Abstract 5245: Adipocyte production of glutamine protects leukemia cells from L-asparaginase

Abstract

Abstract Obesity is associated with increased cancer incidence and mortality. Adipose tissue has been demonstrated to play a role in several types of cancer, including acute lymphoblastic leukemia (ALL). We have shown that obesity impairs the survival of mice transplanted with ALL and treated with the front-line chemotherapy, L-Asparaginase (ASNase), which acts through the deamination of asparagine (ASN) and glutamine (GLN). We have also shown that adipocytes protect ALL cells from ASNase in vitro. We hypothesized that adipocytes protect ALL cells from ASNase via production of GLN. Fat pads were excised from control and diet-induced obese mice. Glutamine synthetase, the rate-limiting enzyme for GLN synthesis, was analyzed by western blot. Amino acids secreted by fat pads were measured using HPLC. ALL cell sensitivity to ASN and/or GLN depletion, and ASNase, was determined by trypan blue exclusion in culture alone and in transwells over 3T3-L1 adipocytes. Mouse fat pads expressed high levels of glutamine synthetase protein, and this was not affected by obesity or 5 days of L-asparaginase treatment (3,000 IU/kg/day). Fat pad explants secreted GLN (251±50 nmoles/100 mg tissue), but not ASN, over 72 hours. Confirming that GLN secretion could protect ALL from ASNase, twice daily addition of 500 nmoles of GLN to murine ALL cells completely reversed ASNase cytotoxicity (64.0x10^4 vs. 7.9x10^4 viable cells after 72 hours of 1 IU/mL ASNase, p<0.01). Testing a panel of human ALL cell lines, we found that proliferation of 3 of 9 cell lines were decreased when ASN was removed from the media (RS4;11, SupB15, Molt4), while 8 of 9 cell lines were decreased when GLN was removed from the media (RS4;11, SupB15, Molt4, SEM, RCH-ACV, BV-173, SD-1, HL60). To test whether blocking adipocyte production of GLN would reverse their ability to protect ALL cells, we pretreated 3T3-L1 adipocytes with methionine sulfoximine (MSO), an irreversible inhibitor of glutamine synthetase. MSO pretreatment blocked GLN secretion by adipocytes and completely reversed adipocyte protection of ALL cells (with no adipocytes: 7.5x10^4; with untreated adipocytes: 14.8x10^4; with MSO treated adipocytes: 6.2x10^4, p=0.03 vs. untreated adipocytes). Finally, we tested whether an alternative preparation of ASNase with a higher glutaminase activity, ERWinase, could overcome adipocyte protection of three GLN dependent cell lines: SD-1, HL60, and Molt-4. Compared to E. coli ASNase, ERWinase treatment resulted in significantly decreased survival of all 3 cell lines in the presence of adipocytes (SD-1 4.2x10^4 vs. 6.7x10^4, p<0.05; HL60 4.1x10^4 vs. 8.8x10^4, p<0.05; Molt-4 16.9x10^4 vs. 26.4x10^4, p<0.05). These studies uncover a novel mechanism of adipocyte protection of leukemia cells from chemotherapy: adipocytes protect ALL cells from ASNase treatment via the production of GLN. Adipocyte production of GLN is both necessary and sufficient to protect ALL cells from ASNase treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5245. doi:1538-7445.AM2012-5245