Evaluation of combined BCL-2/MCL-1 inhibition as a therapeutic approach for synovial sarcoma.

e23561 Background: While Synovial Sarcoma (SS) only accounts for about 10% of soft tissue sarcomas (STS), or ~10,000 cases/year, ~4,000 of these will be fatal. Little benefit has been seen with newer combination chemotherapies and immune checkpoint inhibitors. SS is a transcription factor-driven cancer characterized by an t(X;18) chromosomal translocation resulting in the SS18-SSX fusion gene. SS is also known to overexpress the anti-apoptotic protein BCL-2, which has been used as a diagnostic marker for SS. Venetoclax, the FDA approved BH3 mimetic which specifically inhibits BCL-2, has been used to treat BCL-2-driven hematological cancers. Yet, preclinical studies of venetoclax, currently in clinical use for BCL-2 related hematologic malignancies, have failed in SS, raising the possibility of other BCL-2 family members playing a role in resistance. Methods: Publicly available gene expression databases of SS were queried for expression of BCL-2 family member expression, including the endogenous MCL-1 inhibitor, NOXA. Six SS cell lines, including ASKA, HS-SY-II, Yamato, SW982, SYO.1, and an ex vivo line from a SS patient-derived xenograft (PDX) were tested in vitro with combinations of venetoclax and the MCL-1 inhibitor S63845, using cell viability assays. In vivo testing employed both a patient-derived xenograft (PDX) model of SS and a cell line derived (SYO.1) xenograft model. Results: Queries of published gene expression data for SS demonstrated that SS expresses low levels of the endogenous MCL-1 inhibitor, NOXA, nominating a mechanism for intrinsic venetoclax resistance in these tumors, and suggesting rational combination of venetoclax and the MCL-1 inhibitor, S63845. In vitro, SS all cell lines possessing the SS18-SSX fusion gene demonstrated synergistic sensitivity to combined S63845 and venetoclax, despite generally insensitivity to either drug as monotherapy. Importantly, in an SS PDX, we demonstrated S63845 and venetoclax synergize to induce tumor regression. In the SYO.1 xenograft model, combination therapy significantly reduced tumor growth compared to no treatment or single agent groups. Conclusions: These findings provide the preclinical rationale for translation of the rational combination of BCL-2 and MCL-1 inhibitors into clinical trials for SS.

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Preclinical Investigations Demonstrate Effectiveness of Aplidin in Acute Leukemias and Lymphomas and Provide the Basis for Further Clinical Studies.

Blood ◽

10.1182/blood.v104.11.4496.4496 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4496-4496

Author(s):

Debabrata Banerjee ◽

Guray Saydam ◽

Lata G. Menon ◽

Giuseppe S.A. Longo ◽

Daniel Medina ◽

...

Keyword(s):

Gene Expression ◽

Cell Lines ◽

Phase Ii ◽

Myeloid Leukemia ◽

Xenograft Model ◽

Vascular Endothelial ◽

Endothelial Growth Factor ◽

Leukemias And Lymphomas

Abstract Aplidin (dehydrodidemnin B, C57H89N7O15) (APLD) is a novel antitumor agent isolated from the Mediterranean tunicate (seasquirt) Aplidium albicans. APLD has shown impressive in vitro and in vivo activity against different human cancer cells and has recently entered Phase II clinical trials in a variety of solid tumors following promising toxicity and pharmacological properties seen in Phase I studies. Fatigue and muscular pain were the most prevalent toxicities at 5 mg/m2 iv 3 h every other week or 3.4 mg/m2/wk with little or no bone marrow toxicity. APLD inhibits protein synthesis via GTP-dependent elongation factors 1-alpha and ornithine decarboxylase (ODC) activity, induces rapid p53-independent apoptosis in vitro, cell cycle perturbation and alteration of gene expression at early times after treatment. APLD inhibits vascular endothelial growth factor (VEGF) secretion and vascular endothelial growth factor-receptor 1 (VEGF-R1/flt-1), preventing autocrine stimulation in the human lymphoid leukemic cell line MOLT-4 cells and in AML blasts. APLD is a potent inhibitor of human myeloid leukemia cell lines (K-562, HEL and HL60), as well as fresh blast cells obtained from patients with both ALL and AML and is more potent than Idarubicin. Cytototoxic doses effective against multiple myeloma cells and fresh pediatric and adult ALL/AML blasts are achievable in plasma and are well below the recommended dose, thus a positive therapeutic index is anticipated. Moreover, the lack of cross resistance with conventional agents against fresh pediatric and adult AML/ALL blasts except fludarabine and Gemcitabine makes APLD an attractive therapeutic choice. Characterization of gene expression profile is currently underway in an attempt to generate a molecular fingerprint of sensitivity/resistance to APLD that will be validated in phase II clinical studies. Based on in vitro antileukemic effect of APLD as well as early results of clinical trials, a systematic study of drug combinations with Aplidin (APLD), for use possible in hematologic malignancies was undertaken. Three cell lines viz. K562 (acute myeloid leukemia), CCRF-CEM (acute lymphocytic leukemia), and SKI-DLCL (diffuse large cell lymphoma) were used for combination studies. Cytarabine and mitoxantrone were found to be synergistic in combination with APLD in all 3 cell lines as assessed by the Chou-Talalay combination index analysis. Since cytarabine and APLD produced impressive synergistic cell kill in all three cell culture models, the combination was further tested in the CCRF-CEM ALL xenograft model in SCID mice. APLD (0.7 mg/Kg) potentiated the antitumoral effect of cytarabine (50mg/Kg) in vivo. Addition of APLD to cytarabine treatment in xenograft model resulted in greater than 50% reduction in tumor size as compared to the untreated group. T/C ratios indicated that the effect of the combination was maximal at day 5 but was still maintained on day 8 (T/C on day 3 = 0.614; day 5= 0.403 and day 8= 0.703). The preclinical results with APLD in leukemias and lymphomas, as a single agent and in combination with cytarabine provide the basis for implementation of a phase II program in resistant relapsed leukemias and lymphomas.

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Effect of a combination of epigenetic agents on the malignant phenotype in models of T-cell lymphoma.

Journal of Clinical Oncology ◽

10.1200/jco.2012.30.15_suppl.e13569 ◽

2012 ◽

Vol 30 (15_suppl) ◽

pp. e13569-e13569

Author(s):

Enrica Marchi ◽

Matko Kalac ◽

Danielle Bongero ◽

Christine McIntosh ◽

Laura K Fogli ◽

...

Keyword(s):

Gene Expression ◽

T Cell ◽

Cell Lines ◽

Molecular Level ◽

Cell Lymphoma ◽

Single Agent ◽

T Cell Lymphoma ◽

Cytotoxicity Assays

e13569 Background: CHOP and CHOP-like chemotherapy are the most used regimens for the treatment of peripheral T-cell lymphomas (PTCLs) despite sub-optimal results. Histone deacetylase inhibitors (HDACIs) have shown class activity in PTCLs. The interaction between the HDACIs (depsipeptide (R), belinostat (B), vorinostat (V) and panobinostat (P)) and a DNMT inhibitor (decitabine (D) was investigated in vitro, in vivo and at the molecular level in T-cell lymphoma and leukemia cell lines (H9, HH, P12, PF-382). Methods: For cytotoxicity assays, luminescence cell viability assay was used (CellTiter-Glo). Drug:drug interactions were analyzed with relative risk ratios (RRR) based on the GraphPad software (RRR<1 defining synergism). Apoptosis was assessed by Yo-Pro-1 and propidium iodine followed by FACSCalibur acquisition. Gene expression profiling was analyzed using Illumina Human HT-12 v4 Expression BeadChip microarrays and Gene Spring Software for the analysis. Results: The IC50s for B, R, V, P, D and 5-Azacytidine alone were assessed at 24, 48 and 72 hours. In cytotoxicity assays the combination of D plus B, R, V or P at 72 hours showed synergism in all the cell lines (RRRs 0.0007-0.9). All the cell lines were treated with D, B or R for 72 hours and all the combinations showed significantly more apoptosis than the single drug exposures and controls (RRR < 1). In vivo, HH SCID beige mice were treated i.p. for 3 cycles with the vehicle solution, D or B or their combination at increasing dose. The combination cohort showed statistically significant tumor growth inhibition compared to all the other cohorts. Gene expression analysis revealed differentially expressed genes and modulated pathways for each of the single agent treatment and the combination. The effects of the two drugs were largely different (only 39 genes modified in common). Most of the effects induced by the single agent were maintained in the combination group. Interestingly, 944 genes were modulated uniquely by the combination treatment. Conclusions: The combination of a DNMTI and HDACIs is strongly synergistic in vitro, in vivo and at the molecular level in model of T-cell lymphoma and these data will constitute the basis for a phase I-II clinical trials.

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

Journal of Experimental & Clinical Cancer Research ◽

10.1186/s13046-021-02150-y ◽

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.

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The Role of SMAD7 Downstream of TEL-AML1 Signalling in t(12;21) Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v126.23.4823.4823 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4823-4823 ◽

Cited By ~ 1

Author(s):

Aishwarya Sundaresh ◽

Maurizio Mangolini ◽

Jasper de Boer ◽

Mike Hubank ◽

Nicholas Goulden ◽

...

Keyword(s):

Gene Expression ◽

Cell Lines ◽

Conflicts Of Interest ◽

Fusion Gene ◽

Lymphoblastic Leukemia ◽

Xenograft Model ◽

Direct Role ◽

Hematopoietic Stem ◽

Childhood All

Abstract The single most frequent chromosomal translocation associated with childhood ALL is the t(12;21) rearrangement that creates a fusion gene between TEL (ETV6) and AML1 (RUNX1). Although TEL-AML1+ patients have very good prognoses, relapses occur in up to 20% of patients and many patients face long-term side effects of chemotherapy. Recent data has shown that TEL-AML1 has a direct role in inducing signal transducer and activator of transcription 3 (STAT3) activation in human t(12;21) leukemia. This activation has been shown to transcriptionally induce MYC and is critical for survival of TEL-AML+ leukemia cells. Here, we demonstrate that STAT3 also regulates SMAD7 gene expression. SMAD7 is an antagonist of TGF-β signaling, functioning through a negative feedback mechanism, but is also known to function in other biological pathways. Interestingly, SMAD7 has also been shown to play a role in promoting self-renewal of hematopoietic stem cells. We show that both pharmacological and mechanistic inhibition of STAT3 results in down regulation of SMAD7 gene expression in TEL-AML1+ cell lines. This result was specific to TEL-AML1+ cells and not found in cells of other ALL subtypes. To understand the role played by SMAD7 in TEL-AML1+ cells, we used lentiviral vectors expressing shRNA targeting SMAD7. Interestingly, SMAD7 silencing was found to inhibit proliferation of TEL-AML1+ cell lines, eventually leading to growth arrest and apoptosis. Furthermore, we have established that this effect is not mediated through TGF-β signalling. This poster highlights the results of RNA-seq performed on TEL-AML1+ cells with SMAD7 knockdown and in vivo xenograft model of SMAD7 shRNA in TEL-AML+ ALL. Disclosures No relevant conflicts of interest to declare.

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

Journal of Clinical Oncology ◽

10.1200/jco.2020.38.15_suppl.3582 ◽

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.

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Mouse Models of Human Mantle Cell Lymphoma for the Study of Disease Biology and for Pre-Clinical Assessment of Experimental Treatment Approaches.

Blood ◽

10.1182/blood.v120.21.2759.2759 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2759-2759

Author(s):

Pavel Klener ◽

Magdalena Klanova ◽

Tomas Soukup ◽

Jan Molinsky ◽

Jan Zivny ◽

...

Keyword(s):

Gene Expression ◽

B Cell ◽

Cell Lines ◽

Single Agent ◽

Cell Receptor ◽

Ki 67 ◽

Disease Biology ◽

B Cell Receptor Signaling

Abstract Abstract 2759 Mantle cell lymphoma (MCL) is an aggressive type of B-cell non-Hodgkin lymphoma associated with poor prognosis. MCL animal models for the study of disease biology and for the testing of novel agents are scarce. We established and characterized various in vivo models of metastatic blastoid human MCL by tail vein injection of five MCL cell lines (Jeko-1, HBL-2, Mino, Rec-1, Granta-519) into the NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ immunodeficient mice. Untreated animals were then observed to evaluate differences in the pattern of lymphoma growth and overall survival (OS) between different cell lines. We analyzed infiltration of selected murine organs (i.e. bone marrow [BM], spleen, liver, brain, kidneys, and enlarged lymph nodes [LN]) by immunohistochemistry (IHC) (CD20, Ki-67) at four different time-points related to OS. Extent of organ infiltration with human MCL cells was estimated using the Image-Pro Plus 5.1 software within 20 samples from different organ areas. Subsequently, we analyzed gene expression of Jeko-1 and Mino cells obtained from the xenografted animals (in vivo growing cells) compared to the cells cultured in vitro (controls).MCL cells isolated from various murine organs (the BM, liver, spleen, and LN) or in vitro cultured cells were magnetically sorted by CD45-microbeads. Gene expression analyses were carried out using Illumina BeadChips, and the data were functionally clustered with DAVID Bioinformatics tool. In addition, differences in surface expression of selected antigens were compared between in vivo vs. in vitro grown MCL cells by flow cytometry. Finally, we evaluated the anti-tumor activity of single-agent chemotherapy agents (cytarabine, fludarabine, bendamustine, and cisplatin), monoclonal antibodies (rituximab, ofatumumab, bevacizumab) or targeted agents (bortezomib, temsirolimus) in Jeko-1 and Mino bearing mice. Tumor engraftment was achieved in all the cell lines tested. The median overall survival (OS) of mice xenografted with 1–10×106 MCL cells ranged from 22 to 55 days depending on the cell line used. The principal site of engraftment and proliferation niche for all MCL cell lines was the bone marrow. MCL cells disseminated to other murine organs including the spleen, liver and brain. Development of enlarged lymph nodes (peripheral, intraabdominal) and/or extranodal MCL masses (subcutaneous tumors) were associated with Mino, while infiltration of the ovaries was inconstant finding in Jeko-1 xenografted mice. Mice xenografted with Jeko-1, HBL-2 and Granta-519 showed leukemization of peripheral blood before death. Gene expression studies of Jeko-1 and Mino in vivo growing cells revealed that the genes from the “B-cell receptor signaling” and the “oxidative-phosphorylation” pathways were the most upregulated or downregulated, respectively. In vivo growing Jeko-1 cells showed upregulation of CD31/PECAM, CD37, CD38, CD44, CD164, and downregulation of podoplanin and CXCR4. In vivo growing Mino cells had upregulation of CD23, but downregulation of CD37, CD40, CD44, CD54, CD138, CXCR4, CCR7 and podoplanin. Both Jeko-1 and Mino cells isolated from the BM (but not from the spleen, liver or LN) were significantly more sensitive to cytarabine (2–4 fold) and cisplatin (2 fold) than in vitro growing controls. Single-agent therapy of Jeko-1 and Mino bearing mice with either a chemotherapy agent, monoclonal antibody, or targeted agent resulted in significant prolongation of OS compared to untreated controls. Treatment of Jeko-1 and HBL2 bearing mice with single-agent cisplatin, single-agent cytarabine or combination of both agents revealed that the therapy with single-agent cisplatin was associated with the longest prolongation of OS. Moreover, IHC analyses of the BM, spleen and liver of the treated animals confirmed the most profound suppression of both MCL infiltration (CD20) and proliferation rate (Ki-67) in the single-agent cisplatin cohort compared to the other cohorts. In summary, the mouse models can be used for the study of MCL biology, as well as for preclinical assessment of experimental therapy of MCL including agents that cannot be properly tested in vitro (e.g. monoclonal antibodies, pro-drugs, anti-angiogenic agents, inhibitors of B-cell receptor signaling etc.). Financial Support: IGA-MZ NT13201-4/2012, GAUK 259211/110709, GAUK 446211, UNCE 204021, PRVOUK P24/LF1/3, PRVOUK 1–5101–280002 PVK, SVV-2012–254260507 Disclosures: No relevant conflicts of interest to declare.

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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 ◽

10.1182/blood.v124.21.3743.3743 ◽

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.

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

Blood ◽

10.1182/blood.v110.11.1375.1375 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1375-1375 ◽

Cited By ~ 3

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.

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TAM Inhibitor ONO-7475 Combination with Sorafenib Is Effective Using in Vitro and In Vivo Models of FLT3 ITD Acute Myeloid Leukemia

Blood ◽

10.1182/blood-2018-99-112211 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2642-2642

Author(s):

Peter P. Ruvolo ◽

Huaxian Ma ◽

Vivian Ruvolo ◽

Xiaorui Zhang ◽

Hong Mu ◽

...

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Myeloid Leukemia ◽

Research Funding ◽

Single Agent ◽

Xenograft Model ◽

Cell Cycle Regulators ◽

Flt3 Inhibitor

Abstract Background: Internal tandem duplication (ITD) mutation in Fems-like kinase 3 (FLT3) occur in ~ 25% of newly diagnosed acute myeloid leukemia (AML) patients and are associated with poor survival outcome. We recently demonstrated that highly specific AXL/MER inhibitor ONO-7475 (Ono Pharmaceutical Co, Osaka, Japan) was effective as a single agent against FLT3 ITD AML cells studied with in vitro co-culture and murine xenograft models (Ruvolo et al Haematologica, 2017). Based on expression of TAM kinases in the AML cells tested, mechanism for ONO-7475 killing in the FLT3 ITD AML cells was due to AXL inhibition. Here we report on the in vitro and in vivo effects of ONO-7475 when combined with FLT3 inhibitor Sorafenib in FLT3 ITD AML models. Methods: FLT3 ITD AML cell lines Molm13 and MV4;11 as well as Sorafenib resistant Molm13 cells were treated with varying doses of ONO-7475 in the presence and absence of FLT3 inhibitor Sorafenib and effect on cell viability and induction of apoptosis was assessed by flow cytometry using DAPI, Annexin V, and counting beads. RNA was isolated and gene expression profiling (GEP) analysis using microarray was performed on ONO-7475 treated cells. GEP results for a number of genes including CDK1, PLK1, and other cell cycle regulators were validated by qRT-PCR. The efficacy of ONO-7475 combination with Sorafenib in an in vivo AML xenograft model was tested using Molm13 cells expressing luciferase/GFP in NSG mice. Both drugs were given by oral gavage (ONO-7475 at 10 mg/kg and Sorafenib at 5 mg/kg). Drugs were given 5 days a week. Leukemia burden was assessed by IVIS imaging. Results: Molm13 and MV4;11 cells were highly sensitive to ONO-7475 combination with Sorafenib. A dose of 50 nM ONO-7475 with 25 nM Sorafenib potently induces apoptosis and eliminates > 90% of cells after 72 hours. Sorafenib resistant Molm13 cells were more resistant to either drug compared to parental cells. However, combination of 50 nM ONO-7475 with 100 nM Sorafenib potently induces apoptosis in the Sorafenib resistant cells with nearly all leukemic cells eliminated after 72 hour treatment. GEP analysis of cells treated with the AXL inhibitor revealed suppression of many genes involved in cell cycle control including various CDKs (e.g. CDK1, CDK4), Cyclins (e.g Cyclin B1), and other cell cycle regulators such as PLK1. ONO-7475 inhibition of gene expression of these genes was verified by qRT-PCR. GEP also revealed induction of genes associated with more mature myeloid cells including HLA-DR, CD114, and CD115. Finally, single agent use of ONO-7475 (10 mg/kg) or Sorafenib (5 mg/kg) had limited effect in the FLT3 ITD AML xenograft model; however, the combination of both drugs at single agent dose was effective reducing leukemia burden and significantly enhancing survival of mice bearing the Molm13 leukemia cells. Conclusions: These results suggest that ONO-7475 combination with Sorafenib is effective killing AML cells with FLT3 ITD including those that are resistant to the FLT3 inhibitor. The identification of gene expression of cell cycle regulators as novel targets of the drug suggests for the first time that AXL regulates cell cycle via a complex transcriptional mechanism. The ability of the drug to induce expression of genes associated with mature myeloid cells suggest the drug may have the potential to promote differentiation of FLT3 AML cells. Finally, combination of ONO-7475 with Sorafenib proved effective in an in vivo AML xenograft model suggesting that combination of ONO-7475 with a FLT3 inhibitor could be efficacious for therapy of AML patients with FLT3 ITD. Disclosures Yasuhiro: Ono Pharmaceutical: Employment. Tanaka:Ono Pharmaceutical: Employment. Yoshizawa:Ono Pharmaceutical: Employment. Cortes:Pfizer: Consultancy, Research Funding; Astellas Pharma: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Daiichi Sankyo: Consultancy, Research Funding; Arog: Research Funding. Andreeff:AstraZeneca: Research Funding.

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Anticancer effects of the combined Thai noni juice ethanolic extracts and 5-fluorouracil against cholangiocarcinoma cells in vitro and in vivo

Scientific Reports ◽

10.1038/s41598-021-94049-z ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Jeerati Prompipak ◽

Thanaset Senawong ◽

Banchob Sripa ◽

Albert J. Ketterman ◽

Suppawit Utaiwat ◽

...

Keyword(s):

Nude Mice ◽

Combination Treatment ◽

Synergistic Effects ◽

Single Agent ◽

Xenograft Model ◽

Ethanolic Extract ◽

Model Combination ◽

Mouse Xenograft Model ◽

Ethanolic Extracts

AbstractApplication of 5-fluorouracil (5-FU) in cholangiocarcinoma (CCA) is limited by adverse side effects and chemoresistance. Therefore, the combination therapy of 5-FU with other substances, especially natural products may provide a new strategy for CCA treatment. The aim of this study was to evaluate the combination effects of 5-FU and two ethanolic extracts of Thai noni juice (TNJ) products on CCA cell lines and nude mice xenografts. The results of antiproliferative assay showed the combination treatment of 5-FU and each TNJ ethanolic extract exerted more cytotoxicity on CCA cells than either single agent treatment. Synergistic effects of drug combinations can enable the dose reduction of 5-FU. The mechanism underlying a combination treatment was apoptosis induction through an activation of p53 and Bax proteins. In the nude mouse xenograft model, combination treatments of 5-FU with each TNJ ethanolic extract suppressed the growth of CCA cells implanted mice more than single agent treatments with no effects on mouse body weight, kidney, and spleen. Moreover, low doses of TNJ ethanolic extracts reduced the hepatotoxicity of 5-FU in nude mice. Taken together, these data suggested that the ethanolic extracts of TNJ products can enhance the anti-CCA effect and reduce toxicity of 5-FU.

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