Antitumor effects of combined bortezomib and tipifarnib in head and neck squamous cell carcinoma (HNSCC) cells

5581 Background: The dysregulation of the NF-κB and Ras/PI3K/AKT pathways in HNSCC supports our hypothesis that combined treatment with the proteasome inhibitor (PI) bortezomib (B) and the farnesyl transferase inhibitor (FTI) tipifarnib (T) leads to synergistic growth inhibition of HNSCC cells. Methods: Growth inhibitory effects of single agents (B 2.5–100 nM; T 0.625–5μM), combination (B+T) (B 12.5–17.5 nM; T 0.625–5 μM) or sequential treatment (B→T after 2h or T→B) were examined in three HNSCC lines (Tu212, 686LN and Sqcc/Y1) using a sulforrhodamine B assay after 72 h of drug exposure. Combination effect in cells treated concomitantly or sequentially was assessed using the combination index (C.I.: < 1.0, synergy; >1.0, antagonism; ≈1.0, additivity). Apoptosis was measured by flow cytometry. Relevant protein markers were evaluated by Western blot. Cell cycle analysis after flow cytometry used FlowJo software. Statistical analysis was done using a two-sided t-test. Results: Growth inhibition by B alone is very effective in all three lines (IC50 = 9–22nM). T used alone is active only in μM range (IC50 = 0.625–5 μM). The inhibitory effects of B and T were sequence dependent. Simultaneous treatment with 12.5 nM B and 5μM T showed synergistic growth inhibition in all 3 lines [C.I., 0.281–0.54]. B→T showed synergistic effects in all three cell lines [C.I., 0.36–0.76]. However, T→B was less synergistic in two of the three lines [Sqcc/Y1 C.I. = 0.404; Tu212 C.I. = 0.748]. Apoptosis was also sequence dependent with B+T or B→T treatment showing significantly more apoptosis than T→B (p = 0.03). Apoptosis induced by T→B was not different from treatment with single agent B (p = 0.22). Sqcc/Y1 cells treated with B and T showed accumulation in G2M phase and an increased percentage of cells in sub-G1. The observed synergistic inhibitory effect of B+T was associated with downregulation of p-AKT. Conclusions: We conclude that treatment with B+T and B→T synergistically enhances HNSCC growth inhibition and results in both significantly increased apoptosis and G2M arrest. These studies strongly support the clinical development of the sequential combination of a PI and a FTI. (Supported by Millenium Pharm. and the Georgia Cancer Coalition). [Table: see text]

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Antitumor effects of the small molecule DMAMCL in neuroblastoma via suppressing aerobic glycolysis and targeting PFKL

Cancer Cell International ◽

10.1186/s12935-021-02330-y ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Simeng Zhang ◽

Zhongyan Hua ◽

Gen Ba ◽

Ning Xu ◽

Jianing Miao ◽

...

Keyword(s):

Cell Death ◽

Synergistic Effects ◽

Aerobic Glycolysis ◽

Single Agent ◽

Molecular Compound ◽

Dependent Manner ◽

Antitumor Effects ◽

Atp Production

Abstract Background Neuroblastoma (NB) is a common solid malignancy in children that is associated with a poor prognosis. Although the novel small molecular compound Dimethylaminomicheliolide (DMAMCL) has been shown to induce cell death in some tumors, little is known about its role in NB. Methods We examined the effect of DMAMCL on four NB cell lines (NPG, AS, KCNR, BE2). Cellular confluence, survival, apoptosis, and glycolysis were detected using Incucyte ZOOM, CCK-8 assays, Annexin V-PE/7-AAD flow cytometry, and Seahorse XFe96, respectively. Synergistic effects between agents were evaluated using CompuSyn and the effect of DMAMCL in vivo was evaluated using a xenograft mouse model. Phosphofructokinase-1, liver type (PFKL) expression was up- and down-regulated using overexpression plasmids or siRNA. Results When administered as a single agent, DMAMCL decreased cell proliferation in a time- and dose-dependent manner, increased the percentage of cells in SubG1 phase, and induced apoptosis in vitro, as well as inhibiting tumor growth and prolonging survival in tumor-bearing mice (NGP, BE2) in vivo. In addition, DMAMCL exerted synergistic effects when combined with etoposide or cisplatin in vitro and displayed increased antitumor effects when combined with etoposide in vivo compared to either agent alone. Mechanistically, DMAMCL suppressed aerobic glycolysis by decreasing glucose consumption, lactate excretion, and ATP production, as well as reducing the expression of PFKL, a key glycolysis enzyme, in vitro and in vivo. Furthermore, PFKL overexpression attenuated DMAMCL-induced cell death, whereas PFKL silencing promoted NB cell death. Conclusions The results of this study suggest that DMAMCL exerts antitumor effects on NB both in vitro and in vivo by suppressing aerobic glycolysis and that PFKL could be a potential target of DMAMCL in NB.

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Roles of the pyroptosis signaling pathway in a sepsis-associated encephalopathy cell model

Journal of International Medical Research ◽

10.1177/0300060520949767 ◽

2020 ◽

Vol 48 (8) ◽

pp. 030006052094976

Author(s):

Yan Wang ◽

Xueyan Liu ◽

Qiang Wang ◽

Xin Yang

Keyword(s):

Flow Cytometry ◽

Protective Effect ◽

Signaling Pathway ◽

Cell Model ◽

Cell Damage ◽

Combined Treatment ◽

Therapeutic Effects ◽

Inhibitory Effects ◽

Caspase Cleavage ◽

Associated Proteins

Objectives The inhibition of pyroptosis has a protective effect in sepsis-associated encephalopathy (SAE). However, the mechanisms underlying pyroptosis in SAE remain to be elucidated. Methods Here, we investigated the effects of the caspase inhibitors, Belnacasan (Beln) and Wedelolactone (Wede), on an induced model of SAE in P12 cells, using immunofluorescence, ELISA, western blotting, and flow cytometry. Results The cell viability decreased, IL-1β and IL-18 secretion increased, and the levels of the caspase cleavage products, N-terminal gasdermin D, cleaved caspase-1, and cleaved caspase-11, increased in P12 cells following combined treatment with lipopolysaccharides (LPS) and adenosine triphosphate (ATP). However, treatment with Beln or Wede ameliorated the effects induced by LPS and ATP. Neither Beln nor Wede notably affected the levels of cell apoptosis-associated proteins but these inhibitors regulated the levels of cell pyroptosis-associated proteins. Further, the combination of Beln and Wede exerted greater inhibitory effects on cell pyroptosis than either Beln or Wede alone. Conclusions The results demonstrated that both the canonical and non-canonical signaling pathways of cell pyroptosis are involved in LPS-induced cell damage and that the non-canonical signaling pathway may be involved to a greater extent. This suggests that the inhibition of pyroptosis may exert potential therapeutic effects on SAE.

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Phase IIa Study of the Anti-CXCL12/SDF-1 Spiegelmer® Nox-A12 Alone and Combined with Bendamustine/Rituximab in Patients with Relapsed Chronic Lymphocytic Leukemia (CLL)

Blood ◽

10.1182/blood.v120.21.4593.4593 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4593-4593

Author(s):

Marco Gobbi ◽

Federico Caligaris-Cappio ◽

Marco Montillo ◽

Stephanie Vauléon ◽

Stefan Zöllner ◽

...

Keyword(s):

Flow Cytometry ◽

Plasma Concentration ◽

Chronic Lymphocytic Leukemia ◽

Healthy Volunteers ◽

Peripheral Blood ◽

Combined Treatment ◽

Single Agent ◽

Lymphocytic Leukemia ◽

Leukemic Cells ◽

Cd34 Cells

Abstract Abstract 4593 Background NOX-A12 is a novel, potent, L-aptamer inhibitor of CXCL12/SDF-1, a chemokine which attracts and activates immune- and non-immune cells. The signaling of CXCL12 has been shown to play an important role in the pathophysiology of chronic lymphocytic leukemia (CLL), especially in the interaction of leukemic cells with their tissue microenvironment. The therapeutic concept of NOX-A12 is to inhibit such tumor-supporting pathways and thereby sensitizing the CLL cells towards chemotherapy. Methods The purpose of this phase IIa study is to evaluate the safety and efficacy of NOX-A12 in combination with background chemo-immunotherapy of bendamustine and rituximab (BR) in patients with relapsed CLL. The described study is being performed in compliance with ethical principles based on the Declaration of Helsinki and ICH-GCP guidelines. The study population was split into a pilot and expansion group. In the pilot group, 3 cohorts of 3 patients each received escalating doses of single agent NOX-A12 two weeks prior to the combined treatment of NOX-A12 and BR. Interim data from these patients are reported. Based on previous Phase I studies in healthy volunteers, pilot patients received a dose of 1, 2 or 4 mg/kg body weight (BW) single agent NOX-A12 on day -14, followed by a 2-weeks period of safety, PK and PD assessments prior to the combined treatment with NOX-A12 and BR. To date, the first cohort of the pilot group already progressed to the 2nd cycle of combined treatment. Evaluation criteria included adverse events according to CTCAE V4, flow cytometry of peripheral blood CD34+ cells and CLL cells, pharmacokinetics of NOX-A12, plasma concentration of CXCL12 and tumor response (NCI-WG 1996 criteria, updated 2008). Results To date 3 patients (age range: 58 – 65 years) have been enrolled in the pilot group of this study. They had received 1 or 2 prior therapies, but no bendamustine. Single i.v. doses of 1 mg/kg BW NOX-A12 had no clinically relevant effects on vital signs, 12-lead ECG parameters and laboratory parameters. One patient reported grade 1 pain in the lower limbs two days after treatment with NOX-A12. This event was not dose-limiting and resolved spontaneously on the same day. Flow cytometry of CD34+ cells and CLL cells (CD19+/CD5+high) showed a rapid mobilization of these cells into the peripheral blood on day 1. Interestingly, return to baseline was not complete at the last assessment on day 3 (for details see Figure 1). The NOX-A12 pharmacokinetics in these 3 patients (for concentration-time profile see Figure 2) is very comparable to healthy volunteers receiving i.v. NOX-A12, with a maximum plasma concentration of 1.52 ± 0.14 μM after 1 h (tmax) and a plasma elimination half-life of about 50 h. As seen in healthy volunteers the plasma concentration of CXCL12 increased upon NOX-A12 treatment and reached a maximum of 0.434 ± 0.076 μM at 24 to 72 h p.a. without ever approaching the plasma concentration of NOX-A12 (Figure 2). Conclusion Single i.v. doses of NOX-A12 at 1 mg/kg BW were safe and well tolerated; the maximum tolerated dose was not reached. NOX-A12 induced a long-lasting mobilization of CD34+ cells and leukemic cells in patients with relapsed CLL, consistent with a mechanism of action based on CXCL12 inhibition. Patient accrual and identification of an optimal chemosensitization regimen of NOX-A12 combined with BR is being continued. Disclosures: Vauléon: NOXXON Pharma AG: Employment. Zöllner:NOXXON Pharma AG: Employment. Dümmler:NOXXON Pharma AG: Employment. Kruschinski:NOXXON Pharma AG: Employment. Fliegert:NOXXON Pharma AG: Employment.

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Growth Inhibition or Apoptosis after Treatment with Motexafin Gadolinium and Celecoxib Is Cell Line Dependent and Correlates with p27 Levels.

Blood ◽

10.1182/blood.v106.11.4296.4296 ◽

2005 ◽

Vol 106 (11) ◽

pp. 4296-4296

Author(s):

Jason Ramos ◽

Mint Sirisawad ◽

Jun Chen ◽

Richard A. Miller ◽

Louie Naumovski

Keyword(s):

Growth Inhibition ◽

Cell Lines ◽

Synergistic Effects ◽

Single Agent ◽

Chemotherapeutic Agents ◽

Motexafin Gadolinium ◽

Raji Cells ◽

Cox 2 ◽

Induced Apoptosis ◽

Cox 2 Inhibitors

Abstract Motexafin gadolinium (MGd, Xcytrin®) is a tumor selective expanded porphyrin belonging to the class of compounds known as texaphyrins. MGd is a redox mediator that may be directly cytotoxic or growth inhibitory to various tumor cells and can potentiate the effects of chemotherapeutic agents and radiation therapy. Initial promising activity has been seen in an ongoing phase II study of MGd as a single agent in patients with lymphoma. Celecoxib, a cyclooxygenase-2 (COX-2) inhibitor, has been shown to induce apoptosis and growth inhibition and potentiate the effects of chemotherapeutic agents. To determine potential synergistic effects of MGd and celecoxib, we treated lymphoma cell lines HF-1, Ramos, DHL-4 and Raji with MGd, celecoxib or the combination. Apoptosis was a prominent feature in two of the cell lines (HF-1 and Ramos) treated with MGd/celecoxib, whereas, growth inhibition was more prominent in the other two (DHL-4 and Raji). In Ramos cells, MGd/celecoxib treatment was synergistic in inducing apoptosis and resulted in activation of a caspase cascade as demonstrated by cleavage of caspases-9, -8, -3 and cleavage of substrates Bid and PARP. Two other specific COX-2 inhibitors, rofecoxib and valdecoxib did not synergize with MGd to induce apoptosis in Ramos cells, suggesting that celecoxib potentiation of MGd is a COX-2-independent activity of celecoxib. MGd/celecoxib-induced apoptosis of HF-1 and Ramos correlated with a decrease in p27Kip1 levels whereas MGd/celecoxib-induced growth inhibition of DHL-4 and Raji correlated with increased p27Kip1 levels. Cell cycle profiles of MGd/celecoxib treated Ramos and Raji cells revealed G1 accumulation only in Raji cells, consistent with elevated levels of active p27Kip1. Our pre-clinical data suggest that celecoxib enhances the efficacy of MGd and indicates a role for p27 in the response of cells to MGd and celecoxib.

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Hepatotoxicity During Eltrombopag Administration Might Be Due to Necrosis of Hepatocytes

Blood ◽

10.1182/blood.v118.21.2228.2228 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2228-2228

Author(s):

Kazunori Murai ◽

Shugo Kowata ◽

Akiko Abo ◽

Tatsuo Oyake ◽

Shigeki Ito ◽

...

Keyword(s):

Oxidative Stress ◽

Flow Cytometry ◽

Growth Inhibition ◽

Cell Line ◽

Mtt Assay ◽

Annexin V ◽

Similar Data ◽

Dependent Manner ◽

Inhibitory Effects ◽

Annexin V Assay

Abstract Abstract 2228 Background: Eltrombopag is an oral thrombopoietin receptor agonist for the treatment of immune thrombocytopenic patients who are refractory to the medications including corticosteroid. In RAISE study (The Lancet 2011: 377; 393–402), 79% patients in the eltrombopag group responded to treatment at least once during the study. However, 7% of eltrombopag-treated patients had increase of alanine aminotransferase (ALT) concentration and 4% of total bilirubin. The mechanism of eltrombopag-induced hepatobiliary toxicity remains unknown. In this study, we evaluated the effects of eltrombopag on hepatocytes using HepG2, human hepatocellular carcinoma cell line. Method: Cell proliferation was analyzed by MTT assay. Analysis of apoptosis/necrosis was analyzed by flow cytometry assay using Annexin V and propium iodide (PI) (Annexin-/PI-, viable cells; Annexin+/PI-, early apoptosis cells; Annexin+/PI+, late apoptosis and necrosis cells; Annexin-/PI+, late necrosis cells). Reduced glutathione was measured by DTNB colorimetric method. Murine hepatoma cell line Hepa 1–6 and murine normal liver derived cell line NCTC clone 1469 were also used in this study. Results: HepG2 was incubated with eltrombopag for 72 hrs and then MTT assay was performed. Figure 1a showed the growth inhibition curve of HepG2. HepG2 growth was suppressed by eltrombopag in a dose dependent manner (Figure 1a: without NAC, 12.5 μM 44.3 ± 8.7 %). In the addition of N-acetylcysteine (NAC) canceled the inhibitory effect (Figure 1a: with NAC 10mM: 60.4 ± 22.3 % at 12.5μM eltrombopag, p<0.05). To investigate whether this suppression was due to apoptosis or necrosis, we used PI/ Annexin V assay using flow cytometry. Figure 1b showed that each cell fraction after 72 hrs incubation with eltrombopag. PI positive and PI negative fraction was markedly increased (0μM: 0.7 ± 0.4%, 12.5 μM: 8.0 ± 6.0% p<0.01, 25μM: 39.6 ± 12.2% p<0.001). To confirm that the inhibitory effects of eltrombopag might be due to necrosis, we used IM-54, which is inhibitory molecule against oxidative stress induced necrosis, in MTT assay and PI/ Annexin V assay. As shown in Figure 2 a and 2 b, IM-54 canceled the inhibitory effects of eltrombopag in MTT assay (IM-54 0μM: 18.3 ± 7.5 %, IM-54 10 μM: 33.9 ± 10.6 % at eltrombopag 25μM, p<0.01) and PI/Annexin V assay (IM-54 0μM: 48.3 ± 12.4 %, IM-54: 10 μM 74.2 ± 8.3 % in PI/Annexin V double negative fraction at eltrombopag 25μM, p<0.01). The addition of Caspase-Inhibitor III (Boc-D-FMK) did not cancel the growth inhibition by eltrombopag in MTT assey. We measured GSH concentration of HepG2 cells. After the treatment of eltrombopag for 72 hrs, GSH decreased at 12.5μM of eltrpmbopag, however IM-54 restored the GSH concentration (83 mM at IM-54 0μM and eltrombopag 0 μM, IM-54 0 μM: 42.0 mM and IM-54 10μM: 83.3 mM at eltrombopag 12.5μM). Similar data were obtained in the study using Hepa 1–6 and NCTC clone 1469. Conclusion: Basic structure of eltrombopag is similar to that of 3-bipheyl-carboxylic acid, which is a strong oxidizing agent. Taken together, eltrombopag have an oxidative stress on hepatocytes to result in hepatotoxicity. Disclosures: No relevant conflicts of interest to declare.

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The Heat Shock Protein 32 (Hsp32)/HO-1-Targeting Drug SMA-ZnPP and the Triterpenoid CDDO-Me Exert Synergistic Growth-Inhibitory Effects on TKI-Resistant Leukemic Cells in Ph+ CML

Blood ◽

10.1182/blood.v118.21.4414.4414 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4414-4414

Author(s):

Karoline V. Gleixner ◽

Harald Herrmann ◽

Katharina Blatt ◽

Winfried F Pickl ◽

Marina Konopleva ◽

...

Keyword(s):

Heat Shock ◽

Growth Inhibition ◽

Synergistic Effects ◽

K562 Cells ◽

Leukemic Cells ◽

Drug Resistant ◽

Inhibitory Effects ◽

Growth Inhibitory ◽

Imatinib Resistant ◽

Ic50 Values

Abstract Abstract 4414 Resistance against one or more tyrosine kinase inhibitors (TKI) prevents eradication of Ph+ chronic myeloid leukemia (CML). In many patients BCR/ABL1 mutations are detectable. We have recently identified two targeted drugs that exert major growth-inhibitory effects on drug-resistant CML cells, the triterpenoid CDDO-Me (Bardoxolone-methyl, REATA Pharma) that blocks several signalling molecules including mTOR, Akt, and STAT3, and upregulates expression of heat shock protein 32 (Hsp32 = heme oxygenase 1, HO-1), and styrene-maleic acid-copolymer micelle-encapsulated ZnPP (SMA-ZnPP), a water-soluble inhibitor of Hsp32/HO-1. In the current project, we asked whether CDDO-Me exerts inhibitory effects on growth of TKI-resistant CML cells and whether the combination of CDDO-Me and SMA-ZnPP would produce synergistic effects in drug-resistant CML cells. As determined by 3H-thymidine incorporation, CDDO-Me was found to inhibit the proliferation of imatinib-responsive and imatinib-resistant K562, imatinib-resistant KCL-22, KU812, and Ba/F3 cells transfected with various TKI-resistant mutants of BCR/ABL1 (T315I, E255K, Y253F, H396P). In each case, IC50 values <1 μM were obtained without major differences between imatinib-responsive and imatinib-resistant cells. Growth-inhibition was accompanied by apoptosis as assessed by combined AnnexinV/PI staining as well as by an increase in expression of HO-1 in KU812 and KCL-22 cells. CDDO-Me was also found to inhibit proliferation of leukemic cells in all patients with TKI-resistant CML (n=4), with IC50 values ranging between <0.1 and 0.5 μM. No differences in IC50 values were observed between treatment-naïve and TKI-resistant cells. Next, we applied the combination CDDO-Me+SMA-ZnPP and found that this combination acts highly synergistically on imatinib-responsive and imatinib-resistant K562 cells as well as primary CML cells isolated from imatinib-naïve CML patients (n=2) or from patients with imatinib-resistant CML (n=2), including one patient in whom BCR/ABL1 T315I was detected. We also examined whether CDDO-Me would exert synergistic effects on CML cells when combined with BCR/ABL1 TKI. In these experiments, we applied the combinations CDDO-Me+dasatinib and CDDO-Me+nilotinib on K562 cells. Both combinations were found to synergistically induce growth inhibition. In conclusion, CDDO-Me inhibits the proliferation of imatinib-resistant BCR/ABL1+ cells, including primary CML cells isolated from untreated patients and cells derived from patients with TKI-resistant CML carrying the BCR/ABL1 mutant T315I. Our data also show that CDDO-Me + SMA-ZnPP and CDDO-Me + BCR/ABL1 TKI synergize in producing growth inhibition in CML cells. Whether these drug combinations also produce synergistic effects in vivo in patients with TKI-resistant CML remains to be evaluated. Disclosures: Valent: Novartis: Consultancy, Honoraria, Research Funding.

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Synergistic activity of vertical combinations of agents targeting multiple steps along the RAF/MEK/ERK cascade as a therapeutic strategy in human tumors.

Journal of Clinical Oncology ◽

10.1200/jco.2012.30.15_suppl.e13572 ◽

2012 ◽

Vol 30 (15_suppl) ◽

pp. e13572-e13572 ◽

Cited By ~ 1

Author(s):

Ursula Cesta Incani ◽

Anais Del Curatolo ◽

Cristina Di Sanza ◽

Italia Falcone ◽

Francesco Cognetti ◽

...

Keyword(s):

Growth Inhibition ◽

Molecular Mechanisms ◽

Synergistic Effects ◽

Braf V600e ◽

Fixed Dose ◽

Synergistic Activity ◽

Dose Ratio ◽

Antitumor Effects ◽

Mapk Activation ◽

A549 Lung

e13572 Background: BRAF-selective kinase inhibitors have potent antitumor effects in mutant BRAF(V600E) tumors; however, in BRAF-wt cells, they paradoxically activate MEK/ERK. In addition, MEK blockade may induce compensatory signaling through both upstream pathway elements (RAF) and parallel pathways (PI3K/AKT/mTOR). Methods: We set out to define molecular and functional effects of single and combined BRAF (GSK2118436A, BRAF-I) and MEK (GSK1120212B, MEK-I) inhibition, using WB analysis to dissect signaling and a fixed dose-ratio experimental design to assess functional synergism by conservative isobologram analysis. Results: In A549 lung adenocarcinoma (KRAS G12S), BRAF-I (10 μM) induces hyperphosphorylation of CRAF, MEK, ERK, and p90RSK, while MEK-I (10 nM), alone or in combination with BRAF-I, potently offsets MAPK activation. Combined BRAF-I and MEK-I suppress malignant growth and survival at 72 h with highly synergistic effects in the A549 (lung, KRAS G12S), H1299 (lung, NRAS), HCT116 (colon, KRAS G13D), and MIAPACA (pancreatic, KRAS G12V) models, with combination indexes (CI), ranging from 1.37 to 0.12. Conversely, in other lung cancer models (H460, Calu-1, Calu-3) the combination of BRAF-I and MEK-I produced modestly additive to highly antagonistic antitumor effects. In BRAF-mutant melanoma and colon carcinoma models (M14 and HT29), there was no paradoxical activation of the MEK/ERK module in response to BRAF-I and both BRAF-I and MEK-I had pronounced growth inhibitory effects as single agents, but were frankly antagonistic in combination. Similarly, the pan-RAF inhibitor RAF265 did not cause MAPK activation and did not result in synergistic growth inhibition when combined with the MEK-I in the A549 and MIAPACA cell lines. Conclusions: Overall, our data indicate that combined inhibition of multiple signaling elements along the RAF/MEK/ERK pathway results in strongly synergistic growth inhibition, particularly in tumors with RAS mutations. Additional studies to better define genetic determinants of sensitivity/resistance and molecular mechanisms of therapeutic synergism of combined BRAF-I and MEK-I are currently ongoing.

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An exploratory study to investigate the immunomodulatory activity of radiation therapy in combination with pembrolizumab in patients with renal cell cancer.

Journal of Clinical Oncology ◽

10.1200/jco.2017.35.6_suppl.518 ◽

2017 ◽

Vol 35 (6_suppl) ◽

pp. 518-518

Author(s):

Jianqing Lin ◽

Jean H. Hoffman-Censits ◽

William Kevin Kelly ◽

Madalina Tuluc ◽

Colette Shaw ◽

...

Keyword(s):

Flow Cytometry ◽

Radiation Therapy ◽

Renal Cell Cancer ◽

Renal Cell ◽

Nk Cell ◽

Synergistic Effects ◽

Cell Cancer ◽

Single Agent ◽

Clinical Activity ◽

Immunomodulatory Activity

518 Background: Preclinical data suggest there are synergistic effects of radiation therapy (RT) and check point inhibitors in anticancer immunity. The primary objective of this study was to explore the immunomodulatory activity of RT alone or in combination with Pembrolizumab (pembro) in solid tumors including renal cell cancer (RCC) patients (pts). Methods: RCC pts who progressed after at least one front-line therapy were eligible. Pts were treated with either RT (8Gy x 1 or 4Gy x 5) followed by (f/b) pembro or 1 dose pembro f/b RT f/b pembro. Pre- and post RT tumor biopsy was obtained to evaluate PD-L1 expression (assay by QualTeck). Immune markers from peripheral blood before, during, and after treatment were analyzed using flow cytometry. Treatment response was measured based on modified RECIST criteria. Results: 12 RCC patients were enrolled including 2 with non-clear cell subtype. One pt was not evaluable since pt quickly deteriorated and was taken off study. As of September 30, 2016, 5 pts were still on study with 3 pts having partial response (27.2%, responders) and were on study for at least 9.8 months. 2 responders were treated with 8 Gy f/b pembro while 1 was treated with pembro f/b RT f/b pembro. Six pts (54.5%, non-responders) had PFS between 2 – 4 m and were off study. For adverse events, 1 pt developed grade 4 pneumonitis after 10 cycles of pembro (RT to adrenal mets). Grade 3 AEs include fatigue, nausea, hyperglycemia, lymphopenia, thrombocytopenia, and AST elevation (post RT for liver mets). PDL1 expression and tumor infiltrating lymphocytes presence after RT showed various patterns. Preliminary flow cytometry showed persistent higher numbers of monocytes in non-responders compared with responders. CD4+, CD8+, and NK cells and other markers are under-analyzed and the results will be presented. Conclusions: The combination of RT (8Gy or 20Gy) with pembro is feasible and tolerated, and demonstrates clinical activity. The AE profile is similar to single agent pembro. Monocytes, T, and NK cell kinetics are being examined. Clinical trial information: NCT02318771.

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Tipping Growth Inhibition into Apoptosis by Combining Treatment with MDM2 and WIP1 Inhibitors in p53WT Uterine Leiomyosarcoma

Cancers ◽

10.3390/cancers14010014 ◽

2021 ◽

Vol 14 (1) ◽

pp. 14

Author(s):

Victoria Chamberlain ◽

Yvette Drew ◽

John Lunec

Keyword(s):

Growth Inhibition ◽

Cell Line ◽

Target Genes ◽

Single Agent ◽

Uterine Leiomyosarcoma ◽

Dependent Manner ◽

Inhibitory Effects ◽

Area Of Interest ◽

Growth Inhibitory ◽

Wip1 Phosphatase

As there is no optimal therapeutic strategy defined for women with advanced or recurrent uLMS, there is an urgent need for the discovery of novel, targeted approaches. One such area of interest is the pharmacological inhibition of the MDM2-p53 interaction with small-molecular-weight MDM2 inhibitors. Growth inhibition and cytotoxic assays were used to evaluate uLMS cell line responses to MDM2 inhibitors as single agents and in combination, qRT-PCR to assess transcriptional changes and Caspase-Glo 3/7 assay to detect apoptosis. RG7388 and HDM201 are potent, selective antagonists of the MDM2-p53 interaction that can effectively stabilise and activate p53 in a dose-dependent manner. GSK2830371, a potent and selective WIP1 phosphatase inhibitor, was shown to significantly potentiate the growth inhibitory effects of RG7388 and HDM201, and significantly increase the mRNA expression of p53 transcriptional target genes in a p53WT cell line at a concentration that has no growth inhibitory effects as a single agent. RG7388, HDM201 and GSK2830371 failed to induce apoptosis as single agents; however, a combination treatment tipped cells into apoptosis from senescence. These data present the possibility of MDM2 and WIP1 inhibitor combinations as a potential treatment option for p53WT uLMS patients that warrants further investigation.

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The Combination Of FLT3 Inhibition and Hypomethylation Confers Synergistic Anti-Leukemic Effects On FLT3/ITD Positive AML Cell Lines and Primary Cells

Blood ◽

10.1182/blood.v122.21.3965.3965 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3965-3965 ◽

Cited By ~ 1

Author(s):

Heiko Konig ◽

Mark Levis

Keyword(s):

Elderly Patients ◽

Growth Inhibition ◽

Combined Treatment ◽

Single Agent ◽

Current Phase ◽

Clinical Activity ◽

Primary Cells ◽

Newly Diagnosed ◽

Sequential Administration ◽

Synergistic Cytotoxicity

Abstract Background Effective treatment regimens for elderly FLT3/ITD+ AML patients are lacking. Recent data on the effects of the FLT3 tyrosine kinase inhibitor quizartinib (QUIZ) on FLT3/ITD+ AML showed promising clinical activity, including in elderly patients. Hypomethylating agents (DNMTis), such as Decitabine (DEC) and 5-azacytidine (AZA), demonstrated clinical benefit in AML and are well tolerated in elderly patients, and are associated with minimal increases in FLT3 ligand, which can represent a potential resistance mechanism to FLT3 inhibitors. In addition, both FLT3 inhibition and hypomethylation therapy are associated with the induction of terminal differentiation of myeloid blasts. Therefore, there is a strong theoretical rationale for combining FLT3 inhibition and hypomethylation for FLT3/ITD AML, particularly in the elderly. Here, we investigated whether the combination of DEC or AZA with QUIZ bears synergistic anti-leukemic potential, and if the sequence of administration was important. Methods Molm14 (M14) and primary cells from elderly, newly diagnosed (ND) and relapsed (REL) FLT3/ITD+ AML patients were incubated in culture medium. QUIZ, DEC or AZA were added either as single agents or in combination (simultaneous vs. sequential administration) at the indicated concentrations, in the presence (M14 and primary cells) and absence of stroma (M14). After 48 to 72 hours, cells were assessed for proliferation and apoptosis using MTT assays, annexin-V staining and FACS analysis. A combination index (CI) was calculated using Calcusyn Software. Results We found that the presence of stromal cells significantly blunted the cytotoxic effects of each compound, both when administered as single agent and in combination. In M14, CI values indicated that combined treatment resulted in synergistic effects that were more pronounced in the presence as compared to the absence of stroma (CIstroma: 0.16 [QUIZ 1nM+DEC 1μM] and 0.2 [QUIZ 1nM+AZA 1μM]; CIno stroma: 0.43 [QUIZ 1nM+DEC 1μM] and 0.87 [QUIZ 20nM+AZA 1μM]). Furthermore, a trend towards greater growth inhibition was observed when the drugs were administered simultaneously rather than in a sequential mode. While the pro-apoptotic effects of DEC were less marked, combined treatment (QUIZ +DEC or AZA) did not further induce apoptosis as compared to QUIZ alone. In primary FLT3/ITD+ AML cells, single agent QUIZ, DEC and AZA demonstrated dose dependent, growth inhibitory effects. While AZA was significantly more potent than DEC in ND (n=3) and REL (n=3) cells, REL cells were generally more drug sensitive (ND: 48.7±10.6% [4μM AZA] vs. 6.0±7.9% [4μM DEC]; REL: 71.3±2.7% [4μM AZA] vs. 5.5±3.1% [4μM DEC]; p<.005). Consistent with greater FLT3-addiction, QUIZ was significantly more potent in REL than in ND cells (5nM: 13.5±4.9% [ND] vs. 28.5±5.0% [REL], p<.05; 10nM: 21.8±4.0% [ND] vs. 41.5±4.8% [REL], and 20nM: 24.9±3.0% [ND] vs. 49.2±6.5% [REL]; p<.005). Combined treatment with QUIZ and AZA resulted in significant, additive to synergistic growth inhibition compared to QUIZ alone in ND and REL cells. QUIZ and DEC demonstrated synergistic growth inhibition only in REL (46.0±6.8% [20nM AC], 15.5±3.1% [DEC 4μM], 55.5±6.3% [QUIZ 20nM+ DEC 1uM], CI=0.68) but not in ND cells. The combination of QUIZ/AZA induced a modest degree of apoptosis in both ND and REL primary cells, whereas DEC with or without QUIZ did not induce any meaningful degree of apoptosis. Conclusions 1. Not surprisingly, stroma blunted the cytotoxic effect of all treatments 2. On stroma, 5-azacytidine was considerably more cytotoxic than decitabine, at least from the standpoint of inducing growth inhibition or apoptosis. 3. There was synergistic cytotoxicity in blasts on stroma with quizartinib/5-azacytidine, less so with quizartinib/decitabine. 4. Simultaneous administration was more effective than sequential. 5. We are currently studying the efficacy of these combinations at inducing terminal myeloid differentiation in these FLT3/ITD AML samples. 6. The combination of a hypomethylating agent and FLT3 inhibition is a potentially important new therapeutic regimen for FLT3/ITD AML, particularly for older patients. A current Phase 1 trial with the combination of AZA and QUIZ in newly diagnosed and relapsed patients is currently being explored. Disclosures: Levis: Ambit Biosciences: Consultancy.

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