GPC-100 Is a Novel CXCR4 Inhibitor That Leads to Improved in Vitro Potency and In Vivo Efficacy in Stem Cell Mobilization When Combined with a Beta-Blocker

Successful autologous stem cell transplant (ASCT) in multiple myeloma (MM) patients is often hindered by poor mobilization, with ~ 1 in 7 patients failing to reach adequate number of CD34⁺ cells/kg. Small molecule inhibitors of CXCR4 like GPC-100 and plerixafor (AMD3100) disrupt the CXCL12/CXCR4 axis critical for migration and retention of hematopoietic stem cells (HSC) in bone marrow. Here, we provide evidence that GPC-100 in combination with propranolol (Pro), a beta-2 adrenoceptor (B2AR) blocker (BB) and G-CSF, has the potential to be a best-in-class mobilization therapy for ASCT.

In vitro activity of GPC-100 was investigated in cell-based assays. In the FRET ligand binding assay in HEK cells, GPC-100 more potently inhibited binding of CXCL12 to CXCR4 than AMD3100 with a ~ 30-fold better binding affinity (Ki of 1.6 vs 40 nM, respectively). Potent inhibition of CXCR4 was recapitulated in cell migration assays using a multiple myeloma cell line MM.1S, where GPC-100 inhibited CXCL12-mediated migration with an IC₅₀ of 30 nM compared to an AMD3100 IC₅₀ of 80 nM.

Previous studies indicate that stress hormones like epinephrine and norepinephrine exert stimulatory effects on cancer progression by modulating tumorigenesis, proliferation, and metastasis via B2AR signaling.In a recent study with 208 MM patients, overall survival was significantly longer in 37% of patients who reported BB usage for ≥ 3 months after diagnosis compared to those with no BB usage (107 vs 86 months, Hwa et al. Eur J Haematol 2021). Furthermore, it has been demonstrated that BBs like Pro can shift bone marrow-derived cells to differentiate away from a myeloid bias to a phenotype consistent with CD34⁺ stem cells and genes associated with stem cells (Knight et al. Blood Adv 2020).

To investigate the interplay between CXCR4 and B2AR blockade in vitro, we performed interaction and functional studies. Using a proximity ligation assay (PLA) in the breast cancer cell line MDA-MB-231 endogenously expressing CXCR4 and B2AR, we detect CXCR4 and B2AR heteromers, while knock-out of B2AR expression leads to a decrease in PLA signal, confirming the proximity of CXCR4 and B2AR. We also demonstrate a functional consequence of CXCR4 and B2AR using Ca²⁺ flux assays in MDA-MB-231 cells that demonstrate synergy when co-stimulating with CXCL12 and salmeterol, a B2AR agonist. Inhibition of Ca²⁺ flux by GPC-100, and not AMD3100, is enhanced ~ 30-fold by co-treatment with Pro (1.3 vs 30 nM, respectively). Taken together, our in vitro results suggest that GPC-100 inhibition of CXCR4 can be modulated by Pro.

To obtain pre-clinical proof-of-concept, we determined the mobilization of white blood cells (WBC) with a CBC analysis, progenitor cells with a colony forming unit (CFU) assay and HSC with flow cytometry in C57/BL6 mice following GPC-100 combination treatments. First, administration of GPC-100 alone led to a greater WBC mobilization into the peripheral blood compared to AMD3100. Next, mice were treated with Pro for 7 days followed by a single dose of GPC-100 or AMD3100 on the 7^th day. Propranolol improved mobilization induced by a single administration of both GPC-100 and AMD3100. Finally, we determined whether the triple combination of G-CSF + GPC-100 + Pro was beneficial over the current ASCT standards of care: G-CSF alone or combined with AMD3100. We demonstrate that the triple combination mobilized greater number of WBC as well as progenitor cells compared to the standard of care. In addition, we show that WBC mobilization is a predictor of progenitor stem cell mobilization as we see a correlation between WBC counts, CFU, as well as the mobilized Lin-/sca-1+/c-kit + population indicative of mouse HSC.

Our findings support the use of GPC-100 and Pro with or without G-CSF for stem cell mobilization. This therapeutic strategy allows elimination of repeated daily injections of G-CSF, improving quality of life in patients, as well as providing a therapeutic option to patients that experience adverse effects from G-CSF. Additionally, combination treatment with G-CSF, GPC-100 and Pro may prove to be best-in-class mobilization therapy for ASCT in MM patients, especially those that fail to mobilize with standard of care. As a result, we will initiate a 2 arm, Phase 2 clinical trial evaluating both GPC-100 + Pro, and the triple combination of G-CSF + GPC-100 + Pro in Q4 of 2022.

No relevant conflicts of interest to declare.

Propranolol is typically used as a beta-blocker for cardiac indications, however we plan to use it in combination with a CXCR4 inhibitor for stem cell mobilization.