Divergent HLA variations and heterogeneous expression but recurrent HLA loss-of- heterozygosity and common HLA-B and TAP transcriptional silencing across advanced pediatric solid cancers

The human leukocyte antigen (HLA) system is a major factor controlling cancer immunosurveillance and response to immunotherapy, yet its status in pediatric cancers remains fragmentary. We determined high-confidence HLA genotypes in 576 children, adolescents and young adults with recurrent/refractory solid tumors from the MOSCATO-01 and MAPPYACTS trials, using normal and tumor whole exome and RNA sequencing data and benchmarked algorithms. There was no evidence for narrowed HLA allelic diversity but discordant homozygosity and allele frequencies across tumor types and subtypes, such as in embryonal and alveolar rhabdomyosarcoma, neuroblastoma MYCN and 11q subtypes, and high-grade glioma, and several alleles may represent protective or susceptibility factors to specific pediatric solid cancers. There was a paucity of somatic mutations in HLA and antigen processing and presentation (APP) genes in most tumors, except in cases with mismatch repair deficiency or genetic instability. The prevalence of loss-of-heterozygosity (LOH) ranged from 5.9 to 7.7% in HLA class I and 8.0 to 16.7% in HLA class II genes, but was widely increased in osteosarcoma and glioblastoma (~15-25%), and for DRB1-DQA1-DQB1 in Ewing sarcoma (~23-28%) and low-grade glioma (~33-50%). HLA class I and HLA-DR antigen expression was assessed in 194 tumors and 44 patient-derived xenografts (PDXs) by immunochemistry, and class I and APP transcript levels quantified in PDXs by RT-qPCR. We confirmed that HLA class I antigen expression is heterogeneous in advanced pediatric solid tumors, with class I loss commonly associated with the transcriptional downregulation of HLA-B and transporter associated with antigen processing (TAP) genes, whereas class II antigen expression is scarce on tumor cells and occurs on immune infiltrating cells. Patients with tumors expressing sufficient HLA class I and TAP levels such as some glioma, osteosarcoma, Ewing sarcoma and non-rhabdomyosarcoma soft-tissue sarcoma cases may more likely benefit from T cell-based approaches, whereas strategies to upregulate HLA expression, to expand the immunopeptidome, and to target TAP-independent epitopes or possibly LOH might provide novel therapeutic opportunities in others. The consequences of HLA class II expression by immune cells remain to be established. Immunogenetic profiling should be implemented in routine to inform immunotherapy trials for precision medicine of pediatric cancers

The collagen-like protein gp12 is a temperature-dependent reversible binder of SPP1 viral capsids.

International audienceIcosahedral capsids of viruses are lattices of defined geometry and homogeneous size. The (quasi-)equivalent organization of their protein building blocks provides, in numerous systems, the binding sites to assemble arrays of viral polypeptides organized with nanometer precision that protrude from the capsid surface. The capsid of bacterial virus (bacteriophage) SPP1 exposes, at its surface, the 6.6-kDa viral polypeptide gp12 that binds to the center of hexamers of the major capsid protein. Gp12 forms an elongated trimer with collagen-like properties. This is consistent with the fold of eight internal GXY repeats of gp12 to build a stable intersubunit triple helix in a prokaryotic setting. The trimer dissociates and unfolds at near physiological temperatures, as reported for eukaryotic collagen. Its structural organization is reacquired within seconds upon cooling. Interaction with the SPP1 capsid hexamers strongly stabilizes gp12, increasing its Tm to 54 °C. Above this temperature, gp12 dissociates from its binding sites and unfolds reversibly. Multivalent binding of gp12 trimers to the capsid is highly cooperative. The capsid lattice also provides a platform to assist folding and association of unfolded gp12 polypeptides. The original physicochemical properties of gp12 offer a thermoswitchable system for multivalent binding of the polypeptide to the SPP1 capsid surface

Two-Dimensional Phase Transition of Viral Capsid Gives Insights into Subunit Interactions

International audienc

Characterization of the Assembly and Disassembly of Capsid Proteins Derived from Hepatitis B Virus

International audienc

Atomic level description of the Binding of the Cancer-Related TCTP Protein to the sertraline drug and to the anti-apoptotic Mcl-1 protein

National audienc

Revisiting the molecular interactions between the tumor protein TCTP and the drugs sertraline/thioridazine.

International audienceTCTP protein is a pharmacological target in cancer and TCTP inhibitors such as sertraline have been evaluated in clinical trials. The direct interaction of TCTP with the drugs sertraline and thioridazine has been reported in vitro by SPR experiments to be in the ~30-50 µM Kd range (Amson et al. Nature Med 2012), supporting a TCTP-dependent mode of action of the drugs on tumor cells. However, the molecular details of the interaction remain elusive although they are crucial to improve the efforts of on-going medicinal chemistry. In addition, TCTP can be phosphorylated by the Plk-1 kinase, which is indicative of poor prognosis in several cancers. The impact of phosphorylation on TCTP structure/dynamics and binding with therapeutical ligands remains unexplored. Here, we combined NMR, TSA, SPR, BLI and ITC techniques to probe the molecular interactions between TCTP with the drugs sertraline and thioridazine. We reveal that drug binding is much weaker than reported with an apparent ~mM Kd and leads to protein destabilization that obscured the analysis of the published SPR data. We further demonstrate by NMR and SAXS that TCTP S46 phosphorylation does not promote tighter interaction between TCTP and sertraline. Accordingly, we question the supported model in which sertraline and thioridazine directly interact with isolated TCTP in tumor cells and discuss alternative modes of action for the drugs in light of current literature

Atomic level description of the Binding of the Cancer-Related TCTP Protein to the sertraline drug and to the anti-apoptotic Mcl-1 protein

National audienc

The Phosin PptA plays a negative role in the regulation of antibiotic production in Streptomyces lividansStreptomyces\ lividans

International audienceIn Streptomyces, antibiotic biosynthesis is triggered in phosphate limitation that is usually correlated with energetic stress. Polyphosphates constitute an important reservoir of phosphate and energy and a better understanding of their role in the regulation of antibiotic biosynthesis is of crucial importance. We previously characterized a gene, SLI_4384/ppk, encoding a polyphosphate kinase, whose disruption greatly enhanced the weak antibiotic production of Streptomyces lividans. In the condition of energetic stress, Ppk utilizes polyP as phosphate and energy donor, to generate ATP from ADP. In this paper, we established that ppk is co-transcribed with its two downstream genes, SLI_4383, encoding a phosin called PptA possessing a CHAD domain constituting a polyphosphate binding module and SLI_4382 encoding a nudix hydrolase. The expression of the ppk/pptA/SLI_4382 operon was shown to be under the positive control of the two-component system PhoR/PhoP and thus mainly expressed in condition of phosphate limitation. However, pptA and SLI_4382 can also be transcribed alone from their own promoter. The deletion of pptA resulted into earlier and stronger actinorhodin production and lower lipid content than the disruption of ppk, whereas the deletion of SLI_4382 had no obvious phenotypical consequences. The disruption of ppk was shown to have a polar effect on the expression of pptA, suggesting that the phenotype of the ppk mutant might be linked, at least in part, to the weak expression of pptA in this strain. Interestingly, the expression of phoR/phoP and that of the genes of the pho regulon involved in phosphate supply or saving were strongly up-regulated in pptA and ppk mutants, revealing that both mutants suffer from phosphate stress. Considering the presence of a polyphosphate binding module in PptA, but absence of similarities between PptA and known exo-polyphosphatases, we proposed that PptA constitutes an accessory factor for exopolyphosphatases or general phosphatases involved in the degradation of polyphosphates into phosphate

Structural studies of cyclodipeptide synthases with RNA microhelices mimicking their natural tRNA susbtrates

International audienc