CRM1 Is Highly Expressed in Myeloma Plasma Cells and Its Inhibition by KPT-SINE Induces Cytotoxicity by Increasing p53 in the Nucleus of Multiple Myeloma (MM) Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1852-1852 ◽  
Author(s):  
Malathi Kandarpa ◽  
Stephanie J Kraftson ◽  
Sean P Maxwell ◽  
Dilara McCauley ◽  
Sharon Shacham ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Cell Lines ◽  
Tumor Suppressors ◽  
Nuclear Export ◽  
Plasma Cells ◽  
Annexin V ◽  
Apoptosis Induction ◽  
Advisory Committees ◽  
Tumor Suppressor Proteins ◽  
Rpmi 8226

Abstract Abstract 1852 Background: CRM1 (XPO1, exportin) is a nuclear export protein which controls the nuclear-cytoplasmic localization of multiple tumor suppressor proteins and cell proliferation pathways including p53, p21, PI3K/AKT/FOXO, Wnt/ß-catenin/APC, topoisomerase II, and NF-κB/I-κB. Transport of nuclear proteins to the cytoplasm can render them ineffective as tumor suppressors or as targets for chemotherapy. Small molecule, selective inhibitors of nuclear export (SINE) that block CRM1-dependent nuclear export can force the nuclear retention of tumor suppressor proteins, thus rendering cancer cells more susceptible to apoptosis and responsive to other chemotherapy. In this study we evaluated CRM1 as a potential target in MM and the effect of SINE on the activity of established anti-myeloma agents currently in use in treatment of MM. KPT-276 is the lead CRM1 inhibitor being investigated which will be submitted for IND in 2012. Methods: To evaluate expression of CRM1, bone marrow aspirates from MM patients and tonsil tissue from normal patients were enriched for plasma cells (PC) and proteins from cell lysates were separated by SDS-PAGE followed by immunoblotting with CRM1 antibodies. In functional experiments, isolated fresh MM PCs from patients, and NCI-H929, MM1.S, MM1.R and RPMI-8226 cell lines were cultured in RPMI-1640 with 10–15% serum. Cells were treated for 24–72 hrs with CRM1 inhibitors KPT-SINE compounds with or without bortezomib and dexamethasone and were analyzed for cytotoxicity by MTT assay. Drug concentrations for combination experiments were chosen to be at or below IC50 for each individual drug. Apoptosis induction in primary MM cells and cell lines was studied by Annexin V labeling and flow cytometry. Cell lysates from primary MM PCs and cell lines were prepared after treatment with KPT-SINE and were used to determine the expression of p53 and CRM1. Results: Primary MM plasma cells derived from naïve, previously untreated patients show 4–20 fold higher CRM1 protein expression, compared to normal peripheral blood mononuclear cells (PBMCs) and normal tonsilar PCs. Dose response analysis of KPT-SINE compounds in myeloma cell lines showed potent activity with IC50s in the range of 10–100nM. The lead compound KPT-276 had an IC50 of <100 nM in NCI-H929, MM1.S, MM1.R and RPMI-8226 cells. Functional studies in MM patient plasma cells showed that in vitro inhibition of CRM1 with related SINEs KPT-185, −225 or −276 increase apoptosis induction as measured by Annexin V assay. In addition, the inhibition of CRM1 with KPT-SINE results in a dose-dependent increase in levels of nuclear as well as total p53 in MM patient plasma cells within 48 hrs. When combined with proteasome inhibitors like bortezomib and/or dexamethasone, KPT-SINE compounds potently increase the cellular cytotoxicity of these drugs in MM cell lines. Mechanism of activity of drug combinations is under investigation in MM cell lines and MM patient plasma cells. Conclusions: MM plasma cells express CRM1 that is functionally active and therefore is a valid target in the treatment of myeloma. Moreover, higher expression of CRM1 in malignant plasma cells compared to normal PBMCs and normal PCs suggests possibility of therapeutic index. Early mechanistic studies indicate that CRM1 inhibition can lead to an increased expression of p53 (and other tumor suppressors) and its nuclear localization in myeloma cells and therefore might serve as a mechanism for the activity of CRM1 inhibitors in MM. Potentiation of cytotoxicity of bortezomib and dexamethasone by KPT-SINE suggests that these drugs might be useful in treating MM refractory to currently used agents and provide rationale for combining inhibitors of nuclear transport with other drugs. Disclosures: Off Label Use: KPT-SINE family of drugs are not approved for the treatment of multiple myeloma. These drugs have a novel mechanism and are in pre-clinical development for the treatment of several malignancies. McCauley:Karyopharm Therapeutic Inc.: Employment. Shacham:Karyopharm Therapeutics Inc.: Employment. Kauffman:Karyopharm Therapeutics Inc.: Employment. Jakubowiak:Exelixis: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Onyx Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Millennium Pharmaceuticals, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Ortho Biotech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

A Novel SIRT1 Activator SIRT1720 Triggers In Vitro and In Vivo Cytotoxicity In Multiple Myeloma Via ATM-Dependent Mechanism

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3007-3007
Author(s):  
Dharminder Chauhan ◽  
Madhavi Bandi ◽  
Ajita V Singh ◽  
Teru Hideshima ◽  
Nikhil C. Munshi ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Board Of Directors ◽  
Cell Lines ◽  
Annexin V ◽  
Immunoblot Analysis ◽  
Advisory Committees ◽  
Rpmi 8226 ◽  
Sirt1 Activator

Abstract Abstract 3007 Background and Rationale: SIRT1 belongs to the silent information regulator 2 (Sir2) family of proteins and functions as NAD+-dependent deacetylase. Previous studies showed that resveratrol, a polyphenolic SIRT1 activator, inhibits tumorigenesis in various solid tumor and hematologic malignancies, including human multiple myeloma (MM) cells. This notion led to the discovery and development of more potent and selective pharmacological activators of SIRT1 as potential anti-cancer therapeutics. In this context, a recent medicinal chemistry research using high-throughput screening, and mass spectrometry identified SRT1720, a small molecule activator of SIRT1 that is structurally distinct from resveratrol. Here, we examined the anti-tumor activity of SRT1720 in MM cells using in vitro and in vivo model systems. Methods and Model: We utilized MM.1S, MM.1R, RPMI-8226, U266, KMS12BM, H929, and INA-6 (an IL-6 dependent) human MM cell lines, as well as purified tumor cells from patients with MM relapsing after prior therapies including lenalidomide or bortezomib. Cell viability, proliferation, and apoptosis assays were performed using trypan blue, MTT, thymidine incorporation, and Annexin V staining. Signal transduction pathways were evaluated using immunoblot analysis, ELISA, and enzymology assays. Results: We first confirmed the functional specificity of SRT1720 against SIRT1 using different experimental strategies. First, we utilized Fluor de Lys Deacetylase Assay to measure whether SRT1720 affects the SIRT1 deacetylase enzymatic activity. Treatment of MM.1R and RPMI-8226 MM cells with SRT1720 markedly increased the deacetylating activity; conversely, pre-treatment of cells with nicotinamide (NAM) - an inhibitor of SIRT1 – significantly blocked SRT1720-triggered deacetylating activity. Second, immunoblot analysis using antibodies specific against acetylated p53 (a known substrate of SIRT1) showed a marked decrease in acetylated state of p53 in SRT1720-treated MM cells. These findings in MM cells confirm SIRT1 as a selective target of SRT1720. We next examined the efficacyof SRT1720 in MM cells. Treatment of MM cell lines and primary patient cells for 24h significantly decreased their viability (IC50 range 3–7 uM) (P < 0.005; n=3) without markedly affecting the viability of normal peripheral blood mononuclear cells, suggesting specific anti-MM activity and a favorable therapeutic index for SRT1720. SRT1720-triggered apoptosis was confirmed in MM.1R and RPMI-8226 cells, evidenced by a marked increase in Annexin V+ and PI- cell population (P < 0.001, n=3). Importantly, SRT1720 induced apoptosis in MM cells even in the presence of bone marrow stromal cells. Mechanistic studies showed that SRT1720-triggered apoptosis in MM cells is associated with 1) activation of caspase-8, caspase-9, caspase-3, and PARP; 2) activation of pATM, CHK2, endoplasmic reticulum stress molecules pEIF2, and BIP; as well as an increase in reactive oxygen species (ROS); 3) inhibition of MM cell growth and survival pathway via NF-kB; and 4) inhibition of VEGF-induced migration of MM cells and associated angiogenesis. Importantly, blockade of pATM using a biochemical inhibitor KU-5593 significantly attenuated SRT1720-induced MM cell death (P value < 0.002; n=2). These data suggest that SRT1720-induced MM cell apoptosis is predominantly mediated by an ATM-dependent apoptotic pathway. We next examined the in vivo efficacy of SRT1720 using a human plasmacytoma xenograft mouse model. Treatment of tumor-bearing mice with SRT1720 (200 mg/kg, 5 days a week for three weeks), but not vehicle alone, significantly (P < 0.008) inhibits MM tumor growth in these mice. Finally, the combination of SRT1720 with bortezomib or dexamethasone triggered synergistic anti-MM activity. Conclusions: These preclinical studies provide the rationale for novel therapeutics targeting SIRT1 to improve patient outcome in MM. Disclosures: Munshi: Millennium Pharmaceuticals: Honoraria, Speakers Bureau. Richardson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees. Anderson:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Anti-Myeloma Activity of Enzymatically Activated Melphalan Prodrug J1

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1838-1838
Author(s):  
Dharminder Chauhan ◽  
Madhavi Bandi ◽  
Ajita V Singh ◽  
Klaus Podar ◽  
Paul G Richardson ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Board Of Directors ◽  
Cell Lines ◽  
Annexin V ◽  
Immunoblot Analysis ◽  
Clinical Protocols ◽  
Advisory Committees ◽  
Pre Treatment ◽  
Rpmi 8226

Abstract Abstract 1838 Background and Rationale: The alkylating drug melphalan is routinely used in clinical protocols for the treatment of multiple myeloma (MM). Importantly, clinical trials in MM have effectively utilized combination of melphalan with proteasome inhibitor bortezomib and prednisolone (VMP regimen) to reduce toxicity, overcome drug resistance and enhance cytotoxicity. These findings highlight the utility of conventional alkylating agent, and importantly, provide impetus to develop conventional agents based prodrugs with a potent cytotoxic activity. In this context, pharmacological screening of alkylating oligopeptides led to the identification of a novel melphalan-containing prodrug J1 (L-melphalanyl-p-L-fluoro phenylalanine ethyl ester) - a new molecular entity with a more distinct activity profile than melphalan (Gullbo J, et al., Anticancer Drugs 2003,14:617–24; Gullbo J, et al., Invest New Drugs 2004, 22:411–20; Wickstrom M, et al., Mol Cancer Ther 2007, 6:2409–17). J1 is rapidly incorporated into the tumor cells cytoplasm, followed by intracellular hydrolysis in part mediated by aminopeptidase N (APN), resulting in a 10-fold greater release of free intracellular melphalan than exposure to melphalan at the same molar concentration (Gullbo J, et al., J Drug Target 2003,11:355–63; Wickstrom et al., Biochem Pharmacol 2010, 79(9):1281-90). In vitro studies showed a greater cytotoxic potency of J1 versus melphalan against different human solid cancers; however, its effect in MM is undefined. In the present study, we examined the anti-tumor activity of J1 in MM cells using both in vitro and in vivo model systems. Methods and Models: We utilized MM.1S, MM.1R, RPMI-8226, melphalan-resistant derivative of RPMI-8226 (LR-5), KMS12BM, and INA-6 (an IL-6 dependent) human MM cell lines, as well as purified tumor cells from patients with MM relapsing after prior therapies including lenalidomide or bortezomib. Cell viability-, proliferation-, and apoptosis assays were performed using Trypan blue, MTT, thymidine incorporation, and Annexin V/Propidium iodide staining. Signal transduction pathways were evaluated using immunoblot analysis, ELISA, and enzymologic assays. Statistical significance of data was determined using Student t test. Results: As aminopeptidase N (APN) has been shown to play a key role in conversion of J1 into melphalan in solid tumors, we first examined both expression and enzymatic activity of APN in MM cells. Immunoblot analysis showed a high expression of APN in various MM cell lines. Similarly, colorimetric analysis of APN enzymatic activity using the APN substrate L-alanine-4-nitro-anilide demonstrated elevated APN activity in MM cells. Moreover, pre-treatment of MM cells with APN inhibitor Bestatin showed a moderate, but significant blockade of J1-induced cytotoxicity in MM cells (P < 0.05; n=3). We next examined the effects of J1 in MM cells. Treatment of MM cell lines and primary patient cells for 24h significantly decreased their viability (IC50 range 0.5 – 1.0 uM; P < 0.001; n=3) without markedly affecting the viability of normal peripheral blood mononuclear cells, suggesting specific anti-MM activity and a favorable therapeutic index forJ1. Of note, the IC50 range of melphalan for MM cell lines is 10–40 uM. J1-triggered apoptosis was confirmed in MM.1R and RPMI-8226 cells, evidenced by marked increase in Annexin V+ and PI-cell population (P < 0.001, n=3). Importantly, J1induced apoptosis in MM cells even in the presence of MM bone marrow stromal cells. Mechanistic studies showed that J1-triggered apoptosis in MM cells is associated with 1) activation of caspase-7, caspase-8, caspase-9, caspase-3, and PARP; 2) induction of phospho-c-Jun and phospho-JNK, p53, and p21; 3) release of mitochondrial apoptogenic protein cytochrome-c; 4) inhibition of VEGF-induced migration of MM cells and angiogenesis; and 5) induction of DNA damage response, evidenced by increase in phospho-histone H2AX. Pre-treatment of MM cells with pan-caspase inhibitor Z-VAD-fmk attenuated J1-triggered MM cell apoptosis (P value < 0.001; n=3). Finally, treatment of tumor-bearing mice with J1 (3 mg/kg, twice a week for 2 weeks), but not vehicle alone, significantly (P < 0.008) inhibits MM tumor growth in these mice. Conclusions: Our study provides the rationale for clinical protocols evaluating J1, either alone or in combination, to improve patient outcome in MM. Disclosures: Richardson: Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees. Munshi:Millennium Pharmaceuticals: Honoraria, Speakers Bureau. Spira:Oncopeptide AB: Employment, Equity Ownership. Anderson:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Identification of Biomarkers Associated with MAF-Mediated Resistance to Proteasome Inhibitors in t(14;16) Multiple Myeloma

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3020-3020 ◽  
Author(s):  
Ya-Wei Qiang ◽  
Shiqiao Ye ◽  
Ricky D Edmondson ◽  
Yuhua Huang ◽  
Sarah K. Johnson ◽  
...  
Keyword(s):  
Cell Line ◽  
Board Of Directors ◽  
Cell Lines ◽  
Target Genes ◽  
Plasma Cells ◽  
Proteasome Inhibitors ◽  
Annexin V ◽  
University Of Arkansas ◽  
Medical Sciences ◽  
Advisory Committees

Abstract Introduction : Multiple myeloma (MM) patients with t(14;16)(p16;q32) leading to high expression of C-MAF mRNA and protein have a poor prognosis and have not benefitted from the recent advances in MM therapy. Our previous work showed that posttranslational modification up-regulated C-MAF (ASH Abstract # 281, ASH 2013) and MAF-B proteins (ASH Abstract #2091, 2014) conferred innate resistance to proteasome inhibitors (PI). In order to identify the molecular mechanism underlying this resistance in high C-MAF and MAFB-expressing patients, the present study investigated the downstream targets regulated by C-MAF in MM cells and analyzed the clinical association of target genes using a C-MAF-loss and gain of function cell line model as well as primary plasma cells from MM patients. Methods : To elucidate the molecular mechanism underlying the resistance of MAF expressing cells to PIs, we silenced the C-MAF gene in 3 MM cell lines that highly expressed C-MAF mRNA and protein using lentiviral shRNA. Annexin V staining and analyzed by FACS were used to determine the effect of manipulating MAF on PI-induced apoptosis and activation of the caspase family. Gene expression prolife (GEP) was utilized to determine downstream target genes regulated by MAF. To identify the proteins associated with C-MAF, we used mass spectrometry (MS)-based quantitative proteomics with stable isotope labeling by amino acids in cell culture (SILAC). Results: Our results demonstrated that apoptotic cell numbers were higher in shMAF cells, compared to shControl cells when cultured in the presence of serial concentrations of bortezomib (BZB) or carfilzomib (CFZ), indicating that silencing MAF expression enhances PI-induced apoptosis. The Annexin V + cells numbers were also higher in shMAF compared to shControl when cells were treated with BZB or CFZ in co-culture with bone marrow stromal cells. Furthermore, knockdown of C-MAF expression augmented the PI-induced activation of the caspase family with an increase in the cleavage fragments of caspases-3, -7, -8, PARP and LaminA/C. In contrast, PI-induced apoptotic numbers were lower in XG1/MAF, a stably induced MAF overexpressing MM cell line, than in XG1/EV, the control cell line carrying an empty vector when cultured in serial concentrations of BZB and CFZ. Similarly, the caspase cleavage fragments, PARP and lamin A/C were decreased in XG1/MAF compared with XG1/EV in response to BZB and CFZ. These results suggest that MAF confers MM resistance to PI by preventing the PI-induction of apoptosis. To further identify the downstream target genes regulated by C-MAF, we analyzed the gene expression between shMAF and shControl MM cells by microarray analysis and identified more than 150 genes differentially expressed. Of them, ATP2C1, CCNE2 , CCND2, ELK4 , IGFBP3, SUPT16 and TUBA1A were significantly (p=5.62e-5) lower in shMAF than shControl cells indicating that these genes might be regulated by C-MAF. Furthermore, expression of these genes in primary CD138 plasma cells were significant higher (p<0.05) in the UAMS molecular MF subgroup than other UAMS molecular subgroups. A number of genes were also expressed significantly lower (p=5.62e-5) in shMAF cells compared to shControl cells. The clinical association of these genes was analyzed and showed that the expression of IL10Ra (p<0.05), PAXBP1PAX3 (p=0.0032), USP1 (p=0.0038), BNIP3 (p<0.05), ACSL (p=0.009) and SP3 (p=0.018) in CD138 plasma cells was associated with poor overall survival in patients from the MF subgroup indicating that these targets may play a central role in MAF regulated proliferation of MM cells. To identify the proteins associated with C-MAF, we used MS-SILAC and identified more than 20 proteins as differentially expressed between MAF positive cell lines and shMAF cell lines . Western blotting and flow cytometry analysis are ongoing to confirm the functional role of these proteins in MAF regulated MM cell growth. Conclusions : Taken together, these results suggest that MAF confers MM resistance to PI by preventing the induction of apoptosis. Global gene and protein expression profiling along with correlation with clinical data, has identified a number of relevant biologically relevant candidates. Experiments are ongoing to confirm the function of these MAF regulated candidates in order to identify potential alternate approaches for the treatment of this poor performing molecular subgroup. Disclosures Qiang: University of Arkansas for Medical Sciences: Employment. Ye:University of Arkansas for Medical Sciences: Employment. Huang:University of Arkansas for Medical Sciences: Employment. Johnson:University of Arkansas for Medical Sciences: Employment. Stein:University of Arkansas for Medical Sciences: Employment. Barlogie:University of Arkansas for Medical Sciences: Employment. Epstein:University of Arkansas for Medical Sciences: Employment. Morgan:MMRF: Honoraria; Weismann Institute: Honoraria; University of Arkansas for Medical Sciences: Employment; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; CancerNet: Honoraria; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Davies:Celgene: Consultancy; Janssen: Consultancy; Millenium: Consultancy; Onyx: Consultancy; University of Arkansas for Medical Sciences: Employment.

scholarly journals PSMB4 and PSMD4 Are Correlated with 1q21 Amplification in CD138 + Plasma Cells: New Potential Druggable Targets in Myeloma Patients

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2657-2657
Author(s):  
Jessica Burroughs-Garcia ◽  
Paola Storti ◽  
Luca Agnelli ◽  
Denise Toscani ◽  
Valentina Marchica ◽  
...  
Keyword(s):  
Gene Expression ◽  
Board Of Directors ◽  
Cell Lines ◽  
Copy Number ◽  
Plasma Cells ◽  
Disease Stage ◽  
Advisory Committees ◽  
Expression Levels ◽  
Proteasome Subunits ◽  
Rpmi 8226

Abstract The amplification of the 1q21 (amp1q21) region is one of the most acquired cytogenetic abnormalities (CA) in multiple myeloma (MM) associated with a worse patient outcome and disease progression. Moreover, different studies have demonstrated that the number of copies (CN) 1q21 (gain1q21: three copies or amp1q21: ≥ four copies) have a different impact in the response to anti-MM therapies. Particularly, it has been proposed that in MM patients, additional copies of 1q21 may be associated with the resistance to proteasome inhibitor (PI) treatment as bortezomib. A recent study showed that newly diagnosed MM (MMD) patients carrying amp1q21 but not gain1q21 receiving carfilzomib-based treatment have an early disease progression with shorter overall survival. Previous studies underlined that the amplification of 1q21 can lead to the overexpression and/or dysregulation of several candidate genes associated with cell proliferation, apoptosis, and drug resistance. Here we aim to identify 1q21 target genes possibly correlated to the response to PI therapy. We evaluated a total cohort of 29 primary plasma cells (PCs) purified from bone marrow (BM) blood aspirates from 11 smoldering MM (SMM) and 18 MMD. The median age of our cohort was 70 years (range: 38-86). Fluorescence in situ hybridization (FISH) analysis was performed to access the presence or absence of copy number alteration (CNA) in the 1q21 region in all patients. 14 out of 29 patients carried 1q21 CNA (5 with gain1q21 and 9 with amp1q21). A score reflecting the number of 1q21 copies was calculated based on the hybridization pattern. The transcriptional profiles of the 29 BM PCs samples were generated on GeneChip ClariomD Arrays (Affymetrix Inc., Santa Clara, CA, USA). The samr package was used in R for call genes as differentially expressed between 1q21 CN-altered and wild-type samples. The correlation between the 1q21 copy number score and the gene expression levels was performed. Moreover, we have evaluated by FISH the 1q21 CNA in a panel of human myeloma cell lines (HMCLs): OCY-MY5, JJN3, RPMI-8226, NCI-H929, and OPM2. JJN3 were transfected with a control vector and PSMB4 and PSMD4 short hairpin RNA (shRNA) lentivectors. The gene and protein expression levels of PSMB4 and PSMD4 in MM cell lines were analyzed by qRT-PCR and Western Blot, respectively. Cell viability and proliferation were assessed using MTT assay and flow cytometry. Our bioinformatic analyses highlighted the overexpression of different genes (IL6R, ILF2, BCL9, MCL1, CSk1B, ADAR1, ARNT, ANP32E) in the 1q21 CNA samples with respect to the controls, as already reported in the literature. Our analysis showed a significantly higher expression of two proteasome subunits (PSMB4 and PSMD4) in patients with 1q21 CNA when compared with patients without (PSMB4 p=0.0006; PSMD4 p=&lt;0.0001). Patients with amp1q21 showed a higher expression of PSMB4 when compared to the patients with gain1q21 (p=0,007). In our cohort, gene expression profile analysis also showed a strong positive correlation between gene expression levels and 1q21 CN for the proteasome subunits PSMB4 (p=&lt;0.0001, r=0.5631) and PSMD4 (p=&lt;0.0001, r=0.6391). Interestingly, we found that the PSMB4 and PSMD4 expression level was independent of the disease stage (SMM vs MM) and was only driven by 1q21 CN. We have evaluated PSMB4 and PSMD4 mRNA and protein expression levels in a 1q21 wild-type cell line (OCY-MY5) and in a panel of MM cell lines carrying different degrees of 1q21 CN (in order: JJN3, U266, RPMI-8226, OPM2, and NCI-H929). The mRNA expression level of PSMB4 and PSMD4 was higher in cell lines carrying 1q21 amp, following a 1q21 copy number fashion. Similar results were obtained when protein levels of MM cell lines were analyzed by Western Blot. To further determine the potential role of both proteasome subunits in the pathogenesis of amp1q21, we generated a PSMB4-shRNA and PSMD4-shRNA knockdown stable MM cell lines. Functional studies showed that blockade of PSMB4 and PSMD4 decreased MM cell viability. In conclusion, our study identified proteasome subunits PSMB4 and PSMD4 to be significantly upregulated in MM patients carrying amp1q21, correlated with 1q21 copy number but not with disease stage. In addition, knockdown of both, PSMB4 and PSMD4 decreased MM cell proliferation. Therefore, targeting PSMB4 and PSMD4 could be a strategy to treat MM patients with ampq21 Disclosures Giuliani: Celgene: Membership on an entity's Board of Directors or advisory committees, Other: congress, Research Funding; Bristol Mayers Squibb: Other: congress; GSK: Other: clinical studies; Takeda: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees, Other: Clinical studies, congress, Research Funding; Millenium Pharmaceutical: Other: clincial studies.

ABT-737, an Inhibitor of Bcl-2/Bcl-XL Proteins, Is Cytotoxic in Multiple Myeloma Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1589-1589
Author(s):  
Michael Kline ◽  
Terry Kimlinger ◽  
Michael Timm ◽  
Jessica Haug ◽  
John A. Lust ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Tumor Microenvironment ◽  
Cell Lines ◽  
Plasma Cells ◽  
Annexin V ◽  
Significant Activity ◽  
Rpmi 8226 ◽  
Dose Dependent

Abstract Background: Multiple myeloma (MM) is a plasma cell proliferative disorder that is incurable with the currently available therapeutics. New therapies based on better understanding of the disease biology are urgently needed. MM is characterized by accumulation of malignant plasma cells predominantly in the bone marrow. These plasma cells exhibit a relatively low proliferative rate as well as a low rate of apoptosis. Elevated expression of the anti-apoptotic Bcl-2 family members has been reported in MM cell lines as well as in primary patient samples and may be correlated with disease stage as well as resistance to therapy. ABT-737 (Abbott Laboratories, Abbott Park, IL) is a small-molecule inhibitor designed to specifically inhibit anti-apoptotic proteins of the Bcl-2 family and binds with high affinity to Bcl-XL, Bcl-2, and Bcl-w. ABT-737 exhibits toxicity in human tumor cell lines, malignant primary cells, and mouse tumor models. We have examined the in vitro activity of this compound in the context of MM to develop a rationale for future clinical evaluation. Methods: MM cell lines were cultured in RPMI 1640 containing 10% fetal bovine serum supplemented with L-Glutamine, penicillin, and streptomycin. The KAS-6/1 cell line was also supplemented with 1 ng/ml IL-6. Cytotoxicity of ABT-737 was measured using the MTT viability assay. Apoptosis was measured using flow cytometry upon cell staining with Annexin V-FITC and propidium iodide (PI). Flow cytometry was also used to measure BAX: Bcl-2 ratios after ABT-737 treatment and cell permeabilization with FIX & PERM (Caltag Laboratories, Burlingame, CA) Results: ABT-737 exhibited cytotoxicity in several MM cell lines including RPMI 8226, KAS-6/1, OPM-1, OPM-2, and U266 with an LC50 of 5-10μM. The drug also had significant activity against MM cell lines resistant to conventional agents such as melphalan (LR5) and dexamethasone (MM1.R) with similar LC50 (5-10 μM), as well as against doxorubicin resistant cells (Dox40), albeit at higher doses. Furthermore, ABT-737 retained activity in culture conditions reflective of the permissive tumor microenvironment, namely in the presence of VEGF, IL-6, or in co-culture with marrow-derived stromal cells. ABT-737 was also cytotoxic to freshly isolated primary patient MM cells. Time and dose dependent induction of apoptosis was confirmed using Annexin V/PI staining of the MM cell line RPMI 8226. Flow cytometry analysis of cells treated with ABT-737 demonstrated a time and dose dependent increase in pro-apoptotic BAX protein expression without significant change in the Bcl-XL or Bcl-2 expression. Ongoing studies are examining the parameters and mechanisms of ABT-737 cytotoxicity to MM cells in more detail. Conclusion: ABT-737 has significant activity against MM cell lines and patient derived primary MM cells in vitro. It is able to overcome resistance to conventional anti-myeloma agents suggesting a different mechanism of toxicity that may replace or supplement these therapies. Additionally, it appears to be able to overcome resistance offered by elements of the tumor microenvironment. The results of these studies will form the framework for future clinical evaluation of this agent in the clinical setting.

C/EBPβ Is a Critical Factor for Proliferation and Apoptosis in MM Cells by Controlling Transcription Factors Like IRF-4, PAX5 and Blimp1.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1506-1506
Author(s):  
Rekha Pal ◽  
Martin Janz ◽  
Deborah Galson ◽  
Suzanne Lentzsch
Keyword(s):  
Transcription Factors ◽  
Western Blot ◽  
Cell Lines ◽  
Plasma Cells ◽  
Annexin V ◽  
Critical Factor ◽  
Myeloma Cell ◽  
Down Regulation ◽  
Protein Levels ◽  
Rpmi 8226

Abstract The development and maturation of plasma cells is dictated by multiple interacting transcription factors (TFs). C/EBPb (NF-IL6) is a TF regulated by IL-6 and has profound effects on the regulation of growth, survival and differentiation of B-cells. Mice deficient in C/EBPb show impaired generation of B lymphocytes suggesting that C/EBPb plays an important role in B lymphopoiesis. In this study we delineated the effect of C/EBPb on transcription factors critical for myeloma cell proliferation by over-expressing and inhibiting C/EBPb in myeloma cells. Multiple myeloma (MM) cell lines MM.1S, RPMI-8226 and H929 were transiently transfected with GFP, C/EBPb (pcNF-IL6), and truncated C/EBPb with a deletion of the internal spII-spII fragment [pcmNF-IL6(Dspl)] by using Bio-Rad Gene Pulser Xcell, followed by G418 selection. A pool of transfected cells was selected and subjected to thymidine incorporation, flow cytometry and western blot analysis. We found that transfection of a truncated form of C/EBPb induced a down-regulation of C/EBPb in MM cell lines (MM.1S, RPMI-8226 and H929) as measured by western blot. Down-regulation of C/EBPβ significantly inhibited proliferation and induced apoptosis of MM cell lines analyzed by annexin V-FITC/PI staining. This was accompanied by a complete down-regulation of the anti-apoptotic protein BCL-2. Further, inhibition of C/EBPb completely decreased IRF-4 expression. In contrast, over-expression of C/EBPb increased protein levels of IRF-4 suggesting that IRF-4 is under control of C/EBPb. IRF-4, which was over-expressed in all our tested MM cells lines, is an essential TF for the generation of plasma cells by regulating TFs like Blimp-1 and PAX-5, which are critical for plasma cell differentiation. Our studies showed that down-regulation of IRF-4 resulted in a complete abrogation of Blimp-1 and PAX-5 suggesting that the expression of these factors is C/EBPb/IRF-4 dependent. In conclusion, our data indicate that C/EBPb is an important key regulator for survival and growth of MM cells. We show for the first time that C/EBPb is a critical regulator upstream of IRF-4. Down-regulation of the C/EBPb and consequently IRF-4 results in complete disruption of the network of TFs necessary for MM growth and survival. Targeting C/EBPb may provide a novel therapeutic approach in the treatment of MM.

Dual Targeting of -TORC1 and -TORC2 as a New Strategy In the Treatment of Multiple Myeloma

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 133-133 ◽  
Author(s):  
Patricia Maiso ◽  
AbdelKareem Azab ◽  
Yang Liu ◽  
Yong Zhang ◽  
Feda Azab ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Bone Marrow ◽  
Board Of Directors ◽  
Cell Lines ◽  
Mechanism Of Action ◽  
Plasma Cells ◽  
Bone Marrow Microenvironment ◽  
Advisory Committees

Abstract Abstract 133 Introduction: Mammalian target of rapamycin (mTOR) is a downstream serine/threonine kinase of the PI3K/Akt pathway that integrates signals from the tumor microenvironment such as cytokines and growth factors, nutrients and stresses to regulate multiple cellular processes, including translation, autophagy, metabolism, growth, motility and survival. Mechanistically, mTOR operates in two distinct multi-protein complexes, TORC1 and TORC2. Activation of TORC1 leads to the phosphorylation of p70S6 kinase and 4E-BP1, while activation of TORC2 regulates phosphorylation of Akt and other AGC kinases. In multiple myeloma (MM), PI3K/Akt plays an essential role enhancing cell growth and survival and is activated by the loss of the tumor suppressor gene PTEN and by the bone marrow microenvironment. Rapamycin analogues such as RAD001 and CCI-779 have been tested in clinical trials in MM. Their efficacy as single agents is modest, but when used in combination, they show higher responses. However, total inhibition of Akt and 4E-BP1 signaling requires inactivation of both complexes TORC1 and TORC2. Consequently, there is a need for novel inhibitors that can target mTOR in both signaling complexes. In this study we have evaluated the role of TORC1 and TORC2 in MM and the activity and mechanism of action of INK128, a novel, potent, selective and orally active small molecule TORC1/2 kinase inhibitor. Methods: Nine different MM cell lines and BM samples from MM patients were used in the study. The mechanism of action was investigated by MTT, Annexin V, cell cycle analysis, Western-blotting and siRNA assays. For the in vivo analyses, Luc+/GFP+ MM.1S cells (2 × 106/mouse) were injected into the tail vein of 30 SCID mice and tumor progression was detected by bioluminescence imaging. Nanofluidic proteomic immunoassays were performed in selected tumors. Results: To examine activation of the mTOR pathway in MM, we performed kinase activity assays and protein analyses of mTOR complexes and its downstream targets in nine MM cell lines. We found mTOR, Akt, pS6R and 4E-BP1 are constitutively activated in all cell lines tested independently of the status of Deptor, PTEN, and PI3K. All cell lines expressed either Raptor, Rictor or both; excepting H929 and U266LR7 which were negative for both of them. Moreover, primary plasma cells from several MM patients highly expressed pS6R while normal cells were negative for this protein. We found that INK128 and rapamycin effectively suppressed phosphorylation of p6SR, but only INK128 was able to decrease phosphorylation of 4E-BP1. We observed that INK128 fully suppressed cell viability in a dose and time dependent manner, but rapamycin reached a plateau in efficacy at ± 60%. The IC50 of INK128 was in the range of 7.5–30 nM in the eight cell lines tested. Similar results were observed in freshly isolated plasma cells from MM patients. Besides the induction of apoptosis and cell cycle arrest, INK128 was more potent than rapamycin to induce autophagy, and only INK128 was able to induce PARP and Caspases 3, 8 and 9 cleavage. In the bone marrow microenvironment context, INK128 inhibited the proliferation of MM cells and decreased the p4E-BP1 induction. Importantly, treatment with rapamycin under such conditions did not affect cell proliferation. INK128 also showed a significantly greater effect inhibiting cell adhesion to fibronectin OPM2 MM1S, BMSCs and HUVECs compared to rapamycin. These results were confirmed in vivo. Oral daily treatment of NK128 (1.0 mg/kg) decreased tumor growth and improved survival of mice implanted with MM1S. Conclusion: Dual inhibition of TORC1 and TORC2 represent a new and promising approach in the treatment of MM and its microenvironment. The ability of INK128 to inhibit both TORC1 and TORC2 strongly supports the potential use of this compound in MM patients. Disclosures: Anderson: Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Ghobrial:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees.

Decitabine Priming Enhances The Antileukemic Effects Of The Selective Inhibitor Of Nuclear Export (SINE) CRM1/XPO1 Antagonist (Selinexor) In Acute Myeloid Leukemia (AML)

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1453-1453 ◽  
Author(s):  
Parvathi Ranganathan ◽  
Xueyan Yu ◽  
Jessica Hofstetter ◽  
Ramasamy Santhanam ◽  
Sharon Schacham ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Line ◽  
Board Of Directors ◽  
Cell Lines ◽  
Nuclear Export ◽  
Sequential Treatment ◽  
Great Promise ◽  
Treatment Schedule ◽  
Advisory Committees ◽  
Novel Therapeutic

Abstract AML is a clonal hematopoietic disorder characterized by genetic and epigenetic alterations. The prognosis of AML is poor highlighting the urgent need for novel therapeutic approaches. Targeting aberrant DNA hypermethylation by using hypomethylating drugs such as decitabine has been recently investigated in AML. Our group reported a relatively effective (47% complete response) single agent decitabine treatment schedule for older AML patients. These results suggested the opportunity to capitalize on this relatively effective and non-toxic treatment by incorporating this compound into novel molecularly targeted approaches. Recent data indicate that targeting nuclear exporter proteins is a novel therapeutic strategy to overcome cancer. In particular, CRM1/XPO1 is the only nuclear exporter involved in the active transport of the majority of tumor suppressor proteins (TSP) [e.g. p53 and FOX03A] out of the nucleus resulting in their inactivation. We recently reported the anti-leukemic activity of oral SINE CRM1/XPO1 Antagonists in AML. SINEs displayed potent anti-proliferative properties, induced apoptosis, cell-cycle arrest and myeloid differentiation in AML cell lines and patient blasts. In addition, treatment of leukemic mice with oral SINE significantly prolongs their survival. By crossing the lists of genes known to be regulated by DNA methylation in AML with the ones whose nuclear transport is XPO1 dependent, we identified important TSPs such as FOXO3A and p21. Here we hypothesize that treatment of AML cells with decitabine will increase the transcription and expression of a subset of TSPs (including FOX03A and p21) whose nuclear anti-leukemic effects could be enhanced by blocking their XPO1 mediated nuclear export using the clinical stage oral SINE (Selinexor). Thus, we expect that the sequential treatment of decitabine followed by Selinexor will be more effective than each drug alone. To confirm this hypothesis first we treated the AML line OCI-AML3 cells with decitabine (500nM) overnight (ovn) followed by Selinexor (200nM, 2 fold lower than IC50) for an additional 24 hours (hrs) and measured cell proliferation using WST-1 assay. Controls include: 1) DMSO ovn +DMSO 24 hours, 2) Selinexor ovn +selinexor 24 hours (200 nM) and 3) decitabine ovn + decitabine 24 hrs (500nM). AML cells that were treated first with decitabine followed by Selinexor exhibited a higher cytotoxicity (absorbance (Abs) 0.7) than cells treated with either decitabine (Abs 1.36), Selinexor (Abs 1, p=0.006) or DMSO (Abs 1.7). Similar results were observed with the MV4-11 cell line. Next we measured the candidate TSPs (FOX03A and p21) mRNA and protein expression in OCI-AML-3 and MV4-11 cell lines after 24 hrs of decitabine treatment. We found a significant up-regulation of p21 in decitabine versus DMSO treated cells (MV4-11, Fold change (FC) 4.67±1.4; OCI-AML3, FC 3.98±1.19, p<0.05). We also detected a modest up-regulation of FOXO3A in both cell lines treated with decitabine when compared to the DMSO controls (MV4-11, FC 2.56±0.74 and OCI-AML3, FC 1.5±0.23, p<0.05). These results were confirmed also at the protein level by using western blot. Next, we asked whether ectopic up-regulation of p21 (mimicking decitabine effects) in OCI-AML3 cells could re-capitulate the decitabine enhancing antileukemic effects of Selinexor. Overexpression of p21 followed by Selinexor (200nM) for 24 hrs resulted in a larger decrease of cell proliferation (Abs 0.5) with respect to controls (Abs 0.7, p<0.05) using the WST assay. Similar results were observed for the MV4-11 cell line. Finally, we tested the efficacy of the sequential decitabine–Selinexor in vivo using the MV4-11 xenograft model. Treatment began one week after leukemic cell inoculation in 4 different cohorts; 1) Vehicle, 2) decitabine i.p. twice weekly (BIW) (0.4mg/kg); 3) Selinexor BIW (20mg/kg by oral gavage) and 4) decitabine BIW i.p (0.4 mg/kg) followed by Selinexor (10 mg/kg BIW). We found no difference in median survival time (MST) between vehicle and decitabine only treated mice. As expected, Selinexor alone treated mice have significantly improved MST at 36.5 days vs. 28.5 days, vehicle, p=<0.01). Most importantly, the sequential treatment of decitabine followed by Selinexor significantly improved MST compared to Selinexor alone 47 vs 36.5, p=0.008). These pre-clinical results hold great promise for the use of this combination in human clinical trials in AML. Disclosures: Schacham: Karyopharm: Membership on an entity’s Board of Directors or advisory committees. Kauffman:Karyopharm Therapeutics Inc.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties. Garzon:Karyopharm: Research Funding.

Expression Of Truncated Protein Tyrosine Phosphatase, Receptor Type, O (PTPROt) Influences Multiple Myeloma Sensitivity To Bortezomib Through Akt Signaling, and Is a Favorable Clinical Prognostic Factor

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2520-2520
Author(s):  
Hua Wang ◽  
Veerabhadran Baladandayuthapani ◽  
Zhiqiang Wang ◽  
Jiexin Zhang ◽  
Heather Yan Lin ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Myeloma Cell ◽  
Advisory Committees ◽  
Over Expression ◽  
Resistant Disease ◽  
Drug Naïve ◽  
Rpmi 8226

Abstract Background Proteasome inhibitors such as bortezomib and carfilzomib are an important part of our current chemotherapeutic armamentarium against multiple myeloma, and have improved outcomes in the up-front, relapsed, and relapsed/refractory settings. Their efficacy has been demonstrated both as single agents, and as part of rationally designed combination regimens, but they are at this time used empirically, since biomarkers to identify patients who would most or least benefit from their application have not been clinically validated. Moreover, the vast majority of patients eventually develop drug-resistant disease which precludes further proteasome inhibitor use through mechanisms that have not been fully elucidated. Methods We compared gene expression profiles (GEPs) of a panel of bortezomib-resistant myeloma cell lines and their vehicle-treated, drug-naïve counterparts to identify significant changes associated with drug resistance. The list of genes whose expression was changed by at least 2-fold was compared with independent RNA interference studies whose goal was to identify genes whose suppression conferred drug resistance. Further validation of genes of interest was pursued in a panel of myeloma cell lines, and in clinically annotated GEP databases. Results Suppression of PTPROt expression was noted in bortezomib-resistant RPMI 8226 and ANBL-6 myeloma cells compared to isogenic, drug-naïve controls, and this was confirmed by quantitative PCR. Overexpression of PTRPOt in RPMI 8226, ANBL-6 and other myeloma cell lines was by itself sufficient to increase the level of apoptotic, sub-G0/G1 cells compared to vector controls, or cells expressing a phosphatase-dead PTPROt mutant. Moreover, PTPROt enhanced the ability of bortezomib to reduce myeloma cell viability, in association with increased activation of caspases 8 and 9. Exogenous over-expression of PTPROt was found to reduce the activation status of Akt, a known anti-apoptotic pathway that reduces bortezomib activity, based on Western blotting with antibodies to phospho-Akt (Ser473), and Akt kinase activity assays. Notably, we also found that exogenous over-expression of PTPROt resulted in increased expression levels of p27Kip1. Interestingly, array CGH data from studies of myeloma cell lines and primary cells showed that the PTPROt gene was located in a genomic region with a high propensity for loss. Analysis of the Total Therapy databases of GEP and patient outcomes available on the Multiple Myeloma Genomics Portal showed that higher than median expression of PTPROt was associated with better long-term survival (P=0.0175). Finally, analysis of the Millennium Pharmaceuticals database of studies of bortezomib in the relapsed and relapsed/refractory setting showed high PTRPOt expression was more frequently seen in patients who achieved complete remission (P<0.01), and was associated with a better median overall survival (P=0.0003). Conclusions Taken together, the data support the possibility that high expression of PTPROt is a good prognostic factor for response to bortezomib-containing therapies, and that this may occur through modulation by PTPROt of the Akt pathway. Moreover, they suggest that strategies to enhance the expression of PTPROt should be investigated to restore bortezomib sensitivity in patients with proteasome inhibitor-resistant disease. Disclosures: Orlowski: Bristol-Myers Squibb: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Millennium: The Takeda Oncology Company: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Onyx: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Resverlogix: Research Funding; Array Biopharma: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Genentech: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Merck: Membership on an entity’s Board of Directors or advisory committees.

Novel Inhibitors Of CRM1/XPO1 Nuclear Exporter Exhibit Striking Activity Against AML “primagrafts,” Including AML Leukemia Initiating Cells, While Sparing Normal Hematopoietic Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3932-3932
Author(s):  
Julia Etchin ◽  
Bonnie Thi Le ◽  
Alex Kentsis ◽  
Richard M. Stone ◽  
Dilara McCauley ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Cell Lines ◽  
Nuclear Export ◽  
Hematopoietic Cells ◽  
Complex Karyotype ◽  
Employment Equity ◽  
Advisory Committees ◽  
Hematopoietic Stem ◽  
Nsg Mice ◽  
Equity Ownership

Abstract Current treatments for acute myeloid leukemia (AML) often fail to induce long-term remissions and are also toxic to normal tissues, prompting the need to develop new targeted therapies. One attractive cellular pathway with therapeutic potential is nuclear export, which is mediated in part by nuclear exporter CRM1/XPO1. XPO1 mediates the transport of ∼220 proteins and several mRNAs and is the sole nuclear exporter of the major tumor suppressor and growth regulatory proteins p53, p73, FOXO, IkB/NF-kB, Rb, p21, and NPM. Our findings demonstrate that novel irreversible inhibitors of XPO1, termed Selective Inhibitors of Nuclear Export, or SINE, induce rapid apoptosis in 12 AML and 14 T-ALL cell lines with IC50s of 15-474 nM. In the SINE-sensitive cell lines, BCL2 overexpression suppresses SINE-induced apoptosis, indicating its intrinsic pathway mediation. Oral administration of clinical XPO1 inhibitor, Selinexor (KPT-330), at 15 or 25 mg/kg, induced remarkable growth suppression in MV4-11 human AML cells and MOLT-4 human T-ALL cells engrafted in immunodeficient NSG mice with negligible toxicity to normal mouse hematopoietic cells after 35 days of treatment. Bone marrow biopsies of selinexor - treated mice were remarkable in that they showed normal hematopoietic cell morphology and cellularity after 35 days of treatment. Significant survival benefit was observed in mice treated with selinexor, compared to vehicle-treated mice. Selinexor is now in Phase 1 clinical trial in patients with AML and other hematological malignancies (NCT01607892). Recently, we have established primagraft models of AML, using primary leukemia blasts isolated from AML patients at diagnosis transplanted into immunocompromised NSG mice. We demonstrated that selinexor exhibits striking anti-leukemic activity against different subtypes of primary AML, including AML-M4; FLT3-ITD and complex karyotype subtypes of the disease. To determine whether selinexor targets leukemia-initiating cells (LICs) of primary AML, we re-transplanted serial dilutions of human CD45+ cells isolated from leukemic mice treated with either vehicle or selinexor. The preliminary results of our re-population assays indicate that selinexor greatly diminished LIC frequency in AML-M4; FLT3-ITD AML (∼6 fold) and complex karyotype disease (∼100 fold). These findings demonstrate that selinexor may represent a novel targeted therapy for the treatment of AML, which spares normal hematopoietic stem and progenitor cells. Disclosures: McCauley: Karyopharm Therapeutics Inc.: Employment, Equity Ownership, Patents & Royalties. Kauffman:Karyopharm Therapeutics Inc.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties. Shacham:Karyopharm Therapeutics Inc.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties.

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