PS1130 STEM CELL FACTOR IS IMPLICATED IN MICROENVIRONMENTAL INTERACTIONS AND CELLULAR DYNAMICS OF CHRONIC LYMPHOCYTIC LEUKEMIA
Authors: Gavriilidis G. , Ntoufa S. , Papakonstantinou N. , Kotta K. , Chartomatsidou E. , Stavroyianni N. , Anagnostopoulos A. , Papadaki E. , Tsiftsoglou A.S. , Stamatopoulos K. 
Published: June 2019

Background:

The mitogenic glycoprotein Stem Cell Factor (SCF, ligand of c‐kit receptor) has been implicated as a pro‐oncogenic driver in a variety of human cancers. Increased SCF levels have recently been reported in a small series of patients with chronic lymphocytic leukemia (CLL), however little if any evidence exists regarding its precise role in CLL pathophysiology.

Aims:

To analyze the expression profile of SCF in a well‐characterized cohort of CLL patients and investigate whether SCF is modulated by the CLL milieu.

Methods:

CD19+ primary CLL cells were negatively selected. SCF transcripts were detected by qPCR, SCF protein levels were analyzed using Western Blotting (WB) and SCF+ cells were determined by Flow Cytometry (FC). Cellular proliferation (Ki‐67+ cells) and oxidative stress (mitochondrial mass) were analyzed using FC. CLL cells were cultured in vitro with anti‐IgM, CpG, CD40 ligand and H2O2, or co‐cultured with the mesenchymal cell line HS‐5. Secreted SCF was determined using ELISA in supernatants of CLL cells.

Results:

Initially, we determined through qPCR (n = 14) that CLL cells predominantly express the membrane‐bound isoform of SCF, which is known to form a more stable complex with c‐Kit, compared to the soluble isoform. Next, we found significantly increased SCF protein levels in CLL cells (n = 68), compared to healthy tonsillar B cells (n = 13) (FD:9.8; p < 0.001). Within our CLL cohort, increased SCF protein levels significantly correlated with CD38 positivity (FD:2.45; p < 0.05), ZAP70 positivity (FD: 2; p < 0.05), trisomy 12 (FD:3; p < 0.05), and a shorter time to first treatment (p < 0.01). Moreover, increased SCF protein levels and SCF+ cells strongly correlated with the expression of unmutated IGHV genes (FD:6.7; p < 0.001 and FD:4.8; p < 0.01 respectively).

Next, we investigated whether stimulants mimicking the CLL milieu in vitro could modulate SCF protein expression in CLL cells. Stimulation of the B cell receptor (BCR) significantly increased SCF protein levels (n = 11, FD:1.4; p < 0.001). TLR‐9 stimulation significantly increased SCF+ cells (n = 8, FD:1.5; p < 0.01) and induced the secretion of soluble SCF (n = 7, FD:9.3; p < 0.05). Co‐stimulation of TLR9/CD40 receptors induced Ki‐67+ (FD:7.1; p < 0.01) and SCF+ (FD:3.8; p < 0.01) cells. Of note, 85% of the proliferating CLL cells were also SCF+ (Ki‐67+/SCF+ vs Ki‐67+/SCF−
; p < 0.01). The in vitro modulation of SCF in CLL cells by immune determinants prompted us to investigate whether B cell signaling inhibition might also modulate SCF expression in vivo. Longitudinal profiling of 6 CLL patients under Ibrutinib therapy showed a significant decrease in SCF+ cells at +1 month under treatment, compared to the pre‐treatment samples (FD:2.2; p < 0.05), further highlighting the links between immune signaling and SCF expression in CLL.

Finally, co‐culturing of CLL cells (n = 12) with the HS‐5 cells significantly reduced SCF+ cells (FD:2.2; p < 0.001), in parallel with a decrease in mitochondrial mass (r = 0.73; p < 0.01). Treatment with H2O2 abrogated the anti‐oxidant protection of HS‐5 cells and up‐regulated SCF expression (FD = 1.9; p < 0.01), indicating that SCF expression in CLL cells is modulated by oxidative stress.

Summary/Conclusion:

Overexpression of membrane‐bound SCF in CLL is associated with adverse prognosis and a shorter time‐to‐first treatment. Microenvironmental cues modulate the expression and secretion of SCF isoforms by CLL cells, highlighting a crucial role for this inflammatory growth factor in CLL cell homeostasis.