Faculty Bibliography

The most frequently mutated oncogene in cancer is KRAS, which uses alternative fourth exons to generate two gene products (KRAS4A and KRAS4B) that differ only in their C-terminal membrane-targeting region¹. Because oncogenic mutations occur in exons 2 or 3, two constitutively active KRAS proteins-each capable of transforming cells-are encoded when KRAS is activated by mutation². No functional distinctions among the splice variants have so far been established. Oncogenic KRAS alters the metabolism of tumour cells³ in several ways, including increased glucose uptake and glycolysis even in the presence of abundant oxygen⁴ (the Warburg effect). Whereas these metabolic effects of oncogenic KRAS have been explained by transcriptional upregulation of glucose transporters and glycolytic enzymes^3-5, it is not known whether there is direct regulation of metabolic enzymes. Here we report a direct, GTP-dependent interaction between KRAS4A and hexokinase 1 (HK1) that alters the activity of the kinase, and thereby establish that HK1 is an effector of KRAS4A. This interaction is unique to KRAS4A because the palmitoylation-depalmitoylation cycle of this RAS isoform enables colocalization with HK1 on the outer mitochondrial membrane. The expression of KRAS4A in cancer may drive unique metabolic vulnerabilities that can be exploited therapeutically.

The three human RAS genes encode four proteins that play central roles in oncogenesis by acting as binary molecular switches that regulate signaling pathways for growth and differentiation. Each is subject to a set of posttranslational modifications (PTMs) that modify their activity or are required for membrane targeting. The enzymes that catalyze the various PTMs are potential targets for anti-RAS drug discovery. The PTMs of RAS proteins are the focus of this review.

Isoprenylcysteine carboxyl methyltransferase (ICMT) methylesterifies C-terminal prenylcysteine residues of CaaX proteins and some RAB GTPases. Deficiency of either ICMT or NOTCH1 accelerates pancreatic neoplasia in Pdx1-Cre;LSL-KrasG12D mice, suggesting that ICMT is required for NOTCH signaling. We used Drosophila melanogaster wing vein and scutellar bristle development to screen Rab proteins predicted to be substrates for ICMT (ste14 in flies). We identified Rab7 and Rab8 as ICMT substrates that when silenced phenocopy ste14 deficiency. ICMT, RAB7, and RAB8 were all required for efficient NOTCH1 signaling in mammalian cells. Overexpression of RAB8 rescued NOTCH activation after ICMT knockdown both in U2OS cells expressing NOTCH1 and in fly wing vein development. ICMT deficiency induced mislocalization of GFP-RAB7 and GFP-RAB8 from endomembrane to cytosol, enhanced binding to RABGDI, and decreased GTP loading of RAB7 and RAB8. Deficiency of ICMT, RAB7, or RAB8 led to mislocalization and diminished processing of NOTCH1-GFP. Thus, NOTCH signaling requires ICMT in part because it requires methylated RAB7 and RAB8.

Ras guanosine triphosphatases (GTPases) regulate signaling pathways only when associated with cellular membranes through their C-terminal prenylated regions. Ras proteins move between membrane compartments in part via diffusion-limited, fluid phase transfer through the cytosol, suggesting that chaperones sequester the polyisoprene lipid from the aqueous environment. In this study, we analyze the nature of the pool of endogenous Ras proteins found in the cytosol. The majority of the pool consists of farnesylated, but not palmitoylated, N-Ras that is associated with a high molecular weight (HMW) complex. Affinity purification and mass spectrographic identification revealed that among the proteins found in the HMW fraction is VPS35, a latent cytosolic component of the retromer coat. VPS35 bound to N-Ras in a farnesyl-dependent, but neither palmitoyl- nor guanosine triphosphate (GTP)-dependent, fashion. Silencing VPS35 increased N-Ras's association with cytoplasmic vesicles, diminished GTP loading of Ras, and inhibited mitogen-activated protein kinase signaling and growth of N-Ras-dependent melanoma cells.

We present a 40-year-old woman with asymptomatic, linear, hyperpigmented atrophic plaques in a Blaschkoid distribution on the right back and right upper extremity that is consistent with a diagnosis of linear atrophoderma of Moulin. Clinical lesions developed with a biphasic pattern in late adolescence and in adulthood. The pathogenesis of this acquired, progressive Blaschkolinear dermatosis may hold insight into the pathogenesis of this rare dermatologic condition, as well as other dermotoses, which include those resulting from post-zygotic genetic mosaicism.

We present a 64-year-old man with a three-year history of pruritic, pink papules and nodules of the face who was found to have a clonal lymphoproliferative B-cell disease that was characterized by a clonal IGH rearrangement. Although morphologic features present in the biopsy specimen were consistent with a reactive process, additional clinicopathologic correlation (anatomic presentation of lesions on the face, the absence of t(14:18) translocation, and bcl-2 and MUM1 expression) reinforced suspicion of a cutaneous B-cell lymphoma. Systemic work-up with CT/PET and a bone marrow biopsy ultimately excluded systemic disease and primary cutaneous follicle-center lymphoma (PCFCL) was a strong diagnostic consideration. The patient was treated with systemic rituximab with a partial resolution of the facial lesions. The case demonstrates both clinical and pathologic challenges to the diagnosis of primary cutaneous B-cell lymphoma (PCBCL). Furthermore, despite a newly refined classification system, the case also specifically highlights the persistent requirement for flexible clinical reasoning and pathologic correlation. Such reasoning is necessary to generate individualized strategies for diagnosis and treatment when cutaneous B-cell lymphoma is suspected.

Metabolic labeling with tritiated palmitate is a direct method for monitoring posttranslational modification of Ras proteins with this fatty acid. Advances in intensifying screens have allowed for the easy visualization of tritium without the need for extended exposure times. While more energetic radioisotopes are easier to visualize, the lack of commercial source and need for shielding make them more difficult to work with. Since radiolabeled palmitate is directly incorporated into Ras, its loss can be monitored by traditional pulse-chase experiments that cannot be accomplished with the method of acyl-exchange chemistry. As such, tritiated palmitate remains a readily accessible and direct method for monitoring the palmitoylation status of Ras proteins under a multitude of conditions.

RAS proteins are monomeric GTPases that act as binary molecular switches to regulate a wide range of cellular processes. The exchange of GTP for GDP on RAS is regulated by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs), which regulate the activation state of RAS without covalently modifying it. By contrast, post-translational modifications (PTMs) of RAS proteins direct them to various cellular membranes and, in some cases, modulate GTP-GDP exchange. Important RAS PTMs include the constitutive and irreversible remodelling of its carboxy-terminal CAAX motif by farnesylation, proteolysis and methylation, reversible palmitoylation, and conditional modifications, including phosphorylation, peptidyl-prolyl isomerisation, monoubiquitylation, diubiquitylation, nitrosylation, ADP ribosylation and glucosylation

We studied the feasibility of using single-wall carbon nanotubes (SWNTs) as antigen carriers to improve immune responses to peptides that are weak immunogens, a characteristic typical of human tumor antigens. Binding and presentation of peptide antigens by the MHC molecules of antigen presenting cells (APCs) is essential to mounting an effective immune response. The Wilm's tumor protein (WT1) is upregulated in many human leukemias and cancers and several vaccines directed at this protein are in human clinical trials. WT1 peptide 427 induces human CD4 T cell responses in the context of multiple human HLA-DR.B1 molecules, but the peptide has a poor binding affinity to BALB/c mouse MHC class II molecules. We used novel, spectrally quantifiable chemical approaches to covalently append large numbers of peptide ligands (0.4 mmol/g) onto solubilized SWNT scaffolds. Peptide-SWNT constructs were rapidly internalized into professional APCs (dendritic cells and macrophages) within minutes in vitro, in a dose dependent manner. Immunization of BALB/c mice with the SWNT-peptide constructs mixed with immunological adjuvant induced specific IgG responses against the peptide, while the peptide alone or peptide mixed with the adjuvant did not induce such a response. The conjugation of the peptide to SWNT did not enhance the peptide-specific CD4 T cell response in human and mouse cells, in vitro. The solubilized SWNTs alone were nontoxic in vitro, and we did not detect antibody responses to SWNT in vivo. These results demonstrated that SWNTs are able to serve as antigen carriers for delivery into APCs to induce humoral immune responses against weak tumor antigens.

A cycle of palmitoylation/depalmitoylation of H-Ras mediates bidirectional trafficking between the Golgi apparatus and the plasma membrane, but nothing is known about how this cycle is regulated. We show that the prolyl isomerase (PI) FKBP12 binds to H-Ras in a palmitoylation-dependent fashion and promotes depalmitoylation. A variety of inhibitors of the PI activity of FKBP12, including FK506, rapamycin, and cycloheximide, increase steady-state palmitoylation. FK506 inhibits retrograde trafficking of H-Ras from the plasma membrane to the Golgi in a proline 179-dependent fashion, augments early GTP loading of Ras in response to growth factors, and promotes H-Ras-dependent neurite outgrowth from PC12 cells. These data demonstrate that FKBP12 regulates H-Ras trafficking by promoting depalmitoylation through cis-trans isomerization of a peptidyl-prolyl bond in proximity to the palmitoylated cysteines