Thesis title: BCL-2 INHIBITION AS A NEW THERAPEUTIC STRATEGY TO COUNTERACT MELANOMA PROGRESSION AND INCREASE PHARMACOLOGICAL RESPONSE
RATIONALE: Metastatic cutaneous melanoma is considered one of the most aggressive forms of
skin cancer. The primary therapy strategies that dominate the current treatment consist of the
mitogen-activated protein kinase pathway (MAPKi) and immune checkpoint (ICI) inhibitors. These
innovative therapeutic approaches have achieved long-term survival rates of approximately 50%.
However, the emergence of resistance, the inadequate response to treatments, and the absence of
targeted therapy for BRAF wild-type patients represent a significant challenge for researchers and
clinicians and continue to require further efforts to find new therapeutic opportunities to increase
the survival of metastatic patients. Bcl-2 and Bcl-xL anti-apoptotic proteins play a pivotal role in
the resistance to cancer treatments and orchestrate the crosstalk between melanoma cells and those
of the surrounding microenvironment, thereby promoting tumor progression. BH3 mimetics, small
molecules targeting the anti-apoptotic proteins belonging to the Bcl-2 family, offer a promising
therapeutic approach for cancer treatment. In this context, Venetoclax (ABT-199), the first selective
Bcl-2 inhibitor, has received FDA approval for use in several hematological malignancies.
Recently, we discovered a novel pan BH3 mimetic, IS21, with in vitro and in vivo preclinical
antitumor activity in multiple tumor types.
GENERAL OBJECTIVES: The general objectives of this thesis are: (i) to identify new detrimental
roles of the anti-apoptotic proteins in melanoma mainly in promoting immune escape, and (ii) to
develop new combinatorial approaches based on the targeting of the anti-apoptotic proteins to
improve metastatic melanoma treatment.
EXPERIMENTAL DESIGN AND METHODS: Established and patient-derived melanoma cell
lines were used in this study. The anti-tumor efficacy and cell death induction of a panel of specific
[ABT-199 (Bcl-2), WEHI-539 (Bcl-xL), S63845(Mcl-1)] and pan [ABT-263 (Bcl-2 and Bcl-xL),
IS21(Bcl-2, Bcl-xL and Mcl-1)] BH3 mimetics, both in monotherapy and in combination with
MAPKi, was assessed in vitro by using MTT, CellTiterGlo, flow cytometry, and Western Blot
assays. Genetic (siRNA, overexpression) and pharmacological approaches (BH3 mimetics) were
used to evaluate the modulation of PD-L1 by Bcl-2 and Bcl-xL. Flow cytometry, western blot,
qRT-PCR and ChIP analyses were used to evaluate the molecular mechanism responsible for the
Bcl-2-induced PD-L1 regulation. Xenograft and allograft mouse models were used to assess the
ability of BH3 mimetics to potentiate the efficacy of MAPKi and ICI, respectively.
RESULTS: Our results demonstrated that, in monotherapy, BH3 mimetics reduced the viability and
induced apoptosis of a panel of melanoma cell lines, although with a different extent. In particular,
ABT-199 and ABT-263, were the most effective in inhibiting cell proliferation, regardless of the
BRAF mutational status. In combinatorial regimen, ABT-199 and IS21 synergistically potentiated
the effect of MEK inhibition in BRAF wild-type cells in vitro and that of MAPK (BRAF+MEK)
inhibitors in BRAF mutant cells, both in vitro and in vivo. Next, we identified a positive correlation
between PD-L1 and Bcl-2, but not Bcl-xL, protein levels in melanoma cell lines. In addition, we
found that Bcl-2 silencing by siRNA or pharmacological inhibition by ABT-199 in melanoma cell
lines reduced PD-L1 expression, membrane localization and secretion. Mechanistically, we found
that silencing or inhibiting Bcl-2 in melanoma cells reduced the expression of c-Myc. Concordantly,
we demonstrated a reduction in the recruitment of c-Myc protein to the PD-L1 promoter in Bcl-2
silenced melanoma cells. Next, silencing c-Myc in Bcl-2 overexpressing melanoma cells reduced
PD-L1 expression levels. Finally, in vivo studies demonstrated a significant reduction in tumor
growth and an enhancement of the survival rate in mice treated with ABT-199 and ICI (anti-PD1
antibody) compared with those treated with single agents.
CONCLUSIONS: In conclusion, our findings demonstrated that Bcl-2 regulates PD-L1 expression
in melanoma and that BH3 mimetics potentiate the efficacy of target- and immuno-therapies.