Human Myeloma Xenograft Model Research Articles

SSBP1 is a novel prognostic marker and promotes disease progression via p38MAPK signaling pathway in multiple myeloma.

Multiple myeloma (MM) remains an incurable disease. Identification of meaningful co-expressed gene clusters or representative biomarkers of MM may help to identify new pathological mechanisms and promote the development of new therapies. Here, we performed weighted sgene co-expression network analysis and a series of bioinformatics analysis to identify single stranded DNA binding protein 1 (SSBP1) as novel hub gene associated with MM development and prognosis. In vitro, CRISPR/cas9 mediated knockdown of SSBP1 can significantly inhibit the proliferation of MM cells through inducing apoptosis and cell cycle arrest in G0/G1 phase. We also found that decreased SSBP1 expression significantly increased mitochondrial reactive oxygen species (mtROS) generation and the level of phosphorylated p38MAPK. Furthermore, it was further verified that disruption of SSBP1 expression could inhibit the tumor growth via p38MAPK pathway in a human myeloma xenograft model. In summary, our study is the first to demonstrate that SSBP1 promotes MM development by regulating the p38MAPK pathway.

Activated Leukocyte Cell Adhesion Molecule (ALCAM) Regulate Myeloma Cancer Stem Cell Mediated Myelomagenesis and Chemoresistance

IntroductionMultiple myeloma (MM) is an incurable plasma cell cancer. People introduced the concept of cancer stem cells (CSCs) in MM, known as MM-CSCs, to characterize a small population of malignant cells with distinguishable activities of self-renewal and differentiation into regular MM cells. In this study, we showed that activated leukocyte cell adhesion molecule (ALCAM) interacted with epidermal growth factor receptor (EGFR). ALCAM-EGFR axis regulated MM-CSC mediated myelomagenesis and chemoresistance.MethodsMM-CSCs in MM cell lines and primary MM cells were identified by Hoechst staining or ALDEFLUOR dye, followed by flow cytometry analyses. MM-CSC clonalization activity was examined by soft agar colony formation assay. The protein-protein interaction was identified by co-immunoprecipitation, proximity ligation assay, and proteins' structure simulation. MM-CSC mediated myelomagenesis in vivo was examined in human MM xenograft mouse model and murine adoptive MM model. MM-CSC mediated chemoresistance in vivo was examined using 5T MM model. The clinical significance of ALCAM expression in MM was analyzed using patients' gene expression profiles.ResultsBoth MM cell lines and primary MM cells expressed ALCAM. When cultured with bone marrow stromal cells (BMSCs) or in BMSC-conditioned medium, ALCAM knocked down (AL-KD) MM cells exhibited increased colony formation activity. Hoechst staining showed that under such culture conditions, AL-KD MM cells had increased stemness side population (SP), which was MM-CSC. Depletion of MM-CSC reduced MM colony formation activity. In human MM xenograft model, subcutaneous co-injection of AL-KD MM cells and BMSCs had large amount of MM-CSC and fast tumorigenesis. In murine adoptive MM model, which had tumor derived from viral transformed plasma cells with mandatory expression of MYC, KRAS genes and a shRNA sequence, ALCAM shRNA expression resulted significant more rapid tumor development than non-target shRNA.Mechanistic studies showed that ALCAM and EGFR co-immunoprecipitated in MM cells. Proximity ligation assay showed interaction of ALCAM and EGFR in primary MM cells. Immunofluorescent staining showed that ALCAM colocalized with unphosphorylated EGFR in MM. Epidermal growth factor (EGF), the canonical ligand of EGFR, stimulated EGFR phosphorylation and abolished ALCAM-EGFR colocalization. Protein structure simulation suggested that EGF and ALCAM mutual exclusively bound to the same region of EGFR. EGF addition stimulated MM-CSC in cultured MM. Inhibitors of EGFR, ERK, or hedgehog pathway repressed MM-CSC. EGFR activation led to hedgehog pathway activation via RAS/MEK/ERK cascade, while ALCAM inhibited such pro-MM-CSC cell signaling by binding to EGFR.Chemotherapeutics, such as bortezomib and melphalan, induced MM apoptosis, but also stimulated MM-CSCs, which were resistant to chemotherapeutics. Although CTR-KD and AL-KD MM had similar death rate after the drug treatment, AL-KD MM cells had more MM-CSC generated. MM-CSCs derived in AL-KD MM cells had more active cell proliferation even in the presence of drugs. Further study showed that chemotherapy stimulated EGFR expression and repressed ALCAM expression, therefore switched the balance of ALCAM-EGFR axis to pro-MM-CSC direction. EGFR inhibitors, such as gefitinib, did not have anti-MM activity, but repressed MM-CSCs. In 5T MM model, Gefitinib/melphalan combination therapy resulted prolonged remission period, and significant better mouse survival.We identified MM-CSC in patients BM. ALCAM expression correlated with MM overall survival (OS): ALCAMhigh patients had better OS. In ALCAM-EGFR axis, only ALCAM expression, but not EGFR expression, correlated with MM OS. ALCAM expression varied in different patients, while EGFR expression was more even. Therefore we believe in ALCAM-EGFR axis, it was ALCAM, but not the EGFR, that played the regulatory role. Furthermore, pathway enrichment analysis showed that MM-CSC, identified by Hoechst staining, and MM minimum residual disease (MRD) shared similarities in signaling pathways, as well as pattern of ALCAM expressions.Conclusion and DiscussionALCAM-EGFR axis regulates MM-CSC mediated myelomagenesis and chemoresistance. EGFR inhibitors repress chemoresistant MM-CSC. Combination therapy with conventional chemotherapeutics and an EGFR inhibitor might be promising in MM treatment. DisclosuresNo relevant conflicts of interest to declare.

HSF1 Is Essential for Myeloma Cell Survival and A Promising Therapeutic Target.

Purpose: Myeloma is a plasma cell malignancy characterized by the overproduction of immunoglobulin, and is therefore susceptible to therapies targeting protein homeostasis. We hypothesized that heat shock factor 1 (HSF1) was an attractive therapeutic target for myeloma due to its direct regulation of transcriptional programs implicated in both protein homeostasis and the oncogenic phenotype. Here, we interrogate HSF1 as a therapeutic target in myeloma using bioinformatic, genetic, and pharmacologic means.Experimental Design: To assess the clinical relevance of HSF1, we analyzed publicly available patient myeloma gene expression datasets. Validation of this novel target was conducted in in vitro experiments using shRNA or inhibitors of the HSF1 pathway in human myeloma cell lines and primary cells as well as in in vivo human myeloma xenograft models.Results: Expression of HSF1 and its target genes were associated with poorer myeloma patient survival. ShRNA-mediated knockdown or pharmacologic inhibition of the HSF1 pathway with a novel chemical probe, CCT251236, or with KRIBB11, led to caspase-mediated cell death that was associated with an increase in EIF2α phosphorylation, CHOP expression and a decrease in overall protein synthesis. Importantly, both CCT251236 and KRIBB11 induced cytotoxicity in human myeloma cell lines and patient-derived primary myeloma cells with a therapeutic window over normal cells. Pharmacologic inhibition induced tumor growth inhibition and was well-tolerated in a human myeloma xenograft murine model with evidence of pharmacodynamic biomarker modulation.Conclusions: Taken together, our studies demonstrate the dependence of myeloma cells on HSF1 for survival and support the clinical evaluation of pharmacologic inhibitors of the HSF1 pathway in myeloma. Clin Cancer Res; 24(10); 2395-407. ©2018 AACRSee related commentary by Parekh, p. 2237.

Quercetin induces cell apoptosis of myeloma and displays a synergistic effect with dexamethasone in vitro and in vivo xenograft models.

Quercetin, a kind of dietary flavonoid, has shown its anticancer activity in many kinds of cancers including hematological malignancies (acute myelogenous leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, and MM) in vitro and in vivo. However, its effects on MM need further investigation. In this study, MM cell lines were treated with quercetin alone or in combination with dexamethasone. In order to observe the effects in vivo, a xenograft model of human myeloma was established. Quercetin inhibited proliferation of MM cells (RPMI8226, ARP-1, and MM.1R) by inducing cell cycle arrest in the G2/M phase and apoptosis. Western blot showed that quercetin downregulated c-myc expression and upregulated p21 expression. Quercetin also activated caspase-3, caspase-9, and poly(ADP-ribose)polymerase 1. Caspase inhibitors partially blocked apoptosis induced by quercetin. Furthermore, quercetin combined with dexamethasone significantly increased MM cell apoptosis. In vivo xenograft models, quercetin obviously inhibited tumor growth. Caspase-3 was activated to a greater extent when quercetin was combined with dexamethasone. In conclusion, quercetin alone or in combination with dexamethasone may be an effective therapy for MM.

Evidence for a role of the histone deacetylase SIRT6 in DNA damage response of multiple myeloma cells

AbstractMultiple myeloma (MM) is characterized by a highly unstable genome, with aneuploidy observed in nearly all patients. The mechanism causing this karyotypic instability is largely unknown, but recent observations have correlated these abnormalities with dysfunctional DNA damage response. Here, we show that the NAD+-dependent deacetylase SIRT6 is highly expressed in MM cells, as an adaptive response to genomic stability, and that high SIRT6 levels are associated with adverse prognosis. Mechanistically, SIRT6 interacts with the transcription factor ELK1 and with the ERK signaling-related gene. By binding to their promoters and deacetylating H3K9 at these sites, SIRT6 downregulates the expression of mitogen-activated protein kinase (MAPK) pathway genes, MAPK signaling, and proliferation. In addition, inactivation of ERK2/p90RSK signaling triggered by high SIRT6 levels increases DNA repair via Chk1 and confers resistance to DNA damage. Using genetic and biochemical studies in vitro and in human MM xenograft models, we show that SIRT6 depletion both enhances proliferation and confers sensitization to DNA-damaging agents. Our findings therefore provide insights into the functional interplay between SIRT6 and DNA repair mechanisms, with implications for both tumorigenesis and the treatment of MM.

Impaired Lysophosphatidic Acid Receptor 3 Signaling in Mesenchymal Stromal Cells Promotes Multiple Myeloma Progression through Cellular Senescence and Transdifferentiation into Tumor-Associated Fibroblasts

IntroductionMultiple myeloma (MM) is one of the hematologic malignancies characterized as accumulation of monoclonal tumor of plasma cells. Although novel therapeutic agents have significantly improved the survival of MM patients, MM is still a mostly incurable disease. The most common reason of relapsed and refractory MM is that myeloma cells intimately communicate with bone marrow stroma and acquire drug resistance.Stroma is known to secrete soluble factors and express some adhesion molecules advantageous for progression of MM. Recently, there are accumulating evidences that bone marrow-derived mesenchymal stem cells (MSC) serve as a component of stroma and support growth and drug resistance of myeloma cells. Although the exact etiology has not been clearly defined, it has been suggested that the incidence of MM increases with aging.In this study, we tried to explore the relationship between MM progression and cellular senescence in MSC.MethodsHuman myeloma cell lines used in this study were IM-9, OPM-2, and RPMI-8226. Human MSC used in this study were obtained from Texas A&M Health Science Center for the Preparation and Distribution of Adult Stem Cells. To establish a mouse xenograft model of human MM, 1.0 x 106 of IM-9 cells were co-injected with 4.0 x 105 of human MSC subcutaneously into the flank of BALB/c nude mice. Visible and palpable tumors were formed in all animals after 20 days. Tumor specimens were resected surgically and diagnosed pathologically as MM based on marker expression. For in vivo bioimaging, luciferase-expressing IM-9 (IM-9-luc+) cells were co-injected with human MSC subcutaneously into BALB/c-nude mice, and photon emission was detected with a sensitive CCD camera 10, 15 and 20 days later.ResultsIn our earlier research, we have demonstrated that the signaling of lysophosphatidic acid (LPA), a bioactive lipid mediator, modulates cellular senescence in MSC (Kanehira et al. PLoS One. 2012). MSC produced autotaxin (ATX), a key enzyme in LPA synthesis, in response to myeloma cells via Toll-like receptor 4 (TLR4)/NF-kappaB-dependent pathway. To determine the LPA receptor responsible for cellular senescence in MSC, six LPA receptors (LPAR1-6) were individually knocked down using siRNA for each receptor. In BrdU incorpotaion assay, LPAR3 gene-silenced MSC (siLPAR3-MSC) were less proliferative than control MSC, and in cell cycle analysis with 7-AAD and Pyronin Y, siLPAR3-MSC was arrested in G1 phase. Interestingly, siLPAR3-MSC exhibited some characteristics of cellular senescence, such as up-regulation of senescence-associated beta-galactosidase activity, increased cell size, and flattened morphology. In a mouse xenograft model of MM, siLPAR3-MSC promoted progression of MM and also tumor-associated angiogenesis. In in vitro study, we confirmed that siLPAR3-MSC easily transdifferentiated into alpha-SMA+ tumor-associated fibroblast and secreted FGF2 in response to myeloma cells. The MM promoting effect and elevated tumor-associated angiogenesis observed in siLPAR3-MSC co-injection were both completely cancelled by FGF2 gene-silencing in siLPAR3-MSC (Figure 1 and 2).Conclusion and discussionIn this study, we verified the impairment of LPAR3 signaling may accelerate cellular senescence in MSC. And senesced MSC can provide an advantageous microenvironment for MM progression by FGF2-dependent formation of tumor-stroma milieu. Here, we provide the possibility that LPAR3 signaling could be promising as a therapeutic target in MM. [Display omitted] [Display omitted] DisclosuresFujiwara:Chugai Pharmaceutical CO., LTD: Research Funding. Fukuhara:Gilead Sciences: Research Funding. Harigae:Chugai Pharmaceutical Co., Ltd.: Research Funding.

Abstract 3379: HSF1: Essential for myeloma cell survival and a promising therapeutic target

Abstract Heat shock factor 1 (HSF1), a transcription factor known as the “master regulator” of the heat shock response (HSR), is emerging as an exciting therapeutic target in cancer due to its role in regulating cellular responses to proteotoxic stress as well as various oncogenic processes. Myeloma is hypothesized to be susceptible to therapies targeting protein homeostasis as it is characterised by the overproduction of immunoglobulin which is linked to significant ER stress. However, clinical responses to inhibitors of HSP90, a molecular chaperone overexpressed in myeloma patients, have been modest. This is partly due to HSF1-mediated transcriptional upregulation of anti-apoptotic HSP72. We are seeking to determine whether targeting the HSR through HSF1 may be an effective therapeutic strategy. HSF1 knockdown using shRNA downregulated levels of anti-apoptotic HSP72 and HSP27 and significantly reduced viability in human myeloma cell lines. This was associated with apoptosis as evidenced by increases in caspase 3/7 activity and PARP cleavage which were rescued by treatment with z-VAD-FMK, a pan-caspase inhibitor. We next treated human myeloma cell lines with KRIBB-11, a direct small molecule inhibitor of HSF1, and observed growth inhibition with GI50s of between 3.0 - 8.5 μM. Notably, KRIBB-11 was cytotoxic to myeloma cell lines but not to peripheral blood mononuclear cells from healthy donors. To shed light on the mechanisms by which myeloma cells rely on HSF1 for survival, we carried out microarray analysis on HSF1 knockdown cells and found that XIAP-associated factor 1 (XAF1), a pro-apoptotic tumour suppressor gene known to be transcriptionally repressed by HSF1, was upregulated (>2.63 log-fold increase, p<0.0001). qRT-PCR and Western blotting confirmed XAF1 upregulation with HSF1 knockdown. An increase in XAF1, PARP cleavage and decrease in HSP72 and HSP27 levels were also observed with KRIBB-11 treatment, confirming that the loss or inhibition of HSF1 in myeloma modulates its downstream target genes and leads to caspase-mediated cell death. These results suggest a mechanism whereby HSF1 overexpression in myeloma cells represses XAF1 expression to evade apoptotic stresses and suggest XAF1 as a possible biomarker for HSF1 inhibition in myeloma. Interestingly, unlike HSP90 inhibition, HSF1 knockdown in myeloma cell lines did not modulate other key myeloma protein handling mechanisms such as aggresome formation, autophagy, the ubiquitin-proteasome pathway or ER stress. Efficacy testing and biomarker analyses of HSF1 inhibitors in s.c. human myeloma xenograft models are underway and early results are promising. To summarise, using both genetic and pharmacological inhibition, our data demonstrate that HSF1 maintains myeloma cell survival during proteotoxic stress by protecting cells from apoptosis via XAF1. More importantly, we demonstrate the potential efficacy and therapeutic window in targeting HSF1 for myeloma treatment. Citation Format: Jacqueline H L Fok, Somaieh Hedayat, Lei Zhang, Lauren I. Aronson, Fabio Mirabella, Suzanne A. Eccles, Faith E. Davies. HSF1: Essential for myeloma cell survival and a promising therapeutic target. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3379. doi:10.1158/1538-7445.AM2015-3379

A Novel PI3K Inhibitor Identified By a High Throughput Virtual Screen Displays Potent Activity Against Multiple Myeloma

The phosphatidylinositol-3-kinase (PI3K) has been developed as a promising target for the treatment of many cancers, including hematological malignancies, such as lymphoma, leukemia, and multiple myeloma (MM). Recent clinical trials have demonstrated Idelalisib, a specific PI3Kdelta inhibitor, reached a total 57% of response rate in relapsed indolent lymphoma and 72% of overall complete rate in relapsed chronic lymphocytic leukemia. MM remains fatal as a cancer of plasma cells because of lack of efficient drugs. Therefore, we tried to find novel PI3K inhibitors for MM therapy. To this end, we performed a high throughput virtual screen against 800,000 small molecule compounds using PI3Kγ as the analytic model and identified C98, a function unknown compound, as a potent inhibitor of PI3Ks. The cell-free enzymatic studies showed that C98 inhibited all class I PI3Ks at nano- or low micromolar concentrations but had no effects on AKT or mTOR activity. Molecular docking analysis revealed that C98 interfered with the ATP-binding pockets of PI3Ks by forming H-bonds and arene-H interactions with specific amino residues through which C98 specifically inhibited PI3K/AKT/mTOR signaling pathway, but had no effects on other kinases and proteins including IGF-1R, ERK, p38, c-Src, and STAT3. PTEN is a key negative modulator of the PI3K signaling pathway, but KIF did not show any induction of PTEN, thus further demonstrating that KIF is a selective PI3K inhibitor. Inhibition of PI3K by C98 led to myeloma cell apoptosis as shown activated caspase cascade signaling. Furthermore, oral administration of C98 delayed tumor growth in two independent human myeloma xenograft models in nude mice but did not show overt toxicity. Pharmacokinetic analysis revealed that C98 can be found in MM tissues at an average concentration up to 622.6 ng/g, suggesting that C98 can penetrate into the tumor tissues where it exerts its anti-myeloma activity. It also suggests C98 has a reliable oral activity. Therefore, through a high throughput virtual screen we identified a novel PI3K inhibitor that is orally active against multiple myeloma with great potential for further development. DisclosuresNo relevant conflicts of interest to declare.

A novel PI3K inhibitor PIK-C98 displays potent preclinical activity against multiple myeloma.

Recent clinical trials have demonstrated targeting PI3K pathway is a promising strategy for the treatment of blood cancers. To identify novel PI3K inhibitors, we performed a high throughput virtual screen and identified several novel small molecule compounds, including PIK-C98 (C98). The cell-free enzymatic studies showed that C98 inhibited all class I PI3Ks at nano- or low micromolar concentrations but had no effects on AKT or mTOR activity. Molecular docking analysis revealed that C98 interfered with the ATP-binding pockets of PI3Ks by forming H-bonds and arene-H interactions with specific amino acid residues. The cellular assays demonstrated that C98 specifically inhibited PI3K/AKT/mTOR signaling pathway, but had no effects on other kinases and proteins including IGF-1R, ERK, p38, c-Src, PTEN, and STAT3. Inhibition of PI3K by C98 led to myeloma cell apoptosis. Furthermore, oral administration of C98 delayed tumor growth in two independent human myeloma xenograft models in nude mice but did not show overt toxicity. Pharmacokinetic analyses showed that C98 was well penetrated into myeloma tumors. Therefore, through a high throughput virtual screen we identified a novel PI3K inhibitor that is orally active against multiple myeloma with great potential for further development.

Proteasome Maturation Protein (pomp) Is Associated With Proteasome Inhibitor Resistance In Myeloma, and Its Suppression Enhances The Activity Of Bortezomib and Carfilzomib

BackgroundProteasome inhibition with bortezomib has revolutionized the treatment of multiple myeloma, but the vast majority of patients eventually develop clinical bortezomib resistance through poorly understood mechanisms. One of the most conserved cellular responses to proteasome inhibition is to up-regulate proteasome subunit expression, presumably with the goal of enhancing proteasome activity and restoring intracellular protein homeostasis. We therefore hypothesized that proteasome inhibitor resistance could be associated with enhanced proteasome assembly, and that suppression of this assembly process could help restore drug sensitivity. The current studies focused on POMP, which is involved in addition of catalytically active b subunits to the hemiproteasome ring initially formed by structural a subunits. MethodsWe studied ANBL-6, KAS-6/1, OPM-2, and RPMI 8226 multiple myeloma cell lines which had acquired bortezomib resistance through prolonged exposure to increasing drug concentrations, and compared them to their drug-naïve, vehicle-treated counterparts. In addition, we evaluated primary cells derived from patients with myeloma, and examined an in vivo murine xenograft model of human myeloma. ResultsBortezomib-resistant (V10R) ANBL-6, KAS-6/1, OPM-2, and RPMI 8226 cell lines showed enhanced levels of POMP mRNA by quantitative (q) PCR compared to their drug-sensitive counterparts, which was associated with higher POMP protein levels seen by immunoblotting. Exogenous over-expression of POMP in drug-naïve OPM-2 and KAS-6/1 cells was sufficient by itself to induce resistance to both bortezomib and carfilzomib. Conversely, suppression of POMP with one of two different Lentiviral small hairpin (sh) RNAs restored sensitivity in OPM-2 and KAS-6/1 V10R cells to bortezomib and carfilzomib. Since no known pharmaceuticals directly target POMP, we examined its promoter region, and found a consensus binding site for nuclear factor (erythroid-derived 2)-like (NRF) 2. Consistent with a role of NRF2 in POMP expression, NRF2 mRNA and protein were increased in V10R myeloma cells, and in drug-naïve cells treated with bortezomib. Moreover, transfection of cells with NRF2 cDNA activated a POMP promoter reporter, while chromatin immunoprecipitation with anti-NRF2 antibodies preferentially precipitated sequences near the POMP promoter. Also, NRF2 over-expression induced POMP and enhanced proteasome chymotrypsin-like activity, while its suppression had the opposite effects. All trans-retinoic acid (ATRA) blocked nuclear accumulation of NRF2 in OPM-2 and KAS-6/1 V10R cells, and reduced expression of POMP. Combinations of bortezomib with ATRA showed enhanced activity against these drug-resistant cell lines in association with greater proteasome inhibition, and were synergistic in drug-naïve cells. In primary samples, ATRA with bortezomib induced a greater reduction in viability than did either treatment alone. Finally, in a murine xenograft model with OPM-2 V10R cells, neither ATRA nor bortezomib showed substantial activity, while the combination regimen, by comparison, retained efficacy. ConclusionsTaken together, our data support the hypotheses that NRF-2-influenced POMP over-expression contributes to proteasome inhibitor resistance in multiple myeloma, while approaches targeting POMP hold promise in overcoming resistance. Moreover, they provide a framework for translation of proteasome inhibitors with ATRA to the clinic to enhance activity, and to overcome resistance to this important class of anti-myeloma agents. Disclosures:No relevant conflicts of interest to declare.

Molecular Chaperone gp96 Is a Novel Therapeutic Target of Multiple Myeloma

gp96 (grp94) is a key downstream chaperone in the endoplasmic reticulum (ER) to mediate unfolded protein response (UPR) and the pathogenesis of multiple myeloma is closely linked to dysregulated UPR. In this study, we aimed to determine the roles of gp96 in the initiation and progression of multiple myeloma in vivo and in vitro. We generated a mouse model with overexpression of XBP1s and conditional deletion of gp96 in B-cell compartment simultaneously to identify the roles of gp96 in the development of multiple myeloma in vivo. Using a short hairpin RNA (shRNA) system, we silenced gp96 in multiple human multiple myeloma cells and examined the effect of gp96 knockdown on multiple myeloma cells by cell proliferation, cell-cycle analysis, apoptosis assay, immunohistochemistry, and human myeloma xenograft model. The anticancer activity of gp96 selective inhibitor, WS13, was evaluated by apoptosis assay and MTT assay. Genetic deletion of gp96 in XBP1s-Tg mice attenuates multiple myeloma. Silencing of gp96 causes severe compromise in human multiple myeloma cell growth through inhibiting Wnt-LRP-survivin pathway. We also confirmed that knockdown of gp96 decreased human multiple myeloma growth in a murine xenograft model. The targeted gp96 inhibitor induced apoptosis and blocked multiple myeloma cell growth, but did not induce apoptosis in pre-B leukemic cells. We have demonstrated that myeloma growth is dependent on gp96 both genetically and pharmacologically. gp96 is essential for multiple myeloma cell proliferation and survival, suggesting that gp96 is a novel therapeutic target for multiple myeloma. Clin Cancer Res; 19(22); 6242-51. ©2013 AACR.

Aurora and IKK kinases cooperatively interact to protect multiple myeloma cells from Apo2L/TRAIL

AbstractConstitutive activation of the canonical and noncanonical nuclear factor-κB (NF-κB) pathways is frequent in multiple myeloma (MM) and can compromise sensitivity to TRAIL. In this study, we demonstrate that Aurora kinases physically and functionally interact with the key regulators of canonical and noncanonical NF-κB pathways IκB kinase β (IKKβ) and IKKα to activate NF-κB in MM, and the pharmacological blockade of Aurora kinase activity induces TRAIL sensitization in MM because it abrogates TRAIL-induced activation of NF-κB. We specifically found that TRAIL induces prosurvival signaling by increasing the phosphorylation state of both Aurora and IKK kinases and their physical interactions, and the blockade of Aurora kinase activity by pan-Aurora kinase inhibitors (pan-AKIs) disrupts TRAIL-induced survival signaling by effectively reducing Aurora-IKK kinase interactions and NF-κB activation. Pan-AKIs consistently blocked TRAIL induction of the antiapoptotic NF-κB target genes A1/Bfl-1 and/or Mcl-1, both important targets for TRAIL sensitization in MM cells. In summary, these results identify a novel interaction between Aurora and IKK kinases and show that these pathways can cooperate to promote TRAIL resistance. Finally, combining pan-AKIs with TRAIL in vivo showed dramatic efficacy in a multidrug-resistant human myeloma xenograft model. These findings suggest that combining Aurora kinase inhibitors with TRAIL may have therapeutic benefit in MM.

Molecular Target Characterization and Antimyeloma Activity of the Novel, Insulin-like Growth Factor 1 Receptor Inhibitor, GTx-134

Therapeutic strategies that target insulin-like growth factor 1 receptor (IGF-1R) hold promise in a wide variety of cancers including multiple myeloma (MM). In this study, we describe GTx-134, a novel small-molecule inhibitor of IGF-1R and insulin receptor (IR) and characterized its antitumor activity in preclinical models of MM. The activity of GTx-134 as a single agent and in combination was tested in MM cell lines and primary patient samples. Downstream effector proteins and correlation with apoptosis was evaluated. Cytotoxcity in bone marrow stroma coculture experiments was assessed. Finally, the in vivo efficacy was evaluated in a human myeloma xenograft model. GTx-134 inhibited the growth of 10 of 14 myeloma cell lines (<5 μmol/L) and induced apoptosis. Sensitivity to GTx-134 correlated with IGF-1R signal inhibition. Expression of MDR-1 and CD45 were associated with resistance to GTx-134. Coculture with insulin-growth factor-1 (IGF-1) or adherence to bone marrow stroma conferred modest resistance, but did not overcome GTx-134-induced cytotoxicity. GTx-134 showed in vitro synergies when combined with dexamethasone or lenalidomide. Further, GTx-134 enhanced the activity of PD173074, a fibroblast growth factor receptor 3 (FGFR3) inhibitor, against t(4;14) myeloma cells. Therapeutic efficacy of GTx-134 was shown against primary cells and xenograft tumors. Although dysregulation of glucose homeostasis was observed in GTx-134-treated mice, impairment of glucose tolerance was modest. These studies support the potential therapeutic efficacy of GTx-134 in MM. Further, they provide a rationale for clinical application in combination with established antimyeloma treatments and novel targeted therapies.

Antimyeloma Action of the Multikinase Inhibitor TG02

AbstractAbstract 4062Multiple myeloma (MM) is the second most common hematologic malignancy and remains incurable, despite recent advances in its treatment. Therefore, studies to develop new therapies are still necessary. MM is characterized by genetic instability with numerous chromosomal abnormalities and deregulation of several signalling pathways. Cell cycle control is also dysfunctional in this pathology, and altered functioning of cyclins has been indicated as a universal alteration in MM. Cyclins exert their action by controlling the activity of the cyclin-dependent kinases (CDKs). Therefore, CDKs represent promising novel therapeutic targets for MM. Several other kinases have important functions in MM, one of which - BMK or ERK5 - plays a key role in the proliferation and survival of the myelomatous clone. In this study we describe the preclinical activity of a novel multi-kinase inhibitor, TG02, in MM. This compound combines cell cycle regulatory and transcriptional CDK inhibition with activity against other oncogenic kinases, such as ERK5, JAK2, TYK2, FLT3 and TYRO3. Functional potency was assessed using MTT-based proliferation assays in a panel of MM cell lines. Maximal cytotoxicity was observed in the MM1S cell line with an IC50 value after 24 hours of 64 nM. There were cell lines with a more resistant phenotype, like OPM2, with an IC50 value after 24 hours of 4.7 μ M (although this was reduced to low nM by prolonged exposure). TG02 also inhibited the protective effect of the bone marrow microenvironment as it was able to induce growth inhibition of MM cells grown in direct coculture with bone marrow stromal cells (BMSC). To delineate the underlying mechanism of cytotoxicity induced by this compound, we performed cell cycle and apoptosis assays. There was a slight accumulation of cells in the G2-M phase of the cell cycle but the more marked effect was an increase in Annexin V-staining, indicative of apoptosis, which was detected as early as six hours after treatment of MM1S cells with 100 nM TG02. This process was associated with depletion of the anti-apoptotic proteins Mcl-1 and Bcl-XL, and cleavage of caspases-8,-9,-3 and -7 and PARP. Treatment with TG02 also induced a fall in mitochondrial membrane potential and the release of cytochrome c and AIF into the cytosol. We have also analyzed some substrates of the different kinases that TG02 targets, such as Rb (phosphorylated at Ser-807 by CDK2) and RNA pol II (phosphorylated by CDK9). TG02 dephosphorylated these two proteins in MM1S cells. TG02 was tested for in vivo anti-tumor activity using the human OPM2 MM xenograft model. In this model we observed that TG02 was able to decrease tumor volumes, and synergized with bortezomib. In conclusion, these results show a potent anti-myeloma activity of the multi-kinase inhibitor TG02, and supports additional preclinical studies together with its clinical development for the treatment of MM. Disclosures:Meshow:Charles River: Employment. Cheatham:Charles River: Employment. Burrows:Tragara Pharmaceutical: Employment.

The histone deacetylase inhibitor LBH589 enhances the anti-myeloma effects of chemotherapy in vitro and in vivo

Panobinostat (LBH589) is a potent histone deacetylase inhibitor (HDACi) that has shown anti-tumor activity in preclinical studies in both solid and hematological malignancies. We evaluated the anti-multiple myeloma (MM) effects of LBH589 alone and with melphalan or doxorubicin using MM cell lines and our human MM xenograft model LAGλ-1. LBH589 treatment resulted in increased acetylation of histones, induction of caspase cleavage, inhibition of cell proliferation and synergistic anti-MM effects with melphalan or doxorubicin in vitro. LBH589 with melphalan or doxorubicin also showed significantly enhanced anti-myeloma activity in vivo. These findings provide the basis for clinical development of these combination therapies.

Ascorbic acid inhibits antitumor activity of bortezomib in vivo

Earlier studies have shown that ascorbic acid (vitamin C) inhibits bortezomib-induced cytotoxicity against cancer cells in vitro. However, the clinical significance of vitamin C on bortezomib treatment is unclear. In this study, we examined whether daily oral intake of vitamin C inhibits antimultiple myeloma (MM) activities of bortezomib. Vitamin C, at orally achievable concentrations, inhibited in vitro MM cell cytotoxicity of bortezomib and blocked its inhibitory effect on 20S proteasome activity. Specifically, plasma collected from healthy volunteers taking 1 g/day vitamin C reduced bortezomib-induced MM cell death in vitro. This antagonistic effect of vitamin C against proteasome inhibitors is limited to the boronate class of inhibitors (bortezomib and MG262). In vivo activity of this combination treatment was then evaluated using our xenograft model of human MM in SCID (severe combined immune-deficient) mice. Bortezomib (0.1 mg/kg twice a week for 4 weeks) significantly inhibits in vivo MM cell growth, which was blocked by oral vitamin C (40 mg/kg/day). Therefore, our results for the first time show that vitamin C can significantly reduce the activity of bortezomib treatment in vivo; and importantly, suggest that patients receiving treatment with bortezomib should avoid taking vitamin C dietary supplements.

Antitumor activity of humanized monoclonal antibody against HM1.24 antigen in human myeloma xenograft models

A humanized monoclonal antibody against HM1.24 antigen (AHM), which is highly expressed on multiple myeloma (MM) cells, induced antibody-dependent cellular cytotoxicity (ADCC) in vitro. In this study, we further characterized AHM and evaluated its potency for clinical application. AHM bound to HM1.24 antigen with a dissociation constant of 0.35 nM, and its epitope resided between Leu116 and Leu127 of the HM1.24 antigen. Single intravenous injection of AHM significantly inhibited tumor growth in both orthotopic and ectopic human MM xenograft models. AHM reduced serum M protein levels and prolonged survival of mice intravenously inoculated with KPMM2 and ARH-77 cells. The number of KPMM2 cells in bone marrow or tumor volume of subcutaneously inoculated RPMI 8226 cells was also inhibited by AHM. The antitumor activity of AHM against tumor cells in bone marrow was diminished when the mice were pretreated with anti-Fcgamma receptor III/II antibody, demonstrating that antitumor activity by AHM requires effector cell functions in vivo. Experiments involving in vitro ADCC assays indicated that NK cells and monocytes/macrophages serve as effector cells for AHM-induced ADCC in mouse and human. Thus, AHM will provide an additional treatment option for MM.