Avalilação PREreview de MNT loss in MYC-driven B lymphoma cells enhances apoptosis, inhibits proliferation and increases sensitivity to cancer drugs

Summary of the manuscript

This manuscript explores the therapeutic potential of targeting MNT, a transcription repressor and MYC antagonist, in fully malignant MYC-driven B lymphoma cells using both mouse models (Eµ-Myc lymphoma cell lines) and human Burkitt lymphoma cell lines. The authors present robust preclinical evidence that MNT loss dramatically sensitizes lymphoma cells, including multidrug- or BH3-mimetic-resistant lines, to conventional chemotherapeutics (Cisplatin, Doxorubicin, Vincristine) and BH3 mimetics (S63845 and Venetoclax/ABT-199).

Major comments (methodology, mechanism, and translational framing)

1. Discussion: The Discussion section currently contains redundant summaries of the mechanistic results already detailed in the Results section. The authors should restructure the Discussion to explicitly address how their findings represent a breakthrough compared to existing therapeutic strategies. Additionally, since MNT is ubiquitously expressed and vital for normal development, the authors must discuss the potential systemic safety profile and toxicity risks of a theoretical systemic MNT inhibitor in healthy tissue.

2. Mechanistic disconnect in apoptosis-resistant sublines: In Figure 5, the authors characterize sublines selected for resistance to the MCL-1 inhibitor S63845. While the mechanism of resistance for line #691^R (loss of BAX) and #758^R (upregulation of pro-survival proteins) is clear, the mechanism driving resistance in subline #1344^R remains unknown. To make this modeling robust, the authors should expand their analysis (e.g., by checking phosphorylation states or other BCL-2 relatives such as BFL-1/A1 or BCL-W) to clarify why #1344^R is resistant.

1. Human cell line population variability: In Figure 7, the authors note that while BL-41 cells died rapidly upon MNT loss, apoptosis-resistant but slow-growing BL-2 and Rael-BL clones were successfully isolated. The authors must clarify whether these clones represent natural clonal variation pre-existing within the parental population or whether they acquired secondary compensatory mutations during the prolonged CRISPR selection process.

2. Absence of cell competition assays: The authors conducted only static cell viability assays throughout the experiment. Given the slow growth of the MNT-deficient clones, the authors can perform functional cell competition assays (mixing wild-type and MNT-knockout/inducible cells) to evaluate outgrowth dynamics under the selective pressure of different chemotherapeutics or BH3 mimetics.

3. P53-independent upregulation of puma: The manuscript demonstrates that Mnt deletion upregulates Bbc3 (PUMA) mRNA and protein in both p53 wt and mutant backgrounds, stating that "p53-independent mechanisms also increase PUMA levels". Given that the cited CUT&RUN data imply Bbc3 is a direct target of MNT/MAX complexes, the text should explicitly clarify whether they conclude MNT acts as a direct transcriptional repressor of the PUMA promoter/enhancer (where MNT loss causes direct derepression), or if an alternative transcription factor (e.g., E2F1 or FoxO3a) is actively driving it in MNT's absence.

4. In vivo relapse dynamics: In the transplantation assays (Figure 1E), mice injected with inducible Mnt-deletable cells eventually relapsed. While the authors elegantly show that some tumors retained MNT or upregulated BCL-2/BCL -X_L, it remains unclear whether these escapees arose from acute transcriptional adaptations under tamoxifen pressure or from the expansion of pre-existing clonal variants. Discussing the baseline heterogeneity of the parental lines would strengthen this section.

5. Model limitations vs. Significance claims: The manuscript frequently frames its findings in terms of the immediate development of clinical MNT inhibitors to improve Myc-driven blood cancer outcomes. While this is a highly valuable eventual goal, the current work relies entirely on stable, established cell lines and genetic mouse models. It lacks patient-derived organoids or patient-derived xenograft (PDX) models that accurately recapitulate human tumor pathology and microenvironments. The authors could tone down the translational over-selling and refocus the narrative on the exceptionally strong fundamental biology they have uncovered.

Minor comments (data presentation, blots, and typos)

1. Incomplete stripping and antibody carryover (figure 1d): The legend explicitly states that an "Asterisk indicates MNT band left from previous immunoblot." The residual protein signal introduces ambiguity into quantification. The authors must repeat this panel using a fresh blot or a verified, complete stripping and re-probing protocol.

2. Mnt band visibility and exposure times: In several figures (e.g., Figures 2B and 3F), the target MNT bands are barely visible or faint, making it difficult for the reader to confidently assess the true efficiency of the genetic deletion. Conversely, other blots in the manuscript appear heavily overexposed and dark. The authors should:

   • Provide higher-contrast or longer exposures specifically for panels where the MNT band is too faint to visualize.

   • Confirm consistent protein loading across these highly variable exposures by calculating or verifying protein concentrations from their primary laboratory records.

   • Authors can include non-saturated exposures or full unaltered blot acquisitions in the Supplementary Information to ensure complete transparency.

3. Antibody reference clarity: In the Methods section under Western Blot Analysis, the authors state that no suitable antibody was available to test for mouse NOXA protein, yet the antibody appendix lists "human NOXA." Please ensure the text clearly delineates where NOXA was successfully tracked (human Burkitt lines) versus where it was omitted due to technical limitations (mouse lines).

1. Abstract typographical error: At the very end of the Abstract (Page 2), there is a visible word-count placeholder text fragment: 150w can have 150. This must be deleted prior to publication.

Competing interests

The authors declare that they have no competing interests.

Use of Artificial Intelligence (AI)

The authors declare that they used generative AI to come up with new ideas for their review.