Promiscuous Structural Variants Drive Myeloma Initiation and Progression.

A comprehensive genomic analysis of structural variants in multiple myeloma in this issue highlights the key role of these events, involving primarily the immunoglobulin heavy chain locus in disease initiation and the MYC locus in disease progression. However, the current study reveals the large number of genomic hotspots, oncogenes, tumor suppressor genes, and recombination mechanisms that contribute to multiple myeloma heterogeneity. See related article by Rustad et al., p. 258.

Cooperative Targets of Combined mTOR/HDAC Inhibition Promote MYC Degradation.

Cancer treatments often require combinations of molecularly targeted agents to be effective. mTORi (rapamycin) and HDACi (MS-275/entinostat) inhibitors have been shown to be effective in limiting tumor growth, and here we define part of the cooperative action of this drug combination. More than 60 human cancer cell lines responded synergistically (CI<1) when treated with this drug combination compared with single agents. In addition, a breast cancer patient-derived xenograft, and a BCL-XL plasmacytoma mouse model both showed enhanced responses to the combination compared with single agents. Mice bearing plasma cell tumors lived an average of 70 days longer on combination treatment compared with single agents. A set of 37 genes cooperatively affected (34 downregulated; 3 upregulated) by the combination responded pharmacodynamically in human myeloma cell lines, xenografts, and a P493 model, and were both enriched in tumors, and correlated with prognostic markers in myeloma patient datasets. Genes downregulated by the combination were overexpressed in several untreated cancers (breast, lung, colon, sarcoma, head and neck, myeloma) compared with normal tissues. The MYC/E2F axis, identified by upstream regulator analyses and validated by immunoblots, was significantly inhibited by the drug combination in several myeloma cell lines. Furthermore, 88% of the 34 genes downregulated have MYC-binding sites in their promoters, and the drug combination cooperatively reduced MYC half-life by 55% and increased degradation. Cells with MYC mutations were refractory to the combination. Thus, integrative approaches to understand drug synergy identified a clinically actionable strategy to inhibit MYC/E2F activity and tumor cell growth in vivoMol Cancer Ther; 16(9); 2008-21. ©2017 AACR.

VOLIN and KJON-Two novel hyperdiploid myeloma cell lines.

Multiple myeloma can be divided into two distinct genetic subgroups: hyperdiploid (HRD) or nonhyperdiploid (NHRD) myeloma. Myeloma cell lines are important tools to study myeloma cell biology and are commonly used for preclinical screening and testing of new drugs. With few exceptions human myeloma cell lines are derived from NHRD patients, even though about half of the patients have HRD myeloma. Thus, there is a need for cell lines of HRD origin to enable more representative preclinical studies. Here, we present two novel myeloma cell lines, VOLIN and KJON. Both of them were derived from patients with HRD disease and shared the same genotype as their corresponding primary tumors. The cell lines' chromosomal content, genetic aberrations, gene expression, immunophenotype as well as some of their growth characteristics are described. Neither of the cell lines was found to harbor immunoglobulin heavy chain translocations. The VOLIN cell line was established from a bone marrow aspirate and KJON from peripheral blood. We propose that these unique cell lines may be used as tools to increase our understanding of myeloma cell biology. © 2016 Wiley Periodicals, Inc.

Transcriptional repression by the HDAC4-RelB-p52 complex regulates multiple myeloma survival and growth.

Although transcriptional activation by NF-κB is well appreciated, physiological importance of transcriptional repression by NF-κB in cancer has remained elusive. Here we show that an HDAC4-RelB-p52 complex maintains repressive chromatin around proapoptotic genes Bim and BMF and regulates multiple myeloma (MM) survival and growth. Disruption of RelB-HDAC4 complex by a HDAC4-mimetic polypeptide blocks MM growth. RelB-p52 also represses BMF translation by regulating miR-221 expression. While the NIK-dependent activation of RelB-p52 in MM has been reported, we show that regardless of the activation status of NIK and the oncogenic events that cause plasma cell malignancy, several genetically diverse MM cells including Bortezomib-resistant MM cells are addicted to RelB-p52 for survival. Importantly, RelB is constitutively phosphorylated in MM and ERK1 is a RelB kinase. Phospho-RelB remains largely nuclear and is essential for Bim repression. Thus, ERK1-dependent regulation of nuclear RelB is critical for MM survival and explains the NIK-independent role of RelB in MM.

Rescue of Hippo coactivator YAP1 triggers DNA damage-induced apoptosis in hematological cancers.

Oncogene-induced DNA damage elicits genomic instability in epithelial cancer cells, but apoptosis is blocked through inactivation of the tumor suppressor p53. In hematological cancers, the relevance of ongoing DNA damage and the mechanisms by which apoptosis is suppressed are largely unknown. We found pervasive DNA damage in hematologic malignancies, including multiple myeloma, lymphoma and leukemia, which leads to activation of a p53-independent, proapoptotic network centered on nuclear relocalization of ABL1 kinase. Although nuclear ABL1 triggers cell death through its interaction with the Hippo pathway coactivator YAP1 in normal cells, we show that low YAP1 levels prevent nuclear ABL1-induced apoptosis in these hematologic malignancies. YAP1 is under the control of a serine-threonine kinase, STK4. Notably, genetic inactivation of STK4 restores YAP1 levels, triggering cell death in vitro and in vivo. Our data therefore identify a new synthetic-lethal strategy to selectively target cancer cells presenting with endogenous DNA damage and low YAP1 levels.

Repair of DNA double-strand breaks by templated nucleotide sequence insertions derived from distant regions of the genome.

We used the I-SceI endonuclease to produce DNA double-strand breaks (DSBs) and observed that a fraction of these DSBs were repaired by insertion of sequences, which we termed "templated sequence insertions" (TSIs), derived from distant regions of the genome. These TSIs were derived from genic, retrotransposon, or telomere sequences and were not deleted from the donor site in the genome, leading to the hypothesis that they were derived from reverse-transcribed RNA. Cotransfection of RNA and an I-SceI expression vector demonstrated insertion of RNA-derived sequences at the DNA-DSB site, and TSIs were suppressed by reverse-transcriptase inhibitors. Both observations support the hypothesis that TSIs were derived from RNA templates. In addition, similar insertions were detected at sites of DNA DSBs induced by transcription activator-like effector nuclease proteins. Whole-genome sequencing of myeloma cell lines revealed additional TSIs, demonstrating that repair of DNA DSBs via insertion was not restricted to experimentally produced DNA DSBs. Analysis of publicly available databases revealed that many of these TSIs are polymorphic in the human genome. Taken together, these results indicate that insertional events should be considered as alternatives to gross chromosomal rearrangements in the interpretation of whole-genome sequence data and that this mutagenic form of DNA repair may play a role in genetic disease, exon shuffling, and mammalian evolution.

Complex IGH rearrangements in multiple myeloma: Frequent detection discrepancies among three different probe sets.

Primary IGH translocations involving seven recurrent partner loci and oncogenes are present in about 40% of multiple myeloma tumors. Secondary IGH rearrangements, which occur in a smaller fraction of tumors, usually are complex structures, including insertions or translocations that can involve three chromosomes, and often with involvement of MYC. The main approach to detect IGH rearrangements is interphase-but sometimes metaphase-FISH strategies that use a telomeric variable region probe and a centromeric constant region/ Eα enhancer or 3' flanking probe to detect a separation of these two probes, or a fusion of these probes with probes located at nonrandom partner sites in the genome. We analyzed 18 myeloma cell lines for detection discrepancies among Vysis, Cytocell, and in-house IGH probe sets that hybridize with differing sequences in the IGH locus. There were no detection discrepancies for the three telomeric IGH probes, or for unrearranged IGH loci or primary IGH translocations using the centromeric IGH probes. However, the majority of complex IGH rearrangements had detection discrepancies among the three centromeric IGH probes.

Promiscuous MYC locus rearrangements hijack enhancers but mostly super-enhancers to dysregulate MYC expression in multiple myeloma.

MYC locus rearrangements-often complex combinations of translocations, insertions, deletions and inversions-in multiple myeloma (MM) were thought to be a late progression event, which often did not involve immunoglobulin genes. Yet, germinal center activation of MYC expression has been reported to cause progression to MM in an MGUS (monoclonal gammopathy of undetermined significance)-prone mouse strain. Although previously detected in 16% of MM, we find MYC rearrangements in nearly 50% of MM, including smoldering MM, and they are heterogeneous in some cases. Rearrangements reposition MYC near a limited number of genes associated with conventional enhancers, but mostly with super-enhancers (e.g., IGH, IGL, IGK, NSMCE2, TXNDC5, FAM46C, FOXO3, IGJ, PRDM1). MYC rearrangements are associated with a significant increase of MYC expression that is monoallelic, but MM tumors lacking a rearrangement have biallelic MYC expression at significantly higher levels than in MGUS. We also have shown that germinal center activation of MYC does not cause MM in a mouse strain that rarely develops spontaneous MGUS. It appears that increased MYC expression at the MGUS/MM transition usually is biallelic, but sometimes can be monoallelic if there is an MYC rearrangement. Our data suggest that MYC rearrangements, regardless of when they occur during MM pathogenesis, provide one event that contributes to tumor autonomy.

TORC1 and class I HDAC inhibitors synergize to suppress mature B cell neoplasms.

Enhanced proliferative signaling and loss of cell cycle regulation are essential for cancer progression. Increased mitogenic signaling through activation of the mTOR pathway, coupled with deregulation of the Cyclin D/retinoblastoma (Rb) pathway is a common feature of lymphoid malignancies, including plasmacytoma (PCT), multiple myeloma (MM), Burkitt's lymphoma (BL), and mantle cell lymphoma (MCL). Here we evaluate the synergy of pharmacologically affecting both of these critical pathways using the mTOR inhibitor sirolimus and the histone deacetylase inhibitor entinostat. A dose-matrix screening approach found this combination to be highly active and synergistic in a panel of genetically diverse human MM cell lines. Synergy and activity was observed in mouse PCT and human BL and MCL cell lines tested in vitro, as well as in freshly isolated primary MM patient samples tested ex vivo. This combination had minimal effects on healthy donor cells and retained activity when tested in a co-culture system simulating the protective interaction of cancer cells with the tumor microenvironment. Combining sirolimus with entinostat enhanced cell cycle arrest and apoptosis. At the molecular level, entinostat increased the expression of cell cycle negative regulators including CDKN1A (p21) and CDKN2A (p16), while the combination decreased critical growth and survival effectors including Cyclin D, BCL-XL, BIRC5, and activated MAPK.

Molecular pathogenesis of multiple myeloma and its premalignant precursor.

Multiple myeloma is a monoclonal tumor of plasma cells, and its development is preceded by a premalignant tumor with which it shares genetic abnormalities, including universal dysregulation of the cyclin D/retinoblastoma (cyclin D/RB) pathway. A complex interaction with the BM microenvironment, characterized by activation of osteoclasts and suppression of osteoblasts, leads to lytic bone disease. Intratumor genetic heterogeneity, which occurs in addition to intertumor heterogeneity, contributes to the rapid emergence of drug resistance in high-risk disease. Despite recent therapeutic advances, which have doubled the median survival time, myeloma continues to be a mostly incurable disease. Here we review the current understanding of myeloma pathogenesis and insight into new therapeutic strategies provided by animal models and genetic screens.

MYC addiction: a potential therapeutic target in MM.

In this issue of Blood, Holien et al report that MYC addiction is responsible for rapid death of myeloma cell lines and primary myeloma tumor cells treated with a specific MYC inhibitor.

Mouse models can predict cancer therapy.

In this issue of Blood, Chesi et al show that their genetically engineered mouse model of multiple myeloma can predict positive or negative activity of drugs previously tested in clinical trials.

Comprehensive identification of somatic mutations in chronic lymphocytic leukemia.

Massively parallel sequencing enables the sequencing of whole genomes, exomes, and transcriptomes from many tumor samples. Thus, it now is possible to comprehensively identify somatic mutations, including single base changes, deletions, insertions, and genomic rearrangements. Early results for hematopoietic tumors show great promise, but many questions remain to be answered.

Pathogenesis of monoclonal gammopathy of undetermined significance and progression to multiple myeloma.

Monoclonal gammopathy of undetermined significance (MGUS), including immunoglobulin light chain only MGUS, is an age-dependent premalignant tumor that is present in about 4% of Caucasian individuals over the age of 50 years. It is comprised of two different kinds of tumors: about 15% lymphoid or lymphoplasmacytoid MGUS and the remainder plasma cell MGUS. Plasma cell MGUS is stable but can sporadically progress to multiple myeloma (MM) at an average rate of about 1% per year. Most, if not all, MM tumors are preceded by plasma cell MGUS, which shares four partially overlapping oncogenic features with MM. It presently is not possible to unequivocally distinguish an MGUS tumor cell from an MM tumor cell. However, two models based on clinical laboratory tests indicate that it is possible to stratify MGUS tumors into groups that have average rates of progression as low as 0.26% per year and as high as 12% per year.

A mechanistic rationale for MEK inhibitor therapy in myeloma based on blockade of MAF oncogene expression.

Modulating aberrant transcription of oncogenes is a relatively unexplored opportunity in cancer therapeutics. In approximately 10% of multiple myelomas, the initiating oncogenic event is translocation of musculoaponeurotic fibrosarcoma oncogene homolog (MAF), a transcriptional activator of key target genes, including cyclinD2. Our prior work showed that MAF is up-regulated in an additional 30% of multiple myeloma cases. The present study describes a common mechanism inducing MAF transcription in both instances. The second mode of MAF transcription occurred in myelomas with multiple myeloma SET domain (MMSET) translocation. MMSET knockdown decreased MAF transcription and cell viability. A small-molecule screen found an inhibitor of mitogen-activated protein kinase kinase (MEK), which activates extracellular signal-regulated kinase (ERK)-MAP kinases, reduced MAF mRNA in cells representing MMSET or MAF subgroups. ERK activates transcription of FOS, part of the AP-1 transcription factor. By chromatin immunoprecipitation, FOS bound the MAF promoter, and MEK inhibition decreased this interaction. MEK inhibition selectively induced apoptosis in MAF-expressing myelomas, and FOS inactivation was similarly toxic. Reexpression of MAF rescued cells from death induced by MMSET depletion, MEK inhibition, or FOS inactivation. The data presented herein demonstrate that the MEK-ERK pathway regulates MAF transcription, providing molecular rationale for clinical evaluation of MEK inhibitors in MAF-expressing myeloma.

Molecular and biologic markers of progression in monoclonal gammopathy of undetermined significance to multiple myeloma.

Multiple myeloma (MM) is a malignant plasma cell dyscrasia localized in the bone marrow. Recent studies have shown that MM is preceded in virtually all cases by a premalignant state called monoclonal gammopathy of undetermined significance (MGUS). This review focuses on non-IgM MGUS and its progression to MM. Although certain clinical markers of MGUS progression have been identified, it currently is not possible to accurately determine individual risk of progression. This review focuses on the various biologic and molecular markers that could be used to determine the risk of MM progression. A better understanding of the pathogenesis will allow us to define the biological high-risk precursor disease and, ultimately, to develop early intervention strategies designed to delay and prevent full-blown MM.

A critical role for the NFkB pathway in multiple myeloma.

NFkB transcription factors play a key role in the survival and proliferation of many kinds of B-cell tumors, including multiple myeloma (MM). It was shown that NFkB activation in MM tumors results mainly from extrinsic signaling by APRIL and BAFF ligands that stimulate receptors on normal plasma cells as well as on pre-malignant monoclonal gammopathy of undetermined significance (MGUS) and MM tumors. However, the mutations that occur during MM progression and that constitutively activate NFkB would be expected to decrease dependence of tumor cells on the bone marrow microenvironment. These mutations can activate the classical or alternative NFkB pathways selectively, but usually both pathways are activated in MM. Significantly, activation of either NFkB pathway leads to a similar response of MM cell lines. This frequent activation of the alternative pathway distinguishes MM from other B-cell tumors, which more frequently have mutations that are predicted to activate only the classical NFkB pathway. Given the strong dependence of MGUS and MM tumors on NFkB pathway activation, inhibition by a combination of targeting extrinsic signaling plus both NFkB pathways appears to be an attractive therapeutic approach in MM tumors.

Advances in understanding monoclonal gammopathy of undetermined significance as a precursor of multiple myeloma.

Monoclonal gammopathy of undetermined significance (MGUS) affects at least 3% of the population above the age of 50 and is the precursor to multiple myeloma (MM), an incurable malignancy of plasma cells. Recent advances in MGUS include: an improved understanding of the pathogenesis of MGUS and its progression to MM, involving molecular events intrinsic to the malignant plasma cell as well as the microenvironment; novel techniques to assess risk for progression to MM using serum-free light-chain analysis and immunophenotyping; and a renewed interest in chemoprevention of MM. In the future, continued improvement in our understanding of MGUS will lead to the development of better biomarkers for prognosis and therapies for chemoprevention of MM.