The Spiegelmer^® Nox-A12 Abrogates Homing Of Human CLL Cells To Bone Marrow and Mobilizes Murine CLL Cells In The Eμ-TCL1 Transgenic Mouse Model Of CLL

NOX-A12 is a novel mirror-image oligonucleotide (Spiegelmer^®) that specifically antagonizes the chemokine stromal cell-derived factor 1 (SDF-1, CXCL12). SDF-1 is highly expressed by stromal and endothelial cells, and binding to its classical receptor CXCR4 leads to the activation of multiple signaling pathways regulating cell migration, retention and survival. SDF-1-induced inside-out activation of the integrin VLA-4 (a4ß1, CD49d/CD29) via CXCR4 is essential for homing of various normal and malignant hematopoietic cells to bone marrow (BM). We previously found that VLA-4 is indispensable for BM homing of chronic lymphocytic leukemia (CLL) cells. Hence, CXCR4 binding to SDF-1 may be a prerequisite for VLA-4 mediated trafficking of CLL cells into the protective BM microenvironment and for their retention in lymphoid organs, favoring their survival and chemotherapy-resistance.

In this project we compared the efficacy of NOX-A12 and the CXCR4 antagonist AMD3100 to mobilize CLL cells from their protective niches to the peripheral blood, thereby sensitizing them to cytotoxic agents such as fludarabine, and to prevent their recirculation into the protective lymphoid microenvironment.

To assess the contribution of SDF-1/CXCR4 signals to homing of CLL cells, we performed in vivo short term adoptive transfers of human VLA-4⁺ CLL cells into immune deficient mice. CLL cells (5-10 x 10⁶) were injected into the tail vein of NOD/SCID mice that were treated either with NOX-A12 (20 mg/kg) or AMD3100 (5 mg/kg). After a homing period of 3 hours lymphatic organs were analyzed for human CLL cell count. We found that for the majority of investigated CLL samples (except patient samples bearing the chromosomal aberration trisomy 12), CXCR4/SDF-1 inhibition reduced CLL cell recirculation into BM, thereby redirecting them into the spleen. The mean (± SD) relative homing rate (homed human CLL cells relative to injected cells per 1 x 10⁶ acquired murine cells) of CLL cells to BM decreased upon NOX-A12 treatment from 137 (± 87) to 30 (± 17) (p = 0.047), and upon AMD3100 treatment from 137 (± 71) to 38 (± 24) (p = ns). Further, we determined CXCR4 surface levels of CLL cells before injection and after cells had homed to BM. We noticed that homed CLL cells expressed elevated CXCR4 surface levels as mean MFIR (± SD) rose from 25 (± 13) to 93 (± 11) and that CXCR4 levels were further increased upon CXCR4/SDF-1 inhibition with NOX-A12 to MFIR 225 (± 2) and with AMD3100 to MFIR 213 (± 7).

Next, we used the Tcl1 transgenic transplantation model that mimics human CLL to test NOX-A12 and AMD3100-induced CLL cell mobilization. C57BL/6J wild type mice engrafted with splenocytes (1 x 10⁷) from Tcl1tg mice with established disease were treated with separate doses of NOX-A12 (0, 10, 20 and 50 mg/kg) by single intravenous injections. Separate doses of AMD3100 (0, 3, 5, and 10 mg/kg) were administered subcutaneously. Blood was collected from the tail vein before treatment, 1 hr, 3 hrs, 6 hrs, and 24 hrs post treatment and analyzed for absolute leukocyte counts and numbers of CD5⁺/CD19⁺cells. NOX-A12 demonstrated a considerable potential to mobilize CLL cells and other lymphocytes as the relative CLL cell and absolute lymphocyte blood count raised within 1 hour by 50% when using mice with high tumor load and by 200% when using mice with lower tumor load. Mobilization reached a peak after 6 hours with 100% and 220% relative increase to 0 hrs value. Compared to untreated control mice, CLL cell blood count raised from 80% to 120% within 6 hours. Even low doses (10 mg/kg) of NOX-A12 showed a high degree of mobilization of CLL cells and total lymphocytes. Higher doses lead to a prolonged mobilization duration. AMD3100 treatment did not lead to a significant increase in CLL cell or total lymphocyte blood count in peripheral blood.

In summary our data demonstrate a high efficiency of NOX-A12 to mobilize CLL cells and to prevent their recirculation into protective lymphatic organs. Abrogating SDF-1/CXCR4 signaling in combination with chemotherapy may provide an attractive approach for treatment of CLL. Currently, the effect of combined NOX-A12 and fludarabine treatment on progression and overall survival is evaluated using the Tcl1 transgenic transplantation model.