REPURPOSED AT9283 TRIGGERS ANTI-TUMORAL EFFFECTS BY TARGETING MKK3 ONCOGENIC FUNCTIONS IN COLORECTAL CANCER

Background Colorectal cancer (CRC) is the third most common type of cancer and the second leading cause of cancer-related deaths worldwide, with a survival rate near to 10% when diagnosed at an advanced stage. The lack of clinical improvements for advanced-stage CRC require essential the identification of new molecular targets to design more selective and efficient therapies. The Mitogen activated protein kinase kinase 3 (MKK3) may be a novel and efficient therapeutic target, albeit selective inhibitors are currently unavailable. Methods Transcriptomic based drug repurposing approach identified the multitargeted kinase inhibitor AT9283 as a putative compound with MKK3 depletion-mimicking activity. AT9283 effects were investigate with CRC lines, colonocytes and CRC patient-derived organoids through in vitro and in vivo studies. Results AT9283, like MKK3 silencing, promotes autophagy and cell death and reduces cell migration and invasion in tested CRC lines and patient-derived organoids (PDOs). AT9283 inhibits tumor growth in preclinical models. Interestingly, AT9283 did not affect the growth or survival of primary colonocytes. Mechanistically, results demonstrated that AT9283 abrogates phospho- and total-MKK3 protein levels mainly through the inhibition of aurora kinase A (AURKA), uncovering the relevance of MKK3/AURKA crosstalk in sustaining their own protein stability and activity. Conclusion Overall, we demonstrated that the anti-tumoral effects triggered by AT9283 treatment recapitulated the MKK3 depletion effects in all tested CRC cell lines, suggesting that AT9283 is a repurposed drug. According to its good tolerance when tested with primary colonocytes (CCD-18CO), AT9283 is a promising drug for the development of novel therapeutic strategies to target MKK3 oncogenic functions in late-stage and metastatic CRC patients. Two conditions, sh/MKK3 and sh/scr, were analyzes in biological replicates (three). Gene expression profiles were compared to control (sh/scr). H3, GAPDH, GUSB, RPL19, RNU2 and ACTIN were used as endogenous controls to normalize gene expression. The Affymetrix gene expression data (array HTA_2.0) were background adjusted and quantile normalized. Gene expression values were calculated using the robust multiple-array average (RMA) procedure, and duplicates were averaged. Probes not detected in all the samples were removed. The summarized signals were log2-transformed and quantile normalized. Significantly modulated genes between different conditions were identified with a distribution fold change test (DFC) and a permutation test, adjusting for multiple comparisons.