Reactive Oxygen Species and Metabolic Gene Expression Profiling Further Differentiate CXCR4/CD5 Fractions in Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) clones are made up of slow growing, long-lived mature B cells that accumulate in the blood and secondary lymphoid organs. Studies using deuterium exchange into CLL cell DNA of patients drinking heavy water led to the phenotypic classification of intraclonal subfractions that differ in time since last cell division. Cells that have most recently divided, termed the proliferative fraction (PF), are CXCR4^DimCD5^Bright and older cells that are in a resting state, termed the resting fraction (RF), are CXCR4^BrightCD5^Dim. These studies suggest a model of the life cycle of a CLL cell that involves exiting of the PF from the tissue into the blood and subsequent homing of the RF from the blood to the tissue to survive and/or recapitulate the division cycle.

The CXCL12/CXCR4 axis is central to this trafficking of CLL cells out of and into proliferative niches in the tissue. Moreover, activation of this axis is associated with an increase in the transcriptional activity of sterol response element binding protein-1 (SREBP1) in cancer. SREBP1 regulates genes involved in lipid synthesis and uptake. Since CLL cells preferentially rely on fatty acid (FA) metabolism for energy demands, this concurs with the proposed life cycle of CLL subfractions. CXCL12thereby exerts two functions on the CXCR4^BrightCD5^DimRF: directional trafficking to the tissue and altering the transcriptome to adapt to the lipid rich environment in the lymph node [LN] that the cells in this fraction will soon encounter. Indeed, our microarray analysis has confirmed that SREBP1a and c are more highly expressed in the RF [1.012x] compared to the PF that express higher levels of the transcriptional targets such as lipoprotein lipase [LPL: 1.201x] fatty acid synthase [FASN: 1.17x] glucose-6-phosphate dehydrogenase [G6PD: 1.505x] and pyruvate kinase [PKM2: 1.433x]. Cancer cells are known to have a concomitance to FA synthesis and oxidation during proliferation. This appears to be true in CLL if we infer that the higher expression of genes involved within the most recently divided cells of the PF are a reflection of how actively dividing cells behave in the LN.

Since the main source of reactive oxygen species (ROS) is a by-product of oxidative phosphorylation, we examined the extent that the CLL CXCR4/CD5 subfractions differed in ROS levels as a result of their individual metabolic demands during proliferation. We found that PF has significantly higher levels of ROS compared to the RF, regardless of patient IGHV gene mutation status. Additionally, microarray analysis has revealed that the PF express significantly higher levels of genes involved in response to oxidative stress compared to the RF; these include superoxide dismutase [SOD1: 1.149x], catalase [CAT: 1.462] , hemeoxygenase-1 [HMOX1: 1.695], and glutathione peroxidase [GPX1:1.898] . This suggests that this population is uniquely suited to survive under harsh conditions that result from their distinctive demand for fatty acid oxidation to fuel growth in contrast to normal B-cells. This makes targeting these protective enzymes an attractive therapeutic strategy, as an accumulation of damaging levels of ROS will induce cell death.

Finally, we have noted a rare population of cells that are CD5^BrightCXCR4^Negative that contain the highest levels of ROS in CLL patient PBMCs. Initial studies suggest that these CD5^BrightCXCR4^Negative cells are more enriched in patient LNs and might represent the cells actively dividing in proliferation centers. If so, those few in the peripheral circulation bearing this phenotype may be the closest link to the tissue resident dividing cells. Consistent with this, concurrent quantitation of ROS and cell cycle analysis by flow cytometry confirmed that higher levels of ROS are present in MEC1cells in S, M, and G2 phases. Current studies involve comparing gene expression profiles of various CXCR4/CD5 fractions, including the CD5^Bright CXCR4^Negative subset, from the blood and LN based on high ROS and reciprocal densities of CXCR4 and CD5. By sorting this rare population from PB, we may conveniently study those actively dividing CLL cells in the LN in lieu of obtaining rare biopsy samples.