Lyn Kinase Contributes to the Reprogramming of Fibroblasts Promoting Chronic Lymphocytic Leukemia Progression
Authors: Vom Stein Alexander Frederik , Koch Maximilian , Reinarzt Sebastian , Lucas Anna , Rebollido-Rios Rocio , Oellerich Thomas , Nguyen Phuong Hien , Hallek Michael 
Published: November 2019

Growth of chronic lymphocytic leukemia cells strongly depends on a nurturing microenvironmental niche that is specifically primed by diverse, bi-directional interactions to promote leukemic homing, proliferation and progression. The Src-family kinase Lyn was previously identified by our group as a key factor for the formation of this pro-leukemic niche and for the expansion of CLL cells, using the Eµ-TCL1 mouse model.
In order to attribute the pro-leukemic function of Lyn to a specific cell type, chimeric mice with lineage-specific defects of Lyn within hematopoietic or non-hematopoietic compartments were generated by irradiating BL6J-mice lethally and restoring their hematopoietic system with Lyn-WT or Lyn-KO stem cells. Consecutively mice were xenotransplanted with TCL1+-malignant cells. Lyn deficiency within the non-hematopoietic compartment decelerated leukemic expansion to a higher degree than did Lyn deficiency within the hematopoietic compartment. Completely Lyn deficient mice showed a more prominent retardation of leukemic expansion compared with both lineage specific Lyn deficient mouse strains, suggesting an additive effect of the two distinct compartments for leukemic expansion.
In focusing on the non-hematopoietic fibroblastic bystander cells, primary human CLL cells were cocultured in vitro with Lyn-deficient mouse embryonic fibroblasts as well as Lyn-KO human HS5 cells, generated via the CRISPR-Cas9 system, and leukemic cell survival was assessed over time. All Lyn-deficient fibroblasts showed a significantly reduced feeding capacity for CLL cells compared to WT stroma, indicating the functional relevance of Lyn in leukemia-associated fibroblasts.
Subsequently, transcriptomic, proteomic and phosphoproteomic alterations related to Lyn-KO in HS5 cells were comprehensively analyzed, revealing a surprisingly extensive change in gene and protein expression pattern that appeared to be regulated mainly at the transcriptional level. The differentially expressed genes were remarkably often extracellular matrix (ECM)-, cytoskeleton- or cytokine-associated. GO-term enrichment analysis additionally suggested a correlation with ECM processes. Therefore, we hypothesized that Lyn-deficiency might induce transcriptional changes of the cancer-associated fibroblast (CAF)-like phenotype, thus leading to a reduction in leukemic feeding capacity.
The diminished expression of several CAF-makers congruent with this reduced activation status was validated in Lyn-KO fibroblasts, as well as the transcriptionally regulated differential expression of chosen target genes. Amongst those, the deubiquitinating enzyme UCHL1 was most abundantly reduced in Lyn-KO HS5 cells, showing an almost complete loss of mRNA and protein expression.
Application of a specific UCHL1-inhibitor -in a dose without toxic effects on CLL cells - to CLL-stroma coculture resulted in a significantly hampered feeder effect and reduced CLL cell survival, implying a functional relevance of microenvironmental UCHL1 for stromal support in our system. Additionally, stroma cell death induced by higher drug concentrations in WT cells was completely prohibited in Lyn-KO stroma, illustrating the importance of Lyn for regulating UCHL1 expression and function.
In summary, we propose that the Lyn kinase contributes to the formation of a supportive microenvironment via the transcriptional reprogramming of stroma fibroblasts into a "CAF-like" phenotype, which echances viability of CLL cells. In addition, UCHL1 might be a potentially druggable mediator of this activation process.

Disclosures
Hallek: Roche, Gilead Sciences, Inc., Mundipharma, Janssen, Celgene, Pharmacyclics, AbbVie: Honoraria, Research Funding, Speakers Bureau.