Detecting Förster resonance energy transfer in living cells by conventional and spectral flow cytometry.

Assays based on Förster resonance energy transfer (FRET) can be used to study many processes in cell biology. Although this is most often done with microscopy for fluorescence detection, we report two ways to measure FRET in living cells by flow cytometry. Using a conventional flow cytometer and the "3-cube method" for intensity-based calculation of FRET efficiency, we measured the enzymatic activity of specific kinases in cells expressing a genetically-encoded reporter. For both AKT and protein kinase A, the method measured kinase activity in time-course, dose-response, and kinetic assays. Using the Cytek Aurora spectral flow cytometer, which applies linear unmixing to emission measured in multiple wavelength ranges, FRET from the same reporters was measured with greater single-cell precision, in real time and in the presence of other fluorophores. Results from gene-knockout studies suggested that spectral flow cytometry might enable the sorting of cells on the basis of FRET. The methods we present provide convenient and flexible options for using FRET with flow cytometry in studies of cell biology.

Circulating tumor DNA in Hodgkin lymphoma.

Somatic mutations of genes involved in NF-κB, PI3K/AKT, NOTCH, and JAK/STAT signaling pathways play an important role in the pathogenesis of Hodgkin lymphoma (HL). HL tumor cells form only about 5% of the tumor mass; however, it was shown that HL tumor-derived DNA could be detected in the bloodstream. This circulating tumor DNA (ctDNA) reflects the genetic profile of HL tumor cells and can be used for qualitative and quantitative analysis of tumor-specific somatic DNA mutations within the concept of liquid biopsy. Overall, the most frequently mutated gene in HL is STAT6; however, the exact spectrum of mutations differs between individual HL histological subtypes. Importantly, reduction of ctDNA plasma levels after initial treatment is highly correlated with prognosis. Therefore, ctDNA shows great promise as a novel tool for non-invasive tumor genome analysis for biomarker driven therapy as well as for superior minimal residual disease monitoring and treatment resistance detection. Here, we summarize the recent advancements of ctDNA analysis in HL with focus on ctDNA detection methodologies, genetic profiling of HL and its clonal evolution, and the emerging prognostic value of ctDNA.

Impact of PIK3CA gain and PTEN loss on mantle cell lymphoma biology and sensitivity to targeted therapies.

Besides many other mutations in known cancer driver genes, mantle cell lymphoma (MCL) is characterized by recurrent genetic alterations of important regulators of the phosphoinositol-3-kinase (PI3K) cascade including PIK3CA gains and PTEN losses. To evaluate the biological and functional consequences of these aberrations in MCL, we have introduced transgenic expression of PIK3CA (PIK3CA UP) and performed knockout/knockdown of PTEN gene (PTEN KO / KD) in 5 MCL cell lines. The modified cell lines were tested for associated phenotypes including dependence on upstream B-cell receptor (BCR) signaling (by an additional BCR knockout). PIK3CA overexpression decreased the dependence of the tested MCL on prosurvival signaling from BCR, decreased levels of oxidative phosphorylation, and increased resistance to 2-deoxy-glucose, a glycolysis inhibitor. Unchanged AKT phosphorylation status and unchanged sensitivity to a battery of PI3K inhibitors suggested that PIK3CA gain might impact MCL cells in AKT independent manner. PTEN KO was associated with a more distinct phenotype: AKT hyperphosphorylation and overactivation, increased resistance to multiple inhibitors (most of the tested PI3K inhibitors, BTK inhibitor ibrutinib, and BCL2 inhibitor venetoclax), increased glycolytic rates with resistance to 2-deoxy-glucose, and significantly decreased dependence on prosurvival BCR signaling. Our results suggest that the frequent aberrations of the PI3K pathway may rewire associated signaling with lower dependence on BCR signaling, better metabolic and hypoxic adaptation, and targeted therapy resistance in MCL.