Comprehensive genetic and phenotype analysis of 95 individuals with mosaic tuberous sclerosis complex.

Tuberous sclerosis complex (TSC) is a neurogenetic disorder due to loss-of-function TSC1 or TSC2 variants, characterized by tumors affecting multiple organs, including skin, brain, heart, lung, and kidney. Mosaicism for TSC1 or TSC2 variants occurs in 10%-15% of individuals diagnosed with TSC. Here, we report comprehensive characterization of TSC mosaicism by using massively parallel sequencing (MPS) of 330 TSC samples from a variety of tissues and fluids from a cohort of 95 individuals with mosaic TSC. TSC1 variants in individuals with mosaic TSC are much less common (9%) than in germline TSC overall (26%) (p < 0.0001). The mosaic variant allele frequency (VAF) is significantly higher in TSC1 than in TSC2, in both blood and saliva (median VAF: TSC1, 4.91%; TSC2, 1.93%; p = 0.036) and facial angiofibromas (median VAF: TSC1, 7.7%; TSC2 3.7%; p = 0.004), while the number of TSC clinical features in individuals with TSC1 and TSC2 mosaicism was similar. The distribution of mosaic variants across TSC1 and TSC2 is similar to that for pathogenic germline variants in general TSC. The systemic mosaic variant was not present in blood in 14 of 76 (18%) individuals with TSC, highlighting the value of analysis of multiple samples from each individual. A detailed comparison revealed that nearly all TSC clinical features are less common in individuals with mosaic versus germline TSC. A large number of previously unreported TSC1 and TSC2 variants, including intronic and large rearrangements (n = 11), were also identified.

Mosaicism in Tumor Suppressor Gene Syndromes: Prevalence, Diagnostic Strategies, and Transmission Risk.

A mosaic state arises when pathogenic variants are acquired in certain cell lineages during postzygotic development, and mosaic individuals may present with a generalized or localized phenotype. Here, we review the current state of knowledge regarding mosaicism for eight common tumor suppressor genes-NF1, NF2, TSC1, TSC2, PTEN, VHL, RB1, and TP53-and their related genetic syndromes/entities. We compare and discuss approaches for comprehensive diagnostic genetic testing, the spectrum of variant allele frequency, and disease severity. We also review affected individuals who have no mutation identified after conventional genetic analysis, as well as genotype-phenotype correlations and transmission risk for each tumor suppressor gene in full heterozygous and mosaic patients. This review provides new insight into similarities as well as marked differences regarding the appreciation of mosaicism in these tumor suppressor syndromes.

Glycoprotein Nonmetastatic Melanoma Protein B (GPNMB) Immunohistochemistry Can Be a Useful Ancillary Tool to Identify Perivascular Epithelioid Cell Tumor.

Perivascular epithelioid cell tumors (PEComas) are rare mesenchymal tumors that express smooth muscle and melanocytic makers. Diagnosis of PEComas can be challenging due to focal or lost expression of traditional immunohistochemical markers, limited availability of molecular testing, and morphological overlap with much more common smooth muscle tumors. This study evaluates the use of glycoprotein nonmetastatic melanoma protein B (GPNMB) immunohistochemical staining as a surrogate marker for TSC1/2/MTOR alteration or TFE3 rearrangement to differentiate PEComas from other mesenchymal tumors. Cathepsin K was also assessed for comparison. A total of 399 tumors, including PEComas, alveolar soft part sarcomas, and other histologic PEComa mimics, were analyzed using GPNMB and cathepsin K immunohistochemistry. GPNMB expression was seen in all PEComas and alveolar soft part sarcomas with the majority showing diffuse and moderate-to-strong labeling, whereas other sarcomas were negative or showed focal labeling. When a cutoff of diffuse and at least moderate staining was used, GPNMB demonstrated 95% sensitivity and 97% specificity in distinguishing PEComas from leiomyosarcoma, well-differentiated/dedifferentiated liposarcomas, and undifferentiated pleomorphic sarcomas. Cathepsin K with a cutoff of any labeling had lower sensitivity (78%) and similar specificity (94%) to GPNMB. This study highlights GPNMB as a highly sensitive marker for PEComas and suggests its potential use as an ancillary tool within a panel of markers for accurate classification of these tumors.

Tsc2 mutation rather than Tsc1 mutation dominantly causes a social deficit in a mouse model of tuberous sclerosis complex.

BACKGROUND: Tuberous sclerosis complex (TSC) is an autosomal dominant disorder that is associated with neurological symptoms, including autism spectrum disorder. Tuberous sclerosis complex is caused by pathogenic germline mutations of either the TSC1 or TSC2 gene, but somatic mutations were identified in both genes, and the combined effects of TSC1 and TSC2 mutations have been unknown. METHODS: The present study investigated social behaviors by the social interaction test and three-chambered sociability tests, effects of rapamycin treatment, and gene expression profiles with a gene expression microarray in Tsc1 and Tsc2 double heterozygous mutant (TscD+/-) mice. RESULTS: TscD+/- mice exhibited impairments in social behaviors, and the severity of impairments was similar to Tsc2+/- mice rather than Tsc1+/- mice. Impairments in social behaviors were rescued by rapamycin treatment in all mutant mice. Gene expression profiles in the brain were greatly altered in TscD+/- mice more than in Tsc1+/- and Tsc2+/- mice. The gene expression changes compared with wild type (WT) mice were similar between TscD+/- and Tsc2+/- mice, and the overlapping genes whose expression was altered in mutant mice compared with WT mice were enriched in the neoplasm- and inflammation-related canonical pathways. The "signal transducer and activator of transcription 3, interferon regulatory factor 1, interferon regulatory factor 4, interleukin-2R α chain, and interferon-γ" signaling pathway, which is initiated from signal transducer and activator of transcription 4 and PDZ and LIM domain protein 2, was associated with impairments in social behaviors in all mutant mice. LIMITATIONS: It is unclear whether the signaling pathway also plays a critical role in autism spectrum disorders not caused by Tsc1 and Tsc2 mutations. CONCLUSIONS: These findings suggest that TSC1 and TSC2 double mutations cause autistic behaviors similarly to TSC2 mutations, although significant changes in gene expression were attributable to the double mutations. These findings contribute to the knowledge of genotype-phenotype correlations in TSC and suggest that mutations in both the TSC1 and TSC2 genes act in concert to cause neurological symptoms, including autism spectrum disorder.

Tuberous sclerosis complex in adulthood: focus on epilepsy prognosis.

OBJECTIVE: Typically diagnosed in early childhood or adolescence, TSC is a chronic, multisystemic disorder with age-dependent manifestations posing a challenge for transition and for specific surveillance throughout the lifetime. Data on the clinical features and severity of TSC in adults and on the prognosis of epilepsy are scarce. We analyzed the clinical and genetic features of a cohort of adult patients with TSC, to identify the prognostic predictors of seizure remission after a long follow-up. METHOD: We conducted a retrospective analysis of patients diagnosed with TSC according to the updated international diagnostic criteria. Pearson's chi-square or Fisher's exact test and Mann Whitney U test were used to compare variables among the Remission (R) and Non-Remission (NR) group. Univariate and multivariate logistic regression analyses were performed. RESULTS: We selected 43 patients with TSC and neurological involvement in terms of epilepsy and/or brain lesions, attending the Epilepsy Center of our Institute: of them, 16 (37.2%) were transitioning from the pediatric care and 6 (13.9%) were referred by other specialists. Multiorgan involvement includes cutaneous (86.0%), nephrological (70.7%), hepatic (40.0%), ocular (34.3%), pneumological (28.6%) and cardiac (26.3%) manifestations. Thirty-nine patients (90.7 %) had epilepsy. The mean age at seizure onset was 4 ± 7.3 years: most patients (29, 76.3 %) presented with focal seizures or spasms by age 3 years; only 2 (5.3 %) had seizure onset in adulthood. Twenty-seven patients (69.2 %) experienced multiple seizure types overtime, 23 (59.0 %) had intellectual disability (ID). At last assessment, 14 (35.9 %) were seizure free (R group) and 25 (64.1 %) had drug-resistant seizures (NR group). At logistic regression univariate analysis, ID (OR 7.9, 95 % CI 1.8--34.7), multiple seizure types lifelong (OR 13.2, 95 % CI 2.6- 67.2), spasms/tonic seizures at presentation (OR 6.5, 95 % CI 1.2--35.2), a higher seizure frequency at onset (OR 5.4, 95 % CI 1.2--24.3), abnormal neurological examination (OR 9.8, 95 % CI 1.1--90.6) and pathogenic variants in TSC2 (OR 5.4, 95 % CI 1.2--24.5) were significantly associated with non-remission. In the multivariate analysis, both ID and multiple seizure types lifelong were confirmed as independent predictors of poor seizure outcome. CONCLUSIONS: In our cohort of adult patients with TSC, epilepsy remains one of the main neurological challenges with only 5.3% of cases manifesting in adulthood. Approximately 64% of these patients failed to achieve seizure remission. ID and multiple seizure types were the main predictors of poor outcome. Nephrological manifestations require continuous specific follow-up in adults.

Lymphangioleiomyomatosis as a potent lung cancer risk factor: Insights from a Japanese large cohort study.

BACKGROUND AND OBJECTIVE: Lymphangioleiomyomatosis (LAM) is a rare neoplastic disease associated with the functional tumour suppressor genes TSC1 and TSC2 and causes structural destruction in the lungs, which could potentially increase the risk of lung cancer. However, this relationship remains unclear because of the rarity of the disease. METHODS: We investigated the relative risk of developing lung cancer among patients diagnosed with LAM between 2001 and 2022 at a single high-volume centre in Japan, using data from the Japanese Cancer Registry as the reference population. Next-generation sequencing (NGS) was performed in cases where tumour samples were available. RESULTS: Among 642 patients diagnosed with LAM (sporadic LAM, n = 557; tuberous sclerosis complex-LAM, n = 80; unclassified, n = 5), 13 (2.2%) were diagnosed with lung cancer during a median follow-up period of 5.13 years. All patients were female, 61.5% were never smokers, and the median age at lung cancer diagnosis was 53 years. Eight patients developed lung cancer after LAM diagnosis. The estimated incidence of lung cancer was 301.4 cases per 100,000 person-years, and the standardized incidence ratio was 13.6 (95% confidence interval, 6.2-21.0; p = 0.0008). Actionable genetic alterations were identified in 38.5% of the patients (EGFR: 3, ALK: 1 and ERBB2: 1). No findings suggested loss of TSC gene function in the two patients analysed by NGS. CONCLUSION: Our study revealed that patients diagnosed with LAM had a significantly increased risk of lung cancer. Further research is warranted to clarify the carcinogenesis of lung cancer in patients with LAM.

Tsc1 regulates tight junction independent of mTORC1.

Tuberous sclerosis complex 1 (Tsc1) is a tumor suppressor that functions together with Tsc2 to negatively regulate the mechanistic target of rapamycin complex 1 (mTORC1) activity. Here, we show that Tsc1 has a critical role in the tight junction (TJ) formation of epithelium, independent of its role in Tsc2 and mTORC1 regulation. When an epithelial cell establishes contact with neighboring cells, Tsc1, but not Tsc2, migrates from the cytoplasm to junctional membranes, in which it binds myosin 6 to anchor the perijunctional actin cytoskeleton to ß-catenin and ZO-1. In its absence, perijunctional actin cytoskeleton fails to form. In mice, intestine-specific or inducible, whole-body Tsc1 ablation disrupts adherens junction/TJ structures in intestine or skin epithelia, respectively, causing Crohn's disease-like symptoms in the intestine or psoriasis-like phenotypes on the skin. In patients with Crohn's disease or psoriasis, junctional Tsc1 levels in epithelial tissues are markedly reduced, concomitant with the TJ structure impairment, suggesting that Tsc1 deficiency may underlie TJ-related diseases. These findings establish an essential role of Tsc1 in the formation of cell junctions and underpin its association with TJ-related human diseases.

Specific Features of Focal Cortical Dysplasia in Tuberous Sclerosis Complex.

Patients with tuberous sclerosis complex present with cognitive, behavioral, and psychiatric impairments, such as intellectual disabilities, autism spectrum disorders, and drug-resistant epilepsy. It has been shown that these disorders are associated with the presence of cortical tubers. Tuberous sclerosis complex results from inactivating mutations in the TSC1 or TSC2 genes, resulting in hyperactivation of the mTOR signaling pathway, which regulates cell growth, proliferation, survival, and autophagy. TSC1 and TSC2 are classified as tumor suppressor genes and function according to Knudson's two-hit hypothesis, which requires both alleles to be damaged for tumor formation. However, a second-hit mutation is a rare event in cortical tubers. This suggests that the molecular mechanism of cortical tuber formation may be more complicated and requires further research. This review highlights the issues of molecular genetics and genotype-phenotype correlations, considers histopathological characteristics and the mechanism of morphogenesis of cortical tubers, and also presents data on the relationship between these formations and the development of neurological manifestations, as well as treatment options.

Differential impact of PI3K/AKT/mTOR signaling on tumor initiation and progression in animal models of prostate cancer.

BACKGROUND: The PI3K/AKT/mTOR signaling pathway is essential for initiation and progression of prostate cancer. However, there has been no a comprehensive comparison for the role of these signaling nodes in prostate tumor initiation and progression. METHODS: With genetically engineered animal models, we compared the impact of prostate-specific deletions of Pten, Tsc1, and Tsc2 and activation of Akt1 on tumor initiation and progression. Also, we assessed the expression and genetic alterations of PTEN, AKT1, TSC1, and TSC2 in human primary prostate cancers. RESULTS: For the genetically engineered mice, prostate conditional knockout (cKO) of Pten, Tsc1, and Tsc2 led to initiation and progression of mouse prostatic neoplasia hyperplasia (mPIN). Akt1 transgenic mice developed more aggressive mPINs than mice with Tsc1 or Tsc2 single-cKO, but the effect was more moderate than that for Pten single-cKO or Tsc1/Tsc2 double-cKO mice. Functional analyses showed that Pten single-cKO, AKT1 activation, and Tsc1/Tsc2 double-cKO induced cell proliferation more than Tsc1 or Tsc2 single-cKO, but only Pten single-cKO and AKT1 activation reduced epithelial adhesion. All cKO or AKT1 activation enhanced the phosphorylation of p-S6 (S235/236) but only Pten single-cKO and Tsc1/Tsc2 double-cKO enhanced the phosphorylation of p-AKT (S473) and p-4EBP1 (T37/46/70). In human prostate cancers, PTEN, but not AKT1, TSC1, or TSC2 had frequent genetic alterations. However, as key signaling nodes, AKT1, TSC1, and TSC2 may be responsible for PTEN loss-mediated tumor initiation and progression. CONCLUSION: Our results for genetically engineered mouse models suggest a differential role of the PI3K/AKT/mTOR signaling nodes in prostate cancer initiation and progression, but the underlying molecular mechanisms remain unaddressed.

Tuberous sclerosis complex is associated with a novel human tauopathy.

Tuberous sclerosis complex (TSC) is a neurogenetic disorder leading to epilepsy, developmental delay, and neurobehavioral dysfunction. The syndrome is caused by pathogenic variants in TSC1 (coding for hamartin) or TSC2 (coding for tuberin). Recently, we reported a progressive frontotemporal dementia-like clinical syndrome in a patient with a mutation in TSC1, but the neuropathological changes seen in adults with TSC with or without dementia have yet to be systematically explored. Here, we examined neuropathological findings in adults with TSC (n = 11) aged 30-58 years and compared them to age-matched patients with epilepsy unrelated to TSC (n = 9) and non-neurological controls (n = 10). In 3 of 11 subjects with TSC, we observed a neurofibrillary tangle-predominant "TSC tauopathy" not seen in epilepsy or non-neurological controls. This tauopathy was observed in the absence of pathological amyloid beta, TDP-43, or alpha-synuclein deposition. The neurofibrillary tangles in TSC tauopathy showed a unique pattern of post-translational modifications, with apparent differences between TSC1 and TSC2 mutation carriers. Tau acetylation (K274, K343) was prominent in both TSC1 and TSC2, whereas tau phosphorylation at a common phospho-epitope (S202) was observed only in TSC2. TSC tauopathy was observed in selected neocortical, limbic, subcortical, and brainstem sites and showed a 3-repeat greater than 4-repeat tau isoform pattern in both TSC1 and TSC2 mutation carriers, but no tangles were immunolabeled with MC1 or p62 antibodies. The findings suggest that individuals with TSC are at risk for a unique tauopathy in mid-life and that tauopathy pathogenesis may involve TSC1, TSC2, and related molecular pathways.

Low-grade oncocytic tumour of the kidney is characterised by genetic alterations of TSC1, TSC2, MTOR or PIK3CA and consistent GATA3 positivity.

Low-grade oncocytic tumour (LOT) of the kidney has recently emerged as a potential novel tumour type. Despite similarity to oncocytoma or eosinophilic chromophobe renal cell carcinoma, it shows diffuse keratin 7 immunohistochemistry (IHC) and negative KIT (CD117), which differs from both. We aimed to identify the molecular characteristics of these tumours. Seventeen tumours (one male, 16 female, nine previously published) fitting the original description of this entity (solid eosinophilic cell morphology, often with areas of tumour cells loosely stretched in oedematous stroma, and the above IHC features) were analysed with a next-generation sequencing panel of 324 cancer-associated genes from formalin-fixed, paraffin-embedded tissue. All tumours harboured at least one alteration in either TSC1 (n = 7, 41%), TSC2 (n = 2, 12%), MTOR (n = 5, 29%) or PIK3CA (n = 4, 24%). Four tumours harboured a second alteration, including two NF2, one each in conjunction with MTOR and TSC2 alterations, one PTEN with TSC1 alteration and one tumour with both MTOR and TSC1 alterations. No other renal cancer-related or recurring gene alterations were identified. In addition to the previously described IHC findings, 16 of 16 were positive for GATA3. Eleven patients with follow-up had no metastases or recurrent tumours. Recurrent tuberous sclerosis/MTOR pathway gene alterations in LOT support its consideration as a distinct morphological, immunohistochemical and genetic entity. PIK3CA is another pathway member that may be altered in these tumours. Further study will be necessary to determine whether tumour behaviour or syndromic associations differ from those of oncocytoma and chromophobe carcinoma, warranting different clinical consideration.

miR-222-3p-containing macrophage-derived extracellular vesicles confer gemcitabine resistance via TSC1-mediated mTOR/AKT/PI3K pathway in pancreatic cancer.

Gemcitabine resistance limits the efficacy of chemotherapy and maintains a challenge for treatment outcomes. Therefore, we aimed to clarify the downstream mechanisms underlying the role of miR-222-3p delivered by M2 macrophage-derived extracellular vesicles (M2 MDEs) in the chemoresistance of pancreatic cancer (PCa). We separated the mouse macrophages and polarized them to M2 phenotypes, from which the EVs were derived. miR-222-3p was highly expressed in M2 MDEs. M2 MDEs were internalized by PCa cells. miR-222-3p overexpressing M2 MDEs were treated with gemcitabine and co-cultured with PCa cells for in vitro experiments. Co-culture with M2 MDEs enriched with miR-222-3p suppressed the sensitivity to gemcitabine, accompanied by diminished apoptosis and promoted proliferation. Furthermore, the M2 MDEs and PCa cells were injected to mice with gemcitabine exposure for in vivo substantiation. The delivery of miR-222-3p inhibitor by M2 MDEs suppressed tumor growth and elevated sensitivity of cancer cells to gemcitabine. Moreover, miR-222-3p was indicated to target and suppress TSC1 expression, while miR-222-3p activated the PI3K/AKT/mTOR pathway. Together, miR-222-3p-containing M2 MDEs enhance chemoresistance in PCa through TSC1 inhibition and activation of the PI3K/AKT/mTOR pathway.

Genetic context of oncogenic drivers dictates vascular sarcoma development in aP2-Cre mice.

Angiosarcomas are aggressive vascular sarcomas that arise from endothelial cells and have an extremely poor prognosis. Because of the rarity of angiosarcomas, knowledge of molecular drivers and optimized treatment strategies is lacking, highlighting the need for in vivo models to study the disease. Previously, we generated genetically engineered mouse models of angiosarcoma driven by aP2-Cre-mediated biallelic loss of Dicer1 or conditional activation of KrasG12D with Cdkn2a loss that histologically and genetically resemble human tumors. In the present study, we found that DICER1 functions as a potent tumor suppressor and its deletion, in combination with either KRASG12D expression or Cdkn2a loss, is associated with angiosarcoma development. Independent of the genetic driver, the mTOR pathway was activated in all murine angiosarcoma models. Direct activation of the mTOR pathway by conditional deletion of Tsc1 with aP2-Cre resulted in tumors that resemble intermediate grade human kaposiform hemangioendotheliomas, indicating that mTOR activation was not sufficient to drive the malignant angiosarcoma phenotype. Genetic dissection of the spectrum of vascular tumors identified genes specifically regulated in the aggressive murine angiosarcomas that are also enriched in human angiosarcoma. The genetic dissection driving the transition across the malignant spectrum of endothelial sarcomas provides an opportunity to identify key determinants of the malignant phenotype, novel therapies for angiosarcoma, and novel in vivo models to further explore angiosarcoma pathogenesis. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

GPNMB expression identifies TSC1/2/mTOR-associated and MiT family translocation-driven renal neoplasms.

GPNMB (glycoprotein nonmetastatic B) and other TFE3/TFEB transcriptional targets have been proposed as markers for microphthalmia (MiT) translocation renal cell carcinomas (tRCCs). We recently demonstrated that constitutive mTORC1 activation via TSC1/2 loss leads to increased activity of TFE3/TFEB, suggesting that the pathogenesis and molecular markers for tRCCs and TSC1/2-associated tumors may be overlapping. We examined GPNMB expression in human kidney and angiomyolipoma (AML) cell lines with TSC2 and/or TFE3/TFEB loss produced using CRISPR-Cas9 genome editing as well as in a mouse model of Tsc2 inactivation-driven renal tumorigenesis. Using an automated immunohistochemistry (IHC) assay for GPNMB, digital image analysis was employed to quantitatively score expression in clear cell RCC (ccRCC, n = 87), papillary RCC (papRCC, n = 53), chromophobe RCC (chRCC, n = 34), oncocytoma (n = 4), TFE3- or TFEB-driven tRCC (n = 56), eosinophilic solid and cystic RCC (ESC, n = 6), eosinophilic vacuolated tumor (EVT, n = 4), and low-grade oncocytic tumor (LOT, n = 3), as well as AML (n = 29) and perivascular epithelioid cell tumors (PEComas, n = 8). In cell lines, GPNMB was upregulated following TSC2 loss in a MiT/TFE- and mTORC1-dependent fashion. Renal tumors in Tsc2+/- A/J mice showed upregulation of GPNMB compared with normal kidney. Mean GPNMB expression was significantly higher in tRCC than in ccRCC (p < 0.0001), papRCC (p < 0.0001), and chRCC (p < 0.0001). GPNMB expression in TSC1/2/MTOR alteration-associated renal tumors (including ESC, LOT, AML, and PEComa) was comparable to that in tRCC. The immunophenotype of tRCC and TSC1/2/MTOR alteration-associated renal tumors is highly overlapping, likely due to the increased activity of TFE3/TFEB in both, revealing an important caveat regarding the use of TFE3/TFEB-transcriptional targets as diagnostic markers. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Clinical, pathological, and molecular correlation of folliculocystic and collagen hamartoma: A new potential diagnostic criterion for tuberous sclerosis complex?

Folliculocystic and collagen hamartoma (FCCH) is a rare entity with only 18 reported cases worldwide. Of them, most are found in patients diagnosed with tuberous sclerosis complex (TSC). FCCH has distinctive histopathologic features, including collagen deposition in the dermis, perifollicular fibrosis, and comedones with keratin-containing cysts lined by infundibular epithelium. We report three patients with a definitive TSC clinical diagnosis in whom clinical, histopathologic, and molecular features were studied to establish if there exists a genotype-phenotype correlation. The molecular results showed different heterozygous pathogenic variants (PV) in TSC2 in each patient: NM_000548.4:c.5024C>T, NG_005895.1:c.1599+1G>T, and NM_000548.4:c.2297_2298dup, to our knowledge; the latter PV has not been reported in public databases. The same PVs were identified as heterozygous in the tumor tissue samples, none of which yielded evidence of a TSC2 second hit. Because all FCCH patients with available molecular diagnosis carry a pathogenic genotype in TSC1 or TSC2, we suggest that FCCH should be considered as a new and uncommon diagnostic manifestation in the TSC consensus international diagnostic criteria. The early recognition of FCCH by clinicians could prompt the identification of new TSC cases. Interestingly, our molecular findings suggest that one of the patients described herein is a probable case of somatic mosaicism.

Association Mapping of Resistance to Tan Spot in the Global Durum Panel.

Tan spot, caused by the necrotrophic fungal pathogen Pyrenophora tritici-repentis (Ptr), is an important disease of durum and common wheat worldwide. Compared with common wheat, less is known about the genetics and molecular basis of tan spot resistance in durum wheat. We evaluated 510 durum lines from the Global Durum Wheat Panel (GDP) for sensitivity to the necrotrophic effectors (NEs) Ptr ToxA and Ptr ToxB and for reaction to Ptr isolates representing races 1 to 5. Overall, susceptible durum lines were most prevalent in South Asia, the Middle East, and North Africa. Genome-wide association analysis showed that the resistance locus Tsr7 was significantly associated with tan spot caused by races 2 and 3, but not races 1, 4, or 5. The NE sensitivity genes Tsc1 and Tsc2 were associated with susceptibility to Ptr ToxC- and Ptr ToxB-producing isolates, respectively, but Tsn1 was not associated with tan spot caused by Ptr ToxA-producing isolates, which further validates that the Tsn1-Ptr ToxA interaction does not play a significant role in tan spot development in durum. A unique locus on chromosome arm 2AS was associated with tan spot caused by race 4, a race once considered avirulent. A novel trait characterized by expanding chlorosis leading to increased disease severity caused by the Ptr ToxB-producing race 5 isolate DW5 was identified, and this trait was governed by a locus on chromosome 5B. We recommend that durum breeders select resistance alleles at the Tsr7, Tsc1, Tsc2, and the chromosome 2AS loci to obtain broad resistance to tan spot.

The non-canonical nuclear functions of key players of the PI3K-AKT-MTOR pathway.

The PI3K-AKT-MTOR signal transduction pathway is one of the essential signalling cascades within the cell due to its involvement in many vital functions. The pathway initiates with the recruitment of phosphatidylinositol-3 kinases (PI3Ks) onto the plasma membrane, generating phosphatidylinositol-3,4,5-triphosphate [PtdIns(3,4,5)P3 ] and subsequently activating AKT. Being the central node of the PI3K network, AKT activates the mechanistic target of rapamycin kinase complex 1 (MTORC1) via Tuberous sclerosis complex 2 inhibition in the cytoplasm. Although the cytoplasmic role of the pathway has been widely explored for decades, we now know that most of the effector molecules of the PI3K axis diverge from the canonical route and translocate to other cell organelles including the nucleus. The presence of phosphoinositides (PtdIns) inside the nucleus itself indicates the existence of a nuclear PI3K signalling. The nuclear localization of these signaling components is evident in regulating many nuclear processes like DNA replication, transcription, DNA repair, maintenance of genomic integrity, chromatin architecture, and cell cycle control. Here, our review intends to present a comprehensive overview of the nuclear functions of the PI3K-AKT-MTOR signaling biomolecules.

Genetic analysis of 18 families with tuberous sclerosis complex.

Tuberous sclerosis complex (TSC) is mainly caused by variants in TSC1 and TSC2, which encodes hamartin protein and tuberin protein, respectively. Here, we report clinical and molecular characteristics of 18 families with TSC. High-throughput DNA sequencing was employed to detect variants in all the exons and flanking region of TSC1 and TSC2. TA clone and real-time PCR were performed to verify the pathogenicity of candidate variants. A total of 17 mutations were identified, including 13 mutations in TSC2 and 4 mutations in TSC1. Fifty-six percent (10/18) of the families carried de novo mutations, and 8 of these mutations were not reported previously. Most mutations detected were loss-of-function mutations (15/17). One splice-site mutation (TSC2 c.599 + 5G > A) caused abnormal splicing and was confirmed by in vitro analysis. Facial angiofibromas (94%) and epilepsy (89%) were the most prevalent clinical features in our patients. Treatment with anti-seizure medication (ASM) or in combination with rapamycin results in clinical remission in most patients with TSC-associated seizures (14/15). For genotype-phenotype correlation, patients in our cohort with TSC2 mutations had an earlier onset age and patients with TSC1 showed better response to ASM. Our study has expanded the spectrum of TSC1 and TSC2 causing TSC.

Hepatocytic p62 suppresses ductular reaction and tumorigenesis in mouse livers with mTORC1 activation and defective autophagy.

BACKGROUND & AIMS: Either activation of mTORC1 due to loss of Tsc1 (tuberous sclerosis complex 1) or defective hepatic autophagy due to loss of Atg5 leads to spontaneous liver tumorigenesis in mice. The purpose of this study was to investigate the mechanisms by which autophagy contributes to the hepatic metabolic changes and tumorigenesis mediated by mTORC1 activation. METHODS: Atg5 Flox/Flox (Atg5F/F) and Tsc1F/F mice were crossed with albumin-Cre mice to generate liver-specific Atg5 knockout (L-Atg5 KO), L-Tsc1 KO and L-Atg5/Tsc1 double KO (DKO) mice. These mice were crossed with p62/Sqstm1F/F (p62) and whole body Nrf2 KO mice to generate L-Atg5/Tsc1/p62 and L-Atg5/Tsc1-Nrf2 triple KO mice. These mice were housed for various periods up to 12 months, and blood and liver tissues were harvested for biochemical and histological analysis RESULTS: Deletion of Atg5 in L-Tsc1 KO mice inhibited liver tumorigenesis but increased mortality and was accompanied by drastically enhanced hepatic ductular reaction (DR), hepatocyte degeneration and metabolic reprogramming. Deletion of p62 reversed DR, hepatocyte degeneration and metabolic reprogramming as well as the mortality of L-Atg5/Tsc1 DKO mice, but unexpectedly promoted liver tumorigenesis via activation of a group of oncogenic signaling pathways. Nrf2 ablation markedly improved DR with increased hepatocyte population and improved metabolic reprogramming and survival of the L-Atg5/Tsc1 DKO mice without tumor formation. Decreased p62 and increased mTOR activity were also observed in a subset of human hepatocellular carcinomas. CONCLUSIONS: These results reveal previously undescribed functions of hepatic p62 in suppressing tumorigenesis and regulating liver cell repopulation and metabolic reprogramming resulting from persistent mTORC1 activation and defective autophagy. LAY SUMMARY: Metabolic liver disease and viral hepatitis are common chronic liver diseases and risk factors of hepatocellular carcinoma, which are often associated with impaired hepatic autophagy and increased mTOR activation. Using multiple genetically engineered mouse models of defective hepatic autophagy and persistent mTOR activation, we dissected the complex mechanisms behind this observation. Our results uncovered an unexpected novel tumor suppressor function of p62/Sqstm1, which regulated liver cell repopulation, ductular reaction and metabolic reprogramming in liver tumorigenesis.

Tuberous sclerosis complex is required for tumor maintenance in MYC-driven Burkitt's lymphoma.

The tuberous sclerosis complex (TSC) 1/2 is a negative regulator of the nutrient-sensing kinase mechanistic target of rapamycin complex (mTORC1), and its function is generally associated with tumor suppression. Nevertheless, biallelic loss of function of TSC1 or TSC2 is rarely found in malignant tumors. Here, we show that TSC1/2 is highly expressed in Burkitt's lymphoma cell lines and patient samples of human Burkitt's lymphoma, a prototypical MYC-driven cancer. Mechanistically, we show that MYC induces TSC1 expression by transcriptional activation of the TSC1 promoter and repression of miR-15a. TSC1 knockdown results in elevated mTORC1-dependent mitochondrial respiration enhanced ROS production and apoptosis. Moreover, TSC1 deficiency attenuates tumor growth in a xenograft mouse model. Our study reveals a novel role for TSC1 in securing homeostasis between MYC and mTORC1 that is required for cell survival and tumor maintenance in Burkitt's lymphoma. The study identifies TSC1/2 inhibition and/or mTORC1 hyperactivation as a novel therapeutic strategy for MYC-driven cancers.