Understanding the communication between cancer cells and cancer-associated fibroblasts in chronic lymphocytic leukemia: roles clarification and therapeutic targets exploration

ABSTRACT Chronic lymphocytic leukemia (CLL) is the most common leukemia in adults, accounting for 37% of cases in the US. Current therapies, including small molecular inhibitors targeting B-cell receptor (BCR) signaling and BCL-2 survival pathways, do not frequently induce complete remissions and drug-resistant clones accumulate overtime during these treatments. Thus, the Goal of this proposal is to identify potential therapeutic targets in the CLL tumor microenvironment (TME) to improve treatment. We focus on fibroblastic reticular cells (FRCs), the specialized lymphoid organ fibroblasts that regulate lymph node (LN) cellularity and compartmentalization and support immune responses. Our preliminary data show that CLL cells shift FRC subsets from podoplanin (PDPN) expressing FRCs that regulate dendritic cells and T-cell immunity, to CXCL13 secreting FRCs that interact with B-cells and govern the formation of B-cell clusters and follicles. These findings provide the Rationale to target these CLL-associated FRCs to revert the impaired T cell compartment for better therapeutic outcome. We hypothesize that the tri-directional interactions and effects of CLL cells with T cells and FRCs are key influences on disease progression and drug resistance. To test our central hypothesis, we will pursue two Aims. In AIM 1, we will seek to understand the cellular and molecular mechanisms whereby CLL B cell infiltration changes FRCs. For the hypothesis of “CLL B cells regulate the structure and function of FRC networks in an activation-dependent manner”, we will [1] confirm cellular changes in FRCs upon resting or IL4/BCR activated CLL B cell infiltration in 2-D and 3-D Matrigel systems, [2] perform single-cell RNA sequencing to investigate changes in healthy fibroblasts upon the infiltration of CLL B cells at early and late stage of disease for niche- associated heterogeneity that could contribute to CLL disease progression, [3] verify these in vitro findings in an adoptive transfer model of CLL xenografts using Matrigels mixed with human LN fibroblasts and CLL PBMC in SRG-BA6 mice, [4] we will target IL-4/BCR signaling in CLL B cells to reverse cancer-associated fibroblasts (CAF)-like fibroblasts. For AIM 2, we will investigate how CLL-exposed FRCs create an immune-tolerant TME to accelerate disease progression and support the development of drug resistance. For the Hypothesis of “CLL cell-exposed FRCs modulate T-cell functions and create a pro-tumor TME that supports CLL B cells, both ultimately impacting disease progression”, we will [1] decipher changes in T-cell populations and functions mediated by CLL-exposed FRCs in the co-culture system and in animal models and associate those changes with CLL B cell growth rate; [2] determine the subset(s) of CLL-associated fibroblasts that is responsible for BTKi resistance. And [3] finally identify a therapeutic strategy to target CLL-FRC interplay; application of FRC-targeted anti-FAP immunomodulatory drug will be done in the co-culture systems and animal models. The proposed work is innovative as it will be the first to explore the underlying mechanisms of the dynamic interaction between CLL cells and FRCs and will provide novel therapeutic strategies in B-cell malignancies.