MLL3 regulates the CDKN2A tumor suppressor locus in liver cancer

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.


INTRODUCTION
Hepatocellular carcinoma (HCC) is a deadly primary liver cancer with a 5-year survival rate of only 18% (Jemal et al., 2017).HCC is currently the fourth most frequent cause of cancer-related mortality worldwide with a continuously growing incidence (Llovet et al., 2021).Genomic alterations found in HCC are highly diverse, and are characterized by TERT (telomerase reverse transcriptase) promoter mutations, amplifications or chromosomal gains encompassing the MYC oncogene, activating hotspot mutations in CTNNB1 (β-catenin), and inactivating mutations and deletions in the TP53 and CDKN2A tumor suppressor genes (Schulze et al., 2015;The Cancer Genome Atlas Research Network, David A. Wheeler, Lewis R. Roberts, 2017).Beyond these well-studied drivers, HCC frequently harbors mutations in one or more chromatin modifying enzymes, including MLL3 (KMT2C) (Fujimoto et al., 2012;Kan et al., 2013).
MLL3 is a component of the COMPASS-like complex that has structural and functional similarities to the developmentally essential Drosophila Trithorax-related complex (Schuettengruber et al., 2017).This multiprotein complex controls gene expression through its histone H3 lysine 4 (H3K4) methyltransferase activity that establishes chromatin modifications most often associated with transcriptional activation (Shilatifard, 2012).Interestingly, mutations in another homologous H3K4 methyltransferase of the complex, MLL4 (KMT2D) are also found in HCC (Cleary et al., 2013).KDM6A (UTX), a H3K27 demethylase within the COMPASS-like complex, has been recently identified as a potent tumor suppressor in liver and pancreatic cancers (Revia et al., 2021).While these observations suggest that epigenetic-based mechanisms of gene regulation controlled by the MLL3 complex can constrain HCC development, the molecular targets and biological processes that underlie MLL3's tumor suppressor activities remain poorly understood.

Mll3 is a tumor suppressor in Myc-driven liver cancer
To determine whether Mll3 loss impacts liver cancer development, we applied hydrodynamic tail vein injection (HTVI) in wild-type mice to directly introduce genetic manipulations into a subset of adult hepatocytes in vivo (Bell et al., 2007).This gene delivery method facilitates the study of oncogene-tumor suppressor interactions by combining stable genomic integration of oncogenic cDNAs (transposon vector) and transient expression of plasmids encoding Cas9 and single guide RNAs (sgRNAs) to disrupt tumor suppressor genes (Largaespada, 2009;Moon et al., 2019;Tschaharganeh et al., 2014;Xue et al., 2014).
Since MLL3 mutations co-occur with MYC genomic gains and amplifications in human HCC tumors (Fig. 1A), we used the HTVI approach to test whether disruption of Mll3 by CRISPR could cooperate with Myc overexpression to drive murine liver cancer (Fig. 1B).
Mice injected with a Myc cDNA transposon and two independent Cas9/Mll3 sgRNAs (Myc; sgMll3.1 or Myc; sgMll3.2) combinations developed liver tumors with a slightly later onset compared to mice receiving an sgRNA targeting the tumor suppressor Trp53 (Myc; sgp53) (Fig. 1C-D).Adult livers injected with Myc and a control sgRNA (sgChr8) did not succumb to disease over the observation period (Fig. 1C).These findings were confirmed in a second independent cohort of mice (Fig. S1A).Analyses of tumor-derived genomic DNA revealed insertions and deletions (indels) in either Mll3 or Trp53 (hereafter simply referred to as p53) depending on the genotype of tumor-derived cells (Fig. S1B).DNA sequencing of the CRISPRtargeted region from two independent Myc; sgMll3 tumors revealed either heterozygous or homozygous indels predicted to generate premature stop codons (Fig. S1C).These data imply that even partial suppression of Mll3 can promote tumorigenesis.In support of this, GFP-linked Mll3 shRNAs efficiently cooperated with Myc overexpression to drive liver cancer producing tumors with 50-80% reduction in Mll3 mRNA expression (Fig. S1D-S1G).shMll3.2resulted in less potent knockdown than shMll3.1,yet produced faster tumor formation, suggesting that similar to acute myeloid leukemia (Chen et al., 2014), Mll3 can likely act as a haploinsufficient tumor suppressor in liver cancer (Fig. S1E and S1G).

Mll3 loss alters the chromatin landscape of liver cancer cells
MLL3 and MLL4 are histone methyltransferases that can deposit the H3K4 mono-methylation mark at genomic enhancers and intergenic regions during organ development (Hu et al., 2013).However, more recent studies indicate that MLL3 and MLL4 are also capable of binding to promoter regions (Cheng et al., 2014;Dhar et al., 2016;Wang et al., 2010), especially in the context of cancer (Soto-Feliciano et al., 2021).To determine the genomic binding patterns of MLL3 in HCC, we performed MLL3 ChIPsequencing (ChIP-Seq) analysis in Myc; sgMll3 (sgMll3.1 which generates heterozygous or homozygous indels) and Myc; sgp53 liver cancer cell lines.Compared to sgp53 cells, Mll3 deficiency resulted in a marked reduction in MLL3 chromatin binding at a subset of genomic loci (Fig. 2A).Approximately 40% of the peaks that were selectively lost in Mll3-deficient cells occurred at promoter regions, whereas unchanged MLL3 peaks between the two genotypes were more likely to be within intergenic regions (Fig. 2B, Fig. S2).Of note, the residual signal observed in ChIP-Seq most likely reflects the binding of MLL4 and/or remnant MLL3, since the antibody used in these experiments can recognize both MLL3 and MLL4 proteins (Dorighi et al., 2017).Our data suggest that, beyond the canonical action of MLL3 at gene enhancers, MLL3 and MLL4 can also occupy promoter regions in Myc-induced liver cancer.
Similar to the Drosophila Trithorax-related complex (Schuettengruber et al., 2017), the mammalian MLL3 and MLL4 complexes facilitate gene transcription by establishing permissive modifications on histone H3K4 via the MLL3 and MLL4 methyltransferases (Shilatifard, 2012).While MLL3 can establish H3K4 mono-methylation at enhancers in a redundant fashion with MLL4 (Shilatifard, 2012), Mll3 inactivation also decreases H3K4me3 levels at the promoters of metabolism-related genes in normal murine livers (Valekunja et al., 2013) and human liver cancer cells (Ananthanarayanan et al., 2011).To determine whether Mll3 haploinsufficiency impacts the local or global chromatin landscape of HCC cells, we performed ChIP-Seq analyses for H3K4 methylation and H3K27 acetylation in three independently derived Myc; sgMll3 and Myc; sgp53 tumor cell lines (Fig. 2C).Correlation analyses revealed three distinct clusters that include areas of enrichment and depletion for each tested histone modification between Myc; sgMll3 and Myc; sgp53 tumor cells (Fig. 2C, Fig. S3A-C).Loci associated with cluster 1 (reduced H3K4me3, H3K4me1, and H3K27ac in sgMll3 cells) showed the most pronounced and dynamic differences in chromatin modifications between the two liver tumor genotypes.In contrast, the loci in cluster 2 showed increased H3K4me1 and H3K27ac marks and most of them mapped to the intergenic regions of the genome, and cluster 3 loci with enhanced H3K4me3 likely represent some p53 target genes such as Cdkn1a and Eda2r.
To determine whether Mll3 disruption is associated with these drastic changes in the chromatin landscape, we integrated our MLL3 ChIP-Seq from the Myc; sgp53 vs Myc; sgMll3 tumor cells with the chromatin modifications analyses (Fig. 2C).Interestingly, loci present in cluster 1 that displayed the most substantial changes in histone modifications involved genes that showed high MLL3 enrichment in Myc; sgp53 cells compared to the Myc; sgMll3 genotype.These data support a model whereby MLL3 binding to these loci facilitates the acquisition of a chromatin environment conducive for active gene transcription.
We next determined the output of these chromatin landscape changes by transcriptional profiling of the same set of Myc; sgp53 and Myc; sgMll3 liver cancer cell lines described above.Despite the broad binding of MLL3 across the genome, only 132 differentially expressed genes (DEGs) were significantly up-regulated (P<0.05,log 2 fold-change>2) and 116 DEGs significantly down-regulated (P<0.05,log 2 fold-change<-2) in Myc; sgMll3 liver tumor cells compared to Myc; sgp53 controls.As predicted, transcripts encoding p53 and p53 target genes such as Ccng1, Cdkn1a, and Zmat3 (Bieging-Rolett et al., 2020) were elevated in Myc; sgMll3 cells, consistent with nonsense-mediated decay of truncated p53 transcripts and a concomitant reduction in p53 effector genes.Strikingly, some of the downregulated genes in Myc; sgMll3 lines mapped to loci enriched in cluster 1, including Cdkn2a, Bmp6, and Lrp2 (Fig. 2C-D, Fig. S3D-E).In principle, cluster 1 genes that display: 1) MLL3 binding enrichment, 2) a histone profile associated with gene activation, and 3) reduced transcript levels in Myc; sgMll3 tumor cells should include genes that mediate the molecular and cellular effects downstream of Mll3 disruption during tumor formation.

Genomic inactivations of MLL3 and CDKN2A display mutual exclusivity in human HCC
One genomic locus that stood out in our integrative analysis was Cdkn2a, which encodes for the p16 Ink4a and p19 Arf (p14 ARF in humans) tumor suppressor genes (hereafter referred to as Ink4a and Arf, respectively) (Gil and Peters, 2006).CDKN2A is located on the human chromosome 9p, and is deleted or epigenetically silenced in many cancer types (Sherr, 2012) including HCC (The Cancer Genome Atlas Research Network, David A. Wheeler, Lewis R. Roberts, 2017).While MLL3 likely regulates a plethora of genes that contribute to its tumor suppressive potential, the well-defined and potent anti-tumor functions of Cdkn2a-encoded proteins make them attractive candidates as functionally relevant MLL3 effectors.
To gain further insights into the potential relationship between MLL3 and CDKN2A, we first looked at human cancer datasets to assess whether genomic alterations of MLL3 and CDKN2A display an epistatic relationship that would be indicative of functional redundancy.Consistent with this possibility, an analysis of 1158 HCC samples derived from five independent, publicly available HCC sequencing datasets revealed a mutually exclusive relationship between the presence of MLL3 and CDKN2A inactivating alterations including truncating mutations and deep deletions (Fig. 3A).Of note, our analyses excluded missense mutations owing to their unknown impact on MLL3 functions.Further dissection of transcriptional profiling datasets from human and mouse HCCs harboring known gene alterations using gene set enrichment analysis (GSEA) revealed that human tumors with CDKN2A deletions transcriptionally resemble both mouse and human HCC harboring MLL3 alterations (Fig. 3B-C), but not those harboring RB1 loss (Fig. S4A).Consistent with a degree of context-dependence that is observed for other chromatin regulators such as EZH2 (Ntziachristos et al., 2012;Soto-Feliciano et al., 2017;Souroullas et al., 2016;Tirode et al., 2014), MLL3 and CDKN2A alterations displayed co-occurrence in several tumor types (Fig. S4B).Nevertheless, an overall analysis of a pan-cancer cohort showed a pattern of mutual exclusivity (Zehir et al., 2017) (Fig. S4C), with trends noted in ten individual blood and epithelial cancer types (Fig. S4D).While we cannot rule out the possibility that other factors drive these associations, our results support a biologically meaningful relationship between MLL3 and CDKN2A.

Cdkn2a locus is a genomic and transcriptional target of MLL3 in liver cancer
To explore the relationship between MLL3 and Cdkn2a in more detail, we tested whether genes encoded by Cdkn2a were direct targets of MLL3-regulated transcription.Indeed, Cdkn2a is a cluster 1 locus that display: 1) significant reduction in expression in Myc; sgMll3 cancer cells, with both decreased 2) levels of H3K4me1/3 and H3K27ac and 3) MLL3 binding at the promoters of Ink4a and Arf in sgMll3 tumors compared to sgp53 cells (Fig. 2C-D, Fig. 4A).The differential expression of Ink4a and Arf were also confirmed by qPCR, immunoblotting, and ChIP-qPCR analyses on multiple Myc; sgMll3 and Myc; sgp53 liver cancer lines (Fig. 4B, Fig. S5A-B).These results imply that Cdkn2a locus is a genomic and transcriptional target of MLL3 in liver cancer cells.
Since the Myc; sgp53 and Myc; sgMll3 cells we studied above are not isogenic, we performed a series of additional experiments to demonstrate a direct transcriptional effect of MLL3 on the Cdkn2a locus.
While p53 inactivation can lead to compensatory increases in Ink4a and Arf expression (Stott et al., 1998), p53 suppression in sgMll3 cells produced only a subtle and inconsistent effect on expression of Ink4a and Arf (Fig. S6A), and Mll3 suppression in sgp53 cells attenuated p16 Ink4a and p19 Arf protein levels (Fig.

S6B). In addition, liver cancer cells produced by hydrodynamic delivery of the Myc transposon vector and
Axin1 sgRNAs (Fig. S6C) with or without an Mll3 shRNA retained wild-type p53 and showed reduced Ink4a and Arf mRNA and protein expression (Fig. S6D-E).These data imply that MLL3 supports a chromatin environment at the Cdkn2a locus that facilitates transcription of both Ink4a and Arf products of the Cdkn2a locus, and raises the possibility that these factors contribute to the tumor suppressor activity of MLL3 in liver cancer.
We next set out to determine whether MLL3 binding is sufficient to induce transcriptional activation of the CDKN2A locus and, in doing so, extend our analysis to human liver cancer cells.As the MLL3 transcript is too large (14,733 bp) for cDNA transduction, we turned to the CRISPR activation (CRISPRa) system (Chavez et al., 2015) in a human hepatocellular carcinoma cell line (HLE).Following stable integration of the CRISPRa Cas9, cells were transduced with an sgRNA targeting the human MLL3 promoter or sequences within GFP as a control (Fig. 4C).Cells expressing the MLL3 sgRNA showed a marked and specific increase in the expression of endogenous MLL3, but not of MLL4 or TP53 (Fig. 4D, Fig. S6F), which was accompanied by a concomitant increase in MLL3 binding to the CDKN2A locus (Fig. 4E) and transcriptional upregulation of INK4A and ARF (Fig. 4F-G).Therefore, MLL3 directly binds and coactivates transcription of the CDKN2A locus in human liver cancer cells.

MLL3 mediates oncogene-induced apoptosis in a Cdkn2a-dependent manner
The above results raise the possibility that the Cdkn2a products, Ink4a and Arf, may contribute to the tumor suppressive activity of MLL3.In this regard, Myc overexpression in primary cells (MEFs) often triggers apoptosis (Evan et al., 1992), and this in turn limits tumorigenesis in a manner that is dependent on Cdkn2a and most prominently Arf (Zindy et al., 1998).This pathway also suppresses liver tumorigenesis, since concomitant disruption of Ink4a and Arf using CRISPR, or through germline deletion of Arf alone, cooperated with Myc overexpression to rapidly promote tumor development (Fig. S7A).
Similarly, Mll3 suppression also attenuated MYC-induced apoptosis, as shown by tumor histology and apoptosis by TUNEL assay (Negoescu et al., 1997), 5 days after hydrodynamic delivery of transposon vectors encoding Myc together with GFP-linked shRNAs targeting Mll3 (or Renilla luciferase as a control) (Fig. 5A-B).This dramatic difference in apoptosis correlated with an increase in retention of GFP-shMll3 expressing cells 10 days after injection (Fig. S7B-C).We also observed that apoptotic GFP-shRenillaexpressing hepatocytes were typically surrounded by immune cells, while shMll3-expressing cells formed incipiently transformed clusters that lacked immune infiltration (Fig. S7D).Altogether, these results show that Mll3 suppression impairs Myc-induced apoptosis in vivo in a manner that is reminiscent of the antiapoptotic effects of Cdkn2a loss in the context of aberrant Myc activation (Eischen et al., 1999;Jacobs et al., 1999;Schmitt et al., 1999).
To model the interaction between Myc overexpression, Mll3 function, and Cdkn2a regulation, we transduced liver progenitor cells (LPCs) with retroviral vectors encoding Myc linked to a reverse tetracycline transactivator (rtTA3), together with doxycycline (dox)-inducible Mll3 shRNAs to enable reversible Mll3 silencing (Fig. S8A).Infection of LPCs with Myc in the presence of Mll3 (i.e. cells infected with Myc-rtTA3 and a dox-inducible shRNA targeting Renilla luciferase) acutely activated Ink4a and Arf expression (Fig. S8B) and these cells could not be maintained in culture.Phenocopying the ability of Myc and Mll3 suppression to transform liver cells in vivo, combined Myc and shMll3 expression facilitated the persistent growth of cells maintained on Dox (Fig. S8C-D).By contrast, Dox withdrawal induced Mll3 mRNA expression (Fig. S8C) and H3K4me3 deposition at the Arf and Ink4a promoters, ultimately leading to elevations in Arf and Ink4a mRNA and protein (Fig. 5C-D, Fig. S8F), reduced colony formation, and increased apoptosis (Fig. S8D-E).Furthermore, constitutive shRNA-mediated knockdown of Arf and Ink4a through targeting of the shared exon 2 (shInk4a/Arf) significantly rescued colony forming capacity and prevented cell death following Mll3 restoration as determined by time-lapse microscopy of cells cultured with a fluorescent viability dye (Fig. 5E-G, Fig. S8G).These data support a model whereby a prominent tumor suppressive output of MLL3 in liver cancer involves direct upregulation of Cdkn2a that, when impaired, attenuates the MYC-induced apoptotic program and permits tumor progression.

DISCUSSION
Our study combined genetic, epigenomic, and animal modeling approaches to identify Cdkn2a as an important regulatory target of MLL3 in both mouse and human liver cancers.Our results support a model whereby oncogenic stress, herein produced by MYC, leads to an increase in the binding of MLL3 to the CDKN2A locus, an event that is associated with the accumulation of histone marks linked to the biochemical activity of MLL3-containing complexes and conducive to gene activation (Fig. 6).Accordingly, these events are accompanied by transcriptional upregulation of two key CDKN2A gene products, INK4A and ARF, and suppression of MLL3 phenocopies the effects of CDKN2A inactivation in abrogating MYCinduced apoptosis.Conversely, suppression of CDKN2A abolishes the anti-proliferative effects of MLL3 restoration.As such, our results establish a conserved epistatic relationship between the chromatin modifier MLL3 and a well-characterized tumor suppressor network.While the epistatic relationship described above is supported by the trends towards mutually exclusivity of MLL3 and CDKN2A alterations in liver cancer and several other tumor types, this was not always the case.In some tumor types, this may be due to limited samples sizes, other functionally important components linked to the CDKN2A locus could produce CDKN2A-independent forces that drive selection for chromosome 9p deletions, including type I interferon genes, CDKN2B, and MTAP (Fountain et al., 1992;Schmid et al., 2000;Xia et al., 2021).Alternatively, mutual exclusivity between MLL3 and CDKN2A alterations would only be expected under circumstances where CDKN2A action is the most dominant MLL3 effector, and it seems likely that multiple downstream genes contribute to tumor suppression and their relative importance may vary between cell and tissue types.Such a variable output in cancer relevant gene regulation has been noted for other chromatin regulators that, at the extreme, serve as pro-oncogenic factors in some contexts and tumor suppressors in others (Ntziachristos et al., 2012;Soto-Feliciano et al., 2017;Souroullas et al., 2016;Tirode et al., 2014).UTX (KDM6A), MLL3 (KMT2C), and MLL4 (KMT2D), the core catalytic components of the COMPASSlike complex, are all considered tumor suppressors with frequent loss-of-function genomic alterations found in a broad spectrum of human cancers (Sze and Shilatifard, 2016); (Revia et al., 2021).While each of these components regulate redundant sets of genes (Hu et al., 2013;Lee et al., 2009), it appears that they may exert their tumor suppressive functions through different mechanisms.In liver and pancreas cancer models, UTX can control the expression of negative regulators of mTOR such as DEPTOR, and its disruption prevents their transcription and facilitates tumorigenesis through increased mTORC1 activity (Revia et al., 2021).Additionally, while the mechanisms of MLL4 activity in liver cancer have not been examined, studies suggest that MLL4 suppresses skin carcinogenesis by promoting lineage stability and ferroptosis independently of MLL3 (Egolf et al., 2021).Our study demonstrates that MLL3 is both necessary and sufficient for efficient transcriptional activation of the CDKN2A locus that drives oncogeneinduced apoptosis.The molecular basis for this heterogeneity in effector output remains to be determined, but it seems likely that different subsets of target genes are preferentially disabled by haploinsufficiency of individual components and/or subject to compensation by remaining COMPASS complex activities.
Systematic studies comparing the binding, histone modifications, and transcriptional output of cells across a spectrum of allelic configurations of COMPASS complex factors will be needed to achieve a more holistic understanding of their functions and interactions in different contexts.
The most well-established role for MLL3/4-UTX-containing complexes is the control of H3K4 monomethylation at enhancers during development (Herz et al. 2010;Hu et al. 2013).While our ChIP-Seq studies also revealed binding of MLL3/4 to enhancers in liver tumor cells, an even larger fraction of genes including Cdkn2ashowed MLL3/4 chromatin enrichment at gene promoters and, indeed, transcription of this class of genes was most affected by Mll3 disruption.Interestingly, Mll3 suppression preferentially limited the MLL3/4 enrichment at promoters and shifted residual complex binding towards intergenic regions.Such dynamic regulation of distinct cis-acting elements by the MLL3/4 complex has also been observed in other contexts (Cheng et al., 2014;Soto-Feliciano et al., 2021), where the non-canonical binding of MLL3/4 at promoters is a recurrent tumor suppressive mechanism in cancer cells.Further studies into the action and regulation of MLL3/4 complexes at promoters will be informative and may shed new insights into the actions of the COMPASS-like complex in cancer.
While the dominant role of CDKN2A in mediating the tumor suppressive effects of the broadly acting MLL3 enzyme is surprising, the contribution of a single gene to the functional output of chromatin-complex disruption is not unprecedented.Indeed, Polycomb Repressive Complexes (PRC) broadly repress gene expression in different cell types through the coordinated action of PRC1 and PRC2 complexes that deposit and maintain repressive H3K27me3 marks on the enhancers of target genes, including CDKN2A (Bracken et al., 2007;Kotake et al., 2007).Despite these similarly broad effects, CDKN2A is often the most functionally relevant target of PRC-mediated repression, as genetic deletion of either the PRC1 component Bmi1 or the PRC2 component Ezh2, or treatment with small molecule inhibitors of EZH2, can facilitate Cdkn2a induction in normal and tumor cells.This, in turn, triggers anti-proliferative responses that can be rescued by Cdkn2a deletion (Jacobs et al., 1999;Richly et al., 2011).It is noteworthy that the COMPASS-like complexes are biochemically and functionally similar to Trithorax complexes in Drosophila, which have an evolutionarily conserved antagonistic relationship with Polycomb Repressive Complexes (PRC1 and PRC2) that controls epigenetic memory and cell fate during development (Mills, 2010;Piunti and Shilatifard, 2016).Our findings suggest such antagonism extends to tumor suppression in mammalian cells, likely via regulation of Cdkn2a and other tumor suppressor genes (Soto-Feliciano et al., 2021).

Analysis of CRISPR-directed mutations. CRISPR mediated insertions and deletions were detected by
Surveyor assay as directed by the manufacturer (Transgenomic/IDT).Briefly, genomic DNA was extracted from primary tumors and cell lines by isopropanol precipitation following overnight lysis at 37°C in buffer containing 0.4 mg/ml Proteinase K, 10mM Tris, 100mM NaCl, 10mM EDTA, and 0.5% SDS, pH 8.0.~250-500 bp regions flanking predicted CRISPR cleavage sites were PCR amplified with Herculase II taq polymerase, column purified (Qiagen), heated to 95°C, and slowly cooled to promote annealing of heteroduplexes.Following treatment with Surveyor nuclease, products were analyzed by electrophoresis on a 2% polyacrylamide gel.Primers used for Surveyor Assay are listed in Supplementary Table S1.Amplified PCR products were separately gel purified and ligated into blunt-end digested pBlueScript (Stratagene).Sanger nucleotide sequencing analysis was performed on DNA from 48 transformed colonies using a T7 primer.
Colony assays.For measurement of cell proliferation, 5000 transduced and selected LPCs or MEFs were plated in triplicate in 6 well plates.Tetracycline inducible-shMLL3 expressing LPCs were grown in the presence or absence of doxycycline, and cells were fixed with formalin and methanol and stained with 0.05% crystal violet after 5 days.5000 MEFs were plated in 6-well plates, fixed after 6 days with formalin and methanol, and stained with 0.05% crystal violet.
Apoptosis assays.Apoptosis was measured in LPCs via Annexin V staining according to manufacturer's instructions (eBiosciences, Annexin-V APC).25,000 cells were grown with and without doxycycline for 3 days, trypsinized, washed with Annexin-V binding buffer, and ~100,000 cells were incubated with Annexin-V APC and analyzed on an LSRII flow cytometer (BD).
Live Imaging.1000 LPCs immortalized by linked overexpression of myc and 2 independent, inducible Mll3 shRNAs constitutively expressing shRNAs targeting Renilla luciferase or Cdkn2a (generated as detailed above) were plated on collagen coated, 96 well, clear bottom imaging plates in media supplemented with 300nM Draq7 (Invitrogen) with and without doxycycline, in triplicate by genotype.18 hours after plating cells, Venus (marking all plated cells) and Draq7 fluorescence was collected in 2, 10X fields of each well every 15 minutes for 41 hours using an automated, high content microscope (InCell 6000, General Electric).
High-throughput reads were aligned to mouse genome assembly NCBI37/mm9 as previously described (Barradas et al., 2009).Reads that aligned to multiple loci in the mouse genome were discarded.The ChIP-Seq signal for each gene was quantified as total number of reads per million (RPM) in the region 2 kb upstream to 2 kb downstream of the transcription start site (TSS).The complete dataset is available at NCBI Gene Expression Omnibus (GSE85055).Primers used for ChIP-qPCR of mouse Cdkn2a promoter (Barradas et al., 2009) are included in the Supplementary Table S1.
Chromatin extracts were sonicated for 14 minutes using a Covaris E220 focused ultrasonicator.Lysates were centrifuged at maximum speed for 10 minutes at 4°C and 5% of supernatant was saved as input DNA.Beads were prepared by incubating them in 0.5% BSA in PBS and antibodies overnight (100μL of Dynabeads Protein A or Protein G (Invitrogen) plus 20μL of antibody).Antibody used the anti-MLL3/4 (kindly provided by the Wysocka laboratory (Dorighi et al., 2017)).Antibody-Beads mixes were washed with 0.5% BSA in PBS and then added to the lysates overnight while rotating at 4°C.Beads were then washed six times with RIPA buffer (50mM HEPES pH 7.5, 500mM LiCl, 1mM EDTA, 0.7% sodiumdeoxycholate, 1% NP-40) and once with TE-NaCl Buffer (10mM Tris-HCl pH 8.0, 50mM NaCl, 1mM EDTA).Chromatin was eluted from beads in Elution buffer (50mM Tris-HCl pH 8.0, 10mM EDTA, 1% SDS) by incubating at 65°C for 30 minutes while shaking, supernatant was removed by centrifugation, and crosslinking was reversed by further incubating chromatin overnight at 65°C.The eluted chromatin was then treated with RNaseA (10mg/mL) for 1 hour at 37°C and with Proteinase K (Roche) for 2 hours at 55°C.DNA was purified by using phenol-chloroform extraction followed with ethanol precipitation.The NEBNext Ultra II DNA Library Prep kit was used to prepare samples for sequencing on an Illumina NextSeq500 (75bp read length, single-end, or 37bp read length, paired-end).
Quantitative RT-PCR.Total RNA was isolated using RNeasy Mini Kit, QIAshredder Columns and RNase-Free DNase Set (Qiagen).cDNA synthesis was performed using TaqMan® Reverse Transcription Reagents (Thermo Fisher Scientific).Real-time PCR was carried out using Power SYBR® Green Master Mix (Thermo Fisher Scientific) and the Life Technologies ViiA™ 7 machine.Transcript levels were normalized to the levels of mouse or human Actb mRNA expression and calculated using the ΔΔCt method.Each qRT-PCR was performed in triplicate using gene-specific primers (sequences listed in Supplementary Table S1).
RNA sequencing and differential expression analysis.For RNA sequencing, total RNA from three independent tumor-derived cell lines (Myc; sgp53 and Myc; sgMll3) was isolated using RNeasy Mini Kit, QIAshredder Columns and RNase-Free DNase Set (Qiagen).RNA-Seq library construction and sequencing were performed according to protocols used by the integrated genomics operation (IGO) Core at MSKCC.5-10 million reads were acquired per replicate sample.After removing adaptor sequences with Trimmomatic, RNA-seq reads were aligned to GRCm38.91(mm10) with STAR (Dobin et al., 2013).Genome wide transcript counting was performed by HTSeq to generate a FPKM matrix (Anders et al., 2015).Differentially expressed genes were identified by DESeq2 (v.1.8.2,package in R) and plotted in the volcano plot.The complete dataset is available at NCBI Gene Expression Omnibus (GSE85055).
Human cancer analyses.RNA sequencing data of selected samples with somatic mutations and homozygous deletions of either MLL3/KMT2C, CDKN2A, TP53, and RB1 in the TCGA hepatocellular carcinoma (HCC) dataset were downloaded from Broad Institute TCGA Genome Data Analysis Center.
Gene set enrichment analysis (GSEA) analyses.Gene set enrichment analysis (GSEA) was performed using the GSEAPreranked tool for conducting gene set enrichment analysis of data derived from RNA-seq experiments (version 2.07) against other signatures.The metric scores (normalized enrichment scores and false discovery rate q-values) were calculated using the sign of the fold change multiplied by the inverse of the P-value (Subramanian et al., 2005).Specifically, transcriptional signatures of human HCCs with genomic inactivation of CDKN2A were compared to other human HCCs with MLL3 or RB1 alterations, and mouse HCC cell lines (Myc; sgMll3 vs Myc; sgp53, genes with log 2 (foldchange)>4).
Statistical analysis: Data are presented as mean ± standard deviation (SD) or standard error of the mean (SEM) as specified.The Spearman rank coefficient was used for statistical measure of association as indicated.The statistical comparison between 2 groups was accomplished with the two-tailed student's t-test, or One-way ANOVA followed by post hoc t-tests among 3 or more groups.The analyses for cooccurrence or mutual exclusivity were performed using Fisher Exact Test.All statistical tests were performed using the Prism 8 software.The investigators were not blinded to the groups for the experiments.No samples or animals were excluded from the analysis.

Figure 2 .
Figure 2. Mll3 disruption alters the chromatin and transcriptional landscape of liver cancer cells.

Figure 3 .
Figure 3. Genetic alterations in CDKN2A and MLL3 show mutually exclusive patterns in human HCC and other cancers.

Figure 4 .
Figure 4. CDKN2A locus is a genomic and transcriptional target of MLL3 in liver cancer.

Figure 6 .
Figure 6.Model of MLL3 as a tumor suppressor in liver cancer.