The histone methyltransferase KMT2D maintains cellular glucocorticoid responsiveness by shielding the glucocorticoid receptor from degradation

Because of their ability to induce lymphocyte apoptosis, glucocorticoids (GC) are widely used to treat hematological malignancies such as lymphomas and multiple myeloma. Their effectiveness is often limited, however, due to the development of glucocorticoid resistance by a variety of molecular mechanisms. Here we performed an unbiased genome-wide CRISPR screen with the human T-cell leukemia cell line Jurkat to find previously unidentified genes required for GC-induced apoptosis. One such gene was KMT2D (also known as MLL2 or MLL4), which encodes a histone lysine methyltransferase whose mutations are associated with a variety of cancers, blood malignancies in particular, and are considered markers of poor prognosis. Knockout of KMT2D by CRISPR/Cas9 gene editing in Jurkat and several multiple myeloma cell lines downregulated GR protein expression. Surprisingly, this was not due to a reduction in GR transcripts, but rather to a decrease in the protein's half-life, primarily due to proteasomal degradation. Reconstitution of KMT2D expression restored GR levels. In contrast to the known ability of KMT2D to control gene transcription through covalent histone methylation, KMT2D-mediated upregulation of GR levels did not require its methyltransferase activity. Co-immunoprecipitation and proximity ligation assays found constitutive binding of KMT2D to the GR, which was enhanced in the presence of GC. These observations reveal KMT2D to be essential for the stabilization of cellular GR levels, and suggest a possible mechanism by which KMT2D mutations may lead to GC resistance in some malignancies. Because of their ability to induce lymphocyte apoptosis, glucocorticoids (GC) are widely used to treat hematological malignancies such as lymphomas and multiple myeloma. Their effectiveness is often limited, however, due to the development of glucocorticoid resistance by a variety of molecular mechanisms. Here we performed an unbiased genome-wide CRISPR screen with the human T-cell leukemia cell line Jurkat to find previously unidentified genes required for GC-induced apoptosis. One such gene was KMT2D (also known as MLL2 or MLL4), which encodes a histone lysine methyltransferase whose mutations are associated with a variety of cancers, blood malignancies in particular, and are considered markers of poor prognosis. Knockout of KMT2D by CRISPR/Cas9 gene editing in Jurkat and several multiple myeloma cell lines downregulated GR protein expression. Surprisingly, this was not due to a reduction in GR transcripts, but rather to a decrease in the protein's half-life, primarily due to proteasomal degradation. Reconstitution of KMT2D expression restored GR levels. In contrast to the known ability of KMT2D to control gene transcription through covalent histone methylation, KMT2D-mediated upregulation of GR levels did not require its methyltransferase activity. Co-immunoprecipitation and proximity ligation assays found constitutive binding of KMT2D to the GR, which was enhanced in the presence of GC. These observations reveal KMT2D to be essential for the stabilization of cellular GR levels, and suggest a possible mechanism by which KMT2D mutations may lead to GC resistance in some malignancies. Glucocorticoids (GCs) are steroid hormones primarily produced by the adrenals under the control of the hypothalamic-pituitary-adrenal axis. GCs are secreted rhythmically in a circadian manner, and their levels increase dramatically in response to physical and psychological stressors (1Scheff J.D. Kosmides A.K. Calvano S.E. Lowry S.F. Androulakis I.P. Pulsatile glucocorticoid secretion: origins and downstream effects.IEEE Trans. Biomed. Eng. 2011; 58: 3504-3507Crossref PubMed Scopus (11) Google Scholar). GCs function by binding and activating the glucocorticoid receptor (GR), a ligand-dependent transcription factor belonging to the nuclear receptor family (2Shimba A. Ikuta K. Control of immunity by glucocorticoids in health and disease.Semin. Immunopathol. 2020; 42: 669-680Crossref PubMed Scopus (104) Google Scholar, 3Whitfield G.K. Jurutka P.W. Haussler C.A. Haussler M.R. Steroid hormone receptors: evolution, ligands, and molecular basis of biologic function.J. Cell Biochem. 1999; 32-33: 110-122Crossref Google Scholar). The GR is encoded by the gene NR3C1 and bears the characteristic nuclear receptor structure, an N-terminal transactivation domain, a central DNA binding domain, and a C-terminal ligand binding domain. In the absence of ligand, the GR is predominantly localized in the cytoplasm in complexes with chaperones such as heat-shock protein (HSP) 90, HSP70, p23, and immunophilins (FKBP51, FKBP52, and Cyp44). Upon ligand engagement, the GR undergoes conformational changes, disassociates from the multimeric complex, and translocates into the nucleus where it interacts with co-regulators and other transcription factors to activate or repress transcription of numerous genes in a coordinated fashion (4Desmet S.J. De Bosscher K. Glucocorticoid receptors: finding the middle ground.J. Clin. Invest. 2017; 127: 1136-1145Crossref PubMed Scopus (108) Google Scholar). Transcriptional activation is driven by the binding of GR dimers or tetramers to GC response elements (GRE), which causes conformational changes in the GR transactivation domain that allow the recruitment of co-activators and/or other transcription factors. In addition, the GR binds to negative GREs to inhibit gene transcription (5Taves M.D. Ashwell J.D. Glucocorticoids in T cell development, differentiation and function.Nat. Rev. Immunol. 2021; 21: 233-243Crossref PubMed Scopus (119) Google Scholar, 6Presman D.M. Ganguly S. Schiltz R.L. Johnson T.A. Karpova T.S. Hager G.L. DNA binding triggers tetramerization of the glucocorticoid receptor in live cells.Proc. Natl. Acad. Sci. U. S. A. 2016; 113: 8236-8241Crossref PubMed Scopus (101) Google Scholar). GCs exert pleiotropic physiologic actions and have especially prominent effects on metabolism and immunity. Their most potent effect on immune function, and T cells in particular, is immunosuppression (5Taves M.D. Ashwell J.D. Glucocorticoids in T cell development, differentiation and function.Nat. Rev. Immunol. 2021; 21: 233-243Crossref PubMed Scopus (119) Google Scholar). Ligand-bound GR suppresses antigen-stimulated inflammation mediated by macrophages, dendritic cells, and epithelial cells, and weakens cytotoxic immune responses by diminishing interferon-γ production and inhibiting the development of CD4+ helper T cells, CD8+ cytotoxic T cells, and natural killer cells (2Shimba A. Ikuta K. Control of immunity by glucocorticoids in health and disease.Semin. Immunopathol. 2020; 42: 669-680Crossref PubMed Scopus (104) Google Scholar). GCs also induce apoptosis of lymphoid cells, a phenomenon of considerable biological and clinical significance (7Schmidt S. Rainer J. Ploner C. Presul E. Riml S. Kofler R. Glucocorticoid-induced apoptosis and glucocorticoid resistance: molecular mechanisms and clinical relevance.Cell Death Differ. 2004; 11: S45-S55Crossref PubMed Scopus (300) Google Scholar). Biologically, GC-induced apoptosis has been implicated in the generation of the antigen-specific T-cell repertoire and regulation of immune responses (5Taves M.D. Ashwell J.D. Glucocorticoids in T cell development, differentiation and function.Nat. Rev. Immunol. 2021; 21: 233-243Crossref PubMed Scopus (119) Google Scholar, 7Schmidt S. Rainer J. Ploner C. Presul E. Riml S. Kofler R. Glucocorticoid-induced apoptosis and glucocorticoid resistance: molecular mechanisms and clinical relevance.Cell Death Differ. 2004; 11: S45-S55Crossref PubMed Scopus (300) Google Scholar). Clinically, because of their lympholytic action, GCs are exploited to treat lymphoid malignancies. Synthetic GCs such as dexamethasone (Dex) and prednisolone, combined with other chemotherapeutic and immunosuppressing agents, are cornerstones in the treatment of multiple myeloma, acute lymphoblastic leukemia (ALL), and chronic lymphocytic leukemia (CLL) (8Clarisse D. Offner F. De Bosscher K. Latest perspectives on glucocorticoid-induced apoptosis and resistance in lymphoid malignancies.Biochim. Biophys. Acta Rev. Cancer. 2020; 1874188430Crossref PubMed Scopus (24) Google Scholar, 9Burwick N. Sharma S. Glucocorticoids in multiple myeloma: past, present, and future.Ann. Hematol. 2019; 98: 19-28Crossref PubMed Scopus (77) Google Scholar). GC-based therapies, however, may be hindered by GC-related side effects and GC resistance. GC resistance may be congenital or acquired during prolonged GC treatment (10Wilkinson L. Verhoog N.J.D. Louw A. Disease- and treatment-associated acquired glucocorticoid resistance.Endocr. Connect. 2018; 7: R328-R349Crossref PubMed Scopus (34) Google Scholar). Deciphering the mechanisms of GC-induced apoptosis could yield valuable information for the understanding of GC resistance. Despite much effort, the mechanisms governing GC-induced apoptosis are far from fully understood. GR-mediated gene activation is essential for the initiation of the process, as GC-induced apoptosis is inhibited by actinomycin D and cycloheximide, which block gene transcription and protein synthesis, respectively (8Clarisse D. Offner F. De Bosscher K. Latest perspectives on glucocorticoid-induced apoptosis and resistance in lymphoid malignancies.Biochim. Biophys. Acta Rev. Cancer. 2020; 1874188430Crossref PubMed Scopus (24) Google Scholar). It is generally agreed that GC-induced apoptosis primarily involves the intrinsic mitochondrial apoptotic pathway (8Clarisse D. Offner F. De Bosscher K. Latest perspectives on glucocorticoid-induced apoptosis and resistance in lymphoid malignancies.Biochim. Biophys. Acta Rev. Cancer. 2020; 1874188430Crossref PubMed Scopus (24) Google Scholar). Although no GRE is found in the promoter region, several studies reported that the pro-apoptotic Bcl2 family member BCL2L11 (BIM) was upregulated by GCs and thus implicated in GC-induced apoptosis (11Zhang L. Insel P.A. The pro-apoptotic protein Bim is a convergence point for cAMP/protein kinase A- and glucocorticoid-promoted apoptosis of lymphoid cells.J. Biol. Chem. 2004; 279: 20858-20865Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 12Melarangi T. Zhuang J. Lin K. Rockliffe N. Bosanquet A.G. Oates M. et al.Glucocorticoid resistance in chronic lymphocytic leukaemia is associated with a failure of upregulated Bim/Bcl-2 complexes to activate Bax and Bak.Cell Death Dis. 2012; 3e372Crossref PubMed Scopus (15) Google Scholar, 13Jing D. Bhadri V.A. Beck D. Thoms J.A. Yakob N.A. Wong J.W. et al.Opposing regulation of BIM and BCL2 controls glucocorticoid-induced apoptosis of pediatric acute lymphoblastic leukemia cells.Blood. 2015; 125: 273-283Crossref PubMed Scopus (96) Google Scholar). Several GR target genes such as GILZ (14Ayroldi E. Zollo O. Bastianelli A. Marchetti C. Agostini M. Di Virgilio R. et al.GILZ mediates the antiproliferative activity of glucocorticoids by negative regulation of Ras signaling.J. Clin. Invest. 2007; 117: 1605-1615Crossref PubMed Scopus (138) Google Scholar), SARI (15Huang Y. Zhou J. Huang Y. He J. Wang Y. Yang C. et al.SARI, a novel target gene of glucocorticoid receptor, plays an important role in dexamethasone-mediated killing of B lymphoma cells.Cancer Lett. 2016; 373: 57-66Crossref PubMed Scopus (17) Google Scholar), TXNIP (16Wang Z. Rong Y.P. Malone M.H. Davis M.C. Zhong F. Distelhorst C.W. Thioredoxin-interacting protein (txnip) is a glucocorticoid-regulated primary response gene involved in mediating glucocorticoid-induced apoptosis.Oncogene. 2006; 25: 1903-1913Crossref PubMed Scopus (90) Google Scholar), ZBTB16 (17Tissing W.J. den Boer M.L. Meijerink J.P. Menezes R.X. Swagemakers S. van der Spek P.J. et al.Genomewide identification of prednisolone-responsive genes in acute lymphoblastic leukemia cells.Blood. 2007; 109: 3929-3935Crossref PubMed Scopus (85) Google Scholar), and Noxa (18Lynch J.T. Rajendran R. Xenaki G. Berrou I. Demonacos C. Krstic-Demonacos M. The role of glucocorticoid receptor phosphorylation in Mcl-1 and NOXA gene expression.Mol. Cancer. 2010; 9: 38Crossref PubMed Scopus (28) Google Scholar) have also been linked to GC-mediated cell killing. The proposed mechanisms of GC resistance include reduced GR expression, decreased GR affinity, impaired GR translocation, altered GR post-translational modifications (PTMs), altered GR transcriptional activity, altered expression of GR isoforms, and more broadly epigenetic changes, signaling pathway interactions, and deregulated metabolism (8Clarisse D. Offner F. De Bosscher K. Latest perspectives on glucocorticoid-induced apoptosis and resistance in lymphoid malignancies.Biochim. Biophys. Acta Rev. Cancer. 2020; 1874188430Crossref PubMed Scopus (24) Google Scholar, 19Scheijen B. Molecular mechanisms contributing to glucocorticoid resistance in lymphoid malignancies.Cancer Drug Resist. 2019; 2: 647-664PubMed Google Scholar). In an attempt to find new genes that regulate GC-induced apoptosis, we performed a genome-wide CRISPR screen on Jurkat T-lymphoid leukemia cells. We identified the histone methyltransferase KMT2D to be essential for GC-induced cell death in multiple lymphoma and multiple myeloma cells. Mechanistically, KMT2D acts at the post-translational level by protecting the GR from ubiquitination and proteasomal degradation. These findings uncover a novel GR regulatory mechanism and provide new insight into the acquisition of GC resistance. To identify genes required for GC-induced apoptosis, we performed a genome-wide CRISPR screen using the human T leukemia cell line Jurkat (Fig. 1A). Because native Jurkat cells express low levels of GR that limit their responses to GC (20Helmberg A. Auphan N. Caelles C. Karin M. Glucocorticoid-induced apoptosis of human leukemic cells is caused by the repressive function of the glucocorticoid receptor.EMBO J. 1995; 14: 452-460Crossref PubMed Scopus (249) Google Scholar), we transduced them with a human GR expression vector and selected clones, termed JurkatGR, that uniformly express the GR at high levels (Fig. S1A). The use of cells in which GR transcription is controlled by a heterologous rather than native promoter has the theoretical advantage that genes involved in endogenous GR transcription would likely not be identified in the CRISPR screen. JurkatGR cells were transduced with a Cas9 expression vector to obtain a stable JurkatGR-Cas9 clone to be used for screening. Whereas JurkatWT cells were resistant to even micromolar amounts of dexamethasone (Dex), JurkatGR and JurkatGR-Cas9 cells were efficiently killed by nanomolar concentrations (Fig. S1, B and C), which is consistent with the observation by Helmberg et al. (20Helmberg A. Auphan N. Caelles C. Karin M. Glucocorticoid-induced apoptosis of human leukemic cells is caused by the repressive function of the glucocorticoid receptor.EMBO J. 1995; 14: 452-460Crossref PubMed Scopus (249) Google Scholar). JurkatGR-Cas9 cells were transduced with a Brunello pooled sgRNA CRISPR library that targets nearly every human protein-coding gene with 4 different single guide (sg) RNAs. After 1-week puromycin selection for gRNA-expressing cells, there was a secondary selection with 100 nM Dex for 20 days to eliminate any glucocorticoid-sensitive cells. Sequencing of CRISPR sgRNAs identified 28 unique genes that were enriched >20-fold in Dex-treated compared to untreated cells (Fig. 1B). Three of the four most enriched sgRNA sequences targeted NR3C1, which encodes the GR, validating the selective pressure of glucocorticoid-mediated cell killing. Interestingly, despite being known to be involved in GC-induced apoptosis, very few sgRNAs that target genes encoding BCL-2 family members or caspases had enrichment ratios >3 (Table S1). This may suggest that at least using these selecting strategies, eliminating a single apoptosis-mediating or executing molecule is not sufficient to prevent GC-induced cell death. Reasoning that novel regulators of GC-induced apoptosis would be more likely to be found among genes whose products affect gene transcription or cell viability, CRISPR screen-identified sgRNAs that target tumor suppressors (KMT2D and WT1), the transcription factor (NFAT4C), an autophagy-regulating protein (p62/SQSTM1), and an RNA-binding protein (STAU2) were tested by delivering them individually into JurkatGR-Cas9 cells. Serving as a positive control, pooled cells expressing sgRNA targeting NR3C1 were resistant to Dex-induced apoptosis. Of the gene-specific sgRNAs tested, only pools of JurkatGR-Cas9 cells transduced with sgKMT2D were statistically significantly resistant to Dex-induced apoptosis compared to non-targeting sgRNA-transduced cells (Fig. 2A). The sgKMT2D transfectants were subcloned by limiting dilution and immunoblotted for expression of KMT2D. Three subclones were chosen for study, one that expressed KMT2D (KMT2Dhigh) and two in which KMT2D was undetectable (KMT2Dlow) (Fig. 2B). After 40 h of culture in medium or with 20 nM Dex, the cells were stained with Annexin V-FITC and propidium iodide and analyzed by flow cytometry (Fig. 2, C and D). Dex induced the death of > 60% of KMT2D-expressing cells (sgNT-transduced JurkatGR-Cas9 cells and KMT2Dhigh) but <15% of cells in which KMT2D expression was undetectable, indicating that KMT2D is a positive regulator of GC-induced apoptosis. KMT2D (also known as MLL2 or MLL4) belongs to the COMPASS (COMplex of Proteins ASsociated with Set1) class of lysine methyltransferases (KMT) family, which carries out the methylation of histone H3 lysine 4 (H3K4). The KMT2C/D subfamily catalyzes the monomethylation of H3K4 and regulates gene expression through epigenetic modification of chromatin around enhancer regions (21Froimchuk E. Jang Y. Ge K. Histone H3 lysine 4 methyltransferase KMT2D.Gene. 2017; 627: 337-342Crossref PubMed Scopus (200) Google Scholar). KMT2D is considered a tumor suppressor gene, and its mutations are associated with non-Hodgkin lymphoma, including diffuse large B-cell (DLBCL) and follicular lymphoma, as well as some solid tumors such as small cell lung cancer (22Antunes E.T.B. Ottersbach K. The MLL/SET family and haematopoiesis.Biochim. Biophys. Acta Gene Regul. Mech. 2020; 1863194579Crossref PubMed Scopus (16) Google Scholar, 23Ford D.J. Dingwall A.K. The cancer COMPASS: navigating the functions of MLL complexes in Cancer.Cancer Genet. 2015; 208: 178-191Abstract Full Text Full Text PDF PubMed Google Scholar). It has been reported that KMT2D-mediated epigenetic changes assist in the transcription of estrogen receptor (ER)-dependent genes (24Mo R. Rao S.M. Zhu Y.J. Identification of the MLL2 complex as a coactivator for estrogen receptor alpha.J. Biol. Chem. 2006; 281: 15714-15720Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). Based on these findings, it appeared likely that the requirement for KMT2D in GC-induced death may be due to its effect on GR-transcriptional activity as well. Surprisingly, however, we found that the loss of KMT2D led to reduced expression of the GR protein itself (Fig. 2E). Because GR expression in the JurkatGR cells is predominantly driven by the exogenous viral LTR5 promoter (Fig. S1A), it seemed unlikely that the decrease in GR protein caused by KMT2D deletion was due to the downregulation of transcription. Indeed, qRT-PCR analysis found that NR3C1 mRNA levels were relatively similar between KMT2D-positive and KMT2D-deficient cells (Fig. 2F). To assess a post-transcriptional role for KMT2D, the kinetics of GR protein turnover were determined by assessing its levels over time after blocking new protein synthesis with cycloheximide. The GR level in KMT2Dlow JurkatGR cells was low, as expected, and was almost completely undetectable after 4 h (Fig. 2G). GR expression in KMT2D-sufficient control cells, on the other hand, was stable over this time course. These results indicate that KMT2D does not affect NR3C1 transcription but rather the stability of the GR protein. To address the possibility that the phenotype of cells in which KMT2D was targeted was due to sgRNA off-target effects, KMT2D-deficient Jurkat cells were reconstituted by transfection with codon-optimized KMT2D expression plasmid. We were able to identify and isolate two (of 35 screened) independent KMT2D-expressing subclones for analysis. Despite KMT2D levels being much lower than in not-targeted cells (Fig. 3A), likely due to the size of its coding sequence (16,614 nucleotides), GR expression was partially restored to levels sufficient to render them sensitive to Dex-induced apoptosis, although less so than control cells (Fig. 3, B and C). Together, the results demonstrate that KMT2D supports GR-mediated apoptosis, at least in part, by increasing GR protein levels. To ask if this unexpected relationship between KMT2D and GR occurs in other cells, we took a reciprocal approach, overexpressing rather than targeting KTM2D, in human embryonic kidney (HEK) 293T cells. Unmanipulated 293T cells expressed little KMT2D or GR, and as assessed by immunoblotting there was little change in the latter when KMT2D was introduced (Fig. 4A). Transfection with the GR-expression plasmid alone had a modest effect, but in combination with KMT2D, there was a very large increase in GR levels. The synergy between GR and KMT2D was also observed in individual cells using confocal microscopy (Figs. 4B and S2). As expected, the majority of KMT2D localized in the nucleus, whereas GR expressed alone localized to the cytoplasm. Unexpectedly, the GR colocalized with KMT2D in the nucleus in most GR and KMT2D co-expressing cells even in the absence of Dex (Figs. 4B and S2). To determine if KMT2D enzymatic activity plays a role in the stabilization of GR, WT, or a methyltransferase-inactive KMT2D mutant containing two amino acid substitutions in the catalytic domain (KMT2DY5426A/Y5512A) (25Jang Y. Wang C. Zhuang L. Liu C. Ge K. H3K4 methyltransferase activity is required for MLL4 protein stability.J. Mol. Biol. 2017; 429: 2046-2054Crossref PubMed Scopus (46) Google Scholar) (Fig. 4C) were co-expressed with GR in 293T cells (Fig. 4D). Both WT and mutant proteins were expressed at similar levels and resulted in a comparable increase in co-transfected GR, indicating that the effect of KMT2D is independent of its histone-modifying enzymatic activity. Many of the predicted structural features of KMT2D, including the SET domain that contains the catalytic site, are localized near the C-terminus (23Ford D.J. Dingwall A.K. The cancer COMPASS: navigating the functions of MLL complexes in Cancer.Cancer Genet. 2015; 208: 178-191Abstract Full Text Full Text PDF PubMed Google Scholar) (Fig. 4C). We created a construct in which most of the KMT2D C-terminus (residues 5091–5519) was deleted including the C-terminal PHD, FY-rich, and SET domains (Fig. 4D). This protein still retained the capacity to upregulate GR protein levels to the same extent as the full-length KMT2D (Fig. 4E). Synthetic GCs are an integral component in the treatment of multiple myeloma, and low GR levels in tumor cells are associated with inferior survival outcomes (9Burwick N. Sharma S. Glucocorticoids in multiple myeloma: past, present, and future.Ann. Hematol. 2019; 98: 19-28Crossref PubMed Scopus (77) Google Scholar). To assess if endogenous KMT2D regulates the expression of endogenous GR in these cells, we used CRISPR/Cas9 to target KMT2D in L363 cells, a GC-sensitive multiple myeloma cell line (26Kervoelen C. Menoret E. Gomez-Bougie P. Bataille R. Godon C. Marionneau-Lambot S. et al.Dexamethasone-induced cell death is restricted to specific molecular subgroups of multiple myeloma.Oncotarget. 2015; 6: 26922-26934Crossref PubMed Google Scholar). Knockout of KMT2D in three independent subclones (C5, C8, and C21) corresponded with a marked decrease in GR expression compared to parental L363 cells and the two subclones (C2 and C22) in which KMT2D levels were not affected (Fig. 5, A and B). Cellular GR levels corresponded well with sensitivity to Dex-induced apoptosis (Fig. 5C). KMT2D protein levels are reportedly to diminished or absent in many diffuse large B cell lymphoma (DLBCL) cell lines due to KMT2D mutations (27Zhang J. Dominguez-Sola D. Hussein S. Lee J.E. Holmes A.B. Bansal M. et al.Disruption of KMT2D perturbs germinal center B cell development and promotes lymphomagenesis.Nat. Med. 2015; 21: 1190-1198Crossref PubMed Scopus (342) Google Scholar). We investigated the relationship between KMT2D and GR levels in four DLBCL lines. KMT2D was easily detectable in SUDHL2 and SUDHL5 but absent in SUDHL6 and SUDHL10 cells (Fig. 5D). In agreement with the myeloma cells, expression of endogenous GR was high in the KMT2D+ DLBCL lines and much lower in the KMT2D- DLBCL lines. Taken together, the results indicated that GR protein levels are maintained by endogenously expressed KMT2D in multiple different lymphoid malignancies. It has been reported that the GR is ubiquitinated and degraded in proteasomes following GC stimulation (28Wallace A.D. Cidlowski J.A. Proteasome-mediated glucocorticoid receptor degradation restricts transcriptional signaling by glucocorticoids.J. Biol. Chem. 2001; 276: 42714-42721Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar, 29Blecharz K.G. Burek M. Bauersachs J. Thum T. Tsikas D. Widder J. et al.Inhibition of proteasome-mediated glucocorticoid receptor degradation restores nitric oxide bioavailability in myocardial endothelial cells in vitro.Biol. Cell. 2014; 106: 219-235Crossref PubMed Scopus (9) Google Scholar). Ubiquitination of the GR has also been detected in cells under normal culture conditions, and proteasomal inhibition resulted in increased steady-state GR levels (30Deroo B.J. Rentsch C. Sampath S. Young J. DeFranco D.B. Archer T.K. Proteasomal inhibition enhances glucocorticoid receptor transactivation and alters its subnuclear trafficking.Mol. Cell Biol. 2002; 22: 4113-4123Crossref PubMed Scopus (139) Google Scholar). To ask how KMT2D prolongs GR half-life (Fig. 2G), KMT2D-deficient JurkatGR and L363 cells were cultured with inhibitors of proteasomes (MG-132 or lactacystin), lysosomes (chloroquine), or autophagy (bafilomycin A). Proteasome inhibition was able to partially rescue GR levels in KMT2Dlow1 and KMT2D-deficient L363 cells (Figs. S3A and 6A). Culturing these cells for longer periods with lactacystin or MG-132 was toxic, perhaps due to proteotoxic stress and an excessive unfolded protein response (31Obeng E.A. Carlson L.M. Gutman D.M. Harrington Jr., W.J. Lee K.P. Boise L.H. Proteasome inhibitors induce a terminal unfolded protein response in multiple myeloma cells.Blood. 2006; 107: 4907-4916Crossref PubMed Scopus (955) Google Scholar). In contrast, neither bafilomycin A nor chloroquine affected GR loss (Fig. S3B). GR ubiquitination was assessed by immunoblotting immunoprecipitated GR with anti-ubiquitin antibodies. To eliminate the possibility that the ubiquitination bands may come from co-immunoprecipitated proteins, the cell lysates were heated to 95 °C in RIPA buffer containing 1% SDS to disrupt protein complexes prior to immunoprecipitation. The basal level of GR ubiquitination was unaffected by proteasomal inhibition in L363 cells, suggesting a low rate of steady-state ubiquitination (Fig. 6B). In KMT2D-deficient L363 cells, on the other hand, inhibition of proteasomal degradation resulted in a clear increase in the detection of ubiquitinated GR species, consistent with the notion that KMT2D protects the GR from ubiquitination-dependent turnover. KMT2D is a histone methyltransferase that is active on chromatin in the nucleus, whereas in the absence of ligand, the GR is primarily cytosolic. Indeed, when cytosolic and nuclear fractions from L363 cells were compared more KMT2D was found in the nucleus, but a substantial amount was also detected in the cytosolic fraction (Fig. 6C). As expected, the GR was predominantly cytosolic in unstimulated cells, with a small amount present in the nuclear fraction (Fig. 6C). Similar results were obtained with JurkatGR cells (Fig. S3C). Because ectopic expression of proteins can lead to aberrant trafficking and intracellular localization, we asked if we could detect interactions of endogenous proteins by co-immunoprecipitation (IP). To extract proteins from the nucleus as well as cytoplasm, L363 cells were lysed with RIPA buffer. Immunoblotting of anti-KMT2D IP detected a faint co-immunoprecipitated GR band, which was enhanced when cells were cultured with Dex (Fig. 6D). Considering that RIPA lysis buffer contains 0.1% SDS which tends to disrupt noncovalent protein-protein interactions, we also carried out proximity ligation assays (PLA), which detect protein interactions in situ (32Alam M.S. Proximity ligation assay (PLA).Curr. Protoc. Immunol. 2018; 123e58Crossref PubMed Scopus (141) Google Scholar). For these studies another GC-sensitive multiple myeloma cell line, MM1S, was chosen because unlike L363 it is adherent and easier to observe using fluorescence microscopy. MM1S cells expressed easily detectible GR and KMT2D (Fig. S3D) and are sensitive to GC-induced apoptosis (26Kervoelen C. Menoret E. Gomez-Bougie P. Bataille R. Godon C. Marionneau-Lambot S. et al.Dexamethasone-induced cell death is restricted to specific molecular subgroups of multiple myeloma.Oncotarget. 2015; 6: 26922-26934Crossref PubMed Google Scholar). Few if any PLA signals were detected when single antibodies were used (Fig. S3E). When PLA was performed with anti-KMT2D and anti-GR, a few PLA spots were detected in unstimulated cells, primarily at the nuclear periphery and/or cytoplasm (Fig. 6E). The number of PLA spots markedly increased within the nucleus by stimulation with Dex (Fig. 6, E and F). Because KMT2D mutations have been largely associated with hematological malignancies, our initial studies focused on lymphoma and multiple myeloma cells. To ask if