AKT supports the metabolic fitness of multiple myeloma cells by restricting FOXO activity.

Metabolic alterations are important cancer-associated features that allow cancer cell transformation and survival under stress conditions. Multiple myeloma (MM) plasma cells show increased glycolysis and oxidative phosphorylation (OXPHOS), which are characteristics associated with recurrent genetic aberrations that drive the proliferation and survival of MM cells. The protein kinase B/AKT acts as a central node in cellular metabolism and is constitutively active in MM cells. Despite the known role of AKT in modulating cellular metabolism, little is known about the downstream factors of AKT that control the metabolic adaptability of MM cells. Here, we demonstrate that negative regulation of the forkhead box O (FOXO) transcription factors (TFs) by AKT is crucial to prevent the metabolic shutdown in MM cells, thus contributing to their metabolic adaptability. Our results demonstrate that the expression of several key metabolic genes involved in glycolysis, the tricarboxylic acid (TCA) cycle, and OXPHOS are repressed by FOXO TFs. Moreover, the FOXO-dependent repression of glycolysis- and TCA-associated genes correlates with a favorable prognosis in a large cohort of patients with MM. Our data suggest that repression of FOXO by AKT is essential to sustain glycolysis and the TCA cycle activity in MM cells and, as such, predicts patient survival.

Metabolic Effects of Recurrent Genetic Aberrations in Multiple Myeloma.

Oncogene activation and malignant transformation exerts energetic, biosynthetic and redox demands on cancer cells due to increased proliferation, cell growth and tumor microenvironment adaptation. As such, altered metabolism is a hallmark of cancer, which is characterized by the reprogramming of multiple metabolic pathways. Multiple myeloma (MM) is a genetically heterogeneous disease that arises from terminally differentiated B cells. MM is characterized by reciprocal chromosomal translocations that often involve the immunoglobulin loci and a restricted set of partner loci, and complex chromosomal rearrangements that are associated with disease progression. Recurrent chromosomal aberrations in MM result in the aberrant expression of MYC, cyclin D1, FGFR3/MMSET and MAF/MAFB. In recent years, the intricate mechanisms that drive cancer cell metabolism and the many metabolic functions of the aforementioned MM-associated oncogenes have been investigated. Here, we discuss the metabolic consequences of recurrent chromosomal translocations in MM and provide a framework for the identification of metabolic changes that characterize MM cells.

AKT signaling restrains tumor suppressive functions of FOXO transcription factors and GSK3 kinase in multiple myeloma.

The phosphatidylinositide-3 kinases and the downstream mediator AKT drive survival and proliferation of multiple myeloma (MM) cells. AKT signaling is active in MM and has pleiotropic effects; however, the key molecular aspects of AKT dependency in MM are not fully clear. Among the various downstream AKT targets are the Forkhead box O (FOXO) transcription factors (TFs) and glycogen synthase kinase 3 (GSK3), which are negatively regulated by AKT signaling. Here we show that abrogation of AKT signaling in MM cells provokes cell death and cell cycle arrest, which crucially depends on both FOXO TFs and GSK3. Based on gene expression profiling, we defined a FOXO-repressed gene set that has prognostic significance in a large cohort of patients with MM, indicating that AKT-mediated gene activation is associated with inferior overall survival. We further show that AKT signaling stabilizes the antiapoptotic myeloid cell leukemia 1 (MCL1) protein by inhibiting FOXO- and GSK3-mediated MCL1 turnover. In concordance, abrogation of AKT signaling greatly sensitized MM cells for an MCL1-targeting BH3-mimetic, which is currently in clinical development. Taken together, our results indicate that AKT activity is required to restrain the tumor-suppressive functions of FOXO and GSK3, thereby stabilizing the antiapoptotic protein MCL1 in MM. These novel insights into the role of AKT in MM pathogenesis and MCL1 regulation provide opportunities to improve targeted therapy for patients with MM.

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.