Search

Understanding the biological and clinical impact of copy number aberrations (CNAs) on the development of precision therapies in cancer remains an unmet challenge. Genetic amplification of chromosome 1q (chr1q-amp) is a major CNA conferring an adverse prognosis in several types of cancer, including in the blood cancer multiple myeloma (MM). Although several genes across chromosome 1 (chr1q) portend high-risk MM disease, the underpinning molecular etiology remains elusive. Here, with reference to the 3-dimensional (3D) chromatin structure, we integrate multi-omics data sets from patients with MM with genetic variables to obtain an associated clinical risk map across chr1q and to identify 103 adverse prognosis genes in chr1q-amp MM. Prominent among these genes, the transcription factor PBX1 is ectopically expressed by genetic amplification and epigenetic activation of its own preserved 3D regulatory domain. By binding to reprogrammed superenhancers, PBX1 directly regulates critical oncogenic pathways and a FOXM1-dependent transcriptional program. Together, PBX1 and FOXM1 activate a proliferative gene signature that predicts adverse prognosis across multiple types of cancer. Notably, pharmacological disruption of the PBX1-FOXM1 axis with existing agents (thiostrepton) and a novel PBX1 small molecule inhibitor (T417) is selectively toxic against chr1q-amp myeloma and solid tumor cells. Overall, our systems medicine approach successfully identifies CNA-driven oncogenic circuitries, links them to clinical phenotypes, and proposes novel CNA-targeted therapy strategies in MM and other types of cancer.

Ribosome dysfunction underlies the pathogenesis of many cancers and heritable ribosomopathies. Here, we investigate how mutations in either ribosomal protein large (RPL) or ribosomal protein small (RPS) subunit genes selectively affect erythroid progenitor development and clinical phenotypes in Diamond-Blackfan anemia (DBA), a rare ribosomopathy with limited therapeutic options. Using single-cell assays of patient-derived bone marrow, we delineated two distinct cellular trajectories segregating with ribosomal protein genotypes. Almost complete loss of erythroid specification was observed in RPS-DBA. In contrast, we observed relative preservation of qualitatively abnormal erythroid progenitors and precursors in RPL-DBA. Although both DBA genotypes exhibited a proinflammatory bone marrow milieu, RPS-DBA was characterized by erythroid differentiation arrest, whereas RPL-DBA was characterized by preserved GATA1 expression and activity. Compensatory stress erythropoiesis in RPL-DBA exhibited disordered differentiation underpinned by an altered glucocorticoid molecular signature, including reduced ZFP36L2 expression, leading to milder anemia and improved corticosteroid response. This integrative analysis approach identified distinct pathways of erythroid failure and defined genotype-phenotype correlations in DBA. These findings may help facilitate therapeutic target discovery.

Growth factor receptors (GFRs) and receptor tyrosine kinases (RTK) are groups of proteins mediating a plethora of physiological processes, including cell growth, proliferation, survival, differentiation and migration. Under certain circumstances, expression of GFRs and subsequently their downstream kinase signaling are deregulated by genetic, epigenetic, and somatic changes leading to uncontrolled cell division in many human diseases, most notably cancer. Cancer cells rely on growth factors to sustain the increasing need to cell division and metabolic reprogramming through cancer-associated activating mutations of their receptors (i.e., GFRs). In this review, we highlight the recent advances of selected GFRs and their ligands (growth factors) in cancer with emphasis on structural and functional differences. We also interrogate how overexpression and/or hyperactivation of GFRs contribute to cancer initiation, development, progression, and resistance to conventional chemo- and radiotherapies. Novel approaches are being developed as anticancer agents to target growth factor receptors and their signaling pathways in different cancers. Here, we illustrate how the current knowledge of GFRs biology, and their ligands lead to development of targeted therapies to inhibit and/or block the activity of growth factors, GFRs and downstream kinases to treat diseases such as cancer.

Summary Osteoclast (OC) development in response to nuclear factor kappa-Β ligand (RANKL) is critical for bone homeostasis in health and in disease. The early and direct chromatin regulatory changes imparted by the BET chromatin readers Brd2-4 and OC-affiliated transcription factors (TFs) during osteoclastogenesis are not known. Here, we demonstrate that in response to RANKL, early OC development entails regulation of two alternative cell fate transcriptional programmes, OC vs macrophage, with repression of the latter following activation of the former. Both programmes are regulated in a non-redundant manner by increased chromatin binding of Brd2 at promoters and of Brd4 at enhancers/super-enhancers. Myc, the top RANKL-induced TF, regulates OC development in co-operation with Brd2/4 and Max and by establishing negative and positive regulatory loops with other lineage-affiliated TFs. These insights into the transcriptional regulation of osteoclastogenesis suggest the clinical potential of selective targeting of Brd2/4 to abrogate pathological OC activation., Graphical Abstract, Highlights • Upregulation of osteoclast lineage commitment TFs occurs, Biological Sciences; Developmental Biology; Bioinformatics; Omics; Transcriptomics

Multiple myeloma is a malignancy of plasma cells initiated and driven by primary and secondary genetic events. However, myeloma plasma cell survival and proliferation might be sustained by non-genetic drivers. Z-DNA-binding protein 1 (ZBP1; also known as DAI) is an interferon-inducible, Z-nucleic acid sensor that triggers RIPK3-MLKL-mediated necroptosis in mice. ZBP1 also interacts with TBK1 and the transcription factor IRF3 but the function of this interaction is unclear, and the role of the ZBP1-IRF3 axis in cancer is not known. Here we show that ZBP1 is selectively expressed in late B-cell development in both human and murine cells and it is required for optimal T-cell-dependent humoral immune responses. In myeloma plasma cells, the interaction of constitutively expressed ZBP1 with TBK1 and IRF3 results in IRF3 phosphorylation. IRF3 directly binds and activates cell cycle genes, in part through co-operation with the plasma cell lineage-defining transcription factor IRF4, thereby promoting myeloma cell proliferation. This generates a novel, potentially therapeutically targetable and relatively selective myeloma cell addiction to the ZBP1-IRF3 axis. Our data also show a noncanonical function of constitutive ZBP1 in human cells and expand our knowledge of the role of cellular immune sensors in cancer biology.

ZBP1 is an inducible, non-constitutively expressed cellular nucleic acid sensor that triggers type I interferon (IFN) responses via phosphorylation and activation of the transcription factor (TF) IRF3 by TBK1. However, the role of the ZBP1-IRF3 axis in cancer is not known. Here we show that ZBP1 is selectively and constitutively expressed in late B cell development and it is required for optimal T cell-dependent humoral immune responses. In the plasma cell (PC) cancer multiple myeloma, interaction of constitutively expressed ZBP1 with TBK1 and IRF3 results in IRF3 phosphorylation. Notably, rather than IFN type I response genes, IRF3 directly activates, in part through co-operation with the PC lineage-defining TF IRF4, cell cycle genes thus promoting myeloma cell proliferation. This generates a novel, potentially therapeutically targetable and relatively selective myeloma cell addiction to the ZBP1-IRF3 axis. These data expand our knowledge of the role of cellular immune sensors in cancer biology.

Background: Diamond- Blackfan Anemia (DBA) is a rare, heritable ribosomopathy caused by mutations in ribosomal protein large (RPL) and small (RPS) subunit genes. The diagnostic criteria of DBA include presentation in infancy with virtually no mature erythroblasts (EB) on bone marrow (BM) examination, however atypical presentations in later life with milder haematological manifestations are increasingly reported. The cellular and molecular mechanisms underpinning variable clinical phenotypes, and how they relate to genotype, are yet to be elucidated. Furthermore, as many DBA patients do not respond to corticosteroids, understanding the pathological processes contributing to erythroid failure is a prerequisite for developing novel, precision-medicine therapies. Aim: To elucidate phenotypic and functional differences in erythropoiesis in RPS- and RPL-DBA, using primary BM samples from patients. Methods: We performed single-cell transcriptome profiling (scRNAseq), using the 10X Genomics chromium platform, of 45,488 CD34+ Lineage negative (Lin-) BM hematopoietic stem and progenitor cells (HSPC), purified by fluorescence-activated cell sorting. We included six patients with red cell transfusion-dependent DBA (aged 2-19y) with mutations in RPS19 (n=3), RPL11 (n=1) and RPL5 (n=2) and three healthy donors (aged 3-17y). We validated our findings using bulk RNAseq, functional assays, and deep immunophenotyping-based dissection of the haematopoietic architecture of DBA BM ex vivo. Results: High quality sequencing data was obtained for all nine donors; after quality control, 41,415 single cells were carried forward for analysis. Unsupervised clustering analysis and lineage identification revealed two divergent cellular patterns in DBA compared with age-matched control BM that segregated with genotype: a selective loss of erythromegakaryocyte (E/MK) progenitors in RPS-DBA, but relative preservation of the erythroid developmental trajectory in RPL-DBA, at the expense of megakaryopoiesis (Fig 1a). Gene Set Enrichment Analysis (GSEA) of differentially expressed genes between control and DBA HSPC clusters revealed p53-mediated apoptosis, TNFa-, IFNa- and IFNg-mediated inflammatory pathways in DBA EP. Although these pathways were enriched across all HSPC populations irrespective of genotype, they were detected at an earlier stage of the stem cell hierarchy, and more potently, in RPS- versus RPL-EP. Expression of transcriptional targets of the master E/MK transcription factor, GATA1, was significantly upregulated in RPL- versus RPS-DBA EP (Fig 1b), supporting the maintained erythroid program in RPL-DBA HSPC. However, expression of genes denoting erythroid differentiation, including adult haemoglobin (Hb) genes, was significantly elevated in RPL-DBA, suggesting aberrant accelerated differentiation to EB. These findings were confirmed by immunophenotypic examination of patient BM and bulk RNAseq of RPL-EB. Additionally, single-cell clonogenic assays of RPL-DBA EP showed severe qualitative defects. Thus, although erythroid commitment occurs in RPL-DBA, it is coupled with accelerated maturation beyond EP to functionally impaired mature EB, enriched in inflammatory and p53 pathways. Finally, we analysed the clinical characteristics of the 161 patients comprising the U.K DBA registry. In line with the milder erythroid specification defect, patients with RPL-DBA (n=44) presented with anaemia later (P=0.004), and with a higher average Hb concentration (P=0.04), than those with RPS-DBA (n=63). Furthermore, we identified higher corticosteroid responses in RPL-DBA, assessed at 6 months post initiation (P=0.006), consistent with our findings in RPL-DBA of preservation of the cellular EP populations that are targeted by corticosteroids. Impact: In conclusion, a preserved but distinct erythroid developmental trajectory, characterised by accelerated differentiation, underpins a milder haematological phenotype in RPL-DBA. Furthermore, we reveal the first single-cell transcriptomic dataset from haematopoietic cells in a ribosomopathy, uncovering novel cell intrinsic and extrinsic pathogenetic insights into failing erythropoiesis in DBA. Integration of these data with clinical genomics and phenomics provides a paradigm by which single cell approaches can be used to decipher genotype-phenotype relationships in Mendelian genetic disorders. Disclosures Mead: Novartis: Consultancy, Honoraria, Other: travel, accommodations, expenses, Research Funding, Speakers Bureau; Celgene/BMS: Consultancy, Honoraria, Other: travel, accommodations, expenses, Research Funding; Abbvie: Consultancy; CTI: Consultancy; Gilead: Consultancy.

TFIIA is an important positive regulator of TFIID, the primary promoter recognition factor of the basal RNA polymerase II transcription machinery. TFIIA antagonises negative TFIID regulators such as negative cofactor 2 (NC2), promotes specific binding of the TBP subunit of TFIID to TATA core promoter sequence elements and stimulates the interaction of TBP-associated factors (TAFs) in the TFIID complex with core promoter elements located downstream of TATA, such as the initiator element (INR). Metazoan TFIIA consists of 3 subunits, TFIIAα (35 kDa), β (19 kDa) and γ (12 kDa). TFIIAα and β subunits are encoded by a single gene and result from site-specific cleavage of a 55 kDa TFIIA(α/β) precursor protein by the protease Taspase1. Metazoan cells have been shown to contain variable amounts of TFIIA (55/12 kDa) and Taspase1-processed TFIIA (35/19/12 kDa) depending on cell type, suggesting distinct gene-specific roles of unprocessed and Taspase1-processed TFIIA. How precisely Taspase1 processing affects TFIIA functions is not understood. Here we report that Taspase1 processing alters TFIIA interactions with TFIID and the conformation of TFIID/TFIIA promoter complexes. We further show that Taspase1 processing induces increased sensitivity of TFIID/TFIIA complexes to the repressor NC2, which is counteracted by the presence of an INR core promoter element. Our results provide first evidence that Taspase1 processing affects TFIIA regulation of TFIID and suggest that Taspase1 processing of TFIIA is required to establish INR-selective core promoter activity in the presence of NC2.

ZBP1 is an inducible nucleic acid (NA) sensor that is activated when pathogen NA bind to its Zα and Zβ domains. ZBP1 is required for TBK1-dependent phosphorylation of the transcription factor IRF3 (pIRF3) followed by its direct activation of type I interferon genes. However, the role, if any, of ZBP1 in tumour biology is not known. By searching for genes selectively expressed in multiple myeloma (MM) we identified ZBP1 mRNA expressed in 29 MM cell lines (MMCL) but not in >1000 other cancer cell lines (CCLE dataset); ZBP1 was expressed in all 766 patient myeloma PC (CoMMpass dataset) but not in normal blood cells (Blueprint) or 53 healthy tissues (GTex). We confirmed expression of ZBP1 mRNA and/or protein in MMCL, primary human and murine germinal centre B (GCB) and plasma cells (PC) as well as in myeloma PC. By inducing T cell-dependent humoral immune responses after ip alum-NP-KLH immunisation, we explored the role of selective and constitutive expression of Zbp1 in GCB to PC transition. We found no differences in the frequency of splenic GCB cells and PC between control WT and Zbp1-/- mice and in GCB cell frequency between immunised WT and Zbp1-/- mice. However, compared to WT, the increase in PC frequency in immunised Zbp1-/- mice was 50% lower (n=10/group, p To explore the function of ZBP1 in MM we depleted by 2 lentiviral shRNAs either isoform 1 (contains both Zα and Zβ domains; shRNA1) or both isoform 1 and isoform 2 (latter lacks Zα domain; shRNA2). Both shRNAs were toxic to all 5 MMCL tested suggesting that isoform 1 but not isoform 2 is essential for myeloma cell survival. This effect was specific because survival of K562 cells, which lack expression of ZBP1, was not affected by either shRNA and exogenous ZBP1 cDNA rescued cell death of ZBP1-depleted myeloma cells. Dox-induced ZBP1 depletion was toxic to MMCL in vitro and significantly inhibited myeloma cell growth in a subcutaneous NSG model of the MMCL H929 and MM.1S. Together, these findings reveal a novel myeloma cell-specific ZBP1 dependency. Transcriptome analysis of ZBP1-depleted H929 and MM.1S cells showed amongst the significantly downregulated genes enrichment for the cell cycle control and DNA repair pathways consistent with a critical role of ZBP1 in promoting myeloma cell proliferation. Flow-cytometric analysis of ZBP1-depleted MMCL as well as of patient-derived myeloma PC revealed cell cycle arrest at the G0/1 phase and increasing apoptosis. Exploring potential links with IRF3, we first observed that unlike in non-malignant cells, IRF3 was constitutively phosphorylated in MMCL. Using protein-co-immunoprecipitation we found that endogenous ZBP1 interacts with IRF3 and TBK1 while upon co-transfection with different ZBP1 deletion mutants, ZBP1-IRF3 interaction required primarily the ZBP1, RHIM domain-containing, C-terminus. Further, while in ZBP1-depleted myeloma cells total IRF3 and TBK1 levels were not altered, pIRF3 and pTBK1 levels decreased thus showing a post-translational dependency of constitutive pIRF3 and pTBK1 on ZBP1. Finally, pharmacological inhibition of TBK1 resulted in decrease of pIRF3 without affecting total IRF3. Importantly, shRNA-mediated IRF3 depletion resulted in cell cycle arrest and death of MMCL. By integrating histone mark and in-house IRF3 ChiP-seq with transcriptome of IRF3-depleted MM.1S cells we identified 770 down- and 330 up-regulated genes predicted to be directly regulated by IRF3. Pathway enrichment analysis confirmed cell cycle as the most highly regulated by IRF3. Notably, we observed no direct or indirect regulation of the interferon genes (e.g., IFNA1, IFNB1) by IRF3. As well as the IRF3 motif, IRF3 cistrome analysis revealed significant enrichment for the distinct IRF4 motif. Integration of the IRF3/IRF4 cistromes identified >80% IRF3 binding regions are co-occupied by IRF4 and co-regulation of cell cycle genes. Further we validated IRF3-IRF4 interaction at the IRF4 super-enhancer by ChIP-re-ChIP. These data show a novel dependency in MM comprising constitutive activation of the ZBP1-IRF3 pathway and regulation of cell cycle and proliferation by IRF3 thus providing opportunities for therapeutic targeting. Disclosures Caputo: GSK: Research Funding. Auner:Amgen: Other: Consultancy and Research Funding; Takeda: Consultancy; Karyopharm: Consultancy. Karadimitris:GSK: Research Funding.

BACKGROUND: The biological basis for the adverse prognosis of chr1q gain/amplification (1q+) present in ~30% of patients with multiple myeloma (MM) remains ill-defined. The transcription factor (TF) Pre-B-cell leukaemia homeobox 1 (PBX1), encoded on chr1q, acts as master regulator of early hematopoiesis and as an oncogene in leukemia and other malignancies. Herein, we hypothesized that PBX1 orchestrates proliferative regulatory networks that underpin the poor prognosis associated with 1q+ in MM. METHODS: We employed qPCR for mRNA quantification, western blotting and immunohistochemistry for protein analysis, lentiviral shRNA-mediated knock-down, Hoechst/Annexin V staining and flow-cytometry for apoptosis and cell cycle analysis, ChIPseq for cistrome and RNAseq after knock-down for transcriptome analysis. Additional data were obtained from MMRF/CoMMpass, Blueprint Consortium and Arkansas datasets. Computational analysis of clinical and "-omics" data was performed using standard bioinformatic work-flows; pathway enrichment analysis using EnrichR and GSEA. RESULTS: Combined genomic (WGS/WES) and transcriptomic (RNAseq) analysis of the CoMMpass dataset identified a subgroup of 1q+ MM patients (60%) characterized by aberrant PBX1 overexpression and amplification (PBX1amp); survival analysis revealed significantly worse outcome of this subgroup compared to 1q+ non-PBX1amp (p Depletion of PBX1 using 2 validated shRNAs was toxic to the 1q+ MM1.S t(14;16) and U266 t(11;14) myeloma cell lines (MMCL) in vitro, revealing a novel myeloma cell addiction to PBX1. Transcriptome analysis of PBX1-depleted cells showed significant de-regulation of genes (MM1.S: 753down/320up; U266: 598 down/288up) highly enriched in cell cycle and proteasome/ER stress response pathways. Flow-cytometric analysis confirmed that PBX1-depleted myeloma cells undergo cell cycle arrest (G0/1 phase) and apoptosis (n=3; p Genome-wide binding profiling of PBX1 identified 7,354 significantly enriched peaks shared between MM1.S and U266 MMCL. These peaks were distributed at active promoters (~50%) and active enhancers (~30%), as defined by ChromHMM maps; the PBX1 consensus binding motif was identified as top hit upon motif enrichment analysis. Transcriptome-cistrome integration identified 728 and 282 genes directly activated or repressed, respectively, by PBX1 in MM1.S cells. Again, pathway enrichment analysis of PBX1 direct targets demonstrated significant over-representation of cell cycle and proliferation pathways, as well as positive regulation of various metabolic processes (oxidative phosphorylation, glycolysis, unfolded protein response). Strikingly, enrichment analysis against public ChIPseq datasets revealed significant overlap with gene targets of FOXM1 (200) and E2F (150) TF, suggesting the presence of a united PBX1-FOXM1-E2F regulatory circuitry. In fact, PBX1 binding was detected at an active FOXM1 enhancer and at the E2F1/2 promoters, while FOXM1 and E2F1/2 mRNA levels decreased significantly upon PBX1 depletion in two MMCL. To further explore the PBX1-FOXM1 interaction, we overlaid the FOXM1-depleted transcriptome with the PBX1 network in MM.1S cells. This identified 160 genes to be co-regulated by PBX1 and FOXM1, including genes previously linked to myeloma cell drug resistance (NEK2, TOP2A, AURKA); however, PBX1 expression was not altered in FOXM1-depleted cells, suggesting that PBX1 is functionally upstream of FOXM1. Additional analysis across 814 primary MM patient CoMMpass transcriptomes confirmed significant co-expression of PBX1 with FOXM1, E2F1/2 and another 67 PBX1-FOXM1 highly expressed, co-regulated gene targets, conferring poor OS (p CONCLUSIONS: We found that genetic amplification and overexpression of PBX1 defines an ultra high-risk subgroup of 1q+ MM with proliferative features. Myeloma cells are addicted to aberrant PBX1 expression. As well as essential metabolic pathways, PBX1 regulates cell cycle progression by direct transcriptional regulation of FOXM1 and of its target genes. These data reveal PBX1 as a critical biological link between chr1q gain/amplification and poor prognosis in a subset of MM patients. Disclosures Auner: Karyopharm: Consultancy; Amgen: Other: Consultancy and Research Funding; Takeda: Consultancy. Caputo:GSK: Research Funding. Karadimitris:GSK: Research Funding.

BACKGROUND: Bone disease, a common source of morbidity in multiple myeloma (MM), is caused by RANKL-induced aberrant activation of osteoclasts (OC). RANKL-induced OC lineage commitment requires repression of an Irf-8 dependent macrophage inflammatory transcriptional programme commensurate with activation of an OC lineage-specific programme. Functional data have shown the requirement for the histone acetylation readers Brd2-4 BET proteins and of cMyc for OC lineage development. However, how Brd2-4 and Myc co-operate genome-wide to regulate transcriptome changes that underpin the very early stages of RANKL-induced OC lineage commitment has not been defined. METHODS: The OC progenitor-like murine RAW264.7 cell line was used for osteoclastogenesis. OC were assayed by TRAP staining. We performed RNA-seq for transcriptome analysis and ChIP-seq against Brd2-4, cMyc, and H3K27Ac mark for epigenomic profiling. The pan-Bet inhibitor IBET151 was used alone or in combination with RANKL. ChIP-seq/RNA-seq data were processed using standard bioinformatics pipelines; downstream analyses (pathway and motif enrichment, factor differential binding) were performed by various tools including EnrichR, R packages ChIPpeakAnno/DiffBind, Rose. RESULTS: Transcriptomic profiling of OC progenitors at 0, 4, 14 and 24h post-RANKL treatment identified 12 distinct clusters of expression trends. The 4h activated cluster includes OC master transcription factors (TFs; cMyc, Nfatc1, Fosl), and is enriched in OC-defining pathways. Notably, by 14h the majority of the genes required for mature OC formation and activation are already highly expressed (e.g. Ctsk, Mmp9). The downregulated clusters include monocyte defining TFs (e.g. Irf8, Mafb and Bcl6). These RANKL-dependent transcriptome changes are completely abrogated by iBET151, highlighting the critical role of Brd2-4 in osteoclastogenesis. Differential chromatin binding analysis upon RANKL induction revealed an overall enhanced Brd2-4 binding at already existing or de novo gained sites. This was more pronounced for Brd2&4 and much less for Brd3, with differentially binding sites (DBS) comprising 50% and 20% respectively of all binding sites in RANKL-treated cells. For Brd2&3, DBS were primarily distributed at promoters and for Brd4 at intergenic, candidate enhancers regions. Notably, nearly all gained DBS were sensitive to and abrogated by iBET151. Combinatorial profiling of Brd2 and Brd4 showed that almost half of Brd2 DBS peaks overlap with Brd4 (47%; 897/1896), while only 24% (766/3234) of Brd4 DBS peaks are co-occupied by Brd2. Transcriptome and Brd2&4 DBS integration in combination with motif enrichment analysis, identified genes that are predicted to be regulated by Brd2 and/or Brd4. EnrichR analysis suggests that enhanced binding of Brd2&4, singly or in combination, is required for activation of the critical OC lineage-specific and repression of the macrophage-defining transcriptional programs highlighting the non-redundant roles of Brd2&4 in OC development. Cell lineage commitment often requires 'commissioning' of cell-specific super-enhancers (SE). Combined analysis of genome-wide Brd4/H3K27ac profiles identified 678 RANKL-induced SE and their respective target genes. Further, 110 of these SE showed enhanced Brd4 binding in 2 peaks: 20/110 were linked to significantly up- and 90/100 to down-regulated genes. The repressed genes were significantly enriched to previously described Irf8, MafB and RunX1 targets, suggesting a critical role of SE in the repression of the monocyte/macrophage inflammatory programme during OC lineage commitment. Strikingly, among top hits, we detected a SE linked to the regulation of cMyc. To further investigate its role in OC development, we obtained the cistrome of cMyc after RANKL induction. We identified 560 binding sites which were highly enriched in cMyc, Max, Fli1, Fosl2 and Irf8 motifs. Cistrome-transcriptome integration suggested direct activation of 141 and repression of 52 genes by cMyc in response to RANKL; these are enriched in ribosome biogenesis pathways and Irf8-dependent targets respectively. CONCLUSIONS: Myc and Brd4 mark SE that repress an Irf8-dependent transcriptional programme, a requirement for OC lineage commitment. The non-redundant roles of Brd2&4 suggest that selective targeting of either could inhibit aberrant OC activation associated with MM. Disclosures Caputo: GSK: Research Funding. Auner:Amgen: Other: Consultancy and Research Funding; Takeda: Consultancy; Karyopharm: Consultancy. Karadimitris:GSK: Research Funding.

Overexpression of the transcription factor MAF, as a result of its juxtaposition to the IgH enhancer [MAF-translocated t(14;16)], is a myeloma-initiating event in 3-5% of patients with multiple myeloma (MM) and confers a poor prognosis. MAF is also overexpressed in another 40% of cases, often in co-operation with the oncogene MMSET. The mechanisms by which MAF overexpression impacts on the regulatory genome to generate the MAF-driven oncogenic transcriptome and its direct targets are not known. To address this, we employed a multi-layer -omics approach using primary myeloma plasma cells (PC) as well as myeloma cell lines (MMCL). First, we determined the chromatin accessibility and transcriptome profiles of MAF-translocated myeloma by performing ATAC-seq and RNA-seq, respectively, in purified bone marrow CD138+ PC from two patients with t(14;16) and three healthy donors. We identified 6,640 differentially accessible regions, 87% of which displayed enhanced chromatin accessibility in MAF samples compared to normal PC. Secondary analysis comparing this with ATAC-seq data from a set of 28 other MM samples, including hyperdiploid, MMSET and CCND1-translocated MM, revealed 33% of those regions to be MAF subgroup specific (1,949 regions), with the rest shared between MAF and other cytogenetic groups. Gene annotation and pathway enrichment analysis using GREAT confirmed overrepresentation of the MF myeloma patient signature, as previously identified in microarray datasets. RNA-seq detected significant upregulation of approximately 900 genes in MAF samples compared to normal counterparts, including MAF itself (top 4th hit) as well as its presumed targets (CCND2, ITGB7 and NUAK1). Next, we obtained the MAF cistrome using ChIP-seq in the MAF-translocated MMCL MM1.S and integrated it with the primary PC ATAC-seq data. This revealed that 31% (618/1,949) of the differentially accessible regions in MAF-translocated MM PC are also MAF-bound. Additional overlay with ENCODE ChromHMM epigenome map showed that 47% of MAF binding sites are on active enhancers and 42% on active promoters signifying potential direct regulation of the corresponding genes. Next, we superimposed the accessible and MAF-bound loci on the epigenomic landscapes of normal PC and other B-cell types using their corresponding ChromHMM maps (Blueprint consortium data). Interestingly, 56% (345/618) of the MAF-specific regions were not active in any stage of B cell development. This suggests that aberrant MAF overexpression and chromatin binding in PC is associated with de novo activation of these chromatin regions, over half of which (200/345; 58%) are enhancers; we termed these 'neo-enhancers'. Upon de novo motif analysis of MAF ChIP-seq in MAF-translocated JJN3 and MM1.S MMCL, we confirmed MAF as the first and, interestingly, IRF4 as the second top hit, suggesting a possible MAF-IRF4 functional interaction in myelomagenesis. Indeed, overlay of the accessible MAF-bound loci with IRF4 ChIP-seq data in MM1.S revealed 63% co-occupancy (including 62% of "neo-enhancers"), proposing a novel and extensive co-operative chromatin-based network between the two transcription factors. Final integration of the accessible MAF-bound regions with the paired transcriptomes of primary myeloma PC revealed a set 206 candidate enhancer-gene pairs. Strikingly, we identified two IRF4-cobound "neo-enhancers" linked to overexpression of TLR4 and CCR1, two genes known for their roles in myeloma cell proliferation and migration. We confirmed significant downregulation of both genes upon shRNA-mediated knockdown of MAF in the two MAF-translocated MMCL, MM1.S and JJN3, as well as the lethality of MAF depletion. Further, MAF overexpression in MAF-negative myeloma backgrounds led to transcriptional upregulation of these genes, further validating them as MAF targets. While CRISPR/Cas9i experiments targeting TLR4 are ongoing, preliminary results validated the functional role of the "neo-enhancer" in CCR1 gene expression. In conclusion, we demonstrate for the first time an extensive re-organisation of the PC chromatin conferred by oncogenic MAF in MM; we reveal its extensive co-operation with IRF4 in this process; we validate the directly MAF-regulated genes and functionally characterise neo-enhancers of key MAF-dependent genes that in addition to MAF itself are also critical for myeloma biology. Disclosures Hatjiharissi: Janssen: Honoraria. Caputo:GSK: Research Funding. Karadimitris:GSK: Research Funding.

Proteasome inhibitors (PIs) form the backbone of multiple myeloma (MM) treatment regimens used at diagnosis and relapse, but their clinical benefit is limited by varying degrees of resistance. The mechanisms that protect MM cells (MMCs) from PI-induced cell death are only partly understood, and experimental resistance studies often rely on the prolonged exposure of MMCs to relatively low levels of PIs. However, patients receive PI doses that result in high but short plasma peaks. Given that each PI dose reduces a patient's MMC load until a plateau is reached, one can assume that each PI dose kills some MMCs by triggering overwhelming stress. This proposition implies that a proportion of MMCs not only survive but also resolve PI-induced stress. We hypothesised that the mechanisms of PI-induced stress recovery require a redistribution of cellular resources, and that this process triggers specific recovery-associated and potentially druggable vulnerabilities. To test this hypothesis, we obtained a global systems-level view of the cellular response to proteasome inhibition by performing a multi-omics 10-day time-course experiment. To mimic in vivo pharmacokinetics and anti-MM effects, RPMI-8226 MMCs were treated with a 1h pulse of carfilzomib (CFZ; 750nmol/L), which reduced the number of viable MMCs to a nadir of approximately 50% on day +2, followed by a return to pretreatment baseline levels of viable cells by day +6. Levels of ubiquitinated proteins as a readout of effective proteasome inhibition peaked on day +1 and returned to baseline levels on day +6. Samples for transcriptome analysis by RNA-seq, proteome analysis by a multiplexed tandem mass tag (TMT)-based approach, and global metabolomic profiling by ultrahigh performance liquid chromatography-tandem mass spectroscopy (LC-MS) were collected 2h prior to the CFZ pulse (day 0) and on days +1, +2, +4, +6, +8, and +10. The results demonstrate extensive and kinetically complex changes of the MMC transcriptome, proteome and metabolome that did not fully return to baseline by day +10. Conventional and advanced computational analyses of RNA-seq data (including biological homogeneity score-validated graph-based clustering of temporal patterns combined with gene enrichment and KEGG/GO pathway analysis) revealed different patterns of involvement of multiple and functionally diverse pathways. Perhaps most notably, RNA-seq data revealed extensive metabolic pathway responses while cells were dying and throughout recovery. Consistently, metabolomic profiling by LC-MS showed wide-ranging metabolite changes. These included rapid intracellular depletion of glucose, which was paralleled by the accumulation of lactate and pyruvate and lasted until day +8. Consistently, the major glucose uptake regulator TXNIP stood out as upregulated during recovery, which was accompanied by the predicted downregulation of the glucose transporter GLUT1. These findings were confirmed by real-time PCR and immunoblotting in RPMI-8226 and other MMC lines. Moreover, pharmacological inhibition of glucose and lactate metabolising enzymes and transmembrane transport enhanced MMC killing by CFZ, confirming profound and druggable changes in glucose and energy metabolism during PI stress recovery. We also observed prolonged depletion of a number of amino acids, including glutamine, which was paralleled by depletion of glutamate and TCA cycle intermediates. Moreover, we found RNA-seq and real-time PCR/immunoblot-based evidence for an amino acid response (AAR) during stress recovery that was driven by the amino acid sensing kinase GCN2 (EIF2AK4). The preclinical GCN2 inhibitor GCN2iB was effective at blocking the AAR and enhanced MMC killing in a panel of CFZ-treated MMC lines. This effect was particularly pronounced during the intermediate stages of recovery, in line with the kinetics of amino acid depletion and the AAR. Thus, our observations define the complex and prolonged cellular responses that characterise the recovery of MMCs from proteasome inhibition. They reveal wide-ranging metabolic changes that persist far beyond immediate stress survival and highlight the dependence of MMCs on the GCN2-driven AAR to overcome PI stress. The findings demonstrate that the mechanisms of PI recovery trigger druggable vulnerabilities, providing the basis for ongoing investigations into sequential therapeutic interventions to enhance responses to PIs. Disclosures Caputo: GSK: Research Funding. Kaiser:Abbvie, Celgene, Takeda, Janssen, Amgen, Abbvie, Karyopharm: Consultancy; Celgene, Janssen: Research Funding; Takeda, Janssen, Celgene, Amgen: Honoraria, Other: Travel Expenses. Auner:Karyopharm: Consultancy; Takeda: Consultancy; Amgen: Other: Consultancy and Research Funding.

In multiple myeloma (MM), a malignancy of the bone marrow plasma cells (BMPC), hyperdiploidy (HY) and oncogene over-expression via chromosomal translocation [including CCND1- t(11;14), MAF- t(14;16), MMSET-t(4;14)] are the primary myeloma initiating events (MIE) that drive distinct transcriptional programs. These are further shaped by secondary SNV and CNV events. This genetic heterogeneity converges, in most cases, to a functionally dichotomous state of CCND1 or CCND2 overexpression. The molecular mechanisms underlying each of the distinct myelomagenic transcriptomes and the CCND1 vs CCND2 dichotomy have not been defined. To address these questions, we obtained highly purified BMPC from 3 healthy donors and 30 MM patients (HY: 15; CCND1: 4; MMSET: 5; MAF: 2; other: 4), either at diagnosis or relapse, and mapped their chromatin accessibility and transcriptome profiles by ATAC-seq and RNA-seq, respectively. In total, we obtained ~300K regions with accessible chromatin in either MM or normal PC. Overall chromatin accessibility increased in myeloma compared to normal PC, particularly in MAF- and MMSET-translocated subtypes. Analysis of combined ATAC-seq/RNA-seq by Multi-Omics Factor Analysis (MOFA) resulted in a clearer samples distinction than either ATAC-seq or RNA-seq alone, with altered chromatin accessibility accounting for more of the variance than expression. Of the top five identified factors, the top two (one transcriptome driven, one accessibility driven) distinguished normal from MM samples, whilst two more separated MMSET, MAF and CCND1 subgroups. Ninety seven, 157, 256 and 348 overexpressed genes in the CCND1, HY, MMSET and MAF subgroups, respectively, were predicted to be regulated by differentially accessible enhancers. Twenty percent (165/858) of these genes were overexpressed in >1 subgroup suggesting a process of chromatin accessibility-based convergence evolution. Enrichment analysis suggested direct or indirect involvement of Polycomb and chromatin remodellers; significant enrichment was also found for genes involved in neurogenesis. ATAC-seq footprinting predicted binding sites for 250 expressed transcription factors (TFs), 116 of which displayed higher binding frequency in myeloma than in normal PC and included both known (e.g., XBP1, RELA, IRF4, PRDM1) and potentially novel regulators of myeloma biology (e.g., CXXC1 and NFE2L1). The remaining 134 TF were predicted to be present in at least one MM subgroup, but absent in normal PC. Amongst them, as expected, MAF was active in the MMSET- and more so in the MAF-translocated subgroups. DepMap database analysis suggested myeloma cell dependency on 181/250 TF (CRISPR/Cas9 CERES score < -0.1 in >3/14 MMCL analysed). In dissecting the regulatory basis of CCND2 vs CCND1 dichotomy, one MOFA factor completely separated MAF from CCND1 samples, placing extreme opposite weights on the expression of CCND2 and CCND1 respectively. Interestingly, the same factor identified open-chromatin clusters upstream of CCND2 and linked them to its over-expression. These clusters were also open in the MMSET group and in CCND2-expressing HY samples. Conversely, no accessibility was detected in the CCND1 group, the CCND1-expressing HY samples or in normal PCs. Further, super-enhancer calling using the H3K27ac histone mark in MAF-translocated JJN3 cells identified the region of interest as a bona fide super-enhancer. Chromatin long range interactions, as assessed by Capture-HiC, demonstrated high frequency interactions of the CCND2 promoter with the constituent elements of the putative super-enhancer. Experimental validation using a CRISPR/Cas9i system confirmed the functional role of all 4 super-enhancer constituents tested in the regulation of CCND2 expression, while TF footprinting predicted MAF binding to the super-enhancer in MAF-translocated PC. In conclusion, we show that distinct oncogenic transcriptomes in MM are underpinned by extensive chromatin changes, accompanied by TF activity 're-wiring' that does not necessarily require transcriptional deregulation of the TF themselves. We identify novel, non-oncogene TF dependencies that suggest therapeutic opportunities in MM and we discover and characterise the critical super-enhancer that drives overexpression of the CCND2 oncogene in MM. Disclosures Auner: Amgen: Other: Consultancy and Research Funding; Takeda: Consultancy; Karyopharm: Consultancy. Hatjiharissi:Janssen: Honoraria. Caputo:GSK: Research Funding. Karadimitris:GSK: Research Funding.

Multiple myeloma (MM) is an incurable tumor of the plasma cells, the terminally differentiated immunoglobulin secreting B lineage cells. The genetic make-up of MM has been extensively characterized but its impact on the biology of the disease is incomplete without more precise knowledge of the identity and functional role of cells with multiple myeloma propagating activity (MMPA). We review here recent data that link MMPA with myeloma clonotypic populations organized in a cellular hierarchy that mirrors normal B cell development and also with drug resistance and disease relapse. We further propose a conceptual framework which, with optimal use of recent technological advances in genomics and phenomics, could allow dissection of the cellular and molecular properties of cells with MMPA, drug resistance and in vivo relapse in an integrated and patient-specific manner. There is real hope that these approaches will significantly contribute to further improvements in disease control, overall survival, and possibly cure of patients with MM. Stem Cells 2015;33:3205–3211

A rare point mutation in the core promoter -270GC-rich box of PIGM, a housekeeping gene, disrupts binding of the generic transcription factor (TF) Sp1 and causes inherited glycosylphosphatidylinositol (GPI) deficiency (IGD). We show that whereas PIGM messenger RNA levels and surface GPI expression in IGD B cells are low, GPI expression is near normal in IGD erythroid cells. This divergent phenotype results from differential promoter chromatin accessibility and binding of Sp1. Specifically, whereas PIGM transcription in B cells is dependent on Sp1 binding to the -270GC-rich box and is associated with lower promoter accessibility, in erythroid cells, Sp1 activates PIGM transcription by binding upstream of (but not to) the -270GC-rich box. These findings explain intact PIGM transcription in IGD erythroid cells and the lack of clinically significant intravascular hemolysis in patients with IGD. Furthermore, they provide novel insights into tissue-specific transcriptional control of a housekeeping gene by a generic TF.

HDAC inhibitors (HDACi) increase transcription of some genes through histone hyperacetylation. To test the hypothesis that HDACi-mediated enhanced transcription might be of therapeutic value for inherited enzyme deficiency disorders, we focused on the glycolytic and pentose phosphate pathways (GPPPs). We show that among the 16 genes of the GPPPs, HDACi selectively enhance transcription of glucose 6-phosphate dehydrogenase (G6PD). This requires enhanced recruitment of the generic transcription factor Sp1, with commensurate recruitment of histone acetyltransferases and deacetylases, increased histone acetylation, and polymerase II recruitment to G6PD. These G6PD-selective transcriptional and epigenetic events result in increased G6PD transcription and ultimately restored enzymatic activity in B cells and erythroid precursor cells from patients with G6PD deficiency, a disorder associated with acute or chronic hemolytic anemia. Therefore, restoration of enzymatic activity in G6PD-deficient nucleated cells is feasible through modulation of G6PD transcription. Our findings also suggest that clinical consequences of pathogenic missense mutations in proteins with enzymatic function can be overcome in some cases by enhancement of the transcriptional output of the affected gene.

The bromodomain and extraterminal (BET) protein BRD2-4 inhibitors hold therapeutic promise in preclinical models of hematologic malignancies. However, translation of these data to molecules suitable for clinical development has yet to be accomplished. Herein we expand the mechanistic understanding of BET inhibitors in multiple myeloma by using the chemical probe molecule I-BET151. I-BET151 induces apoptosis and exerts strong antiproliferative effect in vitro and in vivo. This is associated with contrasting effects on oncogenic MYC and HEXIM1, an inhibitor of the transcriptional activator P-TEFb. I-BET151 causes transcriptional repression of MYC and MYC-dependent programs by abrogating recruitment to the chromatin of the P-TEFb component CDK9 in a BRD2-4-dependent manner. In contrast, transcriptional upregulation of HEXIM1 is BRD2-4 independent. Finally, preclinical studies show that I-BET762 has a favorable pharmacologic profile as an oral agent and that it inhibits myeloma cell proliferation, resulting in survival advantage in a systemic myeloma xenograft model. These data provide a strong rationale for extending the clinical testing of the novel antimyeloma agent I-BET762 and reveal insights into biologic pathways required for myeloma cell proliferation.

Abstract 977 Transcriptionally active genes, including housekeeping genes, are characterised by co-occupancy and antagonistic actions of histone deacetylases (HDAC) and acetyltransferases (HAT). Transcription is facilitated through the prevailing action of HAT, which maintain histone acetylation. HDAC inhibitors (HDACi) induce widespread histone hyperacetylation and as a consequence are expected to increase expression of transcriptionally active genes. We have previously demonstrated this effect in inherited glycosylphosphatidylinositol (GPI-anchor) deficiency, an autosomal recessive disorder characterised by histone hypoacetylation and transcriptional repression of PIGM due to an in cis mutation which disrupts binding of the transcription factor Sp1 to its core promoter cognate motif. We surmised HDACi-mediated hyperacetylation might lead to increased transcription of other housekeeping genes, such as those of the anaerobic glycolytic and pentose phosphate pathways (GPPP) disruption of which is ameliorated by relatively modest increases in enzymatic activity. HDACi could therefore be of therapeutic value in these disorders. To address these hypotheses, EBV B cell lines were treated with the HDACi sodium butyrate (NaBu; 3mM) and mRNA levels for GPPP genes assessed by RQ-PCR. Of 9 genes tested (glucose-6-phosphate dehydrogenase, G6PD; glucose-6-phosphate isomerase, GPI; triosephosphate isomerase, TPI; pyruvate kinase, PK; 6-phosphogluconolactonase, PGLS; phosphogluconate dehydrogenase, PGD; ribulose-5-phosphate-3-epimerase, RPE; ribose-5-phosphate isomerase A, RPIA; transketolase, TKT), only G6PD mRNA levels increased, in a time-dependent fashion, in response to NaBu (n=3; p The epigenetic correlates of G6PD mRNA induction were assessed by ChIP-Q-PCR. This revealed a dynamic, time-dependent, 3 to 4-fold increase in levels of histone 3 and 4 acetylation and a 4-fold increase in Sp1 and Polymerase II occupancy in the promoter of G6PD but not TPI or GPI. Preliminary pharmacological and shRNA experiments suggest that HDACi-mediated transcriptional upregulation of G6PD is Sp1-dependent. No differences were observed in baseline levels of histone acetylation or Sp1 occupancy of G6PD, TPI and GPI implying a yet to be defined cis acting determinant is required for the selective increase in Sp1 binding and histone hyperacetylation. Finally, in erythroblasts generated from peripheral blood mononuclear cells of two patients with Class I G6PD deficiency (G6PD Brighton and G6PD Harilaou), we confirmed the ability of NaBu to increase mutant G6PD mRNA and protein levels leading to increased G6PD enzymatic activity and its restoration to normal within 24hrs. In conclusion, we show that even within the same metabolic pathway, transcriptional upregulation of active genes in response to HDACi is selective rather than universal and is underpinned by enhanced Sp1 binding and histone hyperacetylation. Our findings raise the prospect of using HDACi to treat severe Class I G6PD deficiency. Disclosures: No relevant conflicts of interest to declare.