IntroductionMultiple myeloma (MM), a malignancy hallmarked by accumulation of malignant plasma cells in the bone marrow, remains largely incurable despite the use of conventional and novel therapies. 1 The bone marrow (BM) microenvironment promotes tumor cell growth, survival, and confers drug resistance against conventional agents. 2 Although currently available anti-MM strategies have been effective in targeting the bulk of tumor cells, it has been postulated that a tumor-initiating subpopulation or cancer stem cell persists, which may be responsible for eventual relapses. 3 Side population (SP) cells are an enriched source of cancer-initiating cells with stem cell properties, which have been identified in solid tumors, as well as in hematopoietic malignancies. [4][5][6][7][8] The SP cells show a distinct ''low-staining pattern" with the Hoechst 33342 dye. 9 Importantly, SP cells possess the ability to generate non-SP cells both in vitro and in vivo, and are associated with chemoresistance and tumorigenicity in vivo. 4,10 The prevalence and biologic function of SP cells in MM are not fully defined.In the late 1990s, thalidomide was introduced to the treatment of relapsed/refractory MM; however, its effect in patients is associated with dose-and duration-dependent side effects. 11,12 Since then, more potent immunomodulatory drugs (IMiDs), such as lenalidomide, have been introduced. Lenalidomide has been approved for the treatment of both myelodysplasia with deletion of chromosome 5q and for relapsed MM, specifically in combination with dexamethasone. 12,13 Although IMiDs act directly on tumor cells, block adherence to bone marrow stromal cells (BMSCs), modulate angiogenesis and cytokines, and up-regulate host antitumor immunity, the molecular mechanism for their action remains largely undefined, and it is unclear whether they target SP cells in MM. [14][15][16][17][18] In this study, we identified SP cells in MM cell lines as well as in primary MM tumor cells by flow cytometry-based Hoechst 33342 staining, and showed heterogeneity in the percentage of SP cells, as well as the lack of strict correlation between SP fraction and CD138 Ϫ status. SP cells exhibited clonogenic and tumorigenic potential; and importantly, lenalidomide significantly decreased the percentage and clonogenicity of SP cells at clinically relevant concentrations. Moreover, lenalidomide only slightly altered expression of drug-resistant transporter ABCG2 with no effect on functional activity of BCRP1 efflux pump. Modulation of diverse signaling cascades in SP cells by lenalidomide, including changes in Akt, GSK-3␣/␤, MEK1, c-Jun, p53, and p70S6K phosphorylation was observed. Adherence to BMSCs increased the percentage, viability, and proliferation potential of SP cells. Interestingly, both lenalidomide and thalidomide attenuated this stimulatory effect of BMSCs by significantly decreasing SP cell percentages. Therefore, our studies provide insight toward developing novel strategies Submitted February 5, 2010; accepted October 10, 2010. Prepub...

Current models of oncogenesis describe cancer as a progression of genetic mutations in a tumor cell mass. However, tumors are more than a clonal expansion of malignant cells. Tumors are heterogeneous, with a complex 3D structure, analogous to organs comprised of different tissues. In a tumor mass, the component cell types interact with each other and with their microenvironment by exchanging information through cell-cell interactions and/or through interactions with the extracellular matrix (ECM). These synergetic interactions facilitate tumor progression. Furthermore, tumor invasion and metastatic development are accomplished through the breakdown of ECM. Disruption of ECM promotes abnormal inter- and/or intra- cellular signaling, leading to dysregulation of cell proliferation, growth and cytoskeleton reorganization. The disruption of the ECM in turn promotes the overproduction of growth factors, which induce elevated epithelial cell proliferation and other abnormalities including carcinogenesis. In this review we will demonstrate that hyaluronan (HA), a core component of ECM, contributes to certain types of cancer development. Additional to extracellular HA, intracellular and nuclear forms of HA have been detected. Intracellular HA is involved in cell signaling, whereas nuclear HA could promote chromatin condensation and thus facilitate mitosis. HA molecules are synthesized by hyaluronan synthases (HASs)-HAS1, HAS2 and HAS3 enzymes. Dysregulation of HAS genes results in abnormal production of HA and promotion of abnormal biological processes such as transformation and metastasis. The function of HASs appears to be cell and tissue specific. HAS1 maintains a low, basal level of HA. HAS2 is involved in embryonic and cardiac cushion morphogenesis and subsequent development through cell migration and invasion. HAS2 stimulates cell proliferation and angiogenesis. HAS3 appears to favor the malignant phenotype in many types of malignancies. However, the exact function of HAS isoenzymes and their role in cell signaling remains to be elucidated. A better understanding of HA and HASs may facilitate the design of novel therapeutic strategies to counter presumptive cancer-promoting effects of microenvironmental components.

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Key Points• SIRT6 is highly expressed in multiple myeloma cells and blocks expression of ERKregulated genes.• Targeting SIRT6 enzymatic activity sensitizes multiple myeloma cells to DNAdamaging agents.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 1 -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. (Blood. 2016;127(9):1138-1150 IntroductionGenomic instability is a common feature of monoclonal gammopathies, resulting in complex genetic changes associated with disease progression from monoclonal gammopathy of undetermined significance to active multiple myeloma (MM) to plasma cell leukemia.1,2 Although alterations in DNA damage checkpoint proteins are less common (10% to 15%) in blood cancers compared with solid tumors, [3][4][5] MM cells do manifest a dysfunctional DNA-damage response (DDR), a key determinant of their genomic instability. [6][7][8] Identifying proteins and signaling pathways that protect MM cells from cumulative genomic instability may therefore lead to innovative therapeutic opportunities, as exemplified by the clinical efficacy of PARP inhibitors in the context of breast and ovarian tumors lacking functional BRCA1 or BRCA2. 9,10 In MM cells, direct evidence of homozygous loss or mutations in BRCA1/2 or other DDR genes is lacking, but increased DNA repair activity has been reported. 11,12 Thus, identification of adaptive pathways for coping with genomic instability in MM may similarly provide the framework for new therapeutic strategies. Sirtuins (SIRTs) are NAD1 -degrading enzymes involved in a variety of biological processes, ranging from metabolism to lifespan regulation. 13,14 Of the 7 sirtuin family members, only SIRT6 clearly contributes to DNA repair. [15][16][17][18] Consistently, murine SIRT6 knockout cells exhibit genomic instability and hypersensitivity to DNA-damaging agents. 15,17,19 Moreover...