Multiomic characterization of oncogenic signaling mediated by wild-type and mutant RIT1

Lo, April; Holmes, Kristin D.; Kamlapurkar, Shriya; Mundt, Filip; Moorthi, Sitapriya; Fung, Iris; Fereshetian, Shaunt; Watson, Julia; Carr, Steven A.; Mertins, Philipp; Berger, Alice H.

doi:10.1126/scisignal.abc4520

Cited by 9 publications

(12 citation statements)

References 51 publications

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“…The process of mRNA splicing is known to involve regulation of phosphorylation states (Cho et al 2011; Aubol et al 2016). Previously, we generated human AALE lung epithelial cells stably expressing KRAS WT , KRAS G12V , KRAS Q61H , RIT1 WT , and RIT1 M90I , and performed liquid chromatography and tandem mass spectrometry (LC-MS/MS) to profile their proteomes and phosphoproteomes (Lo et al 2021). As expected, phosphorylation of Ras pathway proteins was significantly altered in cells expressing KRAS or RIT1 variants (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Proteomic and phosphoproteomic data from AALE cells was generated by LC-MS/MS and quantified as previously described (Lo et al 2021). Briefly, isogenic AALE cells were generated by transduction with lentiviral vectors encoding wild-type KRAS, KRAS G12V , KRAS Q61H , or wild-type RIT1 or RIT1 M90I .…”

Section: Methodsmentioning

confidence: 99%

“…RNA-seq libraries of KRAS-and RIT1-mutant AALE cells were previously described (Lo et al 2021). Briefly, three replicates per cell line were used for RNA isolation by Direct-zol RNA mini-prep (Zymo) and libraries generated using the TruSeq RNA Library kit (Illumina).…”

Section: Methodsmentioning

confidence: 99%

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Oncogenic KRAS alters splicing factor phosphorylation and alternative splicing in lung cancer

McSharry

Berger

2022

Preprint

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BackgroundAlternative RNA splicing is widely dysregulated in cancers including lung adenocarcinoma, where aberrant splicing events are frequently caused by somatic splice site mutations or somatic mutations of splicing factor genes. However, the majority of mis-splicing in cancers is unexplained by these known mechanisms. We hypothesize that the aberrant Ras signaling characteristic of lung cancers plays a role in promoting the alternative splicing observed in tumors.MethodsWe recently performed transcriptome and proteome profiling of human lung epithelial cells ectopically expressing oncogenic KRAS and another cancer-associated Ras GTPase, RIT1. Unbiased analysis of phosphoproteome data identified altered splicing factor phosphorylation in KRAS-mutant cells, so we performed differential alternative splicing analysis using rMATS to identify significantly altered isoforms in lung epithelial. To determine whether these isoforms were uniquely regulated by KRAS, we performed a large-scale splicing screen in which we generated over 300 unique RNA sequencing profiles of isogenic A549 lung adenocarcinoma cells ectopically expressing 75 different wild-type or variant alleles across 28 genes implicated in lung cancer.ResultsMass spectrometry data showed widespread downregulation of splicing factor phosphorylation in lung epithelial cells expressing mutant KRAS compared to cells expressing wild-type KRAS. We observed alternative splicing in the same cells, with 2196 and 2416 skipped exon events in KRASG12V and KRASQ61H cells, respectively, 997 of which were shared (p < 0.001 by hypergeometric test). In the high-throughput splicing screen, mutant KRAS induced the greatest number of differential alternative splicing events, second only to the RNA binding protein RBM45 and its mutant allele RBM45M126I. We identified ten high confidence cassette exon events across multiple KRAS variants and cell lines. These included differential splicing of the Myc Associated Zinc Finger (MAZ). As MAZ regulates expression of KRAS, this splice variant may be a mechanism for the cell to modulate wild-type KRAS levels in the presence of oncogenic KRAS.ConclusionProteomic and transcriptomic profiling of lung epithelial cells uncovered splicing factor phosphorylation and mRNA splicing events regulated by oncogenic KRAS. These data suggest that in addition to widespread transcriptional changes, Ras signaling pathways in cancer promote post-transcriptional splicing changes that may contribute to oncogenic processes.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Oncogenic KRAS alters splicing factor phosphorylation and alternative splicing in lung cancer

McSharry

Berger

2022

Preprint

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“…Both tumor-suppressive and tumor-promoting properties have been reported for the wild-type RAS proteins in the KRAS -mutant context 4–8 . The increased dosage of wild-type RAS-like-without-CAAX-1 (RIT1) partially phenocopies KRAS in the lung cancer model 9 , even though its activity depends on the classical RAS proteins 10 . Mutant KRAS also leads to an increased expression of MRAS, which is part of a phosphatase complex that cooperates with RAS proteins for efficient MAPK pathway activation 11 .…”

Section: Introductionmentioning

confidence: 99%

Increased dosage of wild-type KRAS protein drivesKRAS-mutant lung tumorigenesis and drug resistance

Ivanisevic,

Magits,

et al. 2024

Preprint

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Almost 30% of lung adenocarcinomas are driven by activating KRAS mutations. The heterogeneous clinical behavior observed in these cancers could be due to the imbalance of wild-type and oncogenic KRAS alleles. However, the role of RAS dysregulation at the protein level needs to be further explored. A genome-wide global protein stability screen identified the CUL3 ubiquitin ligase adaptor LZTR1, as a major proteostatic regulator of wild-type but not mutant KRAS. In KRAS-mutant lung adenocarcinoma, shallow deletion of LZTR1 is observed in up to 50% of patients and is associated with hypoxic signatures and poorer progression-free disease survival. In a Kras-mutant lung cancer mouse model, heterozygous loss of Lztr1 promoted tumor growth, led to peritumoral vascular remodeling, and limited the response to the KRAS-G12D inhibitor MRTX1133. The vascular alteration in LZTR1-depleted lung cancer was mediated by increased wild-type KRAS protein dosage, which promoted mTOR pathway activation and a subsequent increase in VEGFA secretion. The inhibition of the PI3K/mTOR pathway using dactolisib normalized tumor vasculature, improved drug delivery, and overcame resistance to KRAS-G12D inhibitors. In summary, the dysregulation of RAS proteostasis contributes to lung tumorigenesis, and targeting wild-type KRAS signaling is crucial to overcome intrinsic resistance to inhibitors of mutant KRAS.

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“…RIT1 is mutated in 2% of LUAD tumors and amplified in another 14% 4,5 . The biological effect of RIT1 amplifications in lung cancer is not well understood, but recent work suggests that RIT1 amplifications phenocopy mutant RIT1 6 . In addition to LUAD, RIT1 alterations have been identified in other cancers such as myeloid malignancies, uterine carcinosarcoma, and hepatocellular carcinoma [7][8][9] .…”

Section: Introductionmentioning

confidence: 99%

The deubiquitinase USP9X regulates RIT1 protein abundance and oncogenic phenotypes

Riley,

Grant,

Snell

et al. 2023

Preprint

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SUMMARYRIT1is a rare and understudied oncogene in lung cancer. Despite structural similarity to other RAS GTPase proteins such as KRAS, oncogenic RIT1 activity does not appear to be tightly regulated by nucleotide exchange or hydrolysis. Instead, there is a growing understanding that the protein abundance of RIT1 is important for its regulation and function. We previously identified the deubiquitinaseUSP9Xas a RIT1 dependency inRIT1-mutant cells. Here, we demonstrate that both wild-type and mutant forms of RIT1 are substrates of USP9X. Depletion of USP9X leads to decreased RIT1 protein stability and abundance and resensitizes cells to EGFR tyrosine kinase inhibitors. Our work expands upon the current understanding of RIT1 protein regulation and presents USP9X as a key regulator of RIT1-driven oncogenic phenotypes.

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Multiomic characterization of oncogenic signaling mediated by wild-type and mutant RIT1

Cited by 9 publications

References 51 publications

Oncogenic KRAS alters splicing factor phosphorylation and alternative splicing in lung cancer

Oncogenic KRAS alters splicing factor phosphorylation and alternative splicing in lung cancer

Increased dosage of wild-type KRAS protein drivesKRAS-mutant lung tumorigenesis and drug resistance

The deubiquitinase USP9X regulates RIT1 protein abundance and oncogenic phenotypes

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