Hadya M. Khawaja

Genetic alterations determine chemotherapy resistance in childhood T-ALL: modelling in stage-specific cell lines and correlation with diagnostic patient samples.

Acquired drug resistance eventually leads to treatment failure in T‐cell acute lymphoblastic leukaemia (T‐ALL). Immunophenotypic and cytogenetic heterogeneities within T‐ALL influence susceptibility to cytotoxic therapy, and little is known about the mechanisms of drug resistance at specific stages of T‐cell ontogeny. We developed tolerance to therapeutic concentrations of daunorubicin (DNR) and l‐asparaginase (l‐asp) in Jurkat (CD1a−, sCD3+) and Sup T1 (CD1a+, sCD3−) cell lines, having respective ‘mature’ and ‘cortical’ stages of developmental arrest. DNR resistant cells acquired multidrug resistance: 310‐fold increased resistance to vincristine (VCR) and a 120‐fold increased resistance to prednisolone (PRED). Microarray analysis identified upregulation of asparagine synthetase (ASNS) and argininosuccinate synthase 1 (ASS1) to cell lines with acquired resistance to l‐asp, and in the case of DNR, upregulation of ATP‐binding cassette B1 (ABCB1). Suppression of ABCB1, ASNS and ASS1 by RNA interference revealed their functional relevance to acquired drug resistance. Expression profiling of these genes in 80 T‐ALL patients showed correlation with treatment response. This study expands the pool of available drug resistant cell lines having cortical and mature stages of developmental arrest, introduces three new drug resistant T‐ALL cell lines, and identifies gene interactions leading to l‐asp and DNR resistance.

High-throughput screening for daunorubicin-mediated drug resistance identifies mometasone furoate as a novel ABCB1-reversal agent.

The overexpression of P-glycoprotein, encoded by the ATP Binding Cassette B1 (ABCB1) gene, contributes to multidrug resistance (MDR) and is considered one of the major obstacles to successful cancer chemotherapy. The authors previously developed a T-lineage acute lymphoblastic leukemia (T-ALL) cell line that overexpresses ABCB1 and exhibits MDR to daunorubicin (DNR), prednisolone, and vincristine. Using this cell line and the fluorescent probe JC-1, they developed a flow cytometry-based, high-throughput screening (HTS) assay that quantifies ABCB1 efflux. They screened a library of 880 off-patent drugs for their ability to inhibit ABCB1 efflux and then measured the ability of 11 lead compounds to reverse in vitro DNR-mediated drug resistance and the toxic doses for each agent. Seven of the 11 drugs were able to reverse drug resistance at a concentration significantly below its toxic dose. Of the remaining 7, only 1 compound, mometasone furoate, has not been previously described as an ABCB1 antagonist to DNR-mediated drug resistance. On the basis of its high ABC modulator activity and relatively large in vitro therapeutic window, this drug warrants further investigation. In addition, the approach used in this study is useful for identifying off-patent drugs that may be repurposed for novel clinical indications. (Journal of Biomolecular Screening 2008:185-193)

A Selective ATP-Binding Cassette Subfamily G Member 2 Efflux Inhibitor Revealed via High-Throughput Flow Cytometry

Chemotherapeutics tumor resistance is a principal reason for treatment failure, and clinical and experimental data indicate that multidrug transporters such as ATP-binding cassette (ABC) B1 and ABCG2 play a leading role by preventing cytotoxic intracellular drug concentrations. Functional efflux inhibition of existing chemotherapeutics by these pumps continues to present a promising approach for treatment. A contributing factor to the failure of existing inhibitors in clinical applications is limited understanding of specific substrate/inhibitor/pump interactions. We have identified selective efflux inhibitors by profiling multiple ABC transporters against a library of small molecules to find molecular probes to further explore such interactions. In our primary screening protocol using JC-1 as a dual-pump fluorescent reporter substrate, we identified a piperazine-substituted pyrazolo[1,5-a]pyrimidine substructure with promise for selective efflux inhibition. As a result of a focused structure-activity relationship (SAR)–driven chemistry effort, we describe compound 1 (CID44640177), an efflux inhibitor with selectivity toward ABCG2 over ABCB1. Compound 1 is also shown to potentiate the activity of mitoxantrone in vitro as well as preliminarily in vivo in an ABCG2-overexpressing tumor model. At least two analogues significantly reduce tumor size in combination with the chemotherapeutic topotecan. To our knowledge, low nanomolar chemoreversal activity coupled with direct evidence of efflux inhibition for ABCG2 is unprecedented.

ATP binding cassette C1 (ABCC1/MRP1)-mediated drug efflux contributes to disease progression in T-lineage acute lymphoblastic leukemia

PURPOSE In acute lymphoblastic leukemia (ALL), multidrug resistance is often mediated by ATPase Binding Cassette (ABC) proteins, which principally involve ABCB1 (multidrug resistance 1, MDR1) and ABCC1 (multidrug resistance protein 1, MRP1). However, direct comparisons between the differential effects of ABCB1 and ABCC1 have been difficult, since identical cell lines with differential expression of these transporters have not been developed. EXPERIMENTAL DESIGN In this study, we developed and compared the biological profiles of Jurkat cell lines that selectively over-expressed ABCB1 and ABCC1. Vincristine (VCR) plays an important role in the treatment of T-lineage ALL (T-ALL), and is often the first drug given to newly-diagnosed patients. Because of its importance in treatment, we provided escalating, sub-lethal doses of VCR to Jurkat cells, and extended our observations to expression profiling of newly diagnosed patients with T-ALL. RESULTS We found that VCR-resistant cells over-expressed ABCC1 nearly 30-fold. The calcein AM assay confirmed that VCR-resistant cells actively extruded VCR, and that ABCC1-mediated drug resistance conferred a different spectrum of multidrug resistance than other T-ALL induction agents. siRNA experiments that blocked ABCC1 export confirmed that VCR resistance could be reversed in vitro. Analyses of T-lymphoblasts obtained from 92 newly diagnosed T-ALL patients treated on Childrens Oncology Group Phase III studies 8704/9404 showed that induction failure could be explained in all but one case by the over-expression of ABCB1 or ABCC1. CONCLUSIONS Taken together, these results suggest that over-expression of ABC transporters plays a contributing role in mediating treatment failure in T-ALL, and underscore the need to employ alternate treatment approaches in patients for whom induction failed or for those with relapsed disease.