Acute lymphoblastic leukemia (ALL) is the most common form of childhood cancer with excellent treatment outcome where >80% are cured. However, relapse and therapy-related toxicities limit further improvements and greatly increase the cost of therapy. Vincristine (VCR) is cheap, well tolerated, and highly effective. Using VCR optimally will help improve the cost-benefit ratio favorably by allowing us to reduce toxicities like infections from myelosuppression and yet improving cure.
The highly successful BFM-ALL treatment backbone starts with a single intrathecal methotrexate on Day 1 followed by 7 days of oral prednisolone (PRED). The persistence of absolute blasts count >1,000/µL at Day 8 (D8), known as PRED poor response, confers a significantly poorer treatment outcome. To avoid seeding the CNS with leukemia from traumatic taps, the new Ma-Spore ALL 2010 treatment protocol, omitted intrathecal methotrexate at Day 1 and replaced with VCR at Day 0. By June 2013, a total of 133 patients have been enrolled. We found that the number of poor PRED responders was halved from the historical 9.5% in the previous Ma-Spore ALL 2003 study (Yeoh et al. J Clin Oncol 2013) to only 4.7% of patients in the ALL 2010 study. In addition, the percentage of MRD standard risk patients (Day 33 blast count ≤1x10
-4) increased from 38.9% in the Ma-Spore ALL 2003 to 51.8% in the Ma-Spore ALL 2010 study (P<0.001). The 2-year event-free survival (EFS) for good and poor D8 response patients under the Ma-Spore ALL 2010 trial remained similar to the ALL 2003 study despite only half the number of PRED poor responders (
Fig. 1). These data taken together suggests that VCR and PRED combination is highly synergistic and can improve early treatment response.
We investigated VCR and PRED combination in PRED and VCR-resistant (VCR-R) cell lines. Specifically, REH cell line is intrinsically resistant to PRED
in vitro because of a mutation in its glucocorticoid receptor. We exposed the REH cell line to increasing concentrations of VCR over 6 months and generated a VCR resistant REH cell line (
Fig. 2). This VCR-R REH cell line is resistant to both PRED or VCR when exposed individually
in vitro. However when exposed to both PRED and VCR in combination, only 30% of the resistant cells survived (P<0.01).
We found that the drug efflux transporter multi-drug resistance protein 1 (MDR1) was preferentially highly expressed in our VCR-R cell line models. To determine if the highly expressed MDR1 is responsible for treatment resistance, we exposed the VCR-R cell lines to VCR, verapamil (an MDR1 inhibitor) and combination of both VCR and verapamil. The combination of VCR and verapamil increased the G2 cell cycle arrest by 3- folds compared to when VCR was used alone (
Fig. 3), supporting the role of MDR1 in treatment resistance.
Interestingly we also found that the combination of VCR and PRED led to a decrease in levels of MDR1 expression by western blot, suggesting that depletion of MDR1 may be a mechanism through which VCR and PRED combination therapy enhances leukemic cell killing.
We also investigated microenvironment-mediated resistance to VCR and PRED using mesenchymal stromal cells (MSC) co-culture systems. It was found that after co-culture with MSC or in conditional medium containing soluble factors secreted by MSC, leukemic cells were resistant to VCR and PRED mono-treatment, but the resistance was abrogated after combinatorial therapy.
In conclusion, VCR in combination with PRED improves D8 peripheral blood treatment response during early induction in our Ma-Spore 2010 trial. This synergistic combination results from its ability to reverse resistance from intrinsic overexpression of MDR1 in resistant leukemia cells and decrease microenvironment-contributed resistance by mesenchymal cells.
Disclosures:
No relevant conflicts of interest to declare.
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