RX-3117, a Novel Nucleoside Analog Targeting UCK2 Is Active Alone and Synergistic with Ibrutinib and Bendamustine in ABC and DH/DP- Diffuse Large B-Cell Lymphoma

Introduction

RX-3117 is a prodrug activated by uridine-cytidine kinase 2 (UCK-2) which is found predominantly in human cancer cells including non-Hodgkin lymphoma (NHL). Once activated by UCK2, RX-3117 inhibits DNA and RNA synthesis leading to apoptosis. Despite current approved therapies for DLBCL, a significant number of patients fail frontline and subsequent therapies. This is more so for activated B-cell (ABC), double-hit/double protein DLBCL (DH/DP-DLBCL) and Transform Follicular lymphoma. We hypothesized that combining RX-3117 with bendamustine or ibrutinib would be synergistic and a rational means to inhibit proliferation and enhance apoptosis in aggressive types of DLBCL.

Methods

U2932, VAL, OCI-Ly10 and TMD8 DLBCL cells were assessed for UCK-2 expression by Western blotting. Cellular cytotoxicity assays were performed with RX-3117, ibrutinib, bendamustine and their combinations using a CellTiter 96 Cell Proliferation Assay (Promega, Madison, WI) and combination-index (CI) calculated by the principle of Chou and Talalay. Apoptosis was detected by Western blotting for PARP cleavage and flow cytometry. Cellular signaling pathways were investigated by Western blotting analyses. Finally, to establish safety and efficacy, we are evaluating above drug combinations in a U2932 DP-DLBCL SCID mouse xenograft model.

Results

The anti-proliferative activity of RX-3117 correlates with increased UCK-2 expression and activity. In DLBCL cell lines UCK-2 is over-expressed relative to normal B cells indicating a significant therapeutic index. MTS assays demonstrate single agent differential activity for RX-3117, bendamustine and ibrutinib in the 4 DLBCL cell lines respectively. RX-3117 plus bendamustine or ibrutinib were highly synergistic in DLBCL cells. Western blotting showed inhibition of NF-kB expression with RX-3117 plus bendamustine or ibrutinib. This combination also induces apoptosis by increased PARP cleavage. The U2932 mouse xenograft model is ongoing with RX-3117 alone and in combination with bendamustine + rituximab or ibrutinib. Results will be available shortly and presented at the meeting.

Conclusions

RX-3117, a novel nucleoside pro-drug activated by high UCK-2 levels in aggressive DLBCL has single agent activity and synergizes with bendamustine and ibrutinib. Novel combination therapies of RX-3117 are warranted in relapsed and refractory DLBCL.