Vincristine and Prednisolone Combination Reduces MDR1 and Microenvironment-Mediated Treatment Resistance In Acute Lymphoblastic Leukemia
Authors: Jiang Nan , Li Zhenhua , Koh Grace Shimin , Lu Yi , Kham Shirley K.Y. , Juh Yeoh Allen Eng 
Published: October 2019

Abstract
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.