Owing to their low expression of asparagine synthetase (ASNS), acute lymphoblastic leukemia (ALL) cells have low asparagine biosynthesis and are exquisitely sensitive to asparagine depletion caused by L-asparaginase. Differences in susceptibility to L-asparaginase have been attributed to the varying levels of ASNS mRNA in leukemic cells, but recent studies have challenged this concept. We found that among leukemic cells from 288 children with ALL analyzed by Affymetrix U133A GeneChip, ASNS levels were higher in patients with T-lineage ALL (P <0.001), and lower in patients with TEL-AML1 (P = 0.033) or hyperdiploid >50 chromosomes (P <0.001) B-lineage ALL. However, ASNS expression was not significantly related to response to remission induction therapy as determined by minimal residual disease measurements on day 46 of treatment.

ALL cells grow in direct contact with bone marrow mesenchymal cells, which form the microenvironmental niches essential for their expansion. We observed that ASNS levels in mesenchymal cells were, on average, 20 times higher than those expressed by ALL cells by GeneChip analysis, real-time PCR and Western blotting with an anti-ASNS specific monoclonal antibody (gift of Dr. M. Kilberg, U. of Florida). When ALL cell lines (380, REH, RS4;11) were exposed to L-asparaginase in the presence of mesenchymal cells, cytotoxicity significantly decreased. To test whether the protective effect of mesenchymal cells was related to their ASNS expression, we used RNA interference (RNAi) to stably downregulate ASNS expression. This profoundly diminished their capacity to protect ALL cells from L-asparaginase cytotoxicity. We then investigated whether enforced expression of ASNS in mesenchymal cells using a MSCV retroviral vector could augment their protective capacity. Overexpression of ASNS significantly augmented the capacity of mesenchymal cells to protect ALL cells from L-asparaginase cytotoxicity. ASNS expression in mesenchymal cells was related not only to their capacity to protect ALL cells lines but also primary ALL cells obtained from 5 patients with newly diagnosed ALL.

We found that mesenchymal cells secreted asparagine and that levels of asparagine in culture supernatants collected from mesenchymal cells after 24 hours of culture were directly related to levels of ASNS expression in the cells; asparagine in supernatants of mesenchymal cells treated with the RNAi target sequence was nearly undetectable. In line with these results, the protective effects of mesenchymal cells were also detectable when ALL cells were placed on a microporous membrane that prevented contact with mesenchymal, and when they were cultured with mesenchymal cell-conditioned medium. By contrast, addition of a mixture of mesenchymal cell-derived cytokines (IL-1 alpha and beta, IL-3, IL-6, IL-7, IL-11, SCF and Flt3 ligand) instead of mesenchymal cell-conditioned medium had no effect on L-asparaginase cytotoxicity.

These results reveal an unexpected mechanism of drug resistance in ALL and indicate that microenvironmental niches can form a safe haven for leukemia cells, thus sustaining minimal residual disease. The role of mesenchymal cells in the response to other anti-leukemic drugs requires further investigation. A better understanding of the molecular mechanisms involved in the interaction between ALL cells and the bone marrow microenvironment may ultimately lead to innovative ways to enhance anti-leukemia therapy.

Disclosure: No relevant conflicts of interest to declare.