ALBERT

Description: Background: The ubiquitin-proteasome system (UPS) has been validated as a target in multiple myeloma (MM) through the success of proteasome inhibitors such as bortezomib, but drug resistance is an emerging challenge. Targeting some of the upstream components of the UPS, such as the E1 ubiquitin activating enzyme (UAE), could therefore be a promising alternative. TAK-243 (MLN7243) specifically blocks the ubiquitin conjugation cascade through the formation of a TAK-243-ubiquitin adduct, thereby inhibiting the UAE. Our aim was to explore the effectiveness of TAK-243 against pre-clinical myeloma models, and to understand some its mechanisms of action. Methods: We performed pre-clinical studies in myeloma cell lines and mouse models using TAK-243. Downstream effects were evaluated using viability, apoptosis assays, western blotting, gene expression profiling (GEP), and Reverse Phase Protein Array (RPPA) techniques. Results: MM1.S and MOLP-8 TP53 wild-type cell lines were sensitive to TAK-243, with median inhibitory concentrations (IC50) of 25 nM at 24 hours based on viability assays. In otherwise isogenic cell lines in which TP53 was suppressed using genome editing techniques, the IC50 was ~40 nM, but higher TAK-243 concentrations of 100 nM overcame resistance due to TP53 inactivation. Similarly, TAK-243 was able to overcome resistance to both conventional (dexamethasone) and novel (bortezomib, lenalidomide) drugs in paired sensitive and resistant cell line models. After treatment with TAK-243, Annexin V and TO-PRO3 staining determined that viable MM1.S cells were induced into early or late apoptosis. This was accompanied by a significant increase in cleaved caspase-3, -8, and -9 as detected by flow cytometry, and in cleaved caspase-7 detected by RPPA and western blot. Exposure to TAK-243 reduced the cellular content of ubiquitin-protein conjugates, and did not enhance expression levels of a fusion protein degraded by the proteasome in a ubiquitin-independent manner, indicating the lack of direct proteasome inhibition. GEP analysis and RPPA detected enhanced expression of p53-pathway related proteins, including MDM2, TP53, and p21 in TAK-243 treated MM1.S cells. Several mRNAs and proteins in the ER stress pathway, including ATF6, ATF4, IRE1a and XBP1 were also elevated, as were many non-coding RNAs and DNA-damage related genes. Combination experiments in MM cell lines demonstrated synergy between TAK-243 and lenalidomide, pomalidomide, panobinostat, melphalan and doxorubicin. Finally, TAK-243 demonstrated in vivo antitumor activity against MM1.S and MOLP-8 xenograft models when dosed at 12.5 mg/kg IV twice-weekly for 2 weeks (tumor growth inhibition of 60% and 73%, respectively). Elevation of BiP, ATF4, XBP1s and cleaved-caspase 3 was detected within the first 24 hrs after dosing in the sensitive MM1.S xenografts. In contrast, RPMI 8226 cells, which showed a 2000 nM IC50 in cell culture, were also resistant to TAK-243 in vivo, with no tumor growth inhibition detected. Conclusions: TAK-243 is a UAE inhibitor that is active against myeloma cells in vitro and in xenograft models in vivo, overcomes conventional and novel drug resistance, and its action is associated with stimulation of the TP53 and ER stress pathways. Thus, it may deserve further evaluation as an anti-myeloma agent. Disclosures Berger: Takeda Pharmaceuticals: Employment. Hyer:Takeda Pharmaceuticals: Employment. Chattopadhyay:Takeda Pharmaceuticals: Employment. Syed:Takeda Pharmaceuticals: Employment. Shi:Takeda Pharmaceuticals: Employment. Yu:Takeda Pharmaceuticals International Co, Cambridge, MA: Employment. Shinde:Takeda Pharmaceuticals: Employment. Kreshock:Takeda Pharmaceuticals: Employment. Tirrell:Takeda Pharmaceuticals: Employment. Menon:Takeda Pharmaceuticals: Employment. Orlowski:Takeda Pharmaceuticals: Research Funding.

Description: Abstract 1824 Background and Object About 80% patients with multiple myeloma (MM) represent destructive bone disease, which is negatively correlated with life quality and survival. The mechanism of myeloma bone disease involves the enhancement of bone absorption accompanied with inhibition of bone formation, namely, over-activated osteoclasts and suppressed osteoblasts. Our previous data have shown that serum level of stromal cell derived factor 1 alpha (SDF-1 α), secreted by bone marrow stromal cells (BMSCs) for stem cell homing, was positively correlated with the serum level of dickkopf-1 (DKK-1), a soluble inhibitor of Wnt signaling pathway which plays an important role in osteoblast differentiation and proliferation. To further reveal the pathogenesis of MM bone disease, this study was to explore the mechanism how SDF-1α and DKK-1 interact to promote myeloma bone disease. Materials and Methods Forty six untreated MM patients were included in our study. The clinical data, sera and bone marrow aspirates were collected. Serum SDF-1α and DKK-1 were tested using ELISA kits. Plain radiographs were used to assess osteolytic lesions. The relationship between the severity of bone disease and serum values of SDF-1α and DKK-1 was analyzed. Human MM cell line RPMI 8226 was cultured in vitro. Under the condition of SDF-1α 10ng/ml, the transcription level of DKK-1 was detected at 8h and 36h by Realtime PCR relative quantitation SYBR technique. Myeloma cells from patients' bone marrow were sorted by CD138 immunomagnetic beads. The purity of sorted cells was detected by flowcytometry. The transcription level of DKK-1 in primary myeloma cells was tested after stimulation of SDF-1α for 72 hours. BMSCs from MM patients were cultured in vitro. When added with Wnt-3a (200ng/ml) and/or DKK-1 (20ng/ml), the transcription level of SDF-1 alpha was assayed. Statistics was carried out using SPSS Statistics 17.0. P value of less than 0.05 was considered as significance. Results The serum level of SDF-1α in MM patients (n=46) was significantly higher than that of the age-matched health control (n=30) (3275.9±1093.0pg/ml vs 2817.5±419.6pg/ml, P=0.015). The serum level of DKK-1 in MM patients was also significantly higher than that of the health control (3275.9±1093.0pg/ml vs 1494.2±918.7pg/ml, P=0.025). There was a positive relationship between serum SDF-1 alpha and DKK-1 in MM patients (r=0.40, P=0.001). However, such association did not show in health control group(r=0.15,P=0.428). Patients with positive radiological findings had higher level of SDF-1α compared to negative patients (2989±838 pg/ml vs 2460±739 pg/ml, respectively) with no statistically difference. Serum DKK-1 was also higher in patients with positive bone disease, which was 5072±8032pg/ml compared to 1032±720pg/ml in patients with negative findings (P=0.18). Both SDF-1α and DKK-1 had the increasing trend according to more advanced stage of ISS, yet with no statistical difference. After stimulation of SDF-1α for 8h and 36h in RPMI 8226 cell line, the mRNA of DKK-1 was increased by 1.92 and 4.19 folds respectively (P=0.099). The purity of CD138 positive myeloma cells was 99.5%. Primary myeloma cells from 9 MM patients had a wide range of DKK-1 relative expression from 0.06% to 11.09% contrasted to the house keeping gene of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Myeloma cells with higher baseline DKK-1 transcription level (higher than 0.2%) seemed to be more susceptible to extra SDF-1α, which leads to significantly upregulated DKK-1 expression (P=0.043). However, upregulation of DKK-1 was not seen in cells with low baseline DKK-1 expression. When BMSCs exposed to Wnt-3a, it showed that SDF-1α mRNA was significantly down regulated to 29% (P=0.028). However, this effect was reversed with the co-existence of DKK-1 and Wnt-3a. Conclusion The serum levels of both SDF-1α and DKK-1 were elevated in MM patients with positive correlation. The severity of clinical bone disease has the trend to parallel to SDF-1α and DKK-1. SDF-1α enhanced mRNA transcription level of DKK-1 in both myeloma cell line and primary myeloma cells, while DKK-1 promoted the transcription of SDF-1α in BMSCs. SDF-1α and DKK-1 interacted with each other as a vicious cycle to facilitate myeloma bone disease. It would be a new target to interrupt the communication of SDF-1α and DKK-1 for treating myeloma bone disease. Disclosures: No relevant conflicts of interest to declare.

Description: Three proteasome inhibitors have garnered regulatory approvals in various multiple myeloma settings; but drug resistance is an emerging challenge, prompting interest in blocking upstream components of the ubiquitin-proteasome pathway. One such attractive target is the E1 ubiquitin-activating enzyme (UAE); we therefore evaluated the activity of TAK-243, a novel and specific UAE inhibitor. TAK-243 potently suppressed myeloma cell line growth, induced apoptosis, and activated caspases while decreasing the abundance of ubiquitin-protein conjugates. This was accompanied by stabilization of many short-lived proteins, including p53, myeloid cell leukemia 1 (MCL-1), and c-MYC, and activation of the activating transcription factor 6 (ATF-6), inositol-requiring enzyme 1 (IRE-1), and protein kinase RNA-like endoplasmic reticulum (ER) kinase (PERK) arms of the ER stress response pathway, as well as oxidative stress. UAE inhibition showed comparable activity against otherwise isogenic cell lines with wild-type (WT) or deleted p53 despite induction of TP53 signaling in WT cells. Notably, TAK-243 overcame resistance to conventional drugs and novel agents in cell-line models, including bortezomib and carfilzomib resistance, and showed activity against primary cells from relapsed/refractory myeloma patients. In addition, TAK-243 showed strong synergy with a number of antimyeloma agents, including doxorubicin, melphalan, and panobinostat as measured by low combination indices. Finally, TAK-243 was active against a number of in vivo myeloma models in association with activation of ER stress. Taken together, the data support the conclusion that UAE inhibition could be an attractive strategy to move forward to the clinic for patients with relapsed and/or refractory multiple myeloma.

Description: A new human myeloma cell line WuS1 was established from the bone marrow of a 45-year-old Chinese male patient with IgGλ type multiple myeloma (stage IIIB). The growth of WuS1 cells is constitutively independent of exogenous growth factors of feeder cells. The WuS1 cell line proliferated consistently as free-floating single cells in suspension, sometimes in small clusters or slightly adherent on the bottom of the plastic culture flask, without forming clumps. The cell line has been maintained without any external growth factors for over a year, and cells frozen in liquid nitrogen can be revived successfully. The doubling time of the cells was about 11 hours and the colony-forming rate was 55.56±6.33%. WuS1 displayed immature plasma cell features with an obvious heterogeneity in size and a high nuclear-cytoplasmic ratio in Wright-Giemsa staining. Figure Figure They were positive for ALP, CE, ACP(not inhibited by tartrate) and PAS stainings and negative for POX and NBE. By transmission electron microscopy, the cytoplasm of WuS1 contained abundant mitochondria, and parallel endoplasmic reticulum or Golgi apparatus in some cells. The monoclonal immunoglobulin G and λ light chain were positive in cell lysate and not in cell culture supernatants by immnuoelectrophoresis. The cell surface antigens were positive for CD3, CD59, CD106 and CD138, and negative for CD4, CD5, CD8, CD10, CD13, CD14, CD19, CD20, CD22, CD29, CD31, CD33, CD34, CD38, CD44, CD49d, CD45, CD54, CD56 and HLA-DR by flow cytometry. Chromosomal analysis revealed a hypodiploidy and complex karyotype. WuS1 cells were negative for Epstein-Barr virus by PCR using EBV nuclear antigen-1 specific primers. Twelve SCID mice were injected with WuS1 cells intravenously or subcutaneously, and obvious tumor infiltration in bone marrow, liver, spleen, lung, kidney and injection site (subcutaneously group) were observed by pathologic examination. Figure Figure The novel WuS1 cell line will be useful in the study of the biology, etiology and treatment of multiple myeloma.

Description: When cancer cells are resident in bone, they initiate a vicious cycle with osteoclasts (OCs) which perpetuates their growth and aggressive behavior. OCs are critical for the maintenance of the vicious cycle, since they control not only bone destruction associated with cancer, but also the aggressive behavior of tumor cells. It has recently been recognized that tumor cells grow in distant sites because they induce non-malignant cells to establish a “pre-metastatic niche” for tumor cells to later engraft. But nothing is yet known for bone. Primitive bone marrow progenitor cells, called myeloid immune suppressor cells (MISCs), which suppress immune reactivity, are important niche components. MISCs belong to the myelomonocytic lineage with surface markers of Gr-1 and CD11b. We hypothesize that MISCs are precursors of OCs recruited by tumors to assist in the establishment of the vicious cycle. To test this hypothesis, we used the well-characterized 5TGM1 murine myeloma model. 5TGM1-GFP tagged myeloma cells were inoculated via tail vein. The proportion of Gr-1+CD11b+ cells in bone marrow and spleen were assessed by FACS. On week 4 after tumor cell inoculation, %Gr-1+CD11b+ cells were significantly greater in tumor-bearing mice compared with controls (60.9±7.8% vs 37.7±8.6% p

Description: Objective: Observe the effect of normal and myeloma bone marrow stromal cells (BMSC) to the growth of myeloma cells. Evaluate the function of adhesion molecules in the mutual action of BMSC and myeloma cells. Methods: Immunomagnetic beads and anti-CD138 antibody were used to separate myeloma cells from patients’ bone marrow of multiple myeloma (MM). U266 and freshly purified myeloma cells respectively co-cultured with normal BMSC and myeloma BMSC. Single U266 or fresh myeloma cells were cultured as control group. Anti-CD11a, anti-CD29, anti-CD44 and anti-CD49d antibodies were respectively added in the co-cultured system and single myeloma cells. After culture for 72 hours, cell cycle of myeloma cells and the ratio of apoptotic cells were tested by flowcytometry. The level of interleukin 6 (IL-6) in the supernate was measured. Results: Normal BMSC inhibited the growth of myeloma cells while myeloma BMSC improved it. As to U266, S+G2% of myeloma cells in normal BMSC group and myeloma BMSC group were 55.5±4.0% and 65.4±4.5%(p

Description: Background: In multiple myeloma (MM), impact of specific chromosomal translocations involving IgH (14q21 locus, including t(4;14), t(11;14), and t(14;16) etc.) has been explored extensively. However, over 15% MM patients harboring IgH translocation with undefined partners have long been ignored. Methods: A prospective non-randomized cohort study with a total of 715 newly-diagnosed MM cases was conducted, 13.6% of whom were t(14;undefined) positive. The whole cohort was divided into four groups: no IgH split (47.7%); t(14;undefined) (13.6%); t(11;14) (17.6%); and t(4;14) or t(14;16) group (21.1%). Results: Median OS for the four groups were 84.2, not reached (NR), 58.7, and 44.2 months respectively, with p values for t(14;undefined) vs. no IgH split, t(11;14), and t(4;14)/t(14;16) groups of 0.197, 0.022 and 0.001, respectively(Figure B).In bortezomib-based group, the survival advantage gained by t(14;undefined) group was much more significant compared to t(11;14) and t(4;14)/t(14;16) groups. Importantly, t(14;undefined) turned out to be an independent predictive factor for longer OS of MM patients in multivariate analysis, especially in the context of bortezomib-treatment. Similar results were also observed in the PUMCH external validation cohort (Figure C). Conclusion: Collectively, our data confirmed and externally validated the favorable prognosis of the t(14;undefined) groups, especially in the era of novel agents. Disclosures No relevant conflicts of interest to declare.

Description: Background Hereditary intrinsic factor deficiency is a rare disease characterized by cobalamin deficiency with the lack of gastric intrinsic factor because of gastric intrinsic factor (GIF) mutations. Patients usually present with cobalamin deficiency without gastroscopy abnormality and intrinsic factor antibodies. Case presentation A Chinese patient presented with recurrent severe anemia since age 2 with low cobalamin level and a mild elevation of indirect bilirubin. The hemoglobin level normalized each time after intramuscular vitamin B12 injection. Gene test verified a c.776delA frame shift mutation in exon 6 combined with c.585C 〉 A nonsense early termination mutation in exon 5 of GIF which result in the dysfunction of gastric intrinsic factor protein. The hereditary intrinsic factor deficiency in literature was further reviewed and the ancestry of different mutation sites were discussed. Conclusions A novel compound heterozygous mutation of GIF in a Chinese patient of hereditary intrinsic factor deficiency was reported. It was the first identified mutation of GIF in East-Asia and may indicate a new ancestry.