Mechanisms of constitutive NF-kappaB signaling in multiple myeloma are unknown. An inhibitor of IkappaB kinase beta (IKKbeta) targeting the classical NF-kappaB pathway was lethal to many myeloma cell lines. Several cell lines had elevated expression of NIK due to genomic alterations or protein stabilization, while others had inactivating mutations of TRAF3; both kinds of abnormality triggered the classical and alternative NF-kappaB pathways. A majority of primary myeloma patient samples and cell lines had elevated NF-kappaB target gene expression, often associated with genetic or epigenetic alteration of NIK, TRAF3, CYLD, BIRC2/BIRC3, CD40, NFKB1, or NFKB2. These data demonstrate that addiction to the NF-kappaB pathway is frequent in myeloma and suggest that IKKbeta inhibitors hold promise for the treatment of this disease.

To identify genetic events underlying the genesis and progression of multiple myeloma (MM), we conducted a high-resolution analysis of recurrent copy number alterations (CNAs) and expression profiles in a collection of MM cell lines and outcome-annotated clinical specimens. Attesting to the molecular heterogeneity of MM, unsupervised classification using nonnegative matrix factorization (NMF) designed for array comparative genomic hybridization (aCGH) analysis uncovered distinct genomic subtypes. Additionally, we defined 87 discrete minimal common regions (MCRs) within recurrent and highly focal CNAs. Further integration with expression data generated a refined list of MM gene candidates residing within these MCRs, thereby providing a genomic framework for dissection of disease pathogenesis, improved clinical management, and initiation of targeted drug discovery for specific MM patients.

In multiple myeloma malignant plasma cells expand within the bone marrow. Since this site is well-perfused, a rapid dissemination of “fitter” clones may be anticipated. However, an imbalanced distribution of multiple myeloma is frequently observed in medical imaging. Here, we perform multi-region sequencing, including iliac crest and radiology-guided focal lesion specimens from 51 patients to gain insight into the spatial clonal architecture. We demonstrate spatial genomic heterogeneity in more than 75% of patients, including inactivation of CDKN2C and TP53, and mutations affecting mitogen-activated protein kinase genes. We show that the extent of spatial heterogeneity is positively associated with the size of biopsied focal lesions consistent with regional outgrowth of advanced clones. The results support a model for multiple myeloma progression with clonal sweeps in the early phase and regional evolution in advanced disease. We suggest that multi-region investigations are critical to understanding intra-patient heterogeneity and the evolutionary processes in multiple myeloma.

• Hits in driver genes and bi-allelic events affecting tumor suppressors increase apoptosis resistance and proliferation rate-driving relapse.• Excessive biallelic inactivation of tumor suppressors in highrisk cases highlights the need for TP53-independent therapeutic approaches.To elucidate the mechanisms underlying relapse from chemotherapy in multiple myeloma, we performed a longitudinal study of 33 patients entered into Total Therapy protocols investigating them using gene expression profiling, high-resolution copy number arrays, and whole-exome sequencing. The study illustrates the mechanistic importance of acquired mutations in known myeloma driver genes and the critical nature of biallelic inactivation events affecting tumor suppressor genes, especially TP53, the end result being resistance to apoptosis and increased proliferation rates, which drive relapse by Darwinian-type clonal evolution. The number of copy number aberration changes and biallelic inactivation of tumor suppressor genes was increased in GEP70 high risk, consistent with genomic instability being a key feature of high risk. In conclusion, the study highlights the impact of acquired genetic events, which enhance the evolutionary fitness level of myeloma-propagating cells to survive multiagent chemotherapy and to result in relapse. (Blood. 2016;128(13):1735-1744

Multiple myeloma (MM) is characterized by osteolytic bone lesions (OBL) that arise as a consequence of osteoblast inactivation and osteoclast activation adjacent to tumor foci within bone. Wnt signaling in osteoblasts regulates osteoclastogenesis through the differential activation and inactivation of Receptor Activator of Nuclear factor Kappa B Ligand (RANKL) and osteoprotegerin (OPG), positive and negative regulators of osteoclast differentiation, respectively. We demonstrate here that MM cell-derived DKK1, a soluble inhibitor of canonical Wnt signaling, disrupted Wnt3a-regulated OPG and RANKL expression in osteoblasts. Confirmed in multiple independent assays, we show that pretreatment with rDKK1 completely abolished Wnt3a-induced OPG mRNA and protein production by mouse and human osteoblasts. In addition, we show that Wnt3a-induced OPG expression was diminished in osteoblasts cocultured with a DKK1-expressing MM cell line or primary MM cells. Finally, we show that bone marrow sera from 21 MM patients significantly suppressed Wnt3a-induced OPG expression and enhanced RANKL expression in osteoblasts in a DKK1-dependent manner. These results suggest that DKK1 may play a key role in the development of MM-associated OBL by directly interrupting Wnt-regulated differentiation of osteoblasts and indirectly increasing osteoclastogenesis via a DKK1-mediated increase in RANKL-to-OPG ratios. (Blood. 2008;112:196-207) IntroductionBone destruction, a cardinal feature of multiple myeloma (MM), results from an uncoupling of osteoclast and osteoblast activities adjacent to intramedullary tumor foci. [1][2][3] Osteoclasts are activated by binding of the receptor activator of nuclear factor kappa B ligand (RANKL) 416 to its cognate receptor, RANK, whereas osteoprotegerin (OPG), 7 a soluble member of the tumor necrosis receptor super-family, acts as a naturally occurring decoy receptor that competes with RANK for binding of RANKL. 8 The balance of these 2 molecules plays a critical role in the control of osteoclastogenesis. MM cells likely stimulate expression of RANKL and suppress expression of OPG by osteoblasts or their progenitors. 9,10 Increased serum levels of RANKL and decreased levels of OPG have been associated with a poor prognosis in MM. 11 Restoring the RANKL/OPG imbalance by RANKL antagonist or recombinant OPG not only reduces MM-associated bone lesions but also halts disease progression in animal models. 10,[12][13][14] Mechanistically, regulation of osteoclastogenesis by osteoblast-derived OPG [15][16][17]18,19 involves Wnt signaling, a pathway that is regulated by a large number of antagonists, including the Dickkopf family, 20 secreted frizzled-related proteins (sRFPs), 21,22 and sclerostin. 23 DKK1 blocks maturation of osteoblasts and formation of mineralized matrix by antagonizing the canonical Wnt pathway through it binding to LRP5/6 and Kremen. [24][25][26][27][28] Germ line inactivating mutations in the Wnt coreceptor LRP5 causes the osteoporosis-pseudoglioma syndrome (OPPG), 29 whereas a high bone m...