The Novel Proteasome Inhibitor CEP-18770 Inhibits Myeloma Tumor Growth In Vitro and In Vivo and Enhances the Anti-MM Effects of Melphalan

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 843-843
Author(s):  
Eric SancheZ ◽  
Richard A Campbell ◽  
Jeffrey A Steinberg ◽  
Mingjie Li ◽  
Haiming Chen ◽  
...  
Keyword(s):  
Tumor Growth ◽  
Arsenic Trioxide ◽  
Cell Lines ◽  
Proteasome Inhibitor ◽  
Tumor Volume ◽  
Single Agent ◽  
Human Tumor ◽  
The Novel

Abstract Proteasome inhibitors (PI) have been shown to be effective agents for the treatment of multiple myeloma (MM) and enhance the anti-tumor effects of a variety of chemotherapeutic drugs including melphalan and doxorubicin as well as arsenic trioxide (ATO). The novel proteasome inhibitor CEP-18770 has recently been shown to induce cytotoxic effects across a broad panel of human tumor cell lines including MM in vitro. However, little data exists on the in vivo anti-MM effects of this PI either alone or in combination with other active anti-MM drugs. First, we examined the anti-proliferative effects of treating MM cell lines in vitro with CEP-18770 alone and in combination with melphalan, arsenic trioxide (ATO) and doxorubicin. MM cell lines were cultured without fetal bovine serum and incubated in the presence of CEP-18770 alone and in combination with these agents for 48 hours. Cell growth was then measured using an MTS assay. First, RPMI8226 and U266 cells were tested in vitro using a constant concentration of melphalan or doxorubicin in combination with varying concentrations of CEP-18770 or varying concentrations of the chemotherapeutic agent with constant CEP-18770. Although single agent treatment showed marked anti-proliferative effects, combination indexes as calculated by the Chou-Talalay method showed synergistic anti- MM effects of CEP-18770 with either melphalan or doxorubicin in these MM cell lines. In addition, similar experiments were carried out evaluating the combination of ATO plus CEP-18770 in RPMI8226 cells and also showed synergism with this combination. Next, a series of in vivo studies were conducted using our SCID-hu models of MM including LAGλ-1, LAGκ-1A and LAGκ-1B. Mice receiving CEP-18770 at 0.1, 0.3, 1, and 3 mg/kg were injected twice weekly via intravenous injection throughout the study. CEP-18770 dosed at 10 mg/kg was administered via oral gavage twice weekly and mice dosed with melphalan received injections once weekly via intraperitoneal injection. Mice bearing intramuscularly implanted LAGλ-1 were treated with CEP-18770 or vehicle alone. Mice treated with the PI inhibited tumor growth as determined by human immunoglobulin (hIg) G levels and measurement of tumor volume (P = 0.0008) compared to mice receiving vehicle. A significant inhibition of both human paraprotein secretion and reduction of tumor growth was also observed in LAGk-1A-bearing mice treated with CEP-18770 at 1, 3 and 10 mg/kg (hIgG: P = 0.0001, P = 0.0002 and P = 0.0001, respectively; tumor volume: P = 0.0001, P = 0.0001 and P = 0.0001, respectively) and LAGk-1B-bearing mice treated with CEP-18770 at 3 and 10 mg/kg (hIgG: P = 0.0008 and P = 0.0034, respectively; tumor volume: P = 0.0008 and P = 0.0028, respectively) compared to mice receiving vehicle. Finally, the combination of CEP-18770 (1 mg/kg) plus melphalan (3 mg/kg) was tested in LAGk-1B-bearing mice. Mice treated with the combination showed markedly smaller tumors compared to treatment with vehicle (P = 0.0008) or melphalan alone (P = 0.0204). Mice treated with the PI alone or in combination with melphalan did not show any observed toxicity. Thus, these studies provide promising preclinical data to suggest the potent anti-MM effects of CEP-18770 both in vitro and in vivo and also suggest that this new PI may enhance the anti-MM effects of several active anti-MM agents including melphalan, doxorubicin and ATO.

scholarly journals Targeted Inhibition of PI3K α/δ Is Synergistic with BCL-2 Blockade in Genetically Defined Subtypes of DLBCL

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 39-39
Author(s):  
Kamil Bojarczuk ◽  
Kirsty Wienand ◽  
Jeremy A. Ryan ◽  
Linfeng Chen ◽  
Mariana Villalobos-Ortiz ◽  
...  
Keyword(s):  
Tumor Growth ◽  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Research Funding ◽  
Single Agent ◽  
Research Report ◽  
Genetic Bases

Abstract Diffuse large B-cell lymphoma (DLBCL) is a genetically heterogeneous disease that is transcriptionally classified into germinal center B-cell (GCB) and activated B-cell (ABC) subtypes. A subset of both GCB- and ABC-DLBCLs are dependent on B-cell receptor (BCR) signaling. Previously, we defined distinct BCR/PI3K-mediated survival pathways and subtype-specific apoptotic mechanisms in BCR-dependent DLBCLs (Cancer Cell 2013 23:826). In BCR-dependent DLBCLs with low baseline NF-κB activity (GCB tumors), targeted inhibition or genetic depletion of BCR/PI3K pathway components induced expression of the pro-apoptotic HRK protein. In BCR-dependent DLBCLs with high NF-κB activity (ABC tumors), BCR/PI3K inhibition decreased expression of the anti-apoptotic NF-κB target gene, BFL1. Our recent analyses revealed genetic bases for perturbed BCR/PI3K signaling and defined poor prognosis DLBCL subsets with discrete BCR/PI3K/TLR pathway alterations (Nat Med 2018 24:679). Cluster 3 DLBCLs (largely GCB tumors) exhibited frequent PTEN deletions/mutations and GNA13 mutations. Cluster 5 DLBCLs (largely ABC tumors) had frequent MYD88L265P and CD79B mutations that often occurred together. These DLBCL subtypes also had different genetic mechanisms for deregulated BCL2 expression - BCL2 translocations in Cluster 3 and focal (18q21.33) or arm level (18q) BCL2 copy number gains in Cluster 5. These observations prompted us to explore the activity of PI3K inhibitors and BCL2 blockade in genetically defined DLBCLs. We utilized a panel of 10 well characterized DLBCL cell line models, a subset of which exhibited hallmark genetic features of Cluster 3 and Cluster 5. We first evaluated the cytotoxic activity of isoform-specific, dual PI3Kα/δ and pan-PI3K inhibitors. In in vitro assays, the PI3Kα/δ inhibitor, copanlisib, exhibited the highest cytotoxicity in all BCR-dependent DLBCLs. We next assessed the transcriptional abundance of BCL2 family genes in the DLBCLs following copanlisib treatment. In BCR-dependent GCB-DLBCLs, there was highly significant induction of the pro-apoptotic HRK. In BCR-dependent ABC-DLBCLs, we observed significant down-regulation of the anti-apoptotic BFL1 protein and another NF-κB target gene, BCLxL (the anti-apoptotic partner of HRK). We then used BH3 profiling, to identify dependencies on certain BCL2 family members and to correlate these data with sensitivity to copanlisib. BCLxL dependency significantly correlated with sensitivity to copanlisib. Importantly, the BCLxL dependency was highest in DLBCL cell lines that exhibited either transcriptional up-regulation of HRK or down-regulation of BCLxL following copanlisib treatment. In all our DLBCL cell lines, PI3Kα/δ inhibition did not alter BCL2 expression. Given the genetic bases for BCL-2 deregulation in a subset of these DLBCLs, we next assessed the activity of the single-agent BCL2 inhibitor, venetoclax, in in vitro cytotoxicity assays. A subset of DLBCL cell lines was partially or completely resistant to venetoclax despite having genetic alterations of BCL2. We postulated that BCR-dependent DLBCLs with structural alterations of BCL2 might exhibit increased sensitivity to combined inhibition of PI3Kα/δ and BCL2 and assessed the cytotoxic activity of copanlisib (0-250 nM) and venetoclax (0-250 nM) in the DLBCL cell line panel. The copanlisib/venetoclax combination was highly synergistic (Chou-Talalay CI<1) in BCR-dependent DLBCL cell lines with genetic bases of BCL2 deregulation. We next assessed copanlisib and venetoclax activity in an in vivo xenograft model using a DLBCL cell line with PTENdel and BCL2 translocation (LY1). In this model, single-agent copanlisib did not delay tumor growth or improve survival. Single-agent venetoclax delayed tumor growth and improved median survival (27 vs 51 days, p<0.0001). Most notably, we found that the combination of copanlisib and venetoclax delayed tumor growth significantly longer than single-agent venetoclax (p<0.0001). Additionally, the combined therapy significantly increased survival in comparison with venetoclax alone (median survival 51 days vs not reached, p<0.0013). Taken together, these results provide in vitro and in vivo pre-clinical evidence for the rational combination of PI3Kα/δ and BCL2 blockade and set the stage for clinical evaluation of copanlisib/venetoclax therapy in patients with genetically defined relapsed/refractory DLBCL. Disclosures Letai: AbbVie: Consultancy, Other: Lab research report; Flash Therapeutics: Equity Ownership; Novartis: Consultancy, Other: Lab research report; Vivid Biosciences: Equity Ownership; AstraZeneca: Consultancy, Other: Lab research report. Shipp:AstraZeneca: Honoraria; Merck: Research Funding; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Research Funding.

scholarly journals CD38 Is a Key Regulator of Tumor Growth By Modulating the Metabolic Signature of Malignant Plasma Cells

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2652-2652
Author(s):  
Ruxandra Maria Irimia ◽  
Margo Brooke Gerke ◽  
Maya Thakar ◽  
Zhihong Ren ◽  
Eric Helmenstine ◽  
...  
Keyword(s):  
Tumor Growth ◽  
Cell Lines ◽  
Tumor Volume ◽  
Plasma Cells ◽  
Growth Impairment ◽  
Nsg Mice ◽  
Cd38 Expression

Abstract Introduction: Multiple myeloma (MM) is a disease of malignant plasma cells, characterized by high CD38 expression. Although the CD38-targeting monoclonal antibodies are highly effective, resistance invariably arises. Tumor CD38 levels decrease after anti-CD38 therapy, but the expression is rarely permanently silenced. This suggests that CD38 expression may offer a tumor cell survival advantage, but the direct impact of CD38 loss on tumor dynamics has not been extensively characterized. Methods: CD38 knockout (KO) cell lines were generated by CRISPR-Cas9. Immunocompetent Balb/c and immunodeficient NSG mice were injected subcutaneously with either non-targeting (NT) or CD38 KO J558 cells. Stromal adhesion was compared using labeled NT and KO cells, with OP-9 murine stroma cells. Cellular NAD content was quantified using the Promega Glo Assay. Mitochondria were isolated with the Mitochondria Isolation Kit (Thermo Scientific). Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were quantified using the Seahorse Assay. Response to hypoxia was evaluated using a modular hypoxic chamber. Cell cycle was quantified using propidium iodine staining. Results: To examine the role of CD38 in murine models, we utilized the CD38-expressing, murine plasmacytoma cell line J558. Strikingly, CD38 KO cells injected into Balb/c mice demonstrated significantly decreased tumor volume compared to NT (113 mm 3 (KO) vs. 1293 mm 3 (NT) at day 25, p &lt;0.001). In contrast, in vitro cell proliferation and colony formation between KO and NT J558 cells were nearly identical, suggesting that the effects of CD38-loss were highly context dependent. Since tumoral CD38 expression may negatively modulate the immune response, we next compared CD38 KO and NT cells injected into immunodeficient NSG mice. CD38 KOs demonstrated an approximately 2.2-fold decreased tumor volume compared to the NT (708 mm 3 (KO) vs. 1592 mm 3 (NT), p=0.07). Further examination of the role of CD38 on the immune microenvironment are ongoing. Considering that some tumor growth impairment was maintained in immunodeficient mice, we next interrogated the effect of CD38 loss on other aspects of cell proliferation using J558 as well as human MM cell lines RPMI-8226 and NCI-H929. Daratumumab induced CD38 internalization has been shown to reduce stromal adhesion of MM cells. Similarly, CD38 KO cells demonstrated reduced stromal adhesion (2.5-fold decrease for J558, p&lt;0.005 and 2-fold decrease for H929, p&lt;0.005). Although stroma is a known promoter of cell survival and proliferation, we further questioned whether the NAD-metabolizing activity of CD38 modulates tumor growth. CD38 overexpression can drive down intracellular NAD and impair mitochondrial biogenesis. Accordingly, we found significantly higher NAD levels in the KO J558 tumor cells compared to NT (2-fold change, p &lt;0.05). Additionally, CD38 KO cells demonstrated significantly higher levels of mitochondrial protein compared with the NTs (5-fold in J558 and 2-fold in H929). CD38 KO cell lines also showed markedly increased metabolic activity, with nearly 2-fold increase in basal OCR and ECAR, as well as in spare respiratory and glycolytic capacity. Given the contrast between in vivo and in vitro growth capacity, we questioned whether changes in mitochondrial content and metabolic function could confer an advantage for CD38-expressing cells under conditions of hypoxia, which is an important characteristic of the tumor microenvironment. Strikingly, under hypoxia, but not normoxia, CD38 KO MM cells demonstrated significantly more cell cycle arrest, defined by G0/G1 blockage (p=0.003 for H929 and p=0.004 for RPMI). Conclusion: We have shown that CD38 KO cells demonstrate decreased tumor growth in vivo but not in vitro. While the immune modulatory potential of CD38 is recognized, some of the growth impairment we observed may be explained by non-immune mediated mechanisms such as reduced stroma adherence as well as changes in cell metabolism. Loss of CD38 was associated with increased mitochondrial respiration, but also elevated ECAR and glycolytic rate. Higher reliance on mitochondrial respiration could explain impaired CD38 KO proliferation rates under hypoxia, possibly as a result of increased generation of reactive oxygen species. Disclosures Ghiaur: Menarini Richerche: Research Funding; Syros Pharmaceuticals: Consultancy.

The Proteasome Inhibitor Bortezomib Induces Tongue, Pharynx and Salivary Gland Cancer Cells Death in Vitro and Delays Tumor Growth of Salivary Gland Cancer Cells Transplanted in Mice

2021 ◽  
Author(s):  
Monica Benvenuto ◽  
Sara Ciuffa ◽  
Chiara Focaccetti ◽  
Diego Sbardella ◽  
Sara Fazi ◽  
...  
Keyword(s):  
Salivary Gland ◽  
Tumor Growth ◽  
Cancer Cells ◽  
Cell Lines ◽  
Proteasome Inhibitor ◽  
Intraperitoneal Administration ◽  
Erbb Receptors ◽  
Salivary Gland Cancer

Abstract Head and neck cancer (HNC) has frequently an aggressive course for the development of resistance to standard chemotherapy. Thus, the use of innovative therapeutic drugs is being assessed. Bortezomib is a proteasome inhibitor with strong in vitro and in vivo anticancer effects. In vitro antitumoral activity of Bortezomib was investigated employing human pharynx (FaDu), tongue (SCC-15, CAL-27), salivary gland (A-253) cancer cell lines and a murine cell line (SALTO-5) originated from a salivary gland adenocarcinoma arising in BALB-neuT male mice transgenic for the oncogene neu. Bortezomib in vivo effects in BALB-neuT mice transplanted with murine SALTO-5 cells were also examined. Bortezomib inhibited cells proliferation, triggered apoptosis, modulated the expression and activation of pro-survival signal transduction pathways proteins activated by ErbB receptors and inhibited proteasome activity in vitro. Furthermore, intraperitoneal administration of Bortezomib delayed tumor growth of SALTO-5 cells transplanted in BALB-neuT mice and protracted mice survival. Our findings further support the use of Bortezomib for the treatment of HNC and reveal its ineffectiveness in counteracting the activation of deregulated specific signaling pathways in HNC cell lines when resistance to proteasome inhibition is developed.

The Novel Fluoropyrimidine FdUMP[10] Is Highly Active Against Acute Myeloid Leukemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3302-3302
Author(s):  
Timothy Pardee ◽  
Evan Gomes ◽  
Jamie Jennings-Gee ◽  
David L. Caudell ◽  
William Gmeiner
Keyword(s):  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Single Agent ◽  
The Novel ◽  
Highly Active ◽  
Ic50 Values ◽  
Acute Myeloid

Abstract Abstract 3302 Acute Myeloid Leukemia (AML) is an aggressive myeloid malignancy that leads to marrow failure and death. This disease affects approximately 12,000 people per year in the United States, causing 9,000 deaths. Despite decades of research, therapy remains essentially unchanged and outcomes are poor. In patients over the age of 60 less then 10% of patients survive 5 years from diagnosis. There is a desperate need for the identification of new active agents with favorable toxicity profiles. The novel polymeric fluoropyrimidine (FP) FdUMP[10] is an oligodeoxynucleotide pro-drug of the thymidylate synthase (TS)-inhibitory FP metabolite 5-fluoro-2'-deoxyuridine-5`-O-monophosphate (FdUMP). The observation that this compound was highly active against several leukemia lines in the NCI 60 cell line screen prompted us to evaluate its activity in several preclinical models of AML. In vitro, FdUMP[10] exhibited remarkable activity against 3 human acute leukemia cell lines, HL60, Jurkat and THP-1, with IC50 values of 3.378 nM (95% CI 2.984 to 3.825), 5.438 nM (4.609 to 6.417) and 4.093 nM (3.413 to 4.907) respectively. We next tested its efficacy against a more genetically defined murine model of AML driven by expression of MLL-ENL. FdUMP[10] exhibited even greater activity against all murine lines tested. The IC50 values of FdUMP[10] against two MLL-ENL driven murine AML cell lines were 214 pM (95%CI 178.9 to 255.9) and 292.3 pM (251.8 to 339.4). The IC50 values observed for FdUMP[10] for all the murine lines tested were lower than both Ara-C (30-40 nM) and doxorubicin (2-4 nM). We then determined the cytotoxic mechanism for FdUMP[10] in vitro. Upon treatment with FdUMP[10] both the human and murine cell lines undergo extensive apoptosis as indicated by Annexin V and propidium iodide staining. Treated cells developed γH2AX foci, rapid and complete TS inhibition and display trapped Topoisomerase I (Topo I) cleavage complexes. FdUMP[10]-mediated induction of apoptosis was p53 independent as murine AML cells that had p53 knocked down by RNAi demonstrated resistance to both Ara-C and doxorubicin, but not to FdUMP[10]. We next tested the efficacy of FdUMP[10] in vivo. The MLL-ENL driven murine AML model results in blasts that can be transplanted into sublethally irradiated, immunocompetent, syngeneic recipients. The recipients develop a fatal and therapy-resistant AML. Lines were generated that expressed a luciferase reporter. Animals were imaged 6–7 days after injection of the leukemias to ensure engraftment and then began treatment with either the combination of Ara-C plus doxorubicin, single-agent FdUMP[10], or observation. Studies were performed using 2 doses of FdUMP[10] at 150 or 300 mg/kg injected on days 1 and 3 and compared to animals treated with 100 mg/kg Ara-C and 3mg/kg doxorubicin injected on days 1 through 5. Both treatments resulted in a statistically significant survival advantage over observation. A preliminary toxicology study compared FdUMP[10], 150 mg/kg daily, to 5-fluorouracil (5 FU), 150 mg/kg daily, or the combination of Ara-C at 100 mg/kg plus doxorubicin at 3 mg/kg daily. All groups were treated for 3, 4 or 5 days. On day 6 animals were sacrificed and organs harvested, sectioned, and stained. Slides were then reviewed by a veterinary pathologist. Tissues most affected were the small intestine, colon, and the bone marrow. The 5FU-treated animals had severe villous blunting and fusion with crypt necrosis in both large and small intestine. In contrast, FdUMP[10]-treated animals had only mild crypt epithelial apoptosis with mitoses. The 5 FU and Ara-C plus doxorubicin groups had a severe pan-cytopenia in the marrow compared to FdUMP[10] treated animals that showed only minimal to mild apoptosis. These data support the assertion that FdUMP[10] has lower toxicity then either Ara-C plus doxorubicin or identically dosed 5 FU. In summary FdUMP[10] exhibited remarkable activity against AML cells in vitro and in vivo. Additionally, FdUMP[10] had decreased toxicity compared to treatment with either single agent 5 FU or combination treatment with Ara-C plus doxorubicin. Disclosures: Gmeiner: Salzburg Therapeutics: Equity Ownership.

The Combination of PLX3397, a Selective FLT3-ITD Inhibitor, and Decitabine, a Hypomethylating Agent, Demonstrates Benefit in AML Cell Lines in Vitro and in Vivo

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3743-3743
Author(s):  
James Tsai ◽  
Elizabeth A Burton ◽  
Gaston Habets ◽  
Brian West ◽  
Paul Lin ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Tumor Growth ◽  
Cell Lines ◽  
Tumor Regression ◽  
Single Agent ◽  
Xenograft Model ◽  
Bone Marrow Toxicity ◽  
Preclinical Studies

Abstract Introduction: While clinical studies using targeted therapies as single agents in AML have shown promising results in recent years, long-term durable responses in this aggressive cancer may require combination therapies to overcome disease progression and single agent resistance mechanisms. PLX3397 is an orally active, selective small molecule inhibitor of the constitutively activated FLT3-ITD mutant kinase. In cellular assays PLX3397 effectively inhibited FLT3-ITD autophosphorylation and FLT3-ITD driven proliferation with IC50s in the 10-100nM range. A clinical study to evaluate the pharmacokinetics (PK), safety and efficacy of PLX3397 in patients with FLT3-ITD AML is currently ongoing. In order to determine if combination therapy could improve efficacy, we evaluated the combination of PLX3397 with the hypomethylating agent decitabine (DEC; 5-aza-2’-deoxycytidine) in preclinical models of FLT-ITD AML. Decitabine, a drug originally indicated for myelodysplastic syndrome, is approved in Europe for the treatment of adult patients (≥65 years of age) with newly diagnosed or secondary AML. Methods: For the in vitro growth assays, cells were pre-treated with decitabine for 0-3 days prior to the addition of PLX3397. Following a 3-day incubation, cell viability was measured based on quantification of the ATP present. The resulting data were analyzed for synergy and combination indices were calculated using CalcuSyn software. Apoptosis was analyzed by measuring caspase 3/7 activity following a 24h incubation with both compounds. For the in vivo study, MV-4-11 cells were grown as subcutaneously implanted xenografts in SCID mice. When tumors reached a size of ~500 mm3 the mice were randomized into equal-sized treatment groups by body weight and tumor size (the day on which this was done was counted as day 0). Decitabine was dosed at 20mg/kg on days 1, 7, 13 and 20 after randomization. PLX3397 was dosed at 20mg/kg on day 2, and continued for 20 days. The combination followed the same dosing schemes as the two single agents. Results: In vitro viability experiments in two AML cell lines (MV-4-11 and MOLM14) using a dose matrix format demonstrated a combination benefit of PLX3397 and decitabine over a range of concentrations. Pre-incubation with decitabine for 3 days prior to the addition of PLX3397 enhanced the synergy observed. PLX3397 alone was more effective than decitabine at inducing apoptosis. Adding both compounds together slightly enhanced the induction of apoptosis, though there did not appear to be an added benefit to pre-treating the cells with decitabine, as was seen in the viability assays. To confirm the synergy observed in vitro we tested the in vivo efficacy of the two agents in the MV-4-11 xenograft model. By day 19, both decitabine and PLX3397 delayed tumor growth, resulting in tumor growth inhibition (TGI) of 89% and 42%, respectively. The combination of decitabine and PLX3397 showed striking antitumor activity, causing tumor regression and reducing tumor volume by 88%. This tumor suppression was maintained for 15 days after the treatment was stopped. Consistent with clinical experience, decitabine treatment was associated with bone marrow toxicity. This toxicity was not worsened by PLX3397. After 2 weeks of recovery bone marrow cellularity rebounded to pre-dosing levels in the combination, with the exception of red blood cell count. Conclusion: Preclinical studies of PLX3397 and decitabine in FLT3-ITD AML cell lines and a xenograft model demonstrated beneficial effects when used in combination. Single agent treatment inhibited MV-4-11 xenograft tumor growth, while the combination resulted in tumor regression. PLX3397 did not further enhance the bone marrow toxicity induced by decitabine. PLX3397 exposures in these preclinical studies are similar to those achieved in AML patients in the on-going single agent clinical trial. Figure 1. Preclinical combination of PLX3397 and decitabine in an MV-4-11 xenograft model. Figure 1. Preclinical combination of PLX3397 and decitabine in an MV-4-11 xenograft model. Disclosures Zhang: Plexxikon: Employment.

Effect of the combination of the dual mTOR/pI3K inhibitor NVP-BEZ235 with gemcitabine on growth inhibition in pancreatic cancer cells in vitro and in vivo.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. e15070-e15070
Author(s):  
Luise Maute ◽  
Johannes Wicht ◽  
Martin Zoernig ◽  
Manuel Niederhagen ◽  
Lothar Bergmann
Keyword(s):  
Pancreatic Cancer ◽  
Tumor Growth ◽  
Cell Lines ◽  
Mtor Inhibitor ◽  
Single Agent ◽  
Human Pancreatic Cancer ◽  
Pancreatic Cancer Cells ◽  
Sequential Administration

e15070 Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive malignant tumours and is still associated with a very poor prognosis. Therefore new treatment strategies are needed. The PI3K/AKT and mTOR signaling pathways are frequently dysregulated in PDAC. Thus we investigated the effects of NVP-BEZ235, a novel dual PI3K/mTOR inhibitor, alone or in combination with gemcitabine first in vitro and after promising results also in vivo. Methods: We examined the effect of gemcitabine and NVP-BEZ235 (kindly provided by Novartis Pharma) on cell viability as single agents and in combination with sequential administrations in the four human pancreatic cancer cell lines MiaPaCa-2, Panc-1, AsPC-1 and BxPC-3. For in vivo experiments we used NOD SCID Mice, which were injected with BxPc3 into the right flank. Treatments consisted of Gemcitabine alone, NVP-BEZ235 alone, simultaneous application of both, first application of Gemcitabine followed by NVP-BEZ235 and NVP-BEZ235 followed by Gemcitabine. Results: Simultaneous incubation of gemcitabine and NVP-BEZ235 affected the PDAC cell lines significantly better than the single agent administration. But most effective was a sequential administration of gemcitabine followed by NVP-BEZ235. In vivo Gemcitabine and NVP-BEZ235 as single agents showed a slightly reduced tumor growth and the treatment in the sequence NVP-BEZ235 first, followed by Gemcitabine resulted in only a minimal reduction of tumor growth. The most effective results were obtained by simultaneous and even better in the sequence of Gemcitabine followed by NVP-BEZ235, respectively. Conclusions: The combination of gemcitabine with the dual PI3k/mTOR inhibitor NVP-BEZ235 enhanced the efficacy of PDAC treatment via down-regulation of the DDR related gene Survivin in vitro. This combination seems to be significantly more effective than single agent use in vitro and also in vivo. Furthermore we demonstrated that the sequence of administration of these agents could be a relevant issue. These promising results might offer a new and effective option for the treatment of pancreatic cancer in the future.

The mTOR Inhibitor RAD001 (Everolimus) Inhibits Myeloma Tumor Growth In Vivo and Enhances the Anti-MM Effects of Bortezomib and Arsenic Trioxide.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4801-4801 ◽  
Author(s):  
Richard A. Campbell ◽  
Eric Sanchez ◽  
Jeffrey Steinberg ◽  
Michael Share ◽  
Joseph Wang ◽  
...  
Keyword(s):  
Tumor Growth ◽  
Cell Survival ◽  
Arsenic Trioxide ◽  
Tumor Volume ◽  
Single Agent ◽  
Mouse Serum ◽  
Oral Gavage ◽  
Once Daily ◽  
Tumor Bearing

Abstract The mammalian target of rapamycin (mTOR) is an intracellular protein that acts as a central regulator of multiple signaling pathways (IGF, EGF, PDGF, VEGF, amino acids) that mediate abnormal growth, proliferation, survival and angiogenesis in cancer. mTOR is a critical component of the PI3K/Akt pathway, a key cell survival pathway that is dysregulated in many cancers including multiple myeloma (MM). mTOR is an important therapeutic target because it is a “rate-limiting” bottleneck in the key signaling pathway that regulates cell survival, proliferation, and angiogenesis. RAD001 (everolimus) is a novel oral mTOR pathway inhibitor. Recent data suggests that RAD001 has direct effects on tumor cell proliferation and may have antiangiogenic activity due to inhibition of tumoral VEGF production and effects on vascular endothelial and smooth muscle cell biology. We first evaluated the in vivo effects of single agent RAD001 in mice bearing the human MM tumor LAGλ-1. Tumor-bearing mice receiving RAD001 at 3, 10, or 30 mg/kg once daily five times per week (M-F) via oral gavage showed marked inhibition of tumor growth at all doses (P<0.0001) and reduction of paraprotein levels (P<0.0001) compared to mice receiving placebo. Next, we evaluated the in vivo anti-MM effects of RAD001 in combination with arsenic trioxide (ATO) and RAD001 in combination with bortezomib. Each severe combined immunodeficient (SCID) mouse was implanted with a fragment (2 – 4 mm3) of the LAGλ-1 tumor into the left hind limb muscle. The tumors were allowed to grow for 10 days at which time human IgG levels were detectable in the mouse serum, and mice were blindly assigned into treatment groups. Tumor-bearing mice received RAD001 at 1, 3, or 10 mg/kg once daily five times per week via oral gavage, ATO (3 mg/kg) once daily five times per week via intraperitoneal injection, bortezomib (0.5 mg/kg) once daily twice weekly via intravenous injection, or a placebo. Mice receiving RAD001/ATO combination therapy exhibited decreased hIgG levels and tumor volume compared to those mice receiving single agent and placebo therapy. Additionally, those mice receiving the RAD001/bortezomib combination treatment displayed similar MM tumor growth inhibition including decreased hIgG levels and tumor volume as the RAD001/ATO-treated mice. These preliminary in vivo results are encouraging with combination RAD001+ATO and RAD001+bortezomib therapy for the treatment of MM and additional studies are being performed to further optimize the clinical development of RAD001 in combination with other anti-MM treatments for patients with MM.

Cell Non-Autonomous Modulation Of Tumor Cell Responses To Pharmacological Inhibition Of The Proteasome

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4454-4454
Author(s):  
Eugen Dhimolea ◽  
Richard W.J. Groen ◽  
Catriona A. Hayes ◽  
Jana Jakubikova ◽  
Bariteau Megan ◽  
...  
Keyword(s):  
Tumor Growth ◽  
Tumor Cells ◽  
Cell Lines ◽  
Induced Resistance ◽  
Proteasome Inhibitor ◽  
Bioluminescence Imaging ◽  
Proteasome Inhibition ◽  
Bortezomib Treatment

Extensive preclinical studies of several groups using tumor cells co-cultured with bone marrow stromal cells (BMSCs) has documented that the potent anti-MM activity of the proteasome inhibitor bortezomib is not suppressed by BMSCs (e.g. primary and immortalized BMSCs). Using our compartment-specific bioluminescence imaging (CS-BLI) assays, we extended these observations to larger panels of MM cell lines. We observed, however, a recurrent pattern that primary CD138+ MM tumor cells from bortezomib-refractory patients recurrently exhibited substantial in vitro response to clinically-achievable concentrations and durations of bortezomib treatment. To simulate this clinicopathological observation, MM.1R-Luc+ cells were injected i.v. in SCID-beige mice and treated with bortezomib (0.5 mg/kg s.c. twice weekly for 5 weeks): diffuse MM tumors initially responded to bortezomib, but eventually became refractory. These in vivo-resistant MM cells were isolated from the mice and were treated in vitro with bortezomib, exhibiteing similar responsinveness to this agent as their isogenic bortezomib-naive MM cells, To further address the possibility that this represents a previously underexplored form of a microenvironment-induced alteration in bortezomib responsiveness, we studied how MM cells respond to pharmacological proteasome inhibition after variable times of co-culture with BMSCs prior to bortezomib exposure. We observed that prolonged tumor-stromal co-culture (48-96hrs) prior to initiation of bortezomib treatment did not affect drug sensitivity for many of the MM cell lines (OPM2, H929, UM9, KMS11, KMS18 and RPMI-8226) tested. Notably, prolonged co-cultures with BMSCs prior to bortezomib treatment enhanced the activity of this agent for other MM cell lines (e.g. OPM1, Dox40, OCI-My5, KMS12BM or KMS18). However, MM.1S and MM.1R cells, when exposed to extended co-cultures with BMSCs prior to initiation of drug exposure, exhibited significant attenuation (2-3 fold increase of IC50 values) of their response to bortezomib in several independent replicate experiments. In support of these in vitro results, heterotypic s.c. xenografts of Luc+ MM.1S cells co-implanted with Luc-negative BMSCs did not show significant reduction in MM tumor growth with bortezomib treatment (0.5 mg/kg s.c. twice weekly for 5 weeks) compared to vehicle-treated controls (p=0.13), as quantified by bioluminescence imaging. In co-cultures with BMSCs, MM.1S and MM.1R cells also exhibited suppression of their response to carfilzomib (the degree of this stroma-induced resistance was more pronounced that in the case of bortezomib for these 2 cell lines). Consistent with these observations, in vivo administration of carfilzomib in the orthotopic model of diffuse bone lesions of MM.1R-Luc+ cells was associated with less pronounced reduction in tumor growth, compared to bortezomib treatment (p<0.03). These results suggest that the stroma-induced attenuation of activity against a subset of MM cells represents a class-effect for this group of therapeutics, despite quantitative differences between different proteasome inhibitors. Mechanistically, we determined a distinct transcriptional signature of stroma-induced transcripts which are overexpressed in refractory myeloma patients with significantly shorter overall survival (p<0.03, log-rank tests) after bortezomib treatment. Our results in vitro and in vivo support the notion that the responses of MM cells to proteasome inhibition can exhibit substantial plasticity depending on the specific microenvironmental context with which these MM cells interact. We also identify prognostically-relevant candidate molecular mediators of stroma-induced resistance to proteasome-inhibitor based therapy in MM. Disclosures: No relevant conflicts of interest to declare.

Preclinical activity of the heat shock protein 90 inhibitor ganetespib in clear cell renal cell carcinoma.

2014 ◽  
Vol 32 (4_suppl) ◽  
pp. 478-478
Author(s):  
Mansi Parasramka ◽  
David A. Proia ◽  
Richard Wayne Joseph
Keyword(s):  
Tumor Growth ◽  
Cell Lines ◽  
Single Agent ◽  
Mtor Inhibitors ◽  
Heat Shock Protein 90 ◽  
Cell Renal Cell Carcinoma ◽  
Mtor Inhibition ◽  
Ccrcc Cell

478 Background: Resistance invariably develops in all patients with metastatic ccRCC treated with mTOR inhibitors. Previously we demonstrated that dual inhibition of Hsp90 and the mTOR pathway in lung cancer models leads to synergistic reductions in tumor growth. Herein, we tested the efficacy of ganetespib as a single agent and in combination with mTOR inhibition using in vitro and in vivoccRCC models. Methods: For the in vitro work we utilized the following seven ccRCC cell lines: Caki-1, Caki-2, A-498, A-704, 769-P, 786-O, ACHN. For the in vivo work we used A498 xenografts. In vitro, we determined the single agent EC50 of everolimus and ganetespib at 72 hours by assessing percent viability of A498 cells compared to vehicle using the MTS assay. We then performed combinations of ganetespib and everolimus at EC20, EC30, and EC50 in A498 cells. Translating these studies in vivo, we compared the combinatorial activity of ganetespib and temsirolimus to monotherapy in mice bearing A498 tumor xenografts. Results: As a single agent, all ccRCC cell lines tested were sensitive to ganetespib at nanomolar concentration (EC50 15 – 75 nm) and to everolimus at micromolar concentrations (EC50 4 – 54 mm). In vitro, the combination of ganetespib and everolimus also decreased cell viability in an additive fashion. In vivo, ganetespib and temsirolimus demonstrated comparable single agent activity at sub-MTD doses (T/C = 63 and 60, respectively). Combining ganetespib with temsirolimus improved tumor growth suppression by ~30% (T/C = 43). Conclusions: Given the broad in vitro sensitivity of ccRCC cell lines to single agent ganetespib as well as the in vivo activity of the combination of ganetespib and temsirolimus, we believe ganetespib warrants further study in ccRCC. Updated results will be presented at the conference including the in vivo activity of the combination of ganetespib and antivascular endothelial growth factor agents.

ANTITUMOR EFFECT OF COLLOIDAL SILVER BISILICATE IN EXPERIMENTAL IN VITRO AND IN VIVO STUDIES

2019 ◽  
Vol 65 (5) ◽  
pp. 760-765
Author(s):  
Margarita Tyndyk ◽  
Irina Popovich ◽  
A. Malek ◽  
R. Samsonov ◽  
N. Germanov ◽  
...  
Keyword(s):  
Antitumor Activity ◽  
Tumor Growth ◽  
Tumor Cells ◽  
Human Tumor ◽  
Significant Inhibition ◽  
In Vivo Studies ◽  
Ehrlich Carcinoma ◽  
In Vivo Experiments

The paper presents the results of the research on the antitumor activity of a new drug - atomic clusters of silver (ACS), the colloidal solution of nanostructured silver bisilicate Ag6Si2O7 with particles size of 1-2 nm in deionized water. In vitro studies to evaluate the effect of various ACS concentrations in human tumor cells cultures (breast cancer, colon carcinoma and prostate cancer) were conducted. The highest antitumor activity of ACS was observed in dilutions from 2.7 mg/l to 5.1 mg/l, resulting in the death of tumor cells in all studied cell cultures. In vivo experiments on transplanted Ehrlich carcinoma model in mice consuming 0.75 mg/kg ACS with drinking water revealed significant inhibition of tumor growth since the 14th day of experiment (maximally by 52% on the 28th day, p < 0.05) in comparison with control. Subcutaneous injections of 2.5 mg/kg ACS inhibited Ehrlich's tumor growth on the 7th and 10th days of the experiment (p < 0.05) as compared to control.

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