Targeting CXCR4, SDF1 and Beta-Adrenergic Receptors In the AML Microenvironment by Novel Antagonist POL6326, G-CSF and Isoproterenol
Authors: Chen Ye , Zeng Zhihong , Shi Yuexi , Jacamo Rodrigo , Ludin Christian , Dembowsky Klaus , Frenette Paul S. , Konopleva Marina , Andreeff Michael 
Published: October 2019

Abstract 2179

The role of SDF-1α /CXCR4 signaling in leukemia cell/bone marrow microenvironment interactions has recently been established. Activation of CXCR4 induces leukemia cell trafficking and homing into the marrow microenvironment, where the CXCR4 ligand SDF-1α is produced, keeping leukemic cells in close contact with the stromal cells and extracellular matrix that constitutively secrete growth and anti-apoptosis signals. We previously reported that CXCR4 is up-regulated under the hypoxic conditions prevalent in leukemia-harboring bone marrow niches (Fiegl M et al. Blood 2009, Konopleva M et al. ASH 2010) and that tumor-stromal interactions can be disrupted by AMD3465, a CXCR4 antagonist (Zeng et al. Blood 2009). Therefore, targeting the SDF-1α /CXCR4 axis is an attractive investigational therapeutic approach for AML. Here, we demonstrate that POL6326, a new CXCR4 antagonist developed by Protein Epitope Mimetic (PEM) Technology, can effectively antagonize SDF-1α and stromal induced chemotaxis of AML cells at concentrations as low as 200nM. By blocking CXCR4, POL6326 inhibited SDF-1α-induced activation of pro-survival signaling pathways in AML cells in vitro. Furthermore, POL6326-mediated CXCR4 inhibition partially abrogated the protective effects of stromal cells in leukemia/stromal co-culture systems. Granulocyte colony-stimulating factor (G-CSF) is known to reduce SDF-1 levels in bone marrow (Petit I et al. Nature Immunology 2002) and isoproterenol was described to mobilize hematopoietic stem cells through activation of beta-adrenergic receptors (Méndez-Ferrer S et al. Nature 2008). To evaluate the potential of POL6326 in mobilizing leukemia cells, we injected 0.5×105 Ba/F3-ITD/luc/GFP cells into NOD/SCID mice. Ten days later, POL6326 (10mg/kg, alone or in combination with isoproterenol or G-CSF) was administered to the mice subcutaneously 1 hour prior to peripheral blood collection. We found that POL6326 alone mobilized murine AML cells into circulation (21±7% increase in circulating GFP+ cells compared with PBS (p<0.05; n=6 mice per group)). Maximal mobilization was observed in mice treated with POL6326 combined with isoproterenol (2mg/kg, i.p) or G-CSF (10ug/mouse, i.p) at different time points: on day 10, mice treated with POL6326 plus isoproterenol showed a 76±11% increase of circulating GFP+ cells as compared to controls (p<0.01), and on day 18, POL6326 plus G-CSF 379±51% increase (p<0.01). Using the same murine leukemia model, we treated mice with POL6326 (10mg/kg s.c.) 1 hour prior and 1 hour post administration of Ara-C (100mg/kg, i.p.) on days 4, 11, 18, 21 and 25. Randomly selected mice from each group were sacrificed on day 18 for tissue collection, and the extent of leukemic infiltrates of different organs was assessed by both hematoxylin and eosin staining and anti-GFP antibody staining for specifically GFP+ leukemic cells. The combination of POL6326 and Ara-C resulted in significantly reduced leukemia cells in all organs (liver: 15.3±4.1% in POL+Ara-C group vs 50.1±16.7% in control group (p<0.05), bone marrow: 12.3±4.4% vs 88.1±6.5% (p<0.01), spleen: 8.0±3.2% vs 45.9±15.7% (p<0.05)), and extended survival by appropriate 35% (p<0.01 compare with control group). In conclusion, our findings demonstrate for the first time that the PEM CXCR4 antagonist POL6326 can effectively mobilize engrafted leukemia cells from their protective stromal microenvironment into circulation. This strategy enhances the efficacy of chemotherapeutic agents such as Ara-C, resulting in significantly reduced leukemia burden and prolonged survival of the animals. Moreover, we demonstrated strong synergies for leukemia mobilization when POL6326 was combined with isoproterenol or G-CSF. These findings extend the previously reported concept of disrupting the SDF-1α/CXCR4 axis to sensitize leukemia cells to therapy by blocking CXCR4 with a novel peptide, cleaving SDF-1 and activating beta-adrenergic receptors.

Disclosures:

Ludin: Polyphor: Employment. Dembowsky: Polyphor: Employment. Andreeff: Genzyme: Consultancy; Polyphor: Research Funding.