Rho GTPases are critical players in the biology of T cell lymphoma. Recurrent genetic lesions of RhoA and dysregulation have been reported in different T cell lymphoma, including Angioimmunoblastic T-cell lymphoma (AITL) and Peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS). In ALK+ anaplastic large cell lymphoma (ALCL) oncogenic ALK regulates the Rho family GTPases CDC42 and RAC. We have demonstrated that the cytoskeleton regulators WASP and WIP are frequently downregulated resulting in hyperactivation of the RhoGTPase CDC42 and the MAPK pathway. Therefore, hyperactivated CDC42 and MAPK pathway represent a therapeutic vulnerability in ALK+ ALCL. We have previously demonstrated that RhoA activity is repressed by ALK, but its specific role in ALK-driven lymphomagenesis is not yet elucidated.

To explore the contribution of RhoA in NPM-ALK lymphomagenesis, we generated the NPM-ALK/CD4Cre/RhoAfl/fl Tg mouse models from which we derived primary murine cell lines to dissect cell-autonomous alterations in vitro. We also abrogated RhoA expression by the CRISPR/Cas technology in human ALK+ lymphoma cell lines TS, JB6 and COST. We assessed cellular rewiring in the absence of RhoA by measuring cell proliferation and metabolism, analyzing actin organization through immunofluorescence and signaling by Western blot. We also performed RNA Sequencing analysis to evaluate changes of the transcriptome profile in RhoA knock-out cells. We tested in vitro the combination of ALK tyrosine kinase inhibitors (TKIs) and the ROCK inhibitor Y-27632.

In vivo RhoA deletion impaired lymphoma development resulting in significant extension of survival of NPM-ALK/RhoAfl/fl mice. In vitro, in both murine and human cell lines, RhoA deletion affected lymphoma cell proliferation and migration, and caused changes in cell morphology and actin organization. RhoA knock-out ALK+ lymphoma cells underwent transcriptional rewiring to repress genes for mitotic spindle organization, cell cycle checkpoint regulation and reprogram metabolic processes for energy production. Moreover, the ratio of glucose/lactate production and oxygen consumption were significantly decreased in RhoA knock-out cells. In addition, RhoA knock-out ALK+ lymphoma cells showed increased sensitivity to ALK TKI crizotinib and inhibition of the RhoA downstream effector ROCK in combination with ALK-TKI, further potentiated the effects of crizotinib.

Overall, our findings suggest that RhoA plays a critical role in ALK-driven lymphomagenesis, with its deletion impairing lymphoma progression and increasing sensitivity to ALK-targeted therapies. In addition, RhoA deletion alters cell proliferation, migration, and metabolism, suggesting its potential as a therapeutic target in ALK+ lymphomas.