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Supplementary Table S1: Clinical information related to the PDX used in Fig1A

Supplemental Methods, Figures, Figure legends, Table and Movie Legends

Supplementary Movie S1 - 3D representation around the Y axis of A375 cell nucleus

Supplementary Table S3: List of the genes that were identified by RNAseq to be significantly deregulated upon SPANX KD.

Supplementary Table S2: List of SPANX-interacting proteins identified by LC-MS/MS

Supplementary Movie S2 - 3D representation around the X axis of A375 cell nucleus

Mechanisms regulating nuclear organization control fundamental cellular processes, including the cell and chromatin organization. Their disorganization, including aberrant nuclear architecture, has been often implicated in cellular transformation. Here, we identify Lamin A, among proteins essential for nuclear architecture, as SPANX (sperm protein associated with the nucleus on the X chromosome), a cancer testis antigen previously linked to invasive tumor phenotypes, interacting protein in melanoma. SPANX interaction with Lamin A was mapped to the immunoglobulin fold-like domain, a region critical for Lamin A function, which is often mutated in laminopathies. SPANX downregulation in melanoma cell lines perturbed nuclear organization, decreased cell viability, and promoted senescence-associated phenotypes. Moreover, SPANX knockdown (KD) in melanoma cells promoted proliferation arrest, a phenotype mediated in part by IRF3/IL1A signaling. SPANX KD in melanoma cells also prompted the secretion of IL1A, which attenuated the proliferation of naïve melanoma cells. Identification of SPANX as a nuclear architecture complex component provides an unexpected insight into the regulation of Lamin A and its importance in melanoma.Implications:SPANX, a testis protein, interacts with LMNA and controls nuclear architecture and melanoma growth.

Tumour dependency on specific metabolic signals has been demonstrated and often guided numerous therapeutic approaches. We identify melanoma addiction to the mitochondrial protein glutaryl-CoA dehydrogenase (GCDH), which functions in lysine metabolism and controls protein glutarylation. GCDH knockdown induced cell death programmes in melanoma cells, an activity blocked by inhibition of the upstream lysine catabolism enzyme DHTKD1. The transcription factor NRF2 mediates GCDH-dependent melanoma cell death programmes. Mechanistically, GCDH knockdown induces NRF2 glutarylation, increasing its stability and DNA binding activity, with a concomitant transcriptional upregulation of ATF4, ATF3, DDIT3 and CHAC1, resulting in cell death. In vivo, inducible inactivation of GCDH effectively inhibited melanoma tumour growth. Correspondingly, reduced GCDH expression correlated with improved survival of patients with melanoma. These findings identify melanoma cell addiction to GCDH, limiting apoptotic signalling by controlling NRF2 glutarylation. Inhibiting the GCDH pathway could thus represent a therapeutic approach to treat melanoma.

The pyruvate kinase M2 isoform (PKM2) is preferentially expressed in cancer cells to regulate anabolic metabolism. Although PKM2 was recently reported to regulate lipid homeostasis, the molecular mechanism remains unclear. Herein, we discovered an ER transmembrane protein 33 (TMEM33) as a downstream effector of PKM2 that regulates activation of SREBPs and lipid metabolism. Loss of PKM2 leads to up-regulation of TMEM33, which recruits RNF5, an E3 ligase, to promote SREBP-cleavage activating protein (SCAP) degradation. TMEM33 is transcriptionally regulated by nuclear factor erythroid 2-like 1 (NRF1), whose cleavage and activation are controlled by PKM2 levels. Total plasma cholesterol levels are elevated by either treatment with PKM2 tetramer-promoting agent TEPP-46 or by global PKM2 knockout in mice, highlighting the essential function of PKM2 in lipid metabolism. Although depletion of PKM2 decreases cancer cell growth, global PKM2 knockout accelerates allografted tumor growth. Together, our findings reveal the cell-autonomous and systemic effects of PKM2 in lipid homeostasis and carcinogenesis, as well as TMEM33 as a bona fide regulator of lipid metabolism.

Acute myeloid leukemia (AML) remains incurable, largely due to its resistance to conventional treatments. Here, we find that increased abundance of the ubiquitin ligase RNF5 contributes to AML development and survival. High RNF5 expression in AML patient specimens correlates with poor prognosis. RNF5 inhibition decreases AML cell growth in culture, in patient-derived xenograft (PDX) samples and in vivo, and delays development of MLL-AF9–driven leukemogenesis in mice, prolonging their survival. RNF5 inhibition causes transcriptional changes that overlap with those seen upon histone deacetylase (HDAC)1 inhibition. RNF5 induces the formation of K29 ubiquitin chains on the histone-binding protein RBBP4, promoting its recruitment to and subsequent epigenetic regulation of genes involved in AML maintenance. Correspondingly, RNF5 or RBBP4 knockdown enhances AML cell sensitivity to HDAC inhibitors. Notably, low expression of both RNF5 and HDAC coincides with a favorable prognosis. Our studies identify an ERAD-independent role for RNF5, demonstrating that its control of RBBP4 constitutes an epigenetic pathway that drives AML, and highlight RNF5/RBBP4 as markers useful to stratify patients for treatment with HDAC inhibitors. Epigenetic changes are implicated in Acute myeloid leukemia (AML) tumorigenesis. Here, the authors show that the ubiquitin ligase RNF5 and its substrate RBBP4 contribute to AML development by regulating epigenetic-controlled transcription which determines AML sensitivity to HDAC inhibitors.

Acute myeloid leukemia (AML) remains incurable, largely due to its resistance to conventional treatments. Here, we found that increased expression and abundance of the ubiquitin ligase RNF5 contributes to AML development and survival. High RNF5 expression in AML patients correlated with poor prognosis. RNF5 inhibition decreased AML cell growth in culture and in vivo, and blocked development of MLL-AF9–driven leukemogenesis in mice, prolonging their survival. RNF5 inhibition led to transcriptional changes that overlapped with those seen upon HDAC1 inhibition. RNF5 induced the formation of K29 ubiquitin chains on the histone-binding protein RBBP4, promoting its recruitment and subsequent epigenetic regulation of genes involved in AML development and maintenance. Correspondingly, RNF5 or RBBP4 knockdown enhanced the sensitivity of AML cells to histone deacetylase (HDAC) inhibitors. Notably, low expression of RNF5 and HDAC coincided with a favorable prognosis. Our studies identified ERAD-independent role for RNF5, demonstrating that its control of RBBP4 constitutes an epigenetic pathway that drives AML while highlighting RNF5/RBBP4 as markers to stratify patients for treatment with HDAC inhibitors.

Mechanisms regulating nuclear organization control fundamental cellular processes, including the cell and chromatin organization. Their disorganization, including aberrant nuclear architecture, has been often implicated in cellular transformation. Here, we identify Lamin A, among proteins essential for nuclear architecture, as SPANX (sperm protein associated with the nucleus on the X chromosome), a cancer testis antigen previously linked to invasive tumor phenotypes, interacting protein in melanoma. SPANX interaction with Lamin A was mapped to the immunoglobulin fold-like domain, a region critical for Lamin A function, which is often mutated in laminopathies. SPANX downregulation in melanoma cell lines perturbed nuclear organization, decreased cell viability, and promoted senescence-associated phenotypes. Moreover, SPANX knockdown (KD) in melanoma cells promoted proliferation arrest, a phenotype mediated in part by IRF3/IL1A signaling. SPANX KD in melanoma cells also prompted the secretion of IL1A, which attenuated the proliferation of naïve melanoma cells. Identification of SPANX as a nuclear architecture complex component provides an unexpected insight into the regulation of Lamin A and its importance in melanoma. Implications: SPANX, a testis protein, interacts with LMNA and controls nuclear architecture and melanoma growth.

Understanding genetic and epigenetic changes that underlie abnormal proliferation of hematopoietic stem and progenitor cells is critical for development of new approaches to monitor and treat leukemia. The unfolded protein response (UPR) is a conserved adaptive signaling pathway that governs protein folding, secretion, and energy production and serves to maintain protein homeostasis in various cellular compartments. Deregulated UPR signaling, which often occurs in hematopoietic stem cells and leukemia, defines the degree of cellular toxicity and perturbs protein homeostasis, and at the same time, offers a novel therapeutic target. Here, we review current knowledge related to altered UPR signaling in leukemia and highlight possible strategies for exploiting the UPR as treatment for this disease.

Acute myeloid leukemia (AML) remains an incurable blood cancer largely due to rapid emergence of resistance to conventional treatments. Thus, new therapeutic modalities are greatly needed to halt AML development. Here, using genetic and xenograft mouse models, we reveal that inhibition of the ubiquitin ligase RNF5 in human AML cell lines and in MLL-AF9-driven AML severely decreased the leukemogenic potential of those cells and prolonged survival of model leukemic mice. These findings suggest the possibility that targeting a single gene, namely RNF5, could effectively inhibit different AML subtypes. We initially focused on RNF5 as its expression is upregulated in AML patient cohorts as well as in AML-derived cell lines compared with normal hematopoietic cells. Furthermore, high RNF5 expression in AML patient specimens correlated with poor prognosis, relapse and short overall patient survival. By contrast, specimens from AML patients who responded to therapy exhibited low RNF5 levels. In vitro, RNF5 loss impaired the clonogenic potential of MLL-AF9-transduced bone marrow cells and markedly attenuated growth and survival of AML but not CML or T-ALL cell lines, in which RNF5 is also highly expressed. High-throughput screen and bioinformatics analysis identified RNF5 and ER-associated degradation (ERAD) components, as augmenting AML cell sensitivity to histone deacetylase (HDAC) inhibition. Indeed, inhibition of RNF5 sensitized AML cells to HDAC inhibitors. Correspondingly, a favorable prognosis was observed in AML patients exhibiting low expression of RNF5 and HDAC. Collectivity, our studies identify a potential new therapeutic modality based on targeting RNF5 to inhibit AML and suggest that RNF5 expression could serve as a prognostic marker and means to stratify patients for treatment with HDAC inhibitors. Disclosures Ofran: AbbVie: Membership on an entity's Board of Directors or advisory committees. Vuori:Bionano Genomics: Membership on an entity's Board of Directors or advisory committees.

Accumulating evidence points to an important role for the gut microbiome in anti-tumor immunity. Here, we show that altered intestinal microbiota contributes to anti-tumor immunity, limiting tumor expansion. Mice lacking the ubiquitin ligase RNF5 exhibit attenuated activation of the unfolded protein response (UPR) components, which coincides with increased expression of inflammasome components, recruitment and activation of dendritic cells and reduced expression of antimicrobial peptides in intestinal epithelial cells. Reduced UPR expression is also seen in murine and human melanoma tumor specimens that responded to immune checkpoint therapy. Co-housing of Rnf5−/− and WT mice abolishes the anti-tumor immunity and tumor inhibition phenotype, whereas transfer of 11 bacterial strains, including B. rodentium, enriched in Rnf5−/− mice, establishes anti-tumor immunity and restricts melanoma growth in germ-free WT mice. Altered UPR signaling, exemplified in Rnf5−/− mice, coincides with altered gut microbiota composition and anti-tumor immunity to control melanoma growth., RNF5 is a ubiquitin ligase regulating ER stress response. Here the authors show that Rnf5 deficiency potentiates immune response against melanoma via altered microbiota, and isolate bacterial strains that confer the same phenotype to wild type mice.

SUMMARY Inflammatory bowel disease (IBD) is prevalent, but the mechanisms underlying disease development remain elusive. We identify a role for the E3 ubiquitin ligase RNF5 in IBD. Intestinal epithelial cells (IECs) express a high level of RNF5, while the colon of Rnf5−/− mice exhibits activated dendritic cells and intrinsic inflammation. Rnf5−/− mice exhibit severe acute colitis following dextran sodium sulfate (DSS) treatment. S100A8 is identified as an RNF5 substrate, resulting in S100A8 ubiquitination and proteasomal-dependent degradation that is attenuated upon inflammatory stimuli. Loss of RNF5 from IECs leads to enhanced S100A8 secretion, which induces mucosal CD4+ T cells, resulting in Th1 pro-inflammatory responses. Administration of S100A8-neutralizing antibodies to DSS-treated Rnf5−/− mice attenuates acute colitis development and increases survival. An inverse correlation between RNF5 and S100A8 protein expression in IECs of IBD patients coincides with disease severity. Collectively, RNF5-mediated regulation of S100A8 stability in IECs is required for the maintenance of intestinal homeostasis., In Brief Fujita et al. show that RNF5 regulation of S100A8 stability in intestinal epithelial cells defines the degree of pro-inflammatory response, culminating in severe intestinal inflammation following DSS treatment to Rnf5−/− mice. Neutralizing S100A8 antibodies attenuates acute colitis phenotypes, and inverse RNF5/S100A8 expression coincides with clinical severity in IBD patients., Graphical Abstract