ABL001, a Potent Allosteric Inhibitor of BCR-ABL, Prevents Emergence of Resistant Disease When Administered in Combination with Nilotinib in an in Vivo Murine Model of Chronic Myeloid Leukemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 398-398 ◽  
Author(s):  
Andrew Wylie ◽  
Joseph Schoepfer ◽  
Giuliano Berellini ◽  
Hongbo Cai ◽  
Giorgio Caravatti ◽  
...  
Keyword(s):  
Biomedical Research ◽  
Tumor Regression ◽  
Single Agent ◽  
Point Mutations ◽  
Xenograft Model ◽  
Kinase Domain ◽  
X Ray Crystallography ◽  
N Terminus ◽  
Emergence Of Resistance

Abstract Background: Chronic myelogenous leukemia (CML) and a subset of acute lymphoblastic leukemia (ALL) are caused by the t(9;22)(q34;q11.2) chromosome translocation, resulting in fusion of the BCR and ABL1 genes on the Philadelphia chromosome to encode constitutively active ABL1 kinase. Despite the dramatic progress made over the past decade with tyrosine kinase inhibitors (TKIs) in the treatment of CML, allogeneic stem cell transplant is considered the only proven curative therapy. To achieve cure or benefit from treatment-free remissions with pharmacologically-based therapies, it is estimated that patients will likely need to achieve a sustained reduction in tumor burden of 4 logs (MR4) or deeper (MR4.5). Currently, only 39% and 18% of patients achieve MR4 by 24 months of treatment with single agent nilotinib or imatinib, respectively. Furthermore, for a subset of CML patients and the majority of Ph+ ALL patients, resistance develops to current TKI’s as a result of emergence of point mutations in the ATP site of the kinase domain. ABL001 is a potent, selective BCR-ABL inhibitor that maintains activity across most mutations, including T315I, with a distinct, allosteric mechanism of action which recently entered Phase I development for the treatment of patients with CML and Ph+ ALL. ABL001 was developed to be dosed in combination with nilotinib to provide greater pharmacological coverage of BCR-ABL disease and prevent the emergence of resistance. Methods: Based on X-ray crystallography, NMR and molecular modeling, ABL001 is the result of a structure-guided medicinal chemistry program targeting the myristoyl pocket of the ABL1 kinase. In vitro cell based assays were performed using the Ba/F3 isogenic cell system and a panel of over 300 cell lines. KCL-22 cells were used to develop an in vivo xenograft model to assess the efficacy of ABL001 and the PD marker, pSTAT5, was used to monitor the inhibition of BCR-ABL signaling. Results: In contrast to TKIs that bind to the ATP-site of the ABL1 kinase domain, NMR and X-Ray crystallography studies confirmed that ABL001 binds to a pocket on the BCR-ABL kinase domain that is normally occupied by the myristoylated N-terminus of ABL1. Upon fusion with BCR, this myristoylated N-terminus that serves to autoregulate ABL1 activity is lost. ABL001 functionally mimics the role of the myristoylated N-terminus by occupying its vacant binding site and restores the negative regulation of the kinase activity. Cell proliferation studies demonstrate that ABL001 selectively inhibited the growth of CML and Ph+ ALL cells with potencies ranging from 1-10nM range. In contrast, BCR-ABL-negative cell lines remained unaffected at concentrations 1000-fold higher. With resistance emerging in the clinic to current TKI’s as a result of point mutations in the ATP-site, ABL001 was tested for activity against clinically observed mutations and found to be active in the low nM range. In the KCL-22 mouse xenograft model, ABL001 displayed potent anti-tumor activity with complete tumor regression observed and a clear dose-dependent correlation with pSTAT5 inhibition. The KCL-22 xenograft model was also used to compare the dosing of ABL001 and nilotinib as single agents to dosing a combination of ABL001 and nilotinib. Single agent dosing regimens led to tumor regressions; however, despite continuous dosing, all tumors relapsed within 30-60 days with evidence of point mutations in the resistant tumors. In contrast, animals treated with the combination of ABL001 and nilotinib achieved sustained tumor regression with no evidence of disease relapse either during the 70 days of treatment or for > 150 days after treatment stopped. Conclusion: ABL001 selectively inhibited the proliferation of cells expressing the BCR-ABL fusion gene and was active against clinically important mutations that arise with current TKI therapy in CML. In an in vivo model of CML, the combination of ABL001 and nilotinib resulted in complete and sustained tumor regression with no evidence of disease relapse. These results provide proof-of-principle that simultaneous targeting of the myristoyl pocket and ATP-pocket by ABL001 and nilotinib, respectively, promotes a more sustained overall efficacy and prevents the emergence of resistance via acquisition of point mutations in the respective binding sites. ABL001 is currently being evaluated in a Phase 1 study in patients with CML and Ph+ ALL. Disclosures Wylie: Novartis Institutes for Biomedical Research, Inc: Employment. Schoepfer:Novartis Institutes for Biomedical Research: Employment. Berellini:Novartis Institutes for Biomedical Research: Employment. Cai:Novartis Institutes for Biomedical Research: Employment. Caravatti:Novartis Institutes for Biomedical Research: Employment. Cotesta:Novartis Institues for Biomedical Research: Employment. Dodd:Novartis Institutes for Biomedical Research: Employment. Donovan:Novartis Institutes for Biomedical Research: Employment. Erb:Novartis Institutes for Biomedical Research: Employment. Furet:Novartis Institutes for Biomedical Research: Employment. Gangal:Novartis Institutes for Biomedical Research: Employment. Grotzfeld:Novartis Institutes for Biomedical Research: Employment. Hassan:Novartis Institutes for Biomedical Research: Employment. Hood:Novartis Institutes for Biomedical Research: Employment. Iyer:Novartis Institutes for Biomedical Research: Employment. Jacob:Novartis Institutes for Biomedical Research: Employment. Jahnke:Novartis Institutes for Biomedical Research: Employment. Lombardo:Novartis Institutes for Biomedical Research: Employment. Loo:Novartis Institutes for Biomedical Research: Employment. Manley:Novartis Institutes for Biomedical Research: Employment. Marzinzik:Novartis Institutes for Biomedical Research: Employment. Palmer:Novartis Institutes for Biomedical Research: Employment. Pelle:Novartis Institutes for Biomedical Research: Employment. Salem:Novartis Institutes for Biomedical Research: Employment. Sharma:Novartis Institutes for Biomedical Research: Employment. Thohan:Novartis Institutes for Biomedical Research: Employment. Zhu:Novartis Institutes for Biomedical Research: Employment. Keen:Novartis Institutes for Biomedical Research: Employment. Petruzzelli:Novartis Institutes for Biomedical Research: Employment. Vanasse:Novartis: Employment, Equity Ownership. Sellers:Novartis: Employment.

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.

Combined SHP2 and ERK inhibition for the treatment of KRAS-driven Pancreatic Ductal Adenocarcinoma

2021 ◽  
Author(s):  
Katrin J Ciecielski ◽  
Antonio Mulero-Sanchez ◽  
Alexandra Berninger ◽  
Laura Ruiz Canas ◽  
Astrid Bosma ◽  
...  
Keyword(s):  
Pancreatic Ductal Adenocarcinoma ◽  
Tumor Regression ◽  
Mapk Pathway ◽  
Single Agent ◽  
Ductal Adenocarcinoma ◽  
Good Tolerability ◽  
Recent Emergence ◽  
Erk Inhibition

Mutant KRAS is present in over 90% of pancreatic as well as 30-40% of lung and colorectal cancers and is one of the most common oncogenic drivers. Despite decades of research and the recent emergence of isoform-specific KRASG12C-inhibitors, most mutant KRAS isoforms, including the ones frequently associated with pancreatic ductal adenocarcinoma (PDAC), cannot be targeted directly. Moreover, targeting single RAS downstream effectors induces adaptive mechanisms leading to tumor recurrence or resistance. We report here on the combined inhibition of SHP2, a non-receptor tyrosine phosphatase upstream of KRAS, and ERK, a serine/threonine kinase and a key molecule downstream of KRAS in PDAC. This combination shows synergistic anticancer activity in vitro, superior disruption of the MAPK pathway, and significantly increased apoptosis induction compared to single-agent treatments. In vivo, we demonstrate good tolerability and efficacy of the combination. Concurrent inhibition of SHP2 and ERK induces significant tumor regression in multiple PDAC mouse models. Finally, we show evidence that 18F-FDG PET scans can be used to detect and predict early drug responses in animal models. Based on these compelling results, we will investigate this drug combination in a clinical trial (SHERPA, SHP2 and ERK inhibition in pancreatic cancer, NCT04916236), enrolling patients with KRAS-mutant PDAC.

YM155, a Novel Survivin Suppressant, Improves the Antitumor Effect of Rituximab and Rituximab-Containing Regimens in Diffuse Large B Cell Lymphoma (DLBCL) Xenograft Mouse Models.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2718-2718
Author(s):  
Mari Nakata ◽  
Takahito Nakahara ◽  
Aya Kita ◽  
Keisuke Mitsuoka ◽  
Kentaro Yamanaka ◽  
...  
Keyword(s):  
Antitumor Effect ◽  
Cell Lymphoma ◽  
Tumor Regression ◽  
Xenograft Model ◽  
Body Weight Loss ◽  
B Cell Lymphoma ◽  
In Vivo Studies ◽  
Large B Cell Lymphoma ◽  
Large B Cell

Abstract Abstract 2718 Poster Board II-694 Introduction: Survivin is a member of the inhibitor of apoptosis (IAP) family of proteins, and is highly expressed in many tumor types. Given its preferential expression in tumor cells, and its ability to block apoptosis and regulate cancer cell proliferation, survivin appears to be an attractive novel target for cancer therapy. YM155 is a novel, small molecule survivin suppressant (Nakahara et al., Cancer Research. 2007;67:8014–21). In this study, we evaluated the antitumor activity of YM155 alone and in combination with rituximab, R-ICE (rituximab + ifosfamide + carboplatin + etoposide), or [rituximab + cytarabin + cisplatin] in DLBCL xenograft models. Methods: Antiproliferative effect of YM155 in a panel of human DLBCL cell lines (DB, Pfeiffer, SU-DHL5, SU-DHL8, WSU-DLCL-2, and RL) was evaluated by sulforhodamine B assay. In in vivo studies, WSU-DLCL-2 and DB were subcutaneously implanted into male BALB/c nu/nu mice. When tumors reached a volume of 300 to 600 mm3, YM155 was administered as a 7-day continuous sc infusion, and the other drugs were administered via iv bolus. Dose and schedule of each drug were adjusted to clinical equivalent dose. PET imaging studies were performed using a Inveon PET/CT system (Siemens Medical Solusion). WSU-DLCL-2 xenografted mice were intravenously injected with [18F] FLT, and five-minute static PET scans were accqiured at 1h after injection. For each small-animal PET scan, region of interest was drawn over each tumor and over normal tissue on decay-corrected whole-body sagittal imagies. Results: In in vitro proliferation assays, YM155 showed potent antiproliferative activity against all six DLBCL cell lines, with GI50 values of 0.35 to 3.9 nM. In in vivo studies using WSU-DLCL2 xenograft model, YM155 at 1 and 3 mg/kg induced tumor regression without body weight loss. In combination studies using WSU-DLCL2 xenograft model, YM155 2 mg/kg enhanced antitumor effects of rituximab, R-ICE and [rituximab + cytarabin + cisplatin] without enhancement of the body weight loss. Tumor regression in the combination groups was sustained longer than single treatment groups, and even complete regressions were achievable. Moreover, combination of YM155 1 mg/kg and rituximab induced strong tumor regression in the DB xenograft model, while single-agent treatments did not show significant antitumor effect compared to vehicle control. In [18F]FLT-PET imaging, a significant reduction of FLT uptake in tumor was observed in rituximab combination group, which was more sensitive than the reduction in tumor volume. Conclusions: YM155 improves the antitumor effect of rituximab and rituximab-containing regimens in diffuse large B cell lymphoma (DLBCL) xenograft mouse models. Disclosures: Nakata: Astellas Pharma Inc.: Employment. Nakahara:Astellas Pharma Inc.: Employment. Kita:Astellas Pharma Inc.: Employment. Mitsuoka:Astellas Pharma Inc.: Employment. Yamanaka:Astellas Pharma Inc.: Employment. Kaneko:Astellas Pharma Inc.: Employment. Miyoshi:Astellas Pharma Inc.: Employment. Mori:Astellas Pharma Inc.: Employment. Koutoku:Astellas Pharma Inc.: Employment. Sasamata:Astellas Pharma Inc.: Employment.

Azacitidine/Decitabine Synergism with the NEDD8-Activating Enzyme Inhibitor MLN4924 in Pre-Clinical AML Models

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 578-578 ◽  
Author(s):  
Peter G Smith ◽  
Tary Traore ◽  
Steve Grossman ◽  
Usha Narayanan ◽  
Jennifer S Carew ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Cell Death ◽  
Synthetic Lethality ◽  
Single Agent ◽  
Xenograft Model ◽  
S Phase ◽  
Myelogenous Leukemia ◽  
Phase Accumulation

Abstract Abstract 578 MLN4924 is an investigational small molecule inhibitor of NEDD8-activating enzyme that has shown clinical activity in a Phase I clinical trial in Acute Myelogenous Leukemia (AML). To identify potential combination partners of MLN4924 we performed a high-throughput viability screen in AML cells with 40 approved and investigational agents. In vitro characterization of AML cell lines revealed two distinct cell cycle phenotypes suggesting alternate mechanism of action following MLN4924 inhibition of NAE. One group demonstrated moderate S-phase accumulation with greater than 4N DNA content consistent with DNA-rereplication as a result of CDT1 dysregulation. The second group demonstrated distinct and rapid accumulation of subG1 cells without S-phase accumulation or DNA re-replication suggesting induction of apoptosis and cell death. These observations led us to choose two cells lines representative of each mechanism to understand potential for synergy in AML cells. Two hypomethylating agents were included in the screen (decitabine and azacitidine) and were found to be synergistic with MLN4924 by Combination Index and Blending Synergy Analysis. These data were confirmed with a second NAE inhibitor that is structurally dissimilar to MLN4924. The combination of azacitidine and MLN4924 were shown to result in significantly increased DNA-damage and cell death compared to single agent alone as measured by Western Blotting and FACS analysis of cell cycle distributions. In vivo studies were performed in HL-60 and THP-1 xenografts using MLN4924 on a clinically relevant dosing schedule twice weekly. Single agent azacitidine at its Maximum Tolerated Dose (MTD) had minimal activity in the HL-60 model and was combined with a sub-optimal dose of MLN4924 that when combined induced complete and sustained tumor regressions. The mechanism for the apparent synthetic lethality in this in vivo model is currently under evaluation; however it is supported by a dramatic elevation in DNA damage and cleaved caspase-3 in vivo in the combination arm. A second xenograft model (THP-1) that was also insensitive to single agent azacitidine treatment underwent complete and sustained tumor regressions when combined with MLN4924. Thus MLN4924 and azacitidine can combine to produce synergistic antitumor activity in pre-clinical models of AML. Coupled with their non-overlapping clinical toxicities these data suggest the potential for future combination studies in clinical trials. Disclosures: Smith: Millennium Pharmaceuticals: Employment. Traore:Millennium Pharmaceuticals: Employment. Grossman:Millennium Pharmaceuticals: Employment. Narayanan:Millennium Pharmaceuticals: Employment. Carew:Millennium Pharmaceuticals: Research Funding. Lublinksky:Millennium Pharmaceuticals: Employment. Kuranda:Millennium Pharmaceuticals: Employment. Milhollen:Millennium Pharmaceuticals: Employment.

Carfilzomib Platform Study Using The LAGκ-1A Multiple Myeloma Xenograft Model

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4452-4452
Author(s):  
Eric Sanchez ◽  
Mingjie Li ◽  
Suzie Vardanyan ◽  
Jillian Gottlieb ◽  
Kevin Delijani ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Disease Progression ◽  
Tumor Size ◽  
Single Agent ◽  
Xenograft Model ◽  
In Vivo Studies ◽  
Human Igg ◽  
Similar Treatment ◽  
Agent Treatment

Introduction We previously demonstrated that severe combined immunodeficient (SCID) mice bearing the human multiple myeloma (MM) xenograft LAGκ-1A treated with single agent carfilzomib or the alkylating agent (AA) cyclophosphamide (CY) did not show a reduction in tumor growth compared to vehicle-treated mice. In contrast, carfilzomib with CY resulted in a significant decrease in tumor size and IgG levels when compared to mice treated with single agent carfilzomib or CY or vehicle alone. We have also shown that the combination of carfilzomib and another AA, bendamustine, decreased tumor size and IgG levels, when compared to mice treated with single agents or vehicle alone. However, no data is available regarding sequencing of the proteasome inhibitors (PI) carfilzomib or bortezomib with the AA melphalan (MEL). Thus, we used our SCID-hu MM models to evaluate the sequencing of these drugs with MEL. These studies are critical as both PIs are now being used to treat MM. Thus, we evaluated the response, toxicity and survival of animals treated sequentially with these drugs. Methods Each naïve SCID mouse was surgically implanted with a 20 – 40 mm3 MM tumor piece into the left hind limb superficial gluteal muscle. Seven days post–implantation mice were randomized into treatment groups based on human immunoglobulin (Ig) G levels. Carfilzomib stock solution (2 mg/ml) was diluted to 3 mg/kg using 5% dextrose and administered twice weekly on two consecutive days via intravenous (i.v.) injection. Bortezomib stock solution (1 mg/ml) was diluted to 0.25 mg/kg using NaCl and administered twice weekly (Thursdays and Saturdays) via i.v. injection. MEL stock solution (3 mg/ml) was diluted to 1 mg/kg using PBS and administered once weekly via intraperitoneal injection. Mice (n = 10/group) were initially treated with carfilzomib or MEL alone until tumor progression. Progression was defined as an increase in paraprotein equal to or above 25% confirmed on one consecutive assessment. Mice initially treated with carfilzomib were randomized to continue to receive single agent carfilzomib, add in MEL alone or combine it with ongoing carfilzomib, substitute single agent bortezomib, or discontinue treatment altogether. A similar treatment strategy was evaluated with mice treated initially with MEL. At progression, these animals were continued on single agent MEL, carfilzomib added alone or with continuation of MEL, or discontinued treatment. Tumor size was measured using standard calipers and human IgG levels with an ELISA (Bethyl Laboratories, Montgomery, TX). This study was conducted according to protocols approved by the Institutional Animal Care and Use Committee. Results When carfilzomib was administered first, followed by the addition of MEL, a modest nonsignificant reduction in tumor size was observed compared to either drug alone. In addition, substitution of single agent bortezomib for carfilzomib showed no effect on tumor size. However, when MEL was administered first and carfilzomib was added after disease progression, at days 35 and 42 (end of study) post tumor implantation, mice treated with the combination showed a reduction in tumor volume compared to mice that discontinued melphalan (P = 0.0378 and P = 0.0105, respectively) whereas mice treated with carfilzomib alone showed no reduction in tumor size following progression from MEL. Notably, throughout the study there was a trend toward smaller tumors in mice receiving this combination when compared to mice receiving single agent treatment with carfilzomib or MEL alone or vehicle. Similar effects were observed on human IgG levels. Overall, all mice survived combination or single agent treatment with these agents. Conclusions These in vivo studies using our human MM LAGκ–1A SCID–hu model show that animals progressing from initial MEL treatment show a reduction in MM tumor burden when carfilzomib is added to MEL at progression. In contrast, mice progressing from initial carfilzomib treatment did not benefit from the addition of MEL at disease progression. No drug-related deaths occurred in any treatment group. This study demonstrates that using a different MM model (LAGκ-1A), that the PI carfilzomib can produce anti-tumor effects among mice progressing from single-agent MEL treatment, providing further support for the use of this PI as an agent that can help overcome drug resistance in MM. Disclosures: Berenson: Onyx Pharmaceuticals: Consultancy, Honoraria, Research Funding, Speakers Bureau.

scholarly journals Re-engineered p53 activates apoptosis in vivo and causes primary tumor regression in a dominant negative breast cancer xenograft model

Gene Therapy ◽  
2014 ◽  
Vol 21 (10) ◽  
pp. 903-912 ◽  
Author(s):  
A Okal ◽  
K J Matissek ◽  
S J Matissek ◽  
R Price ◽  
M E Salama ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Primary Tumor ◽  
Tumor Regression ◽  
Xenograft Model ◽  
Dominant Negative ◽  
Breast Cancer Xenograft

scholarly journals The CDK9 Inhibitor, Alvocidib, Potentiates the Non-Clinical Activity of Azacytidine or Decitabine in an MCL-1-Dependent Fashion, Supporting Clinical Exploration of a Decitabine and Alvocidib Combination

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4355-4355
Author(s):  
Wontak Kim ◽  
Clifford Whatcott ◽  
Adam Siddiqui-Jain ◽  
Stephen Anthony ◽  
David J. Bearss ◽  
...  
Keyword(s):  
Protein Expression ◽  
Cell Viability ◽  
Single Agent ◽  
Xenograft Model ◽  
Complete Response ◽  
Tumor Growth Inhibition ◽  
Phase 2 ◽  
Phase 1B ◽  
Cdk9 Inhibitor

Abstract The hypomethylating agents (HMAs) azacytidine and decitabine exert biological activity via two distinct mechanisms, namely, DNA damage and inhibition of DNA methyltransferases. Azacytidine and decitabine are indicated in the treatment of patients with myelodysplastic syndromes (MDS). As a result of DNA methyltransferase inhibition, it is hypothesized that HMAs may function by inducing re-expression of key pro-apoptotic proteins such as NOXA, which sequesters the anti-apoptotic protein MCL-1, preventing its association with the mitochondrial pore-forming proteins BAX/BAK. Activity of the potent CDK9 inhibitor, alvocidib, is largely driven by targeting of CDK9-dependent MCL-1 expression. Alvocidib is under active clinical investigation, but has also has demonstrated high complete response rates in newly diagnosed AML patients, particularly when administered as part of a cytarabine and mitoxantrone containing regimen (ACM regimen). Given the dual NOXA/MCL-1-targeting ability of combining alvocidib and azacytidine or decitabine, the combination may synergize therapeutically in the treatment of non-clinical models of AML or MDS by means of transcriptional induction of NOXA and repression of MCL-1 expression. Cell viability and induction of apoptosis was assessed following treatment with alvocidib, azacytidine, and decitabine in cells using the Celltiter-Glo and Caspase-Glo assays. Gene expression changes following treatment were assessed using quantitative RT-PCR. Protein expression changes with treatment were also measured using standard immunoblotting technique. To assess the in vivo anti-tumor activity of these compounds, xenograft studies in the MOLM13 and additional models of MDS, exploring sequencing and scheduling of alvocidib administration with HMAs, were performed. Treatment of AML cell lines with alvocidib inhibited both mRNA and protein expression of MCL-1 in a time and concentration-dependent fashion. Pre-treatment of cells with alvocidib, to repress MCL-1 expression prior to azacytidine treatment, reduced the azacytidine cell viability EC50 more than 2.5-fold, from 1.8 µM to 0.6 µM in MV4-11 cells. The alvocidib/azacytidine combination also resulted in synergistic increases in caspase activity relative to either single agent within the combination, at multiple dose levels. The combination of azacytidine or decitabine with alvocidib was active in the MOLM13 xenograft model, yielding up to 65.7 or 91.1% tumor growth inhibition (%TGI) in the azacytidine or decitabine combination, respectively. Taken together, the in vitro and in vivo studies indicated that decitabine was more effective at re-expressing NOXA and potentiating alvocidib activity compared to azacytidine. These non-clinical data suggest that an alvocidib/HMA combination may constitute a viable therapeutic regimen whose rationale focuses on hypertargeting of NOXA/MCL-1. Based on these non-clinical results, a Phase 1b/2 clinical study of alvocidib administered in sequence after decitabine in patients with intermediate to high risk MDS is being conducted (Zella 102). Patients will be enrolled in cohorts of 3-6 patients with decitabine administered as a 1-hour IV infusion daily on days 1 to 5 at a dose of 20 mg/m2 followed by a single alvocidib treatment on day 8 as a loading dose over 30 minutes followed by a 4-hour infusion. Treatment will be repeated every 28 days until disease progression or unacceptable toxicity. Enrollment will include MDS patients (Phase 1b) with previously untreated MDS and patients who received fewer than six (6) cycles of previous HMAs, as well as (Phase 2) untreated patients with de novo or secondary MDS. The primary objective is to determine the maximum tolerated dose and recommended Phase 2 dose of alvocidib when administered in sequence with decitabine. Key Phase 2 endpoints will include complete response rate and improvement in transfusion dependency. Disclosures Kim: Tolero Pharmaceuticals, Inc: Employment. Whatcott:Tolero Pharmaceuticals, Inc: Employment. Siddiqui-Jain:Tolero Pharmaceuticals, Inc: Employment. Anthony:Tolero Pharmaceuticals, Inc: Employment. Bearss:Tolero Pharmaceuticals, Inc: Employment. Warner:Tolero Pharmaceuticals: Employment.

scholarly journals Upregulation of ERK-EGR1-heparanase axis by HDAC inhibitors provides targets for rational therapeutic intervention in synovial sarcoma

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Cinzia Lanzi ◽  
Enrica Favini ◽  
Laura Dal Bo ◽  
Monica Tortoreto ◽  
Noemi Arrighetti ◽  
...  
Keyword(s):  
Synovial Sarcoma ◽  
Cell Response ◽  
Histone Deacetylases ◽  
Molecular Mechanisms ◽  
Combined Treatment ◽  
Single Agent ◽  
Hdac Inhibitors ◽  
Xenograft Model

Abstract Background Synovial sarcoma (SS) is an aggressive soft tissue tumor with limited therapeutic options in advanced stage. SS18-SSX fusion oncogenes, which are the hallmarks of SS, cause epigenetic rewiring involving histone deacetylases (HDACs). Promising preclinical studies supporting HDAC targeting for SS treatment were not reflected in clinical trials with HDAC inhibitor (HDACi) monotherapies. We investigated pathways implicated in SS cell response to HDACi to identify vulnerabilities exploitable in combination treatments and improve the therapeutic efficacy of HDACi-based regimens. Methods Antiproliferative and proapoptotic effects of the HDACi SAHA and FK228 were examined in SS cell lines in parallel with biochemical and molecular analyses to bring out cytoprotective pathways. Treatments combining HDACi with drugs targeting HDACi-activated prosurvival pathways were tested in functional assays in vitro and in a SS orthotopic xenograft model. Molecular mechanisms underlying synergisms were investigated in SS cells through pharmacological and gene silencing approaches and validated by qRT-PCR and Western blotting. Results SS cell response to HDACi was consistently characterized by activation of a cytoprotective and auto-sustaining axis involving ERKs, EGR1, and the β-endoglycosidase heparanase, a well recognized pleiotropic player in tumorigenesis and disease progression. HDAC inhibition was shown to upregulate heparanase by inducing expression of the positive regulator EGR1 and by hampering negative regulation by p53 through its acetylation. Interception of HDACi-induced ERK-EGR1-heparanase pathway by cell co-treatment with a MEK inhibitor (trametinib) or a heparanase inhibitor (SST0001/roneparstat) enhanced antiproliferative and pro-apoptotic effects. HDAC and heparanase inhibitors had opposite effects on histone acetylation and nuclear heparanase levels. The combination of SAHA with SST0001 prevented the upregulation of ERK-EGR1-heparanase induced by the HDACi and promoted caspase-dependent cell death. In vivo, the combined treatment with SAHA and SST0001 potentiated the antitumor efficacy against the CME-1 orthotopic SS model as compared to single agent administration. Conclusions The present study provides preclinical rationale and mechanistic insights into drug combinatory strategies based on the use of ERK pathway and heparanase inhibitors to improve the efficacy of HDACi-based antitumor therapies in SS. The involvement of classes of agents already clinically available, or under clinical evaluation, indicates the transferability potential of the proposed approaches.

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.

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