Combination of the IGF-1 Receptor Inhibitor Picropodophylin and the BH3 Mimetic ABT-737 Has Synergistic Anti-Myeloma Activity

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4010-4010
Author(s):  
Liesbeth Bieghs ◽  
Ken Maes ◽  
Els Van Valckenborgh ◽  
Eline Menu ◽  
Hans Erik Johnsen ◽  
...  
Keyword(s):  
Growth Factors ◽  
Cell Viability ◽  
Cell Lines ◽  
Kinase Inhibitor ◽  
Conflicts Of Interest ◽  
Differential Sensitivity ◽  
Expression Ratio ◽  
Therapy Strategy ◽  
Induced Apoptosis ◽  
Bh3 Mimetic

Abstract Abstract 4010 Despite intensive research multiple myeloma (MM) is still an incurable disease. MM cells are strongly dependent on the BM micro-environment where growth factors are secreted. IGF-1 is one of the most important growth factors in MM and thus forms an attractive target for anti-cancer therapy. Previously, we demonstrated that picropodophylin (PPP), an IGF-1R kinase inhibitor, indeed has potent anti-MM effects both on human cells and in the 5T2MM and 5T33MM murine models. However, mice eventually relapsed and showed signs of morbidity. Therefore it would be an attractive approach to combine PPP with other cytotoxic drugs. ABT-737 is a BH3 mimetic that binds with high affinity to Bcl-xL, Bcl-2 and Bcl-w, but not Mcl-1. In MM, elevated expression of the Bcl-2 pro-survival family of proteins, especially Mcl-1 and to a lesser extent Bcl-2, has extensively been shown to cause resistance to drug induced apoptosis in MM cells. Consequently, ABT-737 was shown to have potent anti-MM activity but only on a subset of human cell lines. Only very recently, it was shown that the ABT-737 sensitivity appears to be determined both by the Bcl-2/Mcl-1 expression ratio and the interaction of these anti-apoptotic proteins with Bim. Interestingly, we demonstrated that IGF-1R inhibition reduces the expression of Mcl-1 and Bcl-xL and that IGF-1 down-regulates the expression of Bim. In addition, a protective effect of IL-6 and BMSC against ABT-737 has been reported. Together, all the above suggests that it would be beneficial to combine ABT-737 with agents that target growth factors, like PPP. Here, we investigated the potential synergistic anti-MM effects of PPP and ABT-737 and studied the underlying mechanisms using two human myeloma cell lines (OPM-2 and RPMI-8226) and the murine 5T33MM model. Both PPP and ABT-737 (kindly provided by Abbott Laboratories) alone were found to significantly decrease cell viability and induce apoptosis dose and time dependently as evidenced by a decrease in ATP levels and an increase in the number of AnnexV/7'AAD positive cells. However, in agreement with previous reported data, we observed differential sensitivity to ABT-737 between the cell lines used. Nevertheless, treatment with PPP/ABT-737 synergistically decreased cell viability and induced apoptosis in all cell lines. In addition, by western blot analysis we could observe increased cleavage of caspase- 3,- 9 and PARP. Mechanistically, PPP was found to circumvent the adverse effect of ABT-737 by blocking the ABT-737 induced Mcl-1 expression and increasing the expression of Noxa. Interestingly, while CD138+ 5T33MM cells were more sensitive to PPP and the CD138- cells more sensitive to ABT-737, treatment with PPP/ABT-737 targeted both MM cell subpopulations to an equal extent. Finally, we tested the combination of PPP and ABT-737 in the 5T33MM model in a prophylactic setting. Whereas, vehicle and ABT-737 treated mice exhibited progressive MM growth, PPP, and to a significant greater extent, PPP/ABT-737 reduced the tumor burden and prolonged overall survival (p≤0.001). In conclusion, PPP combined with ABT-737 appears to have synergistic anti-MM activity and might thus be a novel and promising therapy strategy for MM. Disclosures: No relevant conflicts of interest to declare.

Induction of Apoptosis In Imatinib-Resistant BCR-ABL1-Positive Leukemia Cell Lines by Bortezomib

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4469-4469
Author(s):  
Hilmar Quentmeier ◽  
Sonja Eberth ◽  
Julia Romani ◽  
Margarete Zaborski ◽  
Hans G. Drexler
Keyword(s):  
Cell Lines ◽  
Kinase Inhibitor ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Philadelphia Chromosome ◽  
Leukemia Cell ◽  
Resistant Cell ◽  
Imatinib Resistance ◽  
Imatinib Resistant ◽  
Induced Apoptosis

Abstract Abstract 4469 The BCR-ABL1 translocation occurs in chronic myeloid leukemia (CML) and in 25% of cases with acute lymphoblastic leukemia (ALL). We screened a panel of BCR-ABL1 positive cell lines to find models for imatinib-resistance studies. Five of 19 BCR-ABL1 positive cell lines were resistant to imatinib-induced apoptosis (KCL-22, MHH-TALL1, NALM-1, SD-1, SUP-B15). None of the five resistant cell lines carried mutations in the kinase domain of BCR-ABL1 and – consequently – all also showed resistance to the second generation kinase inhibitors, nilotinib or dasatinib. All Philadelphia chromosome (Ph)-positive cell lines demonstrated constitutive phosphorylation of STAT5 and S6. Imatinib induced dephosphorylation of both BCR-ABL1 downstream effectors in responsive cell lines, but - remarkably – induced dephosphorylation of STAT5 in resistant cell lines as well. By administering well-described signalling pathway inhibitors we were able to show that activation of mTOR complex 1 was responsible for the constitutive S6 phosphorylation of imatinib-resistant cells. Neither BCR-ABL1 nor Src kinases or Ras/Rac-GTPases underlie tyrosine kinase inhibitor resistance in these cell lines. In conclusion, none of the five TKI-resistant cell lines showed aberrant activation of previously-described oncogenic pathways which would explain their resistance. These findings raise the question whether these cell lines might help to find a novel – alternative – explanation for TKI resistance. Interestingly, the proteasome inhibitor bortezomib induced apoptosis in TKI-resistant and –sensitive Ph+ cell lines. Bortezomib is being used for the treatment of multiple myeloma. Our findings support the notion that bortezomib might also be useful for the treatment of imatinib-resistant CML. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The Role BAFF and BAFF Receptors in Macrophage Induced Drug Resistance of Multiple Myeloma

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5701-5701
Author(s):  
Jing Chen ◽  
Donghua He ◽  
Xing Guo ◽  
Li Yang ◽  
Yi Li ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Drug Resistance ◽  
Cell Viability ◽  
B Cell ◽  
Real Time ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Blood Monocytes ◽  
Healthy Donors ◽  
Induced Apoptosis

Abstract Background: B-cell–activating factor (BAFF) is a member of TNF family that critical for maintenance of B-cell development and homeostasis. BAFF also modulates the proliferation, survival and drug resistance of multiple meyloma (MM) cells. BAFF can be secreted by neutrophils, monocytes,dentritic cells and macrophages. Our previous study showed that, macrophages protect MM cells from drug-induced apoptosis by a cell-to-cell interaction between MM cells and macrophages. We supposed that the interaction of BAFF and BAFF receptors plays a role in macrophages induced MM resistance. Methods: First, we detected the expression levels of BAFF and its three receptors in MM cells were detected by semiquantitative real time-polymerase chain reaction (qPCR) and flow-cytometry. The concentration of BAFF in the culture supernatants of MM cell lines was detected by ELISA. Second, we collect peripheral blood monocytes form healthy donors. Monocytes were induced into macrophages by culturing with M-CSF for 7 days.BAFF expression level in macrophages was detected by qPCR, flow-cytometry and ELISA. Then MM cells were sole-cultured or coculture with macrophages for the indicated time (usually 24 h), after that bortezomib was added to the culture system. Cell viability and apoptosis of MM cells were verified by MTT and flow cytometry.At last,the recombinant human BAFF were added to MM cells, MTT and flow cytometry were used for detection of cell viability and apoptosis of MM cells. Results: Two receptors of BAFF, transmembrane activator and CAML interactor (TACI) and B-cell maturation antigen (BCMA), are highly expressed in various MM cell lines evidenced by real-time PCR and flow cytometry. The expression of BAFF in PBMC-induced macrophages are heterogeneous. We verify macrophage-mediated MM drug resistance by directly coculturing MM cells (ARP-1, 8226 and OPM2) with PBMC-derived macrophages from healthy donors. Functional studies show that recombinant human BAFF rescues RPMI8226 myeloma cells from dexamethasone-induced apoptosis. Conclusions: Our data showed that macrophage might induce drug resistance of MM cells by the interaction of BAFF and BAFF receptors. Further study will focused on the mechanism of interaction between BAFF and BAFF receptors. Disclosures No relevant conflicts of interest to declare.

Tyrosine Kinase Inhibitor Screen Identifies the Multikinase Inhibitor, Foretinib, As a Sensitisor of Treatment-resistant B-Precursor ALL to Core Chemotherapeutic Agents

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1509-1509
Author(s):  
Shaun R Wilson ◽  
Victoria J Weston ◽  
Tatjana Stankovic ◽  
Pamela R Kearns
Keyword(s):  
Cell Viability ◽  
Cell Lines ◽  
Kinase Inhibitor ◽  
Conflicts Of Interest ◽  
Philadelphia Chromosome ◽  
Chemotherapeutic Agents ◽  
Significant Heterogeneity ◽  
Synergistic Activity ◽  
Multikinase Inhibitor ◽  
Childhood All

Abstract Abstract 1509 Acute lymphoblastic leukaemia (ALL) is the most frequent malignancy in childhood with resistance or relapse occurring in up to 20% of patients. The precise mechanisms of resistance to conventional therapy leading to relapse have not been elucidated. Deregulation of tyrosine kinases (TKs) have been implicated in resistant solid tumours and the of aetiology haemopoietic tumours, Philadelphia – chromosome positive ALL (Ph+ ALL), FLT3 in MLL+ infant ALL and FLT3-ITD subset in acute myeloid leukaemia. The role of TK inhibitors (TKIs) has not been extensively investigated in non-Ph + ALL. We screened 5 B-cell precursor ALL cell lines and 20 primary samples with a library of 34 TKIs. Nalm 6 (t(5;12)), Nalm 17 (normal karyotype), REH (t(12;21)), SD1 and Sup15 (Ph+ ALL) and primary cells were tested at 1μM and 10μM and alterations in cell viability assessed with the Promega CellTiter-Glo assay. A drug was considered to be effective if it induced >50% reduction in cell viability at 1μM. While we demonstrated significant heterogeneity in response to many of the TKIs, we observed reduction in viability to lestaurtinib (FLT3/JAK2), dovitinib (FLT3/FGFR/PDGFR/VEGFR) and bosutinib (Abl/Src) in all cell lines. Compared with Nalm 6 and Nalm 17 which only exhibited sensitivity to these 3 TKIs, REH demonstrated additional sensitivity to crizotinib (ALK/Met) and the quinazoline pan-EGFR inhibitors, afatinib and canertinib. The Ph+ cell lines SupB15 and SD1 responded to the highest number of TKIs, 12 and 14 respectively. These included the expected Bcr/Abl and Aurora kinase inhibitors. Activity of the putative PDGFR/VEGFR TKIs axitinib, linifanib, vargatef and also foretinib (MET/VEGFR2/FLT3) appeared limited to Ph+ cell lines. The cell lines, REH and SD-1, which are resistant to ionizing radiation–induced apoptosis, were selectively inhibited by both the quinazolines. Baseline mRNA expression of the ErbB family was present in all cell lines and therefore did not correlate with response. TKIs inducing the greatest reduction in cell viability across the cell lines were those that target class III/IV/V RTKs. Although all cell lines expressed FLT3 mRNA, reduction in cell viability was not universally induced by the specific FLT3 inhibitor tandutinib at doses of up to 10μM. As observed in previous studies, the level of mRNA transcript did not predict or directly correlate with the response to TKI. A panel of 20 primary ALL samples, representative of common biological features, were screened. We found no correlation between cytogenetics, age, white cell count, post – induction MRD status and response to TKI groups or individual inhibitor. Only 5/20 did not respond to any of the tested TKIs. Lestaurtinib, dovitinib and foretinib reduced cell viability in 7/20 primary ALLs. In addition, canertinib reduced cell viabililty in 6/20 primary ALL samples, afatinib and TAE684 (ALK/MET) both in 5/20 ALL samples respectively and vargatef in 4/20 samples. Based on our preliminary screen, the multikinase inhibitor foretinib was selected as one of several promising candidates for further pre-clinical testing. Recent adult phase 1 solid tumor trials have shown limited toxicity and good bioavailability. Foretinib inhibited leukaemia proliferation with LD50 in nanomolar and low micromolar range; SupB15 (333nM ±49), SD-1 (381nM ±239), Nalm 17 (484nM ±124), REH (689nM ±92) and Nalm 6 (1.84μM ±0.25). Annexin/PI staining, DNA fragmentation and PARP protein cleavage confirmed that the mechanism of cell death was apoptosis. We next investigated whether foretinib could sensitise ALL cell lines to dexamethasone, cytarabine, methotrexate, doxorubicin or mitoxantrone. Drug interactions were modelled using the Biosoft Calcusyn software package. We found that the addition of foretinib resulted in predominantly synergistic interactions in all cell lines (CI<1). The most striking example of synergism was in the dexamethasone-resistant cell line, REH. Addition of a sub–LD50 dose of foretinib led to >50% reduction in cell viability when combined with 1nM dexamethasone compared with no response at 10μM dexamethasone alone. Overall these data support further exploration of TKIs as potential therapeutic agents in childhood ALL. Specifically, we are currently investigating the direct anti-leukemic activity of foretinib in childhood ALL and its synergistic activity with dexamethasone in vivo using our NOG mouse primograft model for ALL. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Advantages of Tyrosine Kinase Anti-Angiogenic Cediranib over Bevacizumab: Cell Cycle Abrogation and Synergy with Chemotherapy

2021 ◽  
Vol 14 (7) ◽  
pp. 682
Author(s):  
Jianling Bi ◽  
Garima Dixit ◽  
Yuping Zhang ◽  
Eric J. Devor ◽  
Haley A. Losh ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Endometrial Cancer ◽  
Tyrosine Kinase ◽  
Cell Viability ◽  
Cell Lines ◽  
Cell Cycle Progression ◽  
Kinase Inhibitor ◽  
Cell Cycle Checkpoint ◽  
Mitotic Catastrophe ◽  
M Cell

Angiogenesis plays a crucial role in tumor development and metastasis. Both bevacizumab and cediranib have demonstrated activity as single anti-angiogenic agents in endometrial cancer, though subsequent studies of bevacizumab combined with chemotherapy failed to improve outcomes compared to chemotherapy alone. Our objective was to compare the efficacy of cediranib and bevacizumab in endometrial cancer models. The cellular effects of bevacizumab and cediranib were examined in endometrial cancer cell lines using extracellular signal-related kinase (ERK) phosphorylation, ligand shedding, cell viability, and cell cycle progression as readouts. Cellular viability was also tested in eight patient-derived organoid models of endometrial cancer. Finally, we performed a phosphoproteomic array of 875 phosphoproteins to define the signaling changes related to bevacizumab versus cediranib. Cediranib but not bevacizumab blocked ligand-mediated ERK activation in endometrial cancer cells. In both cell lines and patient-derived organoids, neither bevacizumab nor cediranib alone had a notable effect on cell viability. Cediranib but not bevacizumab promoted marked cell death when combined with chemotherapy. Cell cycle analysis demonstrated an accumulation in mitosis after treatment with cediranib + chemotherapy, consistent with the abrogation of the G2/M checkpoint and subsequent mitotic catastrophe. Molecular analysis of key controllers of the G2/M cell cycle checkpoint confirmed its abrogation. Phosphoproteomic analysis revealed that bevacizumab and cediranib had both similar and unique effects on cell signaling that underlie their shared versus individual actions as anti-angiogenic agents. An anti-angiogenic tyrosine kinase inhibitor such as cediranib has the potential to be superior to bevacizumab in combination with chemotherapy.

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)

Rituximab-Mediated Inhibition of the PI3K-Akt Survival Signaling Pathway in B-NHL Cell Lines Leading to Downregulation of Bclxl Expression and Chemosensitization.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2830-2830
Author(s):  
Eriko Suzuki ◽  
Ali R. Jazirehi ◽  
Benjamin Bonavida
Keyword(s):  
Cell Survival ◽  
Signaling Pathway ◽  
Cell Lines ◽  
Kinase Inhibitor ◽  
Akt Pathway ◽  
Akt Inhibitor ◽  
Drug Induced ◽  
Similar Time ◽  
Cell Lysates ◽  
Induced Apoptosis

Abstract Rituximab (chimeric anti-CD20 monoclonal antibody) has been used in the treatment of B-NHL. We have reported in vitro that rituximab treatment signals B-NHL cell lines Ramos and Daudi and inhibits both the ERK 1/2 MAPK and NF-κB signaling pathways leading to selective inhibition of Bclxl expression and sensitization to drug-induced apoptosis. The inhibition of the NF-κB signaling pathway by rituximab was shown to be due, in part, to the induction of the Raf Kinase Inhibitor Protein (RKIP) (Jazirehi, et al., 2005 Cancer Research 65:264–276). The PI3K-Akt signaling pathway is a key regulator of cell survival and aberrant activation of the PI3K-Akt pathway has been implicated in both drug resistance and resistance to apoptosis-inducing stimuli. Akt can promote cell survival by indirectly activating the proximal transcription factor NF-κB through the phosphorylation of I-kappa B kinase (I-κB) (Ozes et. al. Nature401:82–85, 1999). This study investigated whether NF-κB inhibition by rituximab and downregulation of Bclxl expression was also the result of rituximab-mediated inhibition of the PI3K-Akt pathway. Ramos and Daudi B-NHL cell lines were treated with rituximab (20 ug/ml) and cell lysates were prepared and both Akt and phospho-Akt (p-Akt) expression were examined by western blot. The findings demonstrate that both cell lines show constitutively activated p-Akt and treatment with rituximab significantly inhibited p-Akt but not Akt. Time kinetics analysis demonstrated that inhibition of p-Akt was first detected at 3–6 hours following rituximab treatment and inhibition was maintained up to 24 hours. Concomitantlly, a similar time kinetics revealed inhibition of NF-κB activity as assessed by EMSA. Since the inhibition of NF-kB activity resulted in significant downregulation of Bclxl expression, we also examined the role of the Akt pathway in the regulation of Bclxl expression. Tumor cells were treated with the Akt inhibitor LY294002 and analysis of cell lysates showed significant downregulation of Bclxl expression. Rituximab was previously shown to sensitize B-NHL cells to drug-induced apoptosis via inhibition of NF-κB activity and Bclxl expression. We examined if inhibition of the Akt pathway also chemosensitized the cells. Treatment of Ramos cells with the Akt inhibitor LY294002 significantly sensitized the cells to CDDP-induced apoptosis and synergy was achieved. Altogether, these findings demonstrate, for the first time, that rituximab inhibits the Akt pathway and that this pathway is involved in the regulation of tumor- cell resistance to chemotherapeutic drugs. This study also proposes that the Akt pathway is a potential targeting pathway for therapeutic intervention in the treatment of rituximab and drug-resistant B-NHL.

Serum/Glucocorticoid-Regulated Kinase 1 (SGK1) Is a Prominent Target Gene of the Transcriptional Response to Cytokines In Multiple Myeloma and Supports the Growth of Myeloma Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1915-1915
Author(s):  
Unn-Merete Fagerli ◽  
Thorsten Stühmer ◽  
Toril Holien ◽  
Randi Utne Holt ◽  
Ove Bruland ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Growth Factors ◽  
Cell Lines ◽  
Plasma Cells ◽  
Conflicts Of Interest ◽  
Transcriptional Response ◽  
Myeloma Cell ◽  
Myeloma Cells ◽  
Growth And Survival ◽  
Stat3 Phosphorylation

Abstract Abstract 1915 Multiple myeloma is a paradigm for a malignant disease that exploits external stimuli of the microenvironment for growth and survival. A thorough understanding of the complex interactions between malignant plasma cells and their surrounding requires a detailed analysis of the transcriptional response of myeloma cells to environmental signals. We hypothesized that the intracellular signals evoked by cytokines converge and regulate transcription of a set of genes that are common targets for several growth factors and therefore constitute pivotal mediators of the tumor-promoting effects of autocrine or paracrine stimuli. To identify such targets, we determined the changes in gene expression induced by IL-6, TNFalpha, IL-21 or co-culture with bone marrow stromal cells in myeloma cell lines. Among a limited set of genes that were consistently activated in response to growth factors, a prominent transcriptional target of cytokine-induced signaling in myeloma cells was the gene encoding the serine/threonine kinase SGK1, which is a down-stream effector of PI3-kinase and highly homologous to AKT. We could demonstrate a rapid, strong and sustained induction of SGK1 in the cell lines INA-6, ANBL-6, IH-1, OH-2 and MM.1S as well as in primary myeloma cells. Pharmacologic inhibition of the JAK/STAT pathway abolished STAT3 phosphorylation and SGK1 induction. In addition, shRNA-mediated knock-down of STAT3 reduced basal and induced SGK1 levels, demonstrating the involvement of the JAK/STAT3 signaling pathway in SGK1 induction. Furthermore, down-regulation of SGK1 by shRNAs resulted in decreased proliferation and viability of myeloma cell lines. Our results indicate that SGK1 is a highly cytokine-responsive gene in myeloma cells promoting their growth and survival and represents an attractive candidate for further evaluation as a therapeutic target. Disclosures: No relevant conflicts of interest to declare.

All-Trans-Retinoic Acid (ATRA) Causes Extensive Differentiation In the NPM Mutant, Non-APL Leukemic Cell Line OCI-AML3

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3305-3305 ◽  
Author(s):  
Matthew A. Kutny ◽  
Steven J. Collins ◽  
Keith Loeb ◽  
Roland B. Walter ◽  
Soheil Meshinchi
Keyword(s):  
Cell Viability ◽  
Cell Count ◽  
Cell Line ◽  
Cell Lines ◽  
Time Course ◽  
Conflicts Of Interest ◽  
Live Cell ◽  
Leukemic Cells ◽  
Viable Cell Number ◽  
Nuclear Segmentation

Abstract Abstract 3305 The differentiating agent ATRA has been used successfully in the treatment of acute promyelocytic leukemia (APL). By comparison, non-APL AML has not shown similar sensitivity to ATRA induced differentiation. Recent data has suggested that a subset of de novo AML patients with nucleophosmin (NPM1) mutations may benefit from addition of ATRA to conventional therapy. The NPM1 gene has several functions affecting cell cycle proliferation including regulation of ribosome biogenesis and centrosome duplication and it acts as a histone chaperone. Mutation of the NPM1 gene leads to differentiation arrest contributing to AML pathogenesis. We hypothesized that leukemia cells with NPM1 mutations could be induced to undergo differentiation. We tested this hypothesis with the NPM1 mutant AML cell line OCI-AML3 and compared the results to identical assays using the AML cell line HL-60 which has been previously well documented to differentiate in response to ATRA therapy. OCI-AML3 and HL-60 cell lines were treated for 5 days with control media and four ATRA doses including 0.2 μM, 1 μM, 5 μM, and 25 μM. Cell viability was assessed by flow cytometry. Compared to the control condition, OCI-AML3 cells treated with the lowest dose of ATRA (0.2 μM) had a live cell count 21.6% of the control. HL-60 cells treated at even the highest ATRA dose (25 uM) had a live cell count 79.3% of the control. Due to the sensitivity of OCI-AML3 cells to the toxic effects of ATRA, the experiment was repeated with lower doses of ATRA including 0.001 μM, 0.01 μM and 0.1 μM. At the lowest dose of ATRA (0.001 μM), OCI-AML3 cells demonstrated a cell viability of 49% with further decrease to 26% at 0.1 μM dose of ATRA. At similar ATRA doses, cell viability for HL-60 cells was 91% and 85%, respectively (see table 1). Table 1: Cell viability as a percent of control cells after 5 days of treatment at three different doses of ATRA in OCI-AML3 and HL-60 cell lines. Cell Line: ATRA 0.001 μM ATRA 0.01 μM ATRA 0.1 μM OCI-AML3 49% 33% 26% HL-60 91% 91% 85% We subsequently determined the time course of changes in cell growth and the extent of differentiation at each point was determined by morphologic assessment. Both cell lines were treated with ATRA at doses of 0.001 μM, 0.01 μM, 0.1 μM, and 1 μM for a total of 4 days. Each day viable cell number was determined. In contrast to the HL-60 cells which had continued growth in lower ATRA doses, OCI-AML3 cells demonstrated exquisite sensitivity to growth arrest at the lowest doses of ATRA. Cell morphology was assessed daily with modified Wright-Giemsa staining of cells. Cells were examined for signs of myeloid differentiation including decrease in nuclear to cytoplasmic (N/C) ratio, nuclear segmentation, and cytoplasmic granules and vacuoles. At the lowest dose of ATRA (0.001 μM), after 4 days of exposure, significant number of OCI-AML3 cells demonstrated morphologic evidence of differentiation. At this ATRA dose and exposure interval, HL-60 cells showed no evidence of differentiation. At an ATRA dose of 1 μM (considered a standard dose used for differentiation of HL-60 cells), the OCI-AML3 cells showed differentiation changes as early as day 2 with nuclear segmentation and decreased N/C ratio while HL-60 cells did not show any change at this time point. After 4 days of ATRA exposure, most OCI-AML3 cells showed segmented nuclei and vacuolated cytoplasm, whereas HL-60 cells showed less distinct signs of differentiation with some cytoplasm granules and cup shaped nuclei. This data suggests that leukemic cells with NPM mutations may be susceptible to the pro-differentiating properties of ATRA. Further substantiation of this data with primary human specimens may ultimately provide the rationale for a novel therapeutic option using ATRA-based differentiation therapy for subsets of non-APL AML. Disclosures: No relevant conflicts of interest to declare.

The Kinase Inhibitor Sorafenib Can Disrupt the Survival Support Provided by the Microenvironment and Induce Apoptosis of Chronic Lymphocytic Leukemia Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 47-47
Author(s):  
Jessie-F Fecteau ◽  
Ila Bharati ◽  
Morgan O'Hayre ◽  
Tracy Handel ◽  
Thomas J. Kipps ◽  
...  
Keyword(s):  
Single Dose ◽  
Cell Viability ◽  
Cell Survival ◽  
Kinase Inhibitor ◽  
Vehicle Control ◽  
Lymphocytic Leukemia ◽  
Viable Cells ◽  
Induced Apoptosis

Abstract Abstract 47 Chronic Lymphocytic Leukemia (CLL) is characterized by an accumulation of mature monoclonal B cells in the blood, secondary lymphoid tissue, and marrow. Despite their accumulation in vivo, CLL cells undergo spontaneous apoptosis in vitro unless rescued by extrinsic factors derived from the leukemia-cell microenvironment. Monocyte-derived Nurse-Like Cells (NLCs) and Marrow Stromal Cells (MSCs), representing the leukemic microenvironment, have been show to sustain CLL cell survival and more importantly to protect CLL cells from drug-induced apoptosis in vitro and possibly in vivo. Such protective niches are thought to prevent current therapies from achieving complete remission in patients. Investigating the mechanism(s) by which cells from the microenvironment promote CLL cell survival, particularly the signaling pathways triggered, will allow for the identification of new therapeutic targets aiming to disrupt these protective interactions. NLCs and MSCs have been shown to produce the chemokine SDF-1 (CXCL12), which can enhance CLL cell survival. We recently found that ZAP-70+ aggressive CLL cells responded by an increased survival to this chemokine, compared to ZAP-70- indolent CLL cells, and that this response was accompanied by the activation of the ERK pathway. Attempting to abrogate this survival pathway, we found that sorafenib (BAY 43–9006, Nexavar) a multi-kinase inhibitor targeting among others Raf kinases and thereby the RAF/MEK/ERK pathway, strongly reduced CLL cell viability in a time and dose dependent manner. A regimen of one single dose of 10uM of sorafenib significantly reduced CLL cell viability to 18+/−10% cells after 48hrs compared to vehicle control (DMSO; 100%; n=5). The daily addition of 1uM sorafenib also significantly decreased CLL cell viability, leading to 31+/−21% and 11+/−5% viable cells after 6 and 7 days respectively, compared to DMSO (n=5). More importantly, our results show that sorafenib induces CLL cell death in the presence of NLCs and MSCs. A single dose of sorafenib (10uM) rapidly decreased the fraction of viable CLL cells overtime, passing from 40+/−16% after 1 day to 10+/−3% after 4 days (n=4) in the context of NLCs and to 25+/−3% after 2 days and 14+/−3% after 4 days in the presence of MSCs, when compared to vehicle control (>80%; n=4). In the presence of NLCs, the 1uM daily regimen also uncovered an increased sensitivity of ZAP-70+ CLL cells to this drug, reducing in 6 days their viability to 13+/−2% (n=4), which approximately half the fraction of viable cells remaining in the ZAP-70- group (40+/−16%; n=7). We next studied sorafenib-mediated cytotoxicity by investigating its impact on the expression of pro-survival molecules. We found that Mcl-1, Bcl-2 and Bcl-xL protein expression was reduced in CLL cells compared to vehicle control, when stimulated with CXCL12 (n=3). In the presence of NLCs and MSCs, only Mcl-1 expression was downregulated, which was also associated with a reduction of the active form of the transcription factor CREB, involved in Mcl-1 expression. Because Mcl-1 expression can be regulated by ERK and AKT pathways, we next investigated if they were abrogated by sorafenib. We indeed found that MEK, ERK, and AKT activation were reduced by this inhibitor compared to vehicle control (n=3). We therefore propose that the cytotoxic effect of sorafenib on CLL cells is due to its interference with at least these two major survival pathways. Since sorafenib caused apoptosis of CLL cells in context of the microenvironment, we reasoned that it might also cause apoptosis of chemotherapy resistant CLL cells. To test this hypothesis, we studied cells from fludarabine-refractory patients. In the presence of NLCs, a single dose of 10uM sorafenib induced a significant reduction in CLL cell viability after 2 days: only 4+/−2% viable cells remained compared to 78+/−12% for the vehicle control (n=4). A comparable observation was made in the presence of MSCs: sorafenib potently induced apoptosis, leaving 12+/−3% live cells after 2 days, compared to vehicle control (71+/−16%; n=4). These results are very promising as they suggest that sorafenib could be an effective novel therapeutic for CLL, affecting the viability of the leukemic cells even in protective niches. Since sorafenib has been approved by the FDA in 2007 for the treatment of advanced hepatocellular carcinoma, a pilot study is currently being planned at UCSD to evaluate the potential of this drug in CLL in vivo. Disclosures: No relevant conflicts of interest to declare.

Compensatory Signaling From ROR1 and the Pre-B Cell Receptor Promote Survival of t(1;19) Acute Lymphoblastic Leukemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2466-2466
Author(s):  
Vincent T Bicocca ◽  
Bill H Chang ◽  
Markus Muschen ◽  
Brian J. Druker ◽  
Jeffrey W Tyner
Keyword(s):  
Cell Viability ◽  
B Cell ◽  
Signaling Pathways ◽  
Cell Lines ◽  
Tyrosine Kinases ◽  
Kinase Inhibitor ◽  
Lymphoblastic Leukemia ◽  
Immunoblot Analysis ◽  
Cell Receptor ◽  
Cell Surface Receptor

Abstract Abstract 2466 BACKGROUND: Aberrant tyrosine kinase activity is commonly implicated in the pathogenesis of leukemia and other cancers. Identification of these leukemogenic tyrosine kinases has proven invaluable for diagnostic and prognostic stratification of patients as well as for the development of novel strategies for therapeutic intervention. We previously demonstrated that siRNA screening of mononuclear cells from leukemia patients can determine sensitivity to individual tyrosine kinases. With the goal of uncovering novel viability-dependent tyrosine kinases in leukemia patients, we have employed an RNAi-assisted protein target identification (RAPID) assay to screen cytogenetic subtypes of acute lymphoblastic leukemia (ALL). ALL is the most common pediatric cancer, accounting for one-quarter of all childhood malignancies. Childhood ALL has a primarily B cell precursor phenotype and is characterized by chromosomal abnormalities, primarily translocations and duplications. One of the most common recurring translocations associated with pediatric ALL, t(1;19)(q23;p13.3), generates the E2A-PBX1 fusion product. Here we show unique viability-dependent expression of a receptor tyrosine kinase, ROR1, in the t(1;19) ALL background. In addition, we identify a kinase inhibitor, dasatinib, with significant activity against t(1;19) ALL cells due to its capacity to inhibit tyrosine kinases necessary for transduction of pre-B cell receptor (preBCR) signaling. Finally, we show that ROR1 and the preBCR activate mutually compensatory signaling pathways, suggesting that optimal therapeutic regimens would include agents targeting both pathways. METHODS: To identify targets required for viability of leukemic cells, we screened cell lines as well as primary cells from ALL patients with siRNAs and determined cell viability using an MTS assay. ROR1 expression levels were determined by RT-PCR, immunoblot analysis and flow cytometry. Kinase inhibitor screening was performed on both cells lines and primary ALL cells by treating samples with a library of small-molecule inhibitors and cell viability was assessed by MTS. Signaling pathways disrupted by inhibitor treatment or ROR1 knockdown were interrogated by phospho-protein arrays and confirmed by immunoblot analysis. RESULTS: The RAPID assay identified a unique sensitivity to the cell surface receptor ROR1 in a subject identified with t(1;19) pediatric ALL. Similar sensitivity was not observed in ALL patients of alternative cytogenetic subtypes. Examination of additional ALL patient samples revealed conserved overexpression of the ROR1 transcript in t(1;19)-positive specimens with absence of ROR1 expression in t(1;19)-negative samples. Cell lines and early passage xenograft cells confirmed overexpression and functional dependence of t(1;19)-positive cells on ROR1. A subsequent kinase inhibitor screen of t(1;19) ALL cell lines and patient samples revealed universal sensitivity to the FDA-approved drug dasatinib. Further examination revealed the dasatinib targets, BTK and LYN, which signal downstream of the preBCR as the viability dependent targets of dasatinib in t(1;19) ALL. Inhibition of the preBCR results in transient loss of AKT activity and, surprisingly, upregulation of ROR1. Analysis of signaling pathways after silencing of ROR1 or dasatinib treatment revealed compensatory signaling pathways emanating from ROR1 and the preBCR that both serve to regulate AKT activity. Consequently, combination of ROR1 knockdown and dasatinib treatment resulted in additive impairment of cell viability compared with either treatment alone. CONCLUSION: The cell surface receptor ROR1 is consistently overexpressed in t(1;19) ALL. RNAi mediated downregulation of ROR1 impairs the viability of these cells. Similarly, t(1;19) cells are sensitive to the kinase inhibitor dasatinib due to activity against the preBCR. Combined targeting of ROR1 and the preBCR with dasatinib yields an additive viability effect due to compensatory signaling pathways aimed at regulating AKT. These results demonstrate a novel mechanism of AKT regulation in t(1;19) ALL as well as a therapeutic strategy for treatment of t(1;19) ALL. Disclosures: Druker: MolecularMD: Equity Ownership, OHSU and Dr. Druker have a financial interest in MolecularMD. Technology used in this research has been licensed to MolecularMD. This potential conflict of interest has been reviewed and managed by the OHSU Conflict of Interest in Research Committee and t; Ariad Pharmaceuticals: Consultancy; OHSU patent #843: Mutated ABL Kinase Domains: Patents & Royalties; Bristol-Myers Squibb: OHSU has clinical trial contracts with Bristol-Myers-Squibb to pay for patient costs, nurse and data manager salaries, and institutional overhead. Dr. Druker does not derive salary, nor does his lab receive funds from these contracts.; Novartis: OHSU has clinical trial contracts with Novartis to pay for patient costs, nurse and data manager salaries, and institutional overhead. Dr. Druker does not derive salary, nor does his lab receive funds from these contracts.; Cylene: Consultancy.

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