Apoptotic Blocks in Primary Non-Hodgkin B-Cell Lymphomas Identified By BH3 Profiling
Bibliographic record
Abstract
Abstract Background: The anti-apoptotic protein BCL2 is expressed in most non-Hodgkin lymphomas (NHL) including chronic lymphocytic leukemias (CLL), mantle cell lymphomas (MCL), follicular lymphomas (FL), diffuse large B cell lymphomas (DLBCL) and marginal zone lymphomas (MZL). BCL2 expression is associated with an inferior survival when co-expressed with MYC, an oncogene that induces cellular proliferation, especially in high-grade lymphomas with translocations in MYC and BCL2(HGBL-DH). Venetoclax, a selective BCL2 inhibitor, is effective in CLL, but has modest clinical activity in other NHLs. CLL has been shown to be "primed" and BCL2-dependent, predicting for a favorable response to venetoclax using BH3 profiling. This technique measures mitochondrial outer membrane depolarization (MOMP) after exposure to synthetic pro-apoptotic BH3 peptides that have different affinities to anti-apoptotic proteins BCL2, BCLXL, MCL1 and BCLW. Cells with functional BAX/BAK undergo MOMP after exposure to pro-apoptotic proteins BIM/BID. "Primed" cells undergo MOMP after exposure to BIM, BID and PUMA, where the latter inhibits all anti-apoptotic proteins. Thus, inhibiting the anti-apoptotic proteins would initiate apoptosis. We hypothesized that BH3 profiling of NHLs could help us understand the mechanisms of venetoclax-resistance in BCL2+ NHL. This has not yet been performed due to the rarity of samples archived as a viable cell suspension. Methods: We performed BH3 profiling on 138 B-cell NHLs (36 CLL/SLL, 42 FL, 38 DLBCL, 10 HGBL-DH, 13 MCL, and 3 MZLs) and 34 controls (14 tonsils and 20 peripheral blood mononuclear cells). NHLs were taken prior to (n=107) or after therapy (n= 31). A T-test and ANOVA were performed to determine the differences in MOMP between NHLs subtypes and normal B cells. We also profiled HGBL-DH cell lines to determine if chemotherapy (cyclophosphamide, doxorubicin, vincristine, dexamethasone and bendamustine) could potentiate venetoclax-responses. We measured the levels of anti-apoptotic proteins and MYC by immunoblot, speculating that proteins with short half-lives (MYC and MCL1) may fall upon cell-cycle arrest. Results: Normal B and T lymphocytes were primed and depended on BCL2, MCL1 and BCL-XL. NHLs were primed in 85% (121/138) of cases, 5% (6/138) were unprimed (no PUMA response) and 10% (14/138) were incompetent to undergo MOMP (no BIM/BID response), indicating dysfunctional BAX/BAK. The latter apoptotic defect was mainly seen in DLBCL (10/14). CLL and HGBL-DH had significantly higher venetoclax responses than DLBCL, MCL and FL (all p <0.05). Responses to venetoclax were only observed in samples that expressed BCL2 protein. In BCL2+ NHLs, increased MOMP was achieved by combining venetoclax with the MCL1-specific inhibitor MS1. Inhibiting BCLXL or BCLW did not increase MOMP, suggesting that MCL1 inhibits apoptosis in the absence of BCL2. Profiling HGBL-DH cell lines OCI-Ly8 and Toledo after chemotherapy, only doxorubicin and vincristine increased priming (PUMA response), decreased the levels of MYC and MCL1 proteins and potentiated venetoclax-induced apoptosis (all p <0.05). Conclusion: CLL and HGBL-DH were the most BCL2-dependent NHLs and had the highest responses to venetoclax. Venetoclax-resistance in our NHLs was due to the presence of MCL1, the lack of BCL2 expression and dysfunctional BAX/BAK. In the former scenario, resistance may be overcome by combining venetoclax with vincristine and doxorubicin, two drugs used in standard RCHOP. Disclosures Johnson: Seattle Genetics: Honoraria; Merck: Consultancy, Honoraria; AbbVie Inc.: Consultancy, Honoraria, Research Funding; Lundbeck: Consultancy, Honoraria, Other: travel, Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria; Roche: Consultancy, Honoraria, Other: travel, Research Funding. Steidl:Juno Therapeutics: Consultancy; Roche: Consultancy; Bristol-Myers Squibb: Research Funding; Tioma: Research Funding; Seattle Genetics: Consultancy; Nanostring: Patents & Royalties: patent holding. Scott:Janssen: Research Funding; NanoString: Patents & Royalties: Named Inventor on a patent licensed to NanoString Technologies, Research Funding; Celgene: Consultancy, Honoraria; Roche: Research Funding. Letai:Flash Therapeutics: Equity Ownership; Vivid Biosciences: Equity Ownership; AbbVie: Consultancy, Other: Lab research report; Novartis: Consultancy, Other: Lab research report; AstraZeneca: Consultancy, Other: Lab research report.
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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 | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.002 | 0.000 |
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".