• Selective myeloma cell killing and enhanced effector function of a novel anti-BCMA antibody conjugated with MMAF via noncleavable linker.• Specific multiple myeloma antigen for monoclonal antibody-based immunotherapy.B-cell maturation antigen (BCMA), highly expressed on malignant plasma cells in human multiple myeloma (MM), has not been effectively targeted with therapeutic monoclonal antibodies. We here show that BCMA is universally expressed on the MM cell surface and determine specific anti-MM activity of J6M0-mcMMAF (GSK2857916), a novel humanized and afucosylated antagonistic anti-BCMA antibody-drug conjugate via a noncleavable linker. J6M0-mcMMAF specifically blocks cell growth via G 2 /M arrest and induces caspase 3-dependent apoptosis in MM cells, alone and in coculture with bone marrow stromal cells or various effector cells. It strongly inhibits colony formation by MM cells while sparing surrounding BCMA-negative normal cells. J6M0-mcMMAF significantly induces effector cell-mediated lysis against allogeneic or autologous patient MM cells, with increased potency and efficacy compared with the wild-type J6M0 without Fc enhancement. The antibody-dependent cell-mediated cytotoxicity and apoptotic activity of J6M0-mcMMAF is further enhanced by lenalidomide. Importantly, J6M0-mcMMAF rapidly eliminates myeloma cells in subcutaneous and disseminated mouse models, and mice remain tumor-free up to 3.5 months. Furthermore, J6M0-mcMMAF recruits macrophages and mediates antibody-dependent cellular phagocytosis of MM cells. Together, these results demonstrate that GSK2857916 has potent and selective anti-MM activities via multiple cytotoxic mechanisms, providing a promising next-generation immunotherapeutic in this cancer. (Blood. 2014;123(20):3128-3138) IntroductionAlthough there is no monoclonal antibody (mAb)-based targeted therapy approved to treat patients with multiple myeloma (MM), many mAbs targeting different antigens have been preclinically and clinically evaluated. ) were either moved toward or remain in clinical development based on encouraging results from preclinical studies. However, these antigens still lack specificity and are also expressed in other normal tissues including natural killer (NK) or other effectors, which could limit their clinical utility. Therefore, novel therapeutic mAbs to achieve improved MM selectivity, simultaneously targeting cytotoxic drugs to MM cells, are urgently needed.B-cell maturation antigen (BCMA), a member of the tumor necrosis factor receptor superfamily (TNFRSF17), is selectively induced during plasma cell differentiation and is nearly absent on naive and memory B cells. 13,14 Upon binding to its ligands B-cell activating factor (BAFF) and a proliferation-inducing ligand (APRIL), the survival of bone marrow (BM) plasma cells and plasmablasts is promoted.15,16 BCMA does not maintain normal B-cell homeostasis, but is required for the survival of long-lived plasma cells. 17 In MM, BCMA messenger RNA (mRNA) is commonly expressed at high levels in malignant plasma...

The key nuclear export protein CRM1/XPO1 may represent a promising novel therapeutic target in human multiple myeloma (MM). Here we showed that chromosome region maintenance 1 (CRM1) is highly expressed in patients with MM, plasma cell leukemia cells and increased in patient cells resistant to bortezomib treatment. CRM1 expression also correlates with increased lytic bone and shorter survival. Importantly, CRM1 knockdown inhibits MM cell viability. Novel, oral, irreversible selective inhibitors of nuclear export (SINEs) targeting CRM1 (KPT-185, KPT-330) induce cytotoxicity against MM cells (ED50<200 nM), alone and cocultured with bone marrow stromal cells (BMSCs) or osteoclasts (OC). SINEs trigger nuclear accumulation of multiple CRM1 cargo tumor suppressor proteins followed by growth arrest and apoptosis in MM cells. They further block c-myc, Mcl-1, and nuclear factor κB (NF-κB) activity. SINEs induce proteasome-dependent CRM1 protein degradation; concurrently, they upregulate CRM1, p53-targeted, apoptosis-related, anti-inflammatory and stress-related gene transcripts in MM cells. In SCID mice with diffuse human MM bone lesions, SINEs show strong anti-MM activity, inhibit MM-induced bone lysis and prolong survival. Moreover, SINEs directly impair osteoclastogenesis and bone resorption via blockade of RANKL-induced NF-κB and NFATc1, with minimal impact on osteoblasts and BMSCs. These results support clinical development of SINE CRM1 antagonists to improve patient outcome in MM.

Here we show that overexpression or activation of B-cell maturation antigen (BCMA) by its ligand, a proliferation-inducing ligand (APRIL), promotes human multiple myeloma (MM) progression in vivo. BCMA downregulation strongly decreases viability and MM colony formation; conversely, BCMA overexpression augments MM cell growth and survival via induction of protein kinase B (AKT), MAPK, and nuclear factor (NF)-κB signaling cascades. Importantly, BCMA promotes in vivo growth of xenografted MM cells harboring p53 mutation in mice. BCMA-overexpressing tumors exhibit significantly increased CD31/microvessel density and vascular endothelial growth factor compared with paired control tumors. These tumors also express increased transcripts crucial for osteoclast activation, adhesion, and angiogenesis/metastasis, as well as genes mediating immune inhibition including programmed death ligand 1, transforming growth factor β, and interleukin 10. These target genes are consistently induced by paracrine APRIL binding to BCMA on MM cells, which is blocked by an antagonistic anti-APRIL monoclonal antibody hAPRIL01A (01A). 01A is cytotoxic against MM cells even in the presence of protective bone marrow (BM) myeloid cells including osteoclasts, macrophages, and plasmacytoid dendritic cells. 01A further decreases APRIL-induced adhesion and migration of MM cells via blockade of canonical and noncanonical NF-κB pathways. Moreover, 01A prevents in vivo MM cell growth within implanted human bone chips in SCID mice. Finally, the effect of 01A on MM cell viability is enhanced by lenalidomide and bortezomib. Taken together, these data delineate new molecular mechanisms of in vivo MM growth and immunosuppression critically dependent on BCMA and APRIL in the BM microenvironment, further supporting targeting this prominent pathway in MM

Bruton tyrosine kinase (Btk) has a welldefined role in B-cell development, whereas its expression in osteoclasts (OCs) further suggests a role in osteoclastogenesis.

Malignant cells have a higher nicotinamide adenine dinucleotide (NAD ؉) IntroductionMultiple myeloma (MM) is a clonal B-cell malignancy characterized by excessive bone marrow plasma cells in association with monoclonal protein. 1 The therapeutics currently available improve patients' survival and quality of life, but resistance to therapy and disease progression remain unsolved issues. Therefore, the definition of new aspects of MM biology that can be targeted and exploited from a therapeutic perspective remains a major basic and clinical research goal.Autophagy is a conserved process of normal cell turnover by regulating degradation of its components, which is characterized by the formation of autophagosomes, double-membrane cytoplasmic vesicles engulfing intracellular material including protein, lipids, as well as organelles, such as mitochondria and endoplasmic reticulum. Subsequently autophagosomes fuse with lysosomes, and their contents are degradated by lysosomal enzymes. 2 This selfcannibalization event is a highly conserved response to metabolic stress, in which cellular components are degraded for the maintenance of homeostasis. 3 Intriguingly, the waste removal function of autophagy appears as to be a double-edged sword, because it can either lead to cell survival or death. 4 A series of molecular mechanisms coordinate the autophagy machinery. Specifically, the mammalian target of rapamycin (mTOR) complex 1 (mTORC1) is the major intracellular hub for integrating autophagy-related signals. 5 Upstream of mTORC1 is the cellular energy-sensing pathway. 6 Regulation of autophagy also occurs through the transcription factors EB (TFEB) and forkhead box (FOXO), whose activation leads to transcription of Atg genes. 7,8 Although apoptosis induction has been the major focus of research in novel MM therapies, a recent study documented a pivotal role for autophagy as a prosurvival mechanism in MM cells, suggesting its potential as an additional target for novel therapeutics. 9,10 Intracellular nicotinamide adenine nucleotide (NAD ϩ ) plays a major role in the regulation of several cellular processes. 11,12 In mammals, NAD ϩ is replenished from nicotinamide (Nam), tryptophan or nicotinic acid (NA), with Nam as the most important and widely available precursor. 13 Nicotinamide phosphoribosyltransferase (NAMPT), pre-B colony enhancing factor, is the ratelimiting enzyme in NAD ϩ synthesis from Nam. 14 The expression of this enzyme is up-regulated in activated immune cells, 15 in differentiated myeloid cells, 16 during the circadian clock, 17 in glucose-restriction impaired skeletal myoblast differentiation, 18 and during cytokine production in immune cells. 19 Importantly, Nampt is also overexpressed in cancer cells, which exhibit a significant dependence on NAD ϩ to support their rapid cell proliferation. 20 Importantly, a specific chemical inhibitor of Nampt FK866, also called APO866 or WK175, exhibits a broad antitumor activity both in vitro and in vivo against cell lines derived from several tumors, with a favorabl...

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...

In the last decade, substantial efforts have been made to identify NAD biosynthesis inhibitors, specifically against nicotinamide phosphoribosyltransferase (NAMPT), as preclinical studies indicate their potential efficacy as cancer drugs. However, the clinical activity of NAMPT inhibitors has proven limited, suggesting that alternative NAD production routes exploited by tumors confer resistance. Here, we show the gene encoding nicotinic acid phosphoribosyltransferase (NAPRT), a second NAD-producing enzyme, is amplified and overexpressed in a subset of common types of cancer, including ovarian cancer, where NAPRT expression correlates with a BRCAness gene expression signature. Both NAPRT and NAMPT increased intracellular NAD levels. NAPRT silencing reduced energy status, protein synthesis, and cell size in ovarian and pancreatic cancer cells. NAPRT silencing sensitized cells to NAMPT inhibitors both and; similar results were obtained with the NAPRT inhibitor 2-hydroxynicotinic acid. Reducing NAPRT levels in a BRCA2-deficient cancer cell line exacerbated DNA damage in response to chemotherapeutics. In conclusion, NAPRT-dependent NAD biosynthesis contributes to cell metabolism and to the DNA repair process in a subset of tumors. This knowledge could be used to increase the efficacy of NAMPT inhibitors and chemotherapy. .

Key Points• FK866 combined with bortezomib induces synergistic anti-MM cell death.• Addition of low doses of NAD1-depleting agent FK866 overcomes bortezomib resistance in MM cells.We recently demonstrated that Nicotinamide phosphoribosyltransferase (Nampt) inhibition depletes intracellular NAD 1 content leading, to autophagic multiple myeloma (MM) cell death. Bortezomib has remarkably improved MM patient outcome, but doselimiting toxicities and development of resistance limit its long-term utility. Here we observed higher Nampt messenger RNA levels in bortezomib-resistant patient MM cells, which correlated with decreased overall survival. We demonstrated that combining the NAD 1 depleting agent FK866 with bortezomib induces synergistic anti-MM cell death and overcomes bortezomib resistance. This effect is associated with (1) activation of caspase-8, caspase-9, caspase-3, poly (ADP-ribose) polymerase, and downregulation of Mcl-1; (2) enhanced intracellular NAD 1 depletion; (3) inhibition of chymotrypsin-like, caspase-like, and trypsin-like proteasome activities; (4) inhibition of nuclear factor kB signaling; and (5) inhibition of angiogenesis. Furthermore, Nampt knockdown significantly enhances the anti-MM effect of bortezomib, which can be rescued by ectopically overexpressing Nampt. In a murine xenograft MM model, lowdose combination FK866 and Bortezomib is well tolerated, significantly inhibits tumor growth, and prolongs host survival. Taken together, these findings indicate that intracellular NAD 1 level represents a major determinant in the ability of bortezomib to induce apoptosis in MM cells and provide proof of concept for the combination with FK866 as a new strategy to enhance sensitivity or overcome resistance to bortezomib. (Blood. 2013;122(7):1243-1255