RESUMO
Genomic approaches are widely applied to study the genetic basis of antifungal drug resistance in clinical isolates and experimental studies. Whole-genome sequencing of clinical isolates can comprehensively identify mutations associated with drug resistance and their frequency across fungal populations. In addition, genome comparison of serially collected isolates, such as from patient samples or in vitro drug selection experiments, will identify a small number of changes that can be evaluated for association with drug resistance. Here, we provide a detailed protocol for the computational analysis of genome sequences to identify variants associated with drug resistance.
Assuntos
Farmacorresistência Fúngica , Genômica , Humanos , Mutação , Farmacorresistência Fúngica/genética , Sequenciamento Completo do Genoma/métodos , Antifúngicos/farmacologia , Estudo de Associação Genômica AmplaRESUMO
In chronic lymphocytic leukemia (CLL), B-cell receptor signaling, tumor-microenvironment interactions, and somatic mutations drive disease progression. To better understand the intersection between the microenvironment and molecular events in CLL pathogenesis, we integrated bulk transcriptome profiling of paired peripheral blood (PB) and lymph node (LN) samples from 34 patients. Oncogenic processes were upregulated in LN compared with PB and in immunoglobulin heavy-chain variable (IGHV) region unmutated compared with mutated cases. Single-cell RNA sequencing (scRNA-seq) distinguished 3 major cell states: quiescent, activated, and proliferating. The activated subpopulation comprised only 2.2% to 4.3% of the total tumor bulk in LN samples. RNA velocity analysis found that CLL cell fate in LN is unidirectional, starts in the proliferating state, transitions to the activated state, and ends in the quiescent state. A 10-gene signature derived from activated tumor cells was associated with inferior treatment-free survival (TFS) and positively correlated with the proportion of activated CD4+ memory T cells and M2 macrophages in LN. Whole exome sequencing (WES) of paired PB and LN samples showed subclonal expansion in LN in approximately half of the patients. Since mouse models have implicated activation-induced cytidine deaminase in mutagenesis, we compared AICDA expression between cases with and without clonal evolution but did not find a difference. In contrast, the presence of a T-cell inflamed microenvironment in LN was associated with clonal stability. In summary, a distinct minor tumor subpopulation underlies CLL pathogenesis and drives the clinical outcome. Clonal trajectories are shaped by the LN milieu, where T-cell immunity may contribute to suppressing clonal outgrowth. The clinical study is registered at clinicaltrials.gov as NCT00923507.
Assuntos
Leucemia Linfocítica Crônica de Células B , Camundongos , Animais , Leucemia Linfocítica Crônica de Células B/patologia , Heterogeneidade Genética , Região Variável de Imunoglobulina/genética , Transdução de Sinais , Progressão da Doença , Microambiente Tumoral/genéticaRESUMO
Mitochondrial apoptosis can be effectively targeted in lymphoid malignancies with the FDA-approved B cell lymphoma 2 (BCL-2) inhibitor venetoclax, but resistance to this agent is emerging. We show that venetoclax resistance in chronic lymphocytic leukemia is associated with complex clonal shifts. To identify determinants of resistance, we conducted parallel genome-scale screens of the BCL-2-driven OCI-Ly1 lymphoma cell line after venetoclax exposure along with integrated expression profiling and functional characterization of drug-resistant and engineered cell lines. We identified regulators of lymphoid transcription and cellular energy metabolism as drivers of venetoclax resistance in addition to the known involvement by BCL-2 family members, which were confirmed in patient samples. Our data support the implementation of combinatorial therapy with metabolic modulators to address venetoclax resistance.