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.

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.

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.

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.

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.

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.

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.

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.

The KDM3A-KLF2-IRF4 axis maintains myeloma cell survival.

KDM3A is implicated in tumorigenesis; however, its biological role in multiple myeloma (MM) has not been elucidated. Here we identify KDM3A-KLF2-IRF4 axis dependence in MM. Knockdown of KDM3A is toxic to MM cells in vitro and in vivo. KDM3A maintains expression of KLF2 and IRF4 through H3K9 demethylation, and knockdown of KLF2 triggers apoptosis. Moreover, KLF2 directly activates IRF4 and IRF4 reciprocally upregulates KLF2, forming a positive autoregulatory circuit. The interaction of MM cells with bone marrow milieu mediates survival of MM cells. Importantly, silencing of KDM3A, KLF2 or IRF4 both decreases MM cell adhesion to bone marrow stromal cells and reduces MM cell homing to the bone marrow, in association with decreased ITGB7 expression in MAF-translocated MM cell lines. Our results indicate that the KDM3A-KLF2-IRF4 pathway plays an essential role in MM cell survival and homing to the bone marrow, and therefore represents a therapeutic target.

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.

Combination of a Selective HSP90α/ß Inhibitor and a RAS-RAF-MEK-ERK Signaling Pathway Inhibitor Triggers Synergistic Cytotoxicity in Multiple Myeloma Cells.

Heat shock protein (HSP)90 inhibitors have shown significant anti-tumor activities in preclinical settings in both solid and hematological tumors. We previously reported that the novel, orally available HSP90α/ß inhibitor TAS-116 shows significant anti-MM activities. In this study, we further examined the combination effect of TAS-116 with a RAS-RAF-MEK-ERK signaling pathway inhibitor in RAS- or BRAF-mutated MM cell lines. TAS-116 monotherapy significantly inhibited growth of RAS-mutated MM cell lines and was associated with decreased expression of downstream target proteins of the RAS-RAF-MEK-ERK signaling pathway. Moreover, TAS-116 showed synergistic growth inhibitory effects with the farnesyltransferase inhibitor tipifarnib, the BRAF inhibitor dabrafenib, and the MEK inhibitor selumetinib. Importantly, treatment with these inhibitors paradoxically enhanced p-C-Raf, p-MEK, and p-ERK activity, which was abrogated by TAS-116. TAS-116 also enhanced dabrafenib-induced MM cytotoxicity associated with mitochondrial damage-induced apoptosis, even in the BRAF-mutated U266 MM cell line. This enhanced apoptosis in RAS-mutated MM triggered by combination treatment was observed even in the presence of bone marrow stromal cells. Taken together, our results provide the rationale for novel combination treatment with HSP90α/ß inhibitor and RAS-RAF-MEK-ERK signaling pathway inhibitors to improve outcomes in patients with in RAS- or BRAF-mutated MM.

Lenalidomide Enhances Immune Checkpoint Blockade-Induced Immune Response in Multiple Myeloma.

PURPOSE: PD-1/PD-L1 signaling promotes tumor growth while inhibiting effector cell-mediated antitumor immune responses. Here, we assessed the impact of single and dual blockade of PD-1/PD-L1, alone or in combination with lenalidomide, on accessory and immune cell function as well as multiple myeloma cell growth in the bone marrow (BM) milieu. EXPERIMENTAL DESIGN: Surface expression of PD-1 on immune effector cells, and PD-L1 expression on CD138(+) multiple myeloma cells and myeloid-derived suppressor cells (MDSC) were determined in BM from newly diagnosed (ND) multiple myeloma and relapsed/refractory (RR) multiple myeloma versus healthy donor (HD). We defined the impact of single and dual blockade of PD-1/PD-L1, alone and with lenalidomide, on autologous anti-multiple myeloma immune response and tumor cell growth. RESULTS: Both ND and RR patient multiple myeloma cells have increased PD-L1 mRNA and surface expression compared with HD. There is also a significant increase in PD-1 expression on effector cells in multiple myeloma. Importantly, PD-1/PD-L1 blockade abrogates BM stromal cell (BMSC)-induced multiple myeloma growth, and combined blockade of PD-1/PD-L1 with lenalidomide further inhibits BMSC-induced tumor growth. These effects are associated with induction of intracellular expression of IFNγ and granzyme B in effector cells. Importantly, PD-L1 expression in multiple myeloma is higher on MDSC than on antigen-presenting cells, and PD-1/PD-L1 blockade inhibits MDSC-mediated multiple myeloma growth. Finally, lenalidomide with PD-1/PD-L1 blockade inhibits MDSC-mediated immune suppression. CONCLUSIONS: Our data therefore demonstrate that checkpoint signaling plays an important role in providing the tumor-promoting, immune-suppressive microenvironment in multiple myeloma, and that PD-1/PD-L1 blockade induces anti-multiple myeloma immune response that can be enhanced by lenalidomide, providing the framework for clinical evaluation of combination therapy.

Delineating the mTOR kinase pathway using a dual TORC1/2 inhibitor, AZD8055, in multiple myeloma.

Despite promising preclinical results with mTOR kinase inhibitors in multiple myeloma, resistance to these drugs may arise via feedback activation loops. This concern is especially true for insulin-like growth factor 1 receptor (IGF1R), because IGF1R signaling is downregulated by multiple AKT and mTOR feedback mechanisms. We have tested this hypothesis in multiple myeloma using the novel selective mTOR kinase inhibitor AZD8055. We evaluated p-mTOR S(2481) as the readout for mTORC2/Akt activity in multiple myeloma cells in the context of mTOR inhibition via AZD8055 or rapamycin. We next validated AZD8055 inhibition of mTORC1 and mTORC2 functions in multiple myeloma cells alone or in culture with bone marrow stroma cells and growth factors. Unlike rapamycin, AZD8055 resulted in apoptosis of multiple myeloma cells. AZD8055 treatment, however, induced upregulation of IGF1R phosphorylation in p-Akt S(473)-expressing multiple myeloma cell lines. Furthermore, exposure of AZD8055-treated cells to IGF1 induced p-Akt S(473) and rescued multiple myeloma cells from apoptosis despite mTOR kinase inhibition and TORC2/Akt blockage. The addition of blocking IGF1R antibody resulted in reversing this effect and increased AZD8055-induced apoptosis. Our study suggests that combination treatment with AZD8055 and IGF1R-blocking agents is a promising strategy in multiple myeloma with potential IGF1R/Akt signaling-mediated survival.

Selective and potent Akt inhibition triggers anti-myeloma activities and enhances fatal endoplasmic reticulum stress induced by proteasome inhibition.

The PI3K/Akt pathway plays a crucial role in the pathogenesis of multiple myeloma (MM) in the bone marrow (BM) milieu. However, efficacy of selective and potent Akt inhibition has not yet been fully elucidated. In this study, we, therefore, examined the biologic impact of selective and potent Akt inhibition by a novel allosteric inhibitor TAS-117. TAS-117 induced significant growth inhibition, associated with downregulation of phosphorylated Akt (p-Akt), selectively in MM cell lines with high baseline p-Akt. Cytotoxicity of TAS-117 was also observed in patient MM cells, but not in normal peripheral blood mononuclear cells. Importantly, TAS-117 induced significant cytotoxicity in MM cells even in the presence of BM stromal cells, associated with inhibition of IL6 secretion. Oral administration of TAS-117 significantly inhibited human MM cell growth in murine xenograft models. TAS-117 triggered apoptosis and autophagy, as well as induction of endoplasmic reticulum (ER) stress response with minimal expression of C/EBP homologous protein (CHOP), a fatal ER stress marker. Importantly, TAS-117 enhanced bortezomib-induced cytotoxicity, associated with increased CHOP and PARP cleavage and blockade of bortezomib-induced p-Akt, suggesting that TAS-117 augments bortezomib-induced ER stress and apoptotic signaling. Carfilzomib-induced cytotoxicity was similarly enhanced by TAS-117. Importantly, TAS-117 enhanced bortezomib-induced cytotoxicity in vivo, associated with prolonged host survival. Our results show that selective and potent Akt inhibition by TAS-117 triggers anti-MM activities in vitro and in vivo, as well as enhances cytotoxicity of proteasome inhibition, providing the preclinical framework for clinical evaluation of selective Akt inhibitors, alone and in combination with proteasome inhibitors in MM.