Cell Growth Trajectories of B-Cell Lymphomas Are Defined By Oscillations between MYC- and BCL6-Dependent States

Background: In germinal centers, “centroblasts” and “centrocytes” transition between dark and light zones and thereby change their surface phenotype, proliferation rate and cell size. Likewise, during early stages of development, “large cycling” pre-B cells transition to “small resting” states, associated with profound changes of surface phenotype, proliferation rate and cell size.

Significance: Here we discovered that state transitions during early (bone marrow) and late (germinal centers) stages of B-cell development are tightly controlled by mutually exclusive activity of MYC or BCL6. In germinal center-derived B-cell lymphoma, MYC and BCL6 are frequently targeted by chromosomal translocations, suggesting that disruption of physiological state transitions may be part of the malignant transformation program.

Results: In parallel with dramatic changes of cell mass between BCL6+ MYC- (40 pg dry mass per cell) and BCL6- MYC+ states (90 pg dry mass per cell), we observed profound metabolic changes, suggesting that MYC promotes glycolysis and glutamine uptake, whereas BCL6 represses glycolytic activity and instead promotes phosphatidyl-ethanolamine (PE) and PE-dependent autophagy and cell shrinkage. Interestingly, BCL6 also repressed transcription of MYC, while STAT5, upstream of MYC, not only induced transcriptional activation of MYC but also transcriptional repression of BCL6.

To understand how MYC and BCL6 transition between mutually exclusive states, we developed a dual-knockin mNeonGreen-MYC and mScarlet-BCL6 reporter mouse model to monitor MYC and BCL6 protein expression in normal B-cells and B-cell lymphoma. Time lapse confocal imaging over the course of 14 hours revealed that single B-cell lymphoma cells transitioned through autonomous cycles of MYC and BCL6 phases of 2.5 to 4 hour duration. While these phases did not coincide with cell division, each division was preceded by a MYC phase, suggesting that MYC phases initiate but not always complete cell division. Extending these findings to pre-B cell acute lymphoblastic leukemia, we transduced dual-reporter bone marrow B-cells with BCR-ABL1 and NRAS^G12D to model human B-ALL. MYC+ cells exhibited larger size, higher clonogenicity, and glycolysis-enriched profiles, while BCL6+ cells were smaller with lower clonogenicity. Interestingly, single-cell sorting revealed that MYC+ and BCL6+ clones gave rise to mixed populations that oscillated between MYC and BCL6 states.

Using CRISPR editing, we generated MYC and BCL6 double-reporter human B-ALL PDX and lymphoma models. Remarkably, long-term live-cell imaging unveiled oscillatory expression patterns of MYC and BCL6, with reversed phases. Cells transitioned between MYC-dominant and BCL6-dominant stages, with MYC stages lasting 2-4 hours and BCL6 stages 5-6 hours.

ChIP-Seq data indicated MYC and BCL6 antagonistically regulate genes involved in glycolysis and mitochondrial function. Metabolomics and transcriptomics analyses showed MYC+ cells enriched in translation and protein synthesis, while BCL6+ cells were enriched in phospholipid synthesis and autophagy. Utilizing degron systems for MYC and PROTAC for BCL6 enabled precise degradation of MYC and BCL6 within 2 hours. MYC depletion led to cell shrinkage and amino acid depletion, whereas BCL6 degradation prevented shrinkage and autophagy. These findings suggest MYC and BCL6 oscillation reflects a balance between anabolic and catabolic metabolism, defining transitions between active and quiescent stages.

Conclusion: MYC is involved in biomass accumulation and provides fuel for cell division, while BCL6 confers a quiescent phenotype to cells and protects B cells from DNA damage-induced apoptosis. Our results suggest that normal B-cells and B-cell lymphomas use MYC and BCL6 to coordinate cell growth with quiescence, autophagy and turnover of damaged organelles and misfolded proteins. We propose that MYC phases are associated with a steep slope of cell growth, while BCL6 phases aligned with stalled cell growth. In this scenario, cell growth in normal and malignant B-cells is not linear but more in line with a sequence of risers (MYC) and treads (BCL6) in a stairway. The discovery of MYC-BCL6 state transitions will lead to new conceptual frameworks for the understanding of cell-size fluctuations, how recovery-periods regulate energy-supply and create opportunities for therapeutic intervention.