Targeting Plasma Membrane Phosphatidylserine Content to Inhibit Oncogenic KRAS Function

The small GTPase KRAS, which is frequently mutated in human cancers, must be localized to the plasma membrane (PM) for biological activity. We recently showed that the KRAS C-terminal membrane anchor exhibits exquisite lipid-binding specificity for select species of phosphatidylserine (PtdSer). We therefore investigated whether reducing PM PtdSer content is sufficient to abrogate KRAS oncogenesis. Oxysterol-related binding proteins ORP5 and ORP8 exchange PtdSer synthesized in the ER for phosphatidylinositol-4-phosphate (PI4P) synthesized in the PM. We show that depletion of ORP5 or ORP8 reduced PM PtdSer levels, resulting in extensive mislocalization of KRAS from the PM. Concordantly, ORP5 or ORP8 depletion significantly reduced proliferation and anchorage-independent growth of multiple KRAS-dependent cancer cell lines, and attenuated KRAS signaling in vivo. Similarly, functionally inhibiting ORP5 and ORP8 by inhibiting PI4KIIIα-mediated synthesis of PI4P at the PM selectively inhibited the growth of KRAS-dependent cancer cells over normal cells in vitro and in vivo. Hence, inhibiting KRAS function through regulating PM PtdSer content may represent a viable strategy for KRAS-driven cancers

Acylpeptide hydrolase is a novel regulator of KRAS plasma membrane localization and function

The primary site for KRAS signaling is the inner leaflet of the plasma membrane (PM). We previously reported that oxanthroquinone G01 (G01) inhibited KRAS PM localization and blocked KRAS signaling. In this study, we identified acylpeptide hydrolase (APEH) as a molecular target of G01. APEH formed a stable complex with biotinylated G01, and the enzymatic activity of APEH was inhibited by G01. APEH knockdown caused profound mislocalization of KRAS and reduced clustering of KRAS that remained PM localized. APEH knockdown also disrupted the PM localization of phosphatidylserine (PtdSer), a lipid critical for KRAS PM binding and dustering. The mislocalization of KRAS was fully rescued by ectopic expression of APEH in knockdown cells. APEH knockdown disrupted the endocytic recycling of epidermal growth factor receptor and transferrin receptor, suggesting that abrogation of recycling endosome function was mechanistically linked to the loss of KRAS and PtdSer from the PM. APEH knockdown abrogated RAS-RAF-MAPK signaling in cells expressing the constitutively active (oncogenic) mutant of KRAS (KRASG12V), and selectively inhibited the proliferation of KRAS-transformed pancreatic cancer cells. Taken together, these results identify APEH as a novel drug target for a potential anti-KRAS therapeutic

Novel CYP27B1 Gene Mutations in Patients with Vitamin D-Dependent Rickets Type 1A

The CYP27B1 gene encodes 25-hydroxyvitamin D-1a-hydroxylase. Mutations of this gene cause vitamin D-dependent rickets type 1A (VDDR-IA, OMIM 264700), which is a rare autosomal recessive disorder. To investigate CYP27B1 mutations, we studied 8 patients from 7 unrelated families. All coding exons and intron-exon boundaries of CYP27B1 gene were amplified by PCR from peripheral leukocyte DNA and subsequently sequenced. Homozygous mutations in the CYP27B1 gene were found in all the patients and heterozygous mutations were present in their normal parents. One novel single nucleotide variation (SNV, c. 1215 T>C, p. R379R in the last nucleotide of exon 7) and three novel mutations were identified:, a splice donor site mutation (c. 1215+2T>A) in intron 7, a 16-bp deletion in exon 6 (c. 1022-1037del16), and a 2-bp deletion in exon 5 (c. 934_935delAC). Both c. 1215 T>C and c. 1215+2T>A were present together in homozygous form in two unrelated patients, and caused exon 7 skipping. However, c. 1215 T>C alone has no effect on pre-mRNA splicing. The skipping of exon 7 resulted in a shift of downstream reading frame and a premature stop codon 57 amino acids from L380 (p. L380Afs*57). The intra-exon deletions of c. 1022-1037del16 and c. 934_935delAC also resulted in a frameshift and the creation of premature stop codons at p. T341Rfs*5, and p. T312Rfs*19, respectively, leading to the functional inactivation of the CYP27B1 gene. Clinically, all the patients required continued calcitriol treatment and the clinical presentations were consistent with the complete loss of vitamin D1a-hydroxylase activity. In conclusion, three novel mutations have been identified. All of them caused frameshift and truncated proteins. The silent c. 1215 T>C SNV has no effect on pre-mRNA splicing and it is likely a novel SNP. The current study further expands the CYP27B1 mutation spectrum

Glycolysis regulates KRAS plasma membrane localization and function through defined glycosphingolipids

Oncogenic KRAS expression generates a metabolic dependency on aerobic glycolysis, known as the Warburg effect. We report an effect of increased glycolytic flux that feeds into glycosphingolipid biosynthesis and is directly linked to KRAS oncogenic function. High resolution imaging and genetic approaches show that a defined subset of outer leaflet glycosphingolipids, including GM3 and SM4, is required to maintain KRAS plasma membrane localization, with GM3 engaging in cross-bilayer coupling to maintain inner leaflet phosphatidylserine content. Thus, glycolysis is critical for KRAS plasma membrane localization and nanoscale spatial organization. Reciprocally oncogenic KRAS selectively upregulates cellular content of these same glycosphingolipids, whose depletion in turn abrogates KRAS oncogenesis in pancreatic cancer models. Our findings expand the role of the Warburg effect beyond ATP generation and biomass building to high-level regulation of KRAS function. The positive feedforward loop between oncogenic KRAS signaling and glycosphingolipid synthesis represents a vulnerability with therapeutic potential

Novel splice site mutations in the human <i>CYP27B1</i> gene.

<p>(A) Sequence analysis of genomic DNA from peripheral lymphocytes. A homozygous mutation at the splice donor site of intron 7 (c.1215+2T>A) were found in a patient from family 3. A homozygous silent SNV (c.1215 T>C) at the end of exon 7 was also identified. His parents carry a heterozygous mutation at both these locations, demonstrating they are in cis and not in trans. The mutations are indicated by arrows. (B) Sequence analysis of cDNA from patient’s peripheral lymphocytes. The mutation at the c.1215+2T>A leads to skipping of exon 7, resulting in exons 6 and 8 joined together.</p