Background:

Diffuse Large B cell lymphoma, the most commonly diagnosed type of non-Hodgkin lymphoma is curable in many cases, despite this, up to 30% of patients will relapse after initial therapy, necessitating salvage chemotherapy and transplantation if feasible. There is a pressing need for novel treatment strategies in highly chemo refractory cases. ONC201 is a small molecule that induces p53-independent cell death in tumor cells while sparing normal cells through a number of putative mechanisms, including inactivation of the pro-survival kinases Akt and ERK. ONC201 is currently entering Phase I/II clinical trials as a monoagent in adult advanced cancers. ONC201 promotes up-regulation of TRAIL gene transcription by inactivating AKT and ERK kinases which leads to translocation of transcription factor Foxo3a into the nucleus. It appears to act on a p53 independent pathway.

ABT-199 is a selective, potent and orally bioavailable small molecule that selectively inhibits BCL-2 and triggers apoptosis. The Bcl-2 family of proteins is key regulator of the apoptotic process, comprising proapoptotic and prosurvival proteins. BH3-only proteins (such as Bim) bind to pro-survival proteins and cause increased permeability of mitochondrial membrane, release of cytochrome c and activation of caspases through release of Bax and others.

Methods:

Cell lines Pfeiffer and Toledo were purchased from ATCC and one patient DLBCL cell specimen from the ascitic fluid was cultured for use in experiments. ABT-199 was purchased from MedKoo Biosciences and ONC201 was provided from Oncoceutics.

Cytotoxicity was evaluated by using the CellTiter-Glo Luminescent Cell Viability Assay as per the manufacturerÕs instructions. Cell viability was measured over time in response to treatment with ONC201(1-64μM) and ABT-199(128 nM-4μM).

Western Blotting was performed on treated cells with well-established methodologies, with antibodies to c-Myc, Bcl-2, pAKT, pERK.

Results:

Immunohistochemical Staining Pattern of Cell Lines

Table 1.
PatientToledoPfeiffer
Bcl-2 +++ ++ + 
Bcl-6 + ++ ++ 
c-Myc +++ ++ + 
p-ERK + + ++ 
p-AKT + ++ + 
Bax ++ NA NA 
Bim ++ NA NA 
PatientToledoPfeiffer
Bcl-2 +++ ++ + 
Bcl-6 + ++ ++ 
c-Myc +++ ++ + 
p-ERK + + ++ 
p-AKT + ++ + 
Bax ++ NA NA 
Bim ++ NA NA 
View Large

Mean IC50 Calculated for Cell Lines

Table 2.
Cell LineTherapeutic Agent
ABT199 ONC201
Patient Sample 8 μM 5 nM 
Toledo 9 μM 28 nM 
Pfeiffer 6 μM 2 μM 
Cell LineTherapeutic Agent
ABT199 ONC201
Patient Sample 8 μM 5 nM 
Toledo 9 μM 28 nM 
Pfeiffer 6 μM 2 μM 
View Large

SHAPE

Conclusion/Discussion:

The patient cell line, an ascitic fluid sample of DLBCL was sensitive to both ONC201 and ABT-199 and manifested bright Bcl-2 expression, the target of ABT-199. In this series there was a higher sensitivity to ABT199 in DLBCL cells with higher Bcl-2 expression. ONC201 down regulated pAKT expression, as seen in Western Blots in treated cells, consistent with prior investigation with the agent.

We further found that ONC201 synergizes to potentiate cytotoxicity with ABT199, as demonstrated in the cell viability assay for Toledo cell lines (at the 24 hour time point), which were the least sensitive to ONC201 (highest IC50) when given as a single agent. Yet, when combined with increasing doses of ABT199, there was synergistic lymphoma cell kill with a fixed dose of ONC201. Together these results suggest that ONC201 has potential as an antitumor agent in NHL as monoagent and in combination with ABT-199, which may be further explored in phase Ib/II trials. Further analysis in larger patient sample series may elucidate the biomarkers that predict for greater therapeutic sensitivity to these highly potent lymphoma agents.

Figure 1.
Figure 1.
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Disclosures

Allen:Oncoceutics, Inc: Employment, Equity Ownership. Eldeiry:Oncoceutics, Inc: Equity Ownership.

Topics:
cell lines, diffuse large b-cell lymphoma, mechlorethamine, proto-oncogene proteins c-akt, chemotherapy regimen, cytotoxicity, lymphoma, phosphotransferases, antibodies, antineoplastic agents

Author notes

*

Asterisk with author names denotes non-ASH members.

© 2015 by The American Society of Hematology
2015
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