1026 - Targeting the pro-inflammatory protein S100-A9 in Chronic Lymphocytic Leukemia

Background: During Chronic Lymphocytic Leukemia (CLL) progression, pro-inflammatory pathways are activated, providing leukemic B lymphocytes with survival signals. Recently, our group described the presence of S100-A9 protein in exosomes derived from CLL patients during disease progression. S100-A9 is a potent mediator of inflammation, tumor invasion, and metastasis. This protein plays an important role in the myeloid compartment, where high expression of S100-A9 is associated with the suppressive activity of myeloid-derived suppressor cells (MDSCs). In addition, S100-A9 inhibitors have shown immunomodulatory properties and are currently being evaluated in early-phase clinical trials for multiple myeloma and prostate cancer. We hypothesize that S100-A9 provides B-CLL cells with a survival advantage by activating inflammatory pathways and modulating the tumor microenvironment.

Methods: In vitro stimulation of peripheral blood mononuclear cells (PBMCs) from CLL patients with IL-4/CD40L or IL-4/S100-A9 was made for 72 hours. S100-A9 and Ki-67 expression were assessed by flow cytometry. We generated a new Eµ-TCL1/S100A9-/- murine model by crossing Eµ-TCL1 and S100A9-/- mice. Adoptive transfer (AT) into NOD-scid IL2Rgammanul (NSG) mice was performed using B-CLL isolated from aged Eµ-TCL1 or Eµ-TCL1/S100A9-/-. To evaluate the in vivo effect of S100-A9 inhibitors, we use C57BL/6 mice with adoptively transfer splenocytes from Eµ-TCL1 mice. Paquinimod (PaQ) and Tasquinimod (TasQ) were administered at a dosage of 25 mg/Kg in drinking water ad libitum for 4 weeks. Once the treatment was completed the animals were euthanized for tissue collection. Fluorescence-activated cell sorting (BD FACS Aria II) was used to isolate CD19+ for the Nanostring study. All immunophenotyping analysis was performed using a BD FACSymphony or a BD LSR II cytometer.

Results: S100-A9 expression in B cells from CLL patients increases after stimulation with IL-4/CD40L, likewise IL-4 in combination with rhS100-A9 promotes activation of Ki-67 in primary CLL cells. We also found that CD19+CD5+ lymphocytes from Eµ-TCL1 mice express high levels of S100-A9 protein in comparison with normal B cells from C57BL/6 mice. To better understand the role of S100-A9 in CLL, we developed a novel Eµ-TCL1/S100A9-/- mouse model. Eµ-TCL1/S100A9-/- mice do not show structural abnormalities and develop a CLL phenotype characterized by the accumulation of CD19+/CD5+ cells in PB, BM, and secondary lymphoid tissues. However, these mice show a longer survival rate compared to Eµ-TCL1. Since the Eµ-TCL1/S100A9-/- model is a global knock-out, we aimed to confirm whether the survival advantage was attributed to the deficiency of S100-A9 in the B-CLL cells. We, therefore, isolated CD19+/CD5+ B-CLL cells from Eu-TCL1 and Eu-TCL1/S100A9-/- mouse spleens and adoptively transferred them into NSG mice. Our results show that the S100A9-/- B-CLL cells grow slower than those from Eu-TCL1 mice. Moreover, NSG mice from the Eu-TCL1/S100A9-/- group show accumulation of fewer tumor cells in the spleen and bone marrow. Longer survival was also observed in these mice compared to the Eu-TCL1 controls. Next, we evaluated the in vivo response to the S100-A9 inhibitors PaQ and TasQ. A significantly lower tumor burden and longer survival were found in mice treated with PaQ or TasQ compared to the vehicle group. Interestingly, S100-A9 inhibitors reduced the expression of PD-L1 and impaired the production of IL-10 and IL-6 by B-CLL cells. We also observed a reduction in the total number of patrolling monocytes and MDSCs after the treatment. T cells show a predominantly naïve and central memory phenotype with lower expression of exhaustion markers after being exposed in vivo to S100-A9 inhibitors. To dive deeper into the mechanism of how S100-A9 inhibition impairs CLL progression, we performed a Nanostring PanCancer immune profiling. Our data demonstrated significant downregulation in transcription factors (Fos, Jun, and Egr1) downstream to the MAPK pathway in S100A9-/- B-CLL cells as well as PaQ-treated CD19+ cells.

Conclusion: Genetic and pharmacological targeting of S100-A9 impairs CLL cell growth in vivo, probably through a MAPK pathway-dependent mechanism. This data demonstrates for the first time the significance of targeting S100-A9 as a novel therapeutic approach in CLL.