P4HB TARGETING HALTS TUMOR GROWTH AND RE-SENSITIZES RESISTANT MELANOMA CELLS TO DABRAFENIB AND TRAMETINIB STANDARD-OF-CARE THERAPY VIA IRE1-⍺ ACTIVATION AND AKT DOWNMODULATION

BRAF-mutant melanoma patients account for approximately 50% of all cutaneous melanoma cases. The overall survival rate of metastatic melanoma patients was dramatically improved by the introduction of targeted therapy (BRAF and MEK inhibitors) and immune checkpoints inhibitors (anti-PD1, anti-CTLA4). However, a significant proportion of BRAF-mutant patients develop secondary resistance to targeted therapies, while others exhibit even primary resistance. To either prevent or overcome resistance in BRAF-mutant melanoma patients, new combinations are now being tested, exploiting triple-agent combinations (targeted therapy with immunotherapy), but with unsatisfactory results. Additionally, the use of immunotherapy in neo-adjuvant conditions, has shown promise, being still under investigation. Another potential therapeutic approach involves re-sensitizing resistant melanoma cells to targeted therapies. However, clinical trials exploring this strategy in patients with BRAF/MEK inhibitor resistance are currently limited. Hence, identifying new vulnerabilities in resistant melanoma cells, as well as to establish novel strategies to re-sensitize resistant melanoma cells to targeted therapies is crucial to improving patient outcomes. Herein, I carried out a functional shRNA-based in vivo screen with a library targeting 195 actionable genes (FDAome library), where each gene is linked to specific FDA-approved drugs that can be repurposed for melanoma treatment. A375 parental cells and A375 cells resistant to dabrafenib and trametinib (A375-DT) were used as models to unveil new vulnerabilities. FDAome screen highlighted 37 genes essential for the in vivo tumor growth of DT-resistant cells. I validated two candidates, namely CREB-binding protein (CREBBP) and Prolyl 4-Hydroxylase Subunit Beta (P4HB), whose inhibition reduced tumor growth in vivo and increased overall survival of A375-DT but not of A375 parental cells, suggesting a putative point of intervention in resistant tumors. Additionally, I demonstrated that silencing CREBBP and P4HB reduced the migratory capacity of A375-DT cells but not A375 cells, further corroborating our in vivo findings. I further validated the in vitro results in a different cell line, namely SKMEL-28-DT and in drug resistant patient-derived primary cultures. To further investigate the therapeutic potential of targeting CREBBP and P4HB, I tested the efficacy of CCS1477 and PACMA31, respectively, as monotherapies and in combination with dabrafenib and trametinib. Both compounds effectively inhibited tumor growth in vivo. Notably, PACMA31 demonstrated the ability to re-sensitize resistant tumors to DT. Furthermore, PACMA31 demonstrated significant efficacy in a syngeneic D4M-DT melanoma model, leading to dramatic tumor regression. Mechanistically, I demonstrated that P4HB silencing had an impact on cellular homeostasis, by increasing accumulation of misfolded proteins and activating IRE1⍺ signalling pathway as part of the unfolded proteins response. This resulted in partial cellular death and autophagy induction. Additionally, P4HB knockdown and subsequent ER stress increment reduced AKT phosphorylation, generating a window of opportunity to re-establish sensitivity to dabrafenib and trametinib treatment.