Chemotherapy-Induced CXCR4 Modulation Predicts the In Vivo Efficacy of Plerixafor As a Chemosensitizer in Acute Leukemia

Activation of CXCR4 by the chemokine SDF-1 (CXCL12) results in the migration of leukemia cells to marrow niches that may contribute to chemoresistance and relapse. We previously showed that in vitro chemotherapy (chemo) treatment modulates CXCR4 expression in leukemia cell lines and primary pediatric AML samples, and that chemo-induced increases in surface CXCR4 (s-CXCR4) results in increased chemotaxis toward an SDF-1 gradient and decreased chemo-induced apoptosis when co-cultured with human marrow stroma feeder layers. We hypothesized that 1) CXCR4 inhibition by plerixafor (P) would sensitize leukemias to chemo through the interruption of leukemia-stromal cell signaling and 2) the degree of chemo-induced s-CXCR4 upregulation would be a predictive biomarker of the efficacy of P as a chemosensitizer. Because B-precursor ALL are known to highly express CXCR4, we tested these hypotheses in vitro using ALL cell lines and in vivo using a xenograft model of a high-risk pediatric leukemia, infant ALL.

ALL cell lines were pretreated for 72 hours with araC (A), dauno, vcr, and vehicle control (C). Chemo pretreatment induced upregulation of s-CXCR4 compared to C. Viable cells were then isolated using Ficoll and plated off stroma (O), on stroma (S), or pretreated with P for 30 minutes prior to plating on stroma (P+S). Cells were then treated for an additional 72 hours with full dose ranges of chemo. Apoptosis was measured with Annexin V/7-AAD, and IC50 was calculated. Overall, IC50 values were highest in S, followed by P+S, then O, demonstrating that upregulation of s-CXCR4 leads to stromal protection, and that stromal protection is diminished by treatment with P. Cells with higher levels of s-CXCR4 upregulation had greater differences between S IC50 and O IC50, compared to cells with lower s-CXCR4 upregulation, suggesting that the degree of s-CXCR4 upregulation is predictive of the degree of stromal protection. Cells with higher s-CXCR4 upregulation also had greater differences between S IC50 and P+S IC50, suggesting that P diminishes stromal protection more effectively in leukemias that highly upregulate s-CXCR4 in response to chemo.

Infant ALL patient samples were transplanted into sublethally irradiated NOG mice. After 3 weeks, we treated cohorts (n=5) with single doses of P, A, P followed by A 4 hours later (P+A), or C. We dosed A below the maximal tolerated dose (MTD) to facilitate assessment of P+A synergy. Mice were sacrificed 4 weeks post treatment and cells were isolated from bone marrow (BM), spleen, liver, and peripheral blood (PB) and analyzed by FACS. Leukemic blasts were defined as human CD19+ and CD45+. S-CXCR4 MFI was measured in the blast population. Overall, leukemic burden was similar in C, A, and P, consistent with conservative dosing of A and minimal direct anti-leukemic effect of P. A resulted in increased blasts in spleen and liver compared to C, possibly due to higher levels of s-CXCR4, while P resulted in increased blasts in liver, possibly due to mobilization of blasts. The key finding was that P+A resulted in decreased blasts in BM, spleen, liver, and PB, demonstrating a synergistic effect between P and A. Interestingly, P+A led to a higher reduction in blasts in a sample with A-induced s-CXCR4 upregulation, compared to a sample that did not upregulate s-CXCR4 in response to A. In all treatment cohorts, s-CXCR4 expression was highest in PB blasts, followed by liver, and BM/spleen.

Chemo-induced s-CXCR4 upregulation confers stromal-mediated chemoprotection in vitro that can be reversed by P. In vivo, P is an effective chemosensitizer. S-CXCR4 expression is increased in blasts located outside the BM, suggesting that blasts migrating from BM into PB upregulate s-CXCR4 as they home to new niches. Extramedullary disease may develop as a result of chemo-induced upregulation of s-CXCR4 or through mobilization of blasts by P alone. Importantly, P+A resulted in decreased leukemic burden in our infant ALL xenografts, suggesting that chemo-induced increases in s-CXCR4 and P-induced blast mobilization can be overcome. Finally, the efficacy of P as a chemosensitizer was predicted by the degree of chemo-induced s-CXCR4 upregulation, identifying a biomarker with the potential to identify optimal patients for CXCR4 inhibition. P in combination with chemo may thus prove useful in the treatment of high-risk pediatric ALL.

No relevant conflicts of interest to declare.