Dependent Inhibition Of Cell Growth Research Articles

Abstract 5243: 3-Bromopyruvate in combination with radiation inhibits pancreatic cancer growth by dismantling mitochondria and ATP generation in a preclinical mouse model

Abstract 3-Bromopyruvate (3 BP) is a promising anticancer drug against pancreatic cancer. It exerts potent anticancer effects by inhibiting hexokinase II enzyme of glycolysis pathway and ATP generation in cancer cells. In present investigation we have examined the effect of 3-BromoPyruvate employing panc-2 cancer cells, and syngeneic pancreatic cancer mouse model. The clonogenic survival assay demonstrated dose dependent inhibition of cell growth, and we observed 95% cell death at 15 uM conc. Further, ATP production was severely inhibited in cancer cells treated with same concentration of 3-BP. We also observed that 3-BP in combination with low doses of irradiation was more effective in killing cancer cells than 3-BP alone. Electron microscopy revealed that 3-BromoPyruvate severely damaged mitochondrial integrity which might have severely affected ATP generation in cancer cells. We further tested 3-BromoPyruvate (2.5-10.0 mg/kg) in syngeneic panc-2 tumor mouse model and observed that 3-BP at10.0 mg/kg dose significantly reduced tumor growth (volume) about 35-40%. Immunohistochemistry (IHC) data showed that 3-BP inhibits hexokinase II (HK2) and TGFbeta1 and enhanced active caspase-3 expression in tumor tissues as compared to untreated control. The disruption of the interaction between VDAC1 and HK2 due to 3BP treatment was confirmed by CO-IP. Interestingly, 3 BP at higher dose induces caspase-3 activation in tumor derived organoid without showing any effect on normal pancreatic organoid. Altogether, the results in this study demonstrated that 3 BP could be a promising anti-tumor drug against pancreatic cancer and its efficacy could be improved in combination with radiation therapy. Citation Format: Sanjit K. Roy, Tijana Dukic, Binny Bhandary, Aneesh Acharya, Kevin J. Tu, Young H. Ko, Hem D. Shukla. 3-Bromopyruvate in combination with radiation inhibits pancreatic cancer growth by dismantling mitochondria and ATP generation in a preclinical mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5243.

Combined Inhibition of Chk1 and Wee1 As a New Therapeutic Strategy for High-Risk Multiple Myeloma Patients

Multiple myeloma (MM) is the second most common hematological malignancy characterized by an abnormal clonal proliferation of malignant plasma cells. Despite introduction of novels agents that have significantly improved clinical outcomes, most patients relapse and develop drug resistance. Therefore, novel therapeutic strategies to overcome chemotherapy resistance are needed in clinical settings.MM is characterized by genomic instability and high level of replicative stress that arise during the disease's pathogenesis. In response to replicative and DNA damage stress, MM cells activate various DNA damage signaling pathways that include the kinases ATR, Chk1 and Wee1. Chk1 is activated by ATR after DNA damage or replicative stress and promote cell cycle arrest through the regulation of S and G2 checkpoints. Wee1 kinase plays a central role in the proper timing of cell division cycle controlling entry into mitosis and DNA replication during S phase. Interestingly, experimental studies have identified that Chk1 inhibitors are strongly synergistic with Wee1 inhibitors in various cancer models. Furthermore, Chk1 and Wee1 inhibitors are currently under clinical investigation.Here, we investigated the therapeutic interest of Chk1 and Wee1 in MM.First we investigated the prognostic value of Chk1 and/or Wee1 gene expression in MM patients. Both Chk1 and Wee1 were significantly overexpressed in human myeloma cell lines (HMCLs, n= 40) compared to bone marrow plasma cells (BMPCs, n=5) (P<001 and P<001 respectively). Chk1 and Wee1 expression were significantly higher in the poor prognosis “proliferation” MM subgroup (P<001 and P<001 respectively).High Chk1 expression and high Wee1 expression could predict for shorter overall survival (OS) in 4 independent cohorts of newly-diagnosed patients (P<0.001 and P=0.007 in the HM cohort (N=206), P<0.001 and P=0.008 in UAMS-TT2 cohort (N=345), P<0.001 and P<0.001 in Hovon cohort (N=282) and P=0.002 and P<0.001 in UAMS-TT3 cohort (N=186)). Same results were obtained for event free survival (EFS). Moreover, Chk1 and Wee1 high expression are associated with a poor prognosis in a cohort of 242 patients treated by Bortezomib monotherapy (Mulligan Cohort, P<0.001 and P=0.001 respectively).Interestingly, combined high expression of Chk1 and Wee1 are associated to significantly shorter survival compared to high expression of Chk1 or Wee1 alone in the 4 independent cohorts investigated. Same results were obtained for event free survival (EFS).According, to these results, we investigated the therapeutic interest of Chk1 and Wee1 inhibitors (AZD7762 and MK-1775) alone and in combination in MM.Chk1 inhibitor (AZD7762) induced a dose dependent cell growth inhibition in 13 HMCLs with a median IC50 of 179nM (range 69-366nM). A correlation between HMCLs sensitivity and P53 mutation was observed (r =0.7, P<0.01).Wee1 inhibitor (MK-1775) induced also a dose dependent inhibition of cell growth in all investigated HMCLs (n=10) with a median of IC50 of 450nM (range 100-800nM).Combination treatment of Wee1 inhibitor IC20 with various doses of Chk1 inhibitor was performed in 4 HMCLs. We identified a high synergy (combination index range 0.4-0.7) associated with a significant and remarkable reduction of Chk1 inhibitor (AZD7762) IC50 (198nM to 20nM for XG6; 166nM to 25nM for AMO1; 386nM to 18nM for XG7 and 163nM to 21nM for OPM2) in all the tested HMCLs. Chk1 and Wee1 inhibitors combination demonstrated a significant increase in apoptosis induction, proliferation inhibition and DNA damage induction compared to Chk1i or Wee1i alone in 3 HMCLs.We investigated the effects of Chk1i, Wee1i and Chk1i/Wee1i combination on primary MM cells of patients (n = 5) co-cultured with their bone marrow microenvironment. After 4 days of treatment, cells were enumerated and the fraction of viable myeloma cells (CD138+) and non-myeloma cells (CD138-) were determined by flow cytometry. Chk1i/Wee1i combination significantly and specifically induced a higher toxicity on myeloma cells of patients compared to Chk1i or Wee1i alone. This toxicity is specific of MM cells since normal bone marrow cells were significantly less affected by the combination treatment (P < 0.01).Taken together, these data suggest that association of Chk1i and Wee1i may represent a promising therapeutic approach in high-risk MM patients characterized by high Chk1 and Wee1 expression. DisclosuresBruyer:Diag2tec: Employment. Martin:Diag2tec: Employment. Cartron:Celgene: Consultancy, Employment; Sanofi, BMS, Jansen, celgene, Roche, Gilead: Equity Ownership; Roche: Consultancy, Equity Ownership, Honoraria, Research Funding. Hose:EngMab: Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees; Sanofi: Research Funding.

Abstract B066: Inhibition of farnesyl transferase by tipifarnib leads to isoform specific cell growth inhibition in HRAS-mutated human rhabdomyosarcoma

Abstract Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children and adolescents. A subset of RMS expresses the characteristic PAX3-FOXO1 or PAX7-FOXO1 translocations, thought to be oncogenic drivers. Among the fusion-negative subset of RMS, a substantial proportion express oncogenic mutations in RAS proteins or other elements of RAS signaling pathways. Activating RAS mutations are disproportionately seen in patients with high-risk RMS and attempts to augment combination chemotherapy have not resulted in improvement in overall survival in this poor prognosis group. Given the common finding of dysregulated RAS signaling, targeting RAS represents an attractive therapeutic approach. RAS family members require posttranslational modification for plasma membrane localization and subsequent downstream signaling. One therapeutic strategy, therefore, includes disruption of RAS protein prenylation, and thereby its membrane localization, via inhibition of farnesyl transferase (FTase). NRAS and KRAS may utilize geranylgeranyltransferase prenylation as a bypass when FTase is inhibited, but HRAS is uniquely dependent on FTase, and therefore, tumor cells with HRAS mutations may be preferentially sensitive to the effects of farnesyltransferase inhibition (FTI). We therefore tested human RMS cell lines harboring mutations in HRAS, KRAS or NRAS, and those wild type (WT) for H-, K-, and N-RAS, and found that only tumor cells with oncogenic HRAS mutations demonstrate growth inhibition and decreased ERK signaling in response to the FTI tipifarnib. Using high-throughput cell proliferation assays, dose dependent cell growth inhibition in response to treatment with tipifarnib is observed in HRAS-mutated RMS cell lines. We observe a reduction in phosphorylated MEK and ERK in HRAS mutated cell lines indicating effective RAS signaling inhibition. Activity of PI3K-mTOR pathway elements p70-S6K and RPS6 are inhibited by tipifarnib in cell lines that demonstrate an antiproliferative response to the compound. In vivo exposure to tipifarnib in HRAS-mutated RMS xenografts results in tumor growth inhibition. Therapeutic strategies targeting RAS protein prenylation may therefore provide an effective approach as an alternative to traditional chemotherapy, while improving outcomes and decreasing toxicities. Based on these data, a genomically-selected clinical trial using tipifarnib to treat pediatric patients with HRAS-mutated tumors, including RMS, would be of interest. Citation Format: Patience Obasaju, Kai Pollard, Amy Allen, Jiawan Wang, Christine Pratilas. Inhibition of farnesyl transferase by tipifarnib leads to isoform specific cell growth inhibition in HRAS-mutated human rhabdomyosarcoma [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B066. doi:10.1158/1535-7163.TARG-19-B066

Anti-cancer activity of di- and tri-organotin(IV) compounds with D-(+)-Galacturonic acid on human tumor cells

We have compared the anti-proliferative activity in vitro, of R2SnGala (1-3) [R = Me, n-Bu, Ph] and novel R3SnGala (4, 5) [R = Me, n-Bu] with D-(+)-Galacturonic acid [HGala; Galaq-, q = (2) and (1) for R2SnGala and R3SnGala, respectively] compounds, towards human tumor cell lines of intestinal carcinoma (HCT-116) and breast adenocarcinoma (MCF-7). The new synthesized 4 and 5 compounds were characterized, in solution, by 1H, 13C and 119Sn NMR, that showed that HGala acts as monoanionic moiety and evidenced the dynamic behavior of the compounds, due to inter-conversions involving the anomeric carbon atom of the ligand. Cell viability, apoptosis induction and cell cycle distribution were analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay and flow cytometry, respectively. The cytotoxicity of the compounds, in the micro-submicromolar range, changed in the order of the organotin(IV) moieties, according to 5 > 3 > 2, while 1 and 4, containing MenSn(IV) (n = 2,3) moieties, were ineffective. Compound 5 showed peculiar cytotoxic effects. It did not cause time dependent inhibition of cell growth nor accumulated into the cells. Cell death induced by the active 2, 3, and 5, was shown to be apoptotic by measuring the exposure of phosphatidylserine to the outer membrane and the loss of mitochondrial potential. All the cytotoxic compounds induced an accumulation of cells in the subG0/G1phase, while only 2 and 3 perturbed the cell cycle confining viable cells in G0/G1phase. Finally, none of the compounds investigated affected the viability of normal intestinal or liver cells, indicating selectivity towards tumor cells.

NPM1 directs PIDDosome-dependent caspase-2 activation in the nucleolus.

The PIDDosome (PIDD-RAIDD-caspase-2 complex) is considered to be the primary signaling platform for caspase-2 activation in response to genotoxic stress. Yet studies of PIDD-deficient mice show that caspase-2 activation can proceed in the absence of PIDD. Here we show that DNA damage induces the assembly of at least two distinct activation platforms for caspase-2: a cytoplasmic platform that is RAIDD dependent but PIDD independent, and a nucleolar platform that requires both PIDD and RAIDD. Furthermore, the nucleolar phosphoprotein nucleophosmin (NPM1) acts as a scaffold for PIDD and is essential for PIDDosome assembly in the nucleolus after DNA damage. Inhibition of NPM1 impairs caspase-2 processing, apoptosis, and caspase-2-dependent inhibition of cell growth, demonstrating that the NPM1-dependent nucleolar PIDDosome is a key initiator of the caspase-2 activation cascade. Thus we have identified the nucleolus as a novel site for caspase-2 activation and function.

A Small Molecule That Selectively Targets BLM Helicase Has a Therapeutic Interest in Multiple Myeloma

Hematological malignancies, including MM, are characterized by genomic instability that is possibly related to underlying defects in DNA repair or genomic stability maintenance. RECQ helicases are at the crossroad between replication, recombination, DNA repair and transcription and could represent potent therapeutic targets for cancer therapy.The Bloom’s syndrome helicase, BLM, has DNA annealing and unwinding activities. Through its interaction with TOPOIIIα, BLM unwinds the short stretches of naked duplex DNA and processes homologous recombination (HR) intermediates containing a double holiday junction. It may suppress hyper-sister chromatid exchange (SCE) by disruption of D-loop recombination intermediates and also might be involved in the suppression of crossing over during homology mediated recombination. This helicase facilitates telomere replication by resolving G4 structures. BLM deregulation is also associated with cancer.We recently identified a significant overexpression of BLM in malignant plasma cells compared to normal bone marrow plasma cells. Since BLM, plays important roles in the maintenance of chromosomal stability, we investigated if BLM could play a role in MM pathophysiology and drug resistance.A high BLM expression in MMCs could predict for shorter overall survival (OS) in four independent cohorts of patients (P = 0.003 in the HM cohort (N=206), P = 0.0002 in the UAMS-TT2 cohort (N=345), P = 0.0008 in TT3 cohort (N=186)) and P = 0.04 in Hovon cohort (N=282). A high BLM expression in MMCs could also predict for shorter event free (EFS) survival in the HM and UAMS-TT2 cohorts. BLM expression was significantly higher in the poor prognosis proliferation MM subgroup (P < 0.05). Moreover, we found that BLM was overexpressed in HMCLs (median 1848, range 246 - 10901) compared to primary MMCs or BMPCs (P< .001).The small molecule, ML216, is a membrane permeable selective inhibitor of BLM helicase. ML216 appears to act at the BLM-nucleic acid substrate binding site, inhibiting DNA binding and blocking BLM's helicase activity.We investigated the interest of the BLM inhibitor, ML216, to eradicate MM cells. The effect of ML216 was tested using 10 different human myeloma cell lines (HMCL). ML216 induced a dose dependent inhibition of cell growth in all investigated HMCL with a median IC50 of 4.1 μM (range: 1.2 - 30 μM). Moreover, our tests on HMCL showed that the BLM inhibitor synergizes with Melphalan and Bortezomib, two drugs currently used in myeloma therapy. Furthermore, ML216 induced a significant apoptosis of primary myeloma cells of patients co-cultured with their bone marrow environment and recombinant IL-6 (n=7). BLM inhibitor significantly reduced the median number of viable myeloma cells by 54%, 60% and 74% at respectively 3, 6 and 10 μM (P = 0.001, P < 0.001 and P<0.001 respectively; n=7). Of interest, the normal non myeloma cells present in the culture were significantly less affected by ML216 when used at 3 and 6 μM. These data demonstrated a significant higher toxicity of ML216 on myeloma cells compared to normal bone marrow cells.Taken together, our results underline the therapeutic interest of BLM inhibitor in MM and especially in patients characterized by high BLM expression and a poor prognosis. DisclosuresNo relevant conflicts of interest to declare.

Analysis of the Calyces from the Jamaica Species of Hibiscus Sabdariffa for Adrogenic Compounds

The leaves and flowers of Hibiscus sabdariffa are known to have many pharmacological properties. Hibiscus sabdariffa leaf extracts (HLE) have been documented to have anti‐cancer activities in different cancer cell lines. HLE has been shown to inhibit cell proliferation, migration, and metastasis in human prostate cancer cells suggesting that this flow may be a source for anti‐androgenic compounds. However, the anti‐androgen effect has not been examined in the calyx. The objective of this study was to obtain a unique bioactive fraction from the calyx of H. sabdariffa and to demonstrate its ability to inhibit prostate cancer cell growth. To accomplish our objective, we generated an aqueous and a methanol extraction of the calyces of the H. sabdariffa. The extracts were then examined by HPLC chromatography and for anti‐proliferative activity using the LNCaP cancer cells. Extraction of 22g of the calyces for 48hrs at room temperature resulted in 5.56mg and 2.75mg of analyte in aqueous and methanol extraction respectively. We obtained high yields of extracts (253mg per kg of dry weight) using water as compared to methanol as the solvent. An HPLC chromatogram profile of each extract was generated to characterize the number of respective chemicals present in the analytes. Analysis of the methanol extract revealed the presence of at least two distinct peaks with a retention times of 3.12min and 3.27min. We observed that the aqueous extract resulted only in a single peak with a retention time of 3.02min. Next, we examined the ability of both extracts to inhibit the growth of LNCaP‐GFP cancer cell line. Treatment of these cells for 1–5 days resulted in dose and time dependent inhibition of cell growth. We found that the methanol extract was capable of inducing cytotoxicity in 24hrs as compared to aqueous extracts which induced cytotoxicity after 72hrs. Our results demonstrated that both methanol and aqueous extracts of H. sabdariffa contained growth inhibitory compounds.Support or Funding InformationNIH R15AT006821(WG) and USDA/NIFA 2012‐38821‐20092.

Complementary Targeting of PI3K and the Proteasome Causes Potent Inhibition of mTORC1 and NF-Kappab in Models of B- and T-Cell Lymphoma

Abstract Background: Constitutively activated PI3K/AKT/mTOR pathway plays a critical role in the proliferation and survival of cancer cells, through the expression of numerous pro-survival and proliferative genes. Specific inhibitors of the various isoforms of PI3K have shown promising activity in the treatment of indolent B-cell lymphoma. However, they have not shown similar activity in aggressive lymphoma, potentially because the expression of many PI3K/AKT/mTOR dependent pro-survival genes may be activated by alternative signals, for example, NF-kappaB (NF-kB). While specific inhibitors of NF-kB are currently not available in the clinic, proteasome inhibitors are well known to inhibit NF-kB, among its pleiotropic mechanisms of action. We hypothesize that if cancer cell survival is dependent on a complex protein network involving PI3K and NF-kB, then complimentary inhibition of PI3K and the proteasome will become highly synergistic. Our results provide strong support for the hypothesis, by demonstrating that TGR-1202, a PI3Kdelta inhibitor, and carfilzomib, a proteasome inhibitor, are unique among the combinations of PI3K and proteasome inhibitors in potently suppressing the mTORC1 complex and NF-kB, and are highly effective in models of B- and T-cell lymphoma. Methods: Cytotoxicity of PI3K and proteasome inhibitors was studied in a panel of B and T-cell lymphoma cell lines. Growth inhibition was determined using Cell Titer Glo that measures ATP produced by live and proliferating cells. Cell death was determined by flow cytometry, using the probes Annexin V and propidium iodide. Drug: drug synergy was determined by calculating the combination index (CI) and relative risk ratio (RRR). Values below 1 indicate synergy, with smaller RRR and CI values indicating higher levels of synergy. The mechanistic basis of the synergy is determined through in-depth interrogation of the PI3K/AKT/mTOR pathway and its downstream targets and interacting pathways. Results: We used a high-throughput platform to screen the cytotoxicity of TGR-1202, idelalisib/CAL-101, bortezomib, and carfilzomib, in a panoply of B- and T-cell lymphoma cell lines. We found that TGR-1202 and idelalisib as single agent caused only minimal to mild cytotoxicity in lymphoma cells. Bortezomib and carfilzomib as single agent caused dose dependent inhibition of cell growth. Surprisingly, when TGR-1202 or Cal-101 were combined with bortezomib or carfilzomib in a 2x2 design, the combination of TGR-1202 and carfilzomib was consistently the most synergistic, while the other 3 combinations were either much less or not at all synergistic. For example, Table 1 clearly demonstrated that TGR-1202 and carfilzomib demonstrated much higher inhibitory effect and stronger synergy than Cal-101 and bortezomib in two T-ALL cell lines. We have systematically interrogated the effects of these drug combinations on the PI3K/AKT/mTOR pathway, and found that the combination of TGR-1202 and carfilzomib was able to uniquely and potently inhibit components of the mTORC1 complex, causing inhibition of mTORC1 dependent signals such as phosphorylated 4E-BP1, leading to markedly reduced expression of critical genes such as c-myc (Figure 1). Furthermore, the combination of TGR-1202 and carfilzomib uniquely and potently inhibited activation of NF-kB, resulting in markedly suppressed expression of Bcl-xL. The complementary inhibition of the PI3K/AKT/mTOR and NF-kB pathways resulted in robust growth inhibition and apoptosis (Figure 1), and warrants further clinical evaluation for aggressive B- and T-cell lymphoma. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures Sportelli: TG Therapeutics: Employment, Equity Ownership. Miskin:TG Therapeutics, Inc.: Employment, Equity Ownership. Viswanadha:Incozen: Employment.

Brevilin A, a Novel Natural Product, Inhibits Janus Kinase Activity and Blocks STAT3 Signaling in Cancer Cells

Signal abnormalities in human cells usually cause unexpected consequences for individual health. We focus on these kinds of events involved in JAK-STAT signal pathways, especially the ones triggered by aberrant activated STAT3, an oncoprotein which participates in essential processes of cell survival, growth and proliferation in many types of tumors, as well as immune diseases. By establishing a STAT3 signal based high-throughput drug screening system in human lung cancer A549 cells, we have screened a library from natural products which contained purified compounds from medicinal herbs. One compound, named Brevilin A, exhibited both strong STAT3 signal inhibition and STAT3 signal dependent cell growth inhibition. Further investigations revealed that Brevilin A not only inhibits STAT3 signaling but also STAT1 signaling for cytokines induced phosphorylation of STAT3 and STAT1 as well as the expression of their target genes. In addition, we found Brevilin A could attenuate the JAKs activity by blocking the JAKs tyrosine kinase domain JH1. The levels of cytokine induced phosphorylation of STATs and other substrates were dramatically reduced by treatment of Brevilin A. The roles of Brevilin A targeting on JAKs activity indicate that Brevilin A may not only be used as a STAT3 inhibitor but also a compound blocking other JAK-STAT hyperactivation. Thus, these findings provided a strong impetus for the development of selective JAK-STAT inhibitors and therapeutic drugs in order to improve survival of patients with hyperactivated JAKs and STATs.

γ-Tocotrienol Induces Paraptosis-Like Cell Death in Human Colon Carcinoma SW620 Cells

Colorectal cancer is one of the most serious illnesses among diagnosed cancer. As a new type of anti-cancer composition from tocotrienol-rich fraction of palm oil, γ-tocotrienol is widely used in anti-cancer research. The objectives of this study were to investigate the effects of γ-tocotrienol on human colon cancer SW620 and HCT-8 cells. We showed that treatment with different concentrations of γ-tocotrienol resulted in a dose dependent inhibition of cell growth. Cell death induced by γ-tocotrienol was mediated by a paraptosis-like cell death in SW620 and HCT-8 cells. Real-time RT-PCR and western blot analyses showed that γ-tocotrienol inhibited the expression level of β-catenin, cyclin D1 and c-jun. These data suggest that a paraptosis-like cell death induced by γ-tocotrienol in SW620 cells is associated with the suppression of the Wnt signaling pathway, which offers a novel tool for treating apoptosis-resistance colon cancer.

Delta‐Tocotrienol enhances cisplatin‐induced inhibition of growth and invasion of Non‐Small Cell Lung Cancer Cells

Lung cancer is the leading cause of death among all cancers. Non‐small cell lung cancer (NSCLC) accounts for 80% of lung cancer with a five‐year survival rate of 16%. Although a subset of NSCLC patients benefit from the development of target therapies, cisplatin remains the standard chemotherapeutic drug. Previously, we demonstrated that delta‐tocotrienolinhibits NSCLC cell proliferation, invasion and induces apoptosis by down‐regulation of the Notch‐1 signaling pathway. The objective of this study was to investigate whether delta‐tocotrienol, a naturally occurring isoform of Vitamin E, could enhance the cisplatin‐induced inhibition of growth and invasion of NSCLCcells. Treatment of NSCLCcells with delta‐tocotrienoland cisplatin in combination resulted in a dose dependent inhibition of cell growth, cell migration, tumor cell invasiveness, and induction of apoptosis in NSCLC cells. Real‐time RT‐PCR and western blot analysis showed that antitumor activity by the combination of delta‐tocotrienol and cisplatin was associated with decreases in Notch‐1, Hes‐1, Survivin, and Bcl‐XLand increases in cleaved PARP, Caspase‐3 expressions. In addition, there was a decrease in NF‐κB‐DNA binding activity. These results suggest that down‐regulation of Notch‐1, via inhibition of NF‐κB signaling pathways by delta‐tocotrienol and cisplatin, could provide a potential novel approach for prevention of tumor progressionin non‐small cell lung cancer.This research was supported by funds provided by Wayne State University.Grant Funding Source: Wayne State University

Up-regulation of hypoxia inducible factor-1α by cobalt chloride correlates with proliferation and apoptosis in PC-2 cells

BackgroundThe exact mechanism of the effects of hypoxia on the proliferation and apoptosis in carcinoma cells is still conflicting. This study investigated the variation of hypoxia-inducible factor-1α(HIF-1α) expression and the apoptosis effect of hypoxia stimulated by cobalt chloride (CoCl2) in pancreatic cancer PC-2 cells.MethodsPC-2 cells were cultured with different concentration (50-200 μmol/L) of CoCl2 after 24-120 hours to simulate hypoxia in vitro. The proliferation of PC-2 cells was examined by MTT assay. The cellular morphology of PC-2 cells were observed by light inverted microscope and transmission electron microscope(EM). The expression of HIF-1α on mRNA and protein level was measured by semi-quantitive RT-PCR and Western blot analysis. Apoptosis of PC-2 cells were demonstrated by flow cytometry with Annexin V-FITC/PI double staining.ResultsMTT assay showed that the proliferation of PC-2 cells were stimulated in the first 72 h, while after treated over 72 h, a dose- dependent inhibition of cell growth could be observed. By using transmission electron microscope, swollen chondrosomes, accumulated chromatin under the nuclear membrane and apoptosis bodies were observed. Flow cytometer(FCM) analysis showed the apoptosis rate was correlated with the dosage of CoCl2. RT-PCR and Western blot analysis indicated that hypoxia could up-regulate the expression of HIF-1α on both mRNA and protein levels.ConclusionHypoxic microenvironment stimulated by CoCl2 could effectively induce apoptosis and influence cell proliferation in PC-2 cells, the mechanism could be related to up-expression of HIF-1α.

Abstract B40: The melphalan prodrug melphalan-flufenamide shows cytotoxic effect in bladder cancer cell lines and triggers JNK and caspase-3 mediated apoptotic signaling.

Abstract Background: Bladder cancer is the 4th most common cancer associated death in men in the western world. Cisplatin-based polychemotherapy (1st line) and vinflunine (2nd line) are the only chemotherapy regimens which show survival benefit in patients with metastatic disease. Hence, there is a major need to identify new effective treatment alternatives for patients with this disease. We have developed a prodrug of melphalan, Melphalan-flufenamide (Mel-flufen) which show enhanced cytotoxic efficacy compared to melphalan in several tumour types. Treatment with Mel-flufen gives 10–20 fold higher intracellular drug levels compared to the same dose of melphalan. Mel-flufen is currently undergoing Phase I/II clinical trials. Here we studied if this drug might be a useful in bladder cancer by analyzing its effects in a panel of bladder cancer cell lines. Material and Methods: The bladder cancer cell lines J82, RT4, 5637 and TCC-SUP were profiled for Mel-flufen and melphalan sensitivity using MTT and FMCA cell viability assay. Mel-flufen signaling was measured as caspase-3 and Bak/Bax activation in flow cytometry and PARP cleavage, JNK phosphorylation, p53 and p21WAF1/Cip1 induction on western blot respectively. Results: Mel-flufen caused a dose and time dependent inhibition of cell growth in all the four bladder cancer cell lines studied. Comparison with the parental drug revealed an enhanced cytotoxic effect of Mel-flufen at equimolar doses. Molecular profiling of the apoptotic response showed that Mel-flufen induced activation of the Bcl-2 proteins Bak and Bax followed by caspase-3 activation and induction of apoptotic morphology. Albeit Mel-flufen induced activation of p53, the cytotoxic effect was not dependent on p53 as it was also observed in p53-mutant bladder cancer cell lines. A prominent activation of JNK1/2 was observed in response to Melflufen. Importantly, this JNK activation preceded induction of Melflufen-induced apoptotic signaling and was critical for the induced cytotoxicity in these bladder cancer cell lines. Conclusion: Our data demonstrate that the melphalan prodrug Mel-flufen significantly inhibits bladder cancer cell growth in vitro. Importantly, we show that Mel-flufen is more efficient in inhibiting bladder cancer growth than the parental drug melphalan and causes a more pronounced apoptotic signaling. Taken together these data suggests that Mel-flufen may be a good candidate for bladder cancer treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B40.

Inhibition of cell growth and induction of apoptosis in non-small cell lung cancer cells by delta-tocotrienol is associated with notch-1 down-regulation

Lung cancer is the leading cause of death among all cancers. Non-small cell lung cancer accounts for 80% of lung cancer with a 5-year survival rate of 16%. Notch pathway, especially Notch-1 is up-regulated in a subgroup of non-small cell lung cancer patients. Since Notch-1 signaling plays an important role in cell proliferation, differentiation, and apoptosis, down-regulation of Notch-1 may exert anti-tumor effects. The objective of this study was to investigate whether delta-tocotrienol, a naturally occurring isoform of Vitamin E, inhibits non-small cell lung cancer cell growth via Notch signaling. Treatment with delta-tocotrienol resulted in a dose and time dependent inhibition of cell growth, cell migration, tumor cell invasiveness, and induction of apoptosis. Real-time RT-PCR and western blot analysis showed that antitumor activity by delta-tocotrienol was associated with a decrease in Notch-1, Hes-1, Survivin, MMP-9, VEGF, and Bcl-XL expression. In addition, there was a decrease in NF-κB-DNA binding activity. These results suggest that down-regulation of Notch-1, via inhibition of NF-κB signaling pathways by delta-tocotrienol, could provide a potential novel approach for prevention of tumor progression in non-small cell lung cancer.

Notch‐1 down‐regulation by delta‐tocotrienol is associated with the inhibition of cell growth and the induction of apoptosis in non‐small cell lung cancer cells

Lung cancer is the leading cause of death among all the cancers. Non‐small cell lung cancer accounts for 80% of lung cancer with a five‐year survival rate of 16%. Notch pathway, especially Notch‐1 is upregulated in a subgroup of Non‐small cell lung cancer patients. Since Notch‐1 signaling plays an important role in cell proliferation, differentiation, and apoptosis, down‐regulation of Notch‐1 may exert anti‐tumor effects. The objective of this study was to investigate the effect of delta‐tocotrienol on non‐small cell lung cancer cell lines (A549, H1299) via Notch signaling. Treatment with delta‐tocotrienol resulted in a dose and time dependent inhibition of cell growth (p&lt;0.05), cell migration, tumor cell invasiveness, and induction of apoptosis (p&lt;0.05). Western blot analysis showed that apoptosis induced by delta‐tocotrienol was associated with decrease in Notch‐1, Hes‐1, Survivin, and BCX‐XL expression. This was confirmed by real‐time PCR by a decrease in the gene expression of Notch1, Hes‐1, MMP‐9, and VEGF was observed. In addition, this was accompanied with a decrease in NF‐κB‐DNA binding activity. These results suggest that down‐regulation of Notch‐1, especially by delta‐tocotrienol could provide a novel approach for the prevention of tumor progression, which is likely to be mediated via downregulation of NF‐κB signaling pathways in Non‐small cell lung cancer.Grant Funding Source: Across Societies (EB)

248 APOCYNIN AND DIPHENYLENE IODIUM(DPI) FUNCTION AS INHIBITORS OF CELL MIGRATION, AND PROLIFERATION BUT DO NOT INHIBIT NADPH OXIDASE IN RENAL CELLS CARCINOMA

You have accessJournal of UrologyKidney Cancer: Basic Research1 Apr 2011248 APOCYNIN AND DIPHENYLENE IODIUM(DPI) FUNCTION AS INHIBITORS OF CELL MIGRATION, AND PROLIFERATION BUT DO NOT INHIBIT NADPH OXIDASE IN RENAL CELLS CARCINOMA Jung-Sik Huh, Fernando J. Kim, Hyeon Ju Kim, and Hari Koul Jung-Sik HuhJung-Sik Huh Aurora, CO More articles by this author , Fernando J. KimFernando J. Kim Aurora, CO More articles by this author , Hyeon Ju KimHyeon Ju Kim Aurora, CO More articles by this author , and Hari KoulHari Koul Aurora, CO More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.317AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Renal cell carcinoma, which forms in tubules of the kidney, accounts for approximately 3% of adult malignancies and 90-95% of neoplasms arising from the kidney. Reactive oxygen species(ROS), which originate mainly from NADPH oxidase and are highly reactive O2 metabolites, have physiological and pathological processes. ROX and the oxidative stress have been associated with tumor formation. To respond oxidative stress, inhibition of NADPH oxidase represents a target for the treatment of many diseases. DPI and apocynin has been used as an efficient inhibitor of the complex NADPH oxidase in phagocytic and non-phagocytic cells. We investigated the effects of NOX4 inhibitors in renal cell carcinoma. METHODS We tested the effects of Apocynin and DPI against RCC cell lines (A498, Caki-2, KIKO-12), and the underlying mechanisms. Western Blot analysis was used to measure protein levels of Nox4, Akt and Erk signal molecules. Fluoremetric assays were used to measure ROS generation as well as NADPH oxidase activities. Cell growth and viability as well as migration were measured by standard methods. RESULTS Results show that treatment of RCC cells with apocynin and DPI resulted in a dose and time dependent inhibition of cell growth and proliferation. These treatments however did not induce apoptosis. Apocynin or DPI treatment had no effect on ROS (hydrogen peroxide and superoxide anion) generation in RCC cells. No changes were observed in NOX4, Akt, and Erk signal protein expression or activity change in the presence of apocynin and DPI. But apocynin and DPI interrupted and decreased cell migration of RCC cells. There was no change of actin cytoskeleton in treated and untreated A498 renal cell cancer cell line. CONCLUSIONS Apocynin and DPI, so called specific inhibitors of NADPH oxidase system acts as inhibitors of migration and proliferation but these effects are independent of NADPH oxidase inhibition in RCC cells as these agents did not inhibit Nox activity or ROS generation. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e100 Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information Jung-Sik Huh Aurora, CO More articles by this author Fernando J. Kim Aurora, CO More articles by this author Hyeon Ju Kim Aurora, CO More articles by this author Hari Koul Aurora, CO More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Significant In Vitro Activity of a Novel Inhibitor of Apoptosis (IAP) Inhibitor LC161 In Multiple Myeloma

AbstractAbstract 2998 Background:Multiple myeloma remains incurable with current therapies and novel approaches based on disease biology are needed. Inhibitors of apoptosis (IAP) proteins represent a conserved group of proteins that are important regulators of apoptosis. X-linked IAP (XIAP) is the best studied IAP and inhibits pro-apoptotic caspases 3, 7 and 9. Multiple myeloma (MM) cell lines express high levels of XIAP and levels are further increased when stimulated by cytokines IL6 and IGF-1, both secreted in copious amounts in the myeloma microenvironment. IL6 and IGF1 up-regulate XIAP by activating the NF-κB, MAPK and PI3K signaling pathways that are commonly aberrant in MM and other tumors. XIAP mRNA contains an internal ribosomal entry site (IRES) sequence in the 5‘ untranslated region. IRES sequences enable direct ribosome recruitment and aid in translation that is independent of cap-mediated translation. Thus, molecules like mTOR inhibitors might not lead to XIAP downregulation. This was observed when rapamycin, an mTOR inhibitor, was used on MM cell lines. XIAP protein levels were not reduced due to the IRES sequence which leads to translation that is independent of the 5‘ cap and 4EBP1. Thus XIAP inhibitors might be able to overcome resistance associated with rapamycin treatment. Methods:LC161 was synthesized by Novartis Inc. (Basel, Switzerland). Stock solutions were made in DMSO, and subsequently diluted in RPMI-1640 medium for use. MM cell lines were cultured in RPMI 1640 containing 10% fetal bovine serum (20% serum for primary patient cells) supplemented with L-Glutamine, penicillin, and streptomycin. Cytotoxicity was measured using the MTT viability assay and proliferation using thymidine uptake. Apoptosis was measured using flow cytometry upon cell staining with Annexin V-FITC and propidium iodide (PI). Immunoblotting was done on cell extracts at various time points following incubation with the drug in order to study the cell signaling pathways. Results:LC161 treatment resulted in a dose and time dependent inhibition of cell growth in the MM cell lines tested. Most of the cytotoxicity was evident by 72 hours, with minimal increase seen up to 96 hours of incubation. At 72 hours of incubation, the median inhibitory concentration varied considerably between various cell lines with an IC50 range of 2.5–25μ M. The IC50s were maintained when the cells were treated in co-culture with stromal cells or in the presence of IL6, IGF or VEGF. Dose-dependent decrease in proliferation of the cell lines was evidenced by decreased thymidine incorporation. Apoptotic changes in cells following drug treatment was confirmed by flow cytometry for Annexin and PI. Cleavage of caspases 3, 8 and 9 were confirmed on flow cytometry. Primary myeloma cells from patients were treated with increasing doses of the drug and dose dependent increase in apoptosis was observed. Immunoblotting studies demonstrated dose dependent significant down regulation of Xiap, cIAP1, cIAP2 and surviving and up-regulation of activated caspases 3, 8 and 9 and PARP. Furthermore, LC161 resulted in down regulation of pAkt, canonical and non-canonical NF-κB, pJNK, p-p38MAPK, c-Myc, Bcl-xL and Mcl1 and up-regulation of pErk and Bcl-2. We are currently examining basal levels of expression of the IAP proteins (Xiap, cIAP1 and cIAP2), pAkt and pErk in various MM cell lines to identify marker proteins that might predict response to this class of drug. In addition, our initial studies of LC161 in combination with the proteasome inhibitor bortezomib demonstrated synergy in killing MM cells in vitro. Additional combinations including inhibitors of the Akt/mTOR pathway and MEK/Erk pathway are currently been done. Conclusion:These studies demonstrate significant in vitro activity of LC161 in MM. Our results suggest the presence of two populations one very sensitive to IAP inhibition and one relatively less sensitive. Our current studies will help identify marker proteins that might predict response to LC161 treatment. We are currently testing LC261 in combination with known inhibitors of the other important signaling pathways implicated in MM disease biology as well as in-vivo experiments in mouse models. Performing these experiments will further validate the efficacy of LC161 as an anti-MM agent and form the basis for it to be taken up for clinical evaluation either as a single agent or in combination with other agent(s). Disclosures:Kumar:Celgene: Consultancy, Research Funding; Millennium: Research Funding; Merck: Consultancy, Research Funding; Novartis: Research Funding; Genzyme: Consultancy, Research Funding; Cephalon: Research Funding.

Abstract DD01-04: NVP-BKM120: A selective inhibitor of type I PI3 kinases

Abstract AACR 2010 - poster I Biological characterization of NVP-BKM120, a novel inhibitor of phosphoinosotide 3-kinase in Phase I/II clinical trials The PI3K/Akt/mTor signaling pathway plays an important role in controlling cell growth, proliferation and survival. Through various mechanisms, the pathway is frequently dysregulated in human cancers, suggesting the use of pathway modulators as novel targeted anticancer therapeutic agents. To this end, substantial drug discovery efforts have been devoted both in pharmaceutical companies and in academia to identify and develop therapeutic agents able to specifically down regulate this pathway in tumors cells. Following the discovery of NVP-BEZ235, our first dual pan-PI3K/mTOR clinical compound, we sought to identify additional PI3K inhibitors from different chemical classes with more stringent selectivity profiles. The key to achieve these objectives was to pursue a structure-based design approach coupled with intensive pharmacological evaluation of selected compounds during the medicinal chemistry optimization process. Here we report on the biological characterization of the pan-PI3K pyrimidine-derived inhibitor NVP-BKM120. This compound inhibits all four Class I PI3K isoforms (IC50 values in the 35 to 248 nM range) with at least 50-fold selectivity (compared to p110[[Unsupported Character - Symbol Font ]]) towards protein kinases. The compound is also active against the most common somatic PI3K[[Unsupported Character - Symbol Font ]] mutations (H1047R, E542K and E545K). NVP-BKM120 does not significantly inhibit the related Class III (Vps34) and Class IV (mTOR, DNA-PK) PI3K kinases. Consistent with its mechanism of action, NVP-BKM120 decreases the cellular levels of p-Akt in mechanistic and relevant tumor cell lines (e.g., IC50 for S473P-Akt in Rat1-p110[[Unsupported Character - Symbol Font ]] cells of 93 nM). This biological activity correlates with inhibition of various Akt downstream signaling pathway components, and with its anti-proliferative activity. Thus, the compound demonstrates significant, concentration dependent cell growth inhibition and induction of apoptosis in a variety of tumor cancer cells, particularly for those harboring p110[[Unsupported Character - Symbol Font ]] mutants and/or over-expressing erbB2. In addition, NVP-BKM120 demonstrates significant, dose dependent in vivo pharmacodynamic activity as measured by inhibition of p-Akt in relevant xenograft models. The pharmacological, biological and preclinical safety profile of NVP-BKM120 supports its clinical development and the compound is currently undergoing Phase 1/II clinical trials in cancer patients. Citation Format: William R. Sellers. NVP-BKM120: A selective inhibitor of type I PI3 kinases [abstract]. In: Proceedings of the AACR 101st Annual Meeting 2010; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr DD01-04

Abstract 4498: Biological characterization of NVP-BKM120, a novel inhibitor of phosphoinosotide 3-kinase in Phase I/II clinical trials

Abstract The PI3K/Akt/mTor signaling pathway plays an important role in controlling cell growth, proliferation and survival. Through various mechanisms, the pathway is frequently dysregulated in human cancers, suggesting the use of PI3K inhibitors as novel targeted anticancer therapeutic agents. To this end, substantial drug discovery efforts have been devoted both in pharmaceutical companies and in academia to identify and develop therapeutic agents able to specifically down regulate PI3K or other components of this pathway in tumors cells. Following the discovery of NVP-BEZ235, our first dual pan-PI3K/mTOR clinical compound, we sought to identify additional PI3K inhibitors from different chemical classes with more stringent selectivity profiles. The key to achieve these objectives was to pursue a structure-based design approach coupled with intensive pharmacological evaluation of selected compounds during the medicinal chemistry optimization process. Here we report on the biological characterization of the pan-PI3K pyrimidine-derived inhibitor NVP-BKM120. This compound inhibits all four Class I PI3K isoforms (IC50 values in the 35 to 248 nM range) with at least 50-fold selectivity (compared to p110α) towards protein kinases. The compound is also active against the most common somatic PI3Kα mutations (H1047R, E542K and E545K). NVP-BKM120 does not significantly inhibit the related Class III (Vps34) and Class IV (mTOR, DNA-PK) PI3K kinases. Consistent with its mechanism of action, NVP-BKM120 decreases the cellular levels of p-Akt in mechanistic and relevant tumor cell lines (e.g., IC50 for S473P-Akt in Rat1-p110α cells of 93 nM). This biological activity correlates with inhibition of various Akt downstream signaling pathway components, and with its anti-proliferative activity. Thus, the compound demonstrates significant, concentration dependent cell growth inhibition and induction of apoptosis in a variety of tumor cancer cells, particularly for those harboring p110α mutants and/or over-expressing erbB2. In addition, NVP-BKM120 demonstrates significant, dose dependent in vivo pharmacodynamic activity as measured by inhibition of p-Akt in relevant xenograft models. The pharmacological, biological and preclinical safety profile of NVP-BKM120 supports its clinical development and the compound is currently undergoing Phase 1/II clinical trials in cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4498.

Significant in-Vitro Activity of a Novel JAK2 Inhibitor in Multiple Myeloma Associated with Preferential Cytotoxicity for CD45+ Myeloma Cells.

Abstract 2848Poster Board II-824 Background:Multiple myeloma remains incurable with current therapies and novel approaches based on disease biology are needed. IL-6 is a critical cytokine involved in myeloma cell proliferation and survival and exerts its activity primarily through the JAK/STAT pathway. In addition to IL6, other cytokines are also believed to cross talk with the JAK/STAT pathway, making it a crucial interface for survival signals. It has been implicated in myeloma cell interaction with the microenvironment and resistance to apoptotic stimuli from different drugs, and represents a potential therapeutic target. We examined the pre-clinical activity of a novel JAK2 tyrosine kinase inhibitor TG101209. Methods:TG101209 (N-tert-butyl-3-(5-methyl-2-[4-(4-methyl-piperazin-1-yl)-phenylamino]-pyrimidin-4-ylamino)-benzenesulfonamide) was synthesized by TargeGen Inc. (San Diego, CA, USA). Stock solutions were made in DMSO, and subsequently diluted in RPMI-1640 medium for use. MM cell lines were cultured in RPMI 1640 containing 10% fetal bovine serum (20% serum for primary patient cells) supplemented with L-Glutamine, penicillin, and streptomycin. Cytotoxicity was measured using the MTT viability assay and proliferation using thymidine uptake. Apoptosis was measured using flow cytometry upon cell staining with Annexin V-FITC and propidium iodide (PI) for cell lines and using Apo2.7 in primary patient cells. CD45 expression was estimated using flow cytometry and cells were gated by their CD45 expression to assess differential effects of the drug. Immunoblotting was done on cell extracts at various time points following incubation with the drug in order to study the cell signaling pathways. Results:TG101209 resulted in a dose and time dependent inhibition of cell growth in the MM cell lines tested. Most of the cytotoxicity was evident by 48 hours, with minimal increase seen up to 96 hours of incubation. At 48 hours of incubation, the median inhibitory concentration was between 2 and 4uM with similar IC50 seen for myeloma cell lines sensitive or resistant to conventional therapies. The IC50s were maintained when the cells were treated in co-culture with stromal cells or in the presence of IL6, IGF or VEGF. Increasing doses of IL6 was not able to rescue the cells from the drug. Dose dependent decrease in proliferation of the cell lines was evidenced by decreased thymidine incorporation. Apoptotic changes in cells following drug treatment was confirmed by flow cytometry for Annexin and PI. Cleavage of caspases 3, 8 and 9 were confirmed on flow cytometry. Addition of the pan-caspase inhibitor zvad-fmk did not prevent drug-induced apoptosis confirming non-caspase mediated mechanisms of cell death as well. Primary myeloma cells from several patients were treated with increasing doses of the drug and IC50 similar to cell lines were seen in 8/10 patient samples tested. Interestingly, evaluation of U266 cell lines, which have a mix of CD45+ and negative cells as well as primary patient cells demonstrated more profound cytotoxicity and anti-proliferative activity of the drug on the CD45+ population relative to the CD45- cells. Immunoblotting studies demonstrated significant down regulation of IL-6 induced pSTAT3 with minor effects on the pERK and pAkt. The effect on pSAT3 was sustained compared to that on pERK and pAkt. This was accompanied by significant down regulation of Bcl-xL. Studies in a mouse model of myeloma are planned. Conclusion:These studies demonstrate significant in-vitro activity of JAK2 inhibition in multiple myeloma. In particular, the preferential targeting of CD45 cells, considered to reflect the proliferative compartment in myeloma holds out the promise for more sustained impact on the disease from a therapeutic standpoint. This is likely explained by the increased sensitivity of the CD45 cells to cytokines as a result of higher expression of different cytokine receptors as has been previously shown. This leads to increased activity of and dependence of the cells on the JAK-STAT pathway and likely explains the increased effect of the pathway inhibition. These studies form the framework for clinical evaluation of the drug in the setting of myeloma. Disclosures:Kumar:CELGENE: Research Funding; MILLENNIUM: Research Funding; BAYER: Research Funding; GENZYME: Research Funding; NOVARTIS: Research Funding.