Stepwise engineering of human germinal center B cells to model dark zone high-grade B cell lymphoma driven by sequential BCL2 and MYC deregulation
Bibliographic record
Abstract
Abstract High-grade B-cell lymphomas (HGBCL) encompass a heterogeneous group of aggressive B-cell malignancies. The most common genetically defined subtype, HGBCL with BCL2 and MYC rearrangements (HGBCL-DH-BCL2), is characterized by hallmark chromosomal translocations and poor prognosis, even in the era of improved chemo-immunotherapy, CAR-T cells, and antibody-drug conjugates1-3. Progress in this field is hindered by the lack of robust preclinical models that accurately capture the complex biology of the disease. Our recent work has reported recurrent silencing of the B cell receptor (BCR) in primary cases of HGBCL-DH-BCL2⁴. This phenotype is associated with gene expression programs characteristic of germinal center (GC) dark-zone (DZ) B cells4,5. Histological analyses have also established that HGBCL-DH-BCL2 (particularly those silencing the BCR), display an immune-cold microenvironment that closely resembles that of the GC dark zone5,6. Mutational analyses in HGBCL-DH-BCL2 have revealed recurrent gain-of-function mutations in the GC dark-zone determinant FOXO15. In addition, HGBCL-DH-BCL2 frequently inactivate the TP53 axis and share with Burkitt lymphoma expression of degron-resistant forms of CCND3 (i.e., CCND3T283A)5. HGBCL-DH-BCL2 is often preceded by follicular lymphoma (FL), characterized by BCL2 deregulation as its molecular hallmark. Finally, constitutive expression of an often mutant form of the MYC protein (i.e. MYCT58A) in a BCL2-rearranged FL or t(14;18)+ GC DZ B cell precursor represents the ultimate oncogenic event driving transformation to HGBCL-DH-BCL2. To model this transformation trajectory, we adapted a previously established retroviral transduction system7 for genetic engineering of primary human GC B cells. We constructed retroviral vectors encoding wild-type or mutant forms of lymphoma-relevant genes linked to fluorescent or surface markers for single-cell tracking. Primary GC B cells purified from human tonsils were first transduced with a four-gene cocktail (Oncomix-1) expressing BCL2, BCL6, FOXO1M1L, and dominant-negative TP53 (TP53DD). These genes cooperatively promoted survival and proliferation for > 4 weeks in vitro on CD40L/IL-21-expressing YK6 feeder cells. However, engineered cells remained dependent on exogenous stimulation and failed to form tumors in immunocompromised mice. To mimic the MYC oncogenic event driving HGBCL-DH-BCL2 onset, Oncomix-1-infected cells were superinfected with a second retroviral cocktail (Oncomix-2) expressing mutant MYCT58A and CCND3T283A. This step conferred mitogen independence, rapid proliferation in 2D culture, and tumor formation in NSG xenografts, hallmarks of transformation. In vivo tumors showed histological features of aggressive GCB-like lymphomas. Given that most HGBCL-DH-BCL2 silence their BCR, we sought to reproduce this phenotype. We engineered Oncomix-1-infected cells with an alternative Step-2 retroviral cocktail (Oncomix-3) combining MYCT58A and CCND3T283A, to wild-type KLHL6, a GC DZ-enriched BTB-domain adaptor that targets the BCR subunit CD79B for proteasomal degradation8,9. Deregulated KLHL6 expression supported the emergence of a rapidly expanding, BCR-negative GC immortalized B cell population. These cells showed a GC DZ-like immunophenotype and feeder-independent growth, closely mimicking the phenotype of BCR-silenced HGBCL-DH-BCL2. In summary, we established a stepwise viral transduction protocol to model the progressive transformation of human GC B cells into DZ–like HGBCL, driven by consecutive deregulation of the BCL2 and MYC oncoproteins. This model recapitulates key transcriptional, immunophenotypic, and oncogenic features of HGBCL-DH-BCL2 offering a versatile tool for mechanistic dissection (including immune evasion) and pharmacologic interrogation. References Melani C et al. N Engl J Med. 2024. doi:10.1056/NEJMoa2401532. Phina-Ziebin X et al. Blood Adv. 2025. doi:10.1182/bloodadvances.2024014732 Schneider M et al. Clin Lymphoma Myeloma Leuk. 2025. doi: 10.1016/j.clml.2024.08.010. Hilton LK et al. Blood. 2024. doi: 10.1182/blood.2024024251 Varano G et al. Blood Cancer Discov. 2025. doi: 10.1158/2643-3230.BCD-25-0099 Cancila V et al. J Clin Invest. 2025.doi: 10.1172/JCI187371 Caeser R et al. Nat Commun. 2019. Doi: 10.1038/s41467-019-12494-x Meriranta L et al. Blood Cancer Discov. 2024. doi: 10.1158/2643-3230.BCD-23-0182 Corcoran SR et al. Cancer Discov. 2024. doi: 10.1158/2159-8290.CD-23-0802
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.001 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.001 |
| Insufficient payload (model declined to judge) | 0.001 | 0.001 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".