Differential modulation of mutant CALR and JAK2 V617F-driven oncogenesis by HLA genotype in myeloproliferative neoplasms.

It has been demonstrated previously that human leukocyte antigen class I (HLA-I) and class II (HLA-II) alleles may modulate JAK2 V617F and CALR mutation (CALRmut)-associated oncogenesis in myeloproliferative neoplasms (MPNs). However, the role of immunogenetic factors in MPNs remains underexplored. We aimed to investigate the potential involvement of HLA genes in CALRmut+ MPNs. High-resolution genotyping of HLA-I and -II loci was conducted in 42 CALRmut+ and 158 JAK2 V617F+ MPN patients and 1,083 healthy controls. A global analysis of the diversity of HLA-I genotypes revealed no significant differences between CALRmut+ patients and controls. However, one HLA-I allele (C*06:02) showed an inverse correlation with presence of CALR mutation. A meta-analysis across independent cohorts and healthy individuals from the 1000 Genomes Project confirmed an inverse correlation between the presentation capabilities of the HLA-I loci for JAK2 V617F and CALRmut-derived peptides in both patients and healthy individuals. scRNA-Seq analysis revealed low expression of TAP1 and CIITA genes in CALRmut+ hematopoietic stem and progenitor cells. In conclusion, the HLA-I genotype differentially restricts JAK2 V617F and CALRmut-driven oncogenesis potentially explaining the mutual exclusivity of the two mutations and differences in their presentation latency. These findings have practical implications for the development of neoantigen-based vaccines in MPNs.

Initial Stages of Spontaneous Binding of Folate-Based Vectors to Folate Receptor-α Observed by Unbiased Molecular Dynamics.

Active targeting is a prospective strategy for controlled drug delivery to malignant tumor tissues. One of the approaches relies on recognition of a bioactive ligand by a receptor expressed abundantly on the surface of cancer cell membranes. A promising ligand-receptor pair is folic acid (or its dianionic form, folate) combined with the folate receptor-α (FRα). A number of targeting drug delivery systems based on folate have been suggested, but the mechanism of binding of the ligand or its derivatives to the receptor is not fully known at the molecular level. The current study summarizes the results from unbiased all-atom molecular dynamics simulations at physiological conditions describing the binding of two forms of folate and four of its synthetically available derivatives to FRα. The models (ca. 185,000 atoms) contain one receptor molecule, embedded in the outer leaflet of a lipid bilayer, and one ligand, all immersed in saline. The bilayer represents a human cancer cell membrane and consists of 370 asymmetrically distributed lipid molecules from 35 types. The ability of the vector molecules to bind to the receptor, the position of binding, and the interactions between them are analyzed. Spontaneous binding on the nanosecond scale is observed for all molecules, but its time, position, and persistence depend strongly on the ligand. Only folate, 5-methyltetrahydrofolate, and raltitrexed bind selectively at the active site of the receptor. Two binding poses are observed, one of them (realized by raltitrexed) corresponding qualitatively to that reported for the crystallographic structure of the complex folate-FRα. Pemetrexed adsorbs nonspecifically on the protein surface, while methotrexate and pteroyl ornithine couple much less to the receptor. The molecular simulations reproduce qualitatively correctly the relative binding affinity measured experimentally for five of the ligands. Analysis of the interactions between the ligands and FRα shows that in order to accomplish specific binding to the active site, a combination of hydrogen bonding, π-stacking, and van der Waals and Coulomb attraction should be feasible simultaneously for the vector molecule. The reported results demonstrate that it is possible to observe receptor-ligand binding without applying bias by representing the local environment as close as possible and contain important molecular-level guidelines for the design of folate-based systems for targeted delivery of anticancer drugs.

Characteristics of a Folate Receptor-α Anchored into a Multilipid Bilayer Obtained from Atomistic Molecular Dynamics Simulations.

Thorough computational description of the properties of membrane-anchored protein receptors, which are important for example in the context of active targeting drug delivery, may be achieved by models representing as close as possible the immediate environment of these macromolecules. An all-atom bilayer, including 35 different lipid types asymmetrically distributed among the two monolayers, is suggested as a model neoplastic cell membrane. One molecule of folate receptor-α (FRα) is anchored into its outer leaflet, and the behavior of the system is explored by atomistic molecular dynamics simulations. The total number of atoms in the model is ∼185 000. Three 1-µs-long simulations are carried out, where physiological conditions (310 K and 1 bar) are maintained with three different pressure scaling schemes. To evaluate the structure and the phase state of the membrane, the density profiles of the system, the average area per lipid, and the deuterium order parameter of the lipid tails are calculated. The bilayer is in liquid ordered state, and the specific arrangement varies between the three trajectories. The changes in the structure of FRα are investigated and are found time- and ensemble-dependent. The volume of the ligand binding pocket fluctuates with time, but this variation remains independent of the more global structural alterations. The latter are mostly "waving" motions of the protein, which periodically approaches and retreats from the membrane. The semi-isotropic pressure scaling perturbs the receptor most significantly, while the isotropic algorithm induces rather slow changes. Maintaining constant nonzero surface tension leads to behavior closest to the experimentally observed one.

Molecular simulation of the structure of folate and antifolates at physiological conditions.

The study is focused on description of folate and several antifolates at physiological conditions. Knowledge of the molecular structure and dynamics is important for understanding their biological activity and therapeutic application. They are modelled in saline by atomistic molecular dynamics simulations and characterized in detail. In addition, quantum chemical calculations are used for determining the electronic structure of the six compounds. All molecules are highly flexible and have similar interactions with water. Specifics are found in some of their local backbone conformations, in the molecular shape, and in the electron density distribution. Most of the ligands have fairly folded geometry and prefer U- and Z-shapes. Two of them are quasi-linear. Key to the molecular shape are the bicyclic fragment, its bridge, and the charge of the terminal amino acid residue. Docking into the active site of folate receptor-α predicts a similar best binding pose for four of the ligands, which requires stretching of pterin and bending of glutamate/ornithine relative to the geometry in saline. The chemical modifications in the antifolates induce local electron density redistribution in comparison to folate, leading to increase of the positive charges of the neighboring fragments. The obtained results would help better tuning of the potential usage of the molecules in new bioactive materials, e.g., as vector-ligands for drug delivery.

Identification and computational characterization of isomers with cis and trans amide bonds in folate and its analogues.

Folate and its synthetic analogues, called antifolates, are known to have diverse bio-applications, for example as cell proliferation stimulators or anticancer drugs. Their molecular structure is important for performing the required biological activity. Since all folate-derived ligands contain a peptide-like amide bond, its configuration is one of the key components for the functional fitness of such compounds. During the modelling of folate and three of its derivatives - methotrexate, 5-methyl tetrahydrofolate, and pteroyl ornithine, we registered significant population of the cis isomers along the amide bond. The properties of the cis and trans forms of the ligands in saline are studied in detail by classical atomistic molecular dynamics and by quantum chemical methods. The calculations predict high probability for coexistence of the cis isomers for two of the ligands. The energetic instability of the cis form is explained with a σ-character admixture into the C[double bond, length as m-dash]O(π) bond, while its magnitude is attributed to the pattern of local electron density redistribution. The cis forms of all molecules have markedly slower structural dynamics than the trans ones, which might affect their behavior in vivo.