Proteasome inhibitors
are used to treat blood cancers such as multiple
myeloma (MM) and mantle cell lymphoma. The efficacy of these drugs
is frequently undermined by acquired resistance. One mechanism of
proteasome inhibitor resistance may involve the transcription factor
Nuclear Factor, Erythroid 2 Like 1 (NFE2L1, also referred to as Nrf1),
which responds to proteasome insufficiency or pharmacological inhibition
by upregulating proteasome subunit gene expression. This “bounce-back”
response is achieved through a unique mechanism. Nrf1 is constitutively
translocated into the ER lumen, N-glycosylated, and then targeted
for proteasomal degradation via the ER-associated degradation (ERAD)
pathway. Proteasome inhibition leads to accumulation of cytosolic
Nrf1, which is then processed to form the active transcription factor.
Here we show that the cytosolic enzyme N-glycanase 1 (NGLY1, the human
PNGase) is essential for Nrf1 activation in response to proteasome
inhibition. Chemical or genetic disruption of NGLY1 activity results
in the accumulation of misprocessed Nrf1 that is largely excluded
from the nucleus. Under these conditions, Nrf1 is inactive in regulating
proteasome subunit gene expression in response to proteasome inhibition.
Through a small molecule screen, we identified a cell-active NGLY1
inhibitor that disrupts the processing and function of Nrf1. The compound
potentiates the cytotoxicity of carfilzomib, a clinically used proteasome
inhibitor, against MM and T cell-derived acute lymphoblastic leukemia
(T-ALL) cell lines. Thus, NGLY1 inhibition prevents Nrf1 activation
and represents a new therapeutic approach for cancers that depend
on proteasome homeostasis.
Alternative splicing is regulated by splicing factors that modulate splice site selection. In some cases, however, splicing factors show antagonistic activities by either activating or repressing splicing. Here, we show that these opposing outcomes are based on their binding location relative to regulated 5' splice sites. SR proteins enhance splicing only when they are recruited to the exon. However, they interfere with splicing by simply relocating them to the opposite intronic side of the splice site. hnRNP splicing factors display analogous opposing activities, but in a reversed position dependence. Activation by SR or hnRNP proteins increases splice site recognition at the earliest steps of exon definition, whereas splicing repression promotes the assembly of nonproductive complexes that arrest spliceosome assembly prior to splice site pairing. Thus, SR and hnRNP splicing factors exploit similar mechanisms to positively or negatively influence splice site selection.
Pancreatic ductal adenocarcinoma (PDA) develops through distinct precursor lesions, including pancreatic intraepithelial neoplasia (PanIN) and intraductal papillary mucinous neoplasia (IPMN). However, genetic features resulting in IPMN-associated PDA (IPMN–PDA) versus PanIN-associated PDA (PanIN-PDA) are largely unknown. Here we find that loss of Brg1, a core subunit of SWI/SNF chromatin remodelling complexes, cooperates with oncogenic Kras to form cystic neoplastic lesions that resemble human IPMN and progress to PDA. Although Brg1-null IPMN–PDA develops rapidly, it possesses a distinct transcriptional profile compared with PanIN-PDA driven by mutant Kras and hemizygous p53 deletion. IPMN–PDA also is less lethal, mirroring prognostic trends in PDA patients. In addition, Brg1 deletion inhibits Kras-dependent PanIN development from adult acinar cells, but promotes Kras-driven preneoplastic transformation in adult duct cells. Therefore, this study implicates Brg1 as a determinant of context-dependent Kras-driven pancreatic tumorigenesis and suggests that chromatin remodelling may underlie the development of distinct PDA subsets.
Background:The effects of silent mutations on pre-mRNA splicing are poorly understood. Results: Silent mutations can significantly influence exon inclusion and are under purifying selection. Conclusion: Splicing and coding pressures have co-evolved to maintain sufficient exon inclusion levels. Significance: Modified species alignment approaches can be used to identify silent mutations that may alter exon inclusion.
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