Restricted diet delays accelerated ageing and genomic stress in DNA-repair-deficient mice.

Mice deficient in the DNA excision-repair gene Ercc1 (Ercc1∆/-) show numerous accelerated ageing features that limit their lifespan to 4-6 months. They also exhibit a 'survival response', which suppresses growth and enhances cellular maintenance. Such a response resembles the anti-ageing response induced by dietary restriction (also known as caloric restriction). Here we report that a dietary restriction of 30% tripled the median and maximal remaining lifespans of these progeroid mice, strongly retarding numerous aspects of accelerated ageing. Mice undergoing dietary restriction retained 50% more neurons and maintained full motor function far beyond the lifespan of mice fed ad libitum. Other DNA-repair-deficient, progeroid Xpg-/- (also known as Ercc5-/-) mice, a model of Cockayne syndrome, responded similarly. The dietary restriction response in Ercc1∆/- mice closely resembled the effects of dietary restriction in wild-type animals. Notably, liver tissue from Ercc1∆/- mice fed ad libitum showed preferential extinction of the expression of long genes, a phenomenon we also observed in several tissues ageing normally. This is consistent with the accumulation of stochastic, transcription-blocking lesions that affect long genes more than short ones. Dietary restriction largely prevented this declining transcriptional output and reduced the number of γH2AX DNA damage foci, indicating that dietary restriction preserves genome function by alleviating DNA damage. Our findings establish the Ercc1∆/- mouse as a powerful model organism for health-sustaining interventions, reveal potential for reducing endogenous DNA damage, facilitate a better understanding of the molecular mechanism of dietary restriction and suggest a role for counterintuitive dietary-restriction-like therapy for human progeroid genome instability syndromes and possibly neurodegeneration in general.

Mammary gland-specific ablation of focal adhesion kinase reduces the incidence of p53-mediated mammary tumour formation.

BACKGROUND: Elevated expression of focal adhesion kinase (FAK) occurs in numerous human cancers including colon-, cervix- and breast cancer. Although several studies have implicated FAK in mammary tumour formation induced by ectopic oncogene expression, evidence supporting a role for FAK in spontaneous mammary tumour development caused by loss of tumour suppressor genes such as p53 is lacking. Alterations in the tumour suppressor gene p53 have been implicated in over 50% of human breast cancers. Given that elevated FAK expression highly correlates with p53 mutation status in human breast cancer, we set out to investigate the importance of FAK in p53-mediated spontaneous mammary tumour development. METHODS: To directly assess the role of FAK, we generated mice with conditional inactivation of FAK and p53. We generated female p53(lox/lox)/FAK(+/+)/WapCre, p53(lox/lox)/FAK(flox/+)/WapCre and p53(lox/lox)/FAK(flox/-)/WapCre mice, and mice with WapCre-mediated conditional expression of p53(R270H), the mouse equivalent of human p53(R273H) hot spot mutation, together with conditional deletion of FAK, P53(R270H/+)/FAK(lox/+)/WapCre and p53(R270H/+)/FAK(flox/-)/WapCre mice. All mice were subjected to one pregnancy to induce WapCre-mediated deletion of p53 or expression of p53 R270H, and Fak genes flanked by two loxP sites, and subsequently followed the development of mammary tumours. RESULTS: Using this approach, we show that FAK is important for p53-induced mammary tumour development. In addition, mice with the mammary gland-specific conditional expression of p53 point mutation R270H, the mouse equivalent to human R273H, in combination with conditional deletion of Fak showed reduced incidence of p53(R270H)-induced mammary tumours. In both models these effects of FAK were related to reduced proliferation in preneoplastic lesions in the mammary gland ductal structures. CONCLUSIONS: Mammary gland-specific ablation of FAK hampers p53-regulated spontaneous mammary tumour formation. Focal adhesion kinase deletion reduced proliferative capacity of p53 null and p53(R270H) mammary epithelial cells but did not lead to increased apoptosis in vivo. Our data identify FAK as an important regulator in mammary epithelial cell proliferation in p53-mediated and p53(R270H)-induced mammary tumour development.

Protection against allergic airway inflammation during the chronic and acute phases of Trichinella spiralis infection.

BACKGROUND: Modulation of the host immune response by helminths has been reported to be essential for parasite survival and also to benefit the host by suppressing inflammatory diseases such as allergies. We have previously shown that excretory-secretory products of Trichinella spiralis muscle larvae have immunomodulatory properties and induce in vitro the expansion of CD4(+) CD25(+) FOXP3(+) Treg cells in a TGF-ß-dependent manner. OBJECTIVE: We aimed at determining the effect of the acute (intestinal) and the chronic (muscle) phase of T. spiralis infection on experimental allergic airway inflammation (EAAI) to Ovalbumin (OVA) and the involvement of Treg cells. METHODS: The chronic phase was established before OVA-sensitization/challenge and the acute phase at two-time points, before and after OVA-sensitization. Mice were infected with 400 T. spiralis larvae and after euthanasia different pathological features of EAAI were measured. Adoptive transfer of CD4(+) T cells from Trichinella infected mice to OVA sensitized/challenged recipients was also performed. RESULTS: We found that the chronic as well as the acute phase of Trichinella infection suppress EAAI as indicated by reduction in airway inflammation, OVA-specific IgE levels in sera, Th2-cytokine production and eosinophils in bronchoalveolar lavage. This protective effect was found to be stronger during the chronic phase and to be associated with increased numbers of splenic CD4(+) CD25(+) FOXP3(+) Treg cells with suppressive activity. Adoptive transfer of splenic CD4(+) T cells from chronically infected mice with elevated numbers of Treg cells resulted in partial protection against EAAI. CONCLUSIONS AND CLINICAL RELEVANCE: These results demonstrate that the protective effect of T. spiralis on EAAI increases as infection progresses from the acute to the chronic phase. Here, Treg cells may play an essential role in the suppression of EAAI. Elucidating the mechanisms and molecular helminth structures responsible for this regulatory process is relevant to develop alternative tools for preventing or treating allergic asthma.

A multicentre study on spontaneous in-cage activity and micro-environmental conditions of IVC housed C57BL/6J mice during consecutive cycles of bi-weekly cage-change.

Mice respond to a cage change (CC) with altered activity, disrupted sleep and increased anxiety. A bi-weekly cage change is, therefore, preferred over a shorter CC interval and is currently the prevailing routine for Individually ventilated cages (IVCs). However, the build-up of ammonia (NH3) during this period is a potential threat to the animal health and the literature holds conflicting reports leaving this issue unresolved. We have therefor examined longitudinally in-cage activity, animal health and the build-up of ammonia across the cage floor with female and male C57BL/6 mice housed four per IVC changed every other week. We used a multicentre design with a standardised husbandry enabling us to tease-out features that replicated across sites from those that were site-specific. CC induce a marked increase in activity, especially during daytime (~50%) when the animals rest. A reduction in density from four to two mice did not alter this response. This burst was followed by a gradual decrease till the next cage change. Female but not male mice preferred to have the latrine in the front of the cage. Male mice allocate more of the activity to the latrine free part of the cage floor already the day after a CC. A behaviour that progressed through the CC cycle but was not impacted by the type of bedding used. Reducing housing density to two mice abolished this behaviour. Female mice used the entire cage floor the first week while during the second week activity in the latrine area decreased. Measurement of NH3 ppm across the cage floor revealed x3 higher values for the latrine area compared with the opposite area. NH3 ppm increases from 0-1 ppm to reach ≤25 ppm in the latrine free area and 50-100 ppm in the latrine area at the end of a cycle. As expected in-cage bacterial load covaried with in-cage NH3 ppm. Histopathological analysis revealed no changes to the upper airways covarying with recorded NH3 ppm or bacterial load. We conclude that housing of four (or equivalent biomass) C57BL/6J mice for 10 weeks under the described conditions does not cause any overt discomfort to the animals.

Constitutional 2p16.3 deletion including MSH6 and FBXO11 in a boy with developmental delay and diffuse large B-cell lymphoma.

We describe a case of a boy with neurodevelopmental delay and a diffuse large B-cell lymphoma (DLBCL) in whom we discovered a germline de novo 2p16.3 deletion including MSH6 and part of the FBXO11 gene. A causative role for MSH6 in cancer development was excluded based on tumor characteristics. The constitutional FBXO11 deletion explains the neurodevelopmental delay in the patient. The FBXO11 protein is involved in BCL-6 ubiquitination and BCL-6 is required for the germinal center reaction resulting in B cell differentiation. Somatic loss of function alterations of FBXO11 result in BCL-6 overexpression which is a known driver in DLBCL. We therefore consider that a causative relationship between the germline FBXO11 deletion and the development of DLBCL in this boy is conceivable.

Asymptomatic low ankle-brachial index in vascular surgery patients: a predictor of perioperative myocardial damage.

OBJECTIVES: This study evaluated the prognostic value of asymptomatic low ankle-brachial index (ABI) to predict perioperative myocardial damage, incremental to conventional cardiac risk factors imbedded in cardiac risk indices (Revised Cardiac index and Adapted Lee index). MATERIALS AND METHODS: Preoperative ABI measurements were performed in 627 consecutive vascular surgery patients (carotid artery or abdominal aortic aneurysm repair). An ABI<0.90 was considered abnormal. Patients with ABI>1.40 or (a history of) intermittent claudication were excluded. Serial troponin-T measurements were performed routinely before and after surgery. The main study endpoint was perioperative myocardial damage, the composite of myocardial ischaemia and infarction. Multivariate regression analyses, adjusted for conventional risk factors, evaluated the relation between asymptomatic low ABI and perioperative myocardial damage. RESULTS: In total, 148 (23%) patients had asymptomatic low ABI (mean 0.73, standard deviation+/-0.13). Perioperative myocardial damage was recorded in 107 (18%) patients. Multivariate regression analyses demonstrated that asymptomatic low ABI was associated with an increased risk of perioperative myocardial damage (odds ratio (OR): 2.4, 95% CI: 1.4-4.2) CONCLUSIONS: This study demonstrated that asymptomatic low ABI has a prognostic value to predict perioperative myocardial damage in vascular surgery patients, incremental to risk factors imbedded in conventional cardiac risk indices.

Hepatic extraskeletal chondroblastic osteosarcoma with unusual angioinvasion of the caudal vena cava in a dog.

Extraskeletal osteosarcomas are rare malignant mesenchymal neoplasms that are able to directly produce osteoid, without requiring a cartilage template. The extraskeletal localization indicates that these neoplasms are not associated with pre-existing skeletal elements or periosteum. We describe the gross and histological findings of a 4-year-old male Rottweiler that presented with an extraskeletal chondroblastic osteosarcoma (also known as osteosarcoma of the chondroblastic subtype) originating from the liver and extending into the lumen of the caudal vena cava, passing through the right atrium and terminating in the right ventricle of the heart immediately below the pulmonary valve. In the liver, predominantly fusiform cells grew in loosely packed streams and whorls. In the vena cava, the neoplasm was multilobular with polygonal neoplastic cells scattered within lacunae in a chondroid matrix. In the cardiac lumen, neoplastic cells produced osteoid that showed multifocal mineralization. Immunohistochemical staining showed no cytokeratin and variable S-100 protein and vimentin immunoreactivity. To the best of our knowledge, this is the first report of a chondroblastic osteosarcoma arising in the liver and showing such extensive and unusual extension into the vasculature.

Renal cell carcinoma in young FH mutation carriers: case series and review of the literature.

Hereditary Leiomyomatosis and Renal Cell Cancer (HLRCC) is an autosomal dominant syndrome caused by heterozygous pathogenic germline variants in the fumarate hydratase (FH) gene. It is characterized by cutaneous and uterine leiomyomas and an increased risk of developing renal cell carcinoma (RCC), which is usually adult-onset. HLRCC-related RCC tends to be aggressive and can metastasize even when the primary tumor is small. Data on children and adolescents are scarce. Herein, we report two patients from unrelated Dutch families, with HLRCC-related RCC at the ages of 15 and 18 years, and a third patient with an FH mutation and complex renal cysts at the age of 13. Both RCC's were localized and successfully resected, and careful MRI surveillance was initiated to monitor the renal cysts. One of the patients with RCC subsequently developed an ovarian Leydig cell tumor. A review of the literature identified 10 previously reported cases of HLRCC-related RCC in patients aged younger than 20 years, five of them presenting with metastatic disease. These data emphasize the importance of recognizing HLRCC in young patients to enable early detection of RCC, albeit rare. They support the recommendations from the 2014 consensus guideline, in which genetic testing for FH mutations, and renal MRI surveillance, is advised for HLRCC family members from the age of 8-10 years onwards.

Donor-specific immune regulation by CD8 lymphocytes expanded from rejecting human cardiac allografts.

To assess whether regulatory T cells are present in rejecting human cardiac allografts, we performed functional analyses of graft lymphocytes (GLs) expanded from endomyocardial biopsies (EMB; n = 5) with histological signs of acute cellular rejection. The GL cultures were tested for their proliferative capacity and regulatory activity on allogeneic-stimulated peripheral blood mononuclear cells (PBMC) of the patient (ratio PBMC:GLs = 5:1). Three of these GL cultures were hyporesponsive to donor antigens and suppressed the antidonor proliferative T-cell response of PBMC, but not the anti-third-party response. Interestingly, it was the CD8(+) GL subset of these cultures that inhibited the antidonor response (65-91% inhibition of the proportion of proliferating cells); the CD4(+) GLs of the expanded GL cultures were not suppressive. In conclusion, CD8(+) GLs expanded from rejecting human cardiac allografts can exhibit donor-specific immune regulatory activities in vitro. We suggest that during acute cellular rejection, GLs may not only consist of graft-destructing effector T cells, but also of cells of the CD8(+) type with the potential to specifically inhibit antidonor immune reactivity.

The prevalence of polyvascular disease in patients referred for peripheral arterial disease.

OBJECTIVE: To objectively assess the presence of polyvascular disease in patients with peripheral arterial disease and its relation to inflammation and clinical risk factors. METHODS: A total of 431 vascular surgery patients (mean age 68 years, men 77%) with atherosclerotic disease were enrolled. The presence of atherosclerosis was assessed using ultrasonography. Affected territories were defined as: (1) carotid, stenosis of common or internal carotid artery of >or=50%, (2) cardiac, left ventricular wall motion abnormalities, (3) abdominal aorta, diameter >or=30 mm and (4) lower limb, ankle-brachial pressure index <0.9. Cardiovascular risk factors and high-sensitivity C-reactive protein (hs-CRP) levels were noted in all. RESULTS: One vascular territory was affected in 29% of the patients, whereas polyvascular disease was found in 71%: two affected territories in 45%, three in 23% and four in 3% of patients. Levels of hs-CRP increased with the number of affected vascular territories (p<0.001). Multivariable logistic regression analysis showed age >or=70 years, male gender, body mass index (BMI)>or=25 kg m(-2), and hs-CRP to be independently associated with polyvascular disease. CONCLUSION: Polyvascular disease is a common condition in patients who have undergone vascular surgery. The level of systemic inflammation, reflected by hs-CRP levels, is moderately associated with the extent of polyvascular disease.

Case report: epitheliotropic lymphoma in a zebrafish (Danio rerio).

A proliferative disorder of lymphocytes was observed in a macroscopically normal young adult wild-type zebrafish (Danio rerio) during routine histological screening in a two-generation toxicity study. Proliferating lymphocytes were observed to invade the gill arches, infiltrate the cranial skeletal muscle and inner ear, and accumulate at distant sites in the frontal epidermis. The test compound, tetrabromobisphenol A (TBBPA, a flame retardant), is not known as a carcinogen and no tumours were detected in any of the 53 other fish in the study, including tank mates. Although neoplastic lymphoid proliferation in the thymus region is occasionally observed, we have never seen epitheliotropism in zebrafish during other similar exposure studies or brood stock. Our findings indicate that epitheliotropic lymphoma can occur spontaneously in zebrafish but at a low incidence.

Predisposition to colorectal cancer: exploiting copy number variation to identify novel predisposing genes and mechanisms.

Although cancer is mostly regarded as an acquired disease, familial predisposition plays a significant role in many cancer types. Thus far, several high penetrant cancer predisposing genes have been identified. As yet, however, these genes explain only a fraction of the familial and/or hereditary cases of cancer. This has led to the exploration of the human genome for novel cancer predisposing genes. The identification of such genes will not only increase our understanding of cancer predisposition and development, but will also have direct implications for genetic counseling and personalized management of the patients and their family members. Here we provide an inventory of currently known molecular mechanisms related to familial colorectal cancer development and an outline of copy number analysis-based strategies to identify new predisposing genes. Finally, we discuss a novel copy number-associated epigenetic mechanism underlying the predisposition to colorectal cancer.

High-resolution genomic profiling of childhood ALL reveals novel recurrent genetic lesions affecting pathways involved in lymphocyte differentiation and cell cycle progression.

Gross cytogenetic anomalies are traditionally being used as diagnostic, prognostic and therapeutic markers in the clinical management of cancer, including childhood acute lymphoblastic leukemia (ALL). Recently, it has become increasingly clear that genetic lesions driving tumorigenesis frequently occur at the submicroscopic level and, consequently, escape standard cytogenetic observations. Therefore, we profiled the genomes of 40 childhood ALLs at high resolution. We detected multiple de novo genetic lesions, including gross aneuploidies and segmental gains and losses, some of which were subtle and affected single genes. Many of these lesions involved recurrent (partially) overlapping deletions and duplications, containing various established leukemia-associated genes, such as ETV6, RUNX1 and MLL. Importantly, the most frequently affected genes were those controlling G1/S cell cycle progression (e.g. CDKN2A, CDKN1B and RB1), followed by genes associated with B-cell development. The latter group includes microdeletions of the B-lineage transcription factors PAX5, EBF, E2-2 and IKZF1 (Ikaros), as well as genes with other established roles in B-cell development, that is RAG1 and RAG2, FYN, PBEF1 or CBP/PAG. The fact that we frequently encountered multiple lesions affecting genes involved in cell cycle regulation and B-cell differentiation strongly suggests that both these processes need to be targeted independently and simultaneously to trigger ALL development.

Treatment response assessment with (R)-[11CPAQ PET in the MMTV-PyMT mouse model of breast cancer.

BACKGROUND: The goal of the study was to assess the potential of the vascular endothelial growth factor receptor (VEGFR)-2-targeting carbon-11 labeled (R)-N-(4-bromo-2-fluorophenyl)-6-methoxy-7-((1-methyl-3-piperidinyl)methoxy)-4-quinazolineamine ((R)-[11C]PAQ) as a positron emission tomography (PET) imaging biomarker for evaluation of the efficacy of anticancer drugs in preclinical models. METHODS: MMTV-PyMT mice were treated with vehicle alone (VEH), murine anti-VEGFA antibody (B20-4.1.1), and paclitaxel (PTX) in combination or as single agents. The treatment response was measured with (R)-[11C]PAQ PET as standardized uptake value (SUV)mean, SUVmax relative changes at the baseline (day 0) and follow-up (day 4) time points, and magnetic resonance imaging (MRI)-derived PyMT mammary tumor volume (TV) changes. Expression of Ki67, VEGFR-2, and CD31 in tumor tissue was determined by immunohistochemistry (IHC). Non-parametric statistical tests were used to evaluate the relation between (R)-[11C]PAQ radiotracer uptake and therapy response biomarkers. RESULTS: The (R)-[11C]PAQ SUVmax in tumors was significantly reduced after 4 days in the B20-4.1.1/PTX combinational and B20-4.1.1 monotherapy groups (p < 0.0005 and p < 0.003, respectively). No significant change was observed in the PTX monotherapy group. There was a significant difference in the SUVmax change between the VEH group and B20-4.1.1/PTX combinational group, as well as between the VEH group and the B20-4.1.1 monotherapy group (p < 0.05). MRI revealed significant decreases in TV in the B20-4.1.1/PTX treatment group (p < 0.005) but not the other therapy groups. A positive trend was observed between the (R)-[11C]PAQ SUVmax change and TV reduction in the B20-4.1.1/PTX group. Statistical testing showed a significant difference in the blood vessel density between the B20-4.1.1/PTX combinational group and the VEH group (p < 0.05) but no significant difference in the Ki67 positive signal between treatment groups. CONCLUSIONS: The results of this study are promising. However, additional studies are necessary before (R)-[11C]PAQ can be approved as a predictive radiotracer for cancer therapy response.

Molecular mechanisms underlying the MiT translocation subgroup of renal cell carcinomas.

Renal cell carcinomas (RCCs) represent a heterogeneous group of neoplasms, which differ in histological, pathologic and clinical characteristics. The tumors originate from different locations within the nephron and are accompanied by different recurrent (cyto)genetic anomalies. Recently, a novel subgroup of RCCs has been defined, i.e., the MiT translocation subgroup of RCCs. These tumors originate from the proximal tubule of the nephron, exhibit pleomorphic histological features including clear cell morphologies and papillary structures, and are found predominantly in children and young adults. In addition, these tumors are characterized by the occurrence of recurrent chromosomal translocations, which result in disruption and fusion of either the TFE3 or TFEB genes, both members of the MiT family of basic helix-loop-helix/leucine-zipper transcription factor genes. Hence the name MiT translocation subgroup of RCCs. In this review several features of this RCC subgroup will be discussed, including the molecular mechanisms that may underlie their development.

Differential induction of two p24delta putative cargo receptors upon activation of a prohormone-producing cell.

The p24 family consists of type I transmembrane proteins that are present abundantly in transport vesicles, may play a role in endoplasmic reticulum-to-Golgi cargo transport, and have been classified into subfamilies named p24alpha, -beta, -gamma, and -delta. We previously identified a member of the p24delta subfamily that is coordinately expressed with the prohormone proopiomelanocortin (POMC) in the melanotrope cells of the intermediate pituitary during black background adaptation of the amphibian Xenopus laevis ( approximately 30-fold increase in POMC mRNA). In this study, we report on the characterization of this p24delta member (Xp24delta(2)) and on the identification and characterization of a second member (Xp24delta(1)) that is also expressed in the melanotrope cells and that has 66% amino acid sequence identity to Xp24delta(2). The two p24delta members are ubiquitously expressed, but Xp24delta(2) is neuroendocrine enriched. During black background adaptation, the amount of the Xp24delta(2) protein in the intermediate pituitary was increased approximately 25 times, whereas Xp24delta(1) protein expression was increased only 2.5 times. Furthermore, the level of Xp24delta(2) mRNA was approximately 5-fold higher in the melanotrope cells of black-adapted animals than in those of white-adapted animals, whereas Xp24delta(1) mRNA expression was not induced. Therefore, the expression of Xp24delta(2) specifically correlates with the expression of POMC. Together, our findings suggest that p24delta proteins have a role in selective protein transport in the secretory pathway.

Synergistic functions of E2F7 and E2F8 are critical to suppress stress-induced skin cancer.

E2F transcription factors are important regulators of the cell cycle, and unrestrained activation of E2F-dependent transcription is considered to be an important driver of tumor formation and progression. Although highly expressed in normal skin and skin cancer, the role of the atypical E2Fs, E2F7 and E2F8, in keratinocyte homeostasis, regeneration and tumorigenesis is unknown. Surprisingly, keratinocyte-specific deletion of E2F7 and E2F8 in mice did not interfere with skin development and wound healing. However, the rate for successful isolation and establishment of E2f7/8-deficient primary keratinocyte cultures was much higher than for wild-type keratinocytes. Moreover, E2f7/8-deficient primary keratinocytes proliferate more efficiently under stress conditions, such as low/high confluence or DNA damage. Application of in vivo stress using the DMBA/TPA skin carcinogenesis protocol revealed that combined inactivation of E2f7/8 enhanced tumorigenesis and accelerated malignant progression. Loss of atypical E2Fs resulted in increased expression of E2F target genes, including E2f1. Additional loss of E2f1 did not rescue, but worsened skin tumorigenesis. We show that loss of E2F7/8 triggers apoptosis via induction of E2F1 in response to stress, indicating that the tumor-promoting effect of E2F7/8 inactivation can be partially compensated via E2F1-dependent apoptosis. Importantly, E2F7/8 repressed a large set of E2F target genes that are highly expressed in human patients with skin cancer. Together, our studies demonstrate that atypical E2Fs act as tumor suppressors, most likely via transcriptional repression of cell cycle genes in response to stress.

Germline activating TYK2 mutations in pediatric patients with two primary acute lymphoblastic leukemia occurrences.

The contribution of genetic predisposing factors to the development of pediatric acute lymphoblastic leukemia (ALL), the most frequently diagnosed cancer in childhood, has not been fully elucidated. Children presenting with multiple de novo leukemias are more likely to suffer from genetic predisposition. Here, we selected five of these patients and analyzed the mutational spectrum of normal and malignant tissues. In two patients, we identified germline mutations in TYK2, a member of the JAK tyrosine kinase family. These mutations were located in two adjacent codons of the pseudokinase domain (p.Pro760Leu and p.Gly761Val). In silico modeling revealed that both mutations affect the conformation of this autoregulatory domain. Consistent with this notion, both germline mutations promote TYK2 autophosphorylation and activate downstream STAT family members, which could be blocked with the JAK kinase inhibitor I. These data indicate that germline activating TYK2 mutations predispose to the development of ALL.