PS1130 STEM CELL FACTOR IS IMPLICATED IN MICROENVIRONMENTAL INTERACTIONS AND CELLULAR DYNAMICS OF CHRONIC LYMPHOCYTIC LEUKEMIA
Authors: G. Gavriilidis, S. Ntoufa, N. Papakonstantinou, K. Kotta, E. Chartomatsidou, N. Stavroyianni, A. Anagnostopoulos, E. Papadaki, A.S. Tsiftsoglou, K. Stamatopoulos
Journal: HemaSphere (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 H 2 O 2 , 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 &lt; 0.001 ). Within our CLL cohort, increased SCF protein levels significantly correlated with CD38 positivity (FD:2.45; p &lt; 0.05 ), ZAP70 positivity (FD: 2; p &lt; 0.05 ), trisomy 12 (FD:3; p &lt; 0.05 ), and a shorter time to first treatment ( p &lt; 0.01 ). Moreover, increased SCF protein levels and SCF + cells strongly correlated with the expression of unmutated IGHV genes (FD:6.7; p &lt; 0.001 and FD:4.8; p &lt; 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 &lt; 0.001 ). TLR‐9 stimulation significantly increased SCF + cells (n = 8, FD:1.5 ; p &lt; 0.01 ) and induced the secretion of soluble SCF (n = 7, FD:9.3 ; p &lt; 0.05 ). Co‐stimulation of TLR9/CD40 receptors induced Ki‐67 + (FD:7.1 ; p &lt; 0.01 ) and SCF + (FD:3.8; p &lt; 0.01 ) cells. Of note, 85% of the proliferating CLL cells were also SCF + (Ki‐67 + /SCF + vs Ki‐67 + /SCF − ; p &lt; 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 &lt; 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 &lt; 0.001 ), in parallel with a decrease in mitochondrial mass (r = 0.73; p &lt; 0.01 ). Treatment with H 2 O 2 abrogated the anti‐oxidant protection of HS‐5 cells and up‐regulated SCF expression (FD = 1.9; p &lt; 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.