Regulation Of CLL Growth and Trafficking By The Enzymatic Functions Of CD38: Implications For Therapeutic Targeting

CD38 is a cell surface receptor and enzyme. As a receptor CD38 interacts with CD31 activating a well characterized signaling pathway. As an enzyme, it metabolizes NAD generating Ca²⁺ mobilizing messengers, including cADPR and ADPR. In CLL, CD38 acts as a negative prognostic marker: its expression by the leukemic component is associated with a more aggressive clinical course and a worse overall survival. We and others have shown that CD38 is also a critical element in the pathogenetic network underlying the disease by functioning as a compass which drives CLL cells to growth-favorable niches. This is accomplished through a physical and functional cooperation with chemokine receptors (e.g. CXCR4), integrins (e.g. CD49d) and matrix metalloproteases (MMP-9).

Notwithstanding intense investigation in the field, the functional mechanisms through which CD38 regulates CLL homing remain unknown. To determine the contribution of the enzymatic activities of CD38 in the leukemic environment we took advantage of the Mec-1 cell line as a cellular model. This CLL-like cell line, constitutively CD38^- and CD49d⁺ , was genetically modified by lentiviral infection to obtain CD38⁺ clones (CD38wt), as well as clones that express a mutant CD38 (mutation of glutamic acid at position 226 into aspartic acid, E226D, referred to as CD38M), which results in a complete loss of the enzymatic activities. Despite a similar molecular organization on the cell surface and the presence of an active signaling pathway upon engagement with agonistic mAbs, the two CD38 variants showed markedly different in vivo behaviors. This was shown by xenografting experiments into NOD/SCID/g chain^-/- (NSG) mice: CD38wt cells were characterized by a higher growth rate and metastatic potential, which led to i) a significantly shorter survival as compared to both CD38M and CD38-negative cell lines (median survival of 30 days vs 36 days of CD38M vs 35.5 days of CD38 negative, p=0.0017 and p=0.0024 respectively), ii) a higher weight decrease of mice at day 28 (20% vs 8.45% vs 11.7%, p=0.026 and p=0.001 respectively) and iii) a more pronounced involvement of target organs, such as kidney, as highlighted by measuring organ volume by magnetic resonance imaging (477 mm³vs 294 mm³vs 300 mm³, p=0.028), or by checking for the presence of Mec-1 cells by IHC (19.12 vs 3.89% vs 2.14% of CD20⁺ area, p<0.0001). These results strongly suggested that the enzymatic activities of CD38 are critical for in vivo growth and re-circulation of Mec-1 cells.

Moreover, microarray data confirmed that the genetic signature of the CD38M overlaps with the CD38-negative cell line and that it is clearly distinct from CD38wt cells. The latter cell line showed up-modulation of several genes involved in chemotaxis, adhesion and signaling pathways, such as CXCL10, CXCR3, CXCR7, CADM1.

In line with microarray data, CD38wt clones were characterized by a higher migratory behavior in response to different chemokines (e.g. CXCL12 and CCL19) and adhesive properties in response to VCAM-1. These biological events may be significantly blocked by treatment of CD38wt Mec-1 cells with kuromanin, a natural compound belonging to the flavonoid family and recently described as a specific inhibitor of CD38 enzymatic activities.

Lastly, proof of principle of the validity of the results obtained with the cell line was obtained using freshly purified CD38⁺ CLL cells. In these cells CD38 was enzymatically active and treatment with kuromanin inhibited the production of CD38 metabolites in a dose-dependent manner. Moreover, the chemotactic ability of CLL cells in response to CXCL12 was significantly reduced in the presence of this compound. These in vitro results were confirmed in vivo by showing that pre-treatment of CLL cells with kuromanin before injection in NSG mice significantly impaired leukemic cell homing to spleen and BM.

Considered together, these results support the notion that the enzymatic activities of CD38 play a major role in the regulation of CLL growth and homing to growth-permissive niches. The translational implication is that therapeutic targeting of CD38 could be obtained not only using monoclonal antibodies, but also enzyme inhibitors. The availability of selective and safe inhibitors that can be used in addition to conventional chemotherapy may represent an innovative way to target the tumor-host axis, reducing homing to and retention of CLL cells in the tissue microenvironment.