitinib or tofacitinib. Comprehensive network interrogation using the phosphoproteomic signatures identified significant changes in pathways regulating cell cycle, translation initiation, mitogen-activated protein kinase and phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/AKT signaling, RNA metabolism, as well as epigenetic and apoptotic processes. Key regulatory proteins within pathways that showed altered phosphorylation following JAK inhibition were targeted using selumetinib and trametinib (MEK), buparlisib (PI3K) and ABT-199 (BCL2), and found to be synergistic in combination with JAK kinase inhibitors in primary T-ALL samples harboring JAK3 mutations. These data provide the first detailed molecular characterization of the downstream signaling pathways regulated by JAK3 mutations and provide further understanding into the oncogenic processes regulated by constitutive kinase activation aiding in the development of improved combinatorial treatment regimens.

Several somatic ribosome defects have recently been discovered in cancer, yet their oncogenic mechanisms remain poorly understood. Here we investigated the pathogenic role of the recurrent R98S mutation in ribosomal protein L10 (RPL10-R98S) found in T-ALL. The JAK-STAT signaling pathway is a critical controller of cellular proliferation and survival. A proteome screen revealed overexpression of several Jak-Stat signaling proteins in engineered RPL10-R98S mouse lymphoid cells, which we confirmed in hematopoietic cells from transgenic Rpl10-R98S mice and T-ALL xenograft samples. RPL10-R98S expressing cells displayed JAK-STAT pathway hyper-activation upon cytokine stimulation, as well as increased sensitivity to clinically used JAK-STAT inhibitors like pimozide. A mutually exclusive mutation pattern between RPL10-R98S and JAK-STAT mutations in T-ALL patients further suggests that RPL10-R98S functionally mimics JAK-STAT activation. Mechanistically, besides transcriptional changes, RPL10-R98S caused reduction of apparent programmed ribosomal frameshifting at several ribosomal frameshift signals in mouse and human Jak-Stat genes, as well as decreased Jak1 degradation. Of further medical interest, RPL10-R98S cells showed reduced proteasome activity and enhanced sensitivity to clinical proteasome inhibitors. Collectively, we describe modulation of the JAK-STAT cascade as a novel cancer-promoting activity of a ribosomal mutation, and expand the relevance of this cascade in leukemia.

Objective To identify the underlying genetic defect in a 16‐year‐old girl with severe early‐onset and refractory systemic lupus erythematosus (SLE), IgA deficiency, and mild lower limb spasticity without neuroradiologic manifestations. Methods Whole‐exome sequencing and extensive immunologic analysis were performed on samples from the index patient. Results We identified a de novo p.R779H IFIH1 gain‐of‐function mutation in a patient with severe early‐onset SLE, selective IgA deficiency, and mild lower limb spasticity. The same mutation in IFIH1 was recently identified in patients with Aicardi‐Goutières syndrome, a rare neuroimmunologic disorder associated with elevated levels of type I interferon (IFN). IFN induced with helicase C domain 1 functions as an intracellular innate immune receptor that senses viral nucleic acids and leads to the induction of type I IFN and proinflammatory cytokines. Despite systemic immunosuppressive treatment, disease activity persisted in the patient and was associated with elevated serum levels of IFNα and up‐regulation of IFIH1 itself. Conclusion This finding adds a new genetic causation for Mendelian lupus and greatly extends the disease spectrum associated with mutations in IFIH1 (ranging from inflammatory encephalopathy to prototypic systemic autoimmune disease). This marked phenotypic heterogeneity, despite an identical mutation, demonstrates the importance of modifying factors in type I IFN–dependent pathologies caused by mutations in IFIH1.

Background: Hemophagocytic lymphohistiocytosis (HLH) is a severe inflammatory condition driven by excessive CD8 1 T-cell activation. HLH occurs as both acquired and familial hemophagocytic lymphohistiocytosis (FHL) forms. In both conditions, a sterile or infectious trigger is required for disease initiation, which then becomes self-sustaining and lifethreatening. Recent studies have attributed the key distal event to excessive IFN-g production; however, the proximal events driving immune dysregulation have remained undefined. Objective: We sought to investigate the role of regulatory T (Treg) cells in the pathophysiology of experimental FHL. Methods: Because mutation in perforin is a common cause of FHL, we used an experimental FHL mouse model in which disease in perforin-deficient mice is triggered by lymphocytic choriomeningitis virus (LCMV). We assessed Treg and CD8 1 T-cell homeostasis and activation during the changing systemic conditions in the mice. In addition, human blood samples were collected and analyzed during the HLH episode. Results: We found no primary Treg cell defects in perforindeficient mice. However, Treg cell numbers collapsed after LCMV inoculation. The collapse of Treg cell numbers in LCMV-triggered perforin-deficient, but not wild-type, mice was accompanied by the combination of lower IL-2 secretion by conventional CD4 1 T cells, increased IL-2 consumption by activated CD8 1 T cells, and secretion of competitive soluble CD25. Moreover low Treg cell numbers were observed in untreated patients experiencing HLH flares. Conclusion: These results demonstrate that excessive CD8 1 T-cell activation rewires the IL-2 homeostatic network away

K E Y P O I N T Sl One-third of T-ALL cases with JAK3 mutation harbor 2 JAK3 mutations.l Double JAK3 mutants show stronger signaling than single JAK3 mutants.The Janus kinase 3 (JAK3) tyrosine kinase is mutated in 10% to 16% of T-cell acute lymphoblastic leukemia (T-ALL) cases. JAK3 mutants induce constitutive JAK/STAT signaling and cause leukemia when expressed in the bone marrow cells of mice. Surprisingly, we observed that one third of JAK3-mutant T-ALL cases harbor 2 JAK3 mutations, some of which are monoallelic and others that are biallelic. Our data suggest that wild-type JAK3 competes with mutant JAK3 (M511I) for binding to the common g chain and thereby suppresses its oncogenic potential. We demonstrate that JAK3 (M511I) can increase its limited oncogenic potential through the acquisition of an additional mutation in the mutant JAK3 allele. These double JAK3 mutants show increased STAT5 activation and increased potential to transform primary mouse pro-T cells to interleukin-7-independent growth and were not affected by wild-type JAK3 expression. These data extend our insight into the oncogenic properties of JAK3 mutations and provide an explanation of why progression of JAK3-mutant T-ALL cases can be associated with the accumulation of additional JAK3 mutations. (Blood. 2018;131(4):421-425)

T-cell acute lymphoblastic leukemia (T-ALL) is caused by the accumulation of multiple mutations combined with the ectopic expression of transcription factors in developing T cells. However, the molecular basis underlying cooperation between transcription factor expression and additional oncogenic mutations in driving T-ALL has been difficult to assess due to limited robust T-cell model systems. Here we utilize a new ex vivo pro-T-cell model to study oncogenic cooperation. Using a systems biological approach we first dissect the pro-T-cell signaling network driven by interleukin-7, stem cell factor and Notch1 and identify key downstream Akt, Stat, E2f and Myc genetic signaling networks. Next, this pro-T-cell system was used to demonstrate that ectopic expression of the TAL1 transcription factor and Pten deletion are bona-fide cooperating events resulting in an increased stem cell signature, upregulation of a specific E2f signaling network and metabolic reprogramming with higher influx of glucose carbons into the tricarboxylic acid cycle. This ex vivo pro-T-cell system thereby provides a powerful new model system to investigate how normal T-cell signaling networks are perturbed and/or hijacked by different oncogenic events found in T-ALL.