The deubiquitinase USP9X regulates RIT1 protein abundance and oncogenic phenotypes.

RIT1 is 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 deubiquitinase USP9X as a RIT1 dependency in RIT1-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 epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors in vitro and in vivo. Our work expands upon the current understanding of RIT1 protein regulation and presents USP9X as a key regulator of RIT1-driven oncogenic phenotypes.

The genomic landscape of lung cancer in never-smokers from the Women's Health Initiative.

Over 200,000 individuals are diagnosed with lung cancer in the U.S. every year, with a growing proportion of cases, especially lung adenocarcinoma, occurring in individuals who have never smoked. Women over the age of 50 comprise the largest affected demographic. To understand the genomic drivers of lung adenocarcinoma and therapeutic response in this population, we performed whole genome and/or whole exome sequencing on 73 matched lung tumor/normal pairs from post-menopausal women who participated in the Women's Health Initiative. Somatic copy number alterations showed little variation by smoking status, suggesting that aneuploidy may be a general characteristic of lung cancer regardless of smoke exposure. Similarly, clock-like and APOBEC mutation signatures were prevalent but did not differ in tumors from smokers and never-smokers. However, mutations in both EGFR and KRAS showed unique allelic differences determined by smoking status that are known to alter tumor response to targeted therapy. Mutations in the MYC-network member MGA were more prevalent in tumors from smokers. Fusion events in ALK, RET, and ROS1 were absent, likely due to age-related differences in fusion prevalence. Our work underscores the profound impact of smoking status, age, and sex on the tumor mutational landscape and identifies areas of unmet medical need.

A comprehensive measure of Golgi sphingolipid flux using NBD C6-ceramide: evaluation of sphingolipid inhibitors.

Measurements of sphingolipid metabolism are most accurately performed by LC-MS. However, this technique is expensive, not widely accessible, and without the use of specific probes, it does not provide insight into metabolic flux through the pathway. Employing the fluorescent ceramide analogue NBD-C6-ceramide as a tracer in intact cells, we developed a comprehensive HPLC-based method that simultaneously measures the main nodes of ceramide metabolism in the Golgi. Hence, by quantifying the conversion of NBD-C6-ceramide to NBD-C6-sphingomyelin, NBD-C6-hexosylceramides, and NBD-C6-ceramide-1-phosphate (NBD-C1P), the activities of Golgi resident enzymes sphingomyelin synthase 1, glucosylceramide synthase, and ceramide kinase (CERK) could be measured simultaneously. Importantly, the detection of NBD-C1P allowed us to quantify CERK activity in cells, a usually difficult task. By applying this method, we evaluated the specificity of commonly used sphingolipid inhibitors and discovered that 1-phenyl-2-decanoylamino-3-morpholino-1-propanol, which targets glucosylceramide synthase, and fenretinide (4HPR), an inhibitor for dihydroceramide desaturase, also suppress CERK activity. This study demonstrates the benefit of an expanded analysis of ceramide metabolism in the Golgi, and it provides a qualitative and easy-to-implement method.

The deubiquitinase USP9X regulates RIT1 protein abundance and oncogenic phenotypes.

RIT1 is 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 deubiquitinase USP9X as a RIT1 dependency in RIT1-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.

Somatic mutation but not aneuploidy differentiates lung cancer in never-smokers and smokers.

Lung cancer in never-smokers disproportionately affects older women. To understand the mutational landscape of this cohort, we performed detailed genome characterization of 73 lung adenocarcinomas from participants of the Women’s Health Initiative (WHI). We find enrichment of EGFR mutations in never-/light-smokers and KRAS mutations in heavy smokers as expected, but we also show that the specific variants of these genes differ by smoking status, with important therapeutic implications. Mutational signature analysis revealed signatures of clock, APOBEC, and DNA repair deficiency in never-/light-smokers; however, the mutational load of these signatures did not differ significantly from those found in smokers. Last, tumors from both smokers and never-/light-smokers shared copy number subtypes, with no significant differences in aneuploidy. Thus, the genomic landscape of lung cancer in never-/light-smokers and smokers is predominantly differentiated by somatic mutations and not copy number alterations.

All About That Ras: Novel Fusion Drives Ras Pathway Activation in Lung Cancer.

Lung cancers in never- and light-smokers often harbor targetable oncogenic mutations in Ras pathway genes. Here, a novel OCLN-RASGRF1 fusion is identified in an otherwise Ras wild-type lung tumor. Studying this and other RASGRF1 fusions, the authors show that these fusions lead to malignant phenotypes that can be reversed by MEK inhibition. See related article by Hunihan et al., p. 3091.

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

Aberrant activation of the RAS family of guanosine triphosphatases (GTPases) is prevalent in lung adenocarcinoma, with somatic mutation of KRAS occurring in ~30% of tumors. We previously identified somatic mutations and amplifications of the gene encoding RAS family GTPase RIT1 in lung adenocarcinomas. To explore the biological pathways regulated by RIT1 and how they relate to the oncogenic KRAS network, we performed quantitative proteomic, phosphoproteomic, and transcriptomic profiling of isogenic lung epithelial cells in which we ectopically expressed wild-type or cancer-associated variants of RIT1 and KRAS. We found that both mutant KRAS and mutant RIT1 promoted canonical RAS signaling and that overexpression of wild-type RIT1 partially phenocopied oncogenic RIT1 and KRAS, including induction of epithelial-to-mesenchymal transition. Our findings suggest that RIT1 protein abundance is a factor in its pathogenic function. Therefore, chromosomal amplification of wild-type RIT1 in lung and other cancers may be tumorigenic.

Loss of MGA repression mediated by an atypical polycomb complex promotes tumor progression and invasiveness.

MGA, a transcription factor and member of the MYC network, is mutated or deleted in a broad spectrum of malignancies. As a critical test of a tumor suppressive role, we inactivated Mga in two mouse models of non-small cell lung cancer using a CRISPR-based approach. MGA loss significantly accelerated tumor growth in both models and led to de-repression of non-canonical Polycomb ncPRC1.6 targets, including genes involved in metastasis and meiosis. Moreover, MGA deletion in human lung adenocarcinoma lines augmented invasive capabilities. We further show that MGA-MAX, E2F6, and L3MBTL2 co-occupy thousands of promoters and that MGA stabilizes these ncPRC1.6 subunits. Lastly, we report that MGA loss also induces a pro-growth effect in human colon organoids. Our studies establish MGA as a bona fide tumor suppressor in vivo and suggest a tumor suppressive mechanism in adenocarcinomas resulting from widespread transcriptional attenuation of MYC and E2F target genes mediated by MGA-MAX associated with a non-canonical Polycomb complex.

Whole-genome characterization of lung adenocarcinomas lacking the RTK/RAS/RAF pathway.

RTK/RAS/RAF pathway alterations (RPAs) are a hallmark of lung adenocarcinoma (LUAD). In this study, we use whole-genome sequencing (WGS) of 85 cases found to be RPA(-) by previous studies from The Cancer Genome Atlas (TCGA) to characterize the minority of LUADs lacking apparent alterations in this pathway. We show that WGS analysis uncovers RPA(+) in 28 (33%) of the 85 samples. Among the remaining 57 cases, we observe focal deletions targeting the promoter or transcription start site of STK11 (n = 7) or KEAP1 (n = 3), and promoter mutations associated with the increased expression of ILF2 (n = 6). We also identify complex structural variations associated with high-level copy number amplifications. Moreover, an enrichment of focal deletions is found in TP53 mutant cases. Our results indicate that RPA(-) cases demonstrate tumor suppressor deletions and genome instability, but lack unique or recurrent genetic lesions compensating for the lack of RPAs. Larger WGS studies of RPA(-) cases are required to understand this important LUAD subset.

Regulation of human sphingomyelin synthase 1 translation through its 5'-untranslated region.

Bcr-abl1 oncogene causes a shift in the transcription start site of the SMS1 gene (SGMS1) encoding the sphingomyelin (SM) synthesizing enzyme, sphingomyelin synthase 1 (SMS1). This results in an mRNA with a significantly shorter 5'-UTR, called 7-SGMS1, which is translated more efficiently than another transcript (IIb-SGMS1) with a longer 5'UTR in Bcr-abl1-positive cells. Here, we determine the effects of these alternative 5'UTRs on SMS1 translation and investigate the key features underlying such regulation. First, the presence of the longer IIb 5'UTR is sufficient to greatly impair translation of a reporter gene. Deletion of the upstream open reading frame (-164 nt) or of the predicted stem-loops in the 5'UTR of IIb-SGMS1 has minimal effects on SGMS1 translation. Conversely, deletion of nucleotides -310 to -132 enhanced transcription of IIb-SGMS1 to reach that of 7-SGMS1. We thus suggest that regulatory features within nucleotides -310 and -132 modulate IIb-SGMS1 translation efficiency.

Enzymatic-based cytometry, a sensitive single-cell cytometric method to assess BCR-ABL1 activity in CML.

We developed a simple, rapid and cost-effective enzymatic-based cytometry platform to measure intracellular signaling pathway activity. Our single-cell microwell array platform quantifies protein phosphorylation using enzymatic signal amplification and exploiting Michaelis-Menten kinetics. Our method provides a two-fold increase in resolution compared to conventional flow cytometry.

Role and Function of Sphingomyelin Biosynthesis in the Development of Cancer.

Sphingomyelin (SM) biosynthesis represents a complex, finely regulated process, mostly occurring in vertebrates. It is intimately linked to lipid transport and it is ultimately carried out by two enzymes, SM synthase 1 and 2, selectively localized in the Golgi and plasma membrane. In the course of the SM biosynthetic reaction, various lipids are metabolized. Because these lipids have both structural and signaling functions, the SM biosynthetic process has the potential to affect diverse important cellular processes (such as cell proliferation, cell survival, and migration). Thus defects in SM biosynthesis might directly or indirectly impact the normal physiology of the cell and eventually of the organism. In this chapter, we will focus on evidence supporting a role for SM biosynthesis in specific cellular functions and how its dysregulation can affect neoplastic transformation.

Bcr-Abl regulation of sphingomyelin synthase 1 reveals a novel oncogenic-driven mechanism of protein up-regulation.

Bcr-Abl (break-point cluster region-abelson), the oncogenic trigger of chronic myelogenous leukemia (CML), has previously been shown to up-regulate the expression and activity of sphingomyelin synthase 1 (SMS1), which contributes to the proliferation of CML cells; however, the mechanism by which this increased expression of SMS1 is mediated remains unknown. In the current study, we show that Bcr-Abl enhances the expression of SMS1 via a 30-fold up-regulation of its transcription. Of most interest, the Bcr-Abl-regulated transcription of SMS1 is initiated from a novel transcription start site (TSS) that is just upstream of the open reading frame. This shift in TSS utilization generates an SMS1 mRNA with a substantially shorter 5' UTR compared with its canonical mRNA. This shorter 5' UTR imparts a 20-fold greater translational efficiency to SMS1 mRNA, which further contributes to the increase of its expression in CML cells. Therefore, our study demonstrates that Bcr-Abl increases SMS1 protein levels via 2 concerted mechanisms: up-regulation of transcription and enhanced translation as a result of the shift in TSS utilization. Remarkably, this is the first time that an oncogene-Bcr-Abl-has been demonstrated to drive such a mechanism that up-regulates the expression of a functionally important target gene, SMS1.-Moorthi, S., Burns, T. A., Yu, G.-Q., Luberto, C. Bcr-Abl regulation of sphingomyelin synthase 1 reveals a novel oncogenic-driven mechanism of protein up-regulation.