RPMI-8226 Multiple Myeloma Research Articles

Preclinical and Nonclinical Characterization of HPN217: A Tri-Specific T Cell Activating Construct (TriTAC) Targeting B Cell Maturation Antigen (BCMA) for the Treatment of Multiple Myeloma

About 31,000 new cases of multiple myeloma (MM) will be diagnosed in the US in 2018. In addition to chemotherapeutic agents, several targeted therapies utilizing distinct mechanisms of action, e.g., proteasome inhibitors (bortezomib, carfilzomib, ixazomib), HDAC inhibitors (panobinostat), Cullin-RING E3 ubiquitin ligase activators (thalidomide, lenalidomide, pomalidomide), and antibodies (daratumumab, elotuzumab) have become available for treating MM. However, MM remains an incurable disease. Patients who relapse after or are refractory to standard of care treatments generally have poor prognosis. In 2018, close to 13,000 patients will die of the disease in the US. Targeting the B cell maturation antigen (BCMA), a BAFF/BLyS and APRIL receptor, for treating MM patients can provide a new treatment approach complementary to existing therapies. CAR-T therapies and an antibody-drug conjugate targeting BCMA have demonstrated early clinical success in the treatment of relapsed refractory MM (RRMM).HPN217 is a tri-specific T cell activating construct (TriTAC) consisting of three binding domains: an N-terminal single domain antibody (sdAb) that binds to human BCMA, a middle sdAb that binds to human serum albumin (HSA), and a C-terminal single chain Fv (scFv) that binds to CD3ε of the T cell receptor (TCR) complex. HPN217 is a highly stable single polypeptide of ~ 53 kDa expressed by CHO cells. Simultaneous engagement of BCMA on a target MM cell and CD3 on a T cell results in T cell activation, functional differentiation and the eventual lysis of the target MM cell. Engineering of an HSA binding domain into HPN217 represents a unique strategy in extending serum half-life, giving the TriTAC molecule a small molecular size and flexibility. This approach is different from Fc-engineering applied in other CD3-based bispecific T cell engaging molecules.The KD of HPN217 binding to recombinant human BCMA, HSA, and recombinant human CD3ε was determined to be 5.5 nM, 6 nM, and 17 nM, respectively, as measured by biolayer interferometry. Flow cytometric analysis on a panel of T cells from normal donors and BCMA positive and BCMA negative tumor cell lines confirmed binding of HPN217 to its native targets expressed on cell surface. The in vitro pharmacological activity of HPN217 was evaluated by T cell-dependent cellular cytotoxicity (TDCC) assays. In co-cultures of T cells from normal human or cynomolgus monkey donors, target tumor cells, and HSA, HPN217 mediated dose-dependent and BCMA-dependent cytotoxicity with EC50 values ranging from 0.05 to 0.7 nM. Killing was dependent on expression of BCMA on target tumor cells. Concomitant with target tumor cell killing, HPN217 also mediated dose-dependent upregulation of CD25 and CD69 on T cells in the TDCC co-cultures when BCMA positive tumor cells were presence. Consistent with the mechanism of action of CD3-based T cell engaging molecules, T cell derived cytokines, e.g., TNFα and IFNγ, were detected. Similar T cell activation could be observed using human or cynomolgus monkey whole blood as a source of T cells.Nonclinical in vivo properties of HPN217 were evaluated in xenograft models and a single dose pharmacokinetic (PK) study in cynomolgus monkeys. HPN217 mediated dose-dependent growth suppression against the RPMI-8226 MM model and Jeko-1 mantle cell lymphoma model expressing relatively low levels of 5,600 and 2,200 copies of BCMA per cell, respectively. In the PK study, a single dose of HPN217 at 0.01, 0.1, or 1 mg/kg was given to cynomolgus monkeys. HPN217 exhibited linear PK behavior over this dose range. Serum half-life was in the range of 64 to 85 hours. Serum half-life, volume of distribution, and clearance appeared to be independent of dose. HPN217 was demonstrated to be stable and remained intact up to 3 weeks in vivo as demonstrated by a functional ligand binding assay using recombinant CD3ε and BCMA, respectively, to capture and detect HPN217. Importantly, serum samples collected one week after dosing were as potent as stock HPN217 in MM tumor cell killing in TDCC assays.Collectively, preclinical and nonclinical characterization suggests that HPN217 is an efficacious novel therapeutic candidate that can provide a convenient dosing schedule for patients. A first-in-human phase 1 clinical trial is planned to evaluate HPN217 in RRMM. DisclosuresLaw:Harpoon Therapeutics: Employment. Aaron:Harpoon Therapeutics: Employment. Austin:Harpoon Therapeutics: Employment. Barath:Harpoon Therapeutics: Employment. Callihan:Harpoon Therapeutics: Employment. Evans:Harpoon Therapeutics: Employment. Gamez Guerrero:Harpoon Therapeutics: Employment. Hemmati:Harpoon Therapeutics: Employment. Jones:Harpoon Therapeutics: Employment. Kwant:Harpoon Therapeutics: Employment. Lao:Harpoon Therapeutics: Employment. Lemon:Harpoon Therapeutics: Employment. Patnaik:Harpoon Therapeutics: Employment. Sexton:Harpoon Therapeutics: Employment. Wesche:Harpoon Therapeutics: Employment. Xiao:Harpoon Therapeutics: Employment. Yu:Harpoon Therapeutics: Employment. Yu:Harpoon Therapeutics: Employment.

Evaluation of Oncolytic Viral Therapy Combined with or without Artificial Immunoadjuvant Cell Therapy in Immunocompetent Mouse Tumor Models

Oncolytic viruses have a great potential to provide clinical improvement for cancer patients who do not respond to conventional therapies. The mechanisms of action include direct infection, immune-mediated cell death, and vascular collapse. We previously reported that a miRNA-regulated, thymidine kinase-deleted vaccinia virus (MDVV) specifically infects multiple myeloma (MM) cells and showed that the intravenous injection of MDVV significantly prolonged survival of severe combined immunodeficiency mice harboring a subcutaneous human RPMI8226 MM tumor (Blood 2014:124:2082, Mol Ther Oncolytics 2017;6:57). Although it was highly effective, there remained to be confirmed whether the MDVV would be similarly effective in the immunocompetent setting. To address this question, we performed several in vivo infection using immunocompetent C57BL/6 (B6) models.A vaccinia virus (VV) strain LC16, which was derived from the Lister strain, was widely used in 1970s for the smallpox eradication program with no severe adverse effect. Because its safety is robust, we used the LC16 for the backbone of the MDVV. To improve its safety and tumor specificity, we deleted viral thymidine kinase gene, and introduced miRNA (let-7a)-target sequences into the critical viral B5R gene. Because let-7a is abundant in normal differentiated tissues, and the endogenous let-7a down-regulates viral B5R gene, the MDVV becomes far more tumor specific (Mol Ther Oncolytics 2017;6:57).To observe whether the MDVV protects immunocompetent mice from MM, we generated a mouse MM model by transplanting 1x10^6 Vk*Myc tumor cells into syngeneic B6 mice. After confirming M-proteins in the serum, 1x10^7 pfu of MDVV was injected intravenously. Contrary to our expectation, we could not observe a clearly infected region, nor a prolongation of survival of these mice; probably due to difference in infectivity between species. Although mouse derived cell lines are generally insensitive to vaccinia virus as compared with human cell lines, a mouse lung carcinoma cell line 3LL showed a relatively high infectivity to VV in our screening in vitro infection experiment (Figure), therefore we used the B6/3LL subcutaneous tumor model instead of B6/Vk*Myc model.The B6/3LL mice were injected with mock or 1x10^7 pfu MDVV intravenously when the subcutaneous tumor became more than 5 mm in diameter. Although viral signal (determined by in vivo imaging using luciferase) disappeared 3 days after infection, the therapeutic benefit had remained, and prolongation of survival was observed Because natural killer (NK) cells were thought to play in the clearance of VV in the B6 mice, we attempted to extend the duration of infection by depleting NK cells using anti-asialo GM1 antibody, which, we thought, enhances viral oncolytic activity. As expected, we observed a persistent viral signal for up to 2 weeks in anti-asialo GM1 antibody treated B6/3LL mice. However, the anti-tumor effect seen with anti-asialo GM1 antibody was rather milder than that of without anti-asialo GM1 antibody, indicating the importance of NK activities in anti-tumor immunity.Based on this finding, we, in reverse, tried to stimulate the anti-tumor immunity using artificial immunoadjuvant cells co-expressing CD1d and tumor antigen/GM-CSF fusion protein. The CD1d is one of non-classical MHC molecules and is involved in the presentation of lipid antigens. When α-GlaCer is loaded on the CD1d, it activates natural killer T (NKT) cells through the interaction with invariant T cell receptor on NKT cells. When activated, NKT cells produce Th1 and Th2 cytokines, and activate NK cells. GM-CSF stimulates antigen-presenting cells (APCs), and fusion proteins of tumor antigen and GM-CSF are more efficiently taken up by APCs than tumor antigen alone (Nature 1993, 362:755). We generated the artificial immunoadjuvant cells by transducing 3LL tumor cells with lentiviral vectors expressing CD1d and ovalbumin (OVA)/GM-CSF fusion protein. After loading with α-GlaCer, the immunoadjuvant cells were irradiated with 50Gy, and injected intravenously into B6/3LL-OVA mice. We observed a significant reduction in tumor mass and prolongation of survival as compared with mock-injected mice. Currently, we are exploring the optimal setting of combination with MDVV virotherapy and immunoadjuvant cell therapy. In this meeting, we will present these results. DisclosuresNo relevant conflicts of interest to declare.

Interleukin-6 Adversely Impacts Genomic Stability Via Targeting Multiple Pathways in Multiple Myeloma

In our previous investigation, using whole exome sequencing, we have shown that multiple myeloma (MM) patients display a complex dynamic of clonal evolution and the number of mutations in patient samples correlate with overall and relapse free survival. These results highlight the importance of understanding the mechanisms driving genomic instability in MM. Investigating mechanisms underlying genomic instability, we have shown that dysregulated homologous recombination (HR), nuclease (especially apurinic/apyrimidinic related) and APOBEC deaminase activities contribute to genomic instability in MM. We have also demonstrated that bone marrow microenvironment (BMM) also contributes to genomic instability in MM. So here we have further investigated the soluble factors in BMM that may impact genomic integrity. We treated RPMI8226 MM cells with IL-6, IL-17 and TGFb and evaluated their impact on DNA breaks by monitoring the levels of gH2AX (a DNA break marker). Treatment with all 3 cytokines (IL-6, IL-17 and TGFb) caused DNA breaks, as demonstrated by increase in gH2AX, in MM as well as a solid tumor (esophageal cancer) cell lines. Importantly, among these cytokines, the exposure to IL-6 was associated with the highest induction of gH2AX expression. These observations were confirmed in additional MM cell lines (MM1S, H929, OPM2 and U266) treated with IL-6.Since we have previously demonstrated that elevated HR is a key mechanism of genomic instability in MM, we investigated the role of IL-6 in dysregulation of HR pathway by evaluating its impact on p-RPA32 (a marker of DNA end resection which is a decisive step in the initiation of HR), recombinase (RAD51) expression and HR activity (as assessed by a functional assay). Treatment of MM cells with IL-6 led to increased expression of p-RPA32 and RAD51 (as detected by Western blotting) as well as increased HR activity in MM cells. Increased HR activity was also observed following exposure of MM cells to IL-17, TGFb and IFNb, with the highest induction caused by IL-6. However, the combination of cytokines (IL-6, IL-17 and TGFb) led to a further (> 2-fold) increase in HR activity in these cells, indicating that these soluble factors may interact in inducing mechanisms underlying genomic instability in MM.Based on our data showing important role of apurinic/apyrimidinic (AP) nuclease in regulation of HR and genome stability in MM, we also evaluated the impact of IL-6 on abasic sites, the substrate of AP nuclease activity. An increase in the number of abasic sites (ranging from 1.7- to 2.3-fold increase) was observed with increasing concentration of IL-6 in MM1S cells. To further investigated the impact of IL-6 on number of micronuclei, used as marker of genomic instability. The treatment of MM cell lines with IL-6 for 48 hrs led to a dose-dependent increase (ranging from 2- to > 2.5-fold increase) in the number of micronuclei in MM1S and RPMI cells, indicating increased genomic instability. We have previously shown that inhibition of HR by RAD51 knockdown or ABL kinase inhibitor, nilotinib, significantly reduces genomic instability, as assessed by micronuclei assay as well as direct evaluation of impact on genomewide acquisition of copy number changes over time using SNP arrays. To investigate if HR inhibition can reverse IL-6-induced genomic instability, we treated MM cells with IL-6, nilotinib and combination of both and observed that inhibition of HR by nilotinib can reverse IL-6-induced increase in number of micronuclei (by 48±17%) in MM cells. Similarly, addition of anti-IL-6 antibody also reversed the IL6-induced DNA breaks (as assessed from gH2AX expression) in MM1S cells. Moreover, combination of nilotinib and anti-IL6 antibody resulted in maximum inhibition of IL6-induced DNA breaks in MM1S cells.Taken together, these data suggest that IL-6 significantly contributes to dysregulation of HR and genome stability in MM, and agents targeting HR and/or other mechanisms of genomic instability including anti-IL-6 antibody, have potential to reduce/delay genomic evolution and disease progression. DisclosuresMunshi:OncoPep: Other: Board of director.

The short-term effect of histone deacetylaseinhibitors, chidamide and valproic acid, ontheNF‑κBpathway in multiple myeloma cells.

Research regarding histone deacetylase (HDAC) inhibitors (HDACis) has garnered interest for the treatment of multiple myeloma (MM). In addition, the high expression of nuclear factor (NF)-κB in MM cells is considered an important factor in the occurrence and development of MM. The present study aimed to determine the short-term effects of HDACis, chidamide and valproic acid (VPA), on MM cells, their effects on NF-κB and the underlying mechanisms. The present study measured HDAC activity, and the proliferation and apoptosis of U266 and RPMI8226 MM cells following treatment with various concentrations of chidamide and VPA for 6 and 48 h. Western blotting was used to detect the expression levels of phosphorylated (p)-IκB kinase (IKK)α/β, NF-κB p65 and inhibitor of NF-κB (IκBα) in U266 and RPMI8226 cells at various time points following treatment with chidamide and VPA (0, 2, 4 and 6 h). The results revealed that chidamide and VPA had no significant effect on the HDAC activity, proliferation and apoptosis of cells at 6 h; however, cell HDAC activity and proliferation were inhibited, and apoptosis was induced at 48 h. Furthermore, the expression levels of IκBα were gradually increased over time, whereas the expression levels of NF-κB p65 gradually decreased. These findings indicated that long-term (48 h) treatment with the HDACis chidamide and VPA inhibited the proliferation and promoted the apoptosis of MM cells; however, these HDACis had little effect on cell proliferation and apoptosis in the short term (6 h). Notably, in the short term (2-6 h), hyperactivation of NF-κB was inhibited via the IκBα-NF-κB p65 pathway. These findings indicated that cell growth may be inhibited and drug susceptibility may be promoted by blocking the NF-κB pathway at an early stage, when HDACis are combined with other drugs in the treatment of MM.

Low dose dinaciclib enhances doxorubicin-induced senescence in myeloma RPMI8226 cells by transformation of the p21 and p16 pathways.

Multiple myeloma (MM) is a hematological malignancy that lacks a cure. However, novel combination therapy is a current anti-MM strategy. Doxorubicin (DOX) is a type of anthracycline which is a first-line chemotherapeutic for treating MM and induces senescence in many types of cancer. Dinaciclib is a potent, small molecule CDK inhibitor with promise for treating several types of cancer in I/II phase clinical trials. In the present study the anticancer effects and underlying mechanisms of dinaciclib combined with DOX in MM RPMI-8226 cells were investigated. Results indicated that DOX induced cell viability inhibition, cell cycle arrest and senescence. Furthermore, DOX resulted in increased alterations in DNA damage-related proteins such as p-ATM, p-Chk2, p-p53, p21 and γH2AX, but not p16. Notably, the combination of dinaciclib and DOX inhibited cell growth and promoted senescence by transforming the suppressive effects of the ATM/Chk2/p53/p21 signaling pathway and enhancing the p16 signaling pathway. Thus, low-dose dinaciclib enhanced anti-MM effects mediated by DOX via transformation of p21-p16 signaling pathways, leading to accelerated senescence, but not apoptosis. The present findings suggest this approach may be a promising therapeutic strategy for the treatment of MM.

CAR-T Cells Based on Novel BCMA Monoclonal Antibody Block Multiple Myeloma Cell Growth.

The cell-surface protein B cell maturation antigen (BCMA, CD269) has emerged as a promising target for CAR-T cell therapy for multiple myeloma. In order to create a novel BCMA CAR, we generated a new BCMA monoclonal antibody, clone 4C8A. This antibody exhibited strong and selective binding to human BCMA. BCMA CAR-T cells containing the 4C8A scFv were readily detected with recombinant BCMA protein by flow cytometry. The cells were cytolytic for RPMI8226, H929, and MM1S multiple myeloma cells and secreted high levels of IFN-γ in vitro. BCMA-dependent cytotoxicity and IFN-γ secretion were also observed in response to CHO (Chinese Hamster Ovary)-BCMA cells but not to parental CHO cells. In a mouse subcutaneous tumor model, BCMA CAR-T cells significantly blocked RPMI8226 tumor formation. When BCMA CAR-T cells were given to mice with established RPMI8226 tumors, the tumors experienced significant shrinkage due to CAR-T cell activity and tumor cell apoptosis. The same effect was observed with 3 humanized BCMA-CAR-T cells in vivo. These data indicate that novel CAR-T cells utilizing the BCMA 4C8A scFv are effective against multiple myeloma and warrant future clinical development.

Antiproliferation activities of NK4 on multiple myeloma.

Multiple myeloma (MM) is a plasma cell malignancy. The hepatocyte growth factor (HGF) has been demonstrated to promote MM cell growth. NK4, a splice variant of HGF in which the heavy chain consists of the N-terminal domain and the four kringle domains, is a specific antagonist of HGF that competes with HGF for tyrosine-protein kinase receptor binding. The current study aimed to examine the antiproliferative activity of NK4 on human MM cells and to investigate the underlying mechanism. The results indicated that NK4 suppressed proliferation and induced apoptosis in RPMI 8226 cells. In addition, NK4 altered the expression of cell cycle and apoptosis-associated proteins in RPMI 8226, including cyclin-dependent kinase 4, cyclin D1, cyclin-dependent kinase inhibitor 1B, apoptosis regulator Bcl-2, apoptosis regulator BAX, cleaved caspase-9 and caspase-3. Furthermore, NK4 inhibited the activation of the RAC-α serine/threonine-protein kinase (Akt)/serine/threonine-protein kinase mTOR (mTOR) signaling pathway and reduced the levels of phosphorylated (p)-Akt, p-mTOR, ribosomal protein S6 kinase beta-1 and eukaryotic initiation factor 4E binding protein 1 in RPMI 8226 cells. In conclusion, NK4 inhibited the proliferation of human MM RPMI 8226 cells, which may be attributed to the induction of apoptosis and the inhibition of the Akt/mTOR signaling pathway.

Knockdown of NUPR1 inhibits the growth of U266 and RPMI8226 multiple myeloma cell lines via activating PTEN and caspase activation‑dependent apoptosis.

Nuclear protein‑1 (NUPR1) is a stress response factor that is important in the development of several human malignant tumor cells. However, the role of NUPR1 in multiple myeloma (MM) remains to be fully elucidated. In the present study, it was found that the mRNA levels of NUPR1 were significantly higher in specimens from patients with MM and MM cell lines (U266 and RPMI8226) than in cells of normal human bone marrow. The present study was undertaken to investigate the function of NUPR1 in the growth and apoptosis of MM cell lines. A lentivirus‑mediated short hairpin RNA was used to specifically inhibit the mRNA and protein expression of NUPR1 in the U266 and RPMI8226MM cell lines. Flow cytometry and Cell Counting Kit‑8 assays were applied to examine the apoptosis and proliferation of U266 and RPMI8226 cell lines. The results revealed the inhibitory effect of NUPR1 silencing on the proliferation of U266 and RPMI8226 cells through inducing apoptosis, and arrest of cell cycle at the G0/G1phase. Furthermore, NUPR1 silencing caused activation of caspase‑3,‑8 and‑9 and influenced specific gene expression, including an increase of phosphatase and tensin homolog (PTEN) and decrease of B‑cell lymphoma 2 and proliferating cell nuclear antigen. These findings showed that NUPR1 may be involved in the proliferation and apoptosis of MM cells by adjusting caspase proteins and PTEN, suggesting that NUPR1 may be a novel therapeutic target for MM.

Anti-inflammatory and antiproliferative diterpenoids from Plectranthus scutellarioides

Chemical investigation of the dichloromethane extract of the aerial parts of Plectranthus scutellarioides led to the isolation and characterization of 10 diterpenoids with an abietane skeleton and one cembrane-type diterpenoid. Among them, six have not yet been described in the literature. Their structures were established by 1D and 2D NMR, UV and IR spectroscopy, and HRESIMS. The relative configuration was determined by Gauge-Independent Atomic Orbital NMR chemical shift calculations supported by the advanced statistical method DP4 plus and further confirmed by electronic circular dichroism. The isolated constituents were evaluated for their in vitro NF-κB inhibitory activity, as well as for their cytotoxic effects in human multiple myeloma cancer stem cells and RPMI 8226 tumor cell line. Coleon O, coleon G, lanugone K and 6-acetylfredericone B showed the highest inhibitory effect against NF-κB, displaying IC50 of 11.2, 11.0, 4.5 and 9.7 μM, respectively. Coleon O exhibited also a significant activity towards human multiple myeloma cancer stem cells and RPMI 8226 cells with IC50 of 9.2 and 8.4 μM, respectively.

Anti‐Cancer Effect of 2‐Cyano‐3, 12‐dioxoolean‐1,9‐dien‐28‐oic acid (CDDO) Derivatives on Multiple Myeloma Cells

Multiple myeloma (MM) is a blood cancer that affects 30,000 people yearly in the U.S. It stems from monoclonal plasma cells and can cause symptoms such as bone loss and fatigue. Recent studies have shown that the bone marrow microenvironment may mediate tumor proliferation, drug resistance, and migration of the multiple myeloma cells. Synthetic oleanane triterpenoids have been readily used in research due to their anti‐proliferative and anti‐inflammatory effects. The objective of this study is to examine the anti‐cancer effect of 2‐cyano‐3, 12 dioxoolean‐1,9‐dien‐28‐oic acid (CDDO) derivatives on multiple myeloma cells. Three CDDO derivatives (CDDO‐Methyl Ester (Me), CDDO‐Trifluoethyl Amide (TFEA), and CDDO‐Imidazolide (Im)) with a dose range from 0.1 to 0.5 μM were tested on the growth of MM RPMI 8226 cells within 72 hours. Our current results show that all CDDO derivatives decrease the cell viability and proliferation of the MM 8226 cells in a dose and time‐dependent manner. Based on the potency, CDDO‐Im was selected to study whether its inhibitory effect on MM cell growth is due to the induction of apoptosis. Western blotting was used to check the protein expression of apoptosis‐related factors such as caspase 3, caspase 8, caspase 9 and poly‐(ADP‐ribose) polymerase (PARP). The result indicates that CDDO‐Im may induce the intrinsic apoptotic pathway. To evaluate the effect of CDDO‐Im on MM 8226 cells in the presence of the microenvironment, a Transwell (TW) model using MM cells co‐culturing with bone marrow stromal cells will be applied to mimic the bone marrow microenvironment in vivo. When treating the HS‐5 stromal cells with CDDO‐Im, no significant decrease in proliferation was seen after 24 hours. We expect that after co‐culturing the MM 8226 cells with HS‐5 stromal cells using the TW model, the growth of MM cells will still be prohibited by CDDO‐Im to some extent if not the same as MM cells being treated alone. In conclusion, our data suggest that CDDO derivatives inhibit the growth of MM cells through induction of apoptosis.Support or Funding InformationPCOM Division of Research FundingThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Downregulation of Pim-2 induces cell cycle arrest in the G0/G1 phase via the p53-non-dependent p21 signaling pathway.

Pim-2 is a serine/threonine protein kinase that is highly expressed in various types of cancer, with essential roles in the regulation of signal transduction cascades, which promote cell survival and proliferation. The present study demonstrated that Pim-2 was expressed in cells lines derived from hematopoietic tumors and lung cancer. In vitro, downregulation of Pim-2 by short interfering RNA inhibited proliferation and delayed G0/G1 cell cycle progression in K562 leukemia, RPMI-8226 multiple myeloma, and H1299 and A549 non-small cell lung carcinoma cell lines. Furthermore, downregulation of Pim-2 resulted in upregulation of cyclin-dependent kinase (CDK) inhibitor p21, irrespective of the p53 status. In addition, the present study revealed that CDK2 and phosphorylated retinoblastoma (pRb) were significantly downregulated. This finding suggested that inhibition of CDK2 and pRb expression via upregulated p21 was involved in the downregulation of Pim-2-induced G0/G1 cell cycle arrest in lung cancer and hematopoietic malignancy cells. These results suggested that Pim-2 may serve a role in hematopoietic tumors, lung cancer proliferation and cell cycle progression by regulating the p21 signaling pathway. Downregulation of Pim-2 decreased cancer cell proliferation. Therefore, Pim-2 may be a potential therapy target in clinical cancer therapy.

(N-Hydroxycarbonylbenylamino)quinolines as Selective Histone Deacetylase 6 Inhibitors Suppress Growth of Multiple Myeloma in Vitro and in Vivo.

A series of bicyclic arylamino/heteroarylamino hydroxamic acids (7-31) have been examined as novel histone deacetylase 6 (HDAC6) inhibitors. One compound (13) exhibits remarkable inhibitory activity of HDAC6 with an IC50 value of 0.29 nM, which is 4,000-43,000 times more selective over other HDAC isoforms. Compound 13 was shown to have antiproliferative activity against human multiple myeloma RPMI 8226, U266, and NCI-H929 cells with no effect on normal bone marrow cells. Compound 13, as a single drug, suppresses the growth of tumors by a %TGI factor of 60.4% in human multiple myeloma RPMI 8226 xenograft models and, in combination with bortezomib, shows significant in vivo antitumor activity (%TGI = 86.2%). Compound 13 also demonstrates good human hepatocytic stability and high permeability, without any effect on mutagenicity and cytotoxicity. Thus, compound 13 is a potent HDAC6 inhibitor that could be developed for the treatment of multiple myeloma in the future.

Abstract B057: Plitidepsin inhibits autophagy, the main mechanism of acquired resistance to bortezomib

Abstract Background: Plitidepsin (APL), an antitumor agent isolated from the marine tunicate Aplidium albicans, has been tested with positive results in multiple myeloma (MM) patients in a phase III pivotal trial in combination with dexamethasone (clinicaltrials.gov identifier: NCT01102426) and in a phase I trial in combination with bortezomib and dexamethasone (clinicaltrials.gov identifier: NCT02100657). Plitidepsin targets eEF1A2, one of two isoforms of the alpha subunit of the eEF1 complex. In mammals, eEF1A2 has a selective pattern of expression in those tissues that do not express the A1 isoform, namely brain and muscle. Nonetheless, eEF1A2 is aberrantly expressed in many cancers, including solid tumors (1-3) and MM (4), and has been shown to hold oncogenic properties (5). We have previously demonstrated through several means the interaction of plitidepsin with its target, eEF1A2 and calculated a Kd of around 80 nM for this interaction (6). Indeed, we have found that plitidepsin exclusively binds to the GTP-bound form of eEF1A2 Aims: Here we investigated whether eEF1A2 levels had any effect on the sensitivity of MM cells to both plitidepsin and bortezomib, to better understand the synergistic effect these two drugs yield when combined (7). Methods: RPMI-8226, OPM1, U266-B1, MM1S and NCI-NH929 multiple myeloma cells were cultured in optimal conditions. eEF1A2 (GTX102326) and autophagy (L7543) were followed by Western blot. Dose-response experiments were performed in 96 well plates, treating cells with increasing doses of the compounds for 24 hours and measuring cell growth with MTT. Curves were then fitted with GraphPad Prism5 software. For combination experiments, the Chou-Talalay procedure was used. Results: We first analyzed the level of eEF1A2 protein in a panel of multiple myeloma cell lines. Then, we selected a cell line with low expression, MM-1S, and one with high expression, OPM-1, of the elongation factor and checked the sensitivity to both, bortezomib and plitidepsin through dose-response experiments. Interestingly, the overexpression of eEF1A2, the target of plitidepsin, seems to be related to an increased resistance to bortezomib, a phenomenon usually related to enhanced autophagy since this latter pathway compensates the inhibition of proteasome. eEF1A, in its GTP-bound form, has been shown to inhibit chaperone-mediated autophagy (CMA), essential for the degradation of unfolded proteins (8). Since plitidepsin binds to the GTP-bound eEF1A2, probably stabilizing this form of the elongation factor, it seems likely that, in such a way, it may inhibit CMA. We explored this possibility and, indeed, we observed that plitidepsin inhibited autophagy in MM cells. Summary/Conclusion: Plitidepsin treatment presumably counteracts the main mechanism of acquired resistance to bortezomib, namely autophagy. (1)Sun et al., 2014, Biochem Biophys Res Commun 450:1-6. (2)Xu et al., 2013, Clin Exp Metastasis 30:933-44. (3)Anand et al., 2002, Nat Genet 31:301-5. (4)Li et al., 2010, PLOS One 5, e10755. (5)Lee and Surh, 2009, Ann N Y Acad Sci 1171:87-93. (6)Losada et al., 2004, Br J Cancer 91:1405-13. (7)Mitsiades et al., 2008, Cancer Res 68:5216-25. (8)Bandyopadhyay et al., 2010, Mol Cel 39:535-47. Citation Format: Juan F. Martínez-Leal, Gaëlle Quiniou, Alejandro Losada, María José Muñoz, Rafael Sanchez-Mesa, Patricia Martinez-Rivas, Juan Manuel Dominguez, Carlos M. Galmarini. Plitidepsin inhibits autophagy, the main mechanism of acquired resistance to bortezomib [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B057.

Bioactive metabolites from the leaves of Withania adpressa

Context: Withania (Solanaceae) species are known to be a rich source of withanolides, which have shown several biological properties.Objective: To identify the compounds responsible for Withania adpressa Coss. antioxidant activity and further test them for their NF-κB inhibition and antiproliferative activity in multiple myeloma cells.Materials and methods: Compounds were obtained from the EtOAc extract of W. adpressa leaves. Structure elucidation was carried out mainly by 1D- and 2D-NMR, and mass spectrometry. Isolated compounds were tested in a dose-response for their in vitro NF-κB inhibition and antiproliferative activity in multiple myeloma cells after 5 and 72 h treatment, respectively.Results: The fractionation resulted in the isolation of a new glycowithanolide named wadpressine (5) together with withanolide F, withaferin A, coagulin L, and nicotiflorin. The latter showed a moderate ability to scavenge free radicals in DPPH (IC50 = 35.3 µM) and NO (IC50 = 41.3 µM) assays. Withanolide F and withaferin A exhibited low µM antiproliferative activity against both multiple myeloma cancer stem cells and RPMI 8226 cells. Furthermore, they inhibited NF-κB activity with IC50 values of 1.2 and 0.047 µM, respectively. The other compounds showed a moderate inhibition of cell proliferation in RPMI 8226 cells, but were inactive against cancer stem cells and did not inhibit NF-κB activity.Discussion and conclusions: One new glycowithanolide and four known compounds were isolated. Biological evaluation data gave further insight on the antitumor potential of withanolides for refractory cancers.

CRISPR Genome-Wide Screening Identifies Dependence on the Proteasome Subunit PSMC6 for Bortezomib Sensitivity in Multiple Myeloma.

Bortezomib is highly effective in the treatment of multiple myeloma; however, emergent drug resistance is common. Consequently, we employed CRISPR targeting 19,052 human genes to identify unbiased targets that contribute to bortezomib resistance. Specifically, we engineered an RPMI8226 multiple myeloma cell line to express Cas9 infected by lentiviral vector CRISPR library and cultured derived cells in doses of bortezomib lethal to parental cells. Sequencing was performed on surviving cells to identify inactivated genes responsible for drug resistance. From two independent whole-genome screens, we selected 31 candidate genes and constructed a second CRISPR sgRNA library, specifically targeting each of these 31 genes with four sgRNAs. After secondary screening for bortezomib resistance, the top 20 "resistance" genes were selected for individual validation. Of these 20 targets, the proteasome regulatory subunit PSMC6 was the only gene validated to reproducibly confer bortezomib resistance. We confirmed that inhibition of chymotrypsin-like proteasome activity by bortezomib was significantly reduced in cells lacking PSMC6. We individually investigated other members of the PSMC group (PSMC1 to 5) and found that deficiency in each of those subunits also imparts bortezomib resistance. We found 36 mutations in 19S proteasome subunits out of 895 patients in the IA10 release of the CoMMpass study (https://themmrf.org). Our findings demonstrate that the PSMC6 subunit is the most prominent target required for bortezomib sensitivity in multiple myeloma cells and should be examined in drug-refractory populations. Mol Cancer Ther; 16(12); 2862-70. ©2017 AACR.

Notch1 Gene Silenced by RNAi Inhibits Oncogenicity of Myeloma Cell Line

To investigate the effect of Notch1 gene silencing by RNA interference on oncogenicity of multiple myeloma cells in NOD/SCID mice. Targeting-silenced Notch1 gene was target-sotenced by transfection of Notch1-shRNA in multiple myeloma RPMI8226 cells of NOD/SCID mouse myeloma models, and the change of the volume and speed of oncogenicity in myeloma mouse models were evaluated after Notch1 gene silencing, and ELISA was used to detect the serum expression level of IL-6 and VEGF in the tumor-bearing mice. After Notch1 gene was silenced by Notch1-shRNA, the speed of tumor formation was significantly inhibited and the tumor volume was reduced in the tumor-bearing mice, as compared with the control group, and the difference was statistically significant (P<0.05). The serum level of IL-6 and VEGF in the tumor-bearing mice significantly decreased in comparison with the control group (P<0.05 ). The oncogenicity of myeloma cells in the models NOD/SCID mouse myeloma is significantly inhibited by Notch1 gene-silencing, and its mechanism may relate with the decreased secretory level of IL-6 and VEGF after Notch1 gene silencing. Notch1 gene silencing can be used as a new strategy to treat multiple myeloma.

Targeting Notch1 Gene Inhibits the Proliferation of Multiple Myeloma Cells

To investigate the effects of Notch1 gene silencing on the proliferation and apoptosis of multiple myeloma cells, and to find the new targets for the treatment of multiple myeloma. Notch1-shRNA targeted silencing Notch1 gene was transfected into multiple myeloma RPMI8226 cells, the CCK-8 and flow cytometry were used to detect the proliferation and apoptosis of myeloma cells after Notch1-shRNA transfection, the real-time fluorescence quantitative PCR was used to analyze expression level of Notch1 mRNA, and the Western blot were used to detect the expression level of Notch1 signaling pathway-related protein, such as Hes-1, Jagged-1, Jagged-2, BCL-2, PTEN, AKT and P-AKT. The mRNA and protein expression levels of Notch1-shRNA transfected cells were significantly inhibited in the experimental group assayed by real time fluorescence quantitative PCR and Western blot, the mRNA and protein expression level were down-regulated to 66% + 0.1%, 88% + 3.4% respectively, as compared with the control group(P<0.05). CCK-8 results confirmed that the cell proliferation rate was significantly decreased in the experimental group 48 hours after transfection. Flow cytometry results showed that the cell apoptosis rate was significantly higher in the experimental group than that in the control group. The expression levels of downstream protein Hes1, p-AKT and BCL-2 were decreased, the level of PTEN increased significantly after Notch1 gene silencing. Notch1 gene silencing by transfection of Notch1-shRNA can inhibit the proliferation of myeloma cells and induce their apoptosis, and its mechanism is related to the activation of PTEN gene and p-AKT signaling. Notch1 signal can be used as a potential target for multiple myeloma therapy.

Anti-proliferation Effect of NS-398 in Combination with Bortezomib on Multiple Myeloma RPMI 8226 Cells In Vitro

To investigate if NS-398 could enhance the chemosensitivity of Bortezomib (BOR) on human multiple myeloma RPMI 8226 cells. After the treatment of NS-398 combined with BOR, MTT assay was used to detect the proliferation inhibition effect on human multiply myeloma RPMI 8226 cells in vitro, Flow cytometry was used to analyze their effect of apoptosis and cell cycle; the caspase-3 activity of different treatment group was detected by using ELISA and the activity of Cox-2 was measured by using Cox-2 activity assay kit. The inhibitory rate of NS-398 combined with BOR was higher than that of NS-398 or BOR alone(Q>0.85). After treatment of NS-398 combined with BOR, the percentage of cells arrested in the G0/G1 phase and apoptotic rate were both higher than that of treatment with each drug alone(Q>1.15). The caspase-3 activity in cells treated with combined of NS-398 and BOR was significantly higher than that of treatment of each drug alone(Q>1.15). NS-398 combined with BOR shows a synergistic effect on the growth inhibition of RPMI 8226 cells in vitro.

1,4-Naphthoquinones as inhibitors of Itch, a HECT domain-E3 ligase, and tumor growth suppressors in multiple myeloma

A series of 1,4-naphthoquinones (10a-10q) were synthesized and evaluated for anticancer activity. Compound 10e was identified as an inhibitor of Itch, a HECT domain-E3 ligase. In an evaluation of in vivo efficacy, 10e exhibited remarkable anticancer activity with TGI values of 98.3% and 100% at 25 mg/kg and 50 mg/kg orally daily, respectively, against human RPMI-8226 multiple myeloma xenograft. Treatment with 10e also showed a decrease of Itch level in human RPMI-8226 multiple myeloma cells. Thus 10e is a lead compound for further development of inhibitors targeting E3 ligase for treatment of multiple myeloma.

Anti-proliferative activity of CGK012 against multiple myeloma cells via Wnt/β-catenin signaling attenuation

The aberrant activation of Wnt/β-catenin signaling is involved in the development of multiple myeloma; thus, this signaling pathway is a potential target for the development of therapeutics for this malignancy. Here, we performed cell-based chemical screening and found that CGK012, a pyranocoumarin compound, suppressed the Wnt3a-CM-mediated activation of β-catenin response transcription. CGK012 induced β-catenin phosphorylation at Ser33/Ser37/Thr41, leading to proteasomal degradation and reducing the level of intracellular β-catenin. Furthermore, CGK012 consistently decreased the amount of β-catenin and repressed the expression of cyclin D1, c-myc, and axin-2 (downstream target genes of β-catenin) in RPMI-8226 multiple myeloma cells. In addition, CGK012 inhibited the proliferation of RPMI-8226 cells and promoted apoptosis, as indicated by the increase in the population of Annexin V-FITC-stained cells and caspase-3/7 activity. These findings suggest that CGK012 could exert antiproliferative activity against multiple myeloma cells by attenuating the Wnt/β-catenin pathway; thus, it may have potential as a therapeutic agent for multiple myeloma treatment.