Entinostat, a Novel Histone Deacetylase (HiDAC) Inhibitor Is Active in B-Cell Lymphoma and Enhances the Anti-Tumor Activity of Rituximab, Chemotherapy Agents and Proteasome Inhibitors

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1649-1649
Author(s):  
Sarah Frys ◽  
Matthew J. Barth ◽  
Cory Mavis ◽  
Juan Gu ◽  
John Gibbs ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Mechanism Of Action ◽  
Single Agent ◽  
Mechanisms Of Action ◽  
Tail Vein Injection ◽  
Dual Mechanism ◽  
Chemotherapy Agents ◽  
Anti Tumor Activity

Abstract Abstract 1649 Deacetylases (DACs) are enzymes that remove the acetyl groups from target protein lysines leading to regulation of gene transcription and other cellular processes. Entinostat is a novel and potent class I DAC inhibitor undergoing pre-clinical and clinical testing. We previously demonstrated that entinostat was active against a panel of rituximab-sensitive (RSCL) and –resistant cell lines (RRCL); and enhanced the anti-tumor activity of chemotherapy agents. In the current work, we studied the mechanisms-of-action of entinostat and its effect on rituximab activity and proteasome inhibition. Our results indicate that entinostat potentiates both rituximab and proteasome inhibitor activity in NHL cells through a dual caspase dependent and independent mechanism-of-action. Studies were conducted in RSCL, RRCL and in primary tumor cells isolated from patients with B-cell non-Hodgkin's lymphoma (n = 43). For studies with a proteasome inhibitor, cells were exposed to escalating doses of entinostat +/− bortezomib of up to 72 hrs. Studies were repeated with or without a pan-caspase inhibitor (Q-VD-OPh, 5uM). Changes in mitochondrial potential and ATP synthesis were determined by alamar blue reduction and cell titer glo luminescent assays, respectively. Flow cytometric analysis was used to determine changes in cell cycle. Protein lysates from entinostat +/− BTZ exposed cells were evaluated for changes in members of Bcl-2 and cell cycle family proteins. Microarray analysis was performed to discern differences in gene expression between RRCL and RSCL after incubation with entinostat. Separately, we studied the effects of entinostat in vitro and in vivo on rituximab-associated complement-mediated cytotoxicity (CMC) or antibody-dependent cellular cytotoxicity (ADCC). NHL cells were exposed to entinostat or DMSO 0.01% for 48hrs and then labeled with 51Cr. Labeled target cells were then exposed to rituximab or isotype control (10mg/ml) and human serum (25%) (CMC) or PBMCs (Effector:Target ratio, 40:1) (ADCC). For in vivo studies, 6–8 week old SCID mice were inoculated with 1′106 Raji cells via tail vein injection and after a period of 72 hours animals were divided into six cohorts: control, entinostat alone at 5mg/kg/dose, entinostat alone at 20mg/kg/dose alone, rituximab single agent at 10mg/kg/dose, rituximab and entinostat at 5mg/kg/dose and rituximab + entinostat at 20mg/kg/dose. Entinostat was administered by gastric lavage daily on days +3–8 and +11–16, and rituximab was administered via tail vein injection on days +3, +8, +11, and +16. Difference in survival between treatment groups was performed by Kaplan-Meier analysis. Entinostat exhibited dose-dependent activity as a single agent against RSCL, RRCL and patient-derived primary lymphoma cells. In vitro exposure of lymphoma cells to entinostat resulted in an increase in G1 and a decrease in S phase. Synergistic activity was observed by combining entinostat with BTZ but not rituximab in vitro. Microarray data demonstrated that p21 was up-regulated after exposure to entinostat. Additionally, we found an upregulation of ICAM2, down-regulation of E2F2 but no changes in CD20 mRNA. Findings were confirmed at the protein level. Caspase inhibition diminished entinostat anti-tumor activity in RSCL but not in RRCL suggesting that entinostat has a dual mechanism-of-action and can induce cell death by caspase-dependent and independent pathways (i.e. cell cycle arrest). Furthermore, In vivo treatment of SCID mice with high dose entinostat in combination with rituximab led to prolongation of the mean survival (87 days, 72–103 95% C.I. P=0.05) compared to rituximab (67 days, 52–82 95% C.I.) entinostat 20 mg/kg (19 days, 19–20 95% C.I.) alone. Our data suggests that entinostat is active against RSCL and RRCL and potentiates the anti-tumor activity of BTZ or rituximab. In addition, entinostat appears to have a dual mechanism of action that supports its clinical development in rituximab-sensitive or resistant lymphomas. Ongoing studies are aimed to further define the molecular events responsible for entinostat mechanisms-of-action and optimize its spectrum of anti-tumor activity. (Research, in part, supported by a NIH grant R01 CA136907-01A1 awarded to Roswell Park Cancer Institute). Disclosures: No relevant conflicts of interest to declare.

scholarly journals A Novel Bispecific Antibody Targeting EGFR and VEGFR2 Is Effective against Triple Negative Breast Cancer via Multiple Mechanisms of Action

Cancers ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 1027
Author(s):  
Nishant Mohan ◽  
Xiao Luo ◽  
Yi Shen ◽  
Zachary Olson ◽  
Atul Agrawal ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Tumor Growth ◽  
Mechanisms Of Action ◽  
Breast Cancers ◽  
Single Chain ◽  
Cancer Cell Growth ◽  
Anti Tumor Activity ◽  
Enhance Tumor Growth

Both EGFR and VEGFR2 frequently overexpress in TNBC and cooperate with each other in autocrine and paracrine manner to enhance tumor growth and angiogenesis. Therapeutic mAbs targeting EGFR (cetuximab) and VEGFR2 (ramucirumab) are approved by FDA for numerous cancer indications, but none of them are approved to treat breast cancers. TNBC cells secrete VEGF-A, which mediates angiogenesis on endothelial cells in a paracrine fashion, as well as promotes cancer cell growth in autocrine manner. To disrupt autocrine/paracrine loop in TNBC models in addition to mediating anti-EGFR tumor growth signaling and anti-VEGFR2 angiogenic pathway, we generated a BsAb co-targeting EGFR and VEGFR2 (designated as anti-EGFR/VEGFR2 BsAb), using publicly available sequences in which cetuximab IgG backbone is connected to the single chain variable fragment (scFv) of ramucirumab via a glycine linker. Physiochemical characterization data shows that anti-EGFR/VEGFR2 BsAb binds to both EGFR and VEGFR2 in a similar binding affinity comparable to parental antibodies. Anti-EGFR/VEGFR2 BsAb demonstrates in vitro and in vivo anti-tumor activity in TNBC models. Mechanistically, anti-EGFR/VEGFR2 BsAb not only directly inhibits both EGFR and VEGFR2 in TNBC cells but also disrupts autocrine mechanism in TNBC xenograft mouse model. Furthermore, anti-EGFR/VEGFR2 BsAb inhibits ligand-induced activation of VEGFR2 and blocks paracrine pathway mediated by VEGF secreted from TNBC cells in endothelial cells. Collectively, our novel findings demonstrate that anti-EGFR/VEGFR2 BsAb inhibits tumor growth via multiple mechanisms of action and warrants further investigation as a targeted antibody therapeutic for the treatment of TNBC.

scholarly journals Elacestrant (RAD1901) exhibits anti-tumor activity in multiple ER+ breast cancer models resistant to CDK4/6 inhibitors

2019 ◽  
Vol 21 (1) ◽  
Author(s):  
Hitisha K. Patel ◽  
Nianjun Tao ◽  
Kyung-Min Lee ◽  
Mariela Huerta ◽  
Heike Arlt ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cell Cycle ◽  
Estrogen Receptor ◽  
De Novo ◽  
Single Agent ◽  
Metastatic Breast ◽  
Cancer Resistance ◽  
Treatment Paradigm ◽  
Anti Tumor Activity

Abstract Background Addition of CDK4/6 inhibitors (CDK4/6i) to endocrine therapy significantly increased progression-free survival, leading to their approval and incorporation into the metastatic breast cancer treatment paradigm. With these inhibitors being routinely used for patients with advanced estrogen receptor-positive (ER+) breast cancer, resistance to these agents and its impact on subsequent therapy needs to be understood. Considering the central role of ER in driving the growth of ER+ breast cancers, and thus endocrine agents being a mainstay in the treatment paradigm, the effects of prior CDK4/6i exposure on ER signaling and the relevance of ER-targeted therapy are important to investigate. The objective of this study was to evaluate the anti-tumor activity of elacestrant, a novel oral selective estrogen receptor degrader (SERD), in preclinical models of CDK4/6i resistance. Methods Elacestrant was evaluated as a single agent, and in combination with alpelisib or everolimus, in multiple in vitro models and patient-derived xenografts that represent acquired and “de novo” CDK4/6i resistance. Results Elacestrant demonstrated growth inhibition in cells resistant to all three approved CDK4/6i (palbociclib, abemaciclib, ribociclib) in both ESR1 wild-type and mutant backgrounds. Furthermore, we demonstrated that elacestrant, as a single agent and in combination, inhibited growth of patient-derived xenografts that have been derived from a patient previously treated with a CDK4/6i or exhibit de novo resistance to CDK4/6i. While the resistant lines demonstrate distinct alterations in cell cycle modulators, this did not affect elacestrant’s anti-tumor activity. In fact, we observe that elacestrant downregulates several key cell cycle players and halts cell cycle progression in vitro and in vivo. Conclusions We demonstrate that breast cancer tumor cells continue to rely on ER signaling to drive tumor growth despite exposure to CDK4/6i inhibitors. Importantly, elacestrant can inhibit this ER-dependent growth despite previously reported mechanisms of CDK4/6i resistance observed such as Rb loss, CDK6 overexpression, upregulated cyclinE1 and E2F1, among others. These data provide a scientific rationale for the evaluation of elacestrant in a post-CDK4/6i patient population. Additionally, elacestrant may also serve as an endocrine backbone for rational combinations to combat resistance.

scholarly journals The Mechanism of Action of Cyclophosphamide and Its Consequences for the Development of a New Generation of Oxazaphosphorine Cytostatics

2020 ◽  
Vol 88 (4) ◽  
pp. 42
Author(s):  
Georg Voelcker
Keyword(s):  
Mechanism Of Action ◽  
Active Metabolite ◽  
Dna Alkylation ◽  
In Vivo Metabolism ◽  
Pharmacologically Active ◽  
New Generation ◽  
Anti Tumor Activity ◽  
Oxazaphosphorine Cytostatics

Although cyclophosphamide (CP) has been used successfully in the clinic for over 50 years, it has so far not been possible to elucidate the mechanism of action and to use it for improvement. This was not possible because the basis of the mechanism of action of CP, which was found by lucky coincidence, is apoptosis, the discovery of which was honored with the Nobel Prize only in 2002. Another reason was that results from cell culture experiments were used to elucidate the mechanism of action, ignoring the fact that in vivo metabolism differs from in vitro conditions. In vitro, toxic acrolein is formed during the formation of the cytotoxic metabolite phosphoreamidemustard (PAM), whereas in vivo proapoptotic hydroxypropanal (HPA) is formed. The CP metabolites formed in sequence 4-hydroxycyclophosphamide (OHCP) are the main cause of toxicity, aldophosphamide (ALDO) is the pharmacologically active metabolite and HPA amplifies the cytotoxic apoptosis initiated by DNA alkylation by PAM. It is shown that toxicity is drastically reduced but anti-tumor activity strongly increased by the formation of ALDO bypassing OHCP. Furthermore, it is shown that the anti-tumor activity against advanced solid P388 tumors that grow on CD2F1 mice is increased by orders of magnitude if DNA damage caused by a modified PAM is poorly repairable.

Modulation of Gene Expression Profile and In Vivo Anti-Myeloma Activity Induced by Valproic Acid, a Histone Deacytylase Inhibitor.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4790-4790
Author(s):  
Paola Neri ◽  
Teresa Calimeri ◽  
Mariateresa Di Martino ◽  
Marco Rossi ◽  
Orietta Eramo ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Valproic Acid ◽  
Dna Replication ◽  
Gene Expression Profile ◽  
Cell Cycle Regulation ◽  
Cycle Regulation ◽  
Anti Tumor Activity

Abstract Valproic acid (VPA) is a well-tolerated anticonvulsant drug that has been recently recognized as powerful histone deacetylase (HDCA) inhibitor. VPA induces hyperacetylation of histone H3 and H4 and inhibits both class I and II HDCACs. Recently it has been shown that VPA exerts in vitro and in vivo anti-tumor activity against solid cancers and its in vitro anti-Multiple Myeloma (MM) activity has been previously reported. However, the molecular mechanisms are still unclear. Here we have investigated molecular changes induced by VPA as well as its in vivo activity in murine models of MM. We first studied the in vitro activity of VPA against IL-6 independent as well as IL-6 dependent MM cells. A time- and dose-dependent decrease in proliferation and survival of MM cell lines was observed (IC50 in the range of 1–3 mM). Gene expression profile following treatment with VPA at 2 and 5 mM showed down-regulation of genes involved in cell cycle regulation, DNA replication and transcription as well as up-regulation of genes implicated in apoptosis and chemokine pathways. The signaling pathway analysis performed by Ingenuity Systems Software identified the cell growth, cell cycle, cell death as well as DNA replication and repair as the most important networks modulated by VPA treatment. We next evaluated the in vivo activity of VPA using two xenograft models of human MM. A cohort of SCID mice bearing subcutaneous MM1s or OPM1 were treated i.p. daily with VPA (200 mg/kg, and 300 mg/kg, n=5 mice, respectively), or vehicle alone (n=5 mice) for 16 consecutive days. Tumors were measured every 2 days, and survival was calculated using the Kaplan Mayer method. Following VPA treatment, we found a significant (p=0.006) inhibition of tumor growth in mice bearing subcutaneous MM-1s cells treated with VPA at 200 mg/kg compared to control group, which translated into a significant (p= 0.002) survival advantage in the VPA treated animals. Similar results were obtained in animals bearing subcutaneous OPM1 cells. Flow cytometry analysis performed on retrieved tumor tissues from animals showed reduction of G2-M and S phase in tumor specimens following VPA treatment, versus untreated tumors, strongly suggesting in vivo effects of VPA on cell cycle regulation. Taken together, our data demonstrate the in vitro and in vivo anti-tumor activity of VPA, delineate potential molecular targets triggered by this agent and provide a preclinical rationale for its clinical evaluation, both as a single agent or in combination, to improve patient outcome in MM.

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.

Itraconazole, an Oral Antifungal Drug, Is Active in Chemotherapy Resistant B-Cell Non-Hodgkin Lymphoma and Enhances the Anti-Tumor Activity of Chemotherapy Agents

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5138-5138
Author(s):  
Juan J Gu ◽  
Lianjuan Yang ◽  
Cory Mavis ◽  
Matthew J. Barth ◽  
Francisco J. Hernandez-Ilizaliturri
Keyword(s):  
Cell Cycle ◽  
Membrane Potential ◽  
B Cell ◽  
Mitochondrial Membrane Potential ◽  
Cell Lines ◽  
Mitochondrial Membrane ◽  
In Vitro Exposure ◽  
Chemotherapy Agents ◽  
Anti Tumor Activity

Abstract Background: Relapsed/refractory diffuse large B-cell lymphoma (DLBCL) patients previously treated with rituximab-based therapy have poor clinical outcome, according to the results from collaborative trial in relapsed aggressive lymphoma (CORAL) study. It stresses the need to identify and/or optimize novel targeted agents. To better understand the molecular mechanisms underlining the acquired resistance to rituximab, we generated and characterized several rituximab-resistant DLBCL cell lines (RRCLs). Itraconazole, an oral antifungal agent, was reported had novel anticancer activity in basal cell carcinoma, non-small cell lung cancer and prostate cancer. In our current work, we define and characterize the anticancer activity of itraconazole in preclinical rituximab-sensitive or -resistant lymphoma models. Methods: A panel of rituximab-sensitive (RSCL) and rituximab-resistant (RRCL) cell lines were exposed to escalating doses of itraconazole (0-20μM) for 24, 48 and 72h. Changes in cell viability and cell cycle distribution were evaluated using the Presto Blue assay and flow cytometry respectively. IC50 was calculated by Graphpad Prism6 software. Loss of mitochondrial membrane potential (∆ψm) following itraconazole exposure was assessed by DiOC6 and flow cytometry. Subsequently lymphoma cells were exposed to itraconazole or vehicle and various chemotherapy agents such as doxorubicin (1µM), dexamethasone (1µM), cDDP (20μg/ml), bortezomib (20nM), carfilzomib (20nM) or MLN2238 (20nM) for 48 hours. Coefficient of synergy was calculated using the CalcuSyn software. Changes in hexokinase II (HKII), Voltage dependent anion channel protein (VDAC), LC3 and BCL-xL expression levels were determined by western blotting after exposure cells to itraconazole. VDAC-HKII interactions following in vitro exposure to itraconazole were determined by immunoprecipitation of VDAC and probing for HKII in RSCL and RRCLs. Result:Itraconazole consistently showed potent, specific, dose-and time- dependent inhibition of all our sensitive and resistant lymphoma cell lines. In vitro exposure cells to itraconazole resulted in a loss of mitochondrial membrane potential and caused G2 cell cycle arrest. Itraconazole significantly had a synergistic anti-tumor effect combined with various chemotherapeutic agents, including doxorubicin, dexamethasone, cisplatin and different generations of proteasome inhibitors (bortezomib, carfilzomib or ixazomib) in both RSCL and RRCL. Western blot and immunoprecipitation studies demonstrated that following exposure to itraconazole, HKII bound less to mitochondrial specific protein VDAC. Complete silencing of HKII (using HKII siRNA interference) resulted in a rescue of loss in the mitochondrial membrane potential induced by intraconazole. Conclusion: Taking together, our data suggest that itraconazole had a potent anti-tumor activity against rituximab-sensitive or resistant pre-clinical models. The disruption of HKII from mitochondria following itraconazole exposure may contribute to lower the mitochondrial membrane potential and enhance the chemotherapeutic efficacy. Our finding highlights itraconazole as a potential therapeutic agent in the treatment of B-cell malignancies, and strongly supports clinical translation of its use. Disclosures No relevant conflicts of interest to declare.

scholarly journals Anti-Tumor Activity of ASTX029, a Dual Mechanism Inhibitor of ERK1/2, in Preclinical AML Models

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 7-8
Author(s):  
Christopher J. Hindley ◽  
Lynsey Fazal ◽  
Joanne M. Munck ◽  
Vanessa Martins ◽  
Alpesh D. Shah ◽  
...  
Keyword(s):  
Cell Lines ◽  
Mapk Pathway ◽  
Mapk Signaling ◽  
Activating Mutations ◽  
Ic50 Value ◽  
Ras Family ◽  
Dual Mechanism ◽  
Anti Tumor Activity

Oncogenic mutations in genes such as the RAS family (KRAS, NRAS or HRAS) or receptor tyrosine kinases (RTKs) drive tumor growth through aberrant activation of the mitogen activated protein kinase (MAPK) signaling pathway. Acute myeloid leukemia (AML) patients frequently exhibit activating mutations in MAPK pathway members, such as NRAS and KRAS, suggesting that these malignancies may be driven by aberrant activation of the MAPK pathway. Targeting of the MAPK pathway has been clinically validated in solid tumors, with agents targeting BRAF and MEK approved for the treatment of BRAF-mutant melanoma. However, there is currently no approved therapy directly targeting activated RAS family members and resistance to MAPK pathway inhibitors is frequently associated with reactivation of MAPK signaling. ERK1/2 (ERK) is a downstream node in the MAPK pathway and therefore represents an attractive therapeutic target for inhibition of MAPK signaling in these settings. We have recently described in vivo anti-tumor activity in MAPK-activated solid tumor models following treatment with ASTX029, a highly potent ERK inhibitor developed using fragment-based drug design. ASTX029 has a distinctive ERK binding mode which confers dual mechanism inhibition of ERK, inhibiting both the catalytic activity of ERK and its phosphorylation by MEK. Here, we demonstrate that ASTX029 is also active in AML models and potently inhibits in vitro and in vivo MAPK signaling and growth in these models. Using a panel of 15 AML cell lines, we investigated sensitivity to ASTX029 in vitro. We observed that 8 cell lines bearing mutations leading to increased MAPK pathway signaling were sensitive to treatment with ASTX029 with an average IC50 value of 47 nM, in contrast to an average IC50 value of 1800 nM for cell lines without activating mutations. The phosphorylation of RSK, a direct substrate of ERK, was suppressed for up to 24 h following treatment with ASTX029 in vitro. We have previously demonstrated good oral bioavailability for ASTX029 and once daily dosing resulted in significant tumor growth inhibition in AML cell line xenograft models. To confirm target engagement in vivo, we examined MAPK signaling in xenograft tissue and observed inhibition of the phosphorylation of RSK and of ERK itself, consistent with the dual mechanism of action proposed for ASTX029. In summary, the ERK inhibitor, ASTX029, has potent activity against MAPK-activated tumor models, including AML models, and is now being tested in a Phase 1/2 clinical trial in advanced solid tumors (NCT03520075). These data highlight its therapeutic potential for the treatment of AML in patients with mutations leading to MAPK pathway activation and support further investigation in these patient populations. Disclosures Hindley: Astex Pharmaceuticals: Current Employment. Fazal:Astex Pharmaceuticals: Current Employment. Munck:Astex Pharmaceuticals: Current Employment. Martins:Astex Pharmaceuticals: Current Employment. Shah:Astex Pharmaceuticals: Current Employment. Wilsher:Astex Pharmaceuticals: Current Employment. Wallis:Astex Pharmaceuticals: Current Employment. Keer:Astex Pharmaceuticals, Inc.: Current Employment. Lyons:Astex Pharmaceuticals: Current Employment.

scholarly journals Inhibition of MEK and DDR Pathways Induces Synergistic Killing of Novel Mll-Af4 B-ALL Model Harboring Activated Ras Mutations

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1511-1511
Author(s):  
Evelyn J Song ◽  
S. Haihua Chu ◽  
Janna Minehart ◽  
Jonathan Chabon ◽  
Richard Koche ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Mouse Model ◽  
Small Molecule ◽  
Single Agent ◽  
Short Latency ◽  
Ras Mutations ◽  
In Vivo Treatment

Abstract Childhood B-cell acute lymphoblastic leukemia (B-ALL) that harbor a translocation of the MLL1 and AF4 genes are considered high-risk with poor prognosis (event-free survival (EFS) of 35%-50%), especially when compared to non-MLL-rearranged (MLL-R) childhood ALL (EFS >85%). An important obstacle to developing new therapeutic approaches for this patient population is the lack of models that faithfully recapitulate the short latency and aggressiveness of this disease. Recently, whole genome sequencing of patient childhood MLL-R leukemias revealed that activating mutations of the proto-oncogenes involved in signaling, most prominently, N or K-RAS were found in nearly 50% of patients. Patients with these co-occurring mutations have an even poorer overall survival rate, indicating that a model harboring both mutations is of extreme interest. Here, we report the generation of a highly aggressive, serially transplantable B-ALL by the retroviral overexpression of activating N-RasG12D mutant in bone marrow of an inducible knock-in Mll-Af4 murine model that we have previously published. Recipient mice injected with Mll-Af4/N-RasG12Dpre-leukemic bone marrow cells developed an acute B-ALL (B220+CD43+IgM-) with short latency to development of disease (median 35 days). Furthermore, the resultant primary B-ALL was serially transplantable into sub-lethally irradiated recipients with accelerated latency to secondary and tertiary disease developing at a median of 20 and 12 days, respectively. As our model includes an activating mutation in N-Ras, we wanted to see if the cells would be sensitive to small molecule inhibitors of downstream effectors of Ras. Pre-leukemic Mll-Af4/N-RasG12D cells were sensitive to two different MEK inhibitors, Trametinib or PD901, in vitro. Furthermore, in vivo treatment of tertiary B-ALL mice with Trametinib showed significant reduction in leukemia burden after 7 days of treatment, as well as increase in survival, compared to vehicle controls. However, prolonged in vivo treatment with Trametinib eventually led to loss of sensitivity and development of B-ALL in our mouse model, suggesting that Trametinib alone is insufficient to prevent leukemia progression. As single agent MEK inhibition was insufficient to generate long-term durable responses, we conducted RNA-Sequencing of primary Mll-Af4/N-RasG12D leukemias to discover pathways amenable for therapeutic intervention. Gene set enrichment analysis suggested that targeting the DNA damage response (DDR) pathway as an attractive therapeutic opportunity. We were able to demonstrate an increased basal level of replicative stress in our Mll-Af4/NrasG12D pre-leukemic cells and sensitivity to small molecule inhibition of ATR, a master regulator of the G2 to M transition of cell cycle progression, with AZ20, a selective ATR inhibitor. In vitro and in vivo treatment with AZ20 led to increased leukemia cytotoxicity. However, similar to Trametinib treatment, tertiary B-ALL mice eventually succumbed to disease with prolonged AZ20 treatment in vivo. Since neither single agent MEK nor ATR inhibition could prevent leukemic progression in vivo, we tested the combination and found increased cytotoxicity and cell cycle arrest in vitro at concentrations well below the IC50, as compared to single agent treatment. In vivocombination treatment also demonstrated decreased leukemia burden and significant prolonged survival compared to either AZ20 or Trametinib alone. Lastly, we tested out the efficacy of combination therapy in human B-ALL patient derived xenograft harboring both MLL-AF4 and activating N-RASmutations. 250,000 human primary leukemic blasts were transplanted into non-irradiated immune-compromised mice and treated with vehicle, single agent, or the combination for 14 days. Similar to the results seen in our mouse model, combination treatment with Trametinib and AZ20 led to significant reductions in leukemic burden. In summary, our model of B-ALL faithfully recapitulates the short latency and aggressiveness of this disease and was predictive of response in human patient samples harboring MLL-AF4 and activating N-RAS mutations to small molecule inhibitor therapy to MEK and DDR pathways. In the future, this model can be used as a platform to not only better understand the molecular events governing and sustaining leukemogenesis, but also as a discovery platform for novel therapeutic combinations. Disclosures Armstrong: Epizyme, Inc: Consultancy; Vitae Pharmaceuticals: Consultancy; Imago Biosciences: Consultancy; Janssen Pharmaceutical: Consultancy.

Investigation of a novel irreversible pan-HER inhibitor combined with chemotherapy in bladder cancer models.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. 4545-4545
Author(s):  
Petros Grivas ◽  
Kathleen C. Day ◽  
Stephanie Daignault ◽  
Nazia Shakir ◽  
Alyssa Paul ◽  
...  
Keyword(s):  
Weight Loss ◽  
Bladder Cancer ◽  
Biomarker Discovery ◽  
Single Agent ◽  
Phase Iii ◽  
G1 Arrest ◽  
Cancer Models ◽  
Anti Tumor Activity

4545 Background: Human Epidermal Receptors (HER) play an important role in bladder cancer (BCa) progression and may mediate chemotherapy resistance. Dacomitinib (Dac) is a novel, potent, irreversible pan-HER inhibitor with activity in several solid tumors, currently in phase III trials in NSCLC. We showed that Dac has single agent anti-tumor activity in human BCa models in vitro and in vivo, inducing apoptosis and G1 arrest. We hypothesized that Dac has additive effects with Gemcitabine (G) and Cisplatin (C) in BCa xenografts. Methods: UM-UC-6 (UC6) or UM-UC-9 (UC9) xenografts were established in age-matched NOD/SCID mice. A week after injection, mice had small tumors, were randomized and treated with i. G 50mg/kg + C 2mg/kg via 3 weekly intra-peritoneal injections (IPI) + daily p.o. buffer for 3 weeks; ii. Dac 6mg/kg p.o. daily for 3 weeks + 3 weekly IPI (saline); iii. GC (same dose/schedule) + Dac starting 1 day after GC (based on cell cycle effect and kinetics); iv. no treatment (control). Mice were monitored daily, weighed weekly, sacrificed at 4 weeks and tumors were weighed. 3 tumors/group were stained for EGFR, HER2, Ki67, E-cadherin, ALDH, p-EGFR, p-ERK, p-Akt. 3rd GC dose in UC6 model was given at 50% due to weight loss; all GC doses were given at 50% in UC9 model. Mann-Whitney test with multiple comparison adjustments was used for analysis. Results: Dac- and GC+Dac-treated mice had no significant weight loss. UC6 tumor weights were significantly lower in Dac and GC+Dac vs control (p<0.0001) or GC (p<0.0001), corresponding to decreased p-ERK %cell expression and staining intensity. GC and control had similar tumor weights (p=0.19). 5 Dac and 3 GC+Dac UC6-injected mice had no tumor at 4 weeks. UC9 tumor weights were significantly lower in Dac (p=0.002, 6x reduction) or GC (p=0.0006; 7x reduction) vs control. GC+Dac had significantly lower tumor weights vs GC (p=0.005), Dac (p=0.06) or control (p<0.0001; 17x reduction). Conclusions: Dac had dramatic singe-agent activity in UC6 xenograft that was GC-resistant. Dac+GC was superior to GC in UC9 xenograft, supporting clinical evaluation. Further investigation of Dac anti-tumor activity and predictive biomarker discovery in additional bladder cancer models is pursued.

Entinostat, a Novel Histone Deacetylase (HiDAC) Inhibitor Enhances the Anti-Tumor Activity of Bortezomib (BTZ) in Rituximab-Chemotherapy Sensitive and Resistant Lymphoma Cell Lines,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3734-3734
Author(s):  
Cory Mavis ◽  
Sarah Frys ◽  
Juan Gu ◽  
John Gibbs ◽  
Myron S. Czuczman ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Lines ◽  
Research Funding ◽  
Cell Lymphoma ◽  
Single Agent ◽  
B Cell Lymphoma ◽  
Lymphoma Cells ◽  
In Vitro Exposure ◽  
Anti Tumor Activity

Abstract Abstract 3734 Deacetylases (DACs) are enzymes that remove the acetyl groups from target proteins [histones (class I) and non-histone proteins (class II)], leading to regulation of gene transcription and other cellular processes. Entinostat (MS-275) is a novel and potent DAC class I inhibitor undergoing pre-clinical and clinical testing. In order to better characterize the role of DAC inhibitors in the treatment of refractory/resistant (r/r) B-cell lymphoma, we studied the anti-tumor activity of entinostat as a single agent or in combination with the proteasome inhibitor bortezomib (BTZ) against a panel of rituximab-[chemotherapy]-sensitive cell lines (RSCL), rituximab-[chemotherapy]-resistant cell lines (RRCL), and primary lymphoma cells isolated from patients with treatment-naïve or r/r B-cell lymphoma. In addition, we characterized the mechanisms responsible for entinostat's anti-tumor activity. Non-Hodgkin lymphoma (NHL) cell lines were exposed to escalating doses of entinostat (0.1 to 20uM) +/− BTZ (1–10nM). Changes in mitochondrial potential and ATP synthesis were determined by alamar blue reduction and cell titer glo luminescent assays, respectively. Changes in cell cycle were determined by flow cytometric analysis. Subsequently, protein lysates were isolated from entinostat +/− BTZ exposed cells and changes in members of Bcl-2 and cell cycle family proteins were evaluated by Western blotting. Finally, to characterize entinostat's mechanisms-of-action, lymphoma cells were exposed to entinostat with or without pan-caspase (Q-VD-OPh, 5mM) and changes in cell viability were detected. Entinostat exhibited dose-dependent activity as a single agent against RSCL, RRCL and patient-derived primary tumor cells (N=32). In addition, in vitro exposure of lymphoma cells to entinostat resulted in an increase in G1 and a decrease in S phase. Moreover synergistic activity was observed by combining entinostat with BTZ in vitro. The pharmacological interactions between entinostat and proteasome inhibitor could be explained in part by each agent's effects on the expression levels of cell cycle proteins. In vitro exposure of lymphoma cells to entinostat resulted in p21 upregulation and p53 down-regulation, whereas BTZ exposure lead to up-regulation of Bak and Noxa and downregulation of Mcl-1 and Bcl-XL. Caspase inhibition diminished entinostat anti-tumor activity in RSCL but not in RRCL. Together this data suggests that entinostat has a dual mechanism-of-action and can induce cell death by caspase-dependent and independent pathways. Our data suggests that entinostat as a single agent is active against rituximab-chemotherapy sensitive and resistant lymphoma cells and potentiates the anti-tumor activity of BTZ. A better understanding in the molecular events (caspase-dependent and -independent) triggered by entinostat in combination with proteasome inhibition is important in order to develop optimal combination strategies using these novel agents in future clinical trials. Disclosures: Czuczman: Millennium: Honoraria, Research Funding. Hernandez-Ilizaliturri:Genmab: Research Funding; Amgen: Research Funding; Celgene: Consultancy.

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