Rapamycin sensitizes multiple myeloma cells to apoptosis induced by dexamethasone

Blood ◽  
2004 ◽  
Vol 103 (8) ◽  
pp. 3138-3147 ◽  
Author(s):  
Thomas Strömberg ◽  
Anna Dimberg ◽  
Anna Hammarberg ◽  
Kristina Carlson ◽  
Anders Österborg ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Intracellular Signaling ◽  
Kinase Inhibitor ◽  
Mammalian Target Of Rapamycin ◽  
G1 Arrest ◽  
Cyclin Dependent Kinase Inhibitor ◽  
Induced Apoptosis ◽  
Molecular Mode

Abstract Circumvention of chemoresistance in the B-cell neoplasm multiple myeloma (MM) might be achieved by targeting certain intracellular signaling pathways crucial for survival of the malignant clone. The use of the macrolide rapamycin, selectively inhibiting the phosphoprotein mammalian target of rapamycin (mTOR) downstream of, for example, insulin-like growth factor-I receptor (IGF-IR), possibly represents such a molecular mode of therapy. By using a panel of MM cell lines we showed that rapamycin induced G0/G1 arrest, an effect being associated with an increase of the cyclin-dependent kinase inhibitor p27 and a decrease of cyclins D2 and D3. Interestingly, in primary, mainly noncycling MM cells, rapamycin, at clinically achievable concentrations, induced apoptosis. More important, rapamycin sensitized both MM cell lines and primary MM cells to dexamethasone-induced apoptosis. This effect was associated with a decreased expression of cyclin D2 and survivin. The phosphorylation of the serine/threonine kinase p70S6K at Thr389 and Thr421/Ser424 was down-regulated by rapamycin and/or dexamethasone. Strikingly, the combinatorial treatment with rapamycin and dexamethasone suppressed the antiapoptotic effects of exogenously added IGF-I and interleukin 6 (IL-6) as well as their stimulation of p70S6K phosphorylation. The induction of apoptosis by rapamycin and dexamethasone despite the presence of survival factors was also demonstrated in primary MM cells, thus suggesting this drug combination to be active also in vivo. (Blood. 2004;103:3138-3147)

MTOR Inhibitors Activate the AKT Kinase in Multiple Myeloma Cells by Upregulating the IGF-1/IRS-1/PI-3 Kinase Cascade.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3350-3350 ◽  
Author(s):  
Yijiang Shi ◽  
HuaJun Yan ◽  
Patrick Frost ◽  
Bao Hoang ◽  
Joseph Gera ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Proteasome Inhibitors ◽  
Xenograft Model ◽  
Mtor Inhibitors ◽  
Pi3 Kinase ◽  
Serine Phosphorylation ◽  
G1 Arrest ◽  
Akt Activation ◽  
Induced Apoptosis

Abstract MTOR inhibitors, such as rapamycin and CCI-779, have shown pre-clinical potential as therapy for multiple myeloma (MM). By inhibiting expression of cell cycle proteins, these agents induce G1 arrest. However, by also inhibiting an mTOR-dependent phosphorylation of insulin receptor substrate-1 (IRS-1), they may alter its subcellular localization and/or prevent its degradation which could enhance IGF-1 signaling and downstream PI3-kinase/AKT activation. This may be a particular problem in MM where IGF-1-induced activation of AKT is an important anti-apoptotic cascade. We, thus, studied PI3-kinase/AKT activation in MM cells treated with mTOR inhibitors. Rapamycin enhanced basal AKT activity, AKT phosphorylation and PI3-kinase activity in MM cell lines. Both PTEN-null as well as PTEN-wild type myeloma lines were similarly affected. Rapamycin also significantly prolonged activation of AKT induced by exogenous IGF-1. CCI-779, used in a xenograft model, also resulted in MM cell AKT activation in vivo. Blockade of IGF-1 receptor function prevented rapamycin’s activation of AKT. Furthermore, rapamycin prevented serine phosphorylation of IRS-1 and IRS-1 degradation. Though similarly blocking IRS-1 degradation, proteasome inhibitors did not activate MM cell AKT. Although rapamycin sensitized MM cells for dexamethasone-induced apoptosis, it protected against PS-341-induced apoptosis. Thus, mTOR inhibitors activate PI3-K/AKT in MM cells and activation depends on basal IGF-1/IGF-R signaling. As activated AKT may protect against apoptosis, future use of mTOR inhibitors in myeloma patients will have to carefully consider the types of anti-myeloma agents used in combination.

CHIR258, a Novel Multi-Targeted Tyrosine Kinase Inhibitor, for the Treatment of t(4;14) Multiple Myeloma.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 641-641 ◽  
Author(s):  
Suzanne Trudel ◽  
Zhi Hua Li ◽  
Ellen Wei ◽  
Marion Wiesmann ◽  
Katherine Rendahl ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Mouse Model ◽  
Tyrosine Kinase ◽  
Cell Lines ◽  
Small Molecule ◽  
Kinase Inhibitor ◽  
Wild Type ◽  
Myeloma Cells

Abstract The t(4;14) translocation that occurs uniquely in a subset (15%) of multiple myeloma (MM) patients results in the ectopic expression of the receptor tyrosine kinase, Fibroblast Growth Factor Receptor3 (FGFR3). Wild-type FGFR3 induces proliferative signals in myeloma cells and appears to be weakly transforming in a hematopoeitic mouse model. The subsequent acquisition of FGFR3 activating mutations in some MM is associated with disease progression and is strongly transforming in several experimental models. The clinical impact of t(4;14) translocations has been demonstrated in several retrospective studies each reporting a marked reduction in overall survival. We have previously shown that inhibition of activated FGFR3 causes morphologic differentiation followed by apoptosis of FGFR3 expressing MM cell lines, validating activated FGFR3 as a therapeutic target in t(4;14) MM and encouraging the clinical development of FGFR3 inhibitors for the treatment of these poor-prognosis patients. CHIR258 is a small molecule kinase inhibitor that targets Class III–V RTKs and inhibits FGFR3 with an IC50 of 5 nM in an in vitro kinase assay. Potent anti-tumor and anti-angiogenic activity has been demonstrated in vitro and in vivo. We employed the IL-6 dependent cell line, B9 that has been engineered to express wild-type FGFR3 or active mutants of FGFR3 (Y373C, K650E, G384D and 807C), to screen CHIR258 for activity against FGFR3. CHIR258 differentially inhibited FGF-mediated growth of B9 expressing wild-type and mutant receptors found in MM, with an IC50 of 25 nM and 80 nM respectively as determined by MTT proliferation assay. Growth of these cells could be rescued by IL-6 demonstrating selectivity of CHIR258 for FGFR3. We then confirmed the activity of CHIR258 against FGFR3 expressing myeloma cells. CHIR258 inhibited the viability of FGFR3 expressing KMS11 (Y373C), KMS18 (G384D) and OPM-2 (K650E) cell lines with an IC50 of 100 nM, 250 nM and 80 nM, respectively. Importantly, inhibition with CHIR258 was still observed in the presence of IL-6, a potent growth factors for MM cells. U266 cells, which lack FGFR3 expression, displayed minimal growth inhibition demonstrating that at effective concentrations, CHIR258 exhibits minimal nonspecific cytotoxicity on MM cells. Further characterization of this finding demonstrated that inhibition of cell growth corresponded to G0/G1 cell cycle arrest and dose-dependent inhibition of downstream ERK phosphorylation. In responsive cell lines, CHIR258 induced apoptosis via caspase 3. In vitro combination analysis of CHIR258 and dexamethasone applied simultaneously to KMS11 cells indicated a synergistic interaction. In vivo studies demonstrated that CHIR258 induced tumor regression and inhibited growth of FGFR3 tumors in a plasmacytoma xenograft mouse model. Finally, CHIR258 produced cytotoxic responses in 4/5 primary myeloma samples derived from patients harboring a t(4;14) translocation. These data indicate that the small molecule inhibitor, CHIR258 potently inhibits FGFR3 and has activity against human MM cells setting the stage for a Phase I clinical trial of this compound in t(4;14) myeloma.

Pralatrexate Has Potent Activity Against Multiple Myeloma In Vitro and In Vivo, and Activity Correlates with Tumor RFC-1 and DHFR Expression

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1831-1831 ◽  
Author(s):  
Michael Mangone ◽  
Luigi Scotto ◽  
Enrica Marchi ◽  
Owen A. O'Connor ◽  
Hearn J. Cho
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Research Funding ◽  
Folate Metabolism ◽  
Nog Mice ◽  
Induced Apoptosis ◽  
Dose Dependent ◽  
Functional Biomarkers

Abstract Abstract 1831 Multiple myeloma (MM) is the second most common hematologic malignancy. Although there are effective new agents that can induce remission, relapse is inevitable and the disease is currently incurable. Progress in the treatment of this disease demands development of novel therapeutics and identification of functional biomarkers that may be used to distinguish tumors that are susceptible to specific targeted agents, creating a “personalized” therapeutic strategy for individual patients. We investigated these principles with anti-folates, which are not commonly used in MM but have demonstrated activity in this disease. Pralatrexate (PDX, 10-propargyl 10-deazaaminopterin) is a folate analogue that was rationally designed to have high affinity for Reduced Folate Carrier (RFC)-1, an oncofetal protein expressed in many cancers that actively transports folates into cells. PDX induced dose-dependent apoptotic cell death in a subset of human myeloma cell lines (HMCL) and CD138+ MM cells isolated from a clinical specimen. In sensitive cell lines, PDX exhibited 10-fold greater potency compared to the structurally related drug methotrexate (MTX). PDX induced dose-dependent, intrinsic apoptosis in sensitive HMCLs, characterized by cleavage of caspase-3 and -9 and accompanied by the loss of full-length Mcl-1, a Bcl-2 family protein that plays a critical role in drug-induced apoptosis in MM. Furthermore, the activity of PDX is not abrogated by the presence of exogenous interleukin-6 or by co-culture with HS-5 bone marrow stromal cells, both of which exert powerful survival effects on MM cells and can antagonize apoptosis in response to some cytotoxic chemotherapy drugs. Sensitivity to PDX-induced apoptosis correlated with higher relative levels of RFC-1 mRNA in sensitive compared to resistant HMCL. Resistant HMCL also exhibited a dose-dependent up-regulation of dihydrofolate reductase (DHFR) protein, a primary molecular target for anti-folates, in response to PDX exposure, whereas sensitive HMCL did not. These changes in functional folate metabolism biomarkers, high baseline RFC-1 expression and upregulation of DHFR in response to PDX, appeared to be mutually exclusive to sensitive or resistant HMCL, respectively. Importantly, PDX was also effective against sensitive HMCL in vivo in a novel mouse xenograft model. NOD/Shi-scid/IL-2Rγnull (NOG) mice were inoculated with MM.1s HMCL stably transduced to express both GFP and luciferase (GFP-luc). GFP-luc MM.1s cells engrafted into the long bones, pelvis, and vertebral column of NOG mice within 4–7 days after injection of cells, as assessed by in vivo bioluminescent imaging. Treatment with PDX resulted in a significant reduction in tumor burden after two doses. These results demonstrate that PDX has potent anti-myeloma activity in vitro and in vivo, and that RFC-1 expression and DHFR upregulation are robust functional biomarkers that may identify patients who are likely to benefit from PDX therapy. These data support further exploration of PDX therapy in clinical trials for MM and investigation of folate metabolism biomarkers as indices for treatment with this class of drugs. Improved anti-folates such as PDX are a promising class of agents that may be a valuable addition to the arsenal against MM. Disclosures: O'Connor: Celgene: Consultancy, Research Funding; Merck: Research Funding; Novartis: Research Funding; Spectrum: Research Funding.

scholarly journals Epigenetic Priming with Decitabine Augments the Therapeutic Effect of Cisplatin on Triple-Negative Breast Cancer Cells through Induction of Proapoptotic Factor NOXA

Cancers ◽  
2022 ◽  
Vol 14 (1) ◽  
pp. 248
Author(s):  
Wataru Nakajima ◽  
Kai Miyazaki ◽  
Masahiro Sakaguchi ◽  
Yumi Asano ◽  
Mariko Ishibashi ◽  
...  
Keyword(s):  
Cell Lines ◽  
Triple Negative ◽  
Kinase Inhibitor ◽  
Underlying Mechanism ◽  
Breast Cancers ◽  
Hematopoietic Malignancy ◽  
Cyclin Dependent Kinase Inhibitor ◽  
Tnbc Cell ◽  
Aberrant Dna Methylation ◽  
Induced Apoptosis

Epigenetic alterations caused by aberrant DNA methylation have a crucial role in cancer development, and the DNA-demethylating agent decitabine, is used to treat hematopoietic malignancy. Triple-negative breast cancers (TNBCs) have shown sensitivity to decitabine; however, the underlying mechanism of its anticancer effect and its effectiveness in treating TNBCs are not fully understood. We analyzed the effects of decitabine on nine TNBC cell lines and examined genes associated with its cytotoxic effects. According to the effect of decitabine, we classified the cell lines into cell death (D)-type, growth inhibition (G)-type, and resistant (R)-type. In D-type cells, decitabine induced the expression of apoptotic regulators and, among them, NOXA was functionally involved in decitabine-induced apoptosis. In G-type cells, induction of the cyclin-dependent kinase inhibitor, p21, and cell cycle arrest were observed. Furthermore, decitabine enhanced the cytotoxic effect of cisplatin mediated by NOXA in D-type and G-type cells. In contrast, the sensitivity to cisplatin was high in R-type cells, and no enhancing effect by decitabine was observed. These results indicate that decitabine enhances the proapoptotic effect of cisplatin on TNBC cell lines that are less sensitive to cisplatin, indicating the potential for combination therapy in TNBC.

Lycorine Modulates the Expression of p21 Via a p53-Independent Pathway in HL-60 Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4297-4297
Author(s):  
Jing Liu ◽  
Shu-Ling Wang ◽  
Lin Fang ◽  
Mao Ye ◽  
Zhi-Wei Sun ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Kinase Inhibitor ◽  
Conflicts Of Interest ◽  
Fold Increase ◽  
Alternative Pathways ◽  
Cyclin Dependent Kinase Inhibitor ◽  
M Phase ◽  
Induced Apoptosis

Abstract Abstract 4297 Leukemia is one of the most life-threatening cancers today, and acute promyelogenous leukemia is a common type of leukemia. We have previously shown that lycorine, a natural alkaloid extract from Amaryllidaceae, exhibited anti-leukemia effects in vitro and in vivo. Lycorine treatment of HL-60 cell arrested cell cycle at G2/M phase and induced apoptosis. In the present study, we sought to explore the molecular mechanisms for the anti-leukemia action of lycorine. Gene chip analysis revealed that lycorine treatment of HL-60 cells induced more than 9 fold increase of p21, a cyclin-dependent kinase inhibitor, whose expression is mainly regulated by p53. Since HL-60 cells are p53 null, the above findings suggest that lycorine activates p21 expression through p53-independent pathway. To further explore the alternative pathways for the activation of p21 induced by lycorine, we examined the effect of lycorine on the expression of Rb, pRb, E2F, c-Myc and HDACs which have shown to regulate p21 expression. We show that expression of pRb (ser780) and c-Myc was down-regulated, Rb and E2F were up-regulated, while the expression of HDAC1 and HDAC3 was not changed. Together these findings suggest that lycorine exerts its anti-leukemia effect by activating p21 expression via pRb/E2F and c-Myc pathways. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Siah2 control of T-regulatory cells limits anti-tumor immunity

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Marzia Scortegagna ◽  
Kathryn Hockemeyer ◽  
Igor Dolgalev ◽  
Joanna Poźniak ◽  
Florian Rambow ◽  
...  
Keyword(s):  
Tumor Immunity ◽  
Cell Cycle Progression ◽  
T Regulatory Cells ◽  
Kinase Inhibitor ◽  
G1 Arrest ◽  
Cancer Therapies ◽  
Activated T Cells ◽  
Cyclin Dependent Kinase Inhibitor ◽  
Elevated Expression

AbstractUnderstanding the mechanisms underlying anti-tumor immunity is pivotal for improving immune-based cancer therapies. Here, we report that growth of BRAF-mutant melanoma cells is inhibited, up to complete rejection, in Siah2−/− mice. Growth-inhibited tumors exhibit increased numbers of intra-tumoral activated T cells and decreased expression of Ccl17,Ccl22, and Foxp3. Marked reduction in Treg proliferation and tumor infiltration coincide with G1 arrest in tumor infiltrated Siah2−/− Tregs in vivo or following T cell stimulation in culture, attributed to elevated expression of the cyclin-dependent kinase inhibitor p27, a Siah2 substrate. Growth of anti-PD-1 therapy resistant melanoma is effectively inhibited in Siah2−/− mice subjected to PD-1 blockade, indicating synergy between PD-1 blockade and Siah2 loss. Low SIAH2 and FOXP3 expression is identified in immune responsive human melanoma tumors. Overall, Siah2 regulation of Treg recruitment and cell cycle progression effectively controls melanoma development and Siah2 loss in the host sensitizes melanoma to anti-PD-1 therapy.

scholarly journals Targeting of mTORC1/2 by the mTOR kinase inhibitor PP242 induces apoptosis in AML cells under conditions mimicking the bone marrow microenvironment

Blood ◽  
2012 ◽  
Vol 120 (13) ◽  
pp. 2679-2689 ◽  
Author(s):  
Zhihong Zeng ◽  
Yue Xi Shi ◽  
Twee Tsao ◽  
YiHua Qiu ◽  
Steven M. Kornblau ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mouse Model ◽  
Kinase Inhibitor ◽  
Mammalian Target Of Rapamycin ◽  
Leukemic Cells ◽  
Phosphatidylinositol 3 Kinase ◽  
Mtor Kinase ◽  
Phosphorylated Akt ◽  
Induced Apoptosis

Abstract The interactions between the bone marrow (BM) microenvironment and acute myeloid leukemia (AML) is known to promote survival of AML cells. In this study, we used reverse phase-protein array (RPPA) technology to measure changes in multiple proteins induced by stroma in leukemic cells. We then investigated the potential of an mTOR kinase inhibitor, PP242, to disrupt leukemia/stroma interactions, and examined the effects of PP242 in vivo using a mouse model. Using RPPA, we confirmed that multiple survival signaling pathways, including the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR), were up-regulated in primary AML cells cocultured with stroma. PP242 effectively induced apoptosis in primary samples cultured with or without stroma. Mechanistically, PP242 attenuated the activities of mTORC1 and mTORC2, sequentially inhibited phosphorylated AKT, S6K, and 4EBP1, and concurrently suppressed chemokine receptor CXCR4 expression in primary leukemic cells and in stromal cells cultured alone or cocultured with leukemic cells. In the in vivo leukemia mouse model, PP242 inhibited mTOR signaling in leukemic cells and demonstrated a greater antileukemia effect than rapamycin. Our findings indicate that disrupting mTOR/AKT signaling with a selective mTOR kinase inhibitor can effectively target leukemic cells within the BM microenvironment.

PKC412 Is a Multi-Targeting Kinase Inhibitor with Activity Against Multiple Myeloma In Vitro and In Vivo.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 247-247 ◽  
Author(s):  
Joseph Negri ◽  
Nicholas Mitsiades ◽  
Qingwei Deng ◽  
Zhaoqin Wen ◽  
David C. Geer ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Tyrosine Kinase ◽  
Cell Lines ◽  
Kinase Inhibitor ◽  
De Novo ◽  
Alkylating Agents ◽  
Nod Mice ◽  
Protective Effects

Abstract Multiple myeloma (MM) remains an incurable neoplasia and exhibits high propensity for de novo/acquired refractoriness even to novel agents, e.g. thalidomide (Thal) or proteasome inhibitors. This may be due to complex and evolving patterns of molecular lesions potentially conferring hyperactive antiapoptotic signaling with high degree of redundancy upon inhibition of isolated targets within those pathways. We thus hypothesized that, for genetically complex neoplasias such as MM, patient outcome might improved by addition, in the therapeutic armamentarium, of agents that simultaneously inhibit multiple proliferative/antiapoptotic targets. Towards this effort of multi-targeted therapies, we studied the tyrosine kinase inhibitor PKC412 (midausporin, Novartis, Basel, Switzerland). Low-nM levels of PKC412 selectively inhibit tyrosine kinase receptors, such as FLT3. But higher PKC412 concentrations (in μM range), which presumably inhibit (at least partly) other kinases, are achieved in clinical trials without catastrophic toxicities. This suggests that PKC412 can safely suppress in vivo the activity of, not just FLT3, but a broader spectrum of kinases, some of which (individually or cooperatively) might be critical for survival/proliferation of MM cells. Indeed, in vitro kinase activity assays showed that clinically achievable (high nM or low μM) PKC412 concentrations inhibit by >20% important kinases, including p70S6K, IKK-a and Akt,. Consistent with such multi-targeted effect, in vitro MTT colorimetric survival assays showed activity of PKC412 (at sub-μM levels) against panel of 40 MM cell lines and 10 primary tumor cells from MM patients (including cells resistant to Dex, alkylating agents, anthracyclines, Thal or its immunomodulatory derivatives, bortezomib, and/or Apo2L/TRAIL), and cell lines from hematologic neoplasias, e.g. B- and T-ALL, CML, various non-Hodgkin’s lymphoma subtypes, and solid tumors (e.g. breast, prostate, lung, thyroid, ovarian, renal Ca, retinoblastoma and sarcomas)). Mechanistic studies confirmed that PKC412 blocks key growth/survival pathways (e.g. PI-3K/Akt, IKK-α/NF-κB), coupled with by downstream effects on suppression of diverse inhibitors of apoptosis (e.g. FLIP, XIAP, cIAP-2, survivin). These molecular sequelae explain, at least partly, the ability of PKC412 to sensitize MM cells to other anti-MM agents (such as Dex, cytotoxic chemotherapy or proteasome inhibitor bortezomib) and overcome protective effects of cytokines (e.g. IL-6) or bone marrow stromal cells. Importantly, PKC412 significantly prolonged the overall survival (p<0.03, Kaplan-Meier analysis) of SCID/NOD mice in a clinically relevant model of diffuse MM bones lesions. These studies provide basis for clinical studies of PKC412 in MM and indicate that kinase inhibitors selectively blocking specific targets at low drug levels, may also have potent anti-tumor activities related to inhibition of multiple other, less specific, nonetheless important targets, thus allowing for anti-tumor activity in a much broader spectrum of tumor types than previously appreciated.

Combinations of Kinase Inhibitors May Be An Effective Therapeutic Strategy for Acute Myeloid Leukemia (AML) with MLL-AF9 Expression.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1032-1032
Author(s):  
Xavier Poiré ◽  
Ernesto Diaz-Flores ◽  
Rachel Joy Bergerson ◽  
Dorie Sher ◽  
Olatoyosi Odenike ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Lines ◽  
Intracellular Signaling ◽  
Therapeutic Strategy ◽  
Drug Exposure ◽  
Kinase Inhibitor ◽  
Fusion Gene ◽  
Brdu Incorporation ◽  
Murine Leukemia

Abstract Abstract 1032 Poster Board I-54 MLL-AF9 translocation is the most common MLL fusion gene found in AML and has been associated with a relatively poor prognosis. The acquisition of MLL-AF9 is a crucial event in leukemogenesis but might require additional genetic events to induce AML. Mutations in cell-signaling molecules including FLT3 and RAS, which confer cell survival and proliferation advantages, occur frequently in MLL leukemias and point to a potential crucial pathogenetic role of the receptor tyrosine kinase (RTK) FLT3 and downstream PI3K/AKT, RAS/MAPK and STAT5 pathways. Targeted inhibition of these pathways can be achieved therapeutically, but efficacy of a single kinase inhibitor (KI) is often limited due to activation of compensatory survival pathways. To explore potential synergy and better efficacy, we have tested a number of KI combinations in MLL-AF9 murine leukemia cells. METHODS: AML was induced in MLL-AF9 knock-in mice using the murine leukemia virus MOL4070LTR, and 4 different cell lines have been generated and listed as 467D, 512C, 467B and 417B. The following KIs were tested in various dual combinations: rapamycin, a mTOR inhibitor; PI-103, a PI3K inhibitor; sunitinib, a RTK inhibitor; erlotinib, another RTK inhibitor with off-target effects on STAT5; PD0325901, a MEK inhibitor; and P6, a JAK inhibitor. KIs concentrations for the combination treatments were based on available in vivo pharmacological data. Effects on cell cycle were measured using BrdU incorporation on all 4 cell lines and phospho-protein levels were measured on 512C using flow cytometry 24-hours after drug exposure. RESULTS: Each drug alone modestly affected cell proliferation, but some of the combinations exerted marked anti-proliferative and pro-apoptotic effects, suggesting synergy. Interestingly, some heterogeneity in response to the different combinations was seen in the 4 cell lines. Indeed, the most effective drug duets were PI-103 and P6 in 467D, rapamycin and PI-103 in 512C, and sunitinib and PD0325901 in 467B and 417B. The significant inhibitory effect of some of these combinations on cell cycle progression correlated with a decrease of phosphorylated histone 3 and an increase in cleaved caspase. Furthermore, we were able to demonstrate a correlation with intracellular signaling events, including strong inhibition of pS6, pmTOR, p4EBP1, peIF4E, pErk and Bcl-xl. No effect on pSTAT5 was observed. Despite the heterogeneity in response, 3 different drug combinations were identified as being the most effective for all 4 cell lines: rapamycin and PD0325901, rapamycin and sunitinib, and sunitinib and PD0325901 resulted in a mean decrease in S phase of 24.4 ± 6.4% and a mean increase in apoptosis of 7.2 ± 4.9% after 24 hours exposure in all 4 cell lines. Furthermore, the combination of sunitinib with PD0325901 resulted in a prolonged effect on proliferation and apoptosis after 48 and 72-hour exposure, while the other 2 combinations showed only a transient effect. CONCLUSIONS: The combination of sunitinib and PD0325901 was active in all 4 cell lines, overcoming the genetic heterogeneity, and will now be tested in vivo in these murine MLL-AF9 leukemias. Our results suggest that combined blockade of RTK and MEK signaling may be an effective and attractive therapeutic strategy for MLL-AF9 AML. Disclosures: Sher: invivoscribe: Employment.

CXCR4 Promotes the Tumorogenicity of Multiple Myeloma, Including Increased Motility, Clonogenicity, up-Regulation of VLA-4, Protection From Chemotherapy and Aggressive Tumor Development In Vivo

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1801-1801
Author(s):  
Katia Beider ◽  
Amnon Peled ◽  
Lola Weiss ◽  
Merav Leiba ◽  
Avichai Shimoni ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Molecular Mechanisms ◽  
Conflicts Of Interest ◽  
Critical Role ◽  
Tumor Development ◽  
Soft Agar ◽  
Induced Apoptosis

Abstract Abstract 1801 Background: Multiple myeloma (MM) is by large incurable neoplasm of plasma cells, characterized by accumulation in the bone marrow (BM), in close contact to cellular and extracellular matrix (ECM) components. Chemokine receptor CXCR4 is expressed by the majority of patients' MM cells. It promotes myeloma cell migration and homing to the BM compartment, supports the tumor cells survival and protects the myeloma cells from chemotherapy-induced apoptosis. Further investigation is required to define the specific molecular mechanisms regulated by the CXCR4/CXCL12 axis in MM. However, surface CXCR4 is commonly down-regulated in the MM cell lines. In order to overcome this limitation, the aim of the current study was to produce a reliable model for studying the functional role of high CXCR4 in MM by generating MM cell lines with stable expression of surface CXCR4. Results: To over-express CXCR4, we transduced CXCL12-expressing MM cell lines ARH77 and RPMI8226 with lentiviral vector and generated cell lines with high and stable levels of surface CXCR4. Enhanced CXCR4 expression significantly increased the in vitro survival and growth of the 2 MM cell lines in serum-deprivation conditions (p<0.01). Furthermore, elevated expression of surface CXCR4 prominently increased MM cells motility and promoted CXCL12-dependent transwell migration of the transduced MM cell lines. Highly CXCR4-expressing RPMI8226 and ARH77 cells demonstrated 40% migration in response to CXCL12 (50 ng/ml), versus only 0–5% migration of MM cells with low expression of surface CXCR4 (p<0.01). Furthermore, adhesion of MM cells to either ECM proteins or BMSCs localize the malignant PCs within the BM microenvironment, promote growth and survival of MM cells and play a critical role in myeloma bone disease and tumor invasion. In accordance, we observed induced adhesion of the transfected RPMI8226-CXCR4 cells to ECM components fibronectin and laminin and to BM fibroblasts. Moreover, we found that enhanced CXCR4 not only functionally activates, but rather significantly elevates the surface levels of VLA-4 integrin on the RPMI8226 cells. In addition, we found that CXCR4-expressing MM cells were less sensitive to melphalan- and bortezomib-induced apoptosis, when they were co-cultured with BM fibroblasts. Testing the molecular signaling pathways regulated by CXCR4, we found that elevated CXCR4 increased the basic level of pERK1/2 and pAKT in the MM cells, and promoted their prolonged activation in response to CXCL12 stimulation. Finally, the ability to produce colonies in the soft agar semi-solid culture reflects the tumorigenic capacity of cancer cells and cancer stem cells. Differentiated MM cells thus rarely produce colonies in soft agar. Here, we demonstrate that up regulation of CXCR4 promoted ARH77 and RPMI8226 colony formation, significantly increasing colonies number and size. Lastly, we determined the role of CXCR4 in MM tumor development in vivo. CXCR4-expressing ARH77 and RPMI8226 cells were subcutaneously injected into NOD/SCID mice. CXCR4-expressing cells, but not parental cell lines, produced detectable tumors already 10 days after the injection. Rapid tumor growth was further observed in both CXCR4-expressing cell lines. These findings indicate that CXCR4 provided aggressive phenotype and supported MM growth in vivo. Conclusions: Taken together, our findings clearly demonstrate the important pathophysiologic role of CXCR4 in MM development and progression. Furthermore, for the first time, we provide the evidence for CXCR4 oncogenic potential in MM, showing that CXCR4 promotes the clonogenic growth of MM cells. Our model may further serve to elucidate CXCR4-regulated molecular events potentially involved in the pathogenesis of MM, and strongly support targeting CXCR4 as therapeutic tool in MM. Disclosures: No relevant conflicts of interest to declare.

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