BET protein inhibitor apabetalone (RVX-208) suppresses pro-inflammatory hyper-activation of monocytes from patients with cardiovascular disease and type 2 diabetes.

BACKGROUND: Patients with cardiovascular disease (CVD) and type 2 diabetes (DM2) have a high residual risk for experiencing a major adverse cardiac event. Dysregulation of epigenetic mechanisms of gene transcription in innate immune cells contributes to CVD development but is currently not targeted by therapies. Apabetalone (RVX-208) is a small molecule inhibitor of bromodomain and extra-terminal (BET) proteins-histone acetylation readers that drive pro-inflammatory and pro-atherosclerotic gene transcription. Here, we assess the impact of apabetalone on ex vivo inflammatory responses of monocytes from DM2 + CVD patients. RESULTS: Monocytes isolated from DM2 + CVD patients and matched controls were treated ex vivo with apabetalone, interferon γ (IFNγ), IFNγ + apabetalone or vehicle and phenotyped for gene expression and protein secretion. Unstimulated DM2 + CVD monocytes had higher baseline IL-1α, IL-1ß and IL-8 cytokine gene expression and Toll-like receptor (TLR) 2 surface abundance than control monocytes, indicating pro-inflammatory activation. Further, DM2 + CVD monocytes were hyper-responsive to stimulation with IFNγ, upregulating genes within cytokine and NF-κB pathways > 30% more than control monocytes (p < 0.05). Ex vivo apabetalone treatment countered cytokine secretion by DM2 + CVD monocytes at baseline (GROα and IL-8) and during IFNγ stimulation (IL-1ß and TNFα). Apabetalone abolished pro-inflammatory hyper-activation by reducing TLR and cytokine gene signatures more robustly in DM2 + CVD versus control monocytes. CONCLUSIONS: Monocytes isolated from DM2 + CVD patients receiving standard of care therapies are in a hyper-inflammatory state and hyperactive upon IFNγ stimulation. Apabetalone treatment diminishes this pro-inflammatory phenotype, providing mechanistic insight into how BET protein inhibition may reduce CVD risk in DM2 patients.

Potent lipoprotein(a) lowering following apolipoprotein(a) antisense treatment reduces the pro-inflammatory activation of circulating monocytes in patients with elevated lipoprotein(a).

AIMS: Elevated lipoprotein(a) [Lp(a)] is strongly associated with an increased cardiovascular disease (CVD) risk. We previously reported that pro-inflammatory activation of circulating monocytes is a potential mechanism by which Lp(a) mediates CVD. Since potent Lp(a)-lowering therapies are emerging, it is of interest whether patients with elevated Lp(a) experience beneficial anti-inflammatory effects following large reductions in Lp(a). METHODS AND RESULTS: Using transcriptome analysis, we show that circulating monocytes of healthy individuals with elevated Lp(a), as well as CVD patients with increased Lp(a) levels, both have a pro-inflammatory gene expression profile. The effect of Lp(a)-lowering on gene expression and function of monocytes was addressed in two local sub-studies, including 14 CVD patients with elevated Lp(a) who received apolipoprotein(a) [apo(a)] antisense (AKCEA-APO(a)-LRx) (NCT03070782), as well as 18 patients with elevated Lp(a) who received proprotein convertase subtilisin/kexin type 9 antibody (PCSK9ab) treatment (NCT02729025). AKCEA-APO(a)-LRx lowered Lp(a) by 47% and reduced the pro-inflammatory gene expression in monocytes of CVD patients with elevated Lp(a), which coincided with a functional reduction in transendothelial migration capacity of monocytes ex vivo (-17%, P < 0.001). In contrast, PCSK9ab treatment lowered Lp(a) by 16% and did not alter transcriptome nor functional properties of monocytes, despite an additional reduction of 65% in low-density lipoprotein cholesterol (LDL-C). CONCLUSION: Potent Lp(a)-lowering following AKCEA-APO(a)-LRx, but not modest Lp(a)-lowering combined with LDL-C reduction following PCSK9ab treatment, reduced the pro-inflammatory state of circulating monocytes in patients with elevated Lp(a). These ex vivo data support a beneficial effect of large Lp(a) reductions in patients with elevated Lp(a).

The NEDD8-activating enzyme inhibitor MLN4924 induces DNA damage in Ph+ leukemia and sensitizes for ABL kinase inhibitors.

The BCR-ABL1 fusion gene is the driver oncogene in chronic myeloid leukemia (CML) and Philadelphia-chromosome positive (Ph+) acute lymphoblastic leukemia (ALL). The introduction of tyrosine kinase inhibitors (TKIs) targeting the ABL kinase (such as imatinib) has dramatically improved survival of CML and Ph+ ALL patients. However, primary and acquired resistance to TKIs remains a clinical challenge. Ph+ leukemia patients who achieve a complete cytogenetic (CCR) or deep molecular response (MR) (≥4.5log reduction in BCR-ABL1 transcripts) represent long-term survivors. Thus, the fast and early eradication of leukemic cells predicts MR and is the prime clinical goal for these patients. We show here that the first-in-class inhibitor of the Nedd8-activating enzyme (NAE1) MLN4924 effectively induced caspase-dependent apoptosis in Ph+ leukemia cells, and sensitized leukemic cells for ABL tyrosine kinase inhibitors (TKI) and hydroxyurea (HU). We demonstrate that MLN4924 induced DNA damage in Ph+ leukemia cells by provoking the activation of an intra S-phase checkpoint, which was enhanced by imatinib co-treatment. The combination of MLN4924 and imatinib furthermore triggered a dramatic shift in the expression of MCL1 and NOXA. Our data offers a clear rationale to explore the clinical activity of MLN4924 (alone and in combination with ABL TKI) in Ph+ leukemia patients.

Potential epigenetic therapeutics for atherosclerosis treatment.

Notwithstanding the intense efforts into the understanding and prevention of cardiovascular disease (CVD), its complex pathology remains the leading cause of mortality worldwide. The pivotal role of epigenetic changes in the control of gene expression has been profiled in several diseases, such as cancer and inflammatory disorders. In the last decade, increasing evidence has also linked aberrant epigenetic modulation as a contributor to CVD development. Differential profiles of DNA methylation, histone methylation and acetylation have consistently been observed in tissues and cells (comprising the aortic lesions, vascular endothelium and monocytes) from patients with CVD. This highlights the therapeutic potential of epigenetic drugs for cardiovascular treatment.

The Complex Interplay between DNA Injury and Repair in Enzymatically Induced Mutagenesis and DNA Damage in B Lymphocytes.

Lymphocytes are endowed with unique and specialized enzymatic mutagenic properties that allow them to diversify their antigen receptors, which are crucial sensors for pathogens and mediators of adaptive immunity. During lymphocyte development, the antigen receptors expressed by B and T lymphocytes are assembled in an antigen-independent fashion by ordered variable gene segment recombinations (V(D)J recombination), which is a highly ordered and regulated process that requires the recombination activating gene products 1 & 2 (RAG1, RAG2). Upon activation by antigen, B lymphocytes undergo additional diversifications of their immunoglobulin B-cell receptors. Enzymatically induced somatic hypermutation (SHM) and immunoglobulin class switch recombination (CSR) improves the affinity for antigen and shape the effector function of the humoral immune response, respectively. The activation-induced cytidine deaminase (AID) enzyme is crucial for both SHM and CSR. These processes have evolved to both utilize as well as evade different DNA repair and DNA damage response pathways. The delicate balance between enzymatic mutagenesis and DNA repair is crucial for effective immune responses and the maintenance of genomic integrity. Not surprisingly, disturbances in this balance are at the basis of lymphoid malignancies by provoking the formation of oncogenic mutations and chromosomal aberrations. In this review, we discuss recent mechanistic insight into the regulation of RAG1/2 and AID expression and activity in lymphocytes and the complex interplay between these mutagenic enzymes and DNA repair and DNA damage response pathways, focusing on the base excision repair and mismatch repair pathways. We discuss how disturbances of this interplay induce genomic instability and contribute to oncogenesis.

Detection and Visualization of DNA Damage-induced Protein Complexes in Suspension Cell Cultures Using the Proximity Ligation Assay.

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of programmed DNA breaks in lymphocytes. The Ataxia-Telangiectasia Mutated kinase (ATM), ATM- and Rad3-Related kinase (ATR) and the catalytic subunit of DNA-dependent Protein Kinase (DNA-PKcs) are among the first that are activated upon induction of DNA damage, and are central regulators of a network that controls DNA repair, apoptosis and cell survival. As part of a tumor-suppressive pathway, ATM and ATR activate p53 through phosphorylation, thereby regulating the transcriptional activity of p53. DNA damage also results in the formation of so-called ionizing radiation-induced foci (IRIF) that represent complexes of DNA damage sensor and repair proteins that accumulate at the sites of DNA damage, which are visualized by fluorescence microscopy. Co-localization of proteins in IRIFs, however, does not necessarily imply direct protein-protein interactions, as the resolution of fluorescence microscopy is limited. In situ Proximity Ligation Assay (PLA) is a novel technique that allows the direct visualization of protein-protein interactions in cells and tissues with unprecedented specificity and sensitivity. This technique is based on the spatial proximity of specific antibodies binding to the proteins of interest. When the interrogated proteins are within ~40 nm an amplification reaction is triggered by oligonucleotides that are conjugated to the antibodies, and the amplification product is visualized by fluorescent labeling, yielding a signal that corresponds to the subcellular location of the interacting proteins. Using the established functional interaction between ATM and p53 as an example, it is demonstrated here how PLA can be used in suspension cell cultures to study the direct interactions between proteins that are integral parts of the DNA damage response.

The DNA Damage Response Regulates RAG1/2 Expression in Pre-B Cells through ATM-FOXO1 Signaling.

The recombination activating gene (RAG) 1 and RAG2 protein complex introduces DNA breaks at Tcr and Ig gene segments that are required for V(D)J recombination in developing lymphocytes. Proper regulation of RAG1/2 expression safeguards the ordered assembly of Ag receptors and the development of lymphocytes, while minimizing the risk for collateral damage. The ataxia telangiectasia mutated (ATM) kinase is involved in the repair of RAG1/2-mediated DNA breaks and prevents their propagation. The simultaneous occurrence of RAG1/2-dependent and -independent DNA breaks in developing lymphocytes exposed to genotoxic stress increases the risk for aberrant recombinations. In this study, we assessed the effect of genotoxic stress on RAG1/2 expression in pre-B cells and show that activation of the DNA damage response resulted in the rapid ATM-dependent downregulation of RAG1/2 mRNA and protein expression. We show that DNA damage led to the loss of FOXO1 binding to the enhancer region of the RAG1/2 locus (Erag) and provoked FOXO1 cleavage. We also show that DNA damage caused by RAG1/2 activity in pre-B cells was able to downmodulate RAG1/2 expression and activity, confirming the existence of a negative feedback regulatory mechanism. Our data suggest that pre-B cells are endowed with a protective mechanism that reduces the risk for aberrant recombinations and chromosomal translocations when exposed to DNA damage, involving the ATM-dependent regulation of FOXO1 binding to the Erag enhancer region.

NF-κB and AKT signaling prevent DNA damage in transformed pre-B cells by suppressing RAG1/2 expression and activity.

In developing lymphocytes, expression and activity of the recombination activation gene protein 1 (RAG1) and RAG2 endonuclease complex is tightly regulated to ensure ordered recombination of the immunoglobulin genes and to avoid genomic instability. Aberrant RAG activity has been implicated in the generation of secondary genetic events in human B-cell acute lymphoblastic leukemias (B-ALLs), illustrating the oncogenic potential of the RAG complex. Several layers of regulation prevent collateral genomic DNA damage by restricting RAG activity to the G1 phase of the cell cycle. In this study, we show a novel pathway that suppresses RAG expression in cycling-transformed mouse pre-B cells and human pre-B B-ALL cells that involves the negative regulation of FOXO1 by nuclear factor κB (NF-κB). Inhibition of NF-κB in cycling pre-B cells resulted in upregulation of RAG expression and recombination activity, which provoked RAG-dependent DNA damage. In agreement, we observe a negative correlation between NF-κB activity and the expression of RAG1, RAG2, and TdT in B-ALL patients. Our data suggest that targeting NF-κB in B-ALL increases the risk of RAG-dependent genomic instability.

Systems biology approach identifies the kinase Csnk1a1 as a regulator of the DNA damage response in embryonic stem cells.

In pluripotent stem cells, DNA damage triggers loss of pluripotency and apoptosis as a safeguard to exclude damaged DNA from the lineage. An intricate DNA damage response (DDR) signaling network ensures that the response is proportional to the severity of the damage. We combined an RNA interference screen targeting all kinases, phosphatases, and transcription factors with global transcriptomics and phosphoproteomics to map the DDR in mouse embryonic stem cells treated with the DNA cross-linker cisplatin. Networks derived from canonical pathways shared in all three data sets were implicated in DNA damage repair, cell cycle and survival, and differentiation. Experimental probing of these networks identified a mode of DNA damage-induced Wnt signaling that limited apoptosis. Silencing or deleting the p53 gene demonstrated that genotoxic stress elicited Wnt signaling in a p53-independent manner. Instead, this response occurred through reduced abundance of Csnk1a1 (CK1α), a kinase that inhibits ß-catenin. Together, our findings reveal a balance between p53-mediated elimination of stem cells (through loss of pluripotency and apoptosis) and Wnt signaling that attenuates this response to tune the outcome of the DDR.