DIS3: The Enigmatic Gene in Multiple Myeloma.

Recent studies have revealed the genetic aberrations involved in the initiation and progression of various cancers, including multiple myeloma (MM), via next-generation sequencing analysis. Notably, DIS3 mutations have been identified in approximately 10% of patients with MM. Moreover, deletions of the long arm of chromosome 13, that includes DIS3, are present in approximately 40% of patients with MM. Regardless of the high incidence of DIS3 mutations and deletions, their contribution to the pathogenesis of MM has not yet been determined. Herein, we summarize the molecular and physiological functions of DIS3, focusing on hematopoiesis, and discuss the characteristics and potential roles of DIS3 mutations in MM. Recent findings highlight the essential roles of DIS3 in RNA homeostasis and normal hematopoiesis and suggest that the reduced activity of DIS3 may be involved in myelomagenesis by increasing genome instability.

Water Droplet-in-Oil Digestion Method for Single-Cell Proteomics.

Recent advances in single-cell proteomics highlight the promise of sensitive analyses in limited cell populations. However, technical challenges remain for sample recovery, throughput, and versatility. Here, we first report a water droplet-in-oil digestion (WinO) method based on carboxyl-coated beads and phase transfer surfactants for proteomic analysis using limited sample amounts. This method was developed to minimize the contact area between the sample solution and the container to reduce the loss of proteins and peptides by adsorption. This method increased protein and peptide recovery 10-fold. The proteome profiles obtained from 100 cells using the WinO method highly correlated with those from 10,000 cells using the in-solution digestion method. We successfully applied the WinO method to single-cell proteomics and quantified 462 proteins. Using the WinO method, samples can be easily prepared in a multi-well plate, making it a widely applicable and suitable method for single-cell proteomics.

The JAK-STAT pathway regulates CD38 on myeloma cells in the bone marrow microenvironment: therapeutic implications.

Anti-CD38 monoclonal antibody (MoAb) treatments including daratumumab (DARA) are effective therapies for both newly diagnosed and relapsed multiple myeloma (MM). In this study, we examined the soluble factors that modulate CD38 expression and are associated with sensitivity to DARA-mediated antibody-dependent cellular cytotoxicity (ADCC) in the bone marrow (BM) microenvironment. Importantly, primary BM stromal cell (BMSC) culture supernatant (BMSC-sup) and interleukin-6 (IL-6) downregulated CD38 expression and reduced DARA-mediated ADCC. Both cytokine profiling of the BMSC-sup and genome-scale clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) knockout screening in MM cell lines identified and validated the JAK-STAT3 signaling pathway mediating CD38 downregulation, whereas the JAK-STAT1 pathway mediated CD38 upregulation. STAT3 knockdown abrogated BMSC-sup- and IL-6-induced CD38 downregulation on MM cell lines. We also confirmed that STAT3 and CD38 is negatively correlated in primary MM cells. To assess potential clinical relevance, pharmacological inhibition of the JAK-STAT pathway on BMSC-sup-induced CD38 downregulation was further examined. JAK inhibitor ruxolitinib inhibited STAT3 phosphorylation in MM cell lines, upregulated CD38 expression in MM cell lines and primary patient MM cells, and augmented DARA-mediated ADCC against MM cell lines. Taken together, our results suggest that CD38 expression on MM cells in the BM microenvironment is regulated by both STAT1 (positively) and STAT3 (negatively), and that inhibition of the JAK-STAT3 pathway represents a novel therapeutic option to enhance CD38 expression and anti-CD38 MoAb-mediated MM cytotoxicity.

Downregulation of ERG and FLI1 expression in endothelial cells triggers endothelial-to-mesenchymal transition.

Endothelial cell (EC) plasticity in pathological settings has recently been recognized as a driver of disease progression. Endothelial-to-mesenchymal transition (EndMT), in which ECs acquire mesenchymal properties, has been described for a wide range of pathologies, including cancer. However, the mechanism regulating EndMT in the tumor microenvironment and the contribution of EndMT in tumor progression are not fully understood. Here, we found that combined knockdown of two ETS family transcription factors, ERG and FLI1, induces EndMT coupled with dynamic epigenetic changes in ECs. Genome-wide analyses revealed that ERG and FLI1 are critical transcriptional activators for EC-specific genes, among which microRNA-126 partially contributes to blocking the induction of EndMT. Moreover, we demonstrated that ERG and FLI1 expression is downregulated in ECs within tumors by soluble factors enriched in the tumor microenvironment. These data provide new insight into the mechanism of EndMT, functions of ERG and FLI1 in ECs, and EC behavior in pathological conditions.

p53-related protein kinase confers poor prognosis and represents a novel therapeutic target in multiple myeloma.

p53-related protein kinase (TP53RK, also known as PRPK) is an upstream kinase that phosphorylates (serine residue Ser15) and mediates p53 activity. Here we show that TP53RK confers poor prognosis in multiple myeloma (MM) patients, and, conversely, that TP53RK knockdown inhibits p53 phosphorylation and triggers MM cell apoptosis, associated with downregulation of c-Myc and E2F-1-mediated upregulation of pro-apoptotic Bim. We further demonstrate that TP53RK downregulation also triggers growth inhibition in p53-deficient and p53-mutant MM cell lines and identify novel downstream targets of TP53RK including ribonucleotide reductase-1, telomerase reverse transcriptase, and cyclin-dependent kinase inhibitor 2C. Our previous studies showed that immunomodulatory drugs (IMiDs) downregulate p21 and trigger apoptosis in wild-type-p53 MM.1S cells, Importantly, we demonstrate by pull-down, nuclear magnetic resonance spectroscopy, differential scanning fluorimetry, and isothermal titration calorimetry that IMiDs bind and inhibit TP53RK, with biologic sequelae similar to TP53RK knockdown. Our studies therefore demonstrate that either genetic or pharmacological inhibition of TP53RK triggers MM cell apoptosis via both p53-Myc axis-dependent and axis-independent pathways, validating TP53RK as a novel therapeutic target in patients with poor-prognosis MM.

Discovery of selective small-molecule HDAC6 inhibitor for overcoming proteasome inhibitor resistance in multiple myeloma.

Multiple myeloma (MM) has proven clinically susceptible to modulation of pathways of protein homeostasis. Blockade of proteasomal degradation of polyubiquitinated misfolded proteins by the proteasome inhibitor bortezomib (BTZ) achieves responses and prolongs survival in MM, but long-term treatment with BTZ leads to drug-resistant relapse in most patients. In a proof-of-concept study, we previously demonstrated that blocking aggresomal breakdown of polyubiquitinated misfolded proteins with the histone deacetylase 6 (HDAC6) inhibitor tubacin enhances BTZ-induced cytotoxicity in MM cells in vitro. However, these foundational studies were limited by the pharmacologic liabilities of tubacin as a chemical probe with only in vitro utility. Emerging from a focused library synthesis, a potent, selective, and bioavailable HDAC6 inhibitor, WT161, was created to study the mechanism of action of HDAC6 inhibition in MM alone and in combination with BTZ. WT161 in combination with BTZ triggers significant accumulation of polyubiquitinated proteins and cell stress, followed by caspase activation and apoptosis. More importantly, this combination treatment was effective in BTZ-resistant cells and in the presence of bone marrow stromal cells, which have been shown to mediate MM cell drug resistance. The activity of WT161 was confirmed in our human MM cell xenograft mouse model and established the framework for clinical trials of the combination treatment to improve patient outcomes in MM.

Evidence for a role of the histone deacetylase SIRT6 in DNA damage response of multiple myeloma cells.

Multiple myeloma (MM) is characterized by a highly unstable genome, with aneuploidy observed in nearly all patients. The mechanism causing this karyotypic instability is largely unknown, but recent observations have correlated these abnormalities with dysfunctional DNA damage response. Here, we show that the NAD(+)-dependent deacetylase SIRT6 is highly expressed in MM cells, as an adaptive response to genomic stability, and that high SIRT6 levels are associated with adverse prognosis. Mechanistically, SIRT6 interacts with the transcription factor ELK1 and with the ERK signaling-related gene. By binding to their promoters and deacetylating H3K9 at these sites, SIRT6 downregulates the expression of mitogen-activated protein kinase (MAPK) pathway genes, MAPK signaling, and proliferation. In addition, inactivation of ERK2/p90RSK signaling triggered by high SIRT6 levels increases DNA repair via Chk1 and confers resistance to DNA damage. Using genetic and biochemical studies in vitro and in human MM xenograft models, we show that SIRT6 depletion both enhances proliferation and confers sensitization to DNA-damaging agents. Our findings therefore provide insights into the functional interplay between SIRT6 and DNA repair mechanisms, with implications for both tumorigenesis and the treatment of MM.

Blockade of XBP1 splicing by inhibition of IRE1α is a promising therapeutic option in multiple myeloma.

Multiple myeloma (MM) cells are characterized by high protein synthesis resulting in chronic endoplasmic reticulum (ER) stress, which is adaptively managed by the unfolded protein response. Inositol-requiring enzyme 1α (IRE1α) is activated to splice X-box binding protein 1 (XBP1) mRNA, thereby increasing XBP1s protein, which in turn regulates genes responsible for protein folding and degradation during the unfolded protein response. In this study, we examined whether IRE1α-XBP1 pathway is a potential therapeutic target in MM using a small-molecule IRE1α endoribonuclease domain inhibitor MKC-3946. MKC-3946 triggered modest growth inhibition in MM cell lines, without toxicity in normal mononuclear cells. Importantly, it significantly enhanced cytotoxicity induced by bortezomib or 17-AAG, even in the presence of bone marrow stromal cells or exogenous IL-6. Both bortezomib and 17-AAG induced ER stress, evidenced by induction of XBP1s, which was blocked by MKC-3946. Apoptosis induced by these agents was enhanced by MKC-3946, associated with increased CHOP. Finally, MKC-3946 inhibited XBP1 splicing in a model of ER stress in vivo, associated with significant growth inhibition of MM cells. Taken together, our results demonstrate that blockade of XBP1 splicing by inhibition of IRE1α endoribonuclease domain is a potential therapeutic option in MM.

FOXO1 stimulates tip cell-enriched gene expression in endothelial cells.

Forkhead box O (FOXO) family proteins are expressed in various cells, and play crucial roles in cellular metabolism, apoptosis, and aging. FOXO1-null mice exhibit embryonic lethality due to impaired endothelial cell (EC) maturation and vascular remodeling. However, FOXO1-mediated genome-wide regulation in ECs remains unclear. Here, we demonstrate that VEGF dynamically regulates FOXO1 cytosol-nucleus translocation. FOXO1 re-localizes to the nucleus via PP2A phosphatase. RNA-seq combined with FOXO1 overexpression/knockdown in ECs demonstrated that FOXO1 governs the VEGF-responsive tip cell-enriched genes, and further inhibits DLL4-NOTCH signaling. Endogenous FOXO1 ChIP-seq revealed that FOXO1 binds to the EC-unique tip-enriched genes with co-enrichment of EC master regulators, and the condensed chromatin region as a pioneer factor. We identified new promoter/enhancer regions of the VEGF-responsive tip cell genes regulated by FOXO1: ESM1 and ANGPT2. This is the first study to identify cell type-specific FOXO1 functions, including VEGF-mediated tip cell definition in primary cultured ECs.

Novel antimyeloma therapeutic option with inhibition of the HDAC1-IRF4 axis and PIM kinase.

Multiple myeloma (MM) preferentially expands and acquires drug resistance in the bone marrow (BM). We herein examined the role of histone deacetylase 1 (HDAC1) in the constitutive activation of the master transcription factor IRF4 and the prosurvival mediator PIM2 kinase in MM cells. The knockdown or inhibition of HDAC1 by the class I HDAC inhibitor MS-275 reduced the basal expression of IRF4 and PIM2 in MM cells. Mechanistically, the inhibition of HDAC1 decreased IRF4 transcription through histone hyperacetylation and inhibiting the recruitment of RNA polymerase II at the IRF4 locus, thereby reducing IRF4-targeting genes, including PIM2. In addition to the transcriptional regulation of PIM2 by the HDAC1-IRF4 axis, PIM2 was markedly upregulated by external stimuli from BM stromal cells and interleukin-6 (IL-6). Upregulated PIM2 contributed to the attenuation of the cytotoxic effects of MS-275. Class I HDAC and PIM kinase inhibitors cooperatively suppressed MM cell growth in the presence of IL-6 and in vivo. Therefore, the present results demonstrate the potential of the simultaneous targeting of the intrinsic HDAC1-IRF4 axis plus externally activated PIM2 as an efficient therapeutic option for MM fostered in the BM.

Diverse Functions of KDM5 in Cancer: Transcriptional Repressor or Activator?

Epigenetic modifications are crucial for chromatin remodeling and transcriptional regulation. Post-translational modifications of histones are epigenetic processes that are fine-tuned by writer and eraser enzymes, and the disorganization of these enzymes alters the cellular state, resulting in human diseases. The KDM5 family is an enzymatic family that removes di- and tri-methyl groups (me2 and me3) from lysine 4 of histone H3 (H3K4), and its dysregulation has been implicated in cancer. Although H3K4me3 is an active chromatin marker, KDM5 proteins serve as not only transcriptional repressors but also transcriptional activators in a demethylase-dependent or -independent manner in different contexts. Notably, KDM5 proteins regulate the H3K4 methylation cycle required for active transcription. Here, we review the recent findings regarding the mechanisms of transcriptional regulation mediated by KDM5 in various contexts, with a focus on cancer, and further shed light on the potential of targeting KDM5 for cancer therapy.

Favorable outcome of unrelated cord blood transplantation for Philadelphia chromosome-positive acute lymphoblastic leukemia.

Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph(+)ALL) is one of the highest-risk ALL groups. Whenever possible, patients with Ph(+)ALL should undergo allogeneic hematopoietic stem cell transplantation (HSCT) after induction of remission. Although unrelated cord blood transplantation (CBT) has become a common treatment in adult patients who lack a sibling donor, data on the efficacy of CBT for Ph(+)ALL are limited. We analyzed the clinical outcomes of 20 Ph(+)ALL patients who underwent CBT (n = 8) or unrelated bone marrow transplantation (BMT) (n = 12). The median age was 41 years (range, 17-55 years). All but one of the patients were treated with an imatinib-based regimen before HSCT, and 19 patients were in first complete remission (CR) and 1 patient was in second CR at the time of HSCT. Seventeen patients received a myeloablative conditioning regimen containing 12 Gy of total-body irradiation, and 3 received a reduced-intensity conditioning regimen. After a median of 26 months of follow-up, estimated 3-year overall and leukemia-free survival rates were 100% and 85%, respectively, after CBT, and 49% and 38%, respectively, after unrelated BMT. The CBT group had significantly better overall survival than the BMT group (P = .02). Although BCR-ABL transcript was detected in 4 of 8 CBT patients at transplantation, 7 patients remained in molecular CR. Our findings suggest that CBT may be a viable option as postinduction therapy for Ph(+)ALL in patients lacking a sibling donor.

Inflammation-driven senescence-associated secretory phenotype in cancer-associated fibroblasts enhances peritoneal dissemination.

In the tumor microenvironment, senescent non-malignant cells, including cancer-associated fibroblasts (CAFs), exhibit a secretory profile under stress conditions; this senescence-associated secretory phenotype (SASP) leads to cancer progression and chemoresistance. However, the role of senescent CAFs in metastatic lesions and the molecular mechanism of inflammation-related SASP induction are not well understood. We show that pro-inflammatory cytokine-driven EZH2 downregulation maintains the SASP by demethylating H3K27me3 marks in CAFs and enhances peritoneal tumor formation of gastric cancer (GC) through JAK/STAT3 signaling in a mouse model. A JAK/STAT3 inhibitor blocks the increase in GC cell viability induced by senescent CAFs and peritoneal tumor formation. Single-cell mass cytometry revealed that fibroblasts exist in the ascites of GC patients with peritoneal dissemination, and the fibroblast population shows p16 expression and SASP factors at high levels. These findings provide insights into the inflammation-related SASP maintenance by histone modification and the role of senescent CAFs in GC peritoneal dissemination.

Lysine Demethylase 5A is Required for MYC Driven Transcription in Multiple Myeloma.

Lysine demethylase 5A (KDM5A) is a negative regulator of histone H3K4 trimethylation, a histone mark associated with activate gene transcription. We identify that KDM5A interacts with the P-TEFb complex and cooperates with MYC to control MYC targeted genes in multiple myeloma (MM) cells. We develop a cell-permeable and selective KDM5 inhibitor, JQKD82, that increases histone H3K4me3 but paradoxically inhibits downstream MYC-driven transcriptional output in vitro and in vivo. Using genetic ablation together with our inhibitor, we establish that KDM5A supports MYC target gene transcription independent of MYC itself, by supporting TFIIH (CDK7)- and P-TEFb (CDK9)-mediated phosphorylation of RNAPII. These data identify KDM5A as a unique vulnerability in MM functioning through regulation of MYC-target gene transcription, and establish JQKD82 as a tool compound to block KDM5A function as a potential therapeutic strategy for MM.

Cooperative interaction between hepatocyte nuclear factor 4 alpha and GATA transcription factors regulates ATP-binding cassette sterol transporters ABCG5 and ABCG8.

Cholesterol homeostasis is maintained by coordinate regulation of cholesterol synthesis and its conversion to bile acids in the liver. The excretion of cholesterol from liver and intestine is regulated by ATP-binding cassette half-transporters ABCG5 and ABCG8. The genes for these two proteins are closely linked and divergently transcribed from a common intergenic promoter region. Here, we identified a binding site for hepatocyte nuclear factor 4alpha (HNF4alpha) in the ABCG5/ABCG8 intergenic promoter, through which HNF4alpha strongly activated the expression of a reporter gene in both directions. The HNF4alpha-responsive element is flanked by two conserved GATA boxes that were also required for stimulation by HNF4alpha. GATA4 and GATA6 bind to the GATA boxes, coexpression of GATA4 and HNF4alpha leads to a striking synergistic activation of both the ABCG5 and the ABCG8 promoters, and binding sites for HNF4alpha and GATA were essential for maximal synergism. We also show that HNF4alpha, GATA4, and GATA6 colocalize in the nuclei of HepG2 cells and that a physical interaction between HNF4alpha and GATA4 is critical for the synergistic response. This is the first demonstration that HNF4alpha acts synergistically with GATA factors to activate gene expression in a bidirectional fashion.

The biological significance of histone modifiers in multiple myeloma: clinical applications.

Multiple myeloma (MM) is a clonal plasma cell disorder that is characterized by a variety of genetic alterations. Recent studies have highlighted not only the importance of these genetic events but also epigenetic aberrations including DNA methylation, histone modifications, and non-coding RNAs in the biology of MM. Post-translational modifications of histone, such as methylation and acetylation, contribute to chromatin dynamics, and are modulated by histone modifying enzymes, and dysregulation of these enzymes is implicated in the pathogenesis of cancers, including MM. Histone modifiers also have non-histone substrates and enzymatically independent roles, which are also involved in tumorigenesis. Here we review and provide comprehensive insight into the biologic significance of histone methyl- and acetyl-modifiers in MM, and further provide an overview of the clinical applications of histone modifier inhibitors, especially histone deacetylase inhibitors. These findings underline the emerging roles of histone modifiers in the pathogenesis of MM, and further highlight the possibility of novel epigenetic therapies in MM.

Ribonucleotide Reductase Catalytic Subunit M1 (RRM1) as a Novel Therapeutic Target in Multiple Myeloma.

Purpose: To investigate the biological and clinical significance of ribonucleotide reductase (RR) in multiple myeloma.Experimental Design: We assessed the impact of RR expression on patient outcome in multiple myeloma. We then characterized the effect of genetic and pharmacologic inhibition of ribonucleotide reductase catalytic subunit M1 (RRM1) on multiple myeloma growth and survival using siRNA and clofarabine, respectively, in both in vitro and in vivo mouse xenograft models.Results: Newly diagnosed multiple myeloma patients with higher RRM1 expression have shortened survival. Knockdown of RRM1 triggered significant growth inhibition and apoptosis in multiple myeloma cells, even in the context of the bone marrow microenvironment. Gene expression profiling showed upregulation of DNA damage response genes and p53-regulated genes after RRM1 knockdown. Immunoblot and qRT-PCR analysis confirmed that γ-H2A.X, ATM, ATR, Chk1, Chk2, RAD51, 53BP1, BRCA1, and BRCA2 were upregulated/activated. Moreover, immunoblots showed that p53, p21, Noxa, and Puma were activated in p53 wild-type multiple myeloma cells. Clofarabine, a purine nucleoside analogue that inhibits RRM1, induced growth arrest and apoptosis in p53 wild-type cell lines. Although clofarabine did not induce cell death in p53-mutant cells, it did trigger synergistic toxicity in combination with DNA-damaging agent melphalan. Finally, we demonstrated that tumor growth of RRM1-knockdown multiple myeloma cells was significantly reduced in a murine human multiple myeloma cell xenograft model.Conclusions: Our results therefore demonstrate that RRM1 is a novel therapeutic target in multiple myeloma in the preclinical setting and provide the basis for clinical evaluation of RRM1 inhibitor, alone or in combination with DNA-damaging agents, to improve patient outcome in multiple myeloma. Clin Cancer Res; 23(17); 5225-37. ©2017 AACR.