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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.

We used clustered regularly interspaced short palindromic repeats/Cas9-mediated genomic modification to investigate B-cell receptor (BCR) signaling in cell lines of diffuse large B-cell lymphoma (DLBCL). Three manipulations that altered BCR genes without affecting surface BCR levels showed that BCR signaling differs between the germinal center B-cell (GCB) subtype, which is insensitive to Bruton tyrosine kinase inhibition by ibrutinib, and the activated B-cell (ABC) subtype. Replacing antigen-binding BCR regions had no effect on BCR signaling in GCB-DLBCL lines, reflecting this subtype's exclusive use of tonic BCR signaling. Conversely, Y188F mutation in the immunoreceptor tyrosine-based activation motif of CD79A inhibited tonic BCR signaling in GCB-DLBCL lines but did not affect their calcium flux after BCR cross-linking or the proliferation of otherwise-unmodified ABC-DLBCL lines. CD79A-GFP fusion showed BCR clustering or diffuse distribution, respectively, in lines of ABC and GCB subtypes. Tonic BCR signaling acts principally to activate AKT, and forced activation of AKT rescued GCB-DLBCL lines from knockout (KO) of the BCR or 2 mediators of tonic BCR signaling, SYK and CD19. The magnitude and importance of tonic BCR signaling to proliferation and size of GCB-DLBCL lines, shown by the effect of BCR KO, was highly variable; in contrast, pan-AKT KO was uniformly toxic. This discrepancy was explained by finding that BCR KO-induced changes in AKT activity (measured by gene expression, CXCR4 level, and a fluorescent reporter) correlated with changes in proliferation and with baseline BCR surface density. PTEN protein expression and BCR surface density may influence clinical response to therapeutic inhibition of tonic BCR signaling in DLBCL.

Introduction. Targeting antigen-driven B-cell receptor (BCR) signaling with the BTK inhibitor ibrutinib is clinically effective against most B-cell lymphomas, including activated B-cell diffuse large B-cell lymphoma (ABC-DLBCL), but not germinal center B-cell (GCB) DLBCL. We have formally confirmed that GCB-DLBCL cell lines utilize tonic BCR signaling, by showing: 1) sensitivity (variable) to knockout (KO) of the BCR, SYK, and CD19; 2) dependence on CD79A ITAM phosphorylation; and 3) independence from BCR antigen specificity. However, uncertainty remains about molecular events in upstream parts of tonic BCR signaling, why dependence of GCB-DLBCL cells on tonic BCR signaling is variable, and their clinical relevance. Methods. We used CRISPR/Cas9 methods to modify selected genes by KO and/or knock-in (KI) of the cDNA of a fluorescent protein (FP; e.g., GFP), with the FP serving as a marker of cells with gene KO or modification, or as a gene-fused tag for localization or quantitation. Cells expressing a membrane-targeted Forster resonance energy transfer (FRET) based AKT activity reporter (Lyn-AktAR2) were used to measure AKT activity directly by flow cytometry (FCM). Results. The effect of KI of CD79A Y188F mutation alone was similar to complete BCR KO, implying that CD79A Y188 phosphorylation is essential for tonic BCR signal transduction. Western blot analysis of GCB-DLBCL cell lines after BCR KO showed variable decreases of AKT S473 phosphorylation (frequently used as surrogate measure of AKT activity), but these did not correlate well with the variable decreases in proliferation of GCB-DLBCL cell lines caused by BCR KO. Measuring AKT activity directly (Fig. 1), or by another indirect approach (surface expression of CXCR4, a target gene of FOXO1 inhibited by AKT activity), showed high correlation between decreases in AKT activity and proliferation after BCR KO. In contrast to the variable effect of BCR KO on growth, pan-AKT KO was uniformly growth-slowing in GCB-DLBCL lines (Fig. 2). Interestingly, baseline surface density of BCR units in GCB lines, quantified by FCM using CD79A-GFP KI cells or anti-CD79B staining, correlated highly with reduction in growth or AKT activity caused by BCR KO (Fig. 3). These findings lead us to conclude that the BCR contributes to AKT activation in GCB-DLBCL cell lines, to a variable degree determined by BCR surface density. We also conclude that BCR surface density is determined by cell line-specific factors, as well as immunoglobulin heavy (IgH) and light (IgL) hypervariable region (HVR) sequences, based on measurements of BCR surface levels after exchanging endogenous HVR sequences in OCI-Ly19 and OCI-Ly7 cell lines for HVRs derived from other GCB and ABC-DLBCL cell lines. Reduction of AKT activity after BCR KO (measured by FRET reporter) and baseline BCR surface density in GCB-DLBCL cell lines also correlated well with the sensitivity of GCB-DLBCL lines to the clinically-tested SYK inhibitor (P505-15, PRT062607) or FDA-approved PI3K p110d isoform specific inhibitor (idelalisib). Interestingly, isogenic GCB-DLBCL cell lines with KO of PTEN, a negative regulator of AKT activation, were substantially more resistant to both inhibitors. A crucial role of PTEN deletion in overcoming dependence on tonic BCR signaling in GCB-DLBCL is supported by evidence from two naturally PTEN-deficient cell lines: SUDHL10, which adjusts to BCR KO and resumes normal growth, and HT, which lacks BCR expression, due to a frameshifting deletion in its IgH HVR. Re-expression of the BCR in HT, by KI to correct the IgH sequence, does not affect HT cell line growth. Conclusion. Our findings suggest a biomarker-guided therapeutic strategy in GCB-DLBCL: targeting tonic BCR signaling in BCR-high patients, by inhibiting CD79A phosphorylation, SYK, or PI3K, and downstream targeting of AKT in BCR-low and/or PTEN-deficient patients. Figure 1. Correlation of relative proliferation after BCR KO with decrease of AKT activity (as measured by FRET efficiency of AKT activity reporter) in GCB-DLBCL cell lines. Figure 1. Correlation of relative proliferation after BCR KO with decrease of AKT activity (as measured by FRET efficiency of AKT activity reporter) in GCB-DLBCL cell lines. Figure 2. Effect of BCR KO or pan-AKT KO in GCB-DLBCL cell lines. Figure 2. Effect of BCR KO or pan-AKT KO in GCB-DLBCL cell lines. Figure 3. Correlation of relative proliferation after BCR KO with baseline BCR surface density (as measured by flow cytometry of cells with CD79A-GFP fusion) in GCB-DLBCL cell lines. Figure 3. Correlation of relative proliferation after BCR KO with baseline BCR surface density (as measured by flow cytometry of cells with CD79A-GFP fusion) in GCB-DLBCL cell lines. Disclosures Burger: Pharmacyclics: Research Funding. Westin:Chugai: Membership on an entity's Board of Directors or advisory committees; Spectrum: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; ProNAi: Membership on an entity's Board of Directors or advisory committees.

Introduction. Targeting BCR signaling with the BTK inhibitor ibrutinib is clinically effective against most B-cell lymphomas, including the activated B-cell (ABC) subtype of diffuse large B-cell lymphoma (DLBCL), but not the germinal center B-cell (GCB) subtype. Active BCR signaling in GCB-DLBCL was suggested by studies with a Syk inhibitor and our previous studies using BCR knockout (KO). We addressed these questions: why is the BCR active in DLBCL, and how does it signal in GCB-DLBCL? Methods. We used CRISPR/Cas9 technology to modify selected genes by KO or homologous recombination-mediated knock-in (KI). For some genes KI was used to express a fluorescent protein (FP; e.g., GFP) instead of the targeted gene (KI/KO), or to modify the targeted gene together with KI of an FP, for detection of modified cells. Results. In GCB lines (OCI-Ly7 and OCI-Ly19) and ABC lines (U2932 and HBL-1), we simultaneously replaced the hypervariable region (HVR) exons of both immunoglobulin heavy (IgH) and light chains (IgL) with HVR sequences from normal B cells recognizing tetanus toxoid (TT). GFP and CFP respectively marked KI of IgH and IgL HVRs, and KI of the endogenous HVR sequences in each line served as controls. In CFP+/GFP+ cells, the TT specific BCR (TT-BCR) was expressed at similar or higher levels than the endogenous BCR (endo-BCR) and was functional, as shown by calcium flux in response to TT. The TT-BCR maintained growth of GCB lines (Fig. 1), indicating that they use "tonic", antigen-independent BCR signaling. Other features of tonic signaling were confirmed in more GCB lines: 1) the toxicity of BCR KO, which eliminates AKT S473 phosphorylation, was rescued by PTEN KO or expression of constitutively active AKT (mAKT), showing that BCR signaling serves principally to activate PI3K/AKT; and 2) KO of SYK or CD19, or truncation or ITAM mutation of the cytoplasmic tail of CD79A, none of which affect surface BCR levels, were as toxic as BCR KO but were non-toxic in BCR/PTEN double-KO cells. In contrast, the TT-BCR was as growth-slowing as BCR KO to the ABC line U2932 (Fig. 1), and substantially toxic to HBL-1, indicating that BCR signaling is self antigen-dependent in ABC-DLBCL. Reversion of somatic hypermutations in the U2932 HVRs was also as growth-slowing as BCR KO (Fig. 1), suggesting that self-antigen reactivity developed during BCR affinity maturation. Tonic signaling by the TT-BCR provided a detectable benefit (as compared to BCR KO) in PTEN-expressing HBL-1, whereas there was no difference between TT-HVR BCR and BCR KO in PTEN-deficient U2932. The surface TT-BCR level was higher than the endo-BCR level in ABC lines, and dropped with TT stimulation, suggesting that endo-BCRs in ABC lines undergo constant antigen stimulation with BCR internalization. The presumed self-antigen in ABC lines seems to be cell line-specific, since HVRs from ABC lines TMD8 and HBL-1 did not rescue growth of U2932. BCR KO in ABC lines was also not rescued by PTEN KO or mAKT. In cells whose BCRs were labeled by KI to fuse GFP to CD79A, super-resolution microscopy showed macro-clustering of BCR complexes at the surface of ABC line HBL-1, not seen in GCB lines (Fig. 2). Several findings suggested the clinical potential of targeting tonic BCR signaling in DLBCL: 1) clinical trial-stage inhibitors of SYK (P505-15) and PI3K (idelalisib) were toxic to GCB lines (less so with PTEN KO); 2) GCB lines (6/8) were sensitized by BCR KO to an in vitro CHOP-like regimen; 3) P505-15 or idelalisib sensitized GCB lines (3/3) to CHOP in vitro; and 4) evidence of tonic signaling in ABC line HBL-1 after removing antigen-driven signaling by HVR replacement. Conclusion. The BCR provides antigen-independent tonic signals to activate PI3K/AKT in GCB-DLBCL and antigen-dependent signaling in ABC-DLBCL. Targeting of B-cell specific tonic signling alone or in combination could be clinically effective in both types of DLBCL. Figure 1. Effect of BCR KO or HVR replacement in OCI-LY19 (A) and U2932 (B) cell lines. Endogenous IgH and IgL HVRs were replaced with HVR pairs (TT3 and/or TT6) recognizing tetanus toxoid, reverted to undo the effect of SHM, or restored with original HVRs. Figure 1. Effect of BCR KO or HVR replacement in OCI-LY19 (A) and U2932 (B) cell lines. Endogenous IgH and IgL HVRs were replaced with HVR pairs (TT3 and/or TT6) recognizing tetanus toxoid, reverted to undo the effect of SHM, or restored with original HVRs. Figure 2. Representative super-resolution images of BCR localization in live DLBCL cells. BCR labeled by CD79A-GFP fusion, surface membrane by CellMask staining. (bars = 5 µ m) Figure 2. Representative super-resolution images of BCR localization in live DLBCL cells. BCR labeled by CD79A-GFP fusion, surface membrane by CellMask staining. (bars = 5 µ m) Disclosures Westin: Spectrum: Research Funding.