The BRD4S-LOXL2-MED1 interaction at the forefront of cell cycle transcriptional control in triple-negative breast cancer

  1. Laura Pascual-Reguant
  2. Tian V. Tian
  3. Debayan Datta
  4. Damiano Cianferoni
  5. Savvas Kourtis
  6. Antoni Gañez-Zapater
  7. Chiara Cannatá
  8. Queralt Serra-Camprubi
  9. Lorena Espinar
  10. Maria Guirola
  11. Jessica Querol
  12. Andrea Miró Canturri
  13. Joaquin Arribas
  14. Luis Serrano
  15. Sandra Peiró
  16. Sara Sdelci

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Abstract

Triple-negative breast cancer often develops resistance to single-agent treatments, which can be circumvented with targeted combinatorial approaches. Here, we demonstrate that the simultaneous inhibition of LOXL2 and BRD4 cooperate to reduce triple-negative breast cancer proliferation in vitro and in vivo . Mechanistically, we reveal that LOXL2 interacts in the nucleus with the short isoform of BRD4 and MED1 to control cell cycle progression at the gene expression level via sustaining the formation of BRD4-MED1 nuclear transcriptional foci. Indeed, the pharmacological or transcriptional repression of LOXL2 provokes downregulation of cell cycle gene expression, G1-S cell cycle arrest, and loss of BRD4-MED1 foci. Our results indicate that the BRD4S-LOXL2-MED1 interaction is fundamental for the proliferation of triple-negative breast cancer. Therefore, targeting such interaction holds potential for the development of novel triple-negative breast cancer therapies.

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    Reply to the reviewers

    Reviewer #1 (Evidence, reproducibility and clarity (Required)):

    The manuscript describes that simultaneous inhibition of LOXL2 and BRD4 reduces proliferation of TNBC in vitro and reduces growth in vivo.

    This observation is followed by extensive mechanistic studies that suggest physical interaction between LOXL2 and short isoform of BRD4-MED1. Inferences from Chip-seq analyses suggest that this interaction is involved in regulation of multiple transcriptional programs. Authors focus on differential activation of DREAM complex, to claim that this interaction "is fundamental for proliferation of TNBC". The manuscript is very well written and mechanistic inferences are based on a set of sophisticated epigenetic analyses and bioinformatical inferences. The phenotypic effects from LoxL2 inhibition by itself, or in combination with BRD4 inhibition are relatively modest. These modest effects, as well as many of the reported changes in gene expression are clearly inconsistent with the frequently used adjectives as "dramatic", "fundamental", "deeply affected", "drastically hampered" etc. Given the modest phenotypic effects, many of the key claims and conclusions are not supported by the data.

    We thank the reviewer for appreciating our work, defining the manuscript as well-written, and saying that it comprises extensive mechanistic studies as well as sophisticated epigenetic analysis.

    We apologize if some of our statements seemed exaggerated. In this revised version, we revisited some of our conclusion to moderate them.

    Moreover, we took the reviewer's criticism as an opportunity to strengthen our findings. In the revised version of the manuscript, we included an additional TNBC PDX (PDX-127), and results from this experiment clearly reinforce our claims (Fig. 6D and Fig. EV9E-F). In this new in vivo experiment, we selected a PDX model in which the expression of BRD4L is not detectable, while BRD4S is clearly expressed. Therefore, the treatment with JQ1 would specifically affect the activity of BRD4S, making the treatment selective. Additionally, we reduced by half the dose of JQ1 administrated to limit the effect of BRD4S inhibition alone on tumor growth. The combinatorial treatment (JQ1+PXS) induced a clear superior effect in this setting as compared with single-agent treatments. In addition to this, we discarded that the observed growth reduction is not the result of the sole inhibition of LOXL2, which could affect FAK/Src activity or extracellular Collagen crosslinking. In conclusion, our data show that the combinatorial inhibition of LOXL2 and BRD4S is effective in reducing tumor proliferation in TNBC in vivo models, independently of the inhibition of BRD4S and of other pathways known to be regulated by LOXL2.

    Specifically:

    1. It is unclear why authors generalize their conclusions to TNBC. Figure 1B demonstrates synergy for 1/3 cell lines, which is chosen for the follow up study. Even for MDA231, the synergy is confined to low concentrations of BRD4i (S1c). While MDA231 cell line is frequently used in experimental studies of TNBC, it is quite dissimilar to majority of clinical TNBC, and contains mutant RAS, which is rare in this disease.

    The synergistic effect is observed in MDA-MB-231 cells because only this cell line expresses both BRD4S and LOXL2. Indeed, in Fig. 1C we show that MDA-MB-468 cells do not express LOXL2, while BT549 only express minimal BRD4 levels.

    To corroborate this hypothesis, in the revised version of the manuscript we added:

    1. A new cell line (Cal51) expressing the same LOXL2 and BRD4 levels (Fig. EV8C) but showing greater resistance to JQ1 than MDA-MB-231 (Fig. EV8D). Also, in this cell line, we could show that the combinatorial treatment had a superior effect on cell viability than the single agents’ treatment (Fig. EV8E).
    2. A western blot panel of different TNBC PDXs shows that the majority of them express medium to high levels of both BRD4S and LOXL2 proteins, as is the case of MDA-MB-231 (Fig. EV9E) and Cal51 (Fig. EV8C). This result suggests that the combinatorial treatment could be used in the majority of TNBC patients as they are expected to express both BRD4S and LOXL2.
    3. Finally, as explained above, we performed another in vivo choosing a PDX that expresses BRD4S (but not BRD4L) and LOXL2 (PDX-127) (Fig. 6D and Fig. EV9E-F). Also, in this new model, we could observe that the combinatorial inhibition had a superior effect than single treatments.
    1. In vivo, the effect appears to be modest even in the MDA231 model, selected for evidence of synergy in vitro. In vivo, the combination appears to have an additive effect. Tumor growth rates are reduced, but no shrinkage is occurring. In the PDX model, LOXL2i does not have an effect as a monotherapy, while modestly enhancing the impact of BRD4i. These results are at odds with the claim of the interaction being fundamental for proliferation.

    We agree with the reviewer that the combinatorial inhibition appears to have an additive effect in vivo using the MDA-MB-231 model.

    1. For that reason, we have now performed the in vivo PDX experiment mentioned above (PDX-127; Fig. 6D and Fig. EV9E-F) in which we decreased the dose of JQ1 by half to avoid strong tumor growth effect due to BRD4 inhibition alone. In this new experiment, the synergistic effect is evident. While single-agent treatment showed a very moderate effect (0% or 20% tumor growth reduction for LOXL2 and JQ1, respectively), the combinatorial treatment showed a 50% reduction in tumor volume, further supporting our conclusions.
    2. We also performed either BRD4 or MED1 pull-down experiments in the presence of PXS and JQ1. We show that upon PXS treatment, the interaction between LOXL2 and BRD4S is maintained while the interaction with MED1 is reduced (Fig. 5A-C). However, in the presence of JQ1, the interaction between LOXL2 and MED1 is maintained while BRD4S-LOXL2 and BRD4S-MED1 interactions are impaired (Fig. 5D-F). These new results explain why monotherapy does not have a sufficient effect in vivo and set the rationale for the use of the combinatorial treatment. We believe that these new results corroborate our initial findings and we hope to have been able to satisfy the reviewer comments.
    1. No analysis of cell proliferation was shown in vivo. Authors should have performed BrdU or KI67 staining to support the claim. For in vitro analyses, authors also used indirect assays for proliferation. PI staining by itself does not have sufficient resolution to clearly capture modest effects that authors demonstrate. BrdU-PI double staining would have been much more useful.

    We appreciate the reviewer’s comment. In the revised manuscript we have added Ki67 and H3S10p staining in the tumor samples for the new in vivo PDX experiment (Fig. 6E and Fig. EV10A-C). We show that the combinatorial treatment significantly induces a reduction of both proliferation markers, which is in agreement with a reduced tumor volume. Regarding the in vitro analysis, we did not only use PI staining to show a reduced proliferation state but also H3S10p staining (Fig. 4B) and an SLBP1 fluorescent reporter MDA-MB-231 cell line (Fig. 4D, Fig. EV6B, E, and Movie EV). In the revised version of the manuscript, we included a new FACS-PI analysis (Fig. 4A, C) to better represent the effects we see on the cell cycle.

    Minor points:

    Dose dependent decrease in phosphorylated H3 is not at all obvious from eyeballing the data in S1A; the only effect that I see is a modest reduction at the highest concentration of the inhibitor. Authors need to quantify the results to support the claim.

    We agree with the reviewer and we apologize for the misinterpretation. We have changed the revised manuscript as follows: “The selective LOXL2 inhibitor PXS-538224 (hereafter, PXS) efficiently reduced the levels of oxidized histone H3 (H3K4ox) in MDA-MB-231 cells at 40 μM (Fig. EV6C), indicating an efficient inhibition of LOXL2 catalytic activity in the nucleus.”

    Most of breast cancer cell lines are derived from metastatic disease, including pleural effusion, thus the point that because MDA231 cell line is derived from pleural effusion, it is metastatic does not have sufficient logical foundation.

    Many publications have shown the high metastatic capacity of MDA-MB-231 (e.g. https://doi.org/10.1016/j.bbabio.2011.04.015, doi: 10.1038/s41467-017-01829-1), which are therefore used as TNBC metastatic model. The scope of the analysis reported in Fig. 6C was just to show whether any of the used treatments could reduce the metastatic capacity of this cell line. We believe we do not overstate the results but just report them as they are.

    How is loss of cell-cell junction in vitro consistent with LOXL2 role in modulating ECM? There is no evidence of ECM production in MDA231 in vitro. On the other hand, this loss is associated with EMT.

    We thank the reviewer for identifying this mistake. In the revised manuscript we changed the text as follows: “Gene set enrichment analysis (GSEA) revealed that LOXL2 KD induced upregulation of processes involved in cell morphology, secretion, membrane trafficking, and cell differentiation, with cell-cell junction being one of the most significantly affected pathways (Fig. EV5E). These results agree with the role of LOXL2 in regulating epithelial-to-mesenchymal transition, corroborating the high quality of our dataset.”

    Reviewer #1 (Significance (Required)):

    Discovery and characterization of LOXL2-BRD4 interaction is advancing the ever-deepening understanding of molecular mechanisms of regulation of gene expression. The studies and analyses appear to be sufficiently rigorous and reported with clarity, and the claimed discovery of the biological interaction between LOXL2 and BRD4 is well supported. However, given the magnitude of the reported (rather than claimed) effects of this interaction, and concerns about generalizability of authors conclusions, it is not clear how these results are promising for the development of new therapies in TNBC. Moreover, in contrast to luminal BC, there is no clear evidence for utility of cytostatic drugs in constraining TNBC. Therefore, biological and clinical significance of the authors discovery is unclear and claims in this regard appear to be overblown

    We thank the reviewer for stating that our analysis is rigorous and reported with clarity. We really took the criticisms as an opportunity to strengthen our findings, as explained above.

    For the newly presented in vivo PDX model, we performed immunohistochemistry of Ki67, H3S10p and Cleaved Caspase 3 to check whether the reduction of tumor volume observed in the combinatorial treatment was a result of a cytotoxic and/or a cytostatic effect (Fig. 6E and Fig. EV10A-C). As shown in the figure, the combination of the two inhibitors induced a superior decrease of Ki67, H3S10p, and a clear increase of Cleaved Caspase 3. Therefore, these new data indicate that the combinatorial treatment does not only have a cytostatic effect but also cytotoxic, suggesting a clinical exploitability for the treatment of TNBC patients.

    Reviewer #2 (Evidence, reproducibility and clarity (Required)):

    In their study, Pascual-Reguant et al. show that combined inhibition of BRD4 and LOXL2 can synergize to restrict triple-negative breast cancer (TNBC) proliferation. BRD4 and LOXL2 are transcription regulators that can read and write epigenetic information, respectively. The authors employ three distinct breast cancer cell lines and mouse models with cell line-derived xenografts, and they show that combined inhibition of BRD4 and LOXL2 can be superior to single BRD4/LOXL2 inhibition in these model systems. In an attempt to identify a connection between BRD4 and LOXL2, the authors find that the two proteins can bind to each other. The authors performed most of the experiments in the breast cancer cell line MDA-MB-231. To assess the impact of LOXL2-inhibition on transcription, the authors assessed changes of the transcriptome in MDA-MB-231 cells following LOXL2 knockdown. They found that genes related to cell differentiation and morphology were upregulated, while genes related to the cell cycle were downregulated. ChIP-seq data of BRD4 showed that BRD4 can bind to cell cycle gene promoters and that this binding was enhanced upon loss of LOXL2. The authors found that LOXL2 and BRD4 interacted with the transcriptional cell cycle regulators B-MYB, FOXM1, and LIN9, which are components of the MYB-MuvB-FOXM1 (MMB-FOXM1) complex that is known to promote the expression of late cell cycle genes with important functions during mitosis. The authors conclude that LOXL2/BRD4 interact with each other and with the MMB-FOXM1 complex to drive the expression of cell cycle genes and cell proliferations. Vice versa, they conclude that inhibition of LOXL2/BRD4 reduces cell proliferation through inhibiting the expression of cell cycle genes.

    Major:

    • The data and methods are presented well. The experiments are adequately replicated and analyzed. However, except for the first section, all experiments were performed using only one cell line. It is important to validate key findings in at least a second cell line.

    We thank the reviewer for valuing our work.

    To address the reviewer’s comment, in the revised manuscript we added an additional cell line (Cal-51), that expresses similar levels of LOXL2 and BRD4 as compared to MDA-MB-231 (Fig. EV8C). Even though this cell line is clearly more resistant to JQ1 than the MDA-MB-231 cell line (Fig. EV8D), the combinatorial treatment is significantly more effective as compared with single agents’ treatment (Fig. EV8E).

    Moreover, we have also performed an additional in vivo experiment using another TNBC PDX (PDX-127) that expresses LOXL2 and BRD4S, but not BRD4L. Given that JQ1 can inhibit both BRD4 isoforms, this in vivo system allowed us to demonstrate that the tumor antiproliferative capacity of the combinatorial treatment is due to the simultaneous inhibition of LOXL2 and BRD4S (rather than BRD4S and L) (Fig. 6D and Fig. EV9E-F).

    • There appears to be a misunderstanding of the concept of cell cycle-dependent gene regulation by the DREAM complex and its related factors. Early (G1/S) cell cycle genes contain E2F promoter motifs, while late (G2/M) cell cycle genes contain CHR promoter motifs. The DREAM complex can bind both, while RB-E2F and MuvB recognize only E2F and CHR motifs, respectively. B-MYB and FOXM1 bind to MuvB and regulate late cell cycle genes, but they do not bind to early cell cycle genes. Given this concept, the authors' rationale to connect BRD4/LOXL2 through MuvB/B-MYB/FOXM1 with E2F promoter sequences and early cell cycle genes and the subsequent conclusions must be corrected.

    We thank the reviewer for their expert explanation. We corrected our conclusion in the revised version of the manuscript following the reviewer’s comment.

    • I felt that the suggested functional connection between LOXL2/BRD4 and DREAM is not strongly supported by the authors' data. Figure S6E: A similarity score of Fig. EV6E: We agree with the reviewer that a similarity score of Fig. 4E: We thank the reviewer for this comment. The performed pulldown showed that BRD4S, LOXL2, and MED1 interact with Lin9 and B-Myb, but not with FOXM1, thus FOXM1 itself is an internal negative control of the pulldown. Additionally, BRD4L does not show the same interaction pattern as BRD4S, LOXL2, and MED1, again acting as an internal negative control. We, therefore, believe that the pulldown is properly controlled and that the observed interaction is trustful. We furthermore agree with the reviewer that it would be interesting to characterize the interactions between the DREAM complex and BRD4S, LOXL2, and MED1. However, we believe that the dissection of these interactions at the mechanistic levels would require a deeper study, which can be a project in itself that we aim to explore in the future. For example, it would be interesting to investigate whether either the inhibition or the downregulation of LOXL2 and/or BRD4S specifically impairs the formation of the DREAM complex or the recruitment of specific DREAM complex subunits, as well as how these effects impair the DREAM complex chromatin binding. We are afraid that the suggested pulldowns would not be sufficient to answer these questions, which would require extensive cross-interaction studies in either BRD4/LOXL2 and BRD4+LOXL2 inhibition or downregulation followed by ChIP-seq and transcriptomics for all the conditions. We believe that the provided data, together with the functional characterization (both, in vitro and in vivo), of the phenotypes triggered by BRD4S and LOXL2 inhibition make a strong case for our manuscript and leave out of scope the suggested experiments. We hope the reviewer will understand our explanation and will appreciate that we are planning to pursue this further in the future.

    Fig. 3: We thank the reviewer for this important comment. The ChIP-seq technique very often does not provide exhaustive results due to sequencing depth limits and antibody performance. We believe that the fraction of DREAM target genes found in our dataset as bound by BRD4S is not exhaustive and that the analysis proposed by the reviewer would not lead to clear conclusive results. However, we understand the importance of verifying that DREAM target genes whose promoter is bound by BRD4 are indeed downregulated when LOXL2 is inhibited. To give an answer to this question, in the revised manuscript we added gene expression analysis of selected DREAM target genes upon treatment with JQ1, PXS their combination. We could successfully show that both JQ1 and PXS treatment impairs the transcription of the selected DREAM target genes, however, the combinatorial treatment almost shut down their expression, in agreement with our hypothesis (Fig. 5J).

    • The authors state that it is surprising to find that LOXL2 can promote target gene transcription because it is rather known as a transcriptional repressor. To this point, the authors should perform standard analyses using their RNA-seq and ChIP-seq data. Compare differential expression of genes that are bound by BRD4S/L/S+L and genes not bound by BRD4. Perform motif search and enrichment analyses for transcription factor and co-factor binding data (public ChIP-seq repositories). Such analyses may suggest what gene sets are up- and downregulated by LOXL2 through BRD4S/L and what other factors could be involved in LOXL2-dependent up- and downregulation of gene transcription.

    We thank the reviewer for this valuable comment that certainly provides the rationale for a follow-up project. However, we believe that the proposed study goes beyond the scope of our work at this moment.

    Minor:

    • I felt that background information on the BRD4 isoforms was missing. The short and long isoforms of BRD4 should be introduced briefly.

    We agree with the reviewer. In the revised manuscript, we addressed this by presenting BRD4 isoforms in the introduction part of the manuscript.

    • Given that BRD4 inhibition is known to activate p53 (e.g., PMID 23317504 and 33431824) and p21 (PMID 31265875), the authors should discuss the p53 status of their cell lines (largely mutant). In general, I felt that the authors could better cite and discuss the current literature on BRD4 and LOXL2.

    We appreciate the comment of the reviewer regarding p53. Given the fact that p53 is mutant in MDA-MB-231, we believe that the proliferation defect observed with the combinatorial treatment may be due to the activation of alternative cytostatic or cytotoxic signaling cascades, independently of P53 activation. We have now briefly mentioned this point in the manuscript discussion.

    • It was unclear to me why the authors did not actually test experimentally whether their predicted interaction models 2 or 4 are likely true (Figure 2E+G).

    We understand the reviewer’s comment. The fact that JQ1 treatment almost abrogates the interaction between LOXL2 and BRD4S strongly suggests that models 1 and 3 are likely wrong, therefore pointing towards models 2 and 4 as the correct ones. To test whether models 2 and 4 are indeed the correct models we are now performing extensive mutagenesis studies, which are producing preliminary results suggesting indeed that models 2 and 4 are correct. The reason why we did not include this study in the current manuscript, is that we started a parallel line of investigation aimed at identifying residues fundamental for the interaction that can be exploited in compound screening campaigns to identify molecules able to block the described interaction and thus cancer proliferation. Publishing these preliminary results at this stage could jeopardize the drug discovery campaign and we hope that the reviewer will understand our constraints.

    • The transcription of cell cycle genes depends on the cell cycle (i.e., reduced cell cycle entry correlates with reduced cell cycle gene expression). Given that the authors showed LOXL2 inhibition reduce MDA-MB-231 cell proliferation, they should note that reduced expression of cell cycle-related genes is expected upon LOXL2 knockdown.

    We understand the reviewer’s comment. We believe that we provide sufficient data supporting our hypothesis that LOXL2 controls the expression of cell cycle genes at the transcriptional level together with BRD4S. In addition, the sole inhibition of LOXL2 has practically no effect on tumor proliferation in vivo but largely enhances the antiproliferative effect of low-dose JQ1 (Fig. 6D). We hope these clarifications would satisfy the reviewer.

    • The authors specify in their discussion that their data show a function of LOXL2/BRD4 in the cell cycle interphase, while there were no experiments that support that specific conclusion. At least it is unclear to me why the authors rule out a function in mitosis?

    We thank the reviewer for this comment. We referred to interphase genes because these are the early cell cycle genes, while mitotic genes are the late ones. We do not discard a possible function for BRD4S and LOX2 regulating mitotic progression, however, we believe this would be a consequence of dysregulated G1-S-G2 gene expression, rather than a direct transcriptional effect. This conclusion derives from the fact that while we observe interactions between LOXL2, BRD4S, and MED1 with Lin9 and B-Myb, these are not fully conserved with FOXM1, which is typically required for the transcription of mitotic genes. To avoid confusion, we have now anyway removed the word “interphase” from the text.

    • I felt that the first part of the manuscript (combination of BRD4 and LOXL2 inhibitors in TNBC) was a bit uncoupled from the functional studies on LOXL2 and its connection to BRD4. The transition between these parts and the final discussion on why the joint control of cell cycle genes by LOXL2/BRD4 may be important for the synergistic effect of LOXL2/BRD4 inhibitors. To this point, the authors' model was not clear to me.

    We really appreciate the reviewer’s comment. To better connect the functional studies with the clinical significance of the proposed combinatorial treatment, we restructured the manuscript. In the revised version, the use of the combinatorial treatment is shown in Figure 6. Moreover, to better explain why we focused all the studies on BRD4 and LOXL2, we also included data from the Cancer Cell Line Encyclopedia (CCLE)-associated chemotherapeutics sensitivity (Fig. 1A and Fig. EV1) showing that LOXL2 expression levels can predict the response to BRD4 inhibition, suggesting a functional interaction between BRD4 and LOXL2 and the possibility to exploit it for therapeutical purposes. We believe that these data set the rationale to further explore the connection between LOXL2 and BRD4, both at the mechanistic and functional levels.

    Reviewer #2 (Significance (Required)):

    The study by Pascual-Reguant et al. shows that inhibitors of BRD4 and LOXL2 can be combined to achieve better efficacy in reducing proliferation of breast cancer cell lines and breast tumor growth in xenograft models. They provide strong evidence for a functional interaction between LOXL2 and BRD4 and investigate their common transcriptional targets. Intriguingly, some evidence points towards a direct regulation of the DREAM complex and its cell cycle gene targets.

    The findings are novel and can be the basis for further research on TNBC combination therapy using BRD4 and LOXL2 inhibitors. The link to the DREAM complex is preliminary.

    The study is of interest for a basic research audience with some translational aspects.

    I reviewed this manuscript as a researcher in gene regulatory mechanisms, with cell cycle genes as one focus area. I have no expertise in the computational modeling of protein-protein interactions and I am no expert for breast cancer.

    We thank the reviewer for the positive comments. We also would really like to thank the reviewer for their criticism, which, we believe, contributed to a new and improved manuscript version.

    Reviewer #3 (Evidence, reproducibility and clarity (Required)):

    Summary:

    In this manuscript, Laura Pascual-Reguant et al. identified a novel role of the LOXL2 oxidase in sustaining cell cycle progression through a so far uncharacterized gene-activating function is mediated by the BRD4S epigenetic reader and exerted on key DREAM-target genes in TNBC. Moreover, the authors showed that combinatorial treatment of TNBC with LOXL2- and BRD4-specific inhibitors result in a tremendous anti-tumorigenic effect. For all findings, they leveraged in vitro and in vivo settings as well as high-throughput sequencing approaches. However, the following points should be addressed and explained.

    Major points:

    -The authors on their working hypothesis propose that dual inhibition of BRD4 and LOXL2 is a novel strategy for curing TNBC. For my taste, just because both targets are quite promising for TNBC, the jump to this combinatorial treatment is kind of abrupt. Knowing the difficulty and time-/financial- investment, authors could optionally perform a mass spectrometry analysis on nuclei lysates with LOXL2 pull down to identify physical interactors. Due to the augmented resources and analysis of raw data, authors may necessitate a generous revision period (approx. 4 months for starters). By that, this can provide a more unbiased approached to look at nucleus-specific gene-regulatory functions and particularly at epigenetic readers. It would be also interesting to see if LOXL2 interacts with other members of the BRD family. Selecting BRD4 and no other members of the bromodomain family cannot be the only choice given that other BRD members can also interact with several of these mediator subunits.

    We thank the reviewer for the suggestion and we agree with the fact that the rationale for combining BRD4 and LOXL2 inhibitors was not sufficiently argued in the first version of the manuscript. For that reason, in the revised manuscript, we added new data to explain why we explored this topic. In particular, to better explain why we focused all the studies on BRD4 and LOXL2, we included data from the Cancer Cell Line Encyclopedia (CCLE)-associated chemotherapeutics sensitivity (Fig. 1A and Fig. EV1) showing that LOXL2 expression levels can predict the response to BRD4 inhibition (but not to other approved chemotherapeutic drug), suggesting a functional interaction between BRD4 and LOXL2 and the possibility to exploit it for therapeutical purposes. Moreover, we restructured the manuscript to make the story more linear, explaining first the functionality of BRD4S-LOXL2 interaction at the molecular and cellular levels, and then presenting the in vivo systems in the last part of the manuscript.

    We agree with the reviewer that it may be interesting to explore whether LOXL2 interacts with other BRD family members. However, given the prominent role of BRD4 in promoting cancer proliferation, we believe that understanding the relevance of BRD4S-LOXL2 interaction in TNBC is, per se, of great interest and provide a novel mechanistic understanding of how TNBC proliferation is controlled at the transcription level. In the specific case of TNBC, it has been shown that BRD4S has an oncogenic effect, while BRD4L is an oncosuppressor. In the manuscript, we now showed that LOXL2 downregulation sensitizes cells to JQ1 treatment (Fig. 1D). Additionally, while the downregulation of BRD4L does not have any additional effect on cell treated with PXS, the downregulation of BRD4S sensitize them to LOXL2 inhibition (Fig. EV8B). These results, once again, indicate the relevance of studying the functional interaction between BRD4S and LOXL2.

    -LOXL enzymes have been shown to promote collagen and fibronectin assembly, thereby sustaining the pro-survival effect of the ITG5A/FN1/FAK/SRC signaling cascade and shielding TNBC cells against chemotherapy treatment (32415208). Did authors observe if LOXL2 loss or inhibition decreased the active status of FAK and SRC, which are well known to promote G1-S transition (25381661)?

    Probably the cell cycle defects upon LOXL2 loss may also partially arise from the impairment of this cascade.

    We really appreciate the reviewer’s suggestions. In the revised version of the manuscript, we checked FAK and Src activation status in tumor samples from one of our in vivo experiments (Fig. EV10D). We did not observe any difference in phospho-FAK or phospho-Src upon treatment either with PXS, JQ1, or their combinations, suggesting that alterations in the activity of these factors were not driving the observed proliferation defects.

    -Authors exclusively use JQ1 as a BRD4 inhibitor. As JQ1 may have an unspecific effect on BRD2 as well, authors should consider reproducing key experiments with siControl- and siBRD4-treated cells and increasing doses of PSX as well as repeating the JQ1 dose response assay in Figure 1B using siRNA-mediated silencing of LOXL2. Given that both players are part of the same complex, silencing of one and inhibition of the other should sensitize cells compared to their control counterparts.

    We agree with the reviewer and we addressed this comment in the revised manuscript. In particular, we have added two additional experiments:

    • We transduced MDA-MB-231 cells with isoform-specific shBRD4s (shBRD4L and shBRD4S) (Fig. EV5H) and checked cell sensitivity to PXS treatment (Fig. EV8B). As explained also above, we observed that only when the short isoform of BRD4 was downregulated cells displayed higher sensitivity to PXS treatment. This result corroborates that BRD4S and LOXL2 are required for TNBC proliferation.

    • We transduced MDA-MB-231 cells with shLOXL2 and assessed JQ1 sensitivity (Fig. 1D). We showed that upon LOXL2 downregulation, cells became more sensitive to JQ1 treatment, again corroborating the fact that TNBC proliferation requires BRD4S and LOXL2.

    -Moreover, in Figures 1G and S3D the differential sensitivity of low and high LOXL2 cell lines is unclear. Do authors know if any of these growth kinetic lines represent one of the tested cell lines in Figure 1A-B? Authors should provide respective legends. In addition, authors should take advantage of their homemade data given that they have already selected a panel of TNBC cell lines with various LOXL2 expression at basal state (Figure 1A) for which dose response assays have been performed (Figure 1B). Therefore, I would perform an IC50 graph for JQ1 (without PSX treatment) using the existing data from Figure 1B.

    We apologize if our representation was confusing. In the revised manuscript we have changed the sensitivity plots (Fig. 1A and Fig. EV1) to make them easier to grasp. Additionally, in Figure 1A we included the analysis of CCLE cell lines stratified based on their LOXL2 expression levels. This analysis showed that LOXL2 expression levels could overall predict the response to BETi treatment. As suggested by the reviewer, we also plotted the IC50 of the 3 cell lines tested. However, their JQ1 sensitivity curves did not show any difference that could be attributed to their different LOXL2 levels. Our speculation is that only 3 cell lines do not provide a sufficient size to reach a meaningful conclusion, which, in contrast, can be achieved by comparing the CCLE BETi sensitivity.

    -In Figure 2D, the pull-down assay is inconclusive, as the molecular weight for each construct is not mentioned. I would probably add this information also in all performed western blots. Also, the overexpression of the BD1/BD2-mutated and especially the BD1/BD2-lacking construct is unclear if it still interacts with LOXL2, probably because of the lack of molecular weight reference of each band. Therefore, the authors should make this pull-down assay more descriptive regarding the size of the bands. Also, BD1 mutagenesis at N140 was shown to dislodge the binding of JQ1 to BRD4 (24497639), which implies that BD1 mutagenesis or overexpression of the BD1-deficient construct should abrogate the interaction of LOXL2 with BRD4, reminiscent to the abrogated interaction of BRD4/LOXL2 upon JQ1 that binds to both BDs (Figure 2F). And, what happens if a BD2-deficient construct is expressed?

    We thank the reviewer for spotting this distraction. We apologize for this and in the revised version of the manuscript we included molecular weights for all western blots.

    We acknowledge that BD1 mutagenesis displaces JQ1 binding, however, we respectfully disagree that because of this BD1-N140 mutant should not bind to LOXL2. Our docking analysis indeed showed that none of the poses is impaired either by BD1 or BD2 mutagenesis (Fig. EV4D). The fact that JQ1 disrupts the interaction between BRD4S and LOXL2 (Fig. 2F, G) is not due to the fact that they compete for the same binding residue, but rather for the space occupied by JQ1 inside the AcK binding pocket of either BD1 or BD2, which impedes proper binding to LOXL2. Our pulldown data indeed showed that mutant BD1 and BD2 retain the ability to bind to LOXL2 (Fig. 2C), as predicted by the docking.

    We did not try to express constructs either lacking BD1 or BD2 and we cannot speculate what could happen to the BRD4S-LOXL2 interaction in this scenario. Even though this experiment could help dissect the interaction between LOXL2 and BRD4S, we decided to rather perform mutagenesis of specific residues that have been predicted to be important for the interaction. The reason why we did not include this study in the current manuscript, is that we started a parallel line of investigation aimed at identifying residues fundamental for the interaction that can be exploited in compound screening campaigns to identify molecules able to block the described interaction and thus cancer proliferation. Publishing these preliminary results at this stage could jeopardize the drug discovery campaign and we hope that the reviewer will understand our constraints.

    -If authors support that BRD4S is the predominant isoform driving the expression of DREAM-targets, this means that DREAM-targets are mainly bound by BRD4S, relying on Figure 3E-F. However, based on the author's ChIPseq tracks in Figure 3H, DREAM targets such as EZH2 and HMGB2 are co-occupied by both BRD4 isoforms at the basal state on their promoter region. Also, especially for EZH2 and PLK4, authors should set to 'group auto-scale' both conditions in a smaller scale range for ChIPseq- and RNAseq tracks, although I do not these two genes as good candidates representing your analysis. Therefore, authors should initially show all genes (e.g in a table format) that enrich the 'DREAM-targets' signature and select for a greater panel of genes (like for AURKB and HMGB2) demonstrating a preferential occupancy of the BRD4S at their promoter region. Finally, authors are recommended to perform a ChIP-qPCR on these genomic regions at basal state (no LOXL2 silencing) to validate the predominant occupancy of BRD4S and the low/absent occupancy of BRD4L at these genomic sites.

    We apologize for the confusion. To make the figure more understandable, we now scaled all the panels to the same scale and highlighted in grey the promoter region of each selected DREAM target gene. As the reviewer can appreciate, none of these genes is bound by BRD4L in basal conditions (Fig. 3F).

    To better characterize the differential binding, following the reviewer’s suggestion, we performed ChIP-qPCR using Ab2 (which recognizes both BRD4 isoforms), in cells either downregulated for BRD4L or BRD4S with isoform-specific shRNAs (Fig. EV5H). Results showed that only the downregulation of BRD4S reduced the binding of Ab2 to the promoter of the selected DREAM target genes (Fig. 3D), corroborating our hypothesis and validating our ChIPseq strategy.

    -Authors in Figure 3G should select an equal-sized population of randomly chosen non-DREAM-target genes, otherwise, the comparison of log2FC difference between these two gene cohorts is unreliable and difficult to make. Mann-Whitney test should also be performed.

    We thank the reviewer for this suggestion, which was added to the revised version of the manuscript (Fig. 3E, lower panel).

    -Authors should repeat the cell cycle analysis (Figure 4A) as the number of cells subjected to flow cytometry is quite discrepant between the conditions. Also, it is not clear if the experiment was performed in at least biological triplicates (although in the respective legend, it is stated so). If performed in biological triplicates, authors should make a new graph where each cell cycle phase cell population differs between the two conditions. Moreover, the difference in cell cycle defects in LOXL2-inhibited cells (Figure 4C) is indifferent compared to their control counterpart. Therefore, authors should address these inconsistencies.

    We thank the reviewer for the suggestion. In the revised version of the manuscript, we represent the cell cycle also as a bar plot with statistical analysis (Fig. 4A, C). Even though the number of cells was the same across conditions, the sub-G1 population of the LOXL2 KD cells may have distorted the profile of the cell cycle. To avoid misinterpretations, we repeated the analysis in the revised version of the manuscript. Statistical analysis supports that LOXL2 inhibition or downregulation has a significant effect on cell cycle progression (Fig. 4A, C, right panel).

    -Furthermore, authors should explain what was the rational selecting a mediator subunit and specifically MED1 as a possible interacting partner of LOXL2 and BRD4s since MED12 and MED24 were also highly essential (Figure 4F).

    We selected MED1 as a Mediator Complex proxy. In our essentiality analysis MED 1, 9, 10, 12, 15, 16, 19, 23, 24, 25 score as significant, suggesting a functional interaction between LOXL2 and the Mediator Complex, rather than a specific subunit. MED1 has been previously described as a BRD4 partner and it is often used in immunofluorescence to visualize transcriptional foci, which made it the best candidate for follow-up study in our project.

    -Moreover, do authors also observe this functional relationship of LOXL2 and BRD4S in cell cycle progression in other breast cancer subtypes presenting a high proliferation index e.g HER2+?

    Presumably, the author's proposed mechanism applies to a wide panel of breast cancer entities, for which, only key experiments could be performed.

    We thank the reviewer for the suggestion. We hypothesized that other cancer types expressing LOXL2 and BRD4S could also benefit from the combinatorial treatment. Indeed, the CCLE drug sensitivity panel in Fig. 1A comprises cancer cell lines of different origins, not just TNBC, and corroborates that the relationship between LOXL2 expression levels and BRD4 sensitivity exist also beyond TNBC. Even though it is important to experimentally verify this hypothesis, we decided to pursue it in the future to broaden the applicability of the proposed strategy in preclinical settings.

    -Authors in Figure 5H represent LOXL2 and BRD4s as integral chromatin looping factors together with MED1 at promoter and enhancer regions. However, this illustration is an overrepresentation of their finding because authors did not address the differential occupancy of BRD4S upon LOXL2 loss in DREAM-target-specific enhancer regions. If they wish to do so, they may use the RANK ORDERING OF SUPER-ENHANCERS (ROSE) package to call for super-enhancer regions in the proximity of DREAM-targets and confirm similar results as for their TSS-proximal sites.

    We thank the reviewer for the useful suggestion. In the new version of the manuscript, we have simplified the representation, which now does not show super-enhancers. However, following the reviewer’s suggestion, we performed super enhancer analysis using ROSE. Results showed that BRD4S binds to super-enhancers more than BRD4L, including DREAM target gene super-enhancers. Additionally, while LOXL2 KD did not alter the binding of LOXL2 to DREAM target gene super-enhancers, it decreased the binding of BRD4S to them (Fig. EV7D, E). Overall, these data are in agreement with our hypothesis that BRD4S together with LOXL2 controls the expression of DREAM target genes.

    -In the current manuscript, authors did not address the translational relevance of their proposed mechanism in the context of conventional therapies. Knowing that several BRD-specific compounds currently undergo clinical trials, authors should address if LOXL2 low (MDAMB468) and high (BT549) cells demonstrate a differential sensitivity to increasing doses of chemotherapy, in the presence or absence of BRD4. By doing that, LOXL2 apart from being a therapeutic target could be also used as a prognostic marker to stratify patients and achieve better response to standard therapies.

    We really appreciate the reviewer’s suggestion and we think this is a fundamental point. In the new version of the manuscript, we have performed further analysis using a greater panel of chemotherapeutic agents from the CCLE sensitivity database. We now show that LOXL2 low-expressing cells show significantly more sensitivity to BETi treatments, but not to conventional chemotherapeutic agents (e.g. doxorubicin, Olaparib, 5-fluorouracil, paclitaxel, etc.) (Fig. 1A and Fig. EV1), which set the rationale to further explore the functional relationship between BRD4 and LOXL2.

    Minor points:

    -In Figure 1D, the authors should convert the y-axis to a logarithmic scale to better represent the differences between JQ1, PXS, and combo. Also, One-way Anova should be performed between JQ1, PXS and combo.

    We don’t understand the reviewer’s suggestion since Fig. 1D (Fig. 6B, right panel in the revised version) is a tumor picture for which the y-axis cannot be converted to a logarithmic scale.

    -In Figure S6F, authors did not show the sensitivity of LOXL2 low and high cell lines for BRD4 KO. If LOXL2-proficient cells are less sensitive to JQ1, based on Figure 1B, authors should consider showing something similar from the gene essentiality database.

    We agree with the reviewer and we apologize for this mistake. We have included the sensitivity of LOXL2 low and high cell lines for BRD4 KO and also for MYC KO (Fig. EV6G).

    -Authors failed to discuss the work from Ozge Saatci et al (PMID: 32415208) regarding LOXL2 in TNBC and ECM reorganization as well as in other cancer entities (PMID: 35428659) in the context of ECM remodeling. Authors should realize that these published works and the current ones are not conflicting but complement each other.

    We thank the reviewer for the suggestion. In the revised version of the manuscript, we discussed this work.

    Reviewer #3 (Significance (Required)):

    SIGNIFICANCE

    The conception and findings are of enlightening significance for TNBC therapy, especially given the lack of targeted therapies in this particularly aggressive breast cancer subtype. Hence, I posit this work as highly relevant for the cancer epigenetics research community interested in characterizing unknown factors that facilitate the gene-activating function of epigenetic readers in health and disease.

    My field of expertise is to uncover epigenetic vulnerabilities responsible for transcriptional plasticity driving drug tolerance in aggressive forms of breast cancer.

    We would like to take the opportunity to thank the reviewer for the relevant suggestions. We strongly believe the revised version of the manuscript has been substantially improved by addressing the comments the reviewer made.

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    Referee #3

    Evidence, reproducibility and clarity

    Summary:

    In this manuscript, Laura Pascual-Reguant et al. identified a novel role of the LOXL2 oxidase in sustaining cell cycle progression through a so far uncharacterized gene-activating function is mediated by the BRD4S epigenetic reader and exerted on key DREAM-target genes in TNBC. Moreover, the authors showed that combinatorial treatment of TNBC with LOXL2- and BRD4-specific inhibitors result in a tremendous anti-tumorigenic effect. For all findings, they leveraged in vitro and in vivo settings as well as high-throughput sequencing approaches. However, the following points should be addressed and explained.

    Major points:

    • The authors on their working hypothesis propose that dual inhibition of BRD4 and LOXL2 is a novel strategy for curing TNBC. For my taste, just because both targets are quite promising for TNBC, the jump to this combinatorial treatment is kind of abrupt. Knowing the difficulty and time-/financial- investment, authors could optionally perform a mass spectrometry analysis on nuclei lysates with LOXL2 pull down to identify physical interactors. Due to the augmented resources and analysis of raw data, authors may necessitate a generous revision period (approx. 4 months for starters). By that, this can provide a more unbiased approached to look at nucleus-specific gene-regulatory functions and particularly at epigenetic readers. It would be also interesting to see if LOXL2 interacts with other members of the BRD family. Selecting BRD4 and no other members of the bromodomain family cannot be the only choice given that other BRD members can also interact with several of these mediator subunits.
    • LOXL enzymes have been shown to promote collagen and fibronectin assembly, thereby sustaining the pro-survival effect of the ITG5A/FN1/FAK/SRC signaling cascade and shielding TNBC cells against chemotherapy treatment (32415208). Did authors observe if LOXL2 loss or inhibition decreased the active status of FAK and SRC, which are well known to promote G1-S transition (25381661)? Probably the cell cycle defects upon LOXL2 loss may also partially arise from the impairment of this cascade.
    • Authors exclusively use JQ1 as a BRD4 inhibitor. As JQ1 may have an unspecific effect on BRD2 as well, authors should consider reproducing key experiments with siControl- and siBRD4-treated cells and increasing doses of PSX as well as repeating the JQ1 dose response assay in Figure 1B using siRNA-mediated silencing of LOXL2. Given that both players are part of the same complex, silencing of one and inhibition of the other should sensitize cells compared to their control counterparts.
    • Moreover, in Figures 1G and S3D the differential sensitivity of low and high LOXL2 cell lines is unclear. Do authors know if any of these growth kinetic lines represent one of the tested cell lines in Figure 1A-B? Authors should provide respective legends. In addition, authors should take advantage of their homemade data given that they have already selected a panel of TNBC cell lines with various LOXL2 expression at basal state (Figure 1A) for which dose response assays have been performed (Figure 1B). Therefore, I would perform an IC50 graph for JQ1 (without PSX treatment) using the existing data from Figure 1B.
    • In Figure 2D, the pull-down assay is inconclusive, as the molecular weight for each construct is not mentioned. I would probably add this information also in all performed western blots. Also, the overexpression of the BD1/BD2-mutated and especially the BD1/BD2-lacking construct is unclear if it still interacts with LOXL2, probably because of the lack of molecular weight reference of each band. Therefore, the authors should make this pull-down assay more descriptive regarding the size of the bands. Also, BD1 mutagenesis at N140 was shown to dislodge the binding of JQ1 to BRD4 (24497639), which implies that BD1 mutagenesis or overexpression of the BD1-deficient construct should abrogate the interaction of LOXL2 with BRD4, reminiscent to the abrogated interaction of BRD4/LOXL2 upon JQ1 that binds to both BDs (Figure 2F). And, what happens if a BD2-deficient construct is expressed?
    • If authors support that BRD4S is the predominant isoform driving the expression of DREAM-targets, this means that DREAM-targets are mainly bound by BRD4S, relying on Figure 3E-F. However, based on the author's ChIPseq tracks in Figure 3H, DREAM targets such as EZH2 and HMGB2 are co-occupied by both BRD4 isoforms at the basal state on their promoter region. Also, especially for EZH2 and PLK4, authors should set to 'group auto-scale' both conditions in a smaller scale range for ChIPseq- and RNAseq tracks, although I do not these two genes as good candidates representing your analysis. Therefore, authors should initially show all genes (e.g in a table format) that enrich the 'DREAM-targets' signature and select for a greater panel of genes (like for AURKB and HMGB2) demonstrating a preferential occupancy of the BRD4S at their promoter region. Finally, authors are recommended to perform a ChIP-qPCR on these genomic regions at basal state (no LOXL2 silencing) to validate the predominant occupancy of BRD4S and the low/absent occupancy of BRD4L at these genomic sites.
    • Authors in Figure 3G should select an equal-sized population of randomly chosen non-DREAM-target genes, otherwise, the comparison of log2FC difference between these two gene cohorts is unreliable and difficult to make. Mann-Whitney test should also be performed.
    • Authors should repeat the cell cycle analysis (Figure 4A) as the number of cells subjected to flow cytometry is quite discrepant between the conditions. Also, it is not clear if the experiment was performed in at least biological triplicates (although in the respective legend, it is stated so). If performed in biological triplicates, authors should make a new graph where each cell cycle phase cell population differs between the two conditions. Moreover, the difference in cell cycle defects in LOXL2-inhibited cells (Figure 4C) is indifferent compared to their control counterpart. Therefore, authors should address these inconsistencies.
    • Furthermore, authors should explain what was the rational selecting a mediator subunit and specifically MED1 as a possible interacting partner of LOXL2 and BRD4s since MED12 and MED24 were also highly essential (Figure 4F).
    • Moreover, do authors also observe this functional relationship of LOXL2 and BRD4S in cell cycle progression in other breast cancer subtypes presenting a high proliferation index e.g HER2+? Presumably, the author's proposed mechanism applies to a wide panel of breast cancer entities, for which, only key experiments could be performed.
    • Authors in Figure 5H represent LOXL2 and BRD4s as integral chromatin looping factors together with MED1 at promoter and enhancer regions. However, this illustration is an overrepresentation of their finding because authors did not address the differential occupancy of BRD4S upon LOXL2 loss in DREAM-target-specific enhancer regions. If they wish to do so, they may use the RANK ORDERING OF SUPER-ENHANCERS (ROSE) package to call for super-enhancer regions in the proximity of DREAM-targets and confirm similar results as for their TSS-proximal sites.
    • In the current manuscript, authors did not address the translational relevance of their proposed mechanism in the context of conventional therapies. Knowing that several BRD-specific compounds currently undergo clinical trials, authors should address if LOXL2 low (MDAMB468) and high (BT549) cells demonstrate a differential sensitivity to increasing doses of chemotherapy, in the presence or absence of BRD4. By doing that, LOXL2 apart from being a therapeutic target could be also used as a prognostic marker to stratify patients and achieve better response to standard therapies.

    Minor points:

    • In Figure 1D, the authors should convert the y-axis to a logarithmic scale to better represent the differences between JQ1, PXS, and combo. Also, One-way Anova should be performed between JQ1, PXS and combo.
    • In Figure S6F, authors did not show the sensitivity of LOXL2 low and high cell lines for BRD4 KO. If LOXL2-proficient cells are less sensitive to JQ1, based on Figure 1B, authors should consider showing something similar from the gene essentiality database.
    • Authors failed to discuss the work from Ozge Saatci et al (PMID: 32415208) regarding LOXL2 in TNBC and ECM reorganization as well as in other cancer entities (PMID: 35428659) in the context of ECM remodeling. Authors should realize that these published works and the current ones are not conflicting but complement each other.

    Significance

    Minor points:

    • In Figure 1D, the authors should convert the y-axis to a logarithmic scale to better represent the differences between JQ1, PXS, and combo. Also, One-way Anova should be performed between JQ1, PXS, and combo.
    • In Figure S6F, the authors did not show the sensitivity of LOXL2 low and high cell lines for BRD4 KO. If LOXL2-proficient cells are less sensitive to JQ1, based on Figure 1B, authors should consider showing something similar from the gene essentiality database.
    • Authors failed to discuss the work from Ozge Saatci et al (PMID: 32415208) regarding LOXL2 in TNBC and ECM reorganization as well as in other cancer entities (PMID: 35428659) in the context of ECM remodeling. Authors should realize that these published works and the current ones are not conflicting but complement each other.

    Significance

    The conception and findings are of enlightening significance for TNBC therapy, especially given the lack of targeted therapies in this particularly aggressive breast cancer subtype. Hence, I posit this work as highly relevant for the cancer epigenetics research community interested in characterizing unknown factors that facilitate the gene-activating function of epigenetic readers in health and disease.

    My field of expertise is to uncover epigenetic vulnerabilities responsible for transcriptional plasticity driving drug tolerance in aggressive forms of breast cancer.

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    Referee #2

    Evidence, reproducibility and clarity

    In their study, Pascual-Reguant et al. show that combined inhibition of BRD4 and LOXL2 can synergize to restrict triple-negative breast cancer (TNBC) proliferation. BRD4 and LOXL2 are transcription regulators that can read and write epigenetic information, respectively. The authors employ three distinct breast cancer cell lines and mouse models with cell line-derived xenografts, and they show that combined inhibition of BRD4 and LOXL2 can be superior to single BRD4/LOXL2 inhibition in these model systems. In an attempt to identify a connection between BRD4 and LOXL2, the authors find that the two proteins can bind to each other. The authors performed most of the experiments in the breast cancer cell line MDA-MB-231. To assess the impact of LOXL2-inhibition on transcription, the authors assessed changes of the transcriptome in MDA-MB-231 cells following LOXL2 knockdown. They found that genes related to cell differentiation and morphology were upregulated, while genes related to the cell cycle were downregulated. ChIP-seq data of BRD4 showed that BRD4 can bind to cell cycle gene promoters and that this binding was enhanced upon loss of LOXL2. The authors found that LOXL2 and BRD4 interacted with the transcriptional cell cycle regulators B-MYB, FOXM1, and LIN9, which are components of the MYB-MuvB-FOXM1 (MMB-FOXM1) complex that is known to promote the expression of late cell cycle genes with important functions during mitosis. The authors conclude that LOXL2/BRD4 interact with each other and with the MMB-FOXM1 complex to drive the expression of cell cycle genes and cell proliferations. Vice versa, they conclude that inhibition of LOXL2/BRD4 reduces cell proliferation through inhibiting the expression of cell cycle genes.

    Major:

    • The data and methods are presented well. The experiments are adequately replicated and analyzed. However, except for the first section, all experiments were performed using only one cell line. It is important to validate key findings in at least a second cell line.
    • There appears to be a misunderstanding of the concept of cell cycle-dependent gene regulation by the DREAM complex and its related factors. Early (G1/S) cell cycle genes contain E2F promoter motifs, while late (G2/M) cell cycle genes contain CHR promoter motifs. The DREAM complex can bind both, while RB-E2F and MuvB recognize only E2F and CHR motifs, respectively. B-MYB and FOXM1 bind to MuvB and regulate late cell cycle genes, but they do not bind to early cell cycle genes. Given this concept, the authors' rationale to connect BRD4/LOXL2 through MuvB/B-MYB/FOXM1 with E2F promoter sequences and early cell cycle genes and the subsequent conclusions must be corrected.
    • I felt that the suggested functional connection between LOXL2/BRD4 and DREAM is not strongly supported by the authors' data. Figure S6E: A similarity score of <0.7 is poor support for a 'consensus E2F sequence' and indicates very limited specificity. Figure 4E: IP with BRD4 and LOXL2 is missing as important control. A chromatin-binding control is missing that does not bind to DREAM/LOXL2/BRD4. To test for binding to the actual DREAM complex, the authors should include E2F4 and p130 in their IPs and western blots, perhaps following LOXL2 inhibition/knockdown. Figure 3: The authors' ChIP-seq data indicate that only a fraction of DREAM targets is bound by BRD4. To provide more evidence that LOXL2/BRD4 may be directly involved in regulating DREAM targets, the authors should compare the differential regulation of BRD4-bound DREAM targets upon LOXL2 knockdown with DREAM targets which are not bound by BRD4. If LOXL2/BRD4 acted in a direct manner on those targets, one would expect that loss of LOXL2 affected their transcription more strongly than the other DREAM targets which are affected only indirectly. Such an analysis can be performed readily using the available data.
    • The authors state that it is surprising to find that LOXL2 can promote target gene transcription because it is rather known as a transcriptional repressor. To this point, the authors should perform standard analyses using their RNA-seq and ChIP-seq data. Compare differential expression of genes that are bound by BRD4S/L/S+L and genes not bound by BRD4. Perform motif search and enrichment analyses for transcription factor and co-factor binding data (public ChIP-seq repositories). Such analyses may suggest what gene sets are up- and downregulated by LOXL2 through BRD4S/L and what other factors could be involved in LOXL2-dependent up- and downregulation of gene transcription.

    Minor:

    • I felt that background information on the BRD4 isoforms was missing. The short and long isoforms of BRD4 should be introduced briefly.
    • Given that BRD4 inhibition is known to activate p53 (e.g., PMID 23317504 and 33431824) and p21 (PMID 31265875), the authors should discuss the p53 status of their cell lines (largely mutant). In general, I felt that the authors could better cite and discuss the current literature on BRD4 and LOXL2.
    • It was unclear to me why the authors did not actually test experimentally whether their predicted interaction models 2 or 4 are likely true (Figure 2E+G).
    • The transcription of cell cycle genes depends on the cell cycle (i.e., reduced cell cycle entry correlates with reduced cell cycle gene expression). Given that the authors showed LOXL2 inhibition reduce MDA-MB-231 cell proliferation, they should note that reduced expression of cell cycle-related genes is expected upon LOXL2 knockdown.
    • The authors specify in their discussion that their data show a function of LOXL2/BRD4 in the cell cycle interphase, while there were no experiments that support that specific conclusion. At least it is unclear to me why the authors rule out a function in mitosis?
    • I felt that the first part of the manuscript (combination of BRD4 and LOXL2 inhibitors in TNBC) was a bit uncoupled from the functional studies on LOXL2 and its connection to BRD4. The transition between these parts and the final discussion on why the joint control of cell cycle genes by LOXL2/BRD4 may be important for the synergistic effect of LOXL2/BRD4 inhibitors. To this point, the authors' model was not clear to me.

    Significance

    The study by Pascual-Reguant et al. shows that inhibitors of BRD4 and LOXL2 can be combined to achieve better efficacy in reducing proliferation of breast cancer cell lines and breast tumor growth in xenograft models. They provide strong evidence for a functional interaction between LOXL2 and BRD4 and investigate their common transcriptional targets. Intriguingly, some evidence points towards a direct regulation of the DREAM complex and its cell cycle gene targets.

    The findings are novel and can be the basis for further research on TNBC combination therapy using BRD4 and LOXL2 inhibitors. The link to the DREAM complex is preliminary.

    The study is of interest for a basic research audience with some translational aspects.

    I reviewed this manuscript as a researcher in gene regulatory mechanisms, with cell cycle genes as one focus area. I have no expertise in the computational modeling of protein-protein interactions and I am no expert for breast cancer.

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    Referee #1

    Evidence, reproducibility and clarity

    The manuscript describes that simultaneous inhibition of LOXL2 and BRD4 reduces proliferation of TNBC in vitro and reduces growth in vivo. This observation is followed by extensive mechanistic studies that suggest physical interaction between LOXL2 and short isoform of BRD4-MED1. Inferences from Chip-seq analyses suggest that this interaction is involved in regulation of multiple transcriptional programs. Authors focus on differential activation of DREAM complex, to claim that this interaction "is fundamental for proliferation of TNBC". The manuscript is very well written and mechanistic inferences are based on a set of sophisticated epigenetic analyses and bioinformatical inferences. The phenotypic effects from LoxL2 inhibition by itself, or in combination with BRD4 inhibition are relatively modest. These modest effects, as well as many of the reported changes in gene expression are clearly inconsistent with the frequently used adjectives as "dramatic", "fundamental", "deeply affected", "drastically hampered" etc. Given the modest phenotypic effects, many of the key claims and conclusions are not supported by the data.

    Specifically:

    1. It is unclear why authors generalize their conclusions to TNBC. Figure 1B demonstrates synergy for 1/3 cell lines, which is chosen for the follow up study. Even for MDA231, the synergy is confined to low concentrations of BRD4i (S1c). While MDA231 cell line is frequently used in experimental studies of TNBC, it is quite dissimilar to majority of clinical TNBC, and contains mutant RAS, which is rare in this disease.
    2. In vivo, the effect appears to be modest even in the MDA231 model, selected for evidence of synergy in vitro. In vivo, the combination appears to have an additive effect. Tumor growth rates are reduced, but no shrinkage is occurring. In the PDX model, LOXL2i does not have an effect as a monotherapy, while modestly enhancing the impact of BRD4i. These results are at odds with the claim of the interaction being fundamental for proliferation.
    3. No analysis of cell proliferation was shown in vivo. Authors should have performed BrdU or KI67 staining to support the claim. For in vitro analyses, authors also used indirect assays for proliferation. PI staining by itself does not have sufficient resolution to clearly capture modest effects that authors demonstrate. BrdU-PI double staining would have been much more useful.

    Minor points:

    1. Dose dependent decrease in phosphorylated H3 is not at all obvious from eyeballing the data in S1A; the only effect that I see is a modest reduction at the highest concentration of the inhibitor. Authors need to quantify the results to support the claim.
    2. Most of breast cancer cell lines are derived from metastatic disease, including pleural effusion, thus the point that because MDA231 cell line is derived from pleural effusion, it is metastatic does not have sufficient logical foundation.
    3. How is loss of cell-cell junction in vitro consistent with LOXL2 role in modulating ECM? There is no evidence of ECM production in MDA231 in vitro. On the other hand, this loss is associated with EMT.

    Significance

    Discovery and characterization of LOXL2-BRD4 interaction is advancing the ever-deepening understanding of molecular mechanisms of regulation of gene expression. The studies and analyses appear to be sufficiently rigorous and reported with clarity, and the claimed discovery of the biological interaction between LOXL2 and BRD4 is well supported. However, given the magnitude of the reported (rather than claimed) effects of this interaction, and concerns about generalizability of authors conclusions, it is not clear how these results are promising for the development of new therapies in TNBC. Moreover, in contrast to luminal BC, there is no clear evidence for utility of cytostatic drugs in constraining TNBC. Therefore, biological and clinical significance of the authors discovery is unclear and claims in this regard appear to be overblown.

  5. Version published to 10.1101/2022.05.27.493725v1 on bioRxiv