1056 - LYN kinase and its regulator CSK as key players in CLL-supporting macrophages

The expression of several B-cell receptor-associated kinases such as LYN, BTK, PI3Kδ and PKC-β in bystander cells of the CLL tumor microenvironment (TME) were shown to contribute actively to the leukemia-promoting niche (Nguyen et al., 2016, Dong et al., 2019, Lutzny et al., 2013). LYN kinase was an essential factor for disease progression, although activation of this kinase in B cells seemed to play only a minor role in CLL development in vivo (Nguyen et al., 2016; Kohlhas et al., 2020). In contrast, LYN expression in the microenvironment was essential for CLL growth, which could be attributed to the failure of both LYN-knockout macrophages and stromal fibroblasts to fully support CLL cell survival (vom Stein et al., 2023).

Using CRISPR/Cas9 editing in murine zygotes, we generated a mouse model harboring a point mutation (K275R) in the kinase domain of LYN, leading to an inactivation of its kinase function. LYN-K275R mice exhibit a massive reduction in tyrosine phosphorylation with an alleviated phenotype compared to LYN-knockout mice. In adoptive transfer experiments, CLL progression was significantly delayed in LYN-K275R recipients. The viability of primary CLL cells cultured with LYN-K275R bone marrow-derived macrophages was significantly reduced, indicating that the kinase function of LYN in macrophages is the important mediator of CLL support.

Alongside the in vivo findings, we established a library of tyrosine kinase knockout macrophage cell lines to investigate the potential roles of tyrosine kinases in the CLL-TME dialog. For this purpose, we successfully conducted a functional arrayed CRISPR/Cas9 knockout screen of all known tyrosine kinases in the THP-1 cell line. THP-1 monocytes stably expressing the Cas9 nuclease were transduced with lentiviral particles containing sgRNAs targeting all human tyrosine kinases. Single gene knockout THP-1 monocytes were differentiated into macrophages and cocultured with primary CLL cells. CLL cell survival was measured as a read-out for the supportive capacity of the THP-1 feeder cells.

We discovered a dozen single-gene knockouts with consistently reduced CLL cell survival support, with LYN being identified among the top targets, adding further weight to our in vivo findings. Importantly, we detected a significantly enhanced expression of LYN in macrophages of the CLL lymph node compared to those of healthy individuals, implying the clinical relevance of LYN.
Interestingly, the deletion of CSK, the major negative regulator of SRC family kinases (SFK) including LYN, also led to a robust decrease in CLL-feeding capacity of macrophages. LYN and CSK knockout THP-1 macrophages did not show detectable differences in viability and phagocytosis. Although CSK knockout resulted in the increased activation of other SFK, LYN expression was starkly diminished in CSK-knockout macrophages, which was solely dependent on the kinase activity of CSK. These results indicate that the signaling axis governed by LYN and its negative regulator CSK in macrophages are crucial for CLL cell survival. Further validation of these findings with LYN and CSK knockouts in additional macrophage cell lines and in primary macrophages is ongoing.

Our current studies are focusing on deciphering the molecular mechanisms, by which LYN and its regulator CSK in macrophages influence CLL cell survival using transcriptome, proteome, and secretome analyses. Our acquired results will not only unravel the signaling pathways involving tyrosine kinases that are critical for the CLL-TME dialog but also open new venues for novel combinatory treatment regiments, in which available kinase inhibitors can be redesigned for remodeling of the CLL TME.