Biology subjects

Chiarle, R.

Publications and source records attributed to Chiarle, R..

4 recordsLinked to original sources

bioRxiv2025

Cancers modulate p53 truncal neoantigen display to evade T cell detection

Summary paragraphTP53 mutations are early truncal events across cancers1,2. These are perceived to encode tumour-specific neoantigens representing prime cytotoxic T lymphocyte (CTL) targets3,4. However, studies systematically examining the physical cell surface display of p53 peptides bound to major histocompatibility complex molecules (pMHC), their relative antigenicity, and resultant immunogenicity have yet to be conducted. Here, we develop an epitope discovery platform using p53-reconstituted lung cancer cells as well as various tumour cells as pMHC sources. Combining data-independent acquisition mass spectrometry (MS), nanoscale chromatography, and peptide detection based on probabilistic measure and three-dimensional ion visualization techniques allows attomole sensitivity identification of pMHCs. This approach excluded [~]97% of algorithm-based virtual p53 immunopeptidomes, highlighting that only a few p53 pMHCs can be presented by common human MHC (human leukocyte antigen, [HLA]) alleles. Strikingly, surface expressed neoantigens are restricted to the corresponding set of such limited self-p53 peptide arrays and unaffected by enhancing p53 proteasomal turnover. Further curtailment of MS-validated, high affinity p53 neoepitopes that are structurally deviant from self-pMHC occurs in established tumours due to immune selection against the antigen presenting MHC allele or by a novel mechanism involving p53 neoepitope destruction by endoplasmic reticulum aminopeptidase 1 (ERAP1). In contrast, given the extremely weak MHC affinity and resultant short-lived cell surface pMHC expression, the common p53 neoepitope R175H/HLA-A*02:01 escapes immune selection despite CTL with high quality T-cell receptors. Rigorous tumour-protective immunoediting makes effective truncal neoepitope targeting a challenge, requiring attentive MS analysis and functional vetting to focus protective cytolytic responses.

immunology↗

bioRxiv2025

Structural Identification of Major Molecular Determinants for Phosphotyrosine Recognition in Tyrosine Kinases Reveals Tumour Promoting and Suppressive Functions

Protein tyrosine kinases activate signaling pathways by catalyzing the phosphorylation of tyrosine residues in their substrates. Mounting evidence suggests that, in addition to recognizing phosphorylated tyrosine (pTyr) residues through specific phosphobinding modules, many protein kinases selectively recognize pTyr directly adjacent to the tyrosine residue they phosphorylate and catalyze the formation of twin pTyr-pTyr sites. Here, we demonstrate the importance of this phosphopriming-driven twin pTyr signaling in promoting cell cycle progression through the cell cycle-inhibitory protein p27Kip1. We identify, structurally resolve, and tune two distinct molecular determinants driving the selective recognition of pTyr directly N- and C-terminal to the target phospho-acceptor tyrosine site. We further show structural and biochemical conservation in this recognition, and identify cancer-associated alterations to these determinants that are unable to recognize phosphoprimed substrates. Finally, using an in vivo mouse model of leukemia we show that Bcr-Abl mutants unable to recognize phosphoprimed substrates paradoxically result in enhanced tumor development and progression. These data indicate that Bcr-Abl, like other proto-oncogenes such as Ras or Myc, engages both pro- and anti-oncogenic programs - but in the case of Bcr-Abl, this is accomplished through a mechanism involving traditional and phosphoprimed substrate recognition.

biochemistry↗

bioRxiv2024

Directed evolution-based discovery of ligands for in vivo restimulation of CAR-T cells

Chimeric antigen receptor (CAR) T cell therapy targeting CD19 elicits remarkable clinical efficacy in B-cell malignancies, but many patients relapse due to failed expansion and/or progressive loss of CAR-T cells. We recently reported a strategy to potently restimulate CAR-T cells in vivo, enhancing their functionality by administration of a vaccine-like stimulus comprised of surrogate peptide ligands for a CAR linked to a lymph node-targeting amphiphilic PEG-lipid (termed CAR-T-vax). Here, we demonstrate a general strategy to generate and optimize peptide mimotopes enabling CAR-T-vax generation for any CAR. Using the clinical CD19 CAR (FMC63) as a test case, we employed yeast surface display to identify peptide binders to soluble IgG versions of FMC63, which were subsequently affinity matured by directed evolution. CAR-T vaccines using these optimized mimotopes triggered marked expansion and memory development of CD19 CAR-T cells in both syngeneic and humanized mouse models of B-ALL/Lymphoma, and enhanced control of disease progression. This approach thus enables vaccine boosting to be applied to any clinically relevant CAR-T cell product.

bioengineering↗

bioRxiv2018

Parp3 promotes long-range end-joining in murine cells

Chromosomal rearrangements, including translocations, are early and essential events in the formation of many tumors. Previous studies that defined the genetic requirements for rearrangement formation have identified differences between murine and human cells, most notably in the role of classical- and alternative-nonhomologous end joining factors (NHEJ). We reported that poly(ADP)ribose polymerase 3 (PARP3) promotes chromosomal rearrangements induced by endonucleases in multiple human cell types. In contrast to c-NHEJ factors, we show here that Parp3 also promotes rearrangements in murine cells, including translocations in murine embryonic stem cells (mESCs), class switch recombination in primary B cells and inversions in tail fibroblasts that generate Eml4-Alk fusions. In mESCs, Parp3-deficient cells had shorter deletion lengths at translocation junctions. This was corroborated using next-generation sequencing of Eml4-Alk junctions in tail fibroblasts and is consistent with a role for Parp3 in promoting the processing of DNA double-strand breaks. We confirmed a previous report that Parp1 also promotes rearrangement formation. In contrast with Parp3, rearrangement junctions in the absence of Parp1 had longer deletion lengths, suggesting Parp1 may suppress DSB processing. Together, these data indicate that Parp3 and Parp1 promote rearrangements with distinct phenotypes.

cancer biology↗