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.

Efficacy of Panobinostat (LBH589) in CTCL Cell Lines and a Murine Xenograft Model: Defining Molecular Pathways of Panobinostat Activity in CTCL.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1375-1375 ◽  
Author(s):  
Wenlin Shao ◽  
Joseph D. Growney ◽  
Yun Feng ◽  
Gregory O’Connor ◽  
Minying Pu ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Lines ◽  
Molecular Mechanisms ◽  
Tumor Regression ◽  
Single Agent ◽  
Xenograft Model ◽  
Stat Proteins ◽  
Murine Xenograft Model

Abstract Panobinostat (LBH589) is a highly potent oral pan-deacetylase (DAC) inhibitor currently undergoing clinical development in hematologic and solid malignancies. Panobinostat demonstrated preliminary clinical efficacy in cutaneous T-cell lymphoma (CTCL) patients in a phase I trial, with 6 responders out of 10 patients. Here we report the characterization of the effects of panobinostat on CTCL cells in vitro and in a murine xenograft model of CTCL. Panobinostat was found to potently induce growth inhibition of all CTCL cell lines tested (HuT78, HuT102, MJ, and HH) and exhibited significant cytotoxic activity against two CTCL cell lines (HuT78 and HH). Panobinostat was found to induce activation of caspases 3 and 7 in HuT78 and HH cell lines, consistent with its effects on cell viability in these cells. To investigate the effect of panobinostat in vivo, an HH CTCL xenograft mouse model was treated with vehicle or different doses of panobinostat by iv administration qd×5 for 2 weeks. Treatment with panobinostat at 10 mg/kg resulted in complete tumor regression relative to vehicle-treated animals. To gain a better understanding of panobinostat activity in CTCL, molecular mechanisms underlying cell sensitivity or lack thereof were investigated. Inhibition of DAC activity as measured by hyperacetylation of histones H3, H4, and tubulin was observed equally in all four cell lines. Interestingly, CTCL cells insensitive to panobinostat cytotoxicity (HuT102 and MJ) were found to express significantly higher levels of IL-2 receptor and to secrete high levels of select cytokines, including IFN-α, IFN-γ, and TNF-α, as compared with CTCL cells sensitive to panobinostat-induced cytotoxicity. Contrary to panobinostat-sensitive CTCL cells, cells insensitive to panobinostat-induced cell death were found to contain constitutively active NF-κB signaling and elevated activation of STAT proteins. Panobinostat-insensitive HuT102 and MJ cell lines were also found to express high levels of the pro-survival protein Bcl-2, an anti-apoptotic target whose transcription can be activated by NF-κB signaling. Although inhibition of STAT5 activation using a JAK inhibitor did not confer panobinostat sensitivity in the HuT102 and MJ CTCL cell lines, combination of a Bcl-2 inhibitor with panobinostat revealed a synergistic effect on cytotoxicity in these CTCL cells. Such results suggest that blocking anti-apoptotic signaling in combination with panobinostat treatment is effective in conferring panobinostat sensitivity to CTCL cells refractory to panobinostat-induced cell death. These data demonstrate that panobinostat exhibits significant anti-cancer effects on CTCL cells both in vitro and in vivo at clinically attainable concentrations. In addition, we have identified a cellular mechanism of insensitivity to panobinostat and furthermore provided a potential approach for sensitizing cells to panobinostat treatment in combination with a Bcl-2 inhibitor. Panobinostat, as a single agent or in combination, is a promising therapy for CTCL and these studies support continued clinical evaluation of panobinostat in the treatment of CTCL.

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.

Efficacy of adavosertib therapy against anaplastic thyroid cancer

2021 ◽  
Author(s):  
Yu-Ling Lu ◽  
Yu-Tung Huang ◽  
Ming-Hsien Wu ◽  
Ting-Chao Chou ◽  
Richard J Wong ◽  
...  
Keyword(s):  
Thyroid Cancer ◽  
Tumor Growth ◽  
Single Agent ◽  
Xenograft Model ◽  
Anaplastic Thyroid Cancer ◽  
Therapeutic Effects ◽  
Dependent Manner ◽  
Cancer Tumor

Wee1 is a kinase that regulates the G2/M progression by inhibition of CDK1, which is critical for ensuring DNA damage repair before initiation of mitotic entry. Targeting Wee1 may be a potential strategy in the treatment of anaplastic thyroid cancer, a rare but lethal disease. The therapeutic effects of adavosertib, a Wee1 inhibitor for anaplastic thyroid cancer was evaluated in this study. Adavosertib inhibited cell growth in three anaplastic thyroid cancer cell lines in a dose-dependent manner. Cell cycle analysis revealed cells were accumulated in the G2/M phase. Adavosertib induced caspase-3 activity and led to apoptosis. Adavosertib monotherapy showed significant retardation of the growth of two anaplastic thyroid cancer tumor models. The combination of adavosertib with dabrafenib and trametinib revealed strong synergism in vitro and demonstrated robust suppression of tumor growth in vivo in anaplastic thyroid cancer xenograft models with BRAFV600E mutation. The combination of adavosertib with either sorafenib or lenvatinib also demonstrated synergism in vitro and had strong inhibition of tumor growth in vivo in an anaplastic thyroid cancer xenograft model. No appreciable toxicity appeared in mice treated with either single agent or combination treatment. Our findings suggest adavosertib holds the promise for the treatment of patients with anaplastic thyroid cancer.

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.

Expression of Bcl-2 Pro-Survival Family Proteins Predicts Pharmacological Responses to ABT-199, a Novel and Selective Bcl-2 Antagonist, in Multiple Myeloma Models

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 5028-5028 ◽  
Author(s):  
Deepak Sampath ◽  
Elizabeth Punnoose ◽  
Erwin R. Boghaert ◽  
Lisa Belmont ◽  
Jun Chen ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Death ◽  
Cell Lines ◽  
Single Agent ◽  
Employment Equity ◽  
Bone Marrow Biopsies ◽  
Xenograft Models ◽  
Equity Ownership

Abstract Abstract 5028 Multiple myeloma (MM) is a hematological malignancy of the bone marrow caused by the dysregulated proliferation of monoclonal antibody producing plasma cells. A hallmark feature of cancer is the ability to evade cell death signals induced by stress response cues. The Bcl-2 family of proteins regulates the intrinsic apoptosis pathways and consists of pro-apoptotic (Bax, Bak, Bad, Bim, Noxa, Puma) and pro-survival (Bcl-2, Bcl-xL, Mcl-1); the balance of which dictates the life or death status of MM tumor cells. Thus, there is a strong rationale to target members of the Bcl-2 proteins for the treatment of MM. ABT-199 is a potent BH3-only mimetic that selectively antagonizes Bcl-2 and is currently in phase I clinical trials for the treatment of hematological malignancies. Therefore, we evaluated the efficacy of ABT-199 as a single agent and in combination with standard of care drugs such as Velcade (bortezomib) in preclinical models of MM. A panel of 21 human MM cell lines was evaluated in vitro for to sensitivity to ABT-199. ABT-199 potently inhibited cell viability in a sub-set of MM cell lines (7/21) with EC50 values less than 1 μM. Expression of Bcl-2, Bcl-xL, Mcl-1, Bim and other Bcl-2 family proteins were evaluated by protein and mRNA. Cell line modeling identified thresholds for expression of Bcl-2, Bcl-xL and Mcl-1 that best predicted sensitivity and resistance to ABT-199 and the dual Bcl-2/Bcl-xL antagonist, navitoclax. Consistent with the target inhibition profile of these drugs, we found that MM lines that were Bcl-2high/Bcl-xLlow/Mcl-1low are the most sensitive to ABT-199 treatment. Whereas cell lines that are Bcl-xLhigh remain sensitive to navitoclax but not ABT-199. MM cell lines that are Mcl-1high are less sensitive to both ABT-199 and navitoclax, suggesting that Mcl-1 is a resistance factor to both drugs. Utilizing a novel Mesoscale Discovery based immunoassay we determined that levels of Bcl-2/Bim complexes also correlated with sensitivity of ABT-199 in the MM cell lines tested. In addition, the t(11;14) status in these cell lines associated with sensitivity to ABT-199. The clinical relevance of the Bcl-2 pro-survival expression pattern in MM cell lines, was determined by a collection of bone marrow biopsies and aspirates (n=27) from MM patients by immunohistochemistry for prevalence of Bcl-2 and Bcl-xL. Similar to our in vitro observations, the majority (75%) of the MM bone marrow biopsies and aspirates had high Bcl-2 levels whereas 50% had high Bcl-xL expression. Therefore, a subset of patient samples (33%) were identified with a favorable biomarker profile (Bcl-2high/Bcl-xLlow) that may predict ABT-199 single agent activity. ABT-199 synergized with bortezomib in decreasing cell viability in the majority of MM cell lines tested in vitro based on the Bliss model of independence analyses (Bliss score range = 10 to 40). However the window of combination activity was reduced due to high degree of sensitivity to bortezomib alone. Therefore, the combination efficacy of ABT-199 and bortezomib was further evaluated in vivo in MM xenograft models that expressed high levels of Bcl-2 protein (OPM-2, KMS-11, RPMI-8226, H929 and MM. 1s). Bortezomib treatment alone at a maximum tolerated dose resulted in tumor regressions or stasis in all xenograft models tested. ABT-199 at a maximum tolerated dose was moderately efficacious (defined by tumor growth delay) as a single agent in xenograft models that expressed high protein levels of Bcl-2 but relatively lower levels of Bcl-xL. However, the combination of ABT-199 with bortezomib significantly increased the overall response rate and durability of anti-tumor activity when compared to bortezomib, resulting in increased cell death in vivo. Treatment with bortezomib increased levels of the pro-apoptotic BH3-only protein, Noxa, in MM xenograft models that expressed high levels of Mcl-1. Given that the induction of Noxa by bortezomib results in neutralization of Mcl-1 pro-survival activity in MM models [Gomez-Bougie et al; Cancer Res. 67:5418–24 (2007)], greater efficacy may be achieved when Bcl-2 is antagonized by ABT-199 thereby inhibiting pro-survival activity occurring through either Bcl-2 or Mcl-1 and increasing cell death. Thus, our preclinical data support the clinical evaluation of ABT-199 in combination with bortezomib in MM patients in which relative expression of the Bcl-2 pro-survival proteins may serve as predictive biomarkers of drug activity. Disclosures: Sampath: Genentech: Employment, Equity Ownership. Punnoose:Genentech: Employment, Equity Ownership. Boghaert:Abbott Pharmaceuticals: Employment, Equity Ownership. Belmont:Genentech: Employment, Equity Ownership. Chen:Abbott Pharmaceuticals: Employment, Equity Ownership. Peale:Genentech: Employment, Equity Ownership. Tan:Genentech: Employment, Equity Ownership. Darbonne:Genentech: Employment, Equity Ownership. Yue:Genentech: Employment, Equity Ownership. Oeh:Genentech: Employment, Equity Ownership. Lee:Genentech: Employment, Equity Ownership. Fairbrother:Genentech: Employment, Equity Ownership. Souers:Abbott Pharmaceuticals: Employment, Equity Ownership. Elmore:Abbott Pharmaceuticals: Employment, Equity Ownership. Leverson:Abbott Pharmaceuticals: Employment, Equity Ownership.

scholarly journals Efficacy of the Plk Inhibitor Volasertib in Preclinical Models of AML

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2307-2307
Author(s):  
Dorothea Rudolph ◽  
Christoph Albrecht ◽  
Lena Geiselmann ◽  
Maria Antonietta Impagnatiello ◽  
Pilar Garin-Chesa ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Single Agent ◽  
Preclinical Models ◽  
Subcutaneous Xenograft ◽  
Xenograft Models ◽  
Proliferation Assays ◽  
Anti Tumor Activity

Abstract Background: Polo-like kinase 1 (Plk1), a key regulator of cell cycle progression and accurate spindle assembly, is an attractive target for cancer drug discovery. We have previously shown that volasertib (BI 6727), a potent and selective small-molecule inhibitor of Plk, induces a distinct mitotic arrest phenotype in prometaphase (“polo-arrest”) with subsequent apoptosis in a variety of different cancer cell lines, irrespective of their mutational status. When used in vivo, volasertib administered intravenously shows potent anti-tumor activity in xenograft models of human epithelial cancers at well-tolerated doses. The present study was designed to extend the analysis of volasertib to additional preclinical models of human AML, including bone marrow samples from AML patients. Volasertib is the most advanced Plk inhibitor in clinical development and has demonstrated encouraging results in phase II clinical trials. It is currently being investigated in a phase III clinical trial in patients with previously untreated AML, who are ineligible for intensive remission induction therapy. Methods: A panel of human AML cell lines was used to evaluate pharmacodynamic biomarker modulation and anti-tumor effects of volasertib in vitro using FACS analysis, Western blot analysis and proliferation assays. This in vitro analysis of established AML cell lines was extended to proliferation assays using bone marrow samples from AML patients. In vivo anti-tumor activity of volasertib was tested in subcutaneous xenograft models as well as in multiple disseminated xenograft models of AML. Single-agent efficacy of volasertib and combination therapies were evaluated with existing and emerging AML drugs, including an approved cytotoxic drug (cytarabine), hypomethylating agents (decitabine, azacitidine) and a signal transduction inhibitor targeting FLT3 (quizartinib). Results: Volasertib potently inhibited proliferation of established AML cell lines in vitro with EC50 values of 16-169 nM. Proliferation assays with 15 ex vivo bone marrow samples from AML patients showed EC50 values of 8-8800 nM with a median EC50 of 37 nM. Volasertib showed potent anti-tumor activity at well tolerated doses in 3 subcutaneous xenograft models of AML (MV4-11, Molm-13 and a patient-derived AML model AML-6252). While single-agent volasertib at medium dose level (20 mg/kg q7d i.v. for 2 cycles) and single-agent cytarabine (100 mg/kg q3-4d i.p. for 2 cycles) showed moderate efficacy in the AML-6252 AML model, the combination showed improved efficacy. Moreover, efficacy of single-agent volasertib at high dose level (40 mg/kg q7d i.v. for 2 cycles) could be further improved by adding cytarabine to the treatment regime (Figure 1). A combination of volasertib with decitabine or azacitidine was tested in the MV4-11 subcutaneous AML xenograft model. Either combination therapy showed improved efficacy compared to the respective single-agent treatment groups. Volasertib showed also improved anti-tumor activity when tested in combination with the Flt-3 inhibitor quizartinib (5 or 10 mg/kg qd po for 2 cycles) in the MV4-11 AML model. While tumors in the quizartinib single agent treatment groups started to regrow around day 60 post treatment start, a combination with volasertib could control tumor growth long term until the study was terminated (day 87 post treatment start). Efficacy of volasertib was also tested in 3 disseminated xenograft models of AML (MV4-11, Molm-13 and THP-1). Efficacy read out in these disseminated models was based on tumor load measurements as detected by bioluminescence imaging and increased lifespan. Volasertib prolonged survival compared to vehicle treated animals in all three disseminated models of AML. Conclusions: These results indicate that volasertib is highly efficacious as a single agent in preclinical models of AML and shows potential for improved efficacy and good tolerability in combination with existing and emerging AML drugs. Figure 1: Efficacy of volasertib in combination with cytarabine in a patient-derived AML model (AML-6252) Figure 1:. Efficacy of volasertib in combination with cytarabine in a patient-derived AML model (AML-6252) Disclosures Rudolph: Boehringer Ingelheim RCV: Employment. Off Label Use: Volasertib is an investigational agent. Albrecht:Boehringer Ingelheim RCV GmbH & Co KG: Employment. Geiselmann:Boehringer Ingelheim RCV GmbH & Co KG: Employment. Impagnatiello:Boehringer Ingelheim RCV GmbH & Co KG: Employment. Garin-Chesa:Boehringer Ingelheim RCV: Employment. Wernitznig:Boehringer-Ingelheim: Employment. Moll:Boehringer-Ingelheim: Employment. Kraut:Boehringer Ingelheim RCV: Employment.

scholarly journals The Combination of Entospletinib and Vincristine Demonstrates Synergistic Activity in a Broad Panel of Hematological Cancer Cell Lines and Anti-Tumor Efficacy in a DLBCL Xenograft Model

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 5123-5123 ◽  
Author(s):  
Mark Joseph Axelrod ◽  
Peter Fowles ◽  
Jeff Silverman ◽  
Astrid Clarke ◽  
Jennifer Tang ◽  
...  
Keyword(s):  
Tumor Growth ◽  
Growth Inhibition ◽  
B Cell ◽  
Cell Lines ◽  
Xenograft Model ◽  
Lymphocytic Leukemia ◽  
Employment Equity ◽  
Equity Ownership

Abstract Background Entospletinib (GS-9973) selectively inhibits spleen tyrosine kinase (SYK), a critical signaling component of the BCR pathway that is expressed primarily in cells of hematopoietic lineage including normal and malignant B-lymphocytes. Entospletinib is currently in phase II clinical trials, where it has demonstrated both a high degree of safety as well as efficacy against chronic lymphocytic leukemia (Sharman, J., et al. Blood, 2015) and other B cell malignancies. Despite these successes, new therapeutic options, including combinations with standard of care agents, are needed in order to achieve the goal of curing disease through finite treatment. We show here that the combination of entospletinib and vincristine causes synergistic apoptosis in vitro in a broad panel of cell lines derived from hematological cancers including diffuse large B cell lymphoma (DLBCL), acute lymphocytic leukemia, follicular lymphom), multiple myeloma, and acute myelogenous leukemia. We also evaluated and compared the in vivo efficacy of entospletinib and vincristine as singe agents and in combination in a DLBCL tumor xenograft model using the SU-DHL-10 cell line. Methods In vitro growth inhibition of a panel of malignant hematological cell lines was assessed using CellTiter-Glo™ Assay (Promega) after 72h incubation with entospletinib or vincristine alone or in combination. Synergy was evaluated using the Bliss model of independence (Meletiadis, J., et al., Med Mycol, 2005). In vivo, SU-DHL-10 cells (5 x 106 cells) were implanted subcutaneously in the axilla in male SCID beige mice. All mice were sorted into study groups on Day 16 such that each group's mean tumor volume fell within 10% of the overall mean (197mm3). Dosing was initiated on Day 16 and animals were dosed for 17 days. Plasma concentrations of entospletinib and vincristine were assessed on Day 19, and the entospletinib 75 mg/kg dose was lowered on Day 22 to 50 mg/kg to approximate the human achievable SYK target coverage of EC80. Efficacy and tolerability were evaluated by tumor measurements and body weight monitored three times weekly. Tumor burden data were analyzed by the application of a two-way analysis of variance (ANOVA), with post-hoc analysis. Results In vitro combinations of entospletinib with low concentrations of vincristine resulted in marked inhibition of cell proliferation and induction of apoptosis in a broad panel of 19 tumor cell lines representing major B cell malignancies including DLBCL. The combination of entospletinib with vincristine had a profound inhibitory effect on proliferation in all subtypes of DLBCL. Entospletinib was evaluated at a concentration equivalent to the Cminof the clinical dose and vincristine was used at concentrations (≤ 10 nM) that had little to no significant single agent effect in these cell lines. In vivo in a SU-DHL-10 xenograft model, entospletinib dosed alone at 25 or 75/50 mg/kg significantly inhibited tumor growth, causing 39% and 20% tumor growth inhibition (TGI), respectively, compared to the vehicle-treated control group. Vincristine administered at either 0.15 and 0.5 mg/kg Q7D x 3 also resulted in significant TGI (42% and 85% TGI, respectively). The addition of entospletinib (75/50 mg/kg) to 0.5 mg/kg or 0.15 mg/kg vincristine resulted in a significant increase in TGI from 85% to 96% (p= 0.001) and 42% to 71% (p< 0.0001), respectively. The addition of entospletinib (25 mg/kg) to vincristine did not significantly increase the tumor growth inhibition. While the groups receiving either entospletinib or vincristine as single agents had no complete or partial tumor regression, 50% of the mice receiving the combination of 75/50 mg/kg entospletinib with 0.5 mg/kg vincristine had partial responses, 8% had complete regression and 8% were tumor free at the end of study (Figure 1). Conclusion Entospletinib and vincristine demonstrated efficacy and tolerability both alone and in combination in the SU-DHL-10 DLBCL cell line xenograft model in SCID beige mice. Vincristine combinations with entospletinib showed significantly greater efficacy than vincristine alone. These data support the further clinical development of entospletinib in combination with vincristine for the treatment of DLBCL. a ENTO: PO: Q12H x 2 (Day 16-32) b VCR: IV: Q7D x 3 (Days 18, 25, 32) Figure 1. Tumor Regressions in an Entospletinib/ Vincristine Treated Murine DLBCL Xenograft Figure 1. Tumor Regressions in an Entospletinib/ Vincristine Treated Murine DLBCL Xenograft Disclosures Axelrod: Gilead Sciences: Employment, Equity Ownership. Fowles:Gilead Sciences: Employment, Equity Ownership. Silverman:Gilead Sciences: Employment, Equity Ownership. Clarke:Gilead Sciences: Employment, Equity Ownership. Tang:Gilead Sciences: Employment, Equity Ownership. Rousseau:Gilead Sciences: Employment, Equity Ownership. Webb:Gilead Sciences: Employment, Equity Ownership. Di Paolo:Gilead Sciences: Employment, Equity Ownership.

scholarly journals Effective Lipidoid Nanoparticle Delivery In Vivo of siRNA Targeting Kappa Light Chain Production in a Murine Xenograft Model

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3208-3208
Author(s):  
Xun Ma ◽  
Ping Zhou ◽  
Adin Kugelmass ◽  
Denis Toskic ◽  
Melissa Warner ◽  
...  
Keyword(s):  
Tumor Growth ◽  
Cell Viability ◽  
Cell Lines ◽  
Plasma Cells ◽  
Medical Center ◽  
Xenograft Model ◽  
Al Amyloidosis ◽  
Nsg Mice

Abstract INTRODUCTION : Despite advances in therapy, patients with relapsed AL amyloidosis die of resistant disease. New therapies are needed. siRNA directed at the constant regions of Ig light chains (LC) reduces LC mRNA and protein from patient cells, from human myeloma and AL cell lines, and in a flank plasmacytoma model with in vivo electroporation (Blood 2014;123:3440; Gene Ther 2016;23:727). To deliver siRNA in vivo, we first tested a series of biodegradable lipidoid nanoparticles (LPN) generated through Michael addition of aliphatic acrylates containing disulfide bonds responsive to intracellular glutathione that enhance siRNA transit from endosome to cytoplasm, and identified the 8B-3 LPN as safe and active in vitro. To provide an in vivo model, we tested RPMI8226, ALMC-1, NCI929 and JJN3 human myeloma reporter cell lines stably expressing FFL and GFP in NOD scid γ (NSG) mice using different routes of inoculation. We sought an optimal xenograft model that would provide reliable tumor-take, brief latency for circulating LC, rapid short-term increase in LC levels, measurable β2-microglobulin (β2M) levels and ease of administration of multiple injections of LPN. The NSG JJN3 intraperitoneal (IP) model met these standards. This model not only enables timely testing of this siRNA approach but also provides the significant challenge of rapid tumor growth. We now report the results in this model of delivery by the 8B-3 LPN of siIGKC targeting κ LC production. MATERIALS AND METHODS: LPN/siRNA are formulated using a microfluid based mixer (NanoAssemblr, Precision Nanosystems, Inc), and are controlled for size by varying the relative flow rates of lipid and solvent. For in vivo delivery, cholesterol, DOPE and PEGylated co-lipids are used to form stable LPN with siNT or siIGKC (Dharmacon). Standard QA metrics are applied to each lot of 8B-3/siRNA and lot-to-lot checks for cell viability and in vitro FFL knockdown are performed. Coated loaded 8B-3 LPN are ~100nM in diameter and were tested in vitro at 8B-3:siRNA ratios of 10:1, 5:1 and 1.5:1. The 1.5:1 ratio was superior; cell viability was unaffected and κ LC reduction was 84%. NSG mice with JJN3 tumor implants (107 cells IP on day 1) are injected IP with 200μL 8B-3:siRNA (1.5:1 ratio) mixed with 400μL PBS once daily on days 5, 6 and 7. Luciferin imaging is obtained on day 5 and blood is obtained on day 5 (pre-injection) and on day 8 for ELISA for human κ LC and β2M. RESULTS : Current JJN3 cells make only κ LC without IgA (Br J Haematol 1999;106:669) and in vitro at 48 hours after a single exposure to 8B-3/siIGKC we see reductions of 84% and 25% in κ LC and β2M secretion with no change in cell viability. Neither κ LC nor β2M reductions are seen with 8B3/siNT. The NSG JJN3 IP model has a 90% tumor-take and a 5-day LC latency. IP xenograft CD138+ cells are found in liver (subcapsular) and spleen. On day 5, the mice have median serum levels (Q1-Q3) of κ LC and β2M of 2.37μg/mL (1.68-3.32) and 1.56ρg/mL (0.58-5.38), values that strongly correlate (r=0.76, P<<0.01), as do the day 5 κ LC and FLUX values (r=0.88, P<<0.01). In 3 cohorts of 10 mice each, 5 siNT and 5 siIGKC per cohort, there were no differences in day 5 κ LC, FLUX and β2M, or in day 8 β2M, between the siNT and siIGKC groups (Table 1). On day 8 after 3 IP injections, the ratio of the medians of κ LCday 8/κ LCday 5 x 100% was lower in siIGKC mice (161% versus 264%) and trends towards significant reductions in κ LC with siIGKC were observed (Table 1). In a paired comparison of the means of the groups in the 3 cohorts the 33% reduction in κ LC with siIGKC was significant (Table 1). On day 8 there were no differences in the weights or behaviour of the mice. CONCLUSIONS : We have previously shown that siIGKC, a pool of siRNA directed at consensus sequences in the κ LC constant region gene, can significantly reduce κ LC production in clonal plasma cells from patients, in human myeloma cell lines, and in vivo in a flank plasmacytoma xenograft model. In this work, we show that 8B-3 is a promising LPN for delivery of siRNA to human plasma cells and, when loaded with siIGKC, can with relative safety significantly reduce circulating κ LC in the NSG JJN3 IP model after 3 daily IP injections despite rapid tumor growth. We also show the utility of the NSG JJN3 IP model for the study of κ LC directed therapies. Extensive work lies ahead to identify and optimize a lead candidate for delivery of siRNA to human bone marrow plasma cells in vivo and to begin systematic pre-clinical safety studies. Disclosures Ma: Tufts Medical Center: Patents & Royalties: Patent: 9593332. Wang:Tufts University Medical School: Patents & Royalties: Patent: 9765022. Xu:Tufts University School of Medicine: Patents & Royalties: Patent: 9765022. Comenzo:Tufts Medical Center: Patents & Royalties: Patent: 9593332.

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.

scholarly journals Lycorine Downregulates HMGB1 to Inhibit Autophagy and Enhances Bortezomib Activity in Multiple Myeloma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3265-3265
Author(s):  
Mridul Roy ◽  
Long Liang ◽  
Xiaojuan Xiao ◽  
Yuanliang Peng ◽  
Yuhao Luo ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Conflicts Of Interest ◽  
Single Agent ◽  
Xenograft Model ◽  
Hmgb1 Expression ◽  
Resistant Cells

Abstract Multiple myeloma (MM) is the second most prevalent hematologic malignancy, characterized by the infiltration of malignant plasma cells into bone marrow. In spite of current efficient therapeutic regimens, which have significantly increased patients overall survival, the major features inevitably present in MM are the intrinsic and acquired resistance with nearly universal relapse. In addition, the diverse heterogeneous characteristics of this largely incurable disease emphasize the importance of innovative therapies and identification of more effective drugs. Autophagy removes defective cellular organelles, protein aggregates, and intracellular microbes and is associated with cell survival and tumor maintenance. Inhibition of autophagy enhances sensitivity of a number of anticancer agents and induces cell death in MM. High-mobility group box-1 (HMGB1) protein plays an important subcellular localization-dependent role during autophagy. The importance of HMGB1 for induction of autophagy and tumor development has made this protein as a novel target for cancer therapy. Lycorine is a natural alkaloid with significant anti-cancer activity. While previous studies mainly showed lycorine as a potential apoptosis inducer, recent studies stated that apoptosis is not the primary underlying anti-proliferative mechanism of this compound. This led the interest to investigate the role of lycorine on other cell maintenance systems, such as autophagy. In addition single-agent efficacy of lycorine or in combination with other anti-MM agents has not been evaluated in vivo. Herein we investigated the anti-MM effect of lycorine and the role of this natural agent on regulation of autophagy in vitro and in vivo. We found that lycorine inhibits proliferation and induces apoptosis in MM cells with less sensitivity to the normal B-cell at the same concentrations. We also found that lycorine promisingly inhibits autophagy, the mechanism that MM cells use to survive and defeat treatment. We identified HMGB1, an important regulator of autophagy, as the most aberrantly expressed protein after lycorine treatment. Furthermore, we characterized HMGB1 as a critical mediator of lycorine activity against MM. Gene expression profiling (GEP) analysis showed that higher expression of HMGB1 is linked with the poor prognosis of MM. We further confirmed this correlation in human bone marrow CD138+ primary myeloma cells and MM cell lines. Mechanistically, by activating the proteasomal degradation of HMGB1, lycorine induces a rapid turnover of HMGB1. This led to decreased Bcl-2 phosphorylation by MEK-ERK pathway and increased association of Bcl-2 with Beclin-1 resulting in autophagy inhibition and growth attenuation. In addition, we observed higher HMGB1 expression in bortezomib resistant cells. The combination of bortezomib plus lycorine was highly efficient against MM cells and MM cells grown in bone marrow micro-environment. Lycorine showed the capability of inhibiting bortezomib induced autophagy as well as re-sensitizing resistant cells to bortezomib. In agreement with our in vitro observations, in vivo study using human MM xenograft model showed that lycorine is well tolerated, inhibits HMGB1 expression and thereby autophagy and induces enhanced bortezomib activity. These observations indicated lycorine as an effective autophagy inhibitor and revealed that lycorine alone or in combination with bortezomib is a potential therapeutic strategy. Our study supports the feasibility of lycorine in anti-MM treatment and provides a preclinical framework for combining lycorine with bortezomib in clinical setting. Disclosures No relevant conflicts of interest to declare.

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