Identifying and overcoming mechanisms of resistance to a VpreB1-directed antibody-drug conjugate in the treatment of B-cell acute lymphoblastic leukemia Free

Background:

The treatment landscape of B-cell acute lymphoblastic leukemia (B-ALL) has been transformed by therapies targeting leukemia surface antigens. To expand the range of targetable antigens while sparing healthy B cells, we recently developed a novel antibody-drug conjugate (ADC) against VpreB1 (CD179a), a component of the surrogate light chain of the pre-B cell receptor. VpreB1 is broadly expressed across B-ALL subtypes but absent from mature B cells (Gordon PM et al., Blood Neoplasia, 2025). Our ADC utilizes calicheamicin as its toxin payload. We showed that the VpreB1-ADC exhibits potent anti-leukemic activity in vitro and in vivo across multiple B-ALL cell lines and patient-derived xenograft models. However, a small subset of samples showed relative resistance despite surface VpreB1 expression, suggesting additional resistance mechanisms. Herein, we aimed to identify and overcome mechanisms of VpreB1-ADC resistance to enhance its therapeutic potential.

Methods:

Multidrug resistance (MDR) transporter expression in B-ALL: The mRNA expression of key MDR-associated genes, including P-gp (ABCB1/MDR1), ABCG2 (BCRP), MRP1, and other ABCC/MRP family members, was analyzed in over 800 primary B-ALL samples from the St. Jude Cloud database (https://www.stjude.cloud). Protein expression was subsequently evaluated in B-ALL cell lines and primary samples using fluorescently labeled antibodies and flow cytometry.

Combination therapy testing with VpreB1-ADC: To assess multidrug resistance (MDR) transporters involvement, ALL cells were pre-treated overnight with tariquidar, a third-generation P-glycoprotein inhibitor, followed by calcein-AM staining and flow cytometry to measure retention. In parallel, cells pre-treated with tariquidar were exposed to VpreB1-ADC for 48 hours, and viability was assessed using CellTiter-Glo. For BCL-2 co-targeting, cells were treated with VpreB1-ADC and venetoclax, alone or in combination, for 48 hours. Viability was measured by CellTiter-Glo, and synergy was evaluated using SynergyFinder (ZIP model).

Results:

B-ALL cell lines HAL01 and ALL1807, both carrying the rare t(17;19) translocation producing a TCF3::HLF fusion, exhibited relative resistance to VpreB1-ADC compared to other cell lines and PDX models. This resistance occurred despite VpreB1 surface expression and efficient antibody internalization, suggesting that impaired ADC uptake is not responsible. Based on previously described mechanisms of therapy resistance in t(17;19) B-ALL and other ADCs, we hypothesized that VpreB1-ADC resistance may involve upregulation of multidrug resistance (MDR) transporters and/or anti-apoptotic pathways (Watanabe A et al., Cancer Med, 2019; Godwin CD et al., Leuk Lymphoma, 2020).

HAL01, but not ALL1807, expressed high levels of P-glycoprotein (P-gp/MDR1), an ATP-dependent efflux pump commonly linked to MDR. HAL01 also efficiently effluxed calcein-AM, consistent with active P-gp function. Treatment with the third-generation P-gp inhibitor tariquidar blocked calcein-AM efflux and restored HAL01 sensitivity to VpreB1-ADC, reducing viability from >90% to <5%. In contrast, tariquidar had no effect on ALL1807, consistent with its lack of P-gp expression.

Supporting broader relevance, analysis of the St. Jude Cloud database revealed that mRNA for both P-gp and ABCG2 (BCRP), another MDR transporter, is expressed in a substantial subset of primary B-ALL samples. We further confirmed variable ABCG2 protein expression across several B-ALL cell lines.

Given the absence of MDR activity in ALL1807, we next examined BCL-2–mediated survival. Co-treatment with VpreB1-ADC and the BCL-2 inhibitor venetoclax induced cytotoxicity in ALL1807, with a ZIP synergy score of 12, indicating a true synergistic effect.

Conclusion:

These findings suggest that both MDR transporters and BCL-2 may contribute to VpreB1-ADC resistance, probably through the broad xenobiotic drug extrusion properties of P-gps, and that targeting these pathways could enhance ADC efficacy. While venetoclax has been widely studied in leukemia, early MDR inhibitors failed due to poor specificity, toxicity, and adverse interactions. In contrast, third-generation inhibitors like tariquidar, tested here, offer improved specificity and safety. Together, these results support further development of VpreB1-ADC in rational combination therapies for B-ALL and raise the possibility that similar resistance mechanisms may affect other ADCs in cancer treatment.