The microenvironment of secondary lymphoid organs is composed of various cell types, including fibroblastic reticular cells (FRCs), which play a crucial role in providing signals to promote activation and survival of normal and malignant B cells such as in chronic lymphocytic leukemia (CLL). We have discovered a retinoic acid (RA)-dependent FRC-leukemia crosstalk during CLL progression in mice and demonstrated that pharmacological inhibition of the RA pathway reduces tissue infiltration and CLL progression in preclinical models. Here we further explored the role(s) of the RA pathway in human FRC-CLL crosstalk using several approaches, including a newly established humanized lymph node(LN)-like microenvironment suitable for mechanistic studies under more physiological conditions.

We used 2D and 3D culture systems of human lymph node fibroblasts and primary human CLL cells; RNA-scope, RNA-seq, western blot, flow cytometry and confocal microscopy.

Using the RNA-scope, we detected upregulation of genes involved in RA synthesis, RA degradation and RA signaling in human CLL-LN biopsies compared to control tissues. Specifically, we found that the transcription factor Retinoid X Receptor (RXR) alpha, an RA nuclear receptor is overexpressed at mRNA and protein levels in hCLL cells compared to control B cells. Using 2D co-cultures and 3D spheroids, we demonstrated that inhibition of RXR alpha reduces CLL adhesion and prevents CLL cell aggregation and spheroid formation. To further investigate the functional role of RXR alpha inhibition, we used a 3D lymph node-like microenvironment cultured in a bioreactor and found that inhibition of RXR alpha impaired the interaction of hFRCs and hCLL and promoted the mobilization of leukemic cells from the 3D niche. To study the molecular mechanism underlying this phenotype, we performed bulk RNA-seq on hCLL and hFRCs after treatment with an RXR antagonist and found that RXR alpha inhibition caused deregulation of multiple molecular pathways, including cell adhesion, fatty acid metabolism, and cytokine signaling.

Our results show that the retinoic acid signaling pathway is upregulated in hCLL-LN biopsies in vivo and in hCLL cells from the peripheral blood. We demonstrated a functional role of RXR alpha and RA signaling in promoting interactions of hCLL and hFRCs, and hCLL retention within the microenvironment. We are currently investigating the underlying mechanism of RXR alpha overexpression and function.