CAR-iNKT cells targeting clonal TCRVβ chains as a precise strategy to treat T cell lymphoma

Introduction: Most T cell receptor (TCR)Vβ chain-expressing T cell lymphomas (TCL) including those caused by Human T cell leukaemia virus type-1 (HTLV-1) have poor prognosis. We hypothesised that chimeric antigen receptor (CAR)-mediated targeting of the clonal, lymphoma-associated TCRβ chains would comprise an effective cell therapy for TCL that would minimally impact the physiological TCR repertoire. Methods: As proof of concept, we generated CAR constructs to target four TCRVβ subunits. Efficacy of the CAR constructs was tested using conventional T cells as effectors (CAR-T). Since invariant NKT (iNKT) cell do not incite acute graft-versus-host disease and are suitable for ‘off-the-shelf’ immunotherapy, we generated anti-TCRVβ CAR-iNKT cells. Results: We show that anti-TCRVβ CAR-T cells selectively kill their cognate tumour targets while leaving >90% of the physiological TCR repertoire intact. CAR-iNKT cells inhibited the growth of TCL in vivo, and were also selectively active against malignant cells from Adult T cell leukaemia/lymphoma patients without activating expression of HTLV-1. Discussion: Thus we provide proof-of-concept for effective and selective anti-TCRVβ CAR-T and -iNKT cell-based therapy of TCL with the latter providing the option for ‘off-the-shelf’ immunotherapy

Brd2/4 and Myc regulate alternative cell lineage programmes during early osteoclast differentiation in vitro

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

Single-cell profiling of human megakaryocyte-erythroid progenitors identifies distinct megakaryocyte and erythroid differentiation pathways

Background Recent advances in single-cell techniques have provided the opportunity to finely dissect cellular heterogeneity within populations previously defined by “bulk” assays and to uncover rare cell types. In human hematopoiesis, megakaryocytes and erythroid cells differentiate from a shared precursor, the megakaryocyte-erythroid progenitor (MEP), which remains poorly defined. Results To clarify the cellular pathway in erythro-megakaryocyte differentiation, we correlate the surface immunophenotype, transcriptional profile, and differentiation potential of individual MEP cells. Highly purified, single MEP cells were analyzed using index fluorescence-activated cell sorting and parallel targeted transcriptional profiling of the same cells was performed using a specifically designed panel of genes. Differentiation potential was tested in novel, single-cell differentiation assays. Our results demonstrate that immunophenotypic MEP comprise three distinct subpopulations: “Pre-MEP,” enriched for erythroid/megakaryocyte progenitors but with residual myeloid differentiation capacity; “E-MEP,” strongly biased towards erythroid differentiation; and “MK-MEP,” a previously undescribed, rare population of cells that are bipotent but primarily generate megakaryocytic progeny. Therefore, conventionally defined MEP are a mixed population, as a minority give rise to mixed-lineage colonies while the majority of cells are transcriptionally primed to generate exclusively single-lineage output. Conclusions Our study clarifies the cellular hierarchy in human megakaryocyte/erythroid lineage commitment and highlights the importance of using a combination of single-cell approaches to dissect cellular heterogeneity and identify rare cell types within a population. We present a novel immunophenotyping strategy that enables the prospective identification of specific intermediate progenitor populations in erythro-megakaryopoiesis, allowing for in-depth study of disorders including inherited cytopenias, myeloproliferative disorders, and erythromegakaryocytic leukemias

MAF functions as a pioneer transcription factor that initiates and sustains myelomagenesis

Deregulated expression of lineage-affiliated transcription factors (TFs) is a major mechanism of oncogenesis. However, how the deregulation of nonlineage affiliated TF affects chromatin to initiate oncogenic transcriptional programs 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 the activation of enhancers and superenhancers, previously inactive in healthy B cells and plasma cells, and the cooperation 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 the features of superenhancers, leading to the activation of the MAF-specific oncogenic transcriptome and the 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

Chromatin-based, in cis and in trans regulatory rewiring underpins distinct oncogenic transcriptomes in multiple myeloma

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

Systems medicine dissection of chr1q-amp reveals a novel PBX1-FOXM1 axis for targeted therapy in multiple myeloma

Understanding the biological and clinical impact ofcopy number aberrations (CNA)for the development of precision therapies in cancer remains anunmet challenge. Genetic amplification of chromosome 1q (chr1q-amp) is a major CNAconferring adverse prognosis in several types of cancer, including in the blood cancer multiple myeloma (MM). Although severalgenes across chr1q portend high-risk MM disease, the underpinning molecular aetiology remains elusive. Here, with reference to the 3D chromatin structure, we integrate MMpatient multi-omics datasets with genetic variables to obtain an associated clinical risk map across chr1q and to identify 103 adverse prognosis genes in chr1q-amp MM. Prominent amongst 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 super-enhancers, PBX1 directly regulates critical oncogenic pathways and a FOXM1-dependent transcriptional programme. Together, PBX1 and FOXM1 activate a proliferative gene signature which 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-amplified myeloma and solid tumour cells. Overall, our systems medicine approach successfully identifies CNA-driven oncogenic circuitries, links them to clinical phenotypes and proposes novel CNA-targeted therapystrategies in multiple myeloma and other types of cancer

The innate sensor ZBP1-IRF3 axis regulates cell proliferation in multiple myeloma

Multiple myeloma is a malignancy of plasma cells (PC) initiated and driven by primary and secondary genetic events. Nevertheless, myeloma PC 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 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 mouse cells and it is required for optimal T-cell-dependent humoral immune responses. In myeloma PC, 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 PC lineage-defining transcription factor IRF4, and 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 non-canonical function of constitutive ZBP1 in human cells and expand our knowledge of the role of cellular immune sensors in cancer biology

Poly(ADP-ribose) polymerase family member 14 (PARP14) is a novel effector of the JNK2-dependent pro-survival signal in multiple myeloma

Copyright @ 2013 Macmillan Publishers Limited. This is the author's accepted manuscript. The final published article is available from the link below.Regulation of cell survival is a key part of the pathogenesis of multiple myeloma (MM). Jun N-terminal kinase (JNK) signaling has been implicated in MM pathogenesis, but its function is unclear. To elucidate the role of JNK in MM, we evaluated the specific functions of the two major JNK proteins, JNK1 and JNK2. We show here that JNK2 is constitutively activated in a panel of MM cell lines and primary tumors. Using loss-of-function studies, we demonstrate that JNK2 is required for the survival of myeloma cells and constitutively suppresses JNK1-mediated apoptosis by affecting expression of poly(ADP-ribose) polymerase (PARP)14, a key regulator of B-cell survival. Strikingly, we found that PARP14 is highly expressed in myeloma plasma cells and associated with disease progression and poor survival. Overexpression of PARP14 completely rescued myeloma cells from apoptosis induced by JNK2 knockdown, indicating that PARP14 is critically involved in JNK2-dependent survival. Mechanistically, PARP14 was found to promote the survival of myeloma cells by binding and inhibiting JNK1. Moreover, inhibition of PARP14 enhances the sensitization of MM cells to anti-myeloma agents. Our findings reveal a novel regulatory pathway in myeloma cells through which JNK2 signals cell survival via PARP14, and identify PARP14 as a potential therapeutic target in myeloma.Kay Kendall Leukemia Fund, NIH, Cancer Research UK, Italian Association for Cancer Research and the Foundation for Liver Research

The coordinated action of VCP/p97 and GCN2 regulates cancer cell metabolism and proteostasis during nutrient limitation

VCP/p97 regulates numerous cellular functions by mediating protein degradation through its segregase activity. Its key role in governing protein homoeostasis has made VCP/p97 an appealing anticancer drug target. Here, we provide evidence that VCP/p97 acts as a regulator of cellular metabolism. We found that VCP/p97 was tied to multiple metabolic processes on the gene expression level in a diverse range of cancer cell lines and in patient-derived multiple myeloma cells. Cellular VCP/p97 dependency to maintain proteostasis was increased under conditions of glucose and glutamine limitation in a range of cancer cell lines from different tissues. Moreover, glutamine depletion led to increased VCP/p97 expression, whereas VCP/p97 inhibition perturbed metabolic processes and intracellular amino acid turnover. GCN2, an amino acid-sensing kinase, attenuated stress signalling and cell death triggered by VCP/p97 inhibition and nutrient shortages and modulated ERK activation, autophagy, and glycolytic metabolite turnover. Together, our data point to an interconnected role of VCP/p97 and GCN2 in maintaining cancer cell metabolic and protein homoeostasis

A mutation in a functional Sp1 binding site of the telomerase RNA gene (hTERC) promoter in a patient with Paroxysmal Nocturnal Haemoglobinuria

BACKGROUND: Mutations in the gene coding for the RNA component of telomerase, hTERC, have been found in autosomal dominant dyskeratosis congenita (DC) and aplastic anemia. Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal blood disorder associated with aplastic anemia and characterized by the presence of one or more clones of blood cells lacking glycosylphosphatidylinositol (GPI) anchored proteins due to a somatic mutation in the PIGA gene. METHODS: We searched for mutations in DNA extracted from PNH patients by amplification of the hTERC gene and denaturing high performance liquid chromatography (dHPLC). After a mutation was found in a potential transcription factor binding site in one patient electrophoretic mobility shift assays were used to detect binding of transcription factors to that site. The effect of the mutation on the function of the promoter was tested by transient transfection constructs in which the promoter is used to drive a reporter gene. RESULTS: Here we report the finding of a novel promoter mutation (-99C->G) in the hTERC gene in a patient with PNH. The mutation disrupts an Sp1 binding site and destroys its ability to bind Sp1. Transient transfection assays show that mutations in this hTERC site including C-99G cause either up- or down-regulation of promoter activity and suggest that the site regulates core promoter activity in a context dependent manner in cancer cells. CONCLUSIONS: These data are the first report of an hTERC promoter mutation from a patient sample which can modulate core promoter activity in vitro, raising the possibility that the mutation may affect the transcription of the gene in hematopoietic stem cells in vivo, and that dysregulation of telomerase may play a role in the development of bone marrow failure and the evolution of PNH clones