par Sabbah, Malak
Président du jury Langer, Ingrid
Promoteur Awada, Ahmad
Co-Promoteur Ghanem, Ghanem Elias
Publication Non publié, 2022-03-08
Thèse de doctorat
Résumé : Melanoma is divided into four genomic subtypes: BRAF, NRAS, NF1, and Triple-WT. Integrative analysis reveals enrichment of RTK alterations (mutations, focal amplifications, and complex structural rearrangements) as a feature of the WTBRAF group. Non-BRAF mutant melanoma patients have limited treatment options with only 30% responding to checkpoint inhibitors. Several efforts were dedicated to preventing tumor growth and metastases in WTBRAF melanoma by targeting RTKs that regulate cell proliferation, invasion, and migration. Melanoma had been considered a radioresistant tumor showing broad shoulder in survival fraction assays mainly attributed to high repair efficacy and the presence of pigment. Of note, it was shown in several cancers that ionizing radiation (IR) promotes RTK activity (radiation-induced intracellular translocation of RTKs) which enhances DNA repair. Conversely, RTK inhibition could impede repair kinetics of radiation-induced DNA damage and thus improve tumor response to ionizing radiation. This relation of RTK-RT indicates that a possible combination of RT and RTK inhibition could be more interesting than previously thought. Compared to other DNA damaging or anti-mitotic agents, inhibition of tumor specific-receptor tyrosine kinases could be a rational approach for combined radiotherapy to assume tumor control with lesser side effects and a better quality of life. For this reason, we aimed to assess the benefit of combining RTK inhibition and radiotherapy in non-BRAF mutant melanoma and depict the associated pathways and mechanisms. Firstly, we found that specific inhibition of c-Met (HGFRi, Crizotinib), or DNA repair agent inhibitor (PARPi, Olaparib) resulted in a significant enhancement of melanoma radiosensitivity, indicating the possible association between radioresistance and c-Met activity or PARP activity in melanoma. Moreover, we found that RT mediates nuclear translocation of Met that associates to PARP, two hours following RT in melanoma. Interestingly, the nuclear enzymatic activity of PARP following RT can be reversed when combined with c-Meti (Crizotinib), also Ko of Met mediate enhanced response to PARP inhibitor and RT in melanoma. This indicates, that c-Met translocates to the nucleus under radiotherapy and activates PARP that confers melanoma radioresistance. Consequently, targeting MET and PARP under radiotherapy resulted in a significant synergistic decrease in cell survival and increase of cell death compared to METi or PARPi and/or RT both in vitro and in vivo. Our results shed the light on a new promising combinatorial approach in non-BRAF mutant melanoma that deserves further translational investigation, particularly that RTK inhibition is considered of particular importance in the WTBRAF melanoma subgroups showing a short/limited clinical benefit. While running large screenings with RTK inhibitors, a c-Kit inhibitor, dasatinib, attracted our attention because 1) it is the most active among c-Kit inhibitors, due to its ability to bind both active and inactive conformations of the receptor; 2) despite all expectations, its use as a single agent in melanoma showed limited clinical benefit; 3) unexpectedly and despite complete inhibition of both MAPK and PI3K/AKT pathways, we observed less effect at high dasatinib concentrations but yet can be found in patients’ blood during clinical trials. A deeper investigation of other important pathways in melanoma revealed a significant increase of the main transcription factor in melanocytes, MITF, and of its downstream target, particularly, Bcl-2 under high dasatinib concentrations. As the canonical pathway to activate MITF proceeds through the stem cell factor and its receptor kinase c-Kit but also through cAMP/CREB, we searched for a possible off-target effect for dasatinib and found that it inhibits SIK2, which is known to phosphorylate CRTC3. The latter mediates CREB transcription and activation of MITF. A series of validation studies involving selective Bcl-2 inhibition either by ABT-199 or Bcl-2 knockout, was able to restore melanoma cells sensitivity to dasatinib, associated with pieces of evidence (FSK, pigmentation, acquired resistance model…), highly supporting the involvement of MITF and Bcl-2 axis as a mechanism of resistance induced by dasatinib itself that may have contributed to its modest efficacy in various clinical trials.

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