ALBERT

Description: 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.

Description: Abstract 2920 Background: Nicotinamide adenine dinucleotide (NAD+) is a coenzyme crucially involved in several cellular functions, including energy metabolism, reactive oxygen species scavenging, DNA repair, and gene expression. Intracellular NAD+ stores are continuously replenished through pathways whose activity depends on the tissue and availability of substrates. Nicotinamide phosphoribosyltransferase (Nampt) is the rate-limiting enzyme in the NAD+ salvage pathway from nicotinamide. During neoplastic transformation, Nampt is upregulated to compensate for increased metabolic demands. It promotes myeloid and lymphoid differentiation and increases specific cytokine production (TNF- α, IL-6 and VEGF). Importantly, cancer and leukaemia cells appear to be more sensitive to Nampt inhibitor drugs than normal cells. The reasons for this selectivity are not fully understood, but may include aberrant metabolic demands and increased reliance on NAD+-dependent enzymes. Promising results obtained with Nampt inhibitors (such as FK866) in preclinical cancer models suggest that Nampt activity represents an innovative therapeutic target for novel anticancer agents. Methods: A panel of eighteen different MM cell lines, both sensitive and resistant to conventional and novel anti-myeloma drugs, as well as patient MM cells were used in the study. The mechanism of action of FK866 was investigated by Annexin-V/propidium iodide staining, thymidine incorporation, Western-blotting, and with lentivirus-mediated shRNAs. For the autophagy assay, EGFP-LC3+ cells were treated with FK866 and the number of GFP-LC3 punctae was analyzed and quantified by fluorescence microscopy and flow cytometry, respectivley. Intracellular NAD+ content was measured using a biochemical assay. Angiogenesis was measured in vitro using Matrigel capillary-like tube structure formation assay. Results: To study the role of Nampt in MM cells, we performed a protein analysis of this enzyme in eighteen MM cell lines. Nampt is constitutively activated in all cell lines tested. Moreover, patient MM cells highly express this enzyme whereas normal cells lack this protein. Indeed, the Nampt inhibitor FK866 decreased MM cell line viability in a dose and time dependent manner, with an IC50 ranging from 3–30nM. Similar results were observed in patient MM cells. Importantly, FK866 did not inhibit viability of normal peripheral blood mononuclear cells. Tritiated thymidine uptake assay confirmed the antiproliferative effects of FK866 in MM cell lines and patient cells. To examine the mechansim of action, we showed that intracellular NAD+ levels decreased with FK866 treatment at 24 and 48 hours. Furthermore, knock-down of Nampt by small interfering RNAs caused significant inhibition of MM cell growth. FK866 triggered anti-MM activity in our models of MM in the bone marrow (BM) microenvironment, confirming its ability to overcome the proliferative advantage conferred by the BM milieu. FK866 treatment also inhibited angiogenesis via suppression of pivotal MM pathways PI3K/AKT and ERK. In further studies to delineate its mechanisms of action, no activation of apoptosis was observed in treated-cells. Instead FK866 treatment resulted in a marked increase in autophagy, evidenced by autophagic vacuoles in the cytoplasm and proteolitic processing of endogenous LC3-I to LC3-II. FK866 inhibited mTOR signaling and triggered increased formation of EGFP-LC3 punctae, confirming involvement of autophagic cell death. Finally, combined FK866 with bortezomib (CI 〈 0.6), melphalan (CI 〈 0.9), and dexamethasone (CI 〈 0.8), induced synergistic cytotoxicity against MM cells. Conclusion: Our data therefore show a pivotal role of Nampt in MM cell growth, survival and drug resistance. The ability of FK866 to inhibit Nampt activity strongly supports its clinical evaluation to improve patient outcome in MM. Disclosures: Hideshima: Acetylon: Consultancy. Anderson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees; Merck: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Acetylon: Membership on an entity's Board of Directors or advisory committees.

Description: Abstract 2913 CRM1 (chromosomal region maintenance 1, XPO1) is the major export protein regulating degradation of key tumor suppressors by transporting them from nucleus to cytoplasm, thus abrogating their function. Highly expressed CRM1 is associated with poor prognosis in several solid tumors, and inhibition of CRM1 restores function of tumor suppressors such as p53, p21 and FOXO and IκB (cellular antagonist of NF-κB). Furthermore, CRM1 knockdown enhances sensitivity of human multiple myeloma (MM) cell lines to topoisomerase II inhibitor (Cancer Res 2009 :69, 17). In this study, we investigated a novel selective CRM1 inhibitor, KPT-185, in human MM cells. Gene expression profiling analysis using GEO database showed that CRM1 expression is significantly increased in CD138+ cells from MM patients versus monoclonal gammopathy of undetermined significance (MGUS) patients or normal donors (p

Description: Malignant cells have a higher nicotinamide adenine dinucleotide (NAD+) turnover rate than normal cells, making this biosynthetic pathway an attractive target for cancer treatment. Here we investigated the biologic role of a rate-limiting enzyme involved in NAD+ synthesis, Nampt, in multiple myeloma (MM). Nampt-specific chemical inhibitor FK866 triggered cytotoxicity in MM cell lines and patient MM cells, but not normal donor as well as MM patients PBMCs. Importantly, FK866 in a dose-dependent fashion triggered cytotoxicity in MM cells resistant to conventional and novel anti-MM therapies and overcomes the protective effects of cytokines (IL-6, IGF-1) and bone marrow stromal cells. Nampt knockdown by RNAi confirmed its pivotal role in maintenance of both MM cell viability and intracellular NAD+ stores. Interestingly, cytotoxicity of FK866 triggered autophagy, but not apoptosis. A transcriptional-dependent (TFEB) and independent (PI3K/mTORC1) activation of autophagy mediated FK866 MM cytotoxicity. Finally, FK866 demonstrated significant anti-MM activity in a xenograft-murine MM model, associated with down-regulation of ERK1/2 phosphorylation and proteolytic cleavage of LC3 in tumor cells. Our data therefore define a key role of Nampt in MM biology, providing the basis for a novel targeted therapeutic approach.

Description: Multiple myeloma (MM) is a disease characterized by clonal proliferation of plasma cells with a complex genomic phenotype. We recently demonstrated that MM cells have signs of ongoing DNA damage and activation of DNA damage response, via ATM phosphorylation and nuclear localization of ABL1 (Cottini et al, Nature Medicine, 2014). In normal cells, the activation of ATM/ABL1 pathway eventually results in the death of cells with terminal unrepaired DNA damage. However, MM cells inactivate YAP1, a downstream target of ABL1, by homozygous deletions in 10 percent of patients or by protein/mRNA downregulation. Re-expression of YAP1 induces growth inhibition and cell death in YAP1 deleted-cell lines (KMS-18 and KMS-20), as well as in cell lines with low YAP1 expression (MM.1S), according to an ABL1-dependent mechanism. In the presence of DNA damage, YAP1 stabilizes and binds to TP73, facilitating the activation of TP73-target genes, such as BAX, PUMA, and CDKN1A (p21). TP73 levels are generally low in MM cells, indicating that YAP1 is the main regulator of TP73; low YAP1 therefore blocks DNA damage-mediated apoptosis. We identified that YAP1 expression is tightly regulated by the activation of the Hippo pathway serine/threonine kinase STK4. Upon STK4 silencing, YAP1 expression was increased, causing MM cell death both in in vitro and in vivo settings. A kinase screening was performed and we are currently optimizing different compounds with anti-STK4 activity. One of them (STK4i) showed promising growth inhibitory effects. To characterize the efficacy of STK4i, a panel of MM cell lines was incubated with increasing doses of the compound ranging from 0.1-10 μM; growth inhibitory effects were measured by MTT assay, while Annexin V-PI staining detected apoptosis. KSM-20 MM cells, which bear a homozygous deletion for YAP1 genomic locus, were used as negative control. After 48 hour treatment with 1μM dose of STK4i, 62 percent, 66 percent and 15 percent of MM.1S, H929 and U266 cells were positive for Annexin V and PI markers, while minimal effects were observed in KMS-20 cells. To test the activation of YAP1 axis in MM, cells were treated with STK4i for 48hrs, and western blot analysis followed, which confirmed YAP1 upregulation in MM.1S and H929 cells. Further studies are ongoing to evaluate additional functions of STK4 in MM growth and to better characterize the activity of STK4i in the bone marrow microenvironment. Importantly, STK4 silencing also upregulates TP73, in a YAP1-dependent manner. To investigate synergic effects of TP73 and TP53, we used STK4 silencing or STK4i in combination with Nutlin-3a, a MDM2 inhibitor that activates TP53, in both wild-type and mutant TP53 cells. A cooperative response in terms of toxicity by combined TP73 and TP53 was observed in TP53 wild type MM cell lines (MM.1S and H929) as well as in OPM-2 cells with mutant-TP53. However, the response in mutant-TP53 patient cells may be variable, depending on residual TP53 function or dominant negative activity of mutant-TP53 on TP73. To conclude, STK4 is a new and important target in MM pathogenesis and STK4 inhibitors may represent novel therapeutic options in a broad subset of patients with MM. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 2440 Monoclonal B-cell Lymphocytosis (MBL) is a preclinical hematologic condition wherein small B-cell clones are detectable in the peripheral blood of otherwise healthy individuals. Monoclonal B-cell expansions are rather heterogeneous in terms of phenotype, but in two thirds of cases clonal B-cell populations share the same unique immunophenotypic profile of Chronic Lymphocytic Leukemia (“CLL-like MBL”). Based on the number of B cells per μl, MBL cases might be further split into those associated with lymphocytosis, usually diagnosed in a clinical setting (“Clinical MBL”) when clonal B cells reach a concentration 〉1500/μL, and those detected in the general population (“low-count MBL”), usually characterised by 20% of people older than 60 years using highly sensitive techniques). We took advantage of our cohort of 138 MBL cases previously described among 1779 healthy individuals, living in a rural valley in Northern Italy (Val Borbera Valley) that included 96 CLL-like (69.6%), 20 Non-CLL (14.5%), 22 atypical CLL (15.9%) MBL. Of the 138 originally diagnosed MBL subjects, 76 individuals participated to a second visit after a median follow up of 34 months (range 11–50 months). 93.1% (54/58) of CLL-like MBL clones were confirmed, while only 44.5% and 66.7% of Atypical CLL-like and Non-CLL MBL, respectively, persisted over time. The few CLL-like clones that were not confirmed had a very low concentration at the initial visit (median number of clonal B-cells: 0.46 per μl) being proximal to the detection limit of the flow cytometric technique. Among the confirmed CLL-like cases, 1/54 was a Clinical MBL (1764 cells/μL), 3 subjects had 97, 190 and 265 cells/μl, while the vast majority of participants (50/54, 92.6%), had a number of monoclonal B-cells

Description: Background: Multiple myeloma (MM) cells show a variable combination of chromosomal translocations, copy-number variations, somatic mutations and clonal heterogeneity, which makes every patient unique. We have recently shown that MM cells have signs of ongoing DNA-damage, which activates an ATM/ABL1-dependent DNA damage response (DDR) without overt apoptosis (Cottini et al., Nat Med, 2014). Here we further characterize the mechanisms of DNA damage and replicative stress in MM, which provide the basis for a novel synthetic lethality treatment approach. Results: MM cell lines with active DNA damage have enrichment in pathways of DNA replication and cell cycle. These same MM cell lines also present 53BP1, RPA and RAD51 foci with activated ATR and CHK1. Of note, 53BP1, RPA and RAD51 foci are markers of replicative stress, associated with DNA hyper-replication and stalled replication forks. Importantly, replicative stress markers are also present in primary MM cells. We also demonstrated a gene expression signature specific for increased chromosomal instability and DNA damage in a cohort of MM patients versus normal plasma cells. Remarkably, a subset (20 percent) of patients with myeloma overexpress genes belonging to the instability signature; this group also shows an unfavorable prognosis due to a more aggressive disease. These findings suggest that some patients present a similar phenotype to the cell lines, characterized by extensive replicative stress and activation of hyper-replicative pathways. We therefore hypothesized that MM cells might be sensitive to replicative stress overload, which occurs when cells fail to endure the presence of an excess of unrepaired DNA. To evaluate this hypothesis, we used shRNAs to silence ATR, a protein involved in the control of stalled replication origins, in two myeloma cell lines, one with normal TP53 (H929) and another with mutant TP53 (OPM-2). Inhibition of ATR caused a reduction in cell growth and induction of apoptosis, both more evident in MM cell lines with mutant TP53. A similar phenotype was observed when MM cell lines were incubated with VE-821, a specific ATR inhibitor. The strongest response occurred in TP53 mutant cell lines, which are representative of a model of aggressive MM, consistent with the concept of replicative stress overload. Indeed, p53 is normally phosphorylated and active in MM, while TP53 loss in the context of hyper-replication may prevent activation of salvage checkpoint, thereby favoring cell death in the absence of ATR. ATR inhibition also induces an increase in DNA double strand breaks, as evidenced by the higher number of γ-H2A.X foci. Reactive oxygen species (ROS) can also mediate DNA damage, and treatment with an antioxidant reagent N-Acetylcysteine (NAC), which helps scavenging ROS by replenishing glutathione stores, was indeed capable of reducing DNA double strand breaks and replicative stress markers. Since cancer cells are particularly sensitive to oxidative stress, we then evaluated the anti-MM activity of piperlongumine (PL), a drug that induces ROS accumulation. MM cell lines were sensitive to PL treatment, while PBMCs were minimally affected. As expected, the apoptotic effects of PL were abrogated upon co-incubation with NAC, indicating the specific activity of PL on ROS. We next exploited the possibility of combining replicative and oxidative stress in myeloma cells, hoping to overcome the threshold of tolerance to unrepaired DNA. H929 and OPM-2 cells were transfected with ATR shRNAs or treated with VE-821 and incubated with DMSO or 1-2.5 μM PL; synergic effects by the combination treatment were evident in both myeloma cell lines and also in patient MM cells. Conclusion: Replicative stress is present in a group of MM patients, who have aggressive disease, myeloma cell hyperproliferation and poor prognosis. Strategies aimed to shift the balance towards high DNA damage by ROS production and reduced DNA repair can decrease MM growth and may benefit patients with otherwise unfavorable outcomes. Disclosures No relevant conflicts of interest to declare.

Description: Background: Multiple myeloma (MM) is a clonal B cell neoplasia that comes from growth of malignant B cells in the bone marrow. Stromal cells, including inflammatory cells, in the bone marrow enable MM persistence and growth. MM is characterized by an uncoordinated cytokine system with an increase in proinflammatory cytokines. While proinflammatory cytokines are essential in mounting an anti-tumor response, they can also drive cancer progression. Ultimately, it is the effects of the cytokine milieu in the immune microenvironment that help determine MM development. While the role of IFNγ in MM remains mixed and unclear, our analysis suggests IFNγ plays an oncogenic role in MM and offers other insights to MM pathology. Methods: The National Center for Biotechnology Information (NCBI) GEO is an open database of more than 2 million samples of functional genomics experiments. The Search Tag Analyze Resource for GEO (STARGEO) platform allows for meta-analysis of genomic signatures of disease and tissue. We employed the STARGEO platform to search the Gene Expression Omnibus and performed meta-analysis on 517 peripheral blood samples from multiple myeloma patients using 97 healthy peripheral blood samples as a control. We then analyzed the signature in Ingenuity Pathway Analysis (IPA) to help define the genomic signature of MM and identify disease pathways. We analyzed genes that showed statistical significance disease and control samples (p

Description: Abstract 2932 Multiple Myeloma (MM) is a hematologic malignancy characterized by a complex combination of structural and numerical chromosomal abnormalities. However, the underlying molecular basis of the genomic instability remains largely unknown. The ability to repair DNA damages, especially double-strand breaks (DSBs), is essential to suppress genetic instability. Non-homologous end joining (NHEJ) is one of the most important mechanisms responsible for repair of these breaks. Since both impaired and aberrant NHEJ seem to be linked to genomic instability in solid as well as other hematologic tumors, we have investigated its altered function in MM. To confirm involvement of an aberrant NHEJ pathway in MM genomic instability, we measured the end joining (EJ) capacity of 6 different MM cell lines using a plasmid based assay containing both the test gene (Luciferase - LUC) measuring end joining as well as a reporter gene (Alkaline Phosphatase - SEAP) to control for transfection efficiency. MM and normal control cells were transfected with this plasmid and the LUC and SEAP activity was detected directly in the supernatant of the cells at 24 h. Increased EJ activity was observed in all the MM cell lines tested compared to peripheral blood mononuclear cells (PBMCs) and bone marrow stromal cells (BMSCs) from healthy donor. To confirm the role of the NHEJ pathway in this increased DNA EJ activity, nuclear extracts from 9 different MM cell lines were used to determine the DNA-binding-activity of Ku86, a key protein of this repair mechanism involved in the recognition of the broken DNA ends and in the initiation of the DSBs repair process. As in EJ activity, all the MM cell lines showed an increased Ku86 binding respect to normal cells confirming the aberrant activation of the NHEJ pathway in MM cells. Interestingly, we did not observe significant differences in Ku86 level in nuclear extracts between PBMCs and MM cell lines suggesting that the difference in the Ku86 DNA-binding-activity was likely a functional and not due to disparity in the protein levels. We further investigated the link between this aberrant NHEJ activity and MM genomic instability using an immune-fluorescent based assay for DSBs. We observed an increased constitutive DNA damage in the absence of treatment with DSB-inducing agents in 5 of 6 MM cell lines compared with normal PBMCs. Most importantly, we noticed a direct correlation between the basal level of DSBs and the Ku86-binding-affinity. Furthermore, all the MM cell lines showed little or no ability to repair ionizing radiation (IR)-induced DNA damage compared to normal cells as well as no change in the Ku86-binding-affinity after stimulation suggesting that the aberrant NHEJ pathway in MM might represent a response to the constitutive endogenous DNA damage in these cells. We have also observed that 2 key NHEJ genes (Ku86 and Artemis) are overexpressed in MM compared to MGUS and normal plasma cells and their overexpression correlates with a shortened overall survival in MM suggesting that an hyper-activation of this pathway could have a potential role in MM progression and prognosis. Ongoing experiments are assessing the NHEJ activity in primary MM cells to correlate with clinical outcome. In conclusion, our data suggests that an aberrant NHEJ in the context of a constitutive endogenous DNA damage might contribute to the high frequency of chromosome abnormalities in MM cells, thus potentially playing a central role in the tumor progression and as an important prognostic marker in this disease. Disclosures: Anderson: Onyx: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees. Munshi:Celgene: Consultancy; Millenium: Consultancy; Merck: Consultancy; Onyx: Consultancy.