Tumor-wide RNA splicing aberrations generate immunogenic public neoantigens.

T-cell-mediated immunotherapies are limited by the extent to which cancer-specific antigens are homogenously expressed throughout a tumor. We reasoned that recurrent splicing aberrations in cancer represent a potential source of tumor-wide and public neoantigens, and to test this possibility, we developed a novel pipeline for identifying neojunctions expressed uniformly within a tumor across diverse cancer types. Our analyses revealed multiple neojunctions that recur across patients and either exhibited intratumor heterogeneity or, in some cases, were tumor-wide. We identified CD8+ T-cell clones specific for neoantigens derived from tumor-wide and conserved neojunctions in GNAS and RPL22 , respectively. TCR-engineered CD8 + T-cells targeting these mutations conferred neoantigen-specific tumor cell eradication. Furthermore, we revealed that cancer-specific dysregulation in splicing factor expression leads to recurrent neojunction expression. Together, these data reveal that a subset of neojunctions are both intratumorally conserved and public, providing the molecular basis for novel T-cell-based immunotherapies that address intratumoral heterogeneity.

Oncogenic PKA signaling increases c-MYC protein expression through multiple targetable mechanisms.

Genetic alterations that activate protein kinase A (PKA) are found in many tumor types. Yet, their downstream oncogenic signaling mechanisms are poorly understood. We used global phosphoproteomics and kinase activity profiling to map conserved signaling outputs driven by a range of genetic changes that activate PKA in human cancer. Two signaling networks were identified downstream of PKA: RAS/MAPK components and an Aurora Kinase A (AURKA)/glycogen synthase kinase (GSK3) sub-network with activity toward MYC oncoproteins. Findings were validated in two PKA-dependent cancer models: a novel, patient-derived fibrolamellar carcinoma (FLC) line that expresses a DNAJ-PKAc fusion and a PKA-addicted melanoma model with a mutant type I PKA regulatory subunit. We identify PKA signals that can influence both de novo translation and stability of the proto-oncogene c-MYC. However, the primary mechanism of PKA effects on MYC in our cell models was translation and could be blocked with the eIF4A inhibitor zotatifin. This compound dramatically reduced c-MYC expression and inhibited FLC cell line growth in vitro. Thus, targeting PKA effects on translation is a potential treatment strategy for FLC and other PKA-driven cancers.

Effect of curcumin on amyloid-like aggregates generated from methionine-oxidized apolipoprotein A-I.

Curcumin is a polyphenolic phytonutrient that has antineurodegenerative properties. In this study, we investigated the anti-amyloidogenic properties of curcumin. Following incubation with curcumin, intrinsic tryptophan fluorescence emission of apolipoprotein (apo) A-I was strongly quenched. At the same time, curcumin fluorescence emission was enhanced. The fluorescence emission spectra of curcumin in the presence of amyloid-like aggregates formed by methionine-oxidized (ox) apoA-I varied, depending on whether curcumin was added before, or after, aggregate formation. The impact of curcumin on the structure of the aggregating material was revealed by the lower amount of ß-structure in ox-apoA-I amyloid-like aggregates formed in the presence of curcumin, compared to aggregates formed without curcumin. However, the kinetics of ox-apoA-I amyloid-like aggregate formation was not altered by the presence of curcumin. Moreover, electron microscopy analysis detected no discernable differences in amyloid morphology when ox-apoA-I amyloid-like aggregates were formed in the presence or absence of curcumin. In conclusion, curcumin interacts with apoA-I and alters the structure of ox-apoA-I amyloid-like aggregates yet does not diminish the propensity of ox-apoA-I to form aggregates.

Methionine oxidized apolipoprotein A-I at the crossroads of HDL biogenesis and amyloid formation.

Apolipoprotein A-I (apoA-I) shares with other exchangeable apolipoproteins a high level of structural plasticity. In the lipid-free state, the apolipoprotein amphipathic α-helices interact intra- and intermolecularly, providing structural stabilization by self-association. We have reported that lipid-free apoA-I becomes amyloidogenic upon physiologically relevant (myeloperoxidase-mediated) Met oxidation. In this study, we established that Met oxidation promotes amyloidogenesis by reducing the stability of apoA-I monomers and irreversibly disrupting self-association. The oxidized apoA-I monomers also exhibited increased cellular cholesterol release capacity and stronger association with macrophages, compared to nonoxidized apoA-I. Of physiologic relevance, preformed oxidized apoA-I amyloid fibrils induced amyloid formation in nonoxidized apoA-I. This process was enhanced when self-association of nonoxidized apoA-I was disrupted by thermal treatment. Solid state NMR analysis revealed that aggregates formed by seeded nonoxidized apoA-I were structurally similar to those formed by the oxidized protein, featuring a ß-structure-rich amyloid fold alongside α-helices retained from the native state. In atherosclerotic lesions, the conditions that promote apoA-I amyloid formation are readily available: myeloperoxidase, active oxygen species, low pH, and high concentration of lipid-free apoA-I. Our results suggest that even partial Met oxidation of apoA-I can nucleate amyloidogenesis, thus sequestering and inactivating otherwise antiatherogenic and HDL-forming apoA-I.-Witkowski, A., Chan, G. K. L., Boatz, J. C., Li, N. J., Inoue, A. P., Wong, J. C., van der Wel, P. C. A., Cavigiolio, G. Methionine oxidized apolipoprotein A-I at the crossroads of HDL biogenesis and amyloid formation.

Myeloperoxidase-mediated Methionine Oxidation Promotes an Amyloidogenic Outcome for Apolipoprotein A-I.

High plasma levels of apolipoprotein A-I (apoA-I) correlate with cardiovascular health, whereas dysfunctional apoA-I is a cause of atherosclerosis. In the atherosclerotic plaques, amyloid deposition increases with aging. Notably, apoA-I is the main component of these amyloids. Recent studies identified high levels of oxidized lipid-free apoA-I in atherosclerotic plaques. Likely, myeloperoxidase (MPO) secreted by activated macrophages in atherosclerotic lesions is the promoter of such apoA-I oxidation. We hypothesized that apoA-I oxidation by MPO levels similar to those present in the artery walls in atherosclerosis can promote apoA-I structural changes and amyloid fibril formation. ApoA-I was exposed to exhaustive chemical (H2O2) oxidation or physiological levels of enzymatic (MPO) oxidation and incubated at 37 °C and pH 6.0 to induce fibril formation. Both chemically and enzymatically oxidized apoA-I produced fibrillar amyloids after a few hours of incubation. The amyloid fibrils were composed of full-length apoA-I with differential oxidation of the three methionines. Met to Leu apoA-I variants were used to establish the predominant role of oxidation of Met-86 and Met-148 in the fibril formation process. Importantly, a small amount of preformed apoA-I fibrils was able to seed amyloid formation in oxidized apoA-I at pH 7.0. In contrast to hereditary amyloidosis, wherein specific mutations of apoA-I cause protein destabilization and amyloid deposition, oxidative conditions similar to those promoted by local inflammation in atherosclerosis are sufficient to transform full-length wild-type apoA-I into an amyloidogenic protein. Thus, MPO-mediated oxidation may be implicated in the mechanism that leads to amyloid deposition in the atherosclerotic plaques in vivo.