Stepwise engineering of human germinal center B cells to model dark zone high-grade B cell lymphoma driven by sequential BCL2 and MYC deregulation
Notice bibliographique
Résumé
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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Comment cette classification a été obtenuedéplier
Prédiction machine sur la base complète
Imitation des enseignantsNi prévalence calibrée, ni vérité terrain. Validation humaine à venir. Le volet Gemma est une étiquette directe du modèle pour chaque travail de la base, lue sur la notice réduite au titre. Le volet Codex est un classifieur appris des 10 348 étiquettes directes de Codex et calibré sur les taux pondérés de l'échantillon; les champs sans appui suffisant ne portent aucun appel Codex. Le mode candidate est l'union des deux volets; le consensus est leur intersection. Ces sorties portent le statut machine_predicted_unvalidated et ne sont pas des étiquettes humaines.
Scores du classifieur distillé par catégorie (deux têtes)
| Catégorie | Codex | Gemma |
|---|---|---|
| Métarecherche | 0,000 | 0,000 |
| Méta-épidémiologie (sens strict) | 0,000 | 0,000 |
| Méta-épidémiologie (sens large) | 0,000 | 0,000 |
| Bibliométrie | 0,000 | 0,000 |
| Études des sciences et des technologies | 0,000 | 0,000 |
| Communication savante | 0,001 | 0,000 |
| Science ouverte | 0,000 | 0,000 |
| Intégrité de la recherche | 0,000 | 0,001 |
| Charge utile insuffisante (le modèle a refusé de juger) | 0,001 | 0,001 |
Scores machine (provisoires)
Les deux têtes enseignantes du modèle étudiant, lues sur ce travail. Un score ordonne la base pour la relecture; il n'affirme jamais une catégorie, et le statut de validation accompagne chaque rangée tel quel.
Scores de référence d'un modèle non mature (critères de maturité non atteints, 7 itérations). Un score ordonne; il n'affirme jamais une catégorie.
score_only:v0-immature-baseline · tel quel depuis la passe de notation : score_only signifie que le nombre peut ordonner les travaux, et qu'aucune étiquette de catégorie n'en découleClassification
machine, non validéePrédiction automatique; un appel candidat d’une seule source (Gemma direct ou Codex distillé), pas un consensus.
Le détail, modèle par modèle et score par score, se trouve en fin de page sous « Comment cette classification a été obtenue ».