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The Journal of Clinical Investigation Jun 2024Clinical trials have suggested antitumor activity from PARP inhibition beyond homologous recombination deficiency (HRD). RNASEH2B loss is unrelated to HRD and...
BACKGROUND
Clinical trials have suggested antitumor activity from PARP inhibition beyond homologous recombination deficiency (HRD). RNASEH2B loss is unrelated to HRD and preclinically sensitizes to PARP inhibition. The current study reports on RNASEH2B protein loss in advanced prostate cancer and its association with RB1 protein loss, clinical outcome and clonal dynamics during treatment with PARP inhibition in a prospective clinical trial.
METHODS
Whole tumor biopsies from multiple cohorts of patients with advanced prostate cancer were interrogated using whole-exome sequencing (WES), RNA sequencing (bulk and single nucleus) and immunohistochemistry (IHC) for RNASEH2B and RB1. Biopsies from patients treated with olaparib in the TOPARP-A and TOPARP-B clinical trials were used to evaluate RNASEH2B clonal selection during olaparib treatment.
RESULTS
Shallow co-deletion of RNASEH2B and adjacent RB1, co-located at chromosome 13q14, was common, deep co-deletion infrequent, and gene loss associated with lower mRNA expression. In castration-resistant PC (CRPC) biopsies, RNASEH2B and RB1 mRNA expression correlated, but single nucleus RNA sequencing indicated discordant loss of expression. IHC studies showed that loss of the two proteins often occurred independently, arguably due to stochastic second allele loss. Pre- and post-treatment metastatic CRPC (mCRPC) biopsy studies from BRCA1/2 wildtype tumors, treated on the TOPARP phase II trial, indicated that olaparib eradicates RNASEH2B-loss tumor subclones.
CONCLUSION
PARP inhibition may benefit men suffering from mCRPC by eradicating tumor subclones with RNASEH2B loss.
CLINICALTRIALS
gov NCT01682772FUNDING. AstraZeneca; Cancer Research UK; Medical Research Council; Cancer Research UK; Prostate Cancer UK; Movember Foundation; Prostate Cancer Foundation.
PubMed: 38833311
DOI: 10.1172/JCI178278 -
Peripheral apoptosis and limited clonal deletion during physiologic murine B lymphocyte development.Nature Communications Jun 2024Self-reactive and polyreactive B cells generated during B cell development are silenced by either apoptosis, clonal deletion, receptor editing or anergy to avoid...
Self-reactive and polyreactive B cells generated during B cell development are silenced by either apoptosis, clonal deletion, receptor editing or anergy to avoid autoimmunity. The specific contribution of apoptosis to normal B cell development and self-tolerance is incompletely understood. Here, we quantify self-reactivity, polyreactivity and apoptosis during physiologic B lymphocyte development. Self-reactivity and polyreactivity are most abundant in early immature B cells and diminish significantly during maturation within the bone marrow. Minimal apoptosis still occurs at this site, however B cell receptors cloned from apoptotic B cells show comparable self-reactivity to that of viable cells. Apoptosis increases dramatically only following immature B cells leaving the bone marrow sinusoids, but above 90% of cloned apoptotic transitional B cells are not self-reactive/polyreactive. Our data suggests that an apoptosis-independent mechanism, such as receptor editing, removes most self-reactive B cells in the bone marrow. Mechanistically, lack of survival signaling rather than clonal deletion appears to be the underpinning cause of apoptosis in most transitional B cells in the periphery.
Topics: Animals; Apoptosis; Clonal Deletion; B-Lymphocytes; Mice; Mice, Inbred C57BL; Receptors, Antigen, B-Cell; Cell Differentiation; Bone Marrow; Female; Precursor Cells, B-Lymphoid
PubMed: 38824171
DOI: 10.1038/s41467-024-49062-x -
Biomarker Research May 2024Chimeric antigen receptor T cell (CAR-T) therapy has revolutionized the treatment approach for cancer, autoimmune disease, and heart disease. The integration of CAR into... (Review)
Review
Chimeric antigen receptor T cell (CAR-T) therapy has revolutionized the treatment approach for cancer, autoimmune disease, and heart disease. The integration of CAR into T cells is typically facilitated by retroviral or lentiviral vectors. However, the random insertion of CARs can lead to issues like clonal expansion, oncogenic transformation, variegated transgene expression, and transcriptional silencing. The advent of precise gene editing technology, like Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), allows for controlled and precise genome modification, facilitating the translation of CAR-T research to the clinical applications. This review aims to provide a comprehensive analysis of the application of CRISPR gene editing techniques in the context of precise deletion and insertion methodologies, with a specific focus on their potential for enhancing the development and utilization of CAR-T cell therapy.
PubMed: 38816881
DOI: 10.1186/s40364-024-00602-z -
Cell Reports May 2024Resolving the molecular mechanisms of central B cell tolerance might unveil strategies that prevent autoimmunity. Here, using a mouse model of central B cell tolerance...
Resolving the molecular mechanisms of central B cell tolerance might unveil strategies that prevent autoimmunity. Here, using a mouse model of central B cell tolerance in which Forkhead box protein O1 (Foxo1) is either deleted or over-expressed in B cells, we show that deleting Foxo1 blocks receptor editing, curtails clonal deletion, and decreases CXCR4 expression, allowing high-avidity autoreactive B cells to emigrate to the periphery whereby they mature but remain anergic and short lived. Conversely, expression of degradation-resistant Foxo1 promotes receptor editing in the absence of self-antigen but leads to allelic inclusion. Foxo1 over-expression also restores tolerance in autoreactive B cells harboring active PI3K, revealing opposing roles of Foxo1 and PI3K in B cell selection. Overall, we show that the transcription factor Foxo1 is a major gatekeeper of central B cell tolerance and that PI3K drives positive selection of immature B cells and establishes allelic exclusion by suppressing Foxo1.
PubMed: 38796853
DOI: 10.1016/j.celrep.2024.114283 -
EBioMedicine May 2024Immune therapies represented by immune checkpoint blockade (ICB) have significantly transformed cancer treatment. However, the effectiveness of these treatments depends... (Review)
Review
Immune therapies represented by immune checkpoint blockade (ICB) have significantly transformed cancer treatment. However, the effectiveness of these treatments depends on the status of T cells. T cell exhaustion, characterized by diminished effector function, increased expression of co-inhibitory receptors, and clonal deletion, emerges as a hypofunctional state resulting from chronic exposure to antigens, posing an obstacle to ICB therapy. Several studies have deeply explored T cell exhaustion, providing innovative insights and correlating T cell exhaustion with tertiary lymphoid structures (TLS) formation. TLS, lymphocyte aggregates formed in non-lymphoid tissues amid chronic inflammation, serve as pivotal reservoirs for anti-tumour immunity. Here, we underscore the pivotal role of T cell exhaustion as a signalling mechanism in reinvigorating anti-tumour immunity by turbocharging cancer-immunity (CI) cycle, particularly when tumour becomes unmanageable. Building upon this concept, we summarize emerging immunotherapeutic strategies aimed at enhancing the response rate to ICB therapy and improving patient prognosis.
PubMed: 38749300
DOI: 10.1016/j.ebiom.2024.105154 -
Current Oncology (Toronto, Ont.) Apr 2024Myelodysplastic neoplasms (MDS) are a heterogenous clonal disorder of hemopoietic stem cells characterized by cytomorphologic dysplasia, ineffective hematopoiesis,... (Review)
Review
Myelodysplastic neoplasms (MDS) are a heterogenous clonal disorder of hemopoietic stem cells characterized by cytomorphologic dysplasia, ineffective hematopoiesis, peripheral cytopenias and risk of progression to acute myeloid leukemia (AML). Our understanding of this disease has continued to evolve over the last century. More recently, prognostication and treatment have been determined by cytogenetic and molecular data. Specific genetic abnormalities, such as deletion of the long arm of chromosome 5 (del(5q)), TP53 inactivation and SF3B1 mutation, are increasingly associated with disease phenotype and outcome, as reflected in the recently updated fifth edition of the World Health Organization Classification of Hematolymphoid Tumors (WHO5) and the International Consensus Classification 2022 (ICC 2022) classification systems. Treatment of lower-risk MDS is primarily symptom directed to ameliorate cytopenias. Higher-risk disease warrants disease-directed therapy at diagnosis; however, the only possible cure is an allogenic bone marrow transplant. Novel treatments aimed at rational molecular and cellular pathway targets have yielded a number of candidate drugs over recent years; however few new approvals have been granted. With ongoing research, we hope to increasingly offer our MDS patients tailored therapeutic approaches, ultimately decreasing morbidity and mortality.
Topics: Humans; Myelodysplastic Syndromes
PubMed: 38668051
DOI: 10.3390/curroncol31040148 -
International Journal of Infectious... Jul 2024The accuracy of malaria rapid diagnostic tests is threatened by Plasmodium falciparum with pfhrp2/3 deletions. This study compares gene deletion prevalence determined by... (Comparative Study)
Comparative Study
Comparison of prevalence estimates of pfhrp2 and pfhrp3 deletions in Plasmodium falciparum determined by conventional PCR and multiplex qPCR and implications for surveillance and monitoring.
OBJECTIVES
The accuracy of malaria rapid diagnostic tests is threatened by Plasmodium falciparum with pfhrp2/3 deletions. This study compares gene deletion prevalence determined by multiplex real time polymerase chain reaction (qPCR) and conventional polymerase chain reaction (cPCR) using existing samples with clonality previously determined by microsatellite genotyping.
METHODS
Multiplex qPCR was used to estimate prevalence of pfhrp2/3 deletions in three sets of previously collected patient samples from Eritrea and Peru. The qPCR was validated by multiplex digital polymerase chain reaction. Sample classification was compared with cPCR, and receiver operating characteristic curve analysis was used to determine the optimal ΔCq threshold that aligned the results of the two assays.
RESULTS
qPCR classified 75% (637 of 849) of samples as single, and 212 as mixed-pfhrp2/3 genotypes, with a positive association between clonality and proportion of mixed-pfhrp2/3 genotype samples. The sample classification agreement between cPCR and qPCR was 75.1% (95% confidence interval [CI] 68.6-80.7%) and 47.8% (95% CI 38.9-56.9%) for monoclonal and polyclonal infections. The qPCR prevalence estimates of pfhrp2/3 deletions showed almost perfect (κ = 0.804, 95% CI 0.714-0.895) and substantial agreement (κ = 0.717, 95% CI 0.562-0.872) with cPCR for Peru and 2016 Eritrean samples, respectively. For 2019 Eritrean samples, the prevalence of double pfhrp2/3 deletions was approximately two-fold higher using qPCR. The optimal threshold for matching the assay results was ΔCq = 3.
CONCLUSIONS
Multiplex qPCR and cPCR produce comparable estimates of gene deletion prevalence when monoclonal infections dominate; however, qPCR provides higher estimates where multi-clonal infections are common.
Topics: Plasmodium falciparum; Humans; Malaria, Falciparum; Protozoan Proteins; Multiplex Polymerase Chain Reaction; Prevalence; Antigens, Protozoan; Gene Deletion; Real-Time Polymerase Chain Reaction; Peru; Genotype
PubMed: 38631508
DOI: 10.1016/j.ijid.2024.107061 -
BioRxiv : the Preprint Server For... Apr 2024How genetic lesions drive cell transformation and whether they can be circumvented without compromising function of non-transformed cells are enduring questions in...
How genetic lesions drive cell transformation and whether they can be circumvented without compromising function of non-transformed cells are enduring questions in oncology. Here we show that in mature T cells-in which physiologic clonal proliferation is a cardinal feature- constitutive transcription and loss in mice modeled aggressive human malignancy by reinforcing each other's oncogenic programs. This cooperation was supported by MYC-induced large neutral amino acid transporter chaperone SLC3A2 and dietary leucine, which in synergy with deletion overstimulated mTORC1 to promote mitochondrial fitness and MYC protein overexpression in a positive feedback circuit. A low leucine diet was therapeutic even in late-stage disease but did not hinder T cell immunity to infectious challenge, nor impede T cell transformation driven by constitutive nutrient mTORC1 signaling via loss. Thus, mTORC1 signaling hypersensitivity to leucine as an onco-nutrient enables an onco-circuit, decoupling pathologic from physiologic utilization of nutrient acquisition pathways.
PubMed: 38617314
DOI: 10.1101/2024.04.03.587917 -
BioRxiv : the Preprint Server For... May 2024Pan-genome analysis is a fundamental tool for studying bacterial genome evolution; however, the variety of methods used to define and measure the pan-genome poses...
Pan-genome analysis is a fundamental tool for studying bacterial genome evolution; however, the variety of methods used to define and measure the pan-genome poses challenges to the interpretation and reliability of results. To quantify sources of bias and error related to common pan-genome analysis approaches, we evaluated different approaches applied to curated collection of 151 ( ) isolates. is characterized by its clonal evolution, absence of horizontal gene transfer, and limited accessory genome, making it an ideal test case for this study. Using a state-of-the-art graph-genome approach, we found that a majority of the structural variation observed in originates from rearrangement, deletion, and duplication of redundant nucleotide sequences. In contrast, we found that pan-genome analyses that focus on comparison of coding sequences (at the amino acid level) can yield surprisingly variable results, driven by differences in assembly quality and the softwares used. Upon closer inspection, we found that coding sequence annotation discrepancies were a major contributor to inflated accessory genome estimates. To address this, we developed panqc, a software that detects annotation discrepancies and collapses nucleotide redundancy in pan-genome estimates. When applied to and pan-genomes, panqc exposed distinct biases influenced by the genomic diversity of the population studied. Our findings underscore the need for careful methodological selection and quality control to accurately map the evolutionary dynamics of a bacterial species.
PubMed: 38585972
DOI: 10.1101/2024.03.21.586149 -
The Journal of Pharmacology and... Apr 2024The oncogenic protein tyrosine phosphatase PTP4A3 is frequently overexpressed in human ovarian cancers and is associated with poor patient prognosis. PTP4A3 is thought...
Deletion of PTP4A3 phosphatase in high grade serous ovarian cancer cells decreases tumorigenicity and produces marked changes in intracellular signaling pathways and cytokine release.
The oncogenic protein tyrosine phosphatase PTP4A3 is frequently overexpressed in human ovarian cancers and is associated with poor patient prognosis. PTP4A3 is thought to regulate multiple oncogenic signaling pathways, including STAT3, SRC, and ERK. The objective of this study was to generate ovarian cancer cells with genetically depleted PTP4A3; to assess their tumorigenicity; to examine their cellular phenotype; and to uncover changes in their intracellular signaling pathways and cytokine release profiles. Genetic deletion of PTP4A3 using CRISPR/Cas9 enabled the generation of individual clones derived from single cells isolated from the polyclonal knockout population. We observed a >90% depletion of PTP4A3 protein levels by Western blotting in the clonal cell lines compared to the sham transfected wildtype population. The wildtype and polyclonal knockout cell lines shared similar monolayer growth rates, while the isolated clonal populations 2B4, 3C9, and 3C12 exhibited significantly lower monolayer growth characteristics consistent with their lower PTP4A3 levels. The clonal PTP4A3 knockout cell lines also had substantially lower colony formation efficiencies compared to the wildtype cells and were less tumorigenic The clonal knockout cells were markedly less responsive to IL-6-stimulated migration in a scratch wound assay compared to the wildtype cells. Antibody microarray assays documented differences in cytokine release and intracellular phosphorylation patterns in the PTP4A3 deleted clones. Bioinformatic network analyses indicated alterations in cellular signaling nodes. These biochemical changes could ultimately form the foundation for pharmacodynamic endpoints useful for emerging anti-PTP4A3 therapeutics. Clones of high grade serous ovarian cancer cells were isolated in which the oncogenic phosphatase PTP4A3 was deleted using CRISPR/Cas9 methodologies. The PTP4A3 null cells exhibited loss of proliferation, colony formation, and migration, and reduced tumorigenesis. Marked differences in intracellular protein phosphorylation and cytokine release were seen. The newly developed PTP4A3 knockout cells should provide useful tools to probe the role of PTP4A3 phosphatase in ovarian cancer cell survival, tumorigenicity and cell signaling.
PubMed: 38580450
DOI: 10.1124/jpet.124.002110