Modeling Mechanisms of In Vivo Variability in Methotrexate Accumulation and Folate Pathway Inhibition in Acute Lymphoblastic Leukemia Cells

Methotrexate (MTX) is widely used for the treatment of childhood acute lymphoblastic leukemia (ALL). The accumulation of MTX and its active metabolites, methotrexate polyglutamates (MTXPG), in ALL cells is an important determinant of its antileukemic effects. We studied 194 of 356 patients enrolled on St. Jude Total XV protocol for newly diagnosed ALL with the goal of characterizing the intracellular pharmacokinetics of MTXPG in leukemia cells; relating these pharmacokinetics to ALL lineage, ploidy and molecular subtype; and using a folate pathway model to simulate optimal treatment strategies. Serial MTX concentrations were measured in plasma and intracellular MTXPG concentrations were measured in circulating leukemia cells. A pharmacokinetic model was developed which accounted for the plasma disposition of MTX along with the transport and metabolism of MTXPG. In addition, a folate pathway model was adapted to simulate the effects of treatment strategies on the inhibition of de novo purine synthesis (DNPS). The intracellular MTXPG pharmacokinetic model parameters differed significantly by lineage, ploidy, and molecular subtypes of ALL. Folylpolyglutamate synthetase (FPGS) activity was higher in B vs T lineage ALL (p<0.005), MTX influx and FPGS activity were higher in hyperdiploid vs non-hyperdiploid ALL (p<0.03), MTX influx and FPGS activity were lower in the t(12;21) (ETV6-RUNX1) subtype (p<0.05), and the ratio of FPGS to γ-glutamyl hydrolase (GGH) activity was lower in the t(1;19) (TCF3-PBX1) subtype (p<0.03) than other genetic subtypes. In addition, the folate pathway model showed differential inhibition of DNPS relative to MTXPG accumulation, MTX dose, and schedule. This study has provided new insights into the intracellular disposition of MTX in leukemia cells and how it affects treatment efficacy

Rare Variants in APP, PSEN1 and PSEN2 Increase Risk for AD in Late-Onset Alzheimer's Disease Families

Pathogenic mutations in APP, PSEN1, PSEN2, MAPT and GRN have previously been linked to familial early onset forms of dementia. Mutation screening in these genes has been performed in either very small series or in single families with late onset AD (LOAD). Similarly, studies in single families have reported mutations in MAPT and GRN associated with clinical AD but no systematic screen of a large dataset has been performed to determine how frequently this occurs. We report sequence data for 439 probands from late-onset AD families with a history of four or more affected individuals. Sixty sequenced individuals (13.7%) carried a novel or pathogenic mutation. Eight pathogenic variants, (one each in APP and MAPT, two in PSEN1 and four in GRN) three of which are novel, were found in 14 samples. Thirteen additional variants, present in 23 families, did not segregate with disease, but the frequency of these variants is higher in AD cases than controls, indicating that these variants may also modify risk for disease. The frequency of rare variants in these genes in this series is significantly higher than in the 1,000 genome project (p = 5.09×10−5; OR = 2.21; 95%CI = 1.49–3.28) or an unselected population of 12,481 samples (p = 6.82×10−5; OR = 2.19; 95%CI = 1.347–3.26). Rare coding variants in APP, PSEN1 and PSEN2, increase risk for or cause late onset AD. The presence of variants in these genes in LOAD and early-onset AD demonstrates that factors other than the mutation can impact the age at onset and penetrance of at least some variants associated with AD. MAPT and GRN mutations can be found in clinical series of AD most likely due to misdiagnosis. This study clearly demonstrates that rare variants in these genes could explain an important proportion of genetic heritability of AD, which is not detected by GWAS

The impact of viral mutations on recognition by SARS-CoV-2 specific T cells.

We identify amino acid variants within dominant SARS-CoV-2 T cell epitopes by interrogating global sequence data. Several variants within nucleocapsid and ORF3a epitopes have arisen independently in multiple lineages and result in loss of recognition by epitope-specific T cells assessed by IFN-γ and cytotoxic killing assays. Complete loss of T cell responsiveness was seen due to Q213K in the A∗01:01-restricted CD8+ ORF3a epitope FTSDYYQLY207-215; due to P13L, P13S, and P13T in the B∗27:05-restricted CD8+ nucleocapsid epitope QRNAPRITF9-17; and due to T362I and P365S in the A∗03:01/A∗11:01-restricted CD8+ nucleocapsid epitope KTFPPTEPK361-369. CD8+ T cell lines unable to recognize variant epitopes have diverse T cell receptor repertoires. These data demonstrate the potential for T cell evasion and highlight the need for ongoing surveillance for variants capable of escaping T cell as well as humoral immunity.This work is supported by the UK Medical Research Council (MRC); Chinese Academy of Medical Sciences(CAMS) Innovation Fund for Medical Sciences (CIFMS), China; National Institute for Health Research (NIHR)Oxford Biomedical Research Centre, and UK Researchand Innovation (UKRI)/NIHR through the UK Coro-navirus Immunology Consortium (UK-CIC). Sequencing of SARS-CoV-2 samples and collation of data wasundertaken by the COG-UK CONSORTIUM. COG-UK is supported by funding from the Medical ResearchCouncil (MRC) part of UK Research & Innovation (UKRI),the National Institute of Health Research (NIHR),and Genome Research Limited, operating as the Wellcome Sanger Institute. T.I.d.S. is supported by a Well-come Trust Intermediate Clinical Fellowship (110058/Z/15/Z). L.T. is supported by the Wellcome Trust(grant number 205228/Z/16/Z) and by theUniversity of Liverpool Centre for Excellence in Infectious DiseaseResearch (CEIDR). S.D. is funded by an NIHR GlobalResearch Professorship (NIHR300791). L.T. and S.C.M.are also supported by the U.S. Food and Drug Administration Medical Countermeasures Initiative contract75F40120C00085 and the National Institute for Health Research Health Protection Research Unit (HPRU) inEmerging and Zoonotic Infections (NIHR200907) at University of Liverpool inpartnership with Public HealthEngland (PHE), in collaboration with Liverpool School of Tropical Medicine and the University of Oxford.L.T. is based at the University of Liverpool. M.D.P. is funded by the NIHR Sheffield Biomedical ResearchCentre (BRC – IS-BRC-1215-20017). ISARIC4C is supported by the MRC (grant no MC_PC_19059). J.C.K.is a Wellcome Investigator (WT204969/Z/16/Z) and supported by NIHR Oxford Biomedical Research Centreand CIFMS. The views expressed are those of the authors and not necessarily those of the NIHR or MRC