Caspase-Dependent Anti-Tumor Effects of ONC201/TIC10 on Acute Myeloid Leukemia (AML) and Multiple Myeloma (MM)

Abstract PI3K/Akt and Ras/MAPK pathways are attractive therapeutic targets in almost all tumor types, including AML and MM. Apo2L/TRAIL has been deemed a promising therapeutic given its selectivity towards cancer cells although its clinical development has been hampered by various limitations including short half-life and general shortcoming of protein-based therapeutics. ONC201/TIC10 (Oncoceutics, Inc.) is a first-in-class small molecule inducer of TRAIL expression. ONC201/TIC10 has previously been shown to up-regulate TRAIL and its death inducing receptor DR5 in HCT116 colon cancer cells, in part through the inhibition of Foxo3a phosphorylation mediated by dual inhibition of Akt and ERK (Allen JE et al, Sci Transl Med., 2013). Currently, ONC201/TIC10 is set to enter clinical trials for patients with advanced malignancies after the IND was approved by the FDA in March, 2014. We thus investigated the therapeutic potential of ONC201/TIC10 in AML and MM given a major unmet need when conventional therapy fails. We explored the possibility that ONC201/TIC10 induces apoptosis in MM and AML in part through dual inhibition of the PI3K/Akt and Ras/MAPK pathways. We tested a panel of four human MM cell lines (KMS18, MM.1S, MM.1S 33X and RPMI-8226) and three human AML cell lines (Kasumi-1, HL60, HL60/VCR). The Cell-titer Glo assay demonstrated a time and dose-dependent decrease in viability in the entire panel of MM and AML cells. EC50 values ranged from 1-2.5 µM for the MM and 2-5µM for the AML cell lines, respectively. Bortezomib-resistant cells MM.1S 33X and vincristine- resistant cells HL60/VCR were also significantly sensitive to ONC201/ TIC10 as a single agent with EC50s on par with the corresponding parental cell lines. Given the previously reported pro-apoptotic effects of ONC201/TIC10 against solid tumor cells, we assessed apoptosis by performing Sub-G1 analyses and assessing caspase-3 cleavage as two widely used methods to analyze apoptotic cell death. We observed an average of 10-fold induction of ONC201/TIC10–mediated apoptosis in MM cells at 5 mM at 48 hrs post-treatment. Rescue of ONC201/TIC10-mediated apoptosis was demonstrated using the pan-caspase inhibitor (Z-VAD-FMK). In addition, western blot analysis in MM cells indicated a dose-dependent decrease in the anti-apoptotic protein XIAP which is a key mediator of apoptosis inhibition and is reported to be highly up-regulated in MM cells. Dose and time dependent induction of apoptosis was noted in western blot analysis of caspase-3 cleavage in AML cell lines treated with 2.5 µM or 5 µM of ONC201/TIC10 for 48 hrs prior to analysis. Western Blot analysis further demonstrated inhibition of Akt and Foxo3a phosphorylation in Kasumi-1 cells, in line with the previously proposed mechanism of ONC201/TIC10 against solid tumor cells. To further investigate the therapeutic potential of ONC201/TIC10 in the context of AML, fresh AML cells were treated with ONC201/TIC10. The primary cells were also found to be sensitive to ONC201/TIC10 (60% decrease in cell viability 72 hrs post 5mM ONC201/TIC10 treatment). Similarly, caspase 3/7 activity was significantly increased as assessed by the Caspase Glo 3/7 assay (~5 fold induction in activity 72 hrs post 5mM ONC201/TIC10 treatment). To explore further the therapeutic potential of ONC201/TIC10, we performed combinatorial experiments with bortezomib and vincristine using the MM.1S 33X MM cells and the HL60/VCR AML cell lines. ONC201/ TIC10 showed an additive effect with both these compounds against the MM and AML lines. Our work demonstrates activity of ONC201/TIC10 against AML and MM cell lines including fresh AML tumor cells. The efficacy data with resistant cells is in par with the applicability of TIC10 in patients with refractory/relapsed hematological malignancies. The long-term goal of this project is to provide a rationale for a phase 1b trial of ONC201/TIC10 for refractory/relapsed MM and AML in combination with existing therapies. Figure 1: Efficacy of ONC201/TIC10 in AML and MM cells Figure 1:. Efficacy of ONC201/TIC10 in AML and MM cells Disclosures Allen: Oncoceutics, Inc.: Employment, Equity Ownership, Patents & Royalties. El-Deiry:Oncoceutics, Inc.: Equity Ownership, Patents & Royalties.

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Pan-BCL-2 Family Small Molecule Inhibitor GX015-070 (GeminX, Inc.) Exhibits Single Agent Cytotoxicity, Is Synergistic with Select Anti-Leukemia Cytotoxic Agents, and Induces Apoptosis in Cell Lines with t(4;11)(q21;q23) Translocations.

Blood ◽

10.1182/blood.v106.11.3368.3368 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3368-3368 ◽

Cited By ~ 1

Author(s):

Jessicca M. Rege ◽

Blaine W. Robinson ◽

Manish Gupta ◽

Jeffrey S. Barrett ◽

Peter C. Adamson ◽

...

Keyword(s):

Cell Death ◽

Western Blot ◽

Family Member ◽

Cell Lines ◽

Blot Analysis ◽

Western Blot Analysis ◽

Caspase 3 ◽

Single Agent ◽

Cytotoxic Agents ◽

Response Surface Modeling

Abstract Background: Leukemias with MLL translocations, especially t(4;11), often are resistant to common chemotherapeutic agents, which may be due to abnormal apoptosis regulation. Pro- and anti-apoptotic BCL-2 family member interactions govern initiation of the intrinsic apoptosis pathway. GX015-070, which currently is in Phase I/IIA clinical trials, mimics the BH3 domain on pro-apoptotic BCL-2 family proteins and can bind the BH3 binding pocket of anti-apoptotic BCL-2 family members and modulate apoptosis. We performed comprehensive protein expression profiling of BCL-2 family member proteins and evaluated in vitro activity and mechanism of action of GX015-070 in cell lines with t(4;11). Methods: Baseline expression of BCL-2 family proteins was determined by Western blot analysis. Cytotoxicity was assessed by MTT after a 3 day exposure of RS4:11, SEM-K2 and MV4-11 cells in log phase growth to single agent GX015-070 at concentrations from 5 nM to 7.5 μM. Combined effects of fixed-concentration GX015-070 with cytotoxic agents over a range of concentrations were assayed by MTT, and the results were analyzed by pharmacostatistical response surface modeling. Disruption of specific pro- and anti-apoptotic BCL-2 family member interactions was investigated by co-immunoprecipitation/Western blot analysis. Flow cytometry and/or Western blot analysis of Caspase-3 activation, and a FACS TUNEL assay, were used to assess apoptosis in GX015-070 treated and untreated cells. Results: The three cell lines had similar baseline levels of expression of BCL-2 family proteins. BCL-2 and BAX were most abundant followed by PUMA, BAK, BCL-XL, BIM-EL, MCL-1, BIK and NOXA. Results of assays of GX015-070 activity and mechanism of action are in shown in the table. Conclusions: These data indicate that GX015-070 has potent cytotoxic activity in cell lines with t(4;11) as a single agent and that the cytotoxicity results from apoptosis. Response surface modeling in RS4:11 cells suggested ability to achieve effective doses with GX015-070 combined with cytosine arabinoside (Ara-C), dexamethasone (Dex) or doxorubicin (ADR) that are lower than projected from the single agents, but synergy was not suggested when GX015-070 was combined with etoposide, methotrexate or 6-thioguanine. The co-IP experiments give proof of principle that GX015-070 disrupts pro- and anti-apoptotic BCL-2 family protein interactions in cell lines with t(4;11). Additional pre-clinical experiments directed at overcoming drug resistance from abnormal cell death regulation in leukemias with t(4;11) using GX015-070 are in progress. These studies provide a framework to understand the cell death/survival machinery in primary leukemias with t(4;11) translocations more completely and manipulate that machinery to achieve better treatments. GX015-070 Activity and Mechanism Cell Line Single Agent Activity Synergy Inhibition Caspase-3 Activation TUNEL RS4:11 IC50=43.5 nM Ara-C, Dex, ADR Mcl1:Bak; Bcl2:Bak + + SEM-K2 IC50=156 nM In progress Mcl1:Bak; Bcl2:Bak + In Progress MV4-11 IC50=123 nM In progress Mcl1:Bak In progress +

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Proteasome Inhibitors Can Induce Caspase-3 Activation and Apoptosis in Human CML Cell Lines.

Blood ◽

10.1182/blood.v104.11.4681.4681 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4681-4681

Author(s):

Byung-Su Kim ◽

Chang Up Kim ◽

Young-Ju Kim ◽

Eun Kyung Bae ◽

Jinhee Kim ◽

...

Keyword(s):

Cell Cycle ◽

Western Blot ◽

Cell Lines ◽

Blot Analysis ◽

Proteasome Inhibitor ◽

Western Blot Analysis ◽

Caspase 3 ◽

Expression Patterns ◽

Proteasome Inhibitors ◽

Acridine Orange Staining

Abstract The proteasome is a multi-enzyme complex that provides the ubiquitin-dependent degradation of many cytoplasmic and nuclear proteins involved in cell cycle progression and apoptosis. Inhibition of the proteasome represents a promising approach for the treatment of cancer because it can lead to cell cycle arrest and activation of caspases in tumor cells. There are several proteasome inhibitors that have been reported to induce apoptosis in various tumors. However, the effect of proteasome inhibition in human myeloid leukemia has not been reported so far. In this study, we tested two peptide-aldehyde proteasome inhibitors (MG115, MG132) on two human CML cell lines (K562, KCL22). At first, we treated both cell lines for 24, 48 and 72 hours with different doses of MG115 and MG132 and cell viability was tested by MTT assay. It showed substantial time and dose dependent cytotoxicity in both CML cell lines. Acridine orange staining also revealed DNA fragmentation. We then performed caspase-3 colorimetric assay after treating both cell lines for 6, 12 and 24 hours with 0.78μM of MG115, MG132. K562 showed the continuous rising of caspase-3 activity, while KCL22 exhibited the initial increase and subsequent mild decrease of caspase-3 activity. In addition, western blot analysis showed the reduction of procaspase-3 expression. The expression of Bcl-2 and Bcl-XL was reduced by western blot. p21 expression was slightly increased and that of cyclin D1 was decreased. Additionally, the treatment of proteasome inhibitor in CML cell lines initially induced phosphorylation of Jun kinase. We next examined the expression of heat shock proteins (Hsp70, Hsp90) after treating for 6, 12, 24 hours with the same proteasome inhibitors. Western blot analysis results indicated that expression patterns were different between MG115 and MG132. MG115 induced the slight increase of Hsp70 and Hsp90 in K562, but the reduction of both in KCL22. Meanwhile, MG132 produced the decrease of Hsp70 and Hsp90 in both K562, KCL22. In summary, our work supports that a proteasome inhibitor can induce apoptosis in human CML cell lines. We are currently focusing on the combined effect of proteasome inhibitor and Hsp90 inhibitor on CML. IC50 of Proteasome Inhibitors Cell line Proteasome Inhibitor 24hr 48hr 72hr K562 MG115 3.01 μM 1.14 μM 0.59 μM K562 MG132 μ 2.13 M 1.03 μM 0.57 μM KCL22 MG115 156.92 μM 1.36 μM 0.73 μM KCL22 MG132 1.56 μM 0.93 μM μ 0.75 M

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Single Agent and Combinatorial Efficacy of First-in-Class Small Molecule ONC201 in Acute Leukemia and Multiple Myeloma

Blood ◽

10.1182/blood.v128.22.2759.2759 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2759-2759 ◽

Cited By ~ 1

Author(s):

Varun V Prabhu ◽

Amriti Lulla ◽

Christina L Kline ◽

Peter J Van den Heuvel ◽

Mala K. Talekar ◽

...

Keyword(s):

Western Blot ◽

Cell Viability ◽

Cell Lines ◽

Blot Analysis ◽

Western Blot Analysis ◽

Single Agent ◽

Employment Equity ◽

Equity Ownership ◽

Dose Dependent Decrease ◽

Dose Dependent

Abstract ONC201 is the founding member of the imipridone class of anti-cancer small molecules that possess a unique core chemical structure. ONC201 is currently being evaluated in several Phase I/II clinical trials for advanced cancers. In the current study, we evaluated the single agent and combinatorial efficacy of ONC201 in preclinical models of acute leukemia and multiple myeloma (MM). In acute leukemia, we evaluated ONC201 anti-cancer effects in acute myeloid leukemia (AML) (Kasumi-1, HL60) and acute lymphoblastic leukemia (ALL) (Reh, Jurkat and MOLT-4) cell lines. We observed a time- and dose-dependent decrease in cell viability for every cell line in the panel (EC50 1-5 µM). Vincristine-resistant cells HL60/VCR were also sensitive to single agent ONC201 with EC50 values on par with corresponding vincristine-sensitive parental cells. Dose- and time-dependent induction of apoptosis was noted in Western blot analysis of caspase-3 cleavage in AML cell lines treated with 2.5 µM or 5 µM of ONC201 for 48 hr. Western Blot analysis further demonstrated inhibition of Akt and Foxo3a phosphorylation in Kasumi-1 cells, in line with the previously reported late-stage signaling effects of ONC201 in solid tumor cells (Allen et al, 2013). Sub-G1 analysis indicated that ONC201 induces apoptosis in ALL cells and a pan-caspase inhibitor reduced ONC201-mediated apoptosis. Western blot analysis revealed ONC201-mediated apoptosis involves PARP cleavage and caspase-9 activation in ALL cells. Anti-apoptotic Bcl-2 family members Bcl-2 and Bcl-xl were downregulated while the pro-apoptotic Bcl-2 family member Bim is upregulated in response to ONC201 treatment in ALL cells. ONC201 also downregulates the inhibitor of apoptosis (IAP) family proteins cIAP1 and cIAP2 in ALL cells. We observed inhibition of Akt phosphorylation upon ONC201 treatment of ALL cells. Fresh AML patient cells were also found to be sensitive to ONC201 in cell viability and caspase 3/7 activity assays at 5µM. We observed that independent clones of cancer cells with acquired resistance to ONC201 were more sensitive to cytarabine compared to parental ONC201-sensitive cancer cells. In addition, ONC201 demonstrated synergistic reduction in cell viability in combination with cytarabine in AML cell lines. Determination of combination indices (CI) revealed synergy at several concentrations (CI 0.336-0.75 in CMK cells). Also, ONC201 combined additively with midostaurin in CMK cells and vincristine in HL60/VCR cells. Thus, ONC201 is a promising combinatorial partner for AML therapies based on these preclinical sensitization results. In accordance with ONC201-mediated activation of the integrated stress response that B cells are highly sensitive to (Kline et al and Ishizawa et al, 2016), MM was identified as one of the most ONC201-sensitive tumor types in the Genomics of Drug Sensitivity in Cancer collection of cell lines. Three human MM cell lines were used for validation (KMS18, MM.1S and RPMI-8226), which revealed a time- and dose-dependent decrease in cell viability (EC50 1-2.5 µM). Bortezomib-resistant cells MM.1S 33X were sensitive to ONC201 as a single agent with EC50 values comparable to bortezomib-sensitive parental cells. We observed an average of 10-fold induction of ONC201-mediated apoptosis using Sub-G1 analyses in MM cells at 5 µM, 48 hrs post-treatment. Rescue of ONC201-mediated apoptosis was demonstrated using the pan-caspase inhibitor (Z-VAD-FMK). In addition, Western blot analysis in MM cells indicated a dose-dependent decrease in the anti-apoptotic protein XIAP which is a key mediator of apoptosis inhibition and is reported to be highly up-regulated in MM cells. Furthermore, ONC201 demonstrated synergistic reduction in cell viability at various concentrations in combination with either ixazomib or dexamethasone, which are used in the clinical treatment of MM, in RPMI8226 cells (CI 0.228-0.75). Also, ONC201 combined additively with bortezomib in RPMI8226 and MM.1S 33X cells. In summary, these preclinical studies support the ongoing ONC201 single agent trials in acute leukemias and MM. Our findings suggest that ONC201 may be an important therapeutic option for patients with hematological malignancies who have developed resistance to approved therapies. Additionally, our results point to specific standard-of-care therapies that may be combined with ONC201 to exert durable responses without adding to the burden of toxicity. Disclosures Prabhu: Oncoceutics: Employment. Tarapore:Oncoceutics: Employment, Equity Ownership. Oster:Oncoceutics: Employment, Equity Ownership. Allen:Oncoceutics: Employment, Equity Ownership. El-Deiry:Oncoceutics: Equity Ownership.

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Therapeutic Potential of Axl Blockade in BCR-ABL Negative Myeloproliferative Neoplasms (MPN)

Blood ◽

10.1182/blood-2018-99-118175 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3063-3063

Author(s):

Antonia Beitzen-Heineke ◽

Isabel Ben Batalla ◽

Nikolaus Berenbrok ◽

Sarina Paesler ◽

Victoria Gensch ◽

...

Keyword(s):

Western Blot ◽

Cell Viability ◽

Cell Lines ◽

Blot Analysis ◽

Western Blot Analysis ◽

Therapeutic Potential ◽

Brdu Incorporation ◽

Additive Effects ◽

Healthy Donors

Abstract Axl, a member of the TAM family of receptor tyrosine kinases, mediates survival and therapy resistance of different cancer cells. The Axl ligand growth-arrest specific gene 6 (Gas6) was discovered to promote proliferation of leukemia cells in acute and chronic myeloid leukemia and Axl was identified as a potential therapeutic target in these diseases. Based on these data we investigated the role of Axl in BCR-ABL negative myeloproliferative neoplasms (MPN) and the therapeutic potential of Axl blockade in this group of diseases. We studied the effects of Axl blockade using the small molecule Axl inhibitor BGB324 and performing a lentivirus shRNA mediated knockdown of Axl in human SET-2 and murine BaF3-Jak2V617F MPN cell lines. Pharmacologic Axl blockade resulted in a significant dose dependent decrease in viability of MPN cell lines as measured by WST-1 cell viability assay. Annexin+ staining revealed an increased rate of apoptotic cells upon BGB324 treatment for SET-2 (increase by 15% at 1µM, p<0.001) and BaF3-Jak2V617F cells (increase by 54% at 2µM, p<0.05). Moreover, Western Blot analysis showed higher levels of cleaved caspase 3 in BGB324 treated SET-2 cells and decreased levels of anti-apoptotic bcl-2 in BGB324 treated BaF3-Jak2V617F cells. Additionally, BrdU incorporation assays showed a dose dependent decrease in proliferating cells upon treatment with BGB324 in MPN cell lines (p<0.05). Genetic knockdown of Axl in SET-2 cells decreased cell viability by 75% (p<0.01), increased apoptosis levels as measured by Annexin+ staining by 61% (p<0.05) and decreased proliferation as measured by BrdU incorporation by 35% (p<0.001) compared to control-transduced cells. Furthermore, Western Blot analysis revealed that genetic knockdown of Axl resulted in decreased phosphorylation of Stat3 and Stat5 compared to control-transduced cells. Combined Axl and Jak2 blockade, using BGB324 and the Jak2-inhibitor ruxolitinib, showed additive effects on reducing cell viability in SET-2 and BaF3-Jak2V617F cells (p<0.01 and p<0.001, respectively). Western Blot analysis identified inhibition of Stat5 by BGB324 single treatment in SET-2 cells whereas additive effects of combined Axl and Jak2 blockade resulted from additional inhibition of Stat3. In BaF3-Jak2V617F cells, BGB324 single treatment resulted in downstream inhibition of Akt signaling whereas additive effects of combined Axl and Jak2 blockade were exerted via additional inhibition of Stat5, Stat3 and Erk. The finding that BGB324 inhibits growth of MPN cells was further corroborated in vivo. A xenograft tumor model with SET-2 cells was set up in vivo. SET-2 tumor bearing mice treated with BGB324 50mg/kg showed a slower tumor growth (n=8, p<0.01), with a 60% reduction of tumor weight compared to vehicle treated mice (n=8/8, p<0.01). As a second in vivo model, a systemic model of Jak2V617F driven disease was used. After intravenous injection of BaF3-Jak2V617F cells, mice were treated with 50mg/kg BGB324 or vehicle starting the day after inoculation. BGB324 treated mice had a longer overall survival compared to vehicle treated mice (n=10/11, p*<0.05). Furthermore, to evaluate the potential of BGB324 in primary MPN cells, peripheral blood mononuclear cells (PBMC) were isolated from MPN patients and healthy donors. Western Blot analysis showed higher levels of Axl expression by PBMC from MPN patients compared to PBMC from healthy donors. Moreover, colony-forming assays with PBMC were performed in the presence of different concentrations of BGB324. Here, a higher reduction in the number of colony forming units (BFU-E and CFU-GEMM) was observed in samples from MPN patients compared to healthy donors upon treatment with 1µM (77% vs. 5%, respectively; p<0.001) or 2µM (100% vs. 60%, respectively; p<0.01) of BGB324 (n=5/5). In conclusion, these data indicate therapeutic potential of Axl blockade in BCR-ABL negative MPN as monotherapy and in combination with Jak2-inhibition, supporting the need for clinical investigation. Disclosures von Amsberg: Novartis: Honoraria, Speakers Bureau; Ipson: Honoraria, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Speakers Bureau; Sanofi: Honoraria, Speakers Bureau; Astellas: Honoraria, Speakers Bureau; MSD: Honoraria, Speakers Bureau. Loges:BerGenBio: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

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Sulforaphane and PEITC Augment Activity of Conventional and Novel Anti-Myeloma Drugs

Blood ◽

10.1182/blood.v112.11.2648.2648 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2648-2648

Author(s):

Jana Jakubikova ◽

Jan Sedlak ◽

Steffen Klippel ◽

Merav Leiba ◽

Yu-Tzu Tai ◽

...

Keyword(s):

Western Blot ◽

Cell Lines ◽

Stress Responses ◽

Blot Analysis ◽

Western Blot Analysis ◽

Cytotoxic Effect ◽

Therapeutic Potential ◽

Cruciferous Vegetables ◽

M Cell ◽

Mapk Activation

Abstract Isothiocyanates (ITCs) represent a family of phytochemicals found in cruciferous vegetables. Several epidemiological studies indicated that high intake of diet-derived ITCs may provide chemopreventive effect associated with a reduced risk of renal, prostate, pancreatic and colorectal carcinoma. ITCs have also been reported to enhance the chemosensitivity of diverse types of tumor cells. We therefore evaluated the cytotoxic effect of ITC, sulforaphane (SFN) and phenylethyl isothiocyanate (PEITC) on a panel of human MM cell lines, including cells resistant to doxorubicin (RPMI-Dox40), mitoxantrone (RPMI-MR20), melphalan (RPMI-LR5), and dexamethasone (MM.1R, OPM1 and OPM2); as well as cells with low sensitivity to thalidomide derivatives (RPMI 8226-S) and sensitive cell lines (MM.1S). We evaluated the anti-MM activity of these compounds using both MTT and flow cytometric assays. Our results suggest that all tested MM cell lines are susceptible to the cytotoxic effect of both ITCs at concentrations in the same order of magnitude as those achieved in vivo by dietary consumption of cruciferous vegetables. PEITC (IC50 of 3.5–8.2 μM) was more potent than SFN (IC50 of 5–15 mM) at 48 h. ITCs induce apoptotic death of MM cells, evidenced by Annexin V-FITC staining, increased sub-G1 population measured by flow cytometry, and cleavage of PARP and caspase-3 by western blot analysis, ITCs also induced G2/M cell cycle arrest and depletion of mitochondrial potential in JC-1 probed MM cell lines. Multiplex analysis of phoshorylation signaling pathways, using the Luminex system and confirmed by conventional western blot analysis, revealed that PEITC at 2hrs triggers MAPK activation (MEK1 (4-fold), ERK1/2 (4.4-fold), JNK (25.4-fold) and p38MAPK (6-fold)) at 2h, which likely are stress responses of cells to PEITC treatment. SFN induced less pronounced but a more sustained MAPK activation (up to 24 h) than PEITC. Concentration-dependent increases in phosphorylation of GSK3α/β, c-jun, p70S6 kinase, and p90RSK were also observed at early time-points after ITCs-treatment. In contrast, decreased phoshorylation of Akt was observed in MM cells treated with SFN at 2 h and PEITC at 24 h. Chou-Talalay analysis of the effects of combinations of ITCs with anti-MM drugs (dexamethasone, melphalan and bortezomib) revealed all combinations to have synergistic MM cytotoxicity. Importantly, ITCs treatment, both alone and in combination with the aforementioned agents, also significantly suppressed proliferation of CFSE-labeled MM cell lines co-cultured with the human bone stromal cell line HS-5. These results indicate that SFN and PEITC suppress survival and proliferation of MM cells, both alone and in combination, and suggest their therapeutic potential in MM.

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Mer Receptor Tyrosine Kinase Is a Novel Therapeutic Target In Multiple Myeloma

Blood ◽

10.1182/blood.v122.21.1957.1957 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1957-1957

Author(s):

Sandra Christoph ◽

Silke Maag ◽

Deborah DeRyckere ◽

Douglas K. Graham ◽

Stephen V Frye ◽

...

Keyword(s):

Western Blot ◽

Cell Lines ◽

Receptor Tyrosine Kinase ◽

Blot Analysis ◽

Colony Formation ◽

Western Blot Analysis ◽

Caspase 3 ◽

Downstream Signaling ◽

Tk Inhibitors

Abstract Introduction Although many novel therapeutic agents have been explored, multiple myeloma (MM) remains an incurable hematopoietic malignancy. Mer receptor tyrosine kinase (Mer TK) is a member of the TAM (Tyro3, Axl and Mer) family and is involved in the progression of several human malignancies. Inhibition of Mer expression reduces pro-survival signaling, increases chemosensitivity in vitro, and delays tumor progression in vivo. These observations make Mer TK inhibitors to excellent candidates for targeted therapies. We show here for the first time that Mer TK is ectopically expressed in MM cells and test the response of MM cell lines to our previously described small molecule Mer inhibitor, UNC1062, a substituted pyrazolopyrimidine. Here we demonstrate the biochemical and biologic effects of treatment with UNC1062, which suggests efficacy of Mer inhibitors as a novel strategy for the treatment of MM. Methods: Western blot analysis was used to determine expression of the TAM receptors and to evaluate inhibition of Mer TK activation and downstream signaling by UNC1062 in MM cell lines. UNC1062-mediated anti-myeloma activity with or without bortezomib was determined using short-term (MTT) and long-term (colony-formation) assays. Cleaved PARP and caspase 3 were detected by western blot analysis as indicators of apoptosis. Results: Western blot analysis of protein extract from MM cell lines showed that four (RPMI8226, U226, AMO-1 and KMS-12BM) out of six lines (66.6%) express Mer TK protein at varying levels, with AMO-1 expressing the least and U226 expressing the most. The MM cell lines OPM-2 and NCI-H929 did not express Mer TK. UNC1062 potently inhibits Mer TK activity in vitro (Mer IC50= 1.1 nM, Morrison Ki = 0.33 nM). In cell-based assays, treatment with UNC1062 inhibited accumulation of phospho-Mer and downstream signaling through ERK and Stat5 in U226 cells. UNC1062 also reduced proliferation and/or survival in Mer TK-expressing RPMI8226 cells (IC50 = 0.98 ± 0.14 μM, n=7) and U226 (IC50 = 0.28 ± 0.09 μM, n=3) cells. Treatment with UNC1062 did not lead to a meaningful reduction of metabolically active cells in Mer-negative NCI-H929 and OPM-2 cells. In contrast, UNC1062-treated Mer-positive MM cells exhibited increased levels of cleaved caspase 3 and cleaved PARP confirming apoptosis induction. Treatment with UNC1062 (100 nM) resulted in a statistically significant, dose-dependent decrease in colony-formation in methylcellulose compared to control cultures in Mer-positive RPMI8226 cells (418 ± 23 vs. 291 ± 37 colonies, p = 0.04, n = 3). No significant reduction of colony formation was observed in Mer-negative OPM-2 cells. Treatment of U226 cells with UNC1062 (800 nM) in combination with bortezomib resulted in a statistically significant reduction in relative cell number compared with bortezomib alone (0.61 ± 0.06 vs 0.41 ± 0.06, p = 0.04, n =3). Conclusion: Mer TK is ectopically expressed in the majority of investigated MM cell lines. UNC1062 treatment of Mer TK positive MM cells inhibits Mer TK activity and downstream signaling, mediates anti-neoplastic activity in liquid culture, and decreases colony-formation in methylcellulose. In addition, UNC1062 treatment sensitizes MM cells to bortezomib. Taken together, these data support further development of Mer TK inhibitors as novel MM therapy alone and in combination with bortezomib. Disclosures: No relevant conflicts of interest to declare.

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Synthesis and Characterization of Quinoline-3-Carboxamide Derivatives as Inhibitors of the ATM Kinase

Current Topics in Medicinal Chemistry ◽

10.2174/1568026620666200731174216 ◽

2020 ◽

Vol 20 (23) ◽

pp. 2070-2079

Author(s):

Srimadhavi Ravi ◽

Sugata Barui ◽

Sivapriya Kirubakaran ◽

Parul Duhan ◽

Kaushik Bhowmik

Keyword(s):

Western Blot ◽

Cancer Cells ◽

Cell Line ◽

Cell Lines ◽

Cancer Cell ◽

Blot Analysis ◽

Western Blot Analysis ◽

Cancer Cell Lines ◽

Atm Kinase ◽

Cytotoxicity Studies

Background: The importance of inhibiting the kinases of the DDR pathway for radiosensitizing cancer cells is well established. Cancer cells exploit these kinases for their survival, which leads to the development of resistance towards DNA damaging therapeutics. Objective: In this article, the focus is on targeting the key mediator of the DDR pathway, the ATM kinase. A new set of quinoline-3-carboxamides, as potential inhibitors of ATM, is reported. Methods: Quinoline-3-carboxamide derivatives were synthesized and cytotoxicity assay was performed to analyze the effect of molecules on different cancer cell lines like HCT116, MDA-MB-468, and MDA-MB-231. Results: Three of the synthesized compounds showed promising cytotoxicity towards a selected set of cancer cell lines. Western Blot analysis was also performed by pre-treating the cells with quercetin, a known ATM upregulator, by causing DNA double-strand breaks. SAR studies suggested the importance of the electron-donating nature of the R group for the molecule to be toxic. Finally, Western-Blot analysis confirmed the down-regulation of ATM in the cells. Additionally, the PTEN negative cell line, MDA-MB-468, was more sensitive towards the compounds in comparison with the PTEN positive cell line, MDA-MB-231. Cytotoxicity studies against 293T cells showed that the compounds were at least three times less toxic when compared with HCT116. Conclusion: In conclusion, these experiments will lay the groundwork for the evolution of potent and selective ATM inhibitors for the radio- and chemo-sensitization of cancer cells.

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DIAPH1 Is Upregulated and Inhibits Cell Apoptosis through ATR/p53/Caspase-3 Signaling Pathway in Laryngeal Squamous Cell Carcinoma

Disease Markers ◽

10.1155/2019/6716472 ◽

2019 ◽

Vol 2019 ◽

pp. 1-10 ◽

Cited By ~ 3

Author(s):

Jiechao Yang ◽

Liang Zhou ◽

Yanping Zhang ◽

Juan Zheng ◽

Jian Zhou ◽

...

Keyword(s):

Squamous Cell Carcinoma ◽

Western Blot ◽

Cell Carcinoma ◽

Squamous Cell ◽

Blot Analysis ◽

Western Blot Analysis ◽

Molecular Mechanisms ◽

Caspase 3 ◽

Laryngeal Squamous Cell Carcinoma ◽

Poor Overall Survival

Cancer bioinformatics has been used to screen possible key cancer genes and pathways. Here, through bioinformatics analysis, we found that high expression of diaphanous related formin 1 (DIAPH1) was associated with poor overall survival in head and neck squamous cell carcinoma and laryngeal squamous cell carcinoma (LSCC). The effect of DIAPH1 in LSCC has not been previously investigated. Therefore, we evaluated the expression, function, and molecular mechanisms of DIAPH1 in LSCC. Immunohistochemistry and western blot analysis confirmed the significant upregulation of DIAPH1 in LSCC. We used DIAPH1 RNA interference to construct two DIAPH1-knockdown LSCC cell lines, AMC-HN-8 and FD-LSC-1, and validated the knockdown efficiency. Flow cytometry data showed that DIAPH1 inhibited apoptosis. Further, western blot analysis revealed that DIAPH1 knockdown increased the protein levels of ATR, p-p53, Bax, and cleaved caspase-3, -8, and -9. Thus, DIAPH1 is upregulated in LSCC and may act as an oncogene by inhibiting apoptosis through the ATR/p53/caspase-3 pathway in LSCC cells.

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Abstract 255: Cytoprotective Effects of Erythropoietin and Erythropoietin-derivatives in Ischaemic Human Myotubes and the Role of Pi3k/akt Signalling Pathway

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.33.suppl_1.a255 ◽

2013 ◽

Vol 33 (suppl_1) ◽

Author(s):

Rebekah Sian Hwee Yu ◽

Daryll Baker ◽

David Abraham ◽

Janice Tsui

Keyword(s):

Skeletal Muscle ◽

Western Blot ◽

Blot Analysis ◽

Western Blot Analysis ◽

Caspase 3 ◽

Signalling Pathway ◽

Critical Limb Ischaemia ◽

Human Myotubes ◽

Muscle Biopsies

Objectives Erythropoietin (Epo) has tissue-protective effects in response to injury, acting through the EpoR-βcR heteroreceptor. We have previously demonstrated the presence and interaction of the EpoR and βcR in human skeletal muscle. Here we aim to investigate the potential cytoprotective effects of Epo and an Epo-derivative (ARA-290) in a human in vitro model of skeletal muscle and establish a potential downstream signalling pathway utilised in protecting cells from apoptosis (including Jak-2, PI3k/Akt, NFkB). Methods Gastrocnemius muscle biopsies were obtained from patients with critical limb ischaemia and control samples were obtained from non-ischaemic patients. Human myoblasts were isolated from muscle biopsies, cultured, and allowed to differentiate into myotubes in order to investigate the cytoprotective effects of Epo and ARA-290 on myotubes subjected to simulated ischaemia. The PI3k inhibitors, LY294002 and wortmannin, were then used to determine the role of PI3k/Akt pathway in mediating cytoprotection. Following this, inhibitors against the upstreatm (Jak-2) and downstream (NFkB) molecules were also investigated. Western blot analysis, using the pro-apoptotic marker cleaved caspase-3 was performed and compared with levels of Akt and phosphorylated-Akt, using western blot analysis. Results Exogenous administration of Epo and ARA-290 were able to ameliorate the ischaemia-induced apoptosis on isolated human myotubes as shown by a significant reduction in cleaved caspase-3 expression. Addition of all inhibitors, to ARA-290 or Epo pre-treated cells, abolished the reduction in apoptosis. Conclusion The ability of ARA-290 to attenuate apoptosis in human myotubes undergoing ischaemic insult suggests a potential role in tissue protection in skeletal muscle injury. We propose that the PI3k/Akt signalling pathway is involved in mediating this cytoprotection.

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The Spermatozoa Protein, SLLP1, Is a Novel Cancer-Testis Antigen in Hematologic Malignancies.

Blood ◽

10.1182/blood.v104.11.4281.4281 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4281-4281

Author(s):

Zhiqing Wang ◽

Yana Zhang ◽

Arabinda Mandal ◽

Jian Zhang ◽

Francis J. Giles ◽

...

Keyword(s):

Immune Responses ◽

Western Blot ◽

Tumor Cells ◽

Blot Analysis ◽

Western Blot Analysis ◽

Hematologic Malignancies ◽

Myeloma Cell ◽

Rt Pcr ◽

Healthy Donors ◽

Cancer Testis

Abstract SLLP1 is a unique non-bacteriolytic c-lysozyme-like protein isolated from human spermatozoa. Antisera to SLLP1 blocks binding in the hamster egg penetration assay, suggesting that SLLP1 may be involved in sperm/egg adhesion. A recent study by dot blot analysis on RNA showed that SLLP1 was expressed only in the testis and in Burkitt lymphoma Raji cell line, suggesting that further studies are warranted to determine and characterize SLLP1 expression in tumor cells, in particular, fresh tumor specimens. Using a pair of sequence-specific primers in RT-PCR, we found that SLLP1 transcripts could be detected in 5/8 myeloma cell lines, suggesting that SLLP1 may be expressed in tumor cells from some hematologic malignancies. When we applied the investigations to 52 primary hematologic malignant specimens, SLLP1 transcripts were detected in 6/17 myeloma, 4/14 CML, 3/11 CLL, 2/9 AML and 0/1 hairy cell leukemia. In contrast, SLLP1 transcripts were not detected in the peripheral blood (n=12) or bone marrow (n=3) from any healthy donors. The specificity of the PCR products was confirmed by either sequence analysis or restriction digest with Pvu II. SLLP1 transcripts were translated into its corresponding protein in these tumor cells. Using tumor cell lysate in Western blot analysis, we detected SLLP1 protein in the myeloma cell lines and also in fresh malignant specimens, although positivities were only observed in specimens with high RT-PCR signals. All PCR-negative specimens were also negative in Western blot analysis. The specificity of the Western blot signals were confirmed in all cases by blocking assays with a high concentration of recombinant SLLP1 protein. We next investigated the expression of SLLP1 in a large panel of normal tissues using RT-PCR and real time quantitative PCR. Both approaches showed that SLLP1 is a novel Cancer-Testis antigen in hematologic malignancies. SLLP1 was detected, at a level of 8206 copies/0.25 mcg total RNA, only in normal testis. We also found that the SLLP1 mRNA copy numbers in fresh hematologic tumor specimens were up to 2316 copies/0.25 mcg total RNA, i.e. more than 25% of the level found in normal testis. We cloned and generated SLLP1 recombinant protein from E coli and used the purified recombinant SLLP1 in an ELISA system to detect anti-SLLP1 antibodies. Using sera from 24 healthy donors and the mean + 2SD as the cut-off signal intensities, we found that high titer IgG antibodies directed at SLLP1 could be detected in the sera from 2/9 AML, 5/23 CLL, 6/27 CML and 14/51 myeloma patients. The specificity of the antibodies was confirmed in Western blot analysis. Probably due to the decreased sensitivity of the detection system in Western blot analysis, only 1/2 AML, 3/5 CLL, 4/6 CML and 7/14 myeloma SLLP1 antibody+ sera produced a signal in the Western blot analysis. Interesting, IgG2 was by far the commonest SLLP1 antibodies in these patients. There was a good correlation between SLLP1 gene expression and immune responses. In summary, SLLP1 is a novel CT antigen in hematologic malignancies and is capable of eliciting B-cell immune responses in vivo in cancer-bearing patients. Our results support SLLP1 as a protein target appropriate for further in vitro study to define its suitability for immunotherapy.

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