MLL3 regulates the CDKN2A tumor suppressor locus in liver cancer

  1. Changyu Zhu
  2. Yadira M Soto-Feliciano
  3. John P Morris
  4. Chun-Hao Huang
  5. Richard P Koche
  6. Yu-jui Ho
  7. Ana Banito
  8. Chun-Wei Chen
  9. Aditya Shroff
  10. Sha Tian
  11. Geulah Livshits
  12. Chi-Chao Chen
  13. Myles Fennell
  14. Scott A Armstrong
  15. C David Allis
  16. Darjus F Tschaharganeh
  17. Scott W Lowe

  • Curated by eLife

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    Evaluation Summary:

    This manuscript shows that mutations in the gene encoding an enhancer chromatin-modifying enzyme MLL3 cooperate with Myc overexpression to drive hepatocellular carcinoma in mouse models. The authors identify Cyclin Dependent Kinase Inhibitor 2A (Cdkn2a) as a critical direct target gene of MLL3. Overall, the manuscript makes a compelling case that MLL3 is a hepatocellular carcinoma tumor suppressor that directly binds and activates the Cdkn2a locus. This study provides important insights for cancer biologists and those interested in specific epigenetic mechanisms that regulate liver cancer development. Editorial and some experimental suggestions were made to strengthen the work.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their name with the authors.)

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Abstract

Mutations in genes encoding components of chromatin modifying and remodeling complexes are among the most frequently observed somatic events in human cancers. For example, missense and nonsense mutations targeting the mixed lineage leukemia family member 3 (MLL3, encoded by KMT2C ) histone methyltransferase occur in a range of solid tumors, and heterozygous deletions encompassing KMT2C occur in a subset of aggressive leukemias. Although MLL3 loss can promote tumorigenesis in mice, the molecular targets and biological processes by which MLL3 suppresses tumorigenesis remain poorly characterized. Here, we combined genetic, epigenomic, and animal modeling approaches to demonstrate that one of the mechanisms by which MLL3 links chromatin remodeling to tumor suppression is by co-activating the Cdkn2a tumor suppressor locus. Disruption of Kmt2c cooperates with Myc overexpression in the development of murine hepatocellular carcinoma (HCC), in which MLL3 binding to the Cdkn2a locus is blunted, resulting in reduced H3K4 methylation and low expression levels of the locus-encoded tumor suppressors p16/Ink4a and p19/Arf. Conversely, elevated KMT2C expression increases its binding to the CDKN2A locus and co-activates gene transcription. Endogenous Kmt2c restoration reverses these chromatin and transcriptional effects and triggers Ink4a/Arf-dependent apoptosis. Underscoring the human relevance of this epistasis, we found that genomic alterations in KMT2C and CDKN2A were associated with similar transcriptional profiles in human HCC samples. These results collectively point to a new mechanism for disrupting CDKN2A activity during cancer development and, in doing so, link MLL3 to an established tumor suppressor network.

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  1. Version published to 10.7554/elife.80854 on eLife
  2. eLife

    Author Response

    Reviewer #1 (Public Review):

    In this manuscript, Soto-Feliciano et al. investigate the tumor suppressive role of MLL3 in hepatocellular carcinoma (HCC). The authors used a variety of techniques including hydrodynamic tail vain injection (HTVI), CRISPR deletion, and shRNA to disrupt MLL3 expression in mouse models. They clearly show that MLL3 acts as a tumor suppressor in the context of MYC-induced HCC. They show that MLL3 acts by activating the Cdkn2a locus. Genomic analysis showed that MLL3 binds to enhancers and promoters, and specifically interacts with the Cdkn2a promoter. When MLL3 was downregulated, Cdkn2a levels fell and this corresponded to changes in relevant histone marks targeted by MLL3. The authors were also able to show that reintroduced MLL3 expression in a dox inducible system could rescue CDKN2A locus expression, which in turn reduced colony formation and induced apoptosis. Human genomic correlation showed that MLL3 and Cdkn2a mutations are generally mutually exclusive. Overall, the conclusions of the manuscript are well supported by a logical series of experiments with good controls and orthogonal approaches. While it would be useful to examine another HCC model such a CTNNB1-driven model, the current paper is convincing in its conclusions.

    We thank the reviewer for their positive and constructive comments and suggestions. Our study primarily used MYC as the driving oncogene for two reasons: first, in an initial in vivo screen of 12 candidate tumor suppressors, MLL3 was the strongest hit that its loss cooperated with the Myc oncogene to drive HCC (Figure 1—figure supplement 1); second, in human HCCs, KMT2C (gene encoding MLL3) mutations and deletions co-occur with MYC gains and amplification.

    Based on the reviewer’s suggestion, we examined MLL3 loss in conjunction with CTNNB1 activation, using HTVI of a transposon containing the constitutively active Ctnnb1. However, we did not observe oncogenic cooperation between Ctnnb1 activation and Kmt2c loss; no mice developed liver tumors by the experimental endpoint (5 months post HTVI, Figure 1—figure supplement 3). Additionally, analysis of genomic data from human HCCs showed no significant co-occurrence between CTNNB1 and KMT2C alterations (Figure 1A). These results suggest that, similar to other epigenetic regulators, the tumor suppressive function of MLL3 is likely oncogene-specific. Our in vivo screen results that nominated MLL3 as a tumor suppressor also reinforce this functional interaction with MYC oncogene. We have updated the text to reflect the context specificity of MLL3 as a tumor suppressor in our study.

    Reviewer #2 (Public Review):

    Soto-Feliciano et al. have characterized the function of MLL3 in hepatocellular carcinoma (HCC) suppression. MLL3 is recurrently mutated in human HCC. The authors show that Mll3 mutations cooperate with Myc overexpression to drive HCC cancer in mice. They identify Cdkn2a as a critical direct target of MLL3. Overall, the manuscript makes a compelling case that MLL3 is a bona fide HCC tumor suppressor, that it directly binds and activates the Cdkn2a locus, and that Cdkn2a acts downstream of MLL3 to suppress HCC initiation.

    The strengths of the paper include mouse modeling techniques that clearly demonstrate a role for MLL3 in suppressing Myc-driven HCC, a detailed characterization of MLL3 binding sites and target gene expression, and the combined weight of several functional studies showing that MLL3 induces apoptosis in hepatocytes/HCC by inducing p16 and ARF. The major conclusions appear well-supported by the data.

    The paper does have some weaknesses. Some of the genomic data require clarification. Furthermore, the authors draw broad conclusions about an epistatic relationship between MLL3 and CDKN2A based on mutually exclusive mutation patterns in human cancers. Those conclusions are not as well-supported as the mechanistic conclusions. The incidences of MLL3 and CDKN2A mutations in HCC are both relatively low (1% and 5% respectively), so it seems difficult to draw any conclusions from mutually exclusive profiles.

    One additional criticism is that the paper is a bit reductive. The link to CDKN2A offers a satisfying explanation for how MLL3 suppresses HCC, but the model may oversimplify the functions of MLL3.

    We thank the reviewer for their constructive comments and suggestions, which we addressed as follows with point-by-point response provided below. We agree with the concerns regarding the mutational analyses of KMT2C and CDKN2A in human cancers and the working model of the manuscript. We have removed the majority of the mutational analyses from the Results section. Importantly, our latest integrative analyses of RNA-seq and MLL3 ChIP-seq revealed other potential downstream effectors of MLL3 tumor suppressive functions (Figure 3A and Figure 3—figure supplement 1B). We have modified the Results and the Discussion to reflect this more nuanced view of MLL3 function in cancer. Nonetheless, we believe that other data continue to support our conclusion that CDKN2A is a dominant effector of MLL3 tumor suppressive functions in our model.

    Reviewer #3 (Public Review):

    The enhancer chromatin-modifying enzyme MLL3 functions as a tumor suppressor in multiple human cancers, however, the mechanisms underlying its tumor suppressive function remain unclear. The manuscript of SotoFeliciano et al. focused on Myc-driven liver cancer and aimed to address and fill the gap. The authors used an elegant genetic design and approach to manipulate the overexpression of the Myc oncogene and knockout of the Mll3 tumor suppressor gene in mouse liver cancer models. Their genetic mouse models showed that loss of Mll3 constrains Myc-driven liver tumorigenesis, with tumors having a slightly later onset compared to mice with Myc overexpression in conjunction with p53 inactivation. Because MLL3 is a major histone-modifying enzyme for enhancer-associated H3K4 monomethylation and is responsible for enhancer activation and the following target gene transcription, they performed ChIP-seq analysis to study the roles of Mll3 in Myc-driven mouse liver cancer. Interestingly, their ChIP-seq studies revealed that loss of Mll3 preferentially limits Mll3 enrichments at promoters and thereby attenuates promoter-associated H3K4 trimethylation and target gene transcription, whereas the unchanged Mll3 genomic binding between the two genotypes (Myc;sgTrp53 and Myc;sgKmt2c) is largely located within enhancer (intergenic) regions. They further demonstrated that the cdkn2a locus is a genomic and transcriptional target of Mll3 in Myc-driven mouse liver cancer. Supporting their findings, genomic inactivations of MLL3 and CDKN2A displays mutual exclusivity in human liver cancer and many other cancer types. Furthermore, they described a possible mechanism for MLL3's role in MYC-driven liver cancer that MLL3 mediates MYC-induced apoptosis in a CDKN2A-dependent manner by manipulating Myc overexpression, Mll3 function, and Cdkn2a regulation in their genetic mice models. This manuscript describes a potential function of MLL3 in the control of tumor suppressor gene expression via modulating their promoter chromatin landscapes. More importantly, loss of normal function of MLL3 or the downstream effector CDKN2A may impair MYC-induced apoptosis, and in turn, lead to MYC-induced tumorigenesis.

    Overall, the manuscript is well written, organized, and focused on an interesting topic, and with data presented supports the authors' claims.

    We thank the reviewer for their positive and constructive comments and suggestions.

  3. eLife

    Evaluation Summary:

    This manuscript shows that mutations in the gene encoding an enhancer chromatin-modifying enzyme MLL3 cooperate with Myc overexpression to drive hepatocellular carcinoma in mouse models. The authors identify Cyclin Dependent Kinase Inhibitor 2A (Cdkn2a) as a critical direct target gene of MLL3. Overall, the manuscript makes a compelling case that MLL3 is a hepatocellular carcinoma tumor suppressor that directly binds and activates the Cdkn2a locus. This study provides important insights for cancer biologists and those interested in specific epigenetic mechanisms that regulate liver cancer development. Editorial and some experimental suggestions were made to strengthen the work.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their name with the authors.)

  4. eLife

    Reviewer #1 (Public Review):

    In this manuscript, Soto-Feliciano et al. investigate the tumor suppressive role of MLL3 in hepatocellular carcinoma (HCC). The authors used a variety of techniques including hydrodynamic tail vain injection (HTVI), CRISPR deletion, and shRNA to disrupt MLL3 expression in mouse models. They clearly show that MLL3 acts as a tumor suppressor in the context of MYC-induced HCC. They show that MLL3 acts by activating the Cdkn2a locus. Genomic analysis showed that MLL3 binds to enhancers and promoters, and specifically interacts with the Cdkn2a promoter. When MLL3 was downregulated, Cdkn2a levels fell and this corresponded to changes in relevant histone marks targeted by MLL3. The authors were also able to show that reintroduced MLL3 expression in a dox inducible system could rescue CDKN2A locus expression, which in turn reduced colony formation and induced apoptosis. Human genomic correlation showed that MLL3 and Cdkn2a mutations are generally mutually exclusive. Overall, the conclusions of the manuscript are well supported by a logical series of experiments with good controls and orthogonal approaches. While it would be useful to examine another HCC model such a CTNNB1-driven model, the current paper is convincing in its conclusions.

  5. eLife

    Reviewer #2 (Public Review):

    Soto-Feliciano et al. have characterized the function of MLL3 in hepatocellular carcinoma (HCC) suppression. MLL3 is recurrently mutated in human HCC. The authors show that Mll3 mutations cooperate with Myc overexpression to drive HCC cancer in mice. They identify Cdkn2a as a critical direct target of MLL3. Overall, the manuscript makes a compelling case that MLL3 is a bona fide HCC tumor suppressor, that it directly binds and activates the Cdkn2a locus, and that Cdkn2a acts downstream of MLL3 to suppress HCC initiation.

    The strengths of the paper include mouse modeling techniques that clearly demonstrate a role for MLL3 in suppressing Myc-driven HCC, a detailed characterization of MLL3 binding sites and target gene expression, and the combined weight of several functional studies showing that MLL3 induces apoptosis in hepatocytes/HCC by inducing p16 and ARF. The major conclusions appear well-supported by the data.

    The paper does have some weaknesses. Some of the genomic data require clarification. Furthermore, the authors draw broad conclusions about an epistatic relationship between MLL3 and CDKN2A based on mutually exclusive mutation patterns in human cancers. Those conclusions are not as well-supported as the mechanistic conclusions. The incidences of MLL3 and CDKN2A mutations in HCC are both relatively low (1% and 5% respectively), so it seems difficult to draw any conclusions from mutually exclusive profiles.

    One additional criticism is that the paper is a bit reductive. The link to CDKN2A offers a satisfying explanation for how MLL3 suppresses HCC, but the model may oversimplify the functions of MLL3.

  6. eLife

    Reviewer #3 (Public Review):

    The enhancer chromatin-modifying enzyme MLL3 functions as a tumor suppressor in multiple human cancers, however, the mechanisms underlying its tumor suppressive function remain unclear. The manuscript of Soto-Feliciano et al. focused on Myc-driven liver cancer and aimed to address and fill the gap. The authors used an elegant genetic design and approach to manipulate the overexpression of the Myc oncogene and knockout of the Mll3 tumor suppressor gene in mouse liver cancer models. Their genetic mouse models showed that loss of Mll3 constrains Myc-driven liver tumorigenesis, with tumors having a slightly later onset compared to mice with Myc overexpression in conjunction with p53 inactivation. Because MLL3 is a major histone-modifying enzyme for enhancer-associated H3K4 monomethylation and is responsible for enhancer activation and the following target gene transcription, they performed ChIP-seq analysis to study the roles of Mll3 in Myc-driven mouse liver cancer. Interestingly, their ChIP-seq studies revealed that loss of Mll3 preferentially limits Mll3 enrichments at promoters and thereby attenuates promoter-associated H3K4 trimethylation and target gene transcription, whereas the unchanged Mll3 genomic binding between the two genotypes (Myc;sgp53 and Myc;sgMll3) is largely located within enhancer (intergenic) regions. They further demonstrated that the cdkn2a locus is a genomic and transcriptional target of Mll3 in Myc-driven mouse liver cancer. Supporting their findings, genomic inactivations of MLL3 and CDKN2A displays mutual exclusivity in human liver cancer and many other cancer types. Furthermore, they described a possible mechanism for MLL3's role in MYC-driven liver cancer that MLL3 mediates MYC-induced apoptosis in a CDKN2A-dependent manner by manipulating Myc overexpression, Mll3 function, and Cdkn2a regulation in their genetic mice models. This manuscript describes a potential function of MLL3 in the control of tumor suppressor gene expression via modulating their promoter chromatin landscapes. More importantly, loss of normal function of MLL3 or the downstream effector CDKN2A may impair MYC-induced apoptosis, and in turn, lead to MYC-induced tumorigenesis.

    Overall, the manuscript is well written, organized, and focused on an interesting topic, and with data presented supports the authors' claims.

  7. Version published to 10.1101/2022.06.07.495174v1 on bioRxiv