Antitumor Activity of Amphibian Ribonucleases, Onconase and R-Amhinase, on Tumor Cells From B-Cell Lymphoproliferative Disorders
Authors: Smolewski Piotr , Zwoliñska Malgorzata , Cebula-Obrzut Barbara , Majchrzak Agata , Misiewicz Malgorzata , Jeske Aleksandra , Darzynkiewicz Zbigniew , Ardelt Wojciech , Ardelt Barbara , Saxena Shailendra K , Muniz Charles , Robak Tadeusz 
Published: October 2019

Abstract
Abstract 2842

Background:
Several B-cell-derived malignancies still remains incurable. A promising approach that involves targeting RNA either by the use of specific antisense oligonucleotides or cytostatic/cytotoxic ribonucleases (RNases) is being promoted. Two amphibian RNases, onconase (ONC; ranpirnase) and, more recently, R-Amphinase (r-Amph), have already been developed, but studied so far mainly in solid tumors. In this report we demonstrate ex vivo and in vivo antitumor activity of ONC and, for the first time, R-Amph against tumor cells from different B-cell lymphoid malignancies.

Material and methods:
Pro-apoptotic effects of ONC or r-Amph alone and in combinations with doxorubicin (DOX) or purine analoques, fludarabine (FA) and cladribine (2-chlodeoxyadenozine; 2-CDA) were assessed ex vivo in peripheral blood samples of untreated patients with acute lymphoblastic leukemia; ALL, N=15) and chronic lymphocytic leukemia (CLL, N=45). Lymphocytes obtained from 31 healthy volunteers was also treated. Additionally, we tested in vitro activity of both agents alone or in combination with doxorubicin (DOX) on cell lines derived from Burkitt lymphoma (Raji) and with DOX, vincristin (VCR), dexamethasone (DEX), mafosfamide (MAF) or anti-CD20 antibody, rituximab (RIT) on diffuse large B-cell lymphoma (DLBCL)-derived cells (Toledo). Moreover, multiple myeloma (MM) cells were treated in vitro (RPMI 8226) and ex vivo (bone marrow cells). Drug-induced apoptosis was assessed after 48–72 hr incubation by the flow cytometry Annexin-V assay. Additionally, a drop of mitochondrial potential, activation of caspases -8, -9 and -3, and expression of several apoptosis-regulating proteins were investigated. Compensated apoptotic index (CAI) was calculated as a difference in % of apoptotic cells between the drug-treated sample and the parallel untreated culture.

Results:
Based on preliminary studies on different ex vivo and in vitro experimental models, concentrations 10–20 mg/ml of ONC and 20–60 mg/ml of r-Amph were chosen for final studies as the lowest doses inducing significant pro-apoptotic effect. In similar manner concentrations 50 ng/ml of 2-CdA, 1mg/ml of FA, 10–20 mg/ml of DOX, 20 mg/ml of RIT, 1ng/ml of VCR, 20 mg/ml of DEX and 1 mg/ml of MAF were selected.
In CLL cells, significant effect of both ONC and r-Amph was evident after 72 hrs of treatment (median CAI 17.1% and 13.9%; vs untreated control cells p=0.009 and 0.012, respectively). Importantly, the same doses did not affect survival of healthy lymphocytes. Combination of ONC, but not r-Amph, with 2-CdA or FA exerted significantly stronger effect than single drugs (p<0.005).
In case of ALL cells only ONC+DOX combination showed significantly higher pro-apoptotic effect compared to the single agents (p<0.005).
In MM cells, only r-Amph alone induced significant cytotoxity vs. control (p<0.05 both in vitro and ex vivo).
Both ONC and r-Amph showed significant anti-tumor activity in Burkitt lymphoma-derived Raji cell line (vs. control - p=0.007 and p=0,001, respectively). Moreover, combinations of ONC or r-Amph with DOX induced higher pro-apoptotic effect than single agents (p<0.001).
Interestingly, both ONC and r-Amph were highly active against DLBCL-derived cell line and even at the lowest concentrations induced significant toxicity. We mimicked in vitro DLBCL standard therapeutic model using RIT, MAF, DOX, VCR and DEX (R-CHOP regimen). We found, that addition of either ONC or r-Amph to this scheme significantly amplified pro-apoptotic effect against DLBCL cell (p<0.001 vs. “R-CHOP”).
The mechanism of both ONC and r-Amph cytotoxicity in all examined models was induction of apoptosis, evidently dependent on both, mitochondrial and external caspase-activation pathways (caspase -9, -8, -3; decline of mitochondrial potential). Both ONC and r-Amph induced over-expression of Bax and Bak proteins and down-regulation of Bcl-2 expression.

Conclusions:
Our study revealed strong pro-apoptotic activity of ONC and r-Amph in both CLL and aggressive B-cell lymphomas, with less impact for surviving of ALL or MM cells. Amplification of anti-tumor effect of R-CHOP regimen against DLBCL cells by studied RNases suggests that application of such combination of drugs may be considered to increase the cure rate in this aggressive lymphoma. Certainly, this strategy requires further studies, including an in vivo preclinical models and, eventual clinical trials.

Disclosures:
Ardelt: TAmiR Biotechnology, Inc: Employment, Membership on an entity's Board of Directors or advisory committees. Ardelt:Alfacell: Employment. Saxena:TAmiR Biotechnology, Inc: Employment. Muniz:TAmiR Biotechnology, Inc: Employment, Membership on an entity's Board of Directors or advisory committees.