Scientists say blocking bone marrow stromal cells from converting cystine into cysteine may provide a therapeutic approach against chronic lymphocytic leukemia (CLL), including drug-resistant disease. A team led by researchers at the University of Texas MD Anderson Cancer Center has found that leukemia cells rely on cysteine produced by surrounding stromal cells for their own production of the antioxidant GSH. Because CLL cells can’t take up and utilize cystine on their own, preventing the production of cysteine by stromal cells causes levels of GSH in CLL to drop, and the cells display increased levels of apoptosis and become more susceptible to antileukemic drugs including fludarabine and oxaliplatin.
In vivo studies by Peng Huang, Ph.D., and colleagues further showed that treating a mouse model of leukemia using a chemical inhibitor of the cystine transporter resulted in a reduced leukemia burden and increased sensitivity of the animals’ leukemic cells to conventional drug therapy. These effects were evident at cystine transporter inhibitor levels that didn’t harm the bone marrow stromal cells. The authors report their findings in Nature Cell Biology in a paper titled “Stromal control of cystine metabolism promotes cancer cell survival in chronic lymphocytic leukemia.”
Chronic lymphocytic leukemia (CLL) is characterized by functionally defective B lymphocytes in a number of organs. Despite treatments such as fludarabine, the persistence of residual, drug-resistant leukemia cells means the disease can re-emerge. The fact that CLL cells in vitro demonstrate high levels of spontaneous apoptosis but long survival in vivo indicates that the tissue microenvironment plays a pivotal role in promoting CLL survival. A number of molecules including integrins, interferons, growth factors, and hedgehog-related molecules are known to be involved in stromal-CLL interaction, but the mechanism behind drug resistance of CLL cells has still to be explained.
CLL cells exhibit higher levels of reactive oxygen species (ROS) than healthy lymphotyces. This intrinsic ROS stress means the cells have increased dependency on redox regulatory systems to maintain the redox balance. GSH is the most abundant antioxidant in cells, and recent biochemical studies suggest that the tripeptide plays a key role in maintaining the redox balance of CLL cells and supporting their survival and potentially drug resistance.
When cultured in vitro, CLL cells can’t maintain high enough GSH levels and exhibit increased levels of spontaneous apoptosis. The MD Anderson Cancer Center team thus set out to determine the biochemical basis by which CLL obtains GSH in vivo and whether targeting the cells’ access to or utilization of the peptide could represent a therapeutic strategy against the disease.
The researchers first confirmed that primary leukemia cells from CLL patients cultured in vitro exhibited marked reductions in GSH levels and substantial levels of apoptosis within just three days. In contrast, primary leukemia cells co-cultured with bone marrow stromal cells were able to maintain high levels of GSH and exhibited significantly enhanced cell viability, both in the short term and over a number of weeks. This preserving effect imparted by the presence of stromal cells on CLL cells was evident under ambient oxygen concentration conditions and under conditions of reduced oxygen. Moreover, co-culturing CLL cells with the stromal cells was also associated with lower levels of ROS and boosted resistance to exogenous ROS stress resulting from H2O2 administration.
Notably, the presence of either bone marrow stromal cells , stromal-conditioned medium, a GSH precursor, or glutathione itself in the CLL culture also protected the leukemia cells from death after the administration of F-ara-A (the active form of fludarabine), or oxaliplatin, which are both used in the clinical treatment of CLL. Similarly, the cancer cells were protected against treatment with F-ara-A, oxaliplatin, or H2O2, when cultured with stromal cells behind a membrane to prevent direct contact.
To try and identify what it was that the stromal cells were secreting to enable CLL survival in the face of drug challenge, the team cultured CLLs with either the high-molecular-weight (HMW) or low-molecular-weight (LMW) fractions of stromal-conditioned medium. These results demonstrated that it was something in the LMW fraction that protected CLL cells against drug-induced cytotoxicity and also that it was the LMW fraction that led increased levels of GHS in CLL cells. Interestingly, however, stromal medium itself didn’t contain increased levels of GSH itself but did harbor increased levels of other thiol-containing compounds.
Because cysteine is a thiol-containing compound and also a rate-limiting substrate for GSH synthesis, the investigators postulated that the LMW component in the stromal medium might be cysteine, and the presence of cysteine in conditioned stromal medium was subsequently detected by LC-MS/MS analyses. Moreover, adding cysteine daily to CLL culture promoted cell viability and blocked drug-induced cell death in a dose-dependent manner. Administering cysteine at the highest level found in conditioned medium enhanced CLL GSH levels to those demonstrated by the leukemia cells as a result of stromal co-culture.
Cells usually generate cysteine (which is unstable chemically) for GSH synthesis from di-amino acid cystine, but the authors found that CLL cells exhibited very low expression of the active xCT subunit of the cystine-transporter Xc- and much lower levels of xCT mRNA than normal lymphocytes. Moreover, the CLL cells exhibited very little uptake of radiolabeled cystine, whereas the stromal cells were able to uptake much higher levels.
These data indicated that CLL cells can’t take up cystine itself but instead rely on its conversion to cysteine. Radiolabeling studies demonstrated that in fact the stromal cells carry out this cystine-to-cysteine conversion for uptake by CLL cells. “These data together indicate that bone marrow stromal cells promoted GSH synthesis in CLL cells mainly by converting cystine to cysteine, not by enhancing the expression of the cystine transporter," the authors write.
They moved on to investigate whether their findings might translate into a potential therapeutic approach against leukemia. They found that treating CLL-stromal cell co-cultures with PEITC, which depletes GSH in CLL cells, prevented the protective effects of stromal cells. Encouragingly, combined treatment of the co-cultures with PEITC and oxaliplatin had a marked synergistic effect, even against CLL cells with the loss of p53 due to chromosome 17p deletion, which are particularly drug resistant and associated with poor prognosis in a clinical setting.
Significantly, blocking the uptake of cystine by the co-cultured stromal cells using subtoxic levels of cystine transporter Xc- inhibitors also increased the susceptibility of CLL cells to clnically relevant levels of F-ara-A, or oxaliplatin. Importantly, the concentrations of the two Xc inhibitors tested, (S)-4-carboxyphenylglycine (S-4-CPG) and sulphasalazine (SSZ), caused minimal cytotoxicity in normal bone marrow stromal cells.
In a final series of experiments, the investigators evaluated the use of cystine transporter inhibition in the Tcl-1 transgenic mouse model of CLL. As hoped, blocking stromal cell cystine uptake by treating animals with SSZ led to a significant decrease in the level of GSH in CLL cells and resulted in a significant reduction in leukemia burden in all four mice treated.
Leukemia cell counts in the peritoneal cavity reduced from nearly 33 million to 11.4 million, and there was also a 78% reduction in CLL viability post treatment, compared with before treatment. In vivo SSZ treatment in addition rendered the leukemia cells more sensitive to F-ara-A or oxaliplatin when they were cultured ex vivo with sensitivity being partially reversed by co-culturing the cells with bone marrow stromal cells.
“As CLL cells highly rely on stromal cells to provide cysteine for GSH synthesis, this intercellular metabolic pathway may represent a potentially important target for effective killing of CLL cells in vivo,” the authors conclude. The investigators admit the possibility exists that stromal cells may protect CLL cells through addittional mechanisms. Nevertheless, they state, “further evaluation of this biochemical intervention strategy in preclinical and clinical settings is important for the development of effective therapy to overcome drug resistance in vivo.”