Internal bases in mRNA can be subjected to modifications that influence the fate of mRNA in cells. One of the most prevalent modified bases is found at the 5′ end of mRNA, at the first encoded nucleotide adjacent to the 7-methylguanosine cap. Here we show that this nucleotide, N6,2′-O-dimethyladenosine (m6Am), is a reversible modification that influences cellular mRNA fate. Using a transcriptome-wide map of m6Am we find that m6Am-initiated transcripts are markedly more stable than mRNAs that begin with other nucleotides. We show that the enhanced stability of m6Am-initiated transcripts is due to resistance to the mRNA-decapping enzyme DCP2. Moreover, we find that m6Am is selectively demethylated by fat mass and obesity-associated protein (FTO). FTO preferentially demethylates m6Am rather than N6-methyladenosine (m6A), and reduces the stability of m6Am mRNAs. Together, these findings show that the methylation status of m6Am in the 5′ cap is a dynamic and reversible epitranscriptomic modification that determines mRNA stability.

More than half of human colorectal cancers (CRCs) carry either KRAS or BRAF mutations, and are often refractory to approved targeted therapies. We report that cultured CRC cells harboring KRAS or BRAF mutations are selectively killed when exposed to high levels of vitamin C. This effect is due to increased uptake of the oxidized form of vitamin C, dehydroascorbate (DHA), via the GLUT1 glucose transporter. Increased DHA uptake causes oxidative stress as intracellular DHA is reduced to vitamin C depleting glutathione. Thus, ROS accumulates and inactivates glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Inhibiting GAPDH in highly glycolytic KRAS or BRAF mutant cells leads to an energetic crisis and cell death not seen in KRAS and BRAF wild-type cells. In vivo studies indicate that high-dose vitamin C can impair tumor growth in Apc/KrasG12D mutant mouse intestinal cancers. While it is unclear whether human tumors will respond similarly, our results provide a mechanistic rationale for exploring the therapeutic use of vitamin C to treat CRCs with KRAS or BRAF mutations.

Tubular-fluid reabsorption by specialized cells of the nephron at the junction of the ascending limb of the loop of Henle and the distal convoluted tubule, termed the macula densa, releases compounds causing vasoconstriction of the adjacent afferent arteriole. Activation of this tubuloglomerular feedback response reduces glomerular capillary pressure of the nephron and, hence, the glomerular filtration rate. The tubuloglomerular feedback response functions in a negative-feedback mode to relate glomerular capillary pressure to tubular-fluid delivery and reabsorption. This system has been implicated in renal autoregulation, renin release, and longterm body fluid and blood-pressure homeostasis. Here we report that arginine-derived nitric oxide, generated in the macula densa, is an additional intercellular signaln molecule that is released during tubular-fluid reabsorption and counters the vasoconstriction of the afferent arteriole. Antibody to rat cerebellar constitutive nitric oxide synthase stained rat macula densa cells specifically. Microperfusion of the macula densa segment of single nephrons with N'-methyl-L-arginlne (an inhibitor of nitric oxide synthase) or with pyocyanin (a lipidsoluble inhibitor of endothelium-derived relaxation factor) showed that generation of nitric oxide can vasodilate the afferent arteriole and increase glomerular capillary pressure; this effect was blocked by drugs that prevent tubular-fluid reabsorption. We conclude that nitric oxide synthase in macula densa cells is activated by tubular-fluid reabsorption and mediates a vasodilating component to the tubuloglomerular feedback response. These finding imply a role for argininederived nitric oxide in body fluid-volume and blood-pressure homeostasis, in addition to its established roles in modulation of vascular tone by the endothelium and in neurotransmission.Goormaghtigh (1) suggested that the macula densa is the sensor for a stimulus from tubular fluid that is conveyed to the glomerulus. Subsequently, Thurau and Schnermann (2) identified that the stimulus was the delivery and reabsorption of NaCl by this segment. This tubuloglomerular feedback response functions as a negative-feedback control mechanism, whereby glomerular filtration of NaCI, with delivery to and reabsorption by the macula densa, induces release of mediator(s) that cause afferent-arteriolar vasoconstriction and a reduction in glomerular capillary pressure and glomerular filtration rate (2). Although the signaling mechanisms or molecules inducing afferent-arteriolar vasoconstriction have not been clearly defined, the response is promoted by adenosine acting on adenosine type 1 receptors (3), angiotensin II (4), and thromboxane A2 (5)-Previous studies have established that L-arginine-derived nitric oxide (NO) is produced by several cells within the kidney, including isolated glomerular mesangial (6) and endothelial cells (7), and a renal epithelial cell line (8), but its integrative role in the control ofrenal function is not yet clear (9). In the vessel wall, ...

Most cancers are characterized by multiple molecular alterations, but identification of the key proteins involved in these signaling pathways is currently beyond reach. We show that the inhibitor PU-H71 preferentially targets tumor-enriched Hsp90 complexes and affinity captures Hsp90-dependent oncogenic client proteins. We have used PU-H71 affinity capture to design a proteomic approach that, when combined with bioinformatic pathway analysis, identifies dysregulated signaling networks and key oncoproteins in chronic myeloid leukemia. The identified interactome overlaps with the well-characterized altered proteome in this cancer, indicating that this method can provide global insights into the biology of individual tumors, including primary patient specimens. In addition, we show that this approach can be used to identify previously uncharacterized oncoproteins and mechanisms, potentially leading to new targeted therapies. We further show that the abundance of the PU-H71-enriched Hsp90 species, which is not dictated by Hsp90 expression alone, is predictive of the cell’s sensitivity to Hsp90 inhibition.