Exploring surface interactors promoting viability of CLL cells in contact with stromal fibroblasts

INTRODUCTION Survival and expansion of chronic lymphocytic leukemia (CLL) B cells are strongly shaped by interaction with the tissue microenvironment. This pro-survival activity is sustained by indirect and direct interactions, through soluble and surface factors, respectively. In this study, we aimed at exploring the lesser-known direct cell-to-cell surface interactions between CLL cells and their microenvironmental stroma that support the increased viability in CLL cells. METHODS Primary CD19+/CD5+ cells from 20 untreated patients with CLL (16 with unmutated IGHV) were cultured in the presence or absence of HS-5, a human bone marrow derived fibroblast cell line, that was also grown alone, as control. After 72 hours, viability of the primary CLL cells was assessed and HS-5 cells were harvested from the 2 culture conditions, with and without CLL cells, respectively, to be processed for RNA sequencing. Cell surface protein annotation method was used to identify the most prominent proteins expressed on the cell surface. These genes were used to design the CRISPR/Cas9 screening library encapsulated in lentiviral particles to infect HS-5 cells. RESULTS As expected, direct cellular contact with stroma increased the viability of CLL B cells by ~30% at day 3 (p < 0.0001) compared to CLL cells cultured alone. Using the method of surface protein annotation, we analyzed the RNA-Seq data obtained from HS-5 cells grown in co-culture and identified a list of 120 genes, likely coding for surface proteins, that were differentially expressed as compared to HS-5 cells cultured alone. Pathway enrichment analysis of these genes, particularly including notable names like ALCAM , DDR2 , TM4SF1 , ITGB8 & GJA1 etc., identified few vital cellular activities such as cell-adhesion, extracellular matrix protein binding, integrin binding and kinase binding. We have created a CRISPR/Cas9 library to enable functional testing directly in HS-5 cells to selectively remove each gene of interest. These modified HS-5 cells will be used for co-culture with primary CLL B cells to identify specific genes whose removal may show a significant impact on leukemic viability in our in vitro model. CONCLUSIONS In our work, we focused on the surface interactions, occurring between CLL and stromal cells, supporting the contact-dependent survival of the leukemic clone. By combining RNA-seq-based surface protein annotation with a targeted CRISPR screen, we establish a functional approach to identify a number of surface molecules on stromal cells that are potentially involved in direct surface interactions. This strategy may expose microenvironmental vulnerabilities that can be targeted to disrupt CLL-stroma interactions. Therapeutically, this study may pave the way to define the stromal interface as a tractable target to exploit novel treatment strategies for CLL in future.