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.

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.

A Humanized Anti-Ganglioside GM2 Antibody, BIW-8962, Exhibits ADCC/CDC Activity against Multiple Myeloma Cells and Potent Anti- Tumor Activity in Mouse Xenograft Models.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1718-1718 ◽  
Author(s):  
Toshihiko Ishii ◽  
Asher Alban Chanan-Khan ◽  
Jazur Jafferjee ◽  
Noreen Ersing ◽  
Takeshi Takahashi ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Phase 1 ◽  
Xenograft Model ◽  
Surface Expression ◽  
Scid Mice ◽  
Subcutaneous Xenograft ◽  
Anti Tumor Activity

Abstract BIW-8962 is a humanized anti-ganglioside GM2 (GM2) monoclonal antibody, produced by Poteligent technology to enhance ADCC activity. GM2 is expressed on many cancer cells including multiple myeloma (MM), small cell lung cancer and glioma cells. In this study, we evaluated the anti-myeloma activity of BIW-8962 in preclinical myeloma models both in vitro and in vivo. Expression of GM2 was analyzed in 15 human MM cell lines by FCM. Eleven out of 15 MM cell lines had positive surface expression of GM2. GM2 as a potential target was then verified in primary MM samples obtained from patients. Eleven out of 15 samples were positive for GM2. We then used two GM2 positive MM cell lines (U266B1 and KMS-11) and evaluated ADCC and CDC activity of BIW-8962 in vitro. BIW-8962 exhibited a potent ADCC and less potent CDC activity. In vivo anti-tumor activity of BIW-8962 was then examined using the standard subcutaneous xenograft model; KMS-11 was inoculated in the flank of SCID mice. BIW-8962 (intravenously administered biweekly for 3 weeks) exhibited a potent anti-tumor activity from as low a dose level as 0.1 mg/kg. Furthermore, in a more clinically relevant model, in which OPM-2/GFP (GM2 positive MM cell line) cells were intravenously inoculated into SCID mice with preferentially tumor growth within the bone marrow microenvironment, BIW-8962 (intravenously administered biweekly for 4 weeks, 10 mg/kg) suppressed OPM-2/GFP cell growth and serum M protein elevation, demonstrating in vivo anti-myeloma effect of BIW-8962. Our preclinical investigations rationalize clinical evaluation of BIW-8962 in patients with MM. Currently BIW-8962 is being investigated in a Phase 1 study in patients with multiple myeloma.

Coltuximab Ravtansine (SAR3419) Demonstrates Enhanced Activity in Combination with Bendamustine, Gemcitabine and Novel Targeted Agents Such As PI3K Inhibitors in Pre-Clinical Models of Relapsed and/or Refractory Non-Hodgkins Lymphoma (NHL)

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 5125-5125
Author(s):  
Callum M Sloss ◽  
Katie O'Callaghan ◽  
Jutta Deckert ◽  
Jenny Tsui ◽  
Leanne Lanieri ◽  
...  
Keyword(s):  
Cell Lines ◽  
Single Agent ◽  
Targeted Agents ◽  
Employment Equity ◽  
Pi3k Inhibitors ◽  
Xenograft Models ◽  
Equity Ownership ◽  
Chemotherapy Agents

Abstract Introduction: Relapsed/refractory B-cell NHL remains an area of significant medical need. CD19 is broadly expressed on B-cell malignancies making it an ideal target for antibody-drug conjugate (ADC) based therapy. Coltuximab ravtansine is a CD19-targeting ADC consisting of a CD19-targeting antibody conjugated to the maytansinoid anti-mitotic DM4. In preclinical studies, coltuximab ravtansine has shown potent, targeted activity against NHL cell lines and xenograft models. In early clinical trials, it has been well tolerated and has shown promising signs of efficacy as both a single agent and in combination with rituximab. In the STARLYTE Phase 2 trial coltuximab ravtansine monotherapy resulted in an ORR of 44% in R/R-DLBCL that included an ORR of 21% in hard-to-treat primary refractory patients (NCT01472887). Here we describe studies aimed at the identification of combination partners for coltuximab ravtansine to further optimize clinical benefit to R/R-NHL patients. We are employing a dual approach where we investigate combination of coltuximab ravtansine with multiple, novel targeted therapy partners whilst in parallel also investigating the combination of coltuximab ravtansine with chemotherapies commonly used in the late stage R/R-NHL setting. Methods: Coltuximab ravtansine and the DM4 payload were evaluated in a high throughput screen both as single agents and in combination with a selection of novel, emerging targeted agents across a panel of twenty NHL cell lines. The combinations were evaluated in a dose-response matrix and a statistical method was used to identify combination synergies significantly superseding baseline additivity values. The in vivo efficacy of coltuximab ravtansine was additionally assessed in combination with various clinically relevant chemotherapy agents in subcutaneous xenograft models of NHL. Results: Coltuximab ravtansine and DM4 both showed potent single agent activity against the entire panel of NHL cell lines with median GI50's of 770pM and 100pM, respectively. We observed a significant correlation in the cell line sensitivity of the two compounds suggesting that sensitivity to coltuximab ravtansine is driven, at least in part, by inherent sensitivity of cells to the cytotoxic effects of the DM4 payload. In vitro combination studies for coltuximab ravtansine were performed to identify targets or pathways that result in the most prominent combination effects across the cell line panel. Analysis of the in vitro combination dose-matrix revealed particularly strong synergy between coltuximab ravtansine and various inhibitors of the PI3K/AKT/mTOR axis. Studies to examine the synergism between coltuximab ravtansine and PI3K inhibitors in in vivo models of NHL are ongoing. In order to further determine the utility of coltuximab ravtansine as part of a potential combination regimen for the treatment of R/R-NHL, we assessed the combination of coltuximab ravtansine with the chemotherapy agents bendamustine and gemcitabine in vivo. As gemcitabine is typically used in combination we assessed the efficacy of a coltuximab ravtansine with rituximab and gemcitabine in vivo. In both cases the combination with coltuximab ravtansine was significantly more efficacious than the standard-of-care alone arms. Conclusions: Coltuximab ravtansine demonstrates synergistic activity in combination with multiple PI3K pathway inhibitors across a large panel of NHL cell lines. Additionally, we have shown that combination of coltuximab ravtansine with clinically relevant late stage treatments such as bendamustine and rituximab + gemcitabine is more efficacious than the chemotherapy regimens alone. These results support the continued development of coltuximab ravtansine in R/R-NHL in combination with chemotherapy regimens and suggest that a combination of coltuximab ravtansine with PI3K inhibitors may also be of interest in the clinical setting. Disclosures Sloss: ImmunoGen, Inc.: Employment, Equity Ownership. O'Callaghan:ImmunoGen, Inc.: Employment, Equity Ownership. Deckert:ImmunoGen, Inc.: Employment, Equity Ownership. Tsui:ImmunoGen, Inc.: Employment, Equity Ownership. Lanieri:ImmunoGen, Inc.: Employment, Equity Ownership. Romanelli:ImmunoGen, Inc.: Employment, Equity Ownership.

scholarly journals Combining IMGN779, a Novel Anti-CD33 Antibody-Drug Conjugate (ADC), with the PARP Inhibitor, Olaparib, Results in Enhanced Anti-Tumor Activity in Preclinical Acute Myeloid Leukemia (AML) Models

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1645-1645 ◽  
Author(s):  
Scott Portwood ◽  
Robert A Puchalski ◽  
Russell M Walker ◽  
Eunice S. Wang
Keyword(s):  
Cell Lines ◽  
Combination Treatment ◽  
Parp Inhibitor ◽  
Single Agent ◽  
S Phase ◽  
Complex Karyotype ◽  
Anti Tumor Activity ◽  
Dose Dependent

Abstract Background: New therapies for AML are urgently needed. IMGN779 is a novel CD33-targeting ADC that utilizes as the cytotoxic agent a DNA-alkylatingindolinobenzodiazepine, DGN462. Previous data have shown that IMGN779 exerts dose-dependent activity against human CD33+ AML cells in vitro and in vivo. We hypothesized that combination treatment of AML cells with the poly (ADP-ribose) polymerase (PARP) inhibitor, olaparib, which blocks cellular DNA repair would further enhance the anti-leukemic activity of IMGN779 in preclinical human AML models. Methods:Human CD33+ AML cell lines (HEL, MV4-11, HL60) were treated in vitro with controls, IMGN779, olaparib, and IMGN779 + olaparib. Proliferation was measured by WST-8 reagent. Synergistic/additive effects were calculated using Compusyn software. Flow cytometry was performed to assess apoptosis, viability, and cell cycle effects. SCID mice were engrafted systemically with human AML (HEL-luciferase) cells followed by treatment with IMGN779, olaparib, or both drugs in combination. Changes in disease burden and possible treatment-related toxicities were determined by whole animal bioluminescent imaging, body weights, and time to morbidity, respectively. Primary cells from patients with relapsed/refractory AML characterized by complex karyotype or FLT-3 mutations were plated in methocellulose media supplemented with hematopoietic cytokines for colony formation unit assays (CFU). Vehicle, IMGN779 and/or olaparib in triplicate were added followed by quantification of leukemic CFU 15 days later with a Spot-RT3 camera mounted to an inverted microscope. Results: IMGN779 treatment induced significant growth inhibition in vitro in all CD33+ human AML cell lines tested that was dose dependent. IMGN779 cell killing was CD33 dependent. Olaparib induced cell death in human AML cell lines via reversal of DNA damage repair mechanisms. Combination treatment with IMGN779 (500 pM-1 nM) and olaparib (10-50 μM) significantly enhanced anti-leukemic effects over monotherapy in the same cell lines (representative data in Table 1). Combination indices for IMGN779 and olaparib therapy ranged from 0.7-0.9, consistent with synergistic effects. The combination markedly reduced overall cell viability, increased apoptosis, and induced almost complete S-phase cell cycle arrest as compared with controls and single-agent treatments. Exposure to a combination of IMGN779 and olaparib also significantly inhibited CFU growth of progenitor cells established from bone marrow samples of patients (n=7) with relapsed/refractory, FLT3-ITD, and/or complex karyotype AML. Statistically significant inhibition of viable CFUs was observed following combination IMGN779 (10 pM) and olaparib (1 μM) therapy as compared with monotherapy or vehicle controls (p<0.001). In vivo, IMGN779 administered as a single dose that ranged from 0.5 to 5 mg/kg, by antibody, was overall well tolerated in SCID mice bearing systemic human CD33+ AML (HEL-luciferase) xenografts. Significant dose-dependent anti-leukemic activity, as reflected by decreased leukemia burden and prolonged overall survival, was observed. Combination treatment with IMGN779 (3 mg/kg) and olaparib (100 mg/kg) further significantly decreased leukemic burden when compared with vehicle (p<0.0001), IMGN779 alone (p<0.01) and olaparib alone (p<0.05) on day 14 after dosing (Figure 1). Conclusion:The combination of the CD33-targeting ADC, IMGN779, and the PARP inhibitor, olaparib, enhanced anti-tumor activity in multiple preclinical human CD33+ AML models. The combination increased in vitro DNA damage, apoptosis, S-phase arrest, and cell death effects on human CD33+ AML cells vs. single agent therapy. Combination therapy also markedly inhibited colony formation of primary AML cells representing clinically chemoresistant disease and significantly decreased in vivo leukemic burden in systemic human AML xenograft models. Our results support the future clinical investigation of this novel combinatorial regimen for AML therapy. In vivo IMGN779 + olaparib treatment in systemic HEL AML xenografts In vivo IMGN779 + olaparib treatment in systemic HEL AML xenografts Figure 1 Figure 1. Figure 2 Figure 2. Disclosures Walker: ImmunoGen, Inc.: Employment.

Pronounced Anti-Leukemia Activity in Vivo of the Novel Peptidic CXCR4 Antagonist LY2510924 As a Single Agent and in Combination with Chemotherapy

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3745-3745
Author(s):  
Byung-Sik Cho ◽  
Zhihong Zeng ◽  
Hong Mu ◽  
Zhiqiang Wang ◽  
Teresa McQueen ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Cultured Cells ◽  
Single Agent ◽  
Leukemic Cells ◽  
Cxcr4 Antagonist ◽  
Nsg Mice ◽  
Xenograft Models

Abstract LY2510924 is a novel selective peptidic CXCR4 antagonist that blocks SDF-1α from binding to its receptor. We have demonstrated that LY2510924 at nanomolar concentrations durably disrupts the SDF-1α/CXCR4 axis in acute myeloid leukemia (AML) cells and exerts anti-leukemia effects as a single agent (AACR 2014: #4768). We further investigated the pronounced anti-leukemia activity of LY2510924 and the mechanisms underlying the anti-leukemia effect. To test the efficacy of LY2510924 in combination with chemotherapy, we injected OCI-AML3/luc/GFP cells into NSG mice. Mice were randomized into 4 groups (10 mice per group) on day 8: control, chemotherapy (cytarabine [50 mg/kg, daily for 5 days, intravenous or intraperitoneal]/doxorubicin [1.5 mg/kg, daily for 3 days, co-delivered intravenously]), LY2510924 (2.5 mg/kg, daily for 3 weeks, subcutaneously), or chemotherapy and LY2510924. Bioluminescence imaging demonstrated that LY2510924 exerted an anti-leukemia effect equal to that achieved with chemotherapy (P=0.249), and the combination therapy group had the lowest luciferase activity. LY2510924-treated mice had prolonged survival (Figure 1) compared to controls (52 days vs. 40 days, p=0.006), and combination therapy extended survival even further (62 days vs. 52 days, p=0.004). Next, we examined anti-leukemia efficacy of LY2510924 in primary human AML xenograft models. NSG mice were injected with primary AML cells and randomized into 2 groups on day 25, after engraftment was documented: control (n=13) and treatment with LY2510924 (n=15; 2.5 mg/kg subcutaneously, daily). First, we examined AML cell mobilization by measuring the proportion of circulating leukemic cells after daily LY2510924 administration. Mice treated with LY2519024 had a significant increase of circulating leukemic cells at 3 hours (2.1-fold, P=0.008), and further increases at 24 hours (2.7-fold, P=0.008) and 48 hours (3.0-fold, P=0.009) compared to controls. Flow cytometry showed a sustained inhibition of CXCR4 12G5 surface expression at 3 and 24 hours after the first LY2510924 injection. Thereafter, weekly examination of circulating leukemic cells in both groups revealed slower progression of leukemia in the LY2510924-treated group (54% vs. 86% circulating AML cells on day 45, P<0.001). Additionally, we sacrificed 3 mice per group on days 35 and 45 and demonstrated that LY2510924-treated mice had significantly lower leukemic cell burden in the spleen (22% vs. 51%, P=0.001) on day 35, and in both spleen (20% vs. 60%, P<0.001) and bone marrow (72% vs. 90%, P=0.012) on day 45 by flow cytometry. CXCR4 blockade with LY2510924 was associated with reduced AKT and/or ERK signaling in leukemic cells of spleen, bone marrow, and blood as measured by multi-parametric phospho-flow cytometry. This anti-leukemia effect translated into a significant prolongation of survival in LY2510924-treated mice (56 days vs. 44 days, p<0.001, Figure 2). Our previous study (AACR 2014:#4768) demonstrated that LY2510924 did not induce AML cell death in vitro on its own but inhibited AML cell growth in co-cultures with human marrow stromal cells (hMSC). To explore how CXCR4-mediated signaling in AML cells elicits anti-leukemia effects, we performed whole gene expression profiling of FACS-sorted OCI-AML3 cells co-cultured with hMSC for 48 hours and co-treated with LY2510924, in duplicates. Among genes modified by CXCR4 antagonist, we found that CTNNB1 (human beta-catenin), JARID1C (lysine-specific demethylase 5C), RARA (retinoic acid receptor alpha), RARRES2 (chemerin), and COQ4 (coenzyme Q) were downregulated in co-cultured OCI-AML3 cells treated with LY2510925, when compared to either mono-cultured cells or co-cultured cells without LY2510924. These findings are currently being validated by using functional in vitro assays. In conclusion, our findings demonstrate that CXCR4 antagonist LY2510924 inhibits AML progression in leukemia xenograft models in vivo and has a synergistic anti-leukemia effect in combination with chemotherapy. LY2510924 efficiently inhibits CXCR4 signaling in primary AML cells in vivo and induces mobilization of leukemic cells into circulation. This results in pronounced anti-leukemia activity as a single agent. LY2510924's potency and durable occupancy of CXCR4 receptors will likely translate into greater anti-leukemia potency in future clinical applications. Disclosures Peng: Eli Lilly & Company: Employment. Thornton:Eli Lilly & Company: Employment, stocks Other.

Preclinical evaluation of KZR-261, a novel small molecule inhibitor of Sec61.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. 3582-3582
Author(s):  
Eric Lowe ◽  
R. Andrea Fan ◽  
Jing Jiang ◽  
Henry W. B. Johnson ◽  
Christopher J. Kirk ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Expression Profiling ◽  
Cell Lines ◽  
Therapeutic Target ◽  
Expression Profiling ◽  
Single Agent ◽  
Tumor Types ◽  
Anti Tumor Activity

3582 Background: Secreted and transmembrane proteins play key roles in malignant transformation and growth, including in autocrine growth factor expression, receptor oncogene signaling, and immune system evasion. Biogenesis of these proteins involves translocation of the nascent polypeptides into the endoplasmic reticulum (ER) through the Sec61 channel, providing an untapped therapeutic target for a broad spectrum of malignancies. Here we describe preclinical activity of KZR-261 and related inhibitors of Sec61-dependent protein secretion. Methods: Sec61 inhibition with KZR-261 and related analog KZR-834 were evaluated using cell lines overexpressing proteins of interest tagged with luciferase. In vitro anti-tumor activity was assessed against a panel of 346 cell lines across 25 tumor types. Quantitative proteomic profiling by mass spec and gene expression profiling by RNAseq were conducted following treatment in multiple solid and heme tumor cell lines. Anti-tumor efficacy was evaluated in athymic nude mice implanted with the cancer cell lines H82 (SCLC), HT29 (CRC), BxPC3 (Pancreatic), 22RV1 (Prostate), H929 (Myeloma) and RL (NHL). Activity was also evaluated in a MC38 syngeneic colon tumor model. Results: KZR-261 and KZR-834 exhibited nanomolar potency against many therapeutic targets, including immune checkpoints, VEGF-A, VEGFR and EGFR. Broad in vitro anti-cancer activity was observed with KZR-834, which potently decreased cell viability across both solid and heme tumor types including CRC, Pancreatic, HNSCC, HCC, Lymphoma and Myeloma. Global proteomic analysis observed more than 1.5 fold downregulation of < 10% of detected Sec61 client proteins following treatment, while gene expression profiling revealed upregulation of ER stress response genes in sensitive versus resistant cell lines. Analysis of the TCGA database also found these genes upregulated in a number of different tumor types. In vivo, weekly IV administration was well tolerated and induced a dose dependent anti-tumor response at doses below the MTD in solid and heme xenograft models. In the syngeneic MC38 model, administration of KZR-834 in combination with anti-PD1 antibody resulted in greater anti-tumor activity than either single agent. Conclusions: Novel Sec61 inhibitors potently block expression of secreted and membrane proteins, translating into anti-tumor activity against many tumor types in vitro and in vivo, suggesting broad therapeutic potential. Clinical trials are being planned with KZR-261 to understand safety and early efficacy of this novel compound and therapeutic target.

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 vemurafenib, a selective BRAFV600E inhibitor, in combination with a MEK inhibitor in BRAFV600E colorectal cancer models.

2012 ◽  
Vol 30 (4_suppl) ◽  
pp. 488-488 ◽  
Author(s):  
Brian Higgins ◽  
Kenneth Daniel Kolinsky ◽  
Hong Yang ◽  
Min Jung Kim ◽  
Jia Kui Li ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Single Agent ◽  
Mek Inhibitor ◽  
Apoptosis Induction ◽  
Braf Mutations ◽  
Xenograft Models ◽  
Pathway Inhibition ◽  
Anti Tumor Activity

488 Background: BRAF mutations occur in about 10% of colorectal cancer (CRC). Most BRAF mutations involve the V600E amino acid substitution, resulting in constitutive activation of the MAPK signaling pathway. Vemurafenib (RG7204, PLX4032) is a first-in-class, BRAFV600E-specific small molecule inhibitor that dose-dependently inhibits tumor growth in V600E CRC xenografts. However, unlike responses observed in melanoma, single agent vemurafenib in a Phase I extension trial of 20 patients with previously treated metastatic CRC, resulted in only 5% response rate. The goal here was to explore in vitro and in vivo if addition of a MEK inhibitor (MEKi) could increase effects of vemurafenib on anti-proliferation and anti-tumor activity in BRAFV600E CRC cell lines. Methods: Combo of vemurafenib with a MEKi was tested in MTT assay for antiproliferative effect and combination effect was determined by combination index (CI) calculated by the CalcySyn software. Western analysis and Annexin V staining were utilized to evaluate combo effects on pathway inhibition and apoptosis induction. Optimal doses of both vemurafenib and MEKi were tested as single agent and in combination in the Colo205 and LS411N CRC xenograft models in nude mice. Results: Synergistic anti-proliferative effect was observed with the combo of vemurafenib and a MEKi in the V600E positive CRC cell lines tested. More effective pathway inhibition and apoptosis induction were observed with combo than either agent alone. Combination of vemurafenib and a MEKi delivered greater anti-tumor activity and increased life span of animals in the Colo205 and LS411N CRC xenograft models. Conclusions: These in vitro and in vivo data suggest that combined pharmacologic blockade within the RAS/RAF/MEK/ERK pathway is more effective than either agent alone and may be a way to exploit greater antitumor activity CRC patients in clinic.

The mTOR Inhibitor RAD001 (everolimus) Is Active Against Multiple Myeloma Cells In Vitro and in Vivo.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1496-1496 ◽  
Author(s):  
Nicholas Mitsiades ◽  
Ciaran McMullan ◽  
Vassiliki Poulaki ◽  
Joseph Negri ◽  
Noopur Raje ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Tumor Cells ◽  
Cell Lines ◽  
Stromal Cells ◽  
Mtor Inhibitor ◽  
Alkylating Agents ◽  
Anti Tumor Activity

Abstract We have recently shown that tumor cell proliferation, survival and drug-resistance in multiple myeloma (MM) and a broad range of other tumors is critically influenced by insulin-like growth factors (IGFs) and their receptor (IGF-1R) (Cancer Cell2004;5:221–30). Among the pleiotropic signaling cascades downstream of IGF-1R activation, we focused on the functional implications and therapeutic targeting of the Akt/p70S6K/mTOR axis, particularly of mTOR (mammalian Target of Rapamycin), due to its regulatory role on cellular bioenergetics, a key aspect of tumor pathophysiology. Herein, we describe the in vitro and in vivo profiles of anti-tumor activity of the selective mTOR inhibitor RAD001 (Everolimus, Novartis AG). Using in vitro MTT assays, we observed that RAD001 is active (at nM concentrations) against a broad range of tumor cells, including >40 MM cell lines and >10 primary MM tumor cells (including cell lines or primary cells resistant to Dex, alkylating agents, anthracyclines, thalidomide (Thal), immunomodulatory Thal derivatives, bortezomib, and/or Apo2L/TRAIL), without significant impact on viability of normal hematopoietic cells or other normal tissues (e.g. bone marrow stromal cells), and its anti-MM effect was not blocked by forced overexpression of Bcl-2 or constitutively active Akt. While cytokine- or cell adhesion-mediated interactions with the bone marrow (BM) microenvironment (e.g. BM stromal cells) protects MM cells from conventional therapies (e.g. Dex or cytotoxic chemotherapy), RAD001 was able to overcome this protective effect in co-culture models of MM cells with BM stromal cells or in vitro MM cell exposure to survival factors, e.g. IL-6 or IGF-I. Furthermore, RAD001 sensitized MM cells to other anti-MM therapeutics, e.g. dexamethasone, cytotoxic chemotherapeutics, or the proteasome inhibitor bortezomib, even in cases of primary MM tumor cells refractory to these respective agents. Using hierarchical clustering analyses and relevance network algorithms, we found that the pattern of MM cell dose-response relationships to RAD001 is clearly distinct from the patterns of sensitivity or resistance to other conventional or investigational anti-MM drugs. This further supports the notion that RAD001 confers a constellation of pro-apoptotic/anti-proliferative molecular sequelae distinct from those of currently available anti-MM drugs, and also suggests that RAD001 may have anti-tumor activity even against subgroups of MM which may be resistant to other novel therapies which that are currently in clinical development. Importantly, administration of RAD001 in a SCID/NOD mice model of diffuse MM bone had in vivo anti-tumor activity, including suppression of MM tumor burden and prolongation of survival (p<0.01, log-rank test). These studies highlight an important role for mTOR in growth/survival of human MM cells and provide proof-of-principle for future clinical studies of mTOR inhibitors for the treatment of MM and other plasma cell dyscrasias.

scholarly journals BET bromodomain inhibitor birabresib in mantle cell lymphoma: in vivo activity and identification of novel combinations to overcome adaptive resistance

ESMO Open ◽  
2018 ◽  
Vol 3 (6) ◽  
pp. e000387 ◽  
Author(s):  
Chiara Tarantelli ◽  
Elena Bernasconi ◽  
Eugenio Gaudio ◽  
Luciano Cascione ◽  
Valentina Restelli ◽  
...  
Keyword(s):  
Mantle Cell Lymphoma ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Antitumour Activity ◽  
Single Agent ◽  
Treatment Strategies ◽  
Bet Inhibitor ◽  
Mantle Cell

BackgroundThe outcome of patients affected by mantle cell lymphoma (MCL) has improved in recent years, but there is still a need for novel treatment strategies for these patients. Human cancers, including MCL, present recurrent alterations in genes that encode transcription machinery proteins and of proteins involved in regulating chromatin structure, providing the rationale to pharmacologically target epigenetic proteins. The Bromodomain and Extra Terminal domain (BET) family proteins act as transcriptional regulators of key signalling pathways including those sustaining cell viability. Birabresib (MK-8628/OTX015) has shown antitumour activity in different preclinical models and has been the first BET inhibitor to successfully undergo early clinical trials.Materials and methodsThe activity of birabresib as a single agent and in combination, as well as its mechanism of action was studied in MCL cell lines.ResultsBirabresib showed in vitro and in vivo activities, which appeared mediated via downregulation of MYC targets, cell cycle and NFKB pathway genes and were independent of direct downregulation of CCND1. Additionally, the combination of birabresib with other targeted agents (especially pomalidomide, or inhibitors of BTK, mTOR and ATR) was beneficial in MCL cell lines.ConclusionOur data provide the rationale to evaluate birabresib in patients affected by MCL.

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