Growth Of Multiple Myeloma Cell Lines Research Articles

Therapeutic potential of anti-PIK3CG treatment for multiple myeloma via inhibiting c-Myc pathway

Multiple myeloma (MM) is a malignant plasma cell disease. The activity of PIK3CG (PI3K catalytic subunit γ) is regulated directly by G-protein-coupled receptor and has been confirmed to be highly expressed in MM cells. This study aimed to determine the effect of pharmacological inhibition of PIK3CG on MM. We found that different concentrations of the PIK3CG inhibitor AS-605240 could suppress the growth of MM cell lines and the expression of c-Myc. The combination of PIK3CG inhibitor and the chemotherapy Melphalan could effectively inhibit the proliferation and migration of MM cells, promote the cell apoptosis, and decrease the ratio of Bcl-2/Bax and the expression of vimentin. The expression of proto-oncogene c-Myc was decreased and the sensitivity of cells to chemotherapeutic drugs was enhanced. Collectively, PIK3CG regulates growth of MM via c-Myc pathway, thus emerging as a promising molecular targeted therapy.

Discovery of Prolyl-tRNA Synthetase As a Novel Target in Multiple Myeloma

Discovery of Prolyl-tRNA Synthetase As a Novel Target in Multiple Myeloma

Leflunomide Inhibits c-Myc Signaling and Impairs Myeloma Cell Growth

Introduction:Despite recent advances in treatment that have improved the prognosis for patients with multiple myeloma (MM), the disease remains incurable. There is a need for MM treatments with new mechanisms of action. Leflunomide (Lef), a commercially available oral immunosuppressive agent that has been FDA-approved for treatment of rheumatoid arthritis was evaluated as a potential MM therapeutic. In vitro anti-MM activity of Lef has previously been reported; however, the molecular mechanisms of action of Lef in MM are incompletely understood. Inhibition of Src kinase-family members and blocking of dihydroorotate dehydrogenase, an enzyme involved in pyrimidine synthesis are among the few known targets of Lef. To identify novel direct targets of Lef and to further our understanding of Lefs' mode of action on MM cells, we performed in vitro kinase screening assays and RNA/miRNA sequencing analysis of cells treated with or without Lef.Methods and Results:Our pre-clinical studies with teriflunomide (Ter), the active metabolite of Lef, showed that it inhibited growth of MM cell lines (MM.1S, MM.1R, U266, H929, RPMI-8226, OPM-2, L-363) and primary MM patients' (CD138+) plasma cells at IC50s ranging from 30 uM to 140 uM. Pharmacokinetics analysis accompanying a phase 1 dose-escalation clinical trial in MM at our institute has demonstrated that Ter serum concentrations of 500 uM can be obtained. We also found that Ter induces cell-cycle arrest in both, glucocorticoid-sensitive (MM.1S) and resistant (MM.1R) MM cell lines at <150 uM. At higher in vitro concentrations (100 uM to 200 uM), Ter induces apoptosis in MM cells. Moreover, Ter synergized with dexamethasone as well as lenalidomide in in vitro growth inhibition of MM cell line MM.1S. In vivo mouse studies with Lef as single agent or in combination with the immune modulatory drug lenalidomide or with the proteasome inhibitor ixazomib are currently ongoing and results will be presented at the meeting. To identify novel direct targets of Ter, a screening assay described by Anastassiadis et al. (PMCID: PMC3230241) was applied to test the inhibitory activity of Ter on 600 known kinases. We identified 55 kinases that were inhibited >50% at 200 uM Ter. Subsequent dose-response kinase assays identified, among others, Aurora A and PIM family members, all of which are involved in MM pathogenesis, as novel direct targets of Lef, with IC50s ranging from 35 uM to 53 uM. To identify MM-associated mRNAs and miRNAs whose expression levels are frequently altered upon Lef exposure, 4 MM cell lines (RPMI-8226, U266, MM.1S, NCI-H929) and CD138-enriched primary MM cells were exposed to 200 µM Ter or DMSO control for 24h before extraction, purification and sequencing of mRNA and microRNA. RNA-sequencing analysis and DAVID functional annotation revealed that similar changes were present between patient samples and cell lines. Pathways that were predominantly affected by Ter included purine/pyrimidine metabolism, DNA-replication and cell-cycle regulation. Gene set enrichment analysis revealed that c-Myc, an oncogene that contributes to the malignant phenotype of MM and a known promising target in MM, signaling was impaired upon Ter treatment, in MM.1S cells. Differential expression analysis, real-time PCR and western blot analysis showed that c-Myc mRNA, c-Myc protein and c-Myc-regulated miRNAs belonging to the oncomir-1 cluster miR-17-92 were all down-regulated upon Ter exposure in the majority of the MM cells. Since PIM family members are involved in c-Myc signaling in cancer cells, we performed in silico molecular docking studies using Ter/PIM. Our results suggest that the active metabolite of Lef, Ter, can directly bind to the PIM family of proteins by binding to the ATP-binding site, thereby inhibiting its kinase activity. Since pan-PIM inhibitors such as PIM447 have been reported to be cytotoxic for myeloma cells due to decrease in c-Myc levels, these results suggest that decrease in c-Myc induced by Ter may be due to PIM inhibition.Conclusion:Leflunomide impairs in vitro MM cell growth - at least in part - through inhibition of c-Myc signaling. A clinical trial with leflunomide in MM has been initiated and a separate abstract with encouraging initial results has been submitted for presentation at this meeting. DisclosuresKrishnan:Onyx: Speakers Bureau; Sutro: Consultancy; Janssen: Consultancy, Speakers Bureau; Takeda: Speakers Bureau; Celgene: Consultancy, Equity Ownership, Speakers Bureau. Rosenzweig:Celgene: Speakers Bureau.

HUWE1 Inhibition As a Therapeutic Strategy to Target MYC in MM

BackgroundThe use of proteasome inhibitors to disrupt the ubiquitin proteasome system (UPS) has provided a significant therapeutic advance and highlights the importance of this pathway in Multiple Myeloma (MM). There is increasing interest in developing therapeutic strategies to target enzymes that regulate the UPS, with the aim of gaining more specificity and reducing side-effects. We previously identified elevated expression of the E3 ligase HUWE1 in MM compared to normal cells and demonstrated that inhibition of HUWE1 with shRNA results in growth arrest of MM cell lines (Blood 2016, 128:240). HUWE1 is involved in the regulation of MYC and its expression is deregulated in a number of tumor types. The aim of this study was to elucidate the role and therapeutic potential of inhibiting HUWE1 in MM using small molecule inhibitors.MethodsInhibitors of HUWE1 (BI8622, BI8626) as described in Peter et al. (EMBO Mol Med 2014, 6:1525-1541) were purchased from Syngene. The effect of the inhibitors on MM cell lines (OPM-2, KMS-18, JJN3, MM.1S, MM.1R, ANBL6.WT, ANBL6.BR), patient cells and normal bone marrow (NBM) from healthy donors was assessed using CellTitre-glo and WST1 assays. Cells were analysed for cell cycle status (propidium iodide and EdU staining), apoptosis and β-galactosidase activity. Snapshot Proteomics (AVM Biomed) was performed on HUWE1 knockdown cells to identify novel ubiquitinated substrates of HUWE1. Signalling pathways and substrates of HUWE1 were analysed using UbiQapture-Q (Enzo Life Sciences), Western blotting and RQ-PCR. Calcusyn software was used to calculate a combination index (CI) to assess synergistic anti-MM activity.ResultsUbiQapture assays demonstrated that BI8622 and BI8626 blocked auto-ubiquitination of HUWE1 in a concentration-dependent manner, with an associated decrease in the ubiquitination of MIZ1 (a validated HUWE1 substrate). BI8622 and BI8626 significantly reduced the growth of MM cell lines, including those resistant to dexamethasone (MM.1R) and bortezomib (ANBL6.BR), and patient-derived MM cells (p ≤ 0.01; IC50 range 9-20 µM) without significantly affecting NBM, suggesting a favourable therapeutic index. Furthermore, both compounds inhibit the proliferation of MM cell lines when co-cultured with MM patient-derived bone marrow stromal cells. HUWE1 inhibition induced cell cycle arrest, associated with a reduction of cells undergoing DNA synthesis (p = 0.005), cellular senescence demonstrated by β-galatosidase staining and increased p16 protein expression. HUWE1 inhibition in MM cell lines resulted in a significant decrease in MYC mRNA and protein expression (p < 0.001), accompanied by downregulation of multiple MYC-dependent target genes (CCND2, HSPE1, ODC1, CCNA2, CDC25A; p ≤ 0.02). Proteomic profiling along with ingenuity pathway analysis revealed novel putative substrates of HUWE1 involved in the regulation of MYC. Finally, the combination of HUWE1 inhibitors with proteasome inhibitors (carfilzomib and bortezomib) and dexamethasone results in synergistic anti-MM activity (CI range 0.57 - 1).ConclusionsMYC is frequently dysregulated in MM and represents an attractive therapeutic target. Here we demonstrate that inhibitors of the E3 ligase HUWE1 lead to growth arrest of MM cell lines, associated with decreased expression of MYC and MYC-dependent target genes. While BI8622 and BI8626 have previously been reported to repress MYC activity in models of colorectal cancer, we demonstrate for the first time that in MM this is accompanied with a decrease in MYC expression. Studies are ongoing to elucidate the molecular mechanism of MYC downregulation following HUWE1 inhibition both in vitro and in vivo. In summary, this study demonstrates that HUWE1 is important in the growth and survival of MM cells and presents a novel therapeutic strategy for the inhibition of MYC function in MM. DisclosuresNo relevant conflicts of interest to declare.

Exploring POU2AF1 (BOB-1) Dependency and Transcription Addiction in Multiple Myeloma

Multiple Myeloma (MM) is a complex disease driven by numerous genetic and epigenetic alterations. Recurrent IgH translocations, copy number abnormalities and somatic mutations all contribute to myelomagenesis, yet true drivers of the disease have yet to be identified. In order to investigate the enhancer landscape and identify new dependencies and actionable therapeutic targets in MM, we have generated high-quality active enhancer landscape in MM cell lines as well as in large cohort of primary patient myeloma cells (n=70) and normal plasma cell. We integrated this data with an in-house curated atlas of 600+ active enhancer profile across a wide range of tumor types and normal tissues. Combining this data with gene expression and genetic dependency (CRISPR KO) enables a multidimensional integration of how transcriptional regulation intersects with tumor specific dependencies. We identified genes that are super-enhancer associated, expressed, and required specifically in MM versus other tumor types or normal tissues. Many of the specific and potent dependencies in MM are transcription factors, and especially those establishing plasma cell identity. Among others, the POU2AF1 gene, which encodes the OCA-B/BOB-1, a B cell transcriptional coactivator protein, represented the most striking dependency in MM. BOB-1 is a gene regulatory factor that regulates B-cell development, maturation and GC formation. Although BOB-1 is expressed throughout B-cell development, we found it to be highly expressed in CD138+ plasma cells from patients with precursor conditions (MGUS and SMM) as well and established MM compared to normal plasma cells, giving us the rationale to study the transcriptional program associated with BOB-1 in MM cells. To confirm CRISPR data described above, we have performed loss-of-function (LOF) studies using shRNA, siRNAs as well as antisense GapMers specific for BOB-1. Downregulation of BOB-1 in a panel of MM cell lines caused MM cell growth inhibition in a time-dependent manner which was associated with G2/M cell cycle arrest and induction of MM cell apoptosis. Transcriptomic analysis by RNA-sequencing revealed a set of 72 genes (adj p-value=0.2) commonly modulated in AMO-1 and NCI-929 MM cell lines upon BOB-1 depletion, as compared with scrambled cells. Among the top 5 downregulated genes in BOB-1 knockdown samples, we found AMPD1, KCNN3, BHLHA15, HID1 and XBP1. The modulation of BOB1 target genes was confirmed at the protein level by Western blot analysis in BOB-1 LOF and gain-of-function (GOF) cell systems. Analysis of patient expression datasets revealed that these genes are positively correlated with BOB-1 expression in primary MM cells and overexpressed in MM cells compared to normal plasma cells. One of these 5 genes positively controlled by BOB-1, BHLHA15, is the gene coding for the transcription factor Mist1, which functions as a coregulator of the unfolded protein response (UPR) master transcription factor XBP1. Target genes of the XBP1- BHLHA15 axis are involved in lipid synthesis, protein folding and secretion, and ER-associated protein degradation. As a result, BOB-1 knockdown increased de novo protein synthesis in MM cells compared to control cells. Interestingly, Mist1 gene expression is induced during ER stress by XBP1, but as ER stress subsides, Mist1 serves as a feedback inhibitor. Moreover, we identified that LOF studies in two MM cell lines confirmed increased expression of BOB-1 and XBP1 upon KD of Mist1. However, XBP1 and/or Mist1 are dispensable for MM cell survival, as gene depletion did not affect MM cell viability. Among the genes most significantly upregulated by BOB-1 depletion was heme oxygenase 1 HMOX1 gene, whose expression is significantly lower in-patient MM cells compared to normal plasma cells. Moreover, its lower expression significantly correlated with poor clinical outcome; and siRNA depletion of HMOX1 increased MM cell growth in MM cell lines, suggesting important role in MM. Mechanistic studies aimed at investigating the functions of HMOX1 in MM and its relation with BOB-1 are ongoing and will be presented. In conclusion, we here report BOB1 as an important transcriptional regulator in MM and its two down-stream novel target genes (BHLHA15 and HMOX1) with potential significant biological role in MM. Disclosures Hajek: BMS: Consultancy, Honoraria, Research Funding; Oncopeptides: Consultancy; PharmaMar: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Fulciniti:NIH: Research Funding. Munshi:Janssen: Consultancy; C4: Current equity holder in private company; Karyopharm: Consultancy; Amgen: Consultancy; OncoPep: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents &amp; Royalties; BMS: Consultancy; Legend: Consultancy; Adaptive: Consultancy; AbbVie: Consultancy; Takeda: Consultancy.

PARP1-Mediated Alt-NHEJ Repair Is a Therapeutic Target in Multiple Myeloma

Background- Multiple Myeloma (MM) is a hematologic malignancy strongly characterized by genomic instability, which promotes disease progression and drug resistance. We previously demonstrated that LIG3-dependent Alt-NHEJ repair is involved in genomic instability, drug resistance and survival of MM cells. On these premises, we investigated PARP1 as driver component of Alt-NHEJ pathway and new therapeutic target in MM. Materials and methods- Cell proliferation and apoptosis were evaluated with CellTiter-Glo assay and Annexin V staining. Alt-NHEJ repair was evaluated using EJ2-GFP. PARP1, Caspase 3, MYC and DNA Damage Response protein levels were analyzed by Western blot of whole protein extracts. In vivo anti-MM activity was evaluated in NOD-SCID mice bearing subcutaneous H929 and AMO-1 Bortezomib resistant (ABZB) xenografts, daily treated with Olaparib (Selleckchem) via oral gavage. Results - By interrogating public available datasets, we found significant correlation between higher mRNA expression of PARP1 and shorter survival of MM patients. On these findings, we investigated the effect of available PARP inhibitors (PARPi) on MM cell survival. We found that Olaparib, a clinically available PARPi induced a significant reduction of proliferation and clonogenic growth of MM cell lines at low micromolar concentrations. Importantly, Olaparib impaired viability of MM cell lines or primary malignant plasmacells co-cultured with stromal cells, thus overcoming the bone marrow microenvironment supportive effect for MM survival. As result of PARP-mediated Alt-NHEJ repair inhibition, anti-proliferative effects were associated to increase of DNA double-strand breaks (DSBs), activation of DNA damage response, cell cycle arrest and finally apoptosis. To identify predictive biomarkers for PARPi in MM, a published sensitivity gene expression signature was applied to our MM gene expression profiling (GEP) data. Interestingly, this signature was particularly enriched in TC2 MM and secondary plasma cell leukemia (PCL). Therefore, in order to evaluate concordantly modulated sets of genes that were possibly associated to PARPi signature in MM, PARPi-positive and PARPi-negative MM-TC2 cases were compared by GSEA analysis. Interestingly, groups of genes regulated by MYC or involved in DNA repair resulted among the most significantly up-regulated in PARPi-positive versus PARPi-negative MM-TC2 cases. Accordingly, MYC transcript reached the highest median expression levels in sPCL and HMCLs across PC dyscrasia groups, and in MM-TC2 class. Consistently U266 cells, which was quiet insensitive to PARP knockdown or PARP inhibitor Olaparib, were null for c-MYC as compared to multiple myeloma cell lines evaluated in this study. Conversely, as formal proof of our hypothesis, over-expression of c-MYC in U266 cells (MYC-OE) induced cell death upon PARP silencing or PARP inhibitor treatment. Notably, we found that c-MYC-PARP1 loop was also hyper-activated in Bortezomib resistant cells, thus confirming pivotal role of Alt-NHEJ repair in drug resistance development. Remarkably, to demonstrate the in vivo relevance of our findings, we showed that clinically available Parp-inhibitor Olaparib exerted a significant anti-MM activity on both Bortezomib sensitive (H929) and resistant (ABZB) MM cells injected in immunocompromised mice. Conclusion Taken together, our findings indicate that MM cells are dependent on PARP-mediated Alt-NHEJ repair pathway, which therefore represents a novel druggable target pathway in MYC-driven MM cells. Disclosures No relevant conflicts of interest to declare.

Copanlisib, a novel phosphoinositide 3-kinase inhibitor, combined with carfilzomib inhibits multiple myeloma cell proliferation.

Multiple myeloma (MM) is a uniformly fatal disorder of B cells characterized by the accumulation of abnormal plasma cells. Phosphoinositide 3-kinase (PI3K) signaling pathways play a critical regulatory role in MM pathology. Copanlisib, also known as BAY80-6946, is a potent PI3Kα and δ inhibitor. In this study, we investigated the efficacy of copanlisib and a proteasome inhibitor using MM cell lines and primary samples. The p110α and δ catalytic subunits of the class PI3K increased, and carfilzomib activity reduced in the presence of a supernatant from the feeder cell line, HS-5. Phosphorylation of Akt and activation of caspase 3 and poly (ADP-ribose) polymerase (PARP) partially reduced upon carfilzomib treatment in the presence of HS-5. Apoptosis also decreased. Copanlisib treatment for 72h inhibited growth in MM cell lines and induced apoptosis. Combination treatment of MM cells with carfilzomib and copanlisib caused greater cytotoxicity than that caused by either drug alone and increased apoptosis. Caspase 3 activity increased while that of Akt decreased after combination treatment with copanlisib and carfilzomib. Further, copanlisib inhibited vascular endothelial growth factor (VEGF)-mediated angiogenesis in vitro and in vivo. It also inhibited C-X-C motif chemokine 12 (CXCL12)-mediated chemotaxis. The data suggest that administration of the PI3K inhibitor, copanlisib, may be a powerful strategy against stroma-associated drug resistance of MM cells and can enhance the cytotoxic effects of proteasome inhibitors in such residual MM cells.

PDK1 inhibitor GSK2334470 synergizes with proteasome inhibitor MG‑132 in multiple myeloma cells by inhibiting full AKT activity and increasing nuclear accumulation of the PTEN protein.

Phosphoinositide‑dependent kinase1 (PDK1) is generally active in multiple myeloma (MM) and higher expression than other hematopoietic cells, which is associated with the drug resistance and the disease progression. Previous studies have demonstrated that PDK1 can be targeted therapeutically in MM. In the present study, we examined the combination effect of GSK2334470 (GSK‑470), a novel and highly specific inhibitor of PDK1, with proteasome inhibitor MG‑132 in MM cell lines. GSK‑470 monotherapy significantly inhibited growth of MM cell lines and induced apoptosis that was associated with the activation of both the intrinsic mitochondrial pathway and the extrinsic death receptor pathway. Moreover, GSK‑470 demonstrated synergistic growth inhibitory effects with MG‑132. Notably, treatment with these inhibitors resulted in an almost complete inhibition of phosphorylation of mammalian target of rapamycin on Ser2448 and Ser2481 and full activation of AKT. The combination therapy also caused an upregulation of PTEN and an increased nuclear accumulation of PTEN protein. Collectively, our results provide the rationale for novel combination treatment with PDK1 inhibitor and proteasome inhibitors to improve outcomes in patients with MM.

YM155 exerts potent cytotoxic activity against quiescent (G0/G1) multiple myeloma and bortezomib resistant cells via inhibition of survivin and Mcl-1.

YM155, a novel small molecule inhibitor of survivin, shows broad anticancer activity. Here, we have focused on the cytotoxic activity of YM155 against multiple myeloma (MM) including cytokinetically quiescent (G0/G1) cells and bortezomib resistant cells. YM155 strongly inhibited the growth of MM cell lines with the IC50 value of below 10 nM. YM155 also showed potent anti-myeloma activity in mouse xenograft model. YM155 suppressed the expression of survivin and rapidly directed Mcl-1 protein for proteasome degradation. YM155 abrogated the interleukin-6-induced STAT3 phosphorylation, subsequently blocked Mcl-1 expression and induced apoptosis in MM cells. Triple-color flow cytometric analysis revealed that YM155 potently induced cell death of MM cells in G0 phase. Quiescent primary MM cells were also sensitive to YM155. We established bortezomib-resistant MM cell line, U266/BTZR1, which possess a point mutation G322A. YM155 exhibited similar cytotoxic potency against U266/BTZR1 compared with parental cells. Interestingly, survivin expression was markedly elevated in U266/BTZR1 cells. Treatment with YM155 significantly down-regulated this increased survivin and Mcl-1 expression in U266/BTZR1 cells. In conclusion, our data indicate that YM155 exhibits potent cytotoxicity against quiescent (G0/G1) MM cells and bortezomib-resistant cells. These unique features of YM155 may be beneficial for the development of new therapeutic strategies to eliminate quiescent MM cells and overcome bortezomib resistance.

Small molecule CP-31398 induces reactive oxygen species-dependent apoptosis in human multiple myeloma.

Reactive oxygen species (ROS) are normal byproducts of a wide variety of cellular processes. ROS have dual functional roles in cancer cell pathophysiology. At low to moderate levels, ROS act as signaling transducers to activate cell proliferation, migration, invasion, and angiogenesis. In contrast, high levels of ROS induce cell death. In multiple myeloma (MM), ROS overproduction is the trigger for apoptosis induced by several anticancer compounds, including proteasome inhibitors. However, no drugs for which oxidative stress is the main mechanism of action are currently used for treatment of MM in clinical situations. In this study, we demonstrate that the p53-activating small molecule CP-31398 (CP) effectively inhibits the growth of MM cell lines and primary MM isolates from patients. CP also suppresses the growth of MM xenografts in mice. Mechanistically, CP was found to induce intrinsic apoptosis in MM cells via increasing ROS production. Interestingly, CP-induced apoptosis occurs regardless of the p53 status, suggesting that CP has additional mechanisms of action. Our findings thus indicate that CP could be an attractive candidate for treatment of MM patients harboring p53 abnormalities; this satisfies an unmet clinical need, as such individuals currently have a poor prognosis.

TTI-281, an Orally Bioavailable Bromodomain and Extraterminal Domain (BET) Inhibitor, Is a Promising Candidate for the Treatment of Multiple Myeloma

Although recent advances in the development of multiple myeloma (MM) therapies such as proteasome inhibitors and immunomodulatory agents have improved patient outcomes, MM remains incurable. Additional therapeutic agents with high efficacy, low toxicity and the convenience of oral administration are in high demand. BET inhibitors, such as JQ-1, have been considered as potential therapeutic agents for MM. In the present study, we report that TTI-281, an orally bioavailable BET inhibitor, displays anti-MM activity with a low toxicity profile in preclinical studies.First, TTI-281 was tested for binding and anti-tumor activity in vitro. BROMOscan and AlphaScreen assays demonstrated that TTI-281 bound to bromodomains of BRD2/BRD3/BRD4 with Kd values less than 10 nM. In MTS assays, TTI-281 inhibited the growth of MM cell lines (MM.1s, NCIH929, and RPMI-8826) with cell growth-inhibition (IC50) values less than 300 nM.Next, in vitro ADME screening and in vivo PK studies were conducted. Permeability assays using murine gastrointestinal epithelial cells indicated that TTI-281 had good permeability with little efflux liability (efflux ratio <1), suggesting favorable properties for oral absorption. Indeed, TTI-281 displayed excellent oral bioavailability in both mice and rats (93.1% and 91.8%, respectively). In addition, TTI-281 did not interfere with the metabolism of representative CYP isozyme substrates at concentrations up to 50 μM in pooled human liver microsomes. Data also suggested minimal potential for drug-drug interactions, allowing for the possible combination with first-line therapy to improve therapeutic and survival outcomes.Finally, TTI-281 was tested for anti-myeloma efficacy and tolerability in vivo. NOD-SCID mice (n=10/group) subcutaneously engrafted with the human myeloma cell line MM.1S were treated orally once daily for 21 days with different doses of TTI-281, vehicle control or the benchmark drug carfilzomib. TTI-281 reduced tumor growth in a dose-dependent manner in this MM xenograft model. At 30 mg/kg/day, TTI-281 led to a statistically significant decrease in tumor growth compared with the vehicle control and carfilzomib (reduced tumor volume: 67% after TTI-281 treatment vs 33% after carfilzomib treatment, p<0.0003). Furthermore, TTI-281 treatment was well tolerated, with no effect on body weight or other obvious toxicity.In summary, our preclinical data suggest that the orally available BET inhibitor TTI-281 has an excellent efficacy and safety profile, highlighting its potential as a promising drug candidate for myeloma therapy. DisclosuresWang:Trillium Therapeutics: Employment, Patents & Royalties. Choi:Trillium Therapeutics: Employment. Dove:Trillium Therapeutics: Employment, Patents & Royalties. Wang:Trillium Therapeutics: Employment. Schimmer:Novartis: Honoraria. Petrova:Trillium Therapeutics Inc: Employment, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties. Uger:Trillium Therapeutics: Employment, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties. Slassi:Trillium Therapeutics: Employment, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties.

A Novel Agent SL-401 Triggers Anti-Myeloma Activity By Targeting Plasmacytoid Dendritic Cells: Implications for a Novel Immune-Associated Mechanism

Introduction Multiple myeloma (MM) remains incurable despite novel therapies, highlighting the need for further identification of factors mediating disease progression and resistance. Our studies have identified an integral role of bone marrow (BM) plasmacytoid dendritic cells (pDCs) in MM pathogenesis. The functional significance of increased numbers of pDCs in MM BM is evident from our observations that pDCs: are relatively resistant to novel and conventional therapies; protect tumor cells from therapy-induced cytotoxicity; promote tumor growth and survival; and suppress immune responses (Chauhan et al, Cancer Cell 2009, 16:309-323). Aberrant pDC function is evidenced in their interactions not only with MM cells, but also with other immune effector T cells and NK cells in the MM BM milieu (Ray et al, Leukemia 2015, 29:1441-1444). Directly targeting pDC interactions with MM and immune effector cells in the MM BM milieu will be required to enhance both anti-tumor immunity and cytotoxicity. However, therapies targeting pDCs are lacking. We found that IL-3R is highly expressed on pDCs, and that pDC-MM interactions trigger secretion of IL-3, which in turn, promotes both pDC survival and osteolytic bone disease. Recent efforts have led to the development of a novel therapeutic agent SL-401, which specifically targets IL-3R-expressing pDCs. Here we examined the effect of SL-401 on pDC-induced MM cell growth both in vitro and in vivo, as well as on IL-3R-expressing osteoclasts.Methods Patient MM cells, pDCs, and MNCs were obtained from normal donors or MM patients. Cell growth/viability was analyzed using MTT/WST assays. OCL function and bone resorption were measured using the OsteoAssays and TRAP staining. The RPMI-8226 cell line was used to isolate MM-SPs by flow-cytometry based Hoechst 33342 staining. SL-401 is a recombinant protein expressed in E. coli. The hybrid gene is comprised of human IL-3 fused to truncated diphtheria toxin (DT). The IL-3 domain of SL-401, which replaces the native binding domain of DT, targets SL-401 to cells that overexpress IL-3R. SL-401 was obtained from Stemline Therapeutics, NY; bortezomib, lenalidomide, pomalidomide, and melphalan were purchased from Selleck Chemicals. For animal model studies, SL-401 (16.5 μg/kg) was administered intravenously daily for 2 weeks.Results SL-401 triggered significant apoptosis in pDCs (>95%) at low picomolar concentrations that are well within clinically achievable doses.Higher concentrations of SL-401 trigger a modest apoptosis (30%± 1.3% apoptosis at 83 ng/ml or 1.3 nM) in MM cells due to lower IL-3R expression versus pDCs. Moreover, SL-401 did not significantly induce apoptosis of normal PBMCs (8% ± 0.5% apoptosis at 83 ng/ml), suggesting a favorable therapeutic index for SL-401. SL-401 inhibited pDC-induced growth of MM cell lines and patient MM cells in a dose-dependent manner. Moreover, 6 of 9 MM samples were obtained from patients whose disease was progressing while on bortezomib, dexamethasone, and lenalidomide therapies. Combinations of SL-401 with melphalan, bortezomib, lenalidomide, or pomalidomide induced synergistic anti-MM activity (Combination index < 1). SL-401 blocked monocyte-derived osteoclast formation in a dose-dependent fashion, as well as restored MM patient BM-derived osteoblast formation. Having defined the efficacy of SL-401 in targeting pDCs and pDC-triggered MM cell growth in vitro, we validated these findings in vivo using our murine xenograft model of human MM, under auspices of protocols approved by our institutional animal protection committee. SL-401 inhibited pDC-induced MM cell growth in vivo and prolonged survival in a murine xenograft model of human MM. We also evaluated the efficacy of SL-401 in vivo using our SCID-human (SCID-hu) mouse model, which reflects the human BM milieu with human cytokines and extracellular matrix proteins. SL-401 significantly abrogated pDC-triggered MM cell growth in vivo in SCID-hu model.Conclusions Our data provide the basis for using SL-401 to directly target pDCs and inhibit the pDC-MM interaction as well as target osteolytic bone disease in novel therapeutic strategies in order to enhance MM cytotoxicity, overcome drug resistance, and improve patient outcome. The interactions of immune effector cells in the MM tumor microenvironment also provide a rationale for combining SL-401 with checkpoint inhibitors.Correspondence: Dharminder Chauhan DisclosuresMacri:Stemline Therapeutics, Inc., New York, NY USA: Employment. Brooks:Stemline Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties. Rowinsky:Stemline Therapeutics: Employment, Equity Ownership. Richardson:Millennium Takeda: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gentium S.p.A.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees. Chauhan:Stemline Therapeutics: Consultancy.

The KDM3A-KLF2-IRF4 Axis Maintains Myeloma Cell Survival

Histone methylations are tightly regulated by a balance between methyltransferases and demethylases that mediate the addition and removal of these modifications. Importantly, dysregulation of histone methylation is implicated in pathogenesis of cancers, including multiple myeloma (MM). For example, the t(4;14) (p16;q32) is present in 15 - 20% of MM patients and results in overexpression of WHSC1, a histone H3 lysine 36 (H3K36) methyltransferase. On the other hand, approximately 10% of MM patients without the t(4;14) have inactivating mutations in KDM6A, a H3K27 demethylase.KDM3A is a Jumonji C-domain-containing histone demethylase which catalyzes removal of H3K9 mono- and dimethylation (H3K9me1 and H3K9me2). KDM3A is implicated in pathogenesis of different types of cancers. Here we investigated the biological impact of KDM3A in MM.KDM3A expression was significantly elevated in MM patient samples compared to normal plasma cells in publicly available dataset (GSE5900, GSE6691). To evaluate the functional role of KDM3A, shRNAs targeting KDM3A were transduced into MM cell lines: knockdown of KDM3A significantly inhibited MM cell growth (RPMI8226, MM.1S, U266, H929) in vitro and in xenograft model (MM.1S). Apo2.7 staining showed that apoptotic cells were significantly increased after knockdown of KDM3A.We next examined gene expression profiles after knockdown of KDM3A in RPMI8226 cells. With a cutoff of > 1.5-fold downregulation, a total of 305 probe sets were downregulated in KDM3A-knockdown cells relative to control cells. Among putative KDM3A targets, a gene of particular interest is KLF2 which plays a key role in maintenance of B cell and plasma cell phenotype, and function. Another intriguing gene is IRF4, given its known crucial role in MM cell survival. We confirmed that expression of KLF2 and IRF4 was downregulated after knockdown of KDM3A by quantitative realtime PCR and immunoblots in RPMI82226, MM.1S, and U266 cells. KDM3A binding to KLF2 and IRF4 core promoters was demonstrated by chromatin immunoprecipitation (ChIP) assay in RPMI8226 cells. Moreover, knockdown of KDM3A increased H3K9me1 and me2 levels at both promoter regions, indicating that KDM3A directly regulates KLF2 and IRF4 expression by removing H3K9 methylation marks at their promoters in MM cells.shRNAs targeting KLF2 were next transduced into MM cell lines: silencing of KLF2 significantly reduced cell growth of MM cell lines, associated with decreased IRF4. Promoter reporter assays using human IRF4 promoter showed that KLF2 significantly increased luciferase expression in a dose-dependent manner. Moreover, ChIP assay showed that KLF2 bound to IRF4 promoter in RPMI8226 cells. Since transcription factors could form an autoregulatory feedback loop, we hypothesized that IRF4 might regulate KLF2 expression. As expected, knockdown of IRF4 downregulated KLF2 expression at both the mRNA and protein levels in 3 MM cell lines. In addition, ChIP assays demonstrated that IRF4 bound to KLF2 second intron that contains tandem IRF4 motifs in RPMI8226 cells. Collectively, these results suggest that KLF2 activates IRF4 expression and vice versa, forming an autoregulatory loop in MM cells.KLF2 has been reported to control homing of plasma cells to the bone marrow; we therefore hypothesized that KDM3A-KLF2-IRF4 axis might regulate adhesion and homing of MM cells to the bone marrow. Importantly, knockdown of KDM3A, KLF2, or IRF4 decreased adhesion of 3 MM cell lines to bone marrow stromal cells. Furthermore, bone marrow homing of MM.1S cells was significantly reduced after knockdown of KDM3A, KLF2, or IRF4 in a murine xenograft MM model, indicating that KDM3A-KLF2-IRF4 axis regulates, at least in part, MM cell adhesion and homing to the bone marrow.In conclusion, our study demonstrated that KDM3A is a crucial epigenetic regulator of MM cell survival, and that inhibition of KDM3A represents a novel therapeutic strategy in MM. [Display omitted] DisclosuresRaje:Amgen: Consultancy; Takeda: Consultancy; Novartis: Consultancy; Celgene Corporation: Consultancy; BMS: Consultancy; Acetylon: Research Funding; Eli Lilly: Research Funding; Onyx: Consultancy; AstraZeneca: Research Funding; Millenium: Consultancy. Richardson:Gentium S.p.A.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Millennium Takeda: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees. Harigae:Chugai Pharmaceutical Co., Ltd.: Research Funding. Anderson:Oncopep: Equity Ownership; Gilead: Consultancy; BMS: Consultancy; Millennium: Consultancy; Celgene: Consultancy; Acetylon: Equity Ownership.

CD26 Is a Novel Target for the Treatment of Tumor Progression and Its Related Osteolytic Bone Disease in Multiple Myeloma

Background: CD26, a 110-kDa transmembrane glycoprotein with DPPIV activity, has been implicated in tumorigenesis and shown to be expressed in several tumor cells including malignant lymphoma, whereas its role has not been characterized in plasma cell malignancies, yet. We have recently shown that CD26 is intensely expressed in activated osteoclasts (OCs) in osteolytic bone tumors including multiple myeloma (MM). CD26 is not expressed in hematopoietic stem cells, but M-CSF and sRANKL induced human OC differentiation with the upregulation of CD26 expression in monocyte-macrophage lineage cells, OC precursor cells and OCs. Blockade of CD26 signaling with humanized anti-CD26 monoclonal antibody (YS110) impaired OC differentiation via inhibiting RANKL-induced MKK3/6-p38MAPK-mi/Mitf phosphorylation in OC precursor cells (Nishida et al., 2014). In the present study, we characterize the biological function of CD26 in MM cells in the bone marrow (BM) and investigate the therapeutic potential of YS110 to treat MM cell growth and reduce MM-induced osteolytic lesions.Methods and Results: Although CD26 expression was not detected any of 11 MM cell lines in mono-culture, CD26 expression levels were upregulated in all MM cell lines when co-cultured with OCs for 72 hours by flow cytometry and immunohistochemistry analysis. CD26 protein levels in MM cell lines were also enhanced in co-culture with OCs by immunoblotting. To determine the factors responsible for CD26 upregulation in MM cells, we examined CD26 expression in MM cell lines under transwell co-culture conditions with OCs or under stimulation with anti-apoptotic cytokines produced by OCs. CD26 expression in MM cell lines was reduced under transwell co-culture conditions with OCs. TNFα, BAFF, APRIL and SDF-1 slightly upregulated CD26 expression in MM cell lines. To further explore CD26 expression in BM of MM patients, we performed immunohistochemical stainig on decalcified bone sections of biopsy specimens of MM cases. CD26/CD138 positive plasma cells were detected around CD26 positive OCs and certain endothelial vascular cells in several cases. Next, to clarify the role of CD26 in MM cell survival, we examined the effects of YS110 on MM cell growth and related osteolytic bone lesions in vitro and in vivo. YS110 had no significant effects on viability of MM cell lines in mono-culture, but dose-dependently inhibited growth of MM cell lines in co-culture with OCs. YS110 immediately inhibited p38 activation and its downstream Hsp27 phosphorylation in MM cells. Contitnued treatment of MM cells with YS110 also led to the reduction of total Hsp27 protein at 12 hours and 16 hours after the treatment of YS110, with commensurate decrease of phospho-Hsp27 and to increased MM apoptosis correlated with the induction of p53, increased activation of caspases and PARP, as well as the reduction of Mcl-1 and c-Myc. In parallel, we constructed a xenograft of murine model of human MM. A total of 5x106 CD26 positive MM cell lines by co-culture with OCs were inoculated to mice (NOD/SCID-s.c. mice) or directly injected into the implanted human bone chips in mice (NOD/SCID-hu mice). Mice treated with YS110 (500μg/dose) showed a significant effect in inhibiting MM tumor weight compared with control mice 4 weeks after tumor cell inoculation. To further investigate whether YS110 could reduce MM-induced osteolysis as well as suppress MM cell growth, we used NOD/SCID-hu mice model. Mice were treated with YS110 after the first detection of tumor growth. Continuous YS110 treatment significantly suppressed MM cell growth after 4 weeks. Immunohistochemistry for CD138 and TRAP staining showed decreased numbers of MM cells and TRAP positive OCs, with decreased bone resorption activity in human bones from YS110-treated versus control mice. Lastly, we examined the effects of targeting CD26 with YS110 in CD26 positive myeloma stem-like cells (SP cells). CD26 expression was demonstrated in SP cells of several MM cell lines in co-culture with OCs. YS110 dose-dependently inhibited cell viability of CD26 positive SP cells co-cultured with OCs, indicating targeting CD26 might reduce chemoresistance in MM.Conclusions: Theses results suggest that CD26 may be a promising novel target in MM, strongly supporting YS110 to inhibit MM cell growth in the BM as well as its related osteolytic bone lesions. Our results provide the framework for clinical development of YS110 as a novel therapeutic option in MM. DisclosuresMorimoto:Y's Therapeutics: Consultancy, Research Funding. Yamada:Y's Therapeutics: Consultancy, Research Funding.

Icaritin suppresses multiple myeloma, by inhibiting IL-6/JAK2/STAT3.

Icaritin is an active prenylflavonoid derived from Epimedium genus, a traditional Chinese medicine. Icaritin has a wide range of pharmacological and biological activities, including cardiovascular function improvement, hormone regulation and antitumor activity. Here, we investigated the effect of icaritin on multiple myeloma (MM) in vitro and in vivo. Icaritin inhibited cell growth of MM cell line and primary MM cells. In contrast, icaritin had low or no cytotoxic effect on normal hematopoiesis. We also demonstrated that in MM xenograft mouse models, icaritin suppressed tumor growth and decreased serum IL-6 and IgE levels, but did not show adverse reactions such as body weight loss. The anti-MM activity of icaritin was mainly mediated by inhibiting IL-6/JAK2/STAT3 signaling. We suggest that icaritin can be further tested in clinical trials in MM.

Effect of a novel agent, SL-401, targeting interleukin-3 (IL-3)-receptor on plasmacytoid dendritic cell (pDC)-induced myeloma cell growth and drug resistance.

8582 Background: Multiple myeloma (MM) remains incurable despite novel therapies, highlighting the need for further identification of factors mediating disease progression and drug resistance. The bone marrow (BM) microenvironment confers growth, survival, and drug resistance in MM cells. Our recent study utilized in vitro and in vivo MM xenograft models to show that plasmacytoid dendritic cells (pDCs) were significantly increased in MM BM and promote MM growth (Chauhan et al., Cancer Cell 2009, 16:309). Importantly, we found increased IL-3 levels upon pDC-MM interaction, which in turn, trigger MM cell growth and pDCs survival. IL-3R is highly expressed on pDCs. We utilized SL-401, a novel biologic conjugate that targets IL-3R, to examine whether abrogation of IL-3–IL-3R signaling axis affects pDC-MM interaction and its tumor promoting sequelae. Methods: MM cell lines, patient MM cells, and pDCs from healthy donors or MM patients were utilized to study the anti-MM activity of SL-401. MM cells and pDCs were cultured alone or together in the presence or absence of SL-401, followed by analysis of cell growth or viability. Results: SL-401 significantly decreased the viability of pDCs at low concentrations (IC50: 0.83 ng/ml; P &lt; 0.005, n = 3). SL-401 also decreased the viability of MM cells at clinically achievable doses. Co-culture of pDCs with MM cells induced growth of MM cell lines; and importantly, low doses (0.8 ng/ml) of SL-401 blocked MM cell growth-promoting activity of pDCs. MM patient-derived pDCs induced growth of MM cell lines and primary MM cells as well; conversely, SL-401 inhibited pDC-triggered MM cell growth (P &lt; 0.005, n= 5). Tumor cells from 3 of the 5 patients were from patients whose disease was progressing while on bortezomib, dexamethasone, and lenalidomide therapies. In agreement with these results, SL-401 blocked pDC-induced growth of dexamethasone-resistant MM cell lines. Conclusions: Our study therefore provides the basis for directly targeting pDCs or blocking the pDC-MM interaction, as well as targeting MM, in novel therapeutic strategies with SL-401 to enhance MM cytotoxicity, overcome drug-resistance, and improve patient outcome.

HM1.24/CD317-directed immunotoxin to eliminate malignant plasma cells in vitro and in vivo.

8604 Background: Targeted immunotherapy, based on antibodies against tumor-associated antigens, is a promising approach for the treatment of multiple myeloma (MM). Recently, antibody-based strategies delivering a toxic payload have documented impressive clinical activity in hematological malignancies. In particular, surface molecules overexpressed on malignant plasma cells and efficiently internalized represent promising targets for developing myeloma-directed immunoconstructs. Here, the identification of CD317 (HM1.24) as a potent target structure and the characterization of a novel CD317-directed single-chain immunotoxin, HM1.24-ETA', is described. Methods: Using a novel screening tool, a panel of antibodies against MM-associated antigens was evaluated for their ability to mediate antigen-dependent delivery of a truncated version of Pseudomonas exotoxin A (ETA’) to MM cells. HM1.24-ETA' was generated by genetic fusion of a CD317-specific single-chain Fv antibody and ETA'. The anti-myeloma activity of the E. coli-expressed immunotoxin was evaluated in vitro and in a xenograft mouse model. Results: By screening a panel of antibodies including CD38, CS1, IL-6R, CD138 and CD317, CD317 was identified as a suitable receptor to deliver ETA’ to MM cells. The subsequently designed recombinant HM1.24-ETA' immunotoxin efficiently inhibited growth of MM cell lines with halfmaximal growth inhibition at concentrations of less than 1 nM. Antigen-specific MM cell killing occurred via induction of apoptosis. The proliferation of IL-6 dependent INA-6 cells was completely inhibited by HM1.24-ETA' even in the presence of bone marrow stromal cells that otherwise strongly support tumor cell growth. Importantly, HM1.24-ETA' strongly triggered apoptosis (up to 80%) in freshly isolated tumor cells from 7 out of 7 MM patients. In a xenograft SCID mouse model, establishment of INA-6 plasma cell tumors was efficiently abrogated by treatment with HM1.24-ETA' immunotoxin (p &lt; 0.04). Conclusions: The HM1.24-ETA' immunotoxin in vitro and in the preclinical xenograft model in vivo demonstrates that the CD317 antigen may represent a promising target structure for immunotherapy of MM using immunoconjugates with toxic payloads.

Abstract 4729: MAGE-C2 promotes proliferation and enhances resistance to p53 DNA damage-mediated apoptosis in multiple myeloma.

Abstract Background: MAGE-C2 is member of the MAGE class I family of genes and is commonly and specifically expressed in Multiple Myeloma (MM). Expression of MAGE genes is associated with an aggressive clinical course of MM and resistance to chemotherapy, suggesting that these genes may confer a survival advantage on myeloma cells. Here, we have analysed on a molecular level the influence of MAGE-C2 on the malignant phenotype of myeloma cells. Methods: The biological function of MAGE-C2 was investigated, by lentiviral shRNA stable gene silencing, in two MM cell lines (MOLP-8, IM-9) expressing wild-type (wt) p53 and two MM cell lines (U-266, SK-007) bearing a missense mutant p53 (A161T) that has partially lost its transcriptional activity. The effects of MAGE-C2 silencing were examined by assessing cell viability, proliferation and colony formation. Finally, we investigated the cell cycle distribution and the expression of key molecules of cell cycle progression and apoptosis using a real-time PCR array and western blot. Results: We found that lentivirus-mediated silencing of MAGE-C2 inhibited the proliferation and anchorage-independent growth of MM cell lines. The anti-proliferative effect of MAGE-C2 silencing was due to a 50-80% decrease of cells in the S phase, a cell cycle arrest at both G0/G1 and G2/M transitions, and an increase in the subG0/G1 diploid population based on an initiation of apoptotic cell death. Loss of MAGE-C2 expression was associated with an increase in the phosphorylation of the histone variant H2A.X on Ser139 which is a sensitive indicator of DNA damage. A constitutive phosphorylation on Ser1981 and Ser428, respectively, of the primary sensors of DNA damage ataxia telangiectasia mutant (ATM) and Rad3-related protein kinase (ATR) was detected before MAGE-C2 silencing suggesting the presence of an ongoing DNA damage in MM cell lines. Downstream of ATM and ATR, MAGE-C2 silencing led to the activation of CHK2 and/or CHK1 through phosphorylation at Ser296 and Thr68, respectively, and an increase in the endogenous level of p53 protein. The stabilization and activation of p53, through phosposphorylation at Ser20 by CHK1 and CHK2, correlated with an up-regulation of two transcriptional target genes, p21WAF1 and GADD45A, known to be essential for p53-induced G1 and G2 arrest, respectively. Accordingly, MAGE-C2 silencing induced a more than two-fold increase in the expression of the pro-apoptotic Bcl-2 genes such as BAX and BAK, being the main down-stream mediators of p53-dependant apoptosis following DNA damage. Conclusions: Our findings support an anti-apoptotic function of MAGE-C2 in MM through the regulation of key molecules involved in DNA damage repair pathway and p53-mediated apoptosis. The central role of MAGE-C2 in the biology of myeloma suggests that this CTA represents a promising target for myeloma-specific immunotherapies or other targeted modes of therapy for MM. Citation Format: Nesrine Lajmi, Sara Yousef, Tim Luetkens, Julia Templin, Carsten Bokemeyer, Nicolaus Kröger, Djordje Atanackovic. MAGE-C2 promotes proliferation and enhances resistance to p53 DNA damage-mediated apoptosis in multiple myeloma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4729. doi:10.1158/1538-7445.AM2013-4729

TC11, a Novel Phthalimide Derivative, Directly Binds to NPM1 and Induced Apoptosis of High-Risk Myeloma Cells Via Centrosomal Disruption

Abstract 1841 PURPOSE:Despite recent advances in the treatment of multiple myeloma (MM), patients with high-risk chromosomal changes such as del13q, t(4;14) or del17p revealed significantly shorter survival. In addition, bone disease markedly reduces quality of life of the patients with MM. To overcome these problems, we have designed and screened synthetic phthalimides which significantly inhibited the growth of MM cell lines with high-risk chromosomal abnormalities. The purposes of this study are to explore novel drugs which possess anti-tumor activity against high-risk MM cells and to examine the anti-osteoclastogenic activity and to isolate directly binding molecules. METHODS AND RESULTS:Thirty synthetic phthalimides were screened for anti-proliferative effect on KMS34 cells with t(4;14) and deletion of chromosome 17. A phthalimide derivative, 2-(2,6-diisopropylphenyl)- 5-amino- 1H-isoindole- 1,3- dione (TC11) significantly inhibited growth of KMS34 cells as well as other MM cells lines with high-risk chromosomal abnormalities (IC50 to KMS34 cells= 4μM). TC11 increased annexin V fraction and induced apoptosis in a caspase-dependent manner. In vivo anti-myeloma activity was evaluated using KMS34-bearing lcr/SCID mice by intraperitoneal injection of TC11. Twenty mg/kg of TC11 significantly inhibited growth of TC11-derived tumor cells, and apoptosis of MM cells was observed by histopathological examination. In order to evaluate hematological toxicity of TC11, growth of colony-forming cells was examined. In the presence of 5μM of TC11, formation of CFCs was not suppressed, suggesting low hematopoietic toxicity. In pharmacokinetic study using lcr/SCID mice, the plasma concentrations of TC11 was examined; Cmax=18.1μM, Tmax=1.5hr, T1/2 =4.5hr when 100mg/kg of TC11 was injected, and Cmax=2.1μM Tmax=1.0hr, T1/2 =4.5hr when 20mg/kg was injected. In order to examine efficacy to bone disease, anti-osteoclastogenic activity was examined by adding TC11 to M-CSF/RANK ligand-induced osteoclastogenic culture of mouse bone marrow mononuclear cells. The number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclasts was reduced in the presence of 1μM of TC11. It was also found that TC11 inhibited bone resorption by pit assay. We also tried to isolate directly binding proteins to TC11 by our unique in vitro selection system using mRNA display, in vitro virus (IVV) method. We identified nucleophosmin 1 (NPM1) as a TC11-binding molecule. Knockdown assay introducing siRNA for NPM1 into HeLa cells induced emergence of the cells with multipolar spindles, suggesting centrosomal disruption during cell division. Since NPM1 gene localizes at chromosome 5q, anti-MDS effect of TC11 was also examined. TC11 also inhibited growth of MDS-L cells (IC50=7μM). CONCLUSION:A novel phthalimide derivative, TC11, has anti-MM activity in vivo and is a potentially effective drug for high-risk MM with bone lesions. TC11 directly binds to NPM1 and induces apoptosis of MM and MDS cells with low toxicity to normal hematopoiesis. Disclosures:No relevant conflicts of interest to declare.

Deregulated Cellular Iron Metabolism Factors Mediate Iron Overload in Myeloma Cells and Osteoclasts, and Promote Myeloma Growth and Bone Disease,

Abstract 3941Iron overload is a significant clinical feature in multiple myeloma (MM) and has been implicated in osteoporosis. MM patients also frequently suffer from anemia presumably due to elevated hepcidin secretion and dysfunctional erythropoiesis. The aims of the study were to shed light on molecular mechanisms associated with iron overload in MM cells and study the effect of the novel iron chelator, Dp44mT, on MM cell growth, osteoclastogenesis and MM bone disease in vitro and in vivo. In our clinical global gene expression profiling (GEP) data the main iron transporter gene TFRC (transferrin receptor) was >3 folds higher (p<0.0001) in newly diagnosed MM cells (n=556) than normal plasma cells (n=25) while the iron exporter ferroportin was downregulated in MM cells by >4 folds (p<0.0001). Deregulated TFRC and ferroportin expression were more profound in the molecularly classified proliferation (PR) subtype. Osteoclasts which are known to have abundant mitochondria due to high energy consumption express excessive TFRC (>5 folds higher than highly proliferating MM cells). In primary MM cell-osteoclast cocultures (n=8) TFRC expression was upregulated in cocultured MM cells than baseline MM cells (p<0.03) while ferroportin was lower in cocultured osteoclasts than control osteoclasts (p<0.04). Our GEP, qRT-PCR and immunohistochemistry analyses revealed expression of hepcidin by osteoclasts but not MM cells. Hepcidin was not detected in conditioned media from osteoclasts cultured alone or cocultured with MM cells using ELISA, suggesting an autocrine role of hepcidin in maintaining excess iron in osteoclasts. In vitro, Dp44mT dose dependently inhibited growth of MM cell lines (n=3) at low nanomolar levels (IC50 at 3±0.8 nM, p<0.03, 48 hrs). In contrast, known chelators such as Deferoxamine and Deferasirox inhibited myeloma cell growth at 10–50 micromoles range. At 1nM Dp44mT also suppressed formation of multinucleated osteoclasts by 87% (p<0.001) and bone resorbing activity of mature osteoclasts on dentine slices by 94% (p<0.03). Dp44mT induced upregulation of BMP2 expression in osteoblast precursors and promoted osteoblast differentiation. In vivo, SCID-rab mice engrafted with luciferase-expressing U266 MM line (6 mice/group) or the Hg MM line (maintained through in vivo passaging, 10 mice/group) were subcutaneously treated with vehicle or Dp44mT (1 mg/kg/day) for 2–3 weeks. Using live-animal imaging, Dp44mT reduced growth of U266 cells by 3 folds from pretreatment levels (p<0.01) while in control group tumor burden was increased by 52 folds from pretreatment levels (p<0.002). Dp44mT also inhibited growth of Hg MM cells determined by measurement of circulating human immunoglobulins in mice sera (p<0.01). Osteoclasts numbers were lower by 36% (p<0.003) while osteoblasts numbers were higher by 59% (p<0.017) in myelomatous bones from hosts treated with Dp44mT than control vehicle. Our data suggest that interaction of myeloma cells with osteoclasts alters expression of distinct iron metabolism associated factors which elicit iron overload in both cell types, resulting in increased myeloma cell proliferation and osteoclast activity. This study also suggests that Dp44mT is an effective iron chelator with marked anti-MM activity. Disclosures:Barlogie:Celgene, Genzyme, Novartis, Millennium: Consultancy, Honoraria, Patents & Royalties. Shaughnessy:Myeloma Health, Celgene, Genzyme, Novartis: Consultancy, Employment, Equity Ownership, Honoraria, Patents & Royalties.