1020 - The mechanical properties of CLL cells are linked to the actin cytoskeleton and are target of BTK inhibitors

Chronic lymphocytic leukemia (CLL) is an incurable leukemia characterized by an intense trafficking of the leukemic cells between the peripheral blood and lymphoid tissues. The ability to recirculate between tissues depends on the capacity of those cells to modify and rearrange their cytoskeleton, and adapt to external cues, however not much is known about the differences occurring between CLL and healthy B cells during these processes. Nowadays new target therapies are emerging, in particular the BTK inhibitors, as effective therapies leading to sustained responses, though patients may become resistant and relapse. BTK inhibitors promotes CLL cells mobilization from tissue to peripheral blood, where cells eventually undergo apoptosis, however, what really occurs in the tissues, and which are the processes regulating CLL cells dynamics and resistance to therapy remains to be elucidated.
In the present work we demonstrated that CLL cells at nanoscale resolution, have a specific actomyosin complex organization with longer and more branched actin filaments and altered mechanical properties in comparison to their healthy counterpart. By super resolution microscopy on single cells we observed a low level of colocalization of actin and myosin suggesting that CLLs have a less contractile actomyosin complex, which correlates with a softer cellular cortical stiffness, measured by atomic force microscopy (AFM). On the contrary healthy B cells (HBs) are more contractile and stiffer (CLL n = 19, HB n= 9). To evaluate the clinical relevance of our findings we treated CLL cells with the BTK inhibitors ibrutinib and acalabrutinib. We observed a significant rescue of the physiological heathy phenotype upon treatment, characterized by stiffening of the cellular cortex by AFM (before and after treatment: p value < 0.0001), and activation of the actomyosin complex by increasing myosin phosphorylation and colocalization with actin. We further validate our findings also in vivo on CLL cells isolated from patients undergoing ibrutinib treatment (CLL n=4), confirming our conclusion (p value < 0.0001). Moreover, we were preliminary able to show that CLL cells fail to restore the healthy phenotypes in case of resistance to ibrutinib. Furthermore, by measuring the cortical stiffness of tissue resident CLL cells (CLL from bone marrow n= 2, CLL from lymph node n= 3), we observed a persistent stiffening, confirming a possible regulation of CLL cell’s life cycle via BTK inhibition, by impairing the reentry of CLL cells into the tissues during treatment. Our results suggest that CLL cells' mechanical properties are linked to their actin cytoskeleton organization and might be involved in novel mechanisms of drug resistance thus becoming a new potential therapeutic target aiming at the normalization of the mechanical fingerprints of the leukemic cells.