A distinctive genomic and immunohistochemical profile for NOTCH3 and PDGFRB in infantile myofibroma with diagnostic and therapeutic implications

Myofibromas are rare tumors of pericytic lineage, typically affecting children, and are sometimes aggressive. A subset of sporadic and familial myofibromas have activating variants in PDGFRB. The relationship of myofibroma and PDGFRB to the NOTCH pathway has not yet been described. Methods. Ten myofibroma cases were sequenced with a targeted panel of 447 genes, including copy number variation and selected fusions. Immunohistochemical analysis of total NOTCH3 and activated NOTCH3 was assessed for all 10 myofibroma cases, and a series of histologic mimics (n = 20). Results. Alterations identified by next-generation sequencing included PDGFRB sequence variants in 8/10 cases (80%), a NOTCH3 variant in 1/10 cases (10%), and a NOTCH2 variant in 1/10 cases (10%). All 10 cases also showed a pattern of low-amplitude (1.5- to 2-fold) copy number alterations including gains in PDGFRB and NOTCH3. Ten of 10 myofibromas (100%) showed cytoplasmic staining for total NOTCH3 and 9 of 10 cases (90%) showed nuclear staining for activated NOTCH3. Within the control cohort of histologic mimics, 3 of 3 nodular fasciitis cases (100%) were positive for activated and total NOTCH3, and the remaining 17 cases were negative for pan NOTCH3, while 3 of 3 desmoid-type fibromatosis cases (100%) showed patchy weak nuclear staining for activated NOTCH3. Discussion. Our findings suggest a common pathway of PDGFRB/NOTCH3 activation in myofibromas, even in cases that lack PDGFRB sequence variants. These results support the pericytic lineage of myofibroma. Identification of the characteristic genomic alterations or immunohistochemical staining pattern may facilitate a difficult pathologic diagnosis, and support the use of targeted treatments

Insights into autoregulation of Notch 3 from Structural and Functional Studies of its negative Regulatory Region

Abstract: Notch receptors are transmembrane proteins that undergo activating proteolysis in response to stimulation by Delta- and Serrate-family ligands. A negative regulatory region (NRR), which encompasses three Lin12-Notch repeats (LNRs) and a juxtamembrane heterodimerization domain that houses the ligand-dependent processing site, normally maintains the receptor in a resting state by preventing protease cleavage prior to ligand binding. We report here the X-ray structure of the NRR of human Notch3 in its autoinhibited state, and compare it with the autoinhibited structures of the analogous regions of human Notch1 and Notch2. The overall architecture of the autoinhibited conformation, in which the three LNR modules wrap around the heterodimerization domain, is preserved in the Notch3 NRR. The autoinhibited conformation of the Notch3 NRR is less stable than that of Notch1 or Notch2, and Notch3 exhibits more basal activity that either Notch1 or Notch2 in reporter assays. Disease-associated mutations L1515P and L1519P of the heterodimerization domain lead to increased ligand-independent activation of the receptor. The Notch3 NRR uses a highly conserved surface on the third LNR module to form a dimer in the asymmetric unit of the crystal, and a similar homotypic interface exists in the previously reported Notch1 and Notch2 structures. Together, these studies reveal distinguishing structural features associated with increased basal activity of Notch3, demonstrate increased ligand-independent signaling for disease-associated mutations that map to the Notch3 NRR, and identify a conserved dimerization interface present in multiple Notch receptors

Insights into Autoregulation of Notch3 from Structural and Functional Studies of Its Negative Regulatory Region

SummaryNotch receptors are transmembrane proteins that undergo activating proteolysis in response to ligand stimulation. A negative regulatory region (NRR) maintains receptor quiescence by preventing protease cleavage prior to ligand binding. We report here the X-ray structure of the NRR of autoinhibited human Notch3, and compare it with the Notch1 and Notch2 NRRs. The overall architecture of the autoinhibited conformation, in which three LIN12-Notch repeat (LNR) modules wrap around a heterodimerization domain, is preserved in Notch3, but the autoinhibited conformation of the Notch3 NRR is less stable. The Notch3 NRR uses a highly conserved surface on the third LNR module to form a dimer in the crystal. Similar homotypic interfaces exist in Notch1 and Notch2. Together, these studies reveal distinguishing structural features associated with increased basal activity of Notch3, demonstrate increased ligand-independent signaling for disease-associated mutations that map to the Notch3 NRR, and identify a conserved dimerization interface present in multiple Notch receptors

Blockade of metallothioneins 1 and 2 increases skeletal muscle mass and strength

Abstract Metallothioneins are proteins that are involved in intracellular zinc storage and transport. Their expression levels have been reported to be elevated in several settings of skeletal muscle atrophy. Here, we demonstrate that these genes are also induced coincident with aging. We therefore investigated the effect of metallothionein blockade on skeletal muscle anabolism in vitro and in vivo, and found that concomitant abrogation of metallothionein 1 and 2 activates the Akt pathway, promotes myotube growth, specifically drives type IIb fiber hypertrophy and ultimately increases muscle strength. Silencing of metallothioneins results in elevated cytosolic zinc; increasing intracellular zinc levels are sufficient to mimic the effects of metallothionein blockade on myotube hypertrophy. We thereby provide direct evidence of the mechanisms by which metallothioneins can modulate skeletal muscle mass. Importantly, the beneficial effects of metallothionein blockade on muscle mass and function are preserved in the presence of a strong catabolic stimulus: treatment with glucocorticoids. Taken together, our results suggest that blockade of metallothioneins constitutes a promising approach for the treatment of muscle diseases

Notch2 Is Required for Inflammatory Cytokine-Driven Goblet Cell Metaplasia in the Lung

The balance and distribution of epithelial cell types is required to maintain tissue homeostasis. A hallmark of airway diseases is epithelial remodeling, leading to increased goblet cell numbers and an overproduction of mucus. In the conducting airway, basal cells act as progenitors for both secretory and ciliated cells. To identify mechanisms regulating basal cell fate, we developed a screenable 3D culture system of airway epithelial morphogenesis. We performed a high-throughput screen using a collection of secreted proteins and identified inflammatory cytokines that specifically biased basal cell differentiation toward a goblet cell fate, culminating in enhanced mucus production. We also demonstrate a specific requirement for Notch2 in cytokine-induced goblet cell metaplasia in vitro and in vivo. We conclude that inhibition of Notch2 prevents goblet cell metaplasia induced by a broad range of stimuli and propose Notch2 neutralization as a therapeutic strategy for preventing goblet cell metaplasia in airway diseases

Characterization of activating mutations of NOTCH3 in T cell acute lymphoblastic leukemia and anti-leukemic activity of NOTCH3 inhibitory antibodies

Notch receptors have been implicated as oncogenic drivers in several cancers, the most notable example being NOTCH1 in T-cell acute lymphoblastic leukemia (T-ALL). To characterize the role of activated NOTCH3 in cancer, we generated an antibody that detects the neo-epitope created upon gamma-secretase cleavage of NOTCH3 to release its intracellular domain (ICD3), and sequenced the negative regulatory region (NRR) and PEST domain coding regions of NOTCH3 in a panel of cell lines. We also characterize NOTCH3 tumor-associated mutations that result in activation of signaling and report new inhibitory antibodies. We determined the structural basis for receptor inhibition by obtaining the first co-crystal structure of a NOTCH3 antibody with the NRR protein and defined two distinct epitopes for NRR antibodies. The antibodies exhibit potent anti-leukemic activity in cell lines and tumor xenografts harboring NOTCH3 activating mutations. Screening of primary T-ALL samples reveals that two of 40 tumors examined show active NOTCH3 signaling. We also identified evidence of NOTCH3 activation in 12 of 24 patient-derived orthotopic xenograft models, two of which exhibit activation of NOTCH3 without activation of NOTCH1. Our studies provide additional insights into NOTCH3 activation and offer a path forward for identification of cancers that are likely to respond to therapy with NOTCH3 selective inhibitory antibodies

The cellular ratio of immune tolerance (immunoCRIT) is a definite marker for aggressiveness of solid tumors and may explain tumor dissemination patterns.

The adaptive immune system is involved in tumor establishment and aggressiveness. Tumors of the ovaries, an immune-privileged organ, spread via transceolomic routes and rarely to distant organs. This is contrary to tumors of non-immune privileged organs, which often disseminate hematogenously to distant organs. Epigenetics-based immune cell quantification allows direct comparison of the immune status in benign and malignant tissues and in blood. Here, we introduce the "cellular ratio of immune tolerance" (immunoCRIT) as defined by the ratio of regulatory T cells to total T lymphocytes. The immunoCRIT was analyzed on 273 benign tissue samples of colorectal, bronchial, renal and ovarian origin as well as in 808 samples from primary colorectal, bronchial, mammary and ovarian cancers. ImmunoCRIT is strongly increased in all cancerous tissues and gradually augmented strictly dependent on tumor aggressiveness. In peripheral blood of ovarian cancer patients, immunoCRIT incrementally increases from primary diagnosis to disease recurrence, at which distant metastases frequently occur. We postulate that non-pathological immunoCRIT values observed in peripheral blood of immune privileged ovarian tumor patients are sufficient to prevent hematogenous spread at primary diagnosis. Contrarily, non-immune privileged tumors establish high immunoCRIT in an immunological environment equivalent to the bloodstream and thus spread hematogenously to distant organs. In summary, our data suggest that the immunoCRIT is a powerful marker for tumor aggressiveness and disease dissemination