Apparent Lack of BRAFV600E Derived HLA Class I Presented Neoantigens Hampers Neoplastic Cell Targeting by CD8+ T Cells in Langerhans Cell Histiocytosis.

Langerhans Cell Histiocytosis (LCH) is a neoplastic disorder of hematopoietic origin characterized by inflammatory lesions containing clonal histiocytes (LCH-cells) intermixed with various immune cells, including T cells. In 50-60% of LCH-patients, the somatic BRAFV600E driver mutation, which is common in many cancers, is detected in these LCH-cells in an otherwise quiet genomic landscape. Non-synonymous mutations like BRAFV600E can be a source of neoantigens capable of eliciting effective antitumor CD8+ T cell responses. This requires neopeptides to be stably presented by Human Leukocyte Antigen (HLA) class I molecules and sufficient numbers of CD8+ T cells at tumor sites. Here, we demonstrate substantial heterogeneity in CD8+ T cell density in n = 101 LCH-lesions, with BRAFV600E mutated lesions displaying significantly lower CD8+ T cell:CD1a+ LCH-cell ratios (p = 0.01) than BRAF wildtype lesions. Because LCH-lesional CD8+ T cell density had no significant impact on event-free survival, we investigated whether the intracellularly expressed BRAFV600E protein is degraded into neopeptides that are naturally processed and presented by cell surface HLA class I molecules. Epitope prediction tools revealed a single HLA class I binding BRAFV600E derived neopeptide (KIGDFGLATEK), which indeed displayed strong to intermediate binding capacity to HLA-A*03:01 and HLA-A*11:01 in an in vitro peptide-HLA binding assay. Mass spectrometry-based targeted peptidomics was used to investigate the presence of this neopeptide in HLA class I presented peptides isolated from several BRAFV600E expressing cell lines with various HLA genotypes. While the HLA-A*02:01 binding BRAF wildtype peptide KIGDFGLATV was traced in peptides isolated from all five cell lines expressing this HLA subtype, KIGDFGLATEK was not detected in the HLA class I peptidomes of two distinct BRAFV600E transduced cell lines with confirmed expression of HLA-A*03:01 or HLA-A*11:01. These data indicate that the in silico predicted HLA class I binding and proteasome-generated neopeptides derived from the BRAFV600E protein are not presented by HLA class I molecules. Given that the BRAFV600E mutation is highly prevalent in chemotherapy refractory LCH-patients who may qualify for immunotherapy, this study therefore questions the efficacy of immune checkpoint inhibitor therapy in LCH.

Pharmacogenomic analysis of ATP-sensitive potassium channels coexpressing the common type 2 diabetes risk variants E23K and S1369A.

OBJECTIVES: The common ATP-sensitive potassium (KATP) channel variants E23K and S1369A, found in the KCNJ11 and ABCC8 genes, respectively, form a haplotype that is associated with an increased risk for type 2 diabetes. Our previous studies showed that KATP channel inhibition by the A-site sulfonylurea gliclazide was increased in the K23/A1369 haplotype. Therefore, we studied the pharmacogenomics of seven clinically used sulfonylureas and glinides to determine their structure-activity relationships in KATP channels containing either the E23/S1369 nonrisk or K23/A1369 risk haplotypes. RESEARCH DESIGN AND METHODS: The patch-clamp technique was used to determine sulfonylurea and glinide inhibition of recombinant human KATP channels containing either the E23/S1369 or the K23/A1369 haplotype. RESULTS: KATP channels containing the K23/A1369 risk haplotype were significantly less sensitive to inhibition by tolbutamide, chlorpropamide, and glimepiride (IC50 values for K23/A1369 vs. E23/S1369=1.15 vs. 0.71 µmol/l; 4.19 vs. 3.04 µmol/l; 4.38 vs. 2.41 nmol/l, respectively). In contrast, KATP channels containing the K23/A1369 haplotype were significantly more sensitive to inhibition by mitiglinide (IC50=9.73 vs. 28.19 nmol/l for K23/A1369 vs. E23/S1369) and gliclazide. Nateglinide, glipizide, and glibenclamide showed similar inhibitory profiles in KATP channels containing either haplotype. CONCLUSION: Our results demonstrate that the ring-fused pyrrole moiety in several A-site drugs likely underlies the observed inhibitory potency of these drugs on KATP channels containing the K23/A1369 risk haplotype. It may therefore be possible to tailor existing therapy or design novel drugs that display an increased efficacy in type 2 diabetes patients homozygous for these common KATP channel haplotypes.

The molecular genetics of sulfonylurea receptors in the pathogenesis and treatment of insulin secretory disorders and type 2 diabetes.

Sulfonylurea receptors (SURs) form an integral part of the ATP-sensitive potassium (K(ATP)) channel complex that is present in most excitable cell types. K(ATP) channels couple cellular metabolism to electrical activity and provide a wide range of cellular functions including stimulus secretion coupling in pancreatic ß cells. K(ATP) channels are composed of SURs and inward rectifier potassium channel (Kir6.x) subunits encoded by the ABCC8/9 and KCNJ8/11 genes, respectively. Recent advances in the genetics, molecular biology, and pharmacology of SURs have led to an increased understanding of these channels in the etiology and treatment of rare genetic insulin secretory disorders. Furthermore, common genetic variants in these genes are associated with an increased risk for type 2 diabetes. In this review we summarize the molecular biology, pharmacology, and physiology of SURs and K(ATP) channels, highlighting recent advances in their genetics and understanding of rare insulin secretory disorders and susceptibility to type 2 diabetes.

The molecular mechanisms and pharmacotherapy of ATP-sensitive potassium channel gene mutations underlying neonatal diabetes.

Neonatal diabetes mellitus (NDM) is a monogenic disorder caused by mutations in genes involved in regulation of insulin secretion from pancreatic ß-cells. Mutations in the KCNJ11 and ABCC8 genes, encoding the adenosine triphosphate (ATP)-sensitive potassium (K(ATP)) channel Kir6.2 and SUR1 subunits, respectively, are found in ∼50% of NDM patients. In the pancreatic ß-cell, K(ATP) channel activity couples glucose metabolism to insulin secretion via cellular excitability and mutations in either KCNJ11 or ABCC8 genes alter K(ATP) channel activity, leading to faulty insulin secretion. Inactivation mutations decrease K(ATP) channel activity and stimulate excessive insulin secretion, leading to hyperinsulinism of infancy. In direct contrast, activation mutations increase K(ATP) channel activity, resulting in impaired insulin secretion, NDM, and in severe cases, developmental delay and epilepsy. Many NDM patients with KCNJ11 and ABCC8 mutations can be successfully treated with sulfonylureas (SUs) that inhibit the K(ATP) channel, thus replacing the need for daily insulin injections. There is also strong evidence indicating that SU therapy ameliorates some of the neurological defects observed in patients with more severe forms of NDM. This review focuses on the molecular and cellular mechanisms of mutations in the K(ATP) channel that underlie NDM. SU pharmacogenomics is also discussed with respect to evaluating whether patients with certain K(ATP) channel activation mutations can be successfully switched to SU therapy.