SDF-1 Inhibition Using Spiegelmer® Nox-A12 As a Novel Strategy For Targeting AML Cells Within Their BM Microenvironment
Authors: Jacamo Rodrigo , Zeng Zhihong , Chen Ye , Shi Yuexi , McQueen Teresa , Kruschinski Anna , Konopleva Marina , Ruvolo Peter P. , Andreeff Michael 
Published: October 2019

Abstract
Bone marrow (BM) mesenchymal stromal cells (MSC) protect leukemia cells from chemotherapy-induced apoptosis. CXCR4, the receptor for the stromal-derived factor 1 (SDF-1/CXCL12), is involved in modulating leukemic progenitor cell homing and it is essential for the migration of these cells to the BM niche. As the BM niche imparts favorable survival advantage to leukemia cells, a strategy to disrupt leukemic cell homing to the BM microenvironment may represent a novel way to effectively kill these malignant cells. Strategies to disrupt the SDF-1/CXCR4 axis have relied on inhibition of the CXCR4 receptor using various drugs including plerixafor, which is potent at mobilizing normal progenitor cells. We previously found that plerixafor antagonized SDF-1 and MSC-induced migration of leukemic cells, inhibited SDF-1-induced AKT and ERK activation, and decreased stroma-mediated protection from AraC-induced apoptosis in 11 out of 18 primary AML samples (Zeng et al Blood 2009; 113: 6215). We also found that the inhibition of CXCR4 induces miRNA let-7a expression via the transcription factor Yin Yang1 (Chen et al, JCI, 2013; 123:2395), and induces chemosensitization irrespective of cell mobilization. While inhibition of CXCR4 has shown promise for use in AML therapy (Andreeff, et al ASH, Blood 120(21) (#142), 11/2012), a more effective strategy might be to target the SDF-1 ligand itself since SDF-1 can bind to other receptors (i.e. CXCR7) that are likely not recognized by CXCR4 antibodies. NOXXON (Berlin, Germany) has recently identified a potent inhibitor to SDF-1 that is based on a so-called Spiegelmer®. The Spiegelmer NOX-A12 is a structured l-RNA oligonucleotide, PEGylated at the 5'-end to improve the pharmacokinetics. In healthy volunteers, NOX-A12 displays a half-life of∼38 hours and has demonstrated a long-term and dose-dependent mobilization of WBCs and CD34+ cells. The drug is currently tested in clinical trials in multiple myeloma and chronic lymphocytic leukemia patients (Gobbi et al., ASH 2012 120: 4593). In the current study, the ability of NOX-A12 to suppress survival signaling in leukemic and MSC cells was examined. In addition, we tested the ability of the Spiegelmer to mobilize leukemia cells in a murine xenograft model. NOX-A12 was found to suppress the phosphorylation of ERK in AML cells, and suppress the phosphorylation of ERK, FAK, and STAT3 in MSC. These results suggested that SDF-1 affected diverse survival signaling pathways in the leukemic cells and the critical supporting stromal niche. In vivo activity was examined in NOD/SCID/-g- mice injected intravenously with murine leukemia Baf/ITDluc/GFP cells and monitored by a non-invasive bioluminescent imaging. The in vivo mobilization assay was assessed when the majority of mice had approximately 10% tumor burden. Plerixafor, NOX-A12, or a combination of both agents was subcutaneously administrated and leukemic GFP+ cells were detected by analytical flow cytometry. Plerixafor and NOX-A12 administered separately increased total WBC count by 2- to 3-fold and concomitantly increased the percentage of circulating GFP+ cells in all mice (to 14.2% and 18.5%, respectively). Importantly, the combination of both targeted agents further increased the percentage of circulating GFP+ cells in an approximately additive fashion (to 26.4%).

Conclusion
we demonstrate that the novel SDF-1 Spiegelmer NOX-A12 can suppress survival signaling in both leukemia and supportive MSC cells. Furthermore, increased mobilization of leukemic cells in vivo was observed when NOX-A12 and plerixafor were used in combination. These findings suggest the possibility that targeted disruption of leukemia/stroma interactions by optimized blockade of the pro-survival SDF-1/CXCR4 axis will not only increase leukemia cell mobilization, but also enhance the sensitivity to chemotherapy of both mobilized and non-mobilized the leukemic cells and leukemic stem cells. Experiments are ongoing to further validate this hypothesis.

Disclosures:
Kruschinski: Noxxon: Employment.