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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.

Deregulated expression of lineage-affiliated transcription factors is a major mechanism of oncogenesis. However, how deregulation of non-lineage affiliated TF impacts chromatin to initiate oncogenic transcriptional programmes is not well known. To address this, we studied the chromatin effects imposed by oncogenic MAF as the cancer-initiating driver in the plasma cell cancer multiple myeloma. We found that the ectopically expressed MAF endows myeloma plasma cells with migratory and proliferative transcriptional potential. This potential is regulated by activation of enhancers and super-enhancers, previously inactive in normal B cells and plasma cells, and in co-operation of MAF with the plasma cell-defining TF IRF4. Forced ectopic MAF expression confirms the de novo ability of oncogenic MAF to convert transcriptionally inert chromatin to active chromatin with features of super-enhancers, leading to activation of the MAF-specific oncogenic transcriptome and acquisition of cancer-related cellular phenotypes such as CCR1-dependent cell migration. These findings establish oncogenic MAF as a pioneer transcription factor that can initiate as well as sustain oncogenic transcriptomes and cancer phenotypes. However, despite its pioneer function, myeloma cells remain MAF-dependent thus validating oncogenic MAF as a therapeutic target that would be able to circumvent the challenges of subsequent genetic diversification driving disease relapse and drug resistance.

Introduction The IMPRESS trial revealed that intralesional collagenase clostridium histolyticum, Xiaflex, is safe and efficacious for treating the physical and subjective effects of Peyronie’s Disease (PD). However, little is known about the other manifestations of PD, namely penile shortening and efficacy for patients of diverse racial backgrounds or with severe penile curvatures. Objective To evaluate predictors associated with response to treatment and alterations in penile morphological measurements. Methods Consecutive patients treated with Xiaflex for dorsal, lateral, and ventral penile curvatures ≥ 25°, in the absence of hourglass deformity, were included in this study. Before treatment and eight weeks after the last injection cycle, during penile duplex Doppler ultrasound evaluation, penile curvature was measured with a goniometer after induced artificial erection via injection of prostaglandin E1 (PGE1). IIEF score, PGE1 dose, circumference, and erectile as well as stretched penile length were also assessed at these times. Based on the results of the IMPRESS trial, patients were considered Xiaflex responders if the decrease in their penile curvature were ≥ 15° (n =134) and Xiaflex nonresponders if the decrease in their penile curvature were < 15° (n =89). Initial measurements were in increments of 5°. Intention to treat analysis was performed. Results Two hundred and twenty-three patients with penile curvature ≥ 25° who received XIAFLEX were included in this study. One hundred and thirty-four patients (60.1%) responded to Xiaflex, while 89 (39.9%) patients did not respond. African American men were 3 times more likely to respond to treatment than their non-African American counterparts (Odds Ratio (OR) =3.35 (95% CI, 1.15-10.70), p =0.031). On the other hand, men with an initial penile curvature of greater than 60° were less likely to respond to Xiaflex (OR = 0.34; 95% CI, 0.16-0.69; p=0.003). However, patients with lateral and ventral curvatures responded at comparable rates to those with dorsal curvatures. Subsequently, changes in penile morphologic measurements post Xiaflex were analyzed. Average stretched penile length, erectile length, and erectile circumference prior to treatment for responders were 13.93 cm, 13.39 cm, and 11.81 cm, respectively; for nonresponders, it was 14.27 cm, 13.72 cm, 11.64 cm, respectively. Nonresponders were shown to experience a significant decrease in penile erect length (-0.55 cm) and circumference (-0.58 cm) (p=0.002 and p=0.002, respectively), whereas responders showed no significant alterations in penile morphologic measurements. Conclusions Our analysis revealed that men with penile curvatures ≥ 60° are less likely to respond to Xiaflex injections and thus may be considered for surgical intervention upon initial presentation. Furthermore, men who do not respond may even be at a higher risk for shortening of penile erect length and circumference. The actual clinical significance of a decrease in average erect length of 0.55 cm and circumference of 0.58 cm is difficult to determine and should be considered per patient preferences. Further investigations into predictors for response to Xiaflex and the pathophysiology behind penile shortening in Xiaflex nonresponders are warranted. Disclosure No

Understanding the biological and clinical impact of copy number aberrations (CNA) in cancer remains an unmet challenge. Genetic amplification of chromosome 1q (chr1q-amp) is a major CNA conferring adverse prognosis in several cancers, including the blood cancer, multiple myeloma (MM). Although several chr1q genes portend high-risk MM disease, the underpinning molecular aetiology remains elusive. Here we integrate patient multi-omics datasets with genetic variables to identify 103 adverse prognosis genes in chr1q-amp MM. Amongst these, the transcription factor PBX1 is ectopically expressed by genetic amplification and epigenetic activation of its own preserved 3D regulatory domain. By binding to reprogrammed super-enhancers, PBX1 directly regulates critical oncogenic pathways, whilst in co-operation with FOXM1, activates a proliferative gene signature which predicts adverse prognosis across multiple cancers. Notably, pharmacological disruption of the PBX1-FOXM1 axis, including with a novel PBX1 inhibitor is selectively toxic against chr1q-amp cancer 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 cancer.SignificanceWe provide a comprehensive systems medicine strategy to unveil oncogenic circuitries and inform novel precision therapy decisions against CNA in cancer. This first clinical multi-omic analysis of chr1q-amp in MM identifies a central PBX1-FOXM1 regulatory axis driving high-risk prognosis, as a novel therapeutic target against chr1q-amp in 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.

Multiple myeloma is a genetically heterogeneous cancer of the bone marrow plasma cells (PC). Distinct myeloma transcriptome profiles are primarily driven by myeloma initiating events (MIE) and converge into a mutually exclusive overexpression of the CCND1 and CCND2 oncogenes. Here, with reference to their normal counterparts, we find that myeloma PC enhanced chromatin accessibility combined with paired transcriptome profiling can classify MIE-defined genetic subgroups. Across and within different MM genetic subgroups, we ascribe regulation of genes and pathways critical for myeloma biology to unique or shared, developmentally activated or de novo formed candidate enhancers. Such enhancers co-opt recruitment of existing transcription factors, which although not transcriptionally deregulated per se, organise aberrant gene regulatory networks that help identify myeloma cell dependencies with prognostic impact. Finally, we identify and validate the critical super-enhancer that regulates ectopic expression of CCND2 in a subset of patients with MM and in chronic lymphocytic leukemia., Despite extensive genetic heterogeneity, nearly half of all multiple myeloma (MM) cases are driven by cyclin D2 (CCND2) over-expression. Here the authors dissect the chromatin landscape of MM to provide insights into the transcriptional regulatory landscape driving MM and divergent transcriptomes corresponding to different MM genetic subtypes.

This essay discusses a foundational theory in media studies and uses an experiential approach to learning the concept. Based on the foundational writings of Sapir, Innes, McLuhan, Postman and others, the following is a two-part assignment that appears simple but can be quite complex. When looking at the cultural influence of media, we are often misled by the illusion of content. This assignment requires the student to examine how the form of a medium of communication influences the content. The student is asked to write a brief comparative analysis (two to four pages) of the same story in two different media, e.g., television news story and print journalism; a novel and a film; a blog and an audio story, etc. Again, students find this assignment quite difficult because now they are asked to show how the medium of communication being used actually changes the content.

DRESS/DiHS is a complex and potentially fatal drug reaction. Little is known about risk factors and elements that can help to identify patients with a severe reaction early. The aim of the study was to investigate those factors favoring the disease and its severity by analyzing the clinical conditions and therapies preceding the reaction. We conducted a retrospective analysis on patients admitted to our center between 2010 and 2020 who were discharged with a diagnosis of DRESS. We used the RegiSCAR diagnostic criteria. We defined the severity of DRESS using the criteria of Mizukawa et al. We included 25 patients (15 females) with a median age of 66 years. Skin involvement, eosinophilia, and liver injury were the most important aspects. Allopurinol was found to be the most involved drug. Reaction severity was significantly associated with the number of daily medications (p = 0.0067) and an age of at least 68 years (p = 0.013). In addition, 75% of severe cases had at least three comorbidities in history, and most of the severe cases were female. In our study the advanced age, the high number of comorbidities and home therapies, and the inflammatory state were found to be predisposing elements to the development of the disease and its severity.

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.

Significance Cancer therapies often fail to cure patients because a proportion of tumor cells withstand the toxic effects of chemotherapy. How surviving cancer cells recover from sublethal drug-induced stress is not known, but given that cellular resources are finite, stress resolution may come at the expense of less essential systems. Here, we studied the global cellular events of stress buildup and resolution in the bone marrow cancer, multiple myeloma, after proteasome inhibition, a commonly used therapeutic approach. Using a temporal multiomics approach, we delineate the unexpectedly complex and protracted changes myeloma cells undergo during stress resolution and demonstrate that recovering cells are more vulnerable to specific insults than acutely stressed cells. Thus, the findings may provide avenues for optimizing cancer therapies., Cancer cells can survive chemotherapy-induced stress, but how they recover from it is not known. Using a temporal multiomics approach, we delineate the global mechanisms of proteotoxic stress resolution in multiple myeloma cells recovering from proteasome inhibition. Our observations define layered and protracted programs for stress resolution that encompass extensive changes across the transcriptome, proteome, and metabolome. Cellular recovery from proteasome inhibition involved protracted and dynamic changes of glucose and lipid metabolism and suppression of mitochondrial function. We demonstrate that recovering cells are more vulnerable to specific insults than acutely stressed cells and identify the general control nonderepressable 2 (GCN2)-driven cellular response to amino acid scarcity as a key recovery-associated vulnerability. Using a transcriptome analysis pipeline, we further show that GCN2 is also a stress-independent bona fide target in transcriptional signature-defined subsets of solid cancers that share molecular characteristics. Thus, identifying cellular trade-offs tied to the resolution of chemotherapy-induced stress in tumor cells may reveal new therapeutic targets and routes for cancer therapy optimization.

Natural killer cells possess key regulatory function in various malignant diseases, including acute myeloid leukemia. NK cell activity is driven by signals received through ligands binding activating or inhibitory receptors. Their activity towards elimination of transformed or virally infected cells can be mediated through MICA, MICB and ULBP ligands binding the activating receptor NKG2D. Given the efficiency of NK cells, potential target cells developed multiple protecting mechanisms to overcome NK cells killing on various levels of biogenesis of NKG2D ligands. Targeted cells can degrade ligand transcripts via microRNAs or modify them at protein level to prevent their presence at cell surface via shedding, with added benefit of shed ligands to desensitize NKG2D receptor and avert the threat of destruction via NK cells. NK cells and their activity are also indispensable during hematopoietic stem cell transplantation, crucial treatment option for patients with malignant disease, including acute myeloid leukemia. Function of both NKG2D and its ligands is strongly affected by polymorphisms and particular allelic variants, as different alleles can play variable roles in ligand-receptor interaction, influencing NK cell function and HSCT outcome differently. For example, role of amino acid exchange at position 129 in MICA or at position 98 in MICB, as well as the role of other polymorphisms leading to different shedding of ligands, was described. Finally, match or mismatch between patient and donor in NKG2D ligands affect HSCT outcome. Having the information beyond standard HLA typing prior HSCT could be instrumental to find the best donor for the patient and to optimize effects of treatment by more precise patient-donor match. Here, we review recent research on the NKG2D/NKG2D ligand biology, their regulation, description of their polymorphisms across the populations of patients with AML and the influence of particular polymorphisms on HSCT outcome.

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

Genetic and transcriptional heterogeneity of Chronic lymphocytic leukaemia (CLL) limits prevention of disease progression. Longitudinal single-cell transcriptomics represents the state-of-the-art method to profile the disease heterogeneity at diagnosis and to inform about disease evolution. Here, we apply single-cell RNA-seq to a CLL case, sampled at diagnosis and relapse, that was treated with FCR (Fludarabine, Cyclophosphamide, Rituximab) and underwent a dramatic decrease in CD19 expression during disease progression. Computational analyses revealed a major switch in clones’ dominance during treatment. The clone that expanded at relapse showed 17p and 3p chromosomal deletions, and up-regulation of pathways related to motility, cytokine signaling and antigen presentation. Single-cell RNA-seq uniquely revealed that this clone was already present at low frequency at diagnosis, and it displays feature of plasma cell differentiation, consistent with a more aggressive phenotype. This study shows the benefit of single-cell profiling of CLL heterogeneity at diagnosis, to identify clones that might otherwise not be recognized and to determine the best treatment options.

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.

Multiple myeloma is a genetically heterogeneous cancer of the bone marrow plasma cells (PC). Myeloma initiating genetic events define subgroups (MIE) and drive distinct oncogenic transcriptomes that converge into a mutually exclusive overexpression of CCND1 and CCND2 oncogenes. Here, with reference to normal PC, we dissect how MIE impact the chromatin regulatory landscape of MM. We find that chromatin accessibility combined with transcriptome profiling classifies myeloma genetic subgroups, while in a topologically constrained manner, distal rather than proximal regulatory elements influence myeloma transcriptomes. Across and within MIE-defined subgroups, genes and pathways critical for myeloma biology can be linked to developmentally activated or de novo formed enhancers. We show that existing transcription factors, co-opted to organise highly ordered, aberrant regulatory networks, generate known and novel myeloma cell dependencies and help identify prognostic markers. Finally, we discover and functionally validate the critical enhancer that regulates ectopic expression of CCND2 in MM.

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.

Relapsed acute myeloid leukemia (AML) is a significant post-transplant complication lacking standard treatment and associated with a poor prognosis. Cellular therapy, which is already widely used as a treatment for several hematological malignancies, could be a potential treatment alternative. Natural killer (NK) cells play an important role in relapse control but can be inhibited by the leukemia cells highly positive for HLA class I. In order to restore NK cell activity after their ex vivo activation, NK cells can be combined with conditioning target cells. In this study, we tested NK cell activity against KG1a (AML cell line) with and without two types of pretreatment&mdash, Ara-C treatment that induced NKG2D ligands (increased activating signal) and/or blocking of HLA&ndash, KIR (killer-immunoglobulin-like receptors) interaction (decreased inhibitory signal). Both treatments improved NK cell killing activity. Compared with target cell killing of NK cells alone (38%), co-culture with Ara-C treated KG1a target cells increased the killing to 80%. Anti-HLA blocking antibody treatment increased the proportion of dead KG1a cells to 53%. Interestingly, the use of the combination treatment improved the killing potential to led to the death of 85% of KG1a cells. The combination of Ara-C and ex vivo activation of NK cells has the potential to be a feasible approach to treat relapsed AML after hematopoietic stem cell transplantation.

Diabetic foot ulcers (DFUs), a micro-vascular complication, are associated with a substantial increase in morbidity and mortality. DFUs are a complicated mixture of neuropathy, peripheral arterial diseases, foot deformities, and infections. Foot infections are frequent and potentially devastating complications. Infection prospers in more than half of all foot ulcers and is the factor that most often leads to lower extremity amputation. The complications of microbial flora span the spectrum from superficial cellulitis to chronic osteomyelitis and gangrenous extremity lower limb amputations. Wounds without confirmed soft tissue or bone infections do not require antibiotic therapy. Mild and moderate infections need empiric therapy covering Gram-positive cocci, while severe infections caused by drug-resistant organisms require broad-spectrum anti-microbials targeting aggressive Gram-negative aerobes and obligate anaerobes.

Despite encouraging therapeutic advances, multiple myeloma (MM) remains an incurable malignancy. The exciting results of chimaeric antigen receptor (CAR)-based immunotherapy in CD19(+) B-cell malignancies have spurred a great interest in extending the use of the CAR technology to other cancers, including MM. Availability of a specific, tumour-restricted antigen is crucial for the design of successful antibody-based CAR therapy. However, in MM, as in other malignancies, the relative dearth of such antigens-targets represents the main obstacle for the wider pre-clinical development and clinical application of the CAR technology. Here we provide an overview of the current progress and future promises of CAR technology in MM therapy. We highlight that, owing to its complexity, phenotypic and functional heterogeneity and the impact of the microenvironment, MM poses several challenges for CAR-based therapeutic approaches. Nevertheless, for the same reasons, MM can serve as a paradigm for better understanding, optimization and overall improvement of the CAR technology for the benefit of cancer and myeloma patients.

With the understanding of nature in terms of ecosystem services and the recognition of the vital role these play for human well-being (Millennium Ecosystem Assessment 2005), the value of the natural realm is scientifically and socially defined while at the same time institutionalized. Within this frame of interpretation, nature is a supplier of provisioning, regulating and cultural services, thus becoming not only a life-enabling factor for humanity but also a conceptual construct comparable to cornerstones of democracy, such as equality, freedom, and citizenship. The idea of green infrastructure is another recently coined term envisioning nature in cities in the form of a network and enabling a broad life-furthering vision of society. Standards for green open spaces embedded in some planning frameworks further state the right for all to a common good. Yet, evidence shows that this common right is not always met. Within the current context of advanced and neoliberal capitalism, green areas are sometimes used as an added financial value for real estate, thus increasing restrictions to their free access and full utilization. In developing countries with young democracies, such as Brazil, this process implies another significant factor of social inequality insofar the restricted access to nature by the poorest people means also diminished food safety and the jeopardizing of certain cultural practices. In developed countries, loss of land for food production and movements reclaiming the right to the city by squatting unoccupied open spaces to initiate community gardens demonstrates that the access to green spaces is also problematic, although in different ways if compared to developing countries. This chapter contributes to this topic by discussing the inequality in the provision of green spaces in informal settlements and social housing development in Brazil, as well as in the globalized north. The chapter concludes with recommendations to enhance democracy through a just provision of nature in cities.

Asthma is a chronic inflammatory disease that represents high hospitalizations and deaths in world. Copaiba oil (CO) is popularly used for relieving asthma symptoms and has already been shown to be effective in many inflammation models. This study aimed to investigate the immunomodulatory relationship of CO in ovalbumin (OVA)-induced allergic asthma. The composition of CO sample analyzed by GC and GC–MS and the toxicity test was performed in mice at doses of 50 or 100 mg/kg (by gavage). After, the experimental model of allergic asthma was induced with OVA and mice were orally treated with CO in two pre-established doses. The inflammatory infiltrate was evaluated in bronchoalveolar lavage fluid (BALF), while cytokines (IL-4, IL-5, IL-17, IFN-γ, TNF-α), IgE antibody and nitric oxide (NO) production was evaluated in BALF and lung homogenate (LH) of mice, together with the histology and histomorphometry of the lung tissue. CO significantly attenuated the number of inflammatory cells in BALF, suppressing NO production and reducing the response mediated by TH2 and TH17 (T helper) cells in both BALF and LH. Histopathological and histomorphometric analysis confirmed that CO significantly reduced the numbers of inflammatory infiltrate in the lung tissue, including in the parenchyma area. Our results indicate that CO has an effective in vivo antiasthmatic effect.

Summary Chimeric antigen receptor anti-CD19 (CAR19)-T cell immunotherapy-induced clinical remissions in CD19+ B cell lymphomas are often short lived. We tested whether CAR19-engineering of the CD1d-restricted invariant natural killer T (iNKT) cells would result in enhanced anti-lymphoma activity. CAR19-iNKT cells co-operatively activated by CD1d- and CAR19-CD19-dependent interactions are more effective than CAR19-T cells against CD1d-expressing lymphomas in vitro and in vivo. The swifter in vivo anti-lymphoma activity of CAR19-iNKT cells and their enhanced ability to eradicate brain lymphomas underpinned an improved tumor-free and overall survival. CD1D transcriptional de-repression by all-trans retinoic acid results in further enhanced cytotoxicity of CAR19-iNKT cells against CD19+ chronic lymphocytic leukemia cells. Thus, iNKT cells are a highly efficient platform for CAR-based immunotherapy of lymphomas and possibly other CD1d-expressing cancers., Graphical Abstract, Highlights • Bespoke protocol for CAR19-iNKT cell transduction and clinical scale expansion • Higher CAR19-iNKT than CAR19-T cell expandability and killing of CD19+CD1d+ targets • CAR19-iNKT cell reactivity potentiation by αGalCer and ATRA • Prolonged survival and brain lymphoma eradication of CAR19-iNKT cell-treated mice, Rotolo et al. show that anti-CD19 chimeric antigen receptor (CAR19)-engineered CD1d-restricted invariant NKT cells (iNKT) are more effective than CAR19-T cells against CD1d-expressing lymphomas, including those in the brain. De-repression of CD1d expression further enhances the anti-tumor efficacy of CAR19-iNKT.

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.

Background and aim: The objective of this cross-sectional study was to evaluate the degree of glycaemic control and the frequency of diabetic complications in Italian people with diabetes who were treated with continuous subcutaneous insulin infusion (CSII). Methods and results: Questionnaires investigating the organisation of diabetes care centres, individuals’ clinical and metabolic features and pump technology and its management were sent to adult and paediatric diabetes centres that use CSII for treatment in Italy. Information on standard clinical variables, demographic data and acute and chronic diabetic complications was derived from local clinical management systems. The sample consisted of 6623 people with diabetes, which was obtained from 93 centres. Of them, 98.8% had type 1 diabetes mellitus, 57.2% were female, 64% used a conventional insulin pump and 36% used a sensor-augmented insulin pump. The median glycated haemoglobin (HbA1c) level was 60 mmol/mol (7.6%). The HbA1c target (i.e. 18 years) was achieved in 43.4% of paediatric and 23% of adult participants. Factors such as advanced pump functions, higher rate of sensor use, pregnancy in the year before the study and longer duration of diabetes were associated with lower HbA1c levels. The most common chronic complications occurring in diabetes were retinopathy, microalbuminuria and hypertension. In the year before the study, 5% of participants reported ≥1 episode of severe hypoglycaemic (SH) episodes (SH) and 2.6% reported ≥1 episode of ketoacidosis. Conclusions: Advanced personal skills and use of sensor-based pump are associated with better metabolic control outcomes in Italian people with diabetes who were treated with CSII. The reduction in SH episodes confirms the positive effect of CSII on hypoglycaemia. Clinical trial registration number: NCT 02620917 (ClinicalTrials.gov).

This study investigated how student perceptions of prosocial instructor variables (immediacy, communication satisfaction, interpersonal attraction) and student academic entitlement predict graduate student reports of three dimensions (incompetent, indolent, and offensive) of teacher misbehaviors in online communication courses. Results revealed that communication satisfaction, immediacy, and task-related interpersonal attraction significantly predict student reports of incompetent and indolent teacher misbehaviors, while communication satisfaction and task- and social-related interpersonal attraction significantly predict perceptions of offensive teacher misbehaviors. The implications of these findings are discussed in light of the teacher-student relationship and instructor approaches in the computer-mediated classroom context.

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.

The dynamics of globalization mean we face a reality wherein leaders are called upon to communicate effectively in cultural situations unfamiliar to them. This situation brings new challenges for communication and leadership educators. Our article describes a cultural immersion experience called The Cagli Project, where communication and leadership students learn effective intercultural communication skills for leadership formation while studying in Italy. The philosophical and experiential aspects of the program are described along with leadership outcomes. The article ends with a discussion of the transferability of this model to alternative programs. Perhaps nothing has had a more profound effect on human communication and hence leadership in the past half century than the movement of people around the globe and the growing diversity of world society. Globalization is the mantra that seems to be driving much that is new in higher education today. It has its roots in the “global economy” and seems most relevant to the business disciplines, but there is a sub-text to globalization that resonates throughout all our institutions, and that is diversity (Caputo, 2011).

Sequelles fonctionnelles et qualite de vie apres duodenopancreatectomie cephalique S. Le Page, S. Caputo, F. Kwiatkowski, P. Berard, C. Gouillat But de l’etude : La duodenopancreatectomie cephalique est une intervention lourde dont les consequences physiologiques sont potentiellement deleteres sur la qualite de vie. Ce travail retrospectif unicentrique pilote avait pour but d’evaluer les effets des sequelles fonctionnelles de l’operation sur la qualite de vie des patients traites par duodenopancreatectomie cephalique. Patients et methode : Trente malades indemnes de recidive plus d’un an apres une duodenopancreatectomie cephalique ont repondu au questionnaire GIQLI (Gastro Intestinal Quality of Life Index) et a un questionnaire « specifique » evaluant en detail les sequelles fonctionnelles. Resultats : Le taux d’acceptabilite etait de 100 %. La coherence interne du questionnaire GIQLI etait bonne (coefficient α de Cronbach = 0,85). Le score total moyen du GIQLI etait de 94 (intervalle de confiance a 95 % -IC95 %- = [86-101]) pour un score ideal de 144. La qualite de vie etait significativement alteree par la steatorrhee, la prise d’un traitement antidiarrheique ou d’un traitement enzymatique substitutif. Conclusion : Par rapport a la reference pour la population normale, la duodenopancreatectomie cephalique est associee globalement a une alteration de la qualite de vie de 25 %, bien que plus d’un quart des patients conservent une qualite de vie normale. L’alteration de la qualite de vie apres duodenopancreatectomie cephalique apparait en rapport avec les sequelles digestives de l’operation. Le score GIQLI pourrait etre utilise pour evaluer les differents montages chirurgicaux.

This chapter describes a peacebuilding project that emerges from the context of intercultural and international communication using dialogic theory and practice in Derry, Northern Ireland. To begin with, even the name of the city is in dispute in this conflict situation. The Northern Irish city has been known by two names, Derry and Londonderry. There are historical resonances for both names. Unionists — who want to maintain a union with the United Kingdom — have a strong preference for Londonderry, which it was renamed in recognition of its connections with City of London livery companies during the Plantation of Ulster in the 1600s. Nationalists — who want Northern Ireland to become a part of the Republic of Ireland — want to retain their original name of Derry (Doire). The city’s Visitor’s Bureau calls it ‘Legenderry’. The Guardian Style Guide states: ‘Derry, Co Derry. Not Londonderry’. Speaking with a local in Derry, he says ‘You ask why any “self-respecting” Irish person would only call it Derry when we’re not talking about a city in Ireland?’ Derry IS a city in Ireland, on the island of Ireland, with a history going back to the 6th century. For the purposes of this chapter, the name Derry will be used.

Post-translational modifications of the nucleosomal histone proteins orchestrate chromatin organization and gene expression in normal and cancer cells. Among them, the acetylation of N-terminal histone tails represents the fundamental epigenetic mark of open structure chromatin and active gene transcription. The bromodomain and extra-terminal (BET) proteins are epigenetic readers which utilize tandem bromodomains (BRD) modules to recognize and dock themselves on the acetylated lysine tails. The BET proteins act as scaffolds for the recruitment of transcription factors and chromatin organizers required in transcription initiation and elongation. The recent discovery of small molecules capable of blocking their lysine-binding pocket is the first paradigm of successful pharmacological inhibition of epigenetic readers. JQ1 is a prototype benzodiazepine molecule and a specific BET inhibitor with antineoplastic activity both in solid tumours and haematological malignancies. The quinolone I-BET151 and the suitable for clinical development I-BET762 benzodiazepine were introduced in parallel with JQ1 and have also shown potent antitumour activity in preclinical studies. I-BET762 is currently being tested in early phase clinical trials, along with a rapidly growing list of other BET inhibitors. Unlike older epigenetic therapies, the study of BET inhibitors has offered substantial, context-specific, mechanistic insights of their antitumour activity, which will facilitate optimal therapeutic targeting in future. Here, we review the development of this novel class of epigenetic drugs, the biology of BET protein inhibition, the emerging evidence from preclinical work and early phase clinical studies and we discuss their potential role in the treatment of haematological malignancies.

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.

How the transcription repressing complex Polycomb interacts with transcriptional regulators at housekeeping genes in somatic cells is not well understood. By exploiting a CpG island (CGI) point mutation causing a Mendelian disease, we show that DNA binding of activating transcription factor (TF) determines histone acetylation and nucleosomal depletion commensurate with Polycomb exclusion from the target promoter. Lack of TF binding leads to reversible transcriptional repression imposed by nucleosomal compaction and consolidated by Polycomb recruitment and establishment of bivalent chromatin status. Thus, within a functional hierarchy of transcriptional regulators, TF binding is the main determinant of Polycomb recruitment to the CGI of a housekeeping gene in somatic cells.

Pancreaticoduodenectomy is a major surgical procedure whose physiological effects may weigh heavily on quality of life. The goal of this retrospective unicentric pilot study was to assess the the functional outcome after pancreaticoduodenectomy and its effect on the patient's quality of life.Thirty patients free from tumor recurrence more than one year after pancreaticoduodenectomy responded to the GIQLI questionnaire (Gastro Intestinal Quality of Life Index) and to a specific questionnaire evaluating long-term functional outcome.The acceptability rate was 100%. The internal coherence of the GIQLI questionnaire was good (a Cronbach rate=0.85). The average total score of the GIQLI was 94 (IC-95%=[86-101]) compared to an ideal rate of 144. The quality of life was significantly impaired by steatorrhea, need for treatment of diarrhea, or need for enzymatic substitutive treatment.Compared to the reference for the normal population, patients post-pancreaticoduodenectomy have an average 25% decrease of quality of life scores (although more than 25% of patients experience a normal quality of life). The impairment of quality of life after pancreaticoduodenectomy appears to be related to the functional digestive consequences of the procedure. The GIQLI score could be used to assess the technical surgical variants.

This paper analyzes a pioneering topic in the telecommunications field: how preserving the signal integrity in all-optical ultra-fast signal processing boards with optical interconnects and Semiconductor Optical Amplifiers (SOAs), probably the most well-known active devices used in the Optical Time Domain Multiplexing systems (OTDM). In particular, it investigates the performances of an attractive SOA-based de-multiplexing architecture which, thanks to the potentialities of integration of these components, appears particularly suitable for being adopted in an OTDM interconnection. The analysis presents a criterion for suppressing the signal degradation, known as pattern- dependence effect, which normally affects the probe signal traversing this type of device, and shows an example of SOA de-multiplexer used in a 100 Gbit/s RZ-OTDM interconnection where the 25 Gb/s tributary channels, are extracted without any significant optical signal degradation.

An optical gain pattern-dependence suppression technique in Semiconductor Optical Amplifiers is presented, and its validity numerically demonstrated. It is applied to a simple all-optical ultra-fast de-multiplexer scheme for Optical Time Domain Multiplexed signals.

A non-adiabatic theoretical model including all fast gain dynamics in SOAs is presented. It emerges that an adiabatic theory cannot properly describe the impact of the TPA on high-energy ultrafast pulses propagating in SOAs.