Targeted CRM1-inhibition perturbs leukemogenic NUP214 fusion proteins and exerts anti-cancer effects in leukemia cell lines with NUP214 rearrangements.

Chromosomal translocations fusing the locus of nucleoporin NUP214 each with the proto-oncogenes SET and DEK are recurrent in, largely intractable, acute leukemias. The molecular basis underlying the pathogenesis of SET-NUP214 and DEK-NUP214 are still poorly understood, but both chimeras inhibit protein nuclear export mediated by the ß-karyopherin CRM1. In this report, we show that SET-NUP214 and DEK-NUP214 both disturb the localization of proteins essential for nucleocytoplasmic transport, in particular for CRM1-mediated protein export. Endogenous and exogenous SET-NUP214 and DEK-NUP214 form nuclear bodies. These nuclear bodies disperse upon targeted inhibition of CRM1 and the two fusion proteins re-localize throughout the nucleoplasm. Moreover, SET-NUP214 and DEK-NUP214 nuclear bodies reestablish shortly after removal of CRM1 inhibitors. Likewise, cell viability, metabolism, and proliferation of leukemia cell lines harboring SET-NUP214 and DEK-NUP214 are compromised by CRM1 inhibition, which is even sustained after clearance from CRM1 antagonists. Our results indicate CRM1 as a possible therapeutic target in NUP214-related leukemia. This is especially important, since no specific or targeted treatment options for NUP214 driven leukemia are available yet.

Disclosing the Interactome of Leukemogenic NUP98-HOXA9 and SET-NUP214 Fusion Proteins Using a Proteomic Approach.

The interaction of oncogenes with cellular proteins is a major determinant of cellular transformation. The NUP98-HOXA9 and SET-NUP214 chimeras result from recurrent chromosomal translocations in acute leukemia. Functionally, the two fusion proteins inhibit nuclear export and interact with epigenetic regulators. The full interactome of NUP98-HOXA9 and SET-NUP214 is currently unknown. We used proximity-dependent biotin identification (BioID) to study the landscape of the NUP98-HOXA9 and SET-NUP214 environments. Our results suggest that both fusion proteins interact with major regulators of RNA processing, with translation-associated proteins, and that both chimeras perturb the transcriptional program of the tumor suppressor p53. Other cellular processes appear to be distinctively affected by the particular fusion protein. NUP98-HOXA9 likely perturbs Wnt, MAPK, and estrogen receptor (ER) signaling pathways, as well as the cytoskeleton, the latter likely due to its interaction with the nuclear export receptor CRM1. Conversely, mitochondrial proteins and metabolic regulators are significantly overrepresented in the SET-NUP214 proximal interactome. Our study provides new clues on the mechanistic actions of nucleoporin fusion proteins and might be of particular relevance in the search for new druggable targets for the treatment of nucleoporin-related leukemia.

NUP214 in Leukemia: It's More than Transport.

NUP214 is a component of the nuclear pore complex (NPC) with a key role in protein and mRNA nuclear export. Chromosomal translocations involving the NUP214 locus are recurrent in acute leukemia and frequently fuse the C-terminal region of NUP214 with SET and DEK, two chromatin remodeling proteins with roles in transcription regulation. SET-NUP214 and DEK-NUP214 fusion proteins disrupt protein nuclear export by inhibition of the nuclear export receptor CRM1, which results in the aberrant accumulation of CRM1 protein cargoes in the nucleus. SET-NUP214 is primarily associated with acute lymphoblastic leukemia (ALL), whereas DEK-NUP214 exclusively results in acute myeloid leukemia (AML), indicating different leukemogenic driver mechanisms. Secondary mutations in leukemic blasts may contribute to the different leukemia outcomes. Additional layers of complexity arise from the respective functions of SET and DEK in transcription regulation and chromatin remodeling, which may drive malignant hematopoietic transformation more towards ALL or AML. Another, less frequent fusion protein involving the C terminus of NUP214 results in the sequestosome-1 (SQSTM1)-NUP214 chimera, which was detected in ALL. SQSTM1 is a ubiquitin-binding protein required for proper autophagy induction, linking the NUP214 fusion protein to yet another cellular mechanism. The scope of this review is to summarize the general features of NUP214-related leukemia and discuss how distinct chromosomal translocation partners can influence the cellular effects of NUP214 fusion proteins in leukemia.

Is Low-Dose Radiation Exposure a Risk Factor for Atherosclerotic Disease?

Nontargeted late effects of radiation include an increased risk of cardiovascular disease, although this is still debatable in the context of low-dose radiation. Tinea capitis patients treated in childhood with X rays to induce scalp epilation received a low dose of radiation to their carotids. To better clarify this issue, we evaluated carotid atherosclerosis in a cohort of such patients treated in 1950-1963 in Portugal. A group of 454 individuals randomly chosen from previously observed Portuguese tinea capitis patients and a control group mainly composed of their spouses (n = 280) were enrolled. Cardiovascular risk factors such as waist circumference, body mass index, blood pressure and tobacco consumption, as well as biochemical measurements were obtained. Ultrasound imaging of carotid arteries for intima media thickness and stenosis evaluation were performed according to a standardized protocol. In comparison to the control group, the irradiated cohort members were significantly older, more frequently never smokers, hypertensive, and presented higher glycated hemoglobin and alkaline phosphatase levels. In addition, the irradiated cohort showed a higher frequency of carotid stenosis ≥30% than the nonirradiated group (13.9% vs. 10.7%), although this was not significant ( P = 0.20). Stenosis was ≥50% in 2.9% of the irradiated group and 0.4% of the nonirradiated group ( P = 0.02). Likewise, the frequency of intima media thickness ≥1 mm was significantly higher in the irradiated group (16.8% vs. 10.7%; P = 0.02). Multivariate analysis, including other cardiovascular risk factors, showed that exposure to low-dose radiation increased the risk of carotid stenosis by ≥50% [odds ratio (OR) = 8.85; P = 0.04] and intima media thickness by ≥1 mm (OR = 1.82; P = 0.02). These findings confirm that low-dose exposure is a risk factor of carotid atherosclerotic disease.

TERT promoter mutations: a genetic signature of benign and malignant thyroid tumours occurring in the context of tinea capitis irradiation.

OBJECTIVE: The aim of this study is to evaluate the frequency and molecular characteristics of TERTp mutations in thyroid adenomas and carcinomas occurring in the low-dose radiation exposure tinea capitis setting. DESIGN AND METHODS: Twenty-seven patients with 34 well-differentiated thyroid carcinomas and 28 patients with 29 follicular adenomas diagnosed in a Portuguese tinea capitis cohort were studied. Blood samples were obtained from all the patients. Screening for TERTp mutations was performed by PCR amplification followed by Sanger sequencing. A series of 33 sporadic thyroid adenomas was used as control. RESULTS: TERTp mutations were detected in six of the 28 patients with adenoma (21.4%) and in four of the 27 patients with carcinoma (14.8%). Three tumours (two carcinomas and one adenoma) had the tandem mutation -124/-125 GG>AA (30.0%), whereas the remaining seven had the -124G > A. The 20.7% frequency of TERTp mutations in adenomas contrasts with the absence of mutations in the adenomas from the control group and from most series on record, whereas the one found in carcinomas (11.8%) is similar to those reported in the literature for sporadic carcinomas. CONCLUSION: TERTp mutations, including the tandem mutation -124/-125 GG>AA not described previously in thyroid tumours, appear to represent a genetic signature for thyroid tumours in patients submitted to low-dose X-ray irradiation. The high frequency of TERTp mutations in the adenomas of our cohort contrasts with their absence in sporadically occurring, as well as in adenomas of the Chernobyl series.

IL6-174 G>C Polymorphism (rs1800795) Association with Late Effects of Low Dose Radiation Exposure in the Portuguese Tinea Capitis Cohort.

Head and neck cancers, and cardiovascular disease have been described as late effects of low dose radiation (LDR) exposure, namely in tinea capitis cohorts. In addition to radiation dose, gender and younger age at exposure, the genetic background might be involved in the susceptibility to LDR late effects. The -174 G>C (rs1800795) SNP in IL6 has been associated with cancer and cardiovascular disease, nevertheless this association is still controversial. We assessed the association of the IL6-174 G>C SNP with LDR effects such as thyroid carcinoma, basal cell carcinoma and carotid atherosclerosis in the Portuguese tinea capitis cohort. The IL6-174 G>C SNP was genotyped in 1269 individuals formerly irradiated for tinea capitis. This sampling group included thyroid cancer (n = 36), basal cell carcinoma (n = 113) and cases without thyroid or basal cell carcinoma (1120). A subgroup was assessed for atherosclerosis by ultrasonography (n = 379) and included matched controls (n = 222). Genotypes were discriminated by real-time PCR using a TaqMan SNP genotyping assay. In the irradiated group, we observed that the CC genotype was significantly associated with carotid plaque risk, both in the genotypic (OR = 3.57, CI = 1.60-7.95, p-value = 0.002) and in the recessive (OR = 3.02, CI = 1.42-6.42, p-value = 0.004) models. Irradiation alone was not a risk factor for carotid atherosclerosis. We did not find a significant association of the IL6-174 C allele with thyroid carcinoma or basal cell carcinoma risk. The IL6-174 CC genotype confers a three-fold risk for carotid atherosclerotic disease suggesting it may represent a genetic susceptibility factor in the LDR context.

The Oncogenic Fusion Proteins SET-Nup214 and Sequestosome-1 (SQSTM1)-Nup214 Form Dynamic Nuclear Bodies and Differentially Affect Nuclear Protein and Poly(A)+ RNA Export.

Genetic rearrangements are a hallmark of several forms of leukemia and can lead to oncogenic fusion proteins. One example of an affected chromosomal region is the gene coding for Nup214, a nucleoporin that localizes to the cytoplasmic side of the nuclear pore complex (NPC). We investigated two such fusion proteins, SET-Nup214 and SQSTM1 (sequestosome)-Nup214, both containing C-terminal portions of Nup214. SET-Nup214 nuclear bodies containing the nuclear export receptor CRM1 were observed in the leukemia cell lines LOUCY and MEGAL. Overexpression of SET-Nup214 in HeLa cells leads to the formation of similar nuclear bodies that recruit CRM1, export cargo proteins, and certain nucleoporins and concomitantly affect nuclear protein and poly(A)+ RNA export. SQSTM1-Nup214, although mostly cytoplasmic, also forms nuclear bodies and inhibits nuclear protein but not poly(A)+ RNA export. The interaction of the fusion proteins with CRM1 is RanGTP-dependent, as shown in co-immunoprecipitation experiments and binding assays. Further analysis revealed that the Nup214 parts mediate the inhibition of nuclear export, whereas the SET or SQSTM1 part determines the localization of the fusion protein and therefore the extent of the effect. SET-Nup214 nuclear bodies are highly mobile structures, which are in equilibrium with the nucleoplasm in interphase and disassemble during mitosis or upon treatment of cells with the CRM1-inhibitor leptomycin B. Strikingly, we found that nucleoporins can be released from nuclear bodies and reintegrated into existing NPC. Our results point to nuclear bodies as a means of preventing the formation of potentially insoluble and harmful protein aggregates that also may serve as storage compartments for nuclear transport factors.

Thyroid and parathyroid tumours in patients submitted to X-ray scalp epilation during the tinea capitis eradication campaign in the North of Portugal (1950-1963).

Tinea capitis attained epidemical proportions in the fifth and sixth decades in Portugal, as in other countries. Before starting the utilization of griseofulvin in 1959, the best approach to treat tinea capitis infection was X-ray scalp epilation combined with topical antimycotic ointments. A long-term side effect of this therapy is thyroid disease, namely thyroid cancer; data on parathyroid lesions (hyperplasia, adenoma and carcinoma) are scarce. We observed clinically 1,375 individuals irradiated in childhood for tinea capitis treatment in the North of Portugal with the main purpose of evaluating thyroid and parathyroid tumours as possible sequelae of the irradiation treatment. For each individual, a cervical ultrasound and a serum calcium measurement were proposed. Fine needle aspiration cytology was suggested whenever ultrasound thyroid nodules presented suspicious features. We observed a 54 % frequency of thyroid nodules and a 2.8 % frequency of thyroid carcinoma (38/1,375). Nineteen of the 38 (50 %) carcinomas were diagnosed by us, whereas the remaining 19 carcinomas had been diagnosed and treated prior to our observation. The carcinomas were significantly more frequent in women than in men. Benign excised lesions were also significantly more frequent in women and in patients irradiated at younger ages. Seven women, considered asymptomatic until our clinical observation, had laboratory signs of hyperparathyroidism. The data we have obtained, namely high thyroid cancer frequency, corroborate previous data from childhood irradiated cohorts and highlight the need for the close follow-up of these populations in order to identify and treat early undiagnosed head and neck lesions. No evidence of increased parathyroid disease was found in this cohort of head and neck X-irradiated patients.

Polymorphisms in the TNFA and IL6 genes represent risk factors for autoimmune thyroid disease.

BACKGROUND: Autoimmune thyroid disease (AITD) comprises diseases including Hashimoto's thyroiditis and Graves' disease, both characterized by reactivity to autoantigens causing, respectively, inflammatory destruction and autoimmune stimulation of the thyroid-stimulating hormone receptor. AITD is the most common thyroid disease and the leading form of autoimmune disease in women. Cytokines are key regulators of the immune and inflammatory responses; therefore, genetic variants at cytokine-encoding genes are potential risk factors for AITD. METHODS: Polymorphisms in the IL6-174 G/C (rs1800795), TNFA-308 G/A (rs1800629), IL1B-511 C/T (rs16944), and IFNGR1-56 T/C (rs2234711) genes were assessed in a case-control study comprising 420 Hashimoto's thyroiditis patients, 111 Graves' disease patients and 735 unrelated controls from Portugal. Genetic variants were discriminated by real-time PCR using TaqMan SNP genotyping assays. RESULTS: A significant association was found between the allele A in TNFA-308 G/A and Hashimoto's thyroiditis, both in the dominant (OR = 1.82, CI = 1.37-2.43, p-value = 4.4×10(-5)) and log-additive (OR = 1.64, CI = 1.28-2.10, p-value = 8.2×10(-5)) models. The allele C in IL6-174 G/C is also associated with Hashimoto's thyroiditis, however, only retained significance after multiple testing correction in the log-additive model (OR = 1.28, CI = 1.06-1.54, p-value = 8.9×10(-3)). The group with Graves' disease also registered a higher frequency of the allele A in TNFA-308 G/A compared with controls both in the dominant (OR = 1.85, CI = 1.19-2.87, p-value = 7.0×10(-3)) and log-additive (OR = 1.69, CI = 1.17-2.44, p-value = 6.6×10(-3)) models. The risk for Hashimoto's thyroiditis and Graves' disease increases with the number of risk alleles (OR for two risk alleles is, respectively, 2.27 and 2.59). CONCLUSIONS: This study reports significant associations of genetic variants in TNFA and IL6 with the risk for AITD, highlighting the relevance of polymorphisms in inflammation-related genes in the etiopathogenesis of AITD.

TERT promoter mutations in skin cancer: the effects of sun exposure and X-irradiation.

The reactivation or reexpression of telomerase (TERT) is a widespread feature of neoplasms. TERT promoter mutations were recently reported that were hypothesized to result from UV radiation. In this retrospective study, we assessed TERT promoter mutations in 196 cutaneous basal cell carcinomas (BCCs), including 102 tumors from X-irradiated patients, 94 tumors from patients never exposed to ionizing radiation treatment, and 116 melanomas. We sought to evaluate the effects of UV and X-ray irradiation on TERT mutation frequency. TERT mutations were detected in 27% of BCCs from X-irradiated patients, 51% of BCCs from nonirradiated patients, and 22% of melanoma patients. TERT mutations were significantly increased in non-X-irradiated BCC patients compared with X-irradiated BCC patients; the mutations also presented a different mutation signature. In nonirradiated patients, TERT mutations were more frequent in BCCs of sun-exposed skin, supporting a possible causative role of UV radiation. In melanoma, TERT promoter mutations were generally restricted to intermittent sun-exposed areas and were associated with nodular and superficial spreading subtypes, increased thickness, ulceration, increased mitotic rate, and BRAFV600E mutations. Our results suggest that various carcinogenic factors may cause distinct TERT promoter mutations in BCC and that TERT promoter mutations might be associated with a poorer prognosis in melanoma.

Mitochondrial D310 D-Loop instability and histological subtypes in radiation-induced cutaneous basal cell carcinomas.

BACKGROUND: Basal cell carcinoma (BCC) is the most frequent skin cancer. An elevated prevalence of BCC has been associated with radiation, namely after the Tinea capitis epilation treatment, being these tumors described as more aggressive. Mitochondrial DNA (mtDNA) mutations have been reported in many human tumors, but their occurrence in BCC is poorly documented. OBJECTIVE: The purpose of this work was to evaluate BCC histological subtypes in individuals subjected to X-ray epilation for Tinea capitis treatment when compared to non-irradiated patients. Moreover we also wanted to evaluate mitochondrial D-Loop instability in both groups of BCCs in order to compare the frequency of D-Loop mutations in post-irradiation BCC versus sporadic BCC. METHODS: 228 histological specimens corresponding to BCCs from 75 irradiated patients and 60 non-irradiated patients were re-evaluated for histological subtype. Subsequently, we sequenced the D-Loop 310 repeat in blood, oral mucosa, tumor lesions and, whenever available, non-tumoral adjacent tissue from these patients. RESULTS: The infiltrative subtype of BCC, considered to be more aggressive, was significantly more frequent in irradiated patients. BCC D-Loop D310 mutation rate was significantly higher in irradiated BCCs than in the non-irradiated ones. Moreover, it was associated with a higher irradiation dose. The presence of mtDNA heteroplasmy in patients' blood was associated with a higher mutation rate in the BCCs suggesting that a more unstable genotype could predispose to mtDNA somatic mutation. CONCLUSIONS: Our results suggest that radiation-induced BCCs may be considered to be more aggressive tumors. Further studies are needed to clarify the role of mtDNA D-Loop mutations in tumors from irradiated patients.

Genetic alterations in thyroid tumors from patients irradiated in childhood for tinea capitis treatment.

OBJECTIVE: Exposure to ionizing radiation at young age is the strongest risk factor for the occurrence of papillary thyroid carcinoma (PTC). RET/PTC rearrangements are the most frequent genetic alterations associated with radiation-induced PTC, whereas BRAF and RAS mutations and PAX8-PPARG rearrangement have been associated with sporadic PTC. We decided to search for such genetic alterations in PTCs of patients subjected in childhood to scalp irradiation. DESIGN: We studied 67 thyroid tumors from 49 individuals irradiated in childhood for tinea capitis scalp epilation: 36 malignant (12 cases of conventional PTC (cPTC), two cPTC metastases, 20 cases of follicular variant PTC (FVPTC), one oncocytic variant of PTC and one follicular carcinoma) and 31 follicular thyroid adenomas. METHODS: The lesions were screened for the BRAF(V600E) and NRAS mutations and for RET/PTC and PAX8-PPARG rearrangements. RESULTS: BRAF(V600E) mutation was detected in seven of 14 (50%) cPTC and two of 20 FVPTC (10%) (P=0.019). NRAS mutation was present in one case of FVPTC (5%). RET/PTC1 rearrangement was found, by RT-PCR, in one of 17 cases (5.9%) and by fluorescence in situ hybridization in two of six cases (33%). PAX8-PPARG rearrangement was not detected in any carcinoma. None of the follicular adenomas presented any of the aforementioned genetic alterations. CONCLUSIONS: The prevalence of BRAF(V600E) mutation in our series is the highest reported in series of PTCs arising in radiation-exposed individuals. The prevalence of RET/PTC1 rearrangement fits with the values recently described in a similar setting.