Abstract
in English, Spanish Background: The P21 activated kinases (PAK) are frequently dysregulated in cancer and have central roles in oncogenic signalling, prompting the development of PAK inhibitors (PAKi) as anticancer agents. However, such compounds have not reached clinical use because, at least partially, there is a limited mechanistic understanding of their mode of action. Here, we aimed to characterize functional and molecular responses to PAKi (PF-3758309, FRAX-486 and IPA-3) in multiple acute myeloid leukaemia (AML) models to gain insights on the biochemical pathways affected by these inhibitors in this disease and identify determinants of response in patient samples. Methods: We mined phosphoproteomic datasets of primary AML, and used proteomics and phosphoproteomics to profile PAKi impact in immortalized (P31/Fuj and MV4-11), and primary AML cells from 8 AML patients. These omics datasets were integrated with gene dependency data to identify which proteins targeted by PAKi are necessary for the proliferation of AML. We studied the effect PAKi on cell cycle progression, proliferation, differentiation and apoptosis. Finally, we used phosphoproteomics data as input for machine learning models that predicted ex vivo response in two independent datasets of primary AML cells (with 36 and 50 cases, respectively) to PF-3758309 and identify markers of response. Results: We found that PAK1 activation- measured from phosphoproteomics data- was predictive of poor prognosis in primary AML cases. PF-3758309 was the most effective PAKi in reducing proliferation and inducing apoptosis in AML cell lines. In cell lines and primary cells, PF-3758309 inhibited PAK, AMPK and PKCA activities, reduced c-MYC transcriptional activity and the expression of ribosomal proteins, and targeted the FLT3 pathway in FLT3-ITD mutated cells. In primary cells, PF-3758309 reduced STAT5 phosphorylation at Tyr699. Functionally, PF-3758309 reduced cell-growth, induced apoptosis, blocked cell cycle progression and promoted differentiation in a model-dependent manner. ML modelling accurately classified primary AML samples as sensitive or resistant to PF-3758309 ex vivo treatment, and highlighted PHF2 phosphorylation at Ser705 as a robust response biomarker. Conclusions: In summary, our data define the proteomic, molecular and functional responses of primary and immortalised AML cells to PF-3758309 and suggest a route to personalise AML treatments based on PAK inhibitors. Current treatments for acute myeloid leukaemia (AML) are ineffective for most patients. Therefore, new treatments for patients that do not respond to approved drugs or who relapse after initial treatment are needed. A group of proteins, named P21 activated kinases (PAK), are frequently overactivated in AML and compounds that inhibit them are effective in killing AML cells. The identification of patients that will respond to PAK inhibitors would be crucial for the translation of these compounds into the clinic by identifying patients more likely to respond. In this work, we studied how three PAK inhibitors modulate protein phosphorylation and expression in AML cell lines and in cells directly obtained from patients. We focused on PF-3758309 because it was the most effective PAK inhibitor. PF-3758309 inhibited the activities of PAK and other kinases and reduced the phosphorylation and expression of key proteins, like c-MYC, which are known to be important for the proliferation of cancer cells. Mathematical modelling using phosphoproteomics data classified leukaemia cells from AML patients as sensitive or resistant to PF-3758309 treatment and identified the phosphorylation of the lysine demethylase PHF2 at Ser705 as an indicator of whether or not patients may respond to PF-3758309, with potential clinical and diagnostic utility. Overall, our data define the molecular and functional response of AML cells to PAK inhibitors and identify molecular indicators that can be used to predict which patients will respond to these treatments. Our findings can therefore contribute to the translation of PAK inhibitors into clinical use.