Association of Asparagine Synthetase Expression and Sensitivity to L-Asparaginase in Cell Lines and Primary Pediatric Acute Lymphoblastic Leukemia Samples.

Abstract 2738

Poster Board II-714

L-Asparaginase (L-Asp) is an important component in the combined chemotherapy for childhood acute lymphoblastic leukemia (ALL). Administration of L-Asp leads to depletion of plasmatic asparagine and consequently causes loss of intracellular asparagine. As a non-essential amino acid, asparagine is synthesized from aspartate and glutamine by asparagine synthetase (ASNS). Primary ALL cells are believed to have low ASNS expression and therefore to be sensitive to asparagine depletion. Although increased ASNS level was shown to be connected with L-Asp resistance the exact relationship between ASNS expression and L-Asp sensitivity is not clear. We and others have previously shown TEL/AML1[+] ALL blasts express more ASNS mRNA than TEL/AML[-] do although primary TEL/AML[+] cell are in vitro more sensitive to treatment with L-Asp. Hutson et al (1997) showed that amino acid deprivation led to increased expression of ASNS on mRNA and protein level as well as to increased biological activity. On the other hand, Nan Su et al described negative correlation between L-Asp sensitivity and ASNS protein rather than mRNA levels. Therefore, in our studies we concentrated on protein expression of ASNS in patients' samples. So far, there has been no reproducible published data on ASNS protein detection by Western blot in primary patients' samples. Despite using 3 different antibodies and precise optimization we were not able to detect ASNS protein in patients' samples in contrast to cell lines. Transcripts' levels confirmed significantly lower (2 log) expression of ASNS in patients' leukemic cells compared to leukemic cell lines. Therefore, for further studies on gene and protein relation we had to rely on cell lines as a model. We detected ASNS gene expression and ASNS protein content in four ALL cell lines: REH (TEL/AML1[+]), UOCB6 (TEL/AML1[+]), NALM6 (TEL/PDGFRB[+]) and RS4;11 (MLL/AF4[+]). ASNS mRNA levels were in accord with sensitivity to L-Asp. UOCB6 as the most resistant cell line (IC50=0.04U/ml) had the highest expression of ASNS (normalized ASNS, nASNS=4.946), then NALM6 (IC50=0.01U/ml; nASNS=1.8), REH (IC50=0.6.10⁻⁴; nASNS=1.176) and RS4;11 (IC50<0.3.10⁻⁴; nASNS=0.024). ASNS protein levels significantly differed through passages in REH cells, likely due to rapid turnover. For the remaining three cell lines L-Asp sensitivity correlated also with protein content.

We have previously shown that different basal expression levels do not affect short-term dynamics of ASNS expression after L-Asp administration. Here we were interested to see the changes of sensitivity to L-Asp using gradient silencing of ASNS by RNAi in two cell lines with different basal expression: REH cell line with intermediate ASNS mRNA expression and RS4;11 cell line with very low mRNA expression. Gradient silencing revealed that L-Asp sensitivity correlated with ASNS expression till 50% decrease; further silencing did not potentiate the effect. The same response was seen in both cell lines despite different basal ASNS expression and sensitivity to L-Asp.

The ASNS is glutamine dependent enzyme therefore we also studied expression of glutamate dehydrogenase (GDH), an enzyme necessary for glutamine synthesis. We found significantly lower GDH mRNA expression in primary TEL/AML1[+] blasts in comparison with TEL/AML[-] blasts (p=0.019), which might lead to deficiency of glutamine in these cells and consequently higher sensitivity to L-Asp. Accordingly, silencing of ASNS in REH tended to increase GDH expression levels.

Our data confirm that generally, both ASNS mRNA and protein expression inversely correlate with the sensitivity to L-Asp in the cell lines. However, it may be misleading to draw conclusions for the patients' cells directly from the results obtained in cell line models. The expression patterns of ASNS in primary leukemic cells differ even from those of genotypically identical cell lines. The control of basal levels of ASNS in leukemic cells remains to be elucidated. Our results implicate an important role of GDH and glutamine metabolic pathway in the regulation of ASNS activity.

This work was supported by MSM0021620813 and GAUK 7835.