1Sbarro Institute for Cancer Research and Molecular Medicine, Temple University, Philadelphia, United States
2The ISOPROG-Somatolink EPFP Research network, Philadelphia, USA 19104 and Caltanissetta, Italy
3Skin Cancer Unit, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori, IRCCS Meldola, Italy
4Clinica Dermatologica, Department of Medicine and Surgery, University of Parma, Italy
5Dept of Medical Biotechnology, University of Siena, Italy
6Medical Oncology Department, San Vincenzo Hospital, Taormina, Messina, Italy
7Unità Operativa Complessa di Chirurgia Oncologica - Dipartimento di Oncologia Azienda di RilievoNazionale e di Alta Specializzazione (ARNAS), Garibaldi – Catania, Italy
Corresponding author details:
Pierluigi Scalia
Sbarro Institute for Cancer Research and Molecular Medicine
Temple University
Philadelphia,United States
Copyright: © 2020 Scalia P, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 international License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Malignant melanoma is an aggressive cancer of the melanocytic cellular components
of the skin which is responsible for 55,000 annual fatalities worldwide with more than 3
million people affected. Although genetics, age, and cumulative UV radiation exposure are
all known risk factors, the knowledge of melanoma genomic profile at the individual patient
level is becoming a more stringent factor towards its clinical management. Especially, since
a number of molecular therapies have revolutionized the life expectancy of these patients
over the last few decades. The present review, summarizes our current knowledge of the
melanoma oncogenomic landscape through its commonly altered gene products, in which
clearly emerges the critical role of tumor microenvironment as a distinctive factor for
the recurrence and the underlying drug resistance linked to molecular target therapies.
BRAF pathway-dependent and independent defects, the T-Cell Receptor (TCR) responsemodulating systems involved in tumoral immune-evasion, the alterations in Cancerassociated fibroblasts (CAFs)-generated signals and the underlying cellular paracrine
and autocrine network are discussed. Such scenario further strengthens the importance
of genotranscriptomic profiling towards designing effective and durable therapeutic
strategies.
A consistent and growing number of advances in the biological knowledge of
Malignant Melanoma (MM) have been observed in the scientific literature following
the initial discovery of its key driver mutation BRAFV600E and the studies uncovering the
alternative and/or compensative circuitry leading to MAPK reactivation in response to
its kinase activation inhibitor, vemurafenib [1-3]. Furthermore, The research focusing on
the role of T-Cell Receptor associated factors on the regulation of cell-mediated immune
response involved in tumor surveillance led in the late nineties to the discovery of the role
of TCR coreceptors molecules such as CTLA-4 (and its counterpart ligand B7) in tumor
surveillance [4]. This line of research was further strengthened by the discovery of the
parallel role of the PD-1 and PD-L1 ligand receptor system linking the T-Cell mediated
antigen recognition to down-modulation of T-Cell activity and to tumor immune evasion in those cancer expressing PD-L1 [5-7] and has provided the rational
for the development of the second generation immune therapies that
have revolutionized the treatment options of melanoma and other
tumor types by extending life expectations from a few months to (for
some) several years [8,9]. The above discoveries in the biological and
pharmacological realm, though, have been speedup by the parallel
advances in geno-transcriptomic analysis with widespread use of NGS
technologies. In fact, the cumulative results obtained by qualitative
and quantitative sequencing analysis on the several genes involved
in the above biological processes in each individual cancer patient
have confirmed the molecular heterogeneity of cancer involved in
the individual sensitivity and responsivity to such treatment and
have clearly pointed at the value of pathway-driven transcriptomic
profiling as an effective decisional tool towards selecting the most
effective molecular therapies tailored to the biologic asset of a patient
cancer [10]. A further game-shifting paradigm has been provided by
a growing amount of investigations in the last decade shedding light
on the mechanistic role of tumor microenvironment to melanoma
progression and responsivity to smart drugs. In particular, due to
the paracrine signals network between the melanoma cell, stromal
active elements (known as cancer-activated fibroblasts, CAFs) and
tumor infiltrating lymphocytes (TILs). Such complexity and the
additional inter-cellular circuitry underlying the up-regulation
and downregulation of the molecular and genetic discriminants of
melanoma’s drug responsivity, support the growth in single-cell base
profiling technologies and dedicated computational tools. These are
likely to become the gold standard to guide the most therapeutic
strategies in the next few years. The present review summarizes
the functional knowledge gained so far in the role of both intrinsic
(melanoma) and extrinsic (CAFs and TILs) molecular pathways
which should be taken in consideration in the melanoma patient
genotranscriptomic profiling.
The implication of BRAF mutations in Malignant Melanoma (of which BRAFV600E is the most commonly observed) and the experience gained with the clinical use of the first BRAF kinase-targeting inhibitor in subjects carrying the BRAF mutant have revolutionized our views on the tumor-regression effects achievable with a molecular targeting drug on an otherwise rapidly lethal neoplastic condition such as Melanoma. In fact, it has been established that approximately 40– 60% of Malignant Melanoma and 70-80% of Acquired Melanocytic Nevi contain a BRAF mutation, the vast majority of which (86%) result in a single amino acid change at codon 600 (BRAFV600E) [11,12]. The development of the ATP-competitive specific BRAF inhibitor PLX4032 (Vemurafenib) [13] has been instrumental in modifying the progression history of this disease. This, due to the sensitivity to inhibition by vemurafenib conferred by the presence of the V600E mutation on the full length BRAF protein which is translated in the clinical setting with a dramatic reduction in tumor size, and a statistically significant increase in disease free survival. This finding, has provided the rational for BRAF mutant pharmacological targeting in malignant melanomas and other cancer types carrying such mutation. However, an almost immediate finding in the treatment of BRAFV600E mutation-carrying patients using BRAF kinase inhibitor as a monotherapy regimen, was the universal recurrence of the disease (measured by PFS) with a median time to disease recurrence of 6.8 months [14,15]. The mechanistic studies performed in cases undergoing disease progression following BRAF inhibition has shown MAPK pathway reactivation mechanisms in 70% of cases, with PI3K-PTENAKT- upregulating geno-transcriptomic alterations present in 22% of the recurrences [16]. As molecular discriminants of the observed MAPK-pathway reactivation in treated patients, two major studies ([16,17], have found BRAF splicing alterations (13%), gene amplification (19%), kinase duplication (10%, [18]) BRAF gene fusion products secondary to gene structural rearrangements [17], RAS and RAS homologous mutations (NRAS 18%, KRAS 7%), MEK1/2 (3%), CDKN2A(7%) and other quali-quantitative defects at the level of NF1, RB1, P53, ATK1, PTEN, RAC1, ARID2, IDH1 and novel candidate genes (DDX3X, MRPS31 and RPS27) [16,17]. Importantly, among the reported signature mutations in melanoma, BRAFV600E and NRAS(Q61) have been shown to be mutually exclusive in their expression pattern [17,19]. A particular consideration, towards understanding Melanoma and BRAF molecular biology, deserves BRAF dimerization. This is required for normal Ras-dependent RAF activation and is impaired in RAF mutants with moderate, low, or impaired kinase activity [20]. In particular, it has been demonstrated that BRAF homologous dimerization as well as CRAF heterologous dimerization in response to RAS activation are mediated by the BRAF N-terminal domain, and that this is required towards downstream MEK trans-activation under physiological circumstances [21,22]. An additional layer of complexity to this basic axiom is provided by the conditional effect of BRAFV600EBRAF V600E depending on whether the cells expresses its full length V600E mutant protein or the p61 isoform lacking the RAS-binding region. In fact, the full length BRAF mutant, differently from its wild type counterpart, is not able to dimerize, and his MEK activation ability is strongly inhibited by vemurafenib. On the contrary, p61BRAF-V600E (missing exons 4-8 coding for its RAS binding domain) displays enhanced dimerization and high intrinsic catalytic activity in a RAS independent fashion, but dimerization per se does not affect downstream MEK activation and most importantly does not respond to vemurafenib inhibition ([23] and Figure 1). Ultimately, dimerization of the RAF kinases may contribute to several mechanisms that mediate Raf-inhibitor resistance, including mutational activation of N-RAS and K-RAS [2,24], upregulation of receptor tyrosine kinases (RTKs) that drive RAS activation [2], and expression of a V600E-BRAF splice variant with enhanced dimerization/MEK trans-activation potential [23]. Our current knowledge of the effects of BRAF dimerization on Vemurafenib responsivity [25] is graphically summarized below (Figures 1).
Although these studies have provided specific actionable knowledge towards identifying those patients responsive to
pharmacological targeting of BRAF they have also raised additional
challenges. In fact, in spite of the extreme sensitivity of the BRAF
mutant to Vemurafenib block, the very same inhibitor displays
opposite effects on wild-type BRAF as well as in NRAS-bearing
melanomas (not displaying BRAF mutation) in terms of both
dimerization and MEK transactivating activity [23,26]. Also, in
addition to the short PFS (less than 7 months) achieved with
Vemurafenib monotherapy in BRAF mutant-carrying Melanoma
patients, the MAPK hyper-activation mechanisms triggered in
response to Vemurafenib sustained treatment has been directly
linked to the onset of secondary melanomas [27] This had led
to the development of dual targeting strategies in the attempt to
either prevent and/or circumvent the paradoxical hyper-activation
effects on wild type BRAF supported by RTKs up-regulation loops
[28] and further overcome or minimize the observed rapid MAPK pathway reactivation and the mid-long term negative effects linked to
Vemurafenib monotherapy treatment. In this regard, the BRAF/MEK
combined block has been found to bear practical advantages in the
clinical setting over monotherapy [27,29] in which it exerts a small
but statistically significant survival improvement over anti-BRAF
monotherapy in BRAF mutant melanomas ranging 2.4 to 4.1 months
measured as Progression Free Survival (PFS) in phase II randomized
clinical trials [30,31]. In other cases, where the PI3K or relateddownstream pathways activation are involved in vemurafenibacquired resistance, the use of MEK/PI3K or MEK/CDK4/6 combo
therapies have been proposed. Indeed, preclinical studies have
experimentally proven their ability to delay MEK inhibitor resistance
[32]. Furthermore, this dual block for BRAF-downstream and
modulating parallel effectors has been shown to work in 2.6-6.7%
of melanomas carrying BRAF fusion products [33]. One of the most
impactful attempts to functionally categorize Melanoma has come from a multi-dimensional genomic study published in 2015 by the
Cancer Genome Atlas Network [17]. The study was performed
on more than three hundred patient-derived matched specimen
from both primary and metastatic melanomas. Besides confirming
and refining previously identified geno-transcriptomic alterations
and identifying the novel cancer-driver DDX3X (Table I), the study
stands out for the original functional framework provided which
allows to classify all melanomas according to: (a) four main genomic
sub-types based upon their main (cancer) driver defect, and (b)
three transcriptomic sub-classes. Specifically, at the genomic level
melanomas were classified in (1) the BRAF mutant sub-type (52%),
(2) the RAS sub-type (28%), (3) the NF1 sub-type (14%), and
(4) a Triple negative wild-type (6%) with the latter group being
characterized by lack of specific hotspot mutations for BRAF, N/H/KRAS or NF1. In this regard, under the proposed genomic clustering
most hotspot mutations attributable to UV signature (affecting
both cancer drivers coding regions and TERT promoter) were
mostly found in the first three sub-types (BRAF 90.7%, RAS 93.5%
and NF1 92.9%, respectively, against a 30% in the Triple negative
sub-type) while somatic copy number alterations and structural
rearrangements were exclusively found in the triple negative subtype (the fourth group). Additionally, based upon transcriptomic
clustering of 1,500 genes corresponding to the most represented
mRNA transcript variants from 329 sample cases, the study proposes
three novel transcriptomically-defined new functional classes named
after each cluster as: (a) “Immune” type, (b) “Keratin” type, and (c)
“MITF low” type, accounting respectively for: (a) 51%, (b) 31% and
(c) 18%, of the screened sample cases. The clinical significance of
this last classifications bears high practical value, since the immune
transcriptomic sub-class was associated with improved postaccession survival in patients with regional metastatic melanoma.
This was assessed via (1) a novel Lymphocyte Score, LS, and (2) the
associated high levels of the non-receptor tyrosine kinase LCK, which
was also strongly associated to this sub-class with favorable outcome.
Another more recent study analyzed 1000 exomes out of 470 patients
from the cancer genome ATLAS [34]. The study conclusions support
the 2015 study and further deepen the previous findings [17]. Among
these, the authors confirm the role of DDX3X, an X-Chromosome
linked tumor suppressor gene which codifies for an ATPase,-RNA
Helicase protein, as a significant mutated gene (SMG) in melanoma.
Using both (a) transcriptomic signature between the DDX3X mutated
and wild type population and (b) DDX3X binding analysis on
putative target transcripts, they strengthen the link between DDX3X mutations and its Loss of Function (LoF) in melanoma with ~75%
of such mutations attributable to UVR (by NMF analysis). They also
find its LoF to be linked to dysregulation of RAS, PI3K and b-Catenin
pathways in the analyzed melanoma samples and ultimately suggest
that DDX3X loss my play a role in de-differentiation, invasiveness
and reduced proliferation in agreement with a published functional
study [35]. Additional novelties of the study, relate to the finding that
specific members of the SWI/SNF transcriptional modulators family
(ARID2, ARID1A, ARID1B, PBRM1, BRD7) and PRKAR1A, a catalytic
subunit of PKA with tumor suppressor ability, are significantly
enriched in the LoF melanoma population. Finally, the study confirms
the significance of the tumor mutational burden, TMB (higher in
males) with the patient post-accession survival (survival relative
to time of tumor sample procurement) along with UVR signature,
Immune signature, age (at time of sample procurement), tumor site
(with Immune signature, UVR signature and TMB carrying the best
predictors of overall survival).
Figure 1: Effects of BRAF dimerization of Vemurafenib block
(modified from Molina-Arcas M et al. [25])
An intense and specific tumorigenic stage-dependent and
sequentially occurring cross-talk among the stromal elements, the
melanoma cells, and immune system cellular elements in the tumor
microenvironment of melanoma has emerged in the last decade.
Among the evidences supporting this view, it has been demonstrated
that melanoma cells carrying BRAFV600E mutation activate stromal
fibroblasts and enhance tumorigenicity through secretion of MMP1 [36]. A study has linked the secretion of active matrix metalloproteinases by CAFs to the decreased NK mediated oncolytic targeting
of melanoma cells via downmodulation of the NKG2D ligands MICA/B [37]. Among the membrane tyrosine kinase receptors and their
autocrine and paracrine networks modulating melanoma tumor
microenvironment, Notch1 autocrine stimulation and signaling
in CAFs [38] has been shown to suppress aggressive and stem-like
features in melanoma cells [39,40]. Furthermore, CAF-expressed
Neuregulin-1 has been found to promote vemurafenib-treated/BRAF
mutated melanoma cells proliferation via paracrine stimulation of
ErbB3/ErbB2 signaling; this effect was reversed by ErbB3 antibodymediated neutralization [41-43]. Interestingly, PEDF (also known as
SerpinF1) which has been shown to be a tumor suppressive factor in
normal fibroblasts, is downregulated by melanoma-secreted PDGFBB and TGF-β in order to favor CAF conversion [44]. A similar stromal
conversion effect to CAF feature is exerted by Nodal, a cancerexpressed TGF superfamily member, along with activation of the TGF-β
- Snail signaling axis in fibroblasts [45]. The immunosuppressive
role and targetable value of TGF-β in the tumor microenvironment
of Malignant Melanoma has been further strengthened by the
observation that TGF-β inhibits tumor infiltration of CD8+ T cells and
their oncolytic effect in vivo. This, through inhibition of their CXCR3
expression which impairs their migration towards melanoma cells
expressing CXCL10 [46]. More recently, a study has shown that loss of
HAPLN1, a secreted proteoglycan component of the ECM which is lost
in aged fibroblasts, is a permissive event in melanoma metastasis.
Notably, its experimental reconstitution selectively stimulates the
motility of mononuclear immune cells, ultimately restoring the
immune microenvironment [47]. A schematics of the relationship
between the key cellular components affecting malignant melanoma
response to current therapies (including CAFs) is provided in Figure
2 below. The findings linking such mechanisms to drug-resistance in
melanoma are summarized in Table I.
Table 1: Genes and related gene products involved in MM growth, molecular target- and ICI- drugs resistance and post-treatment survival
Although the idea of triggering the immune system against
tumor cells with the use of traditional vaccination is not new, the
failure of those initial strategies has provided biological answers
and immediate alternatives with the discovery of the role of T-Cell
receptor, co-receptors and associated modulating molecules in
the context of processed antigen presentation by macrophages. In
fact, the possibility to guide an endogenous effective anti-tumoral
immune response strongly relies on the functionality of these T-Cell
membrane molecules and their ligands counterparts expressed in the
targeted antigen-carrying cancer cell. The T-cell gene products that
have been proven crucial to step-up immunotherapies in the last few
decades include the CTLA-4/B7 system [48,49] and the PD-1/PD-L1
coreceptor/ ligand system [50] with PD-L1 being co-expressed with
the antigen carrying target cells. Indeed, the expression of PD-L1 in
cancer has been associated to a worse outcome in agreement with its
role in tumor immune-evasion [51]. The success of the animal studies
with the first human anti-CTLA-4 monoclonal antibody (Ipilimumab)
and the following phase II and III clinical trials displaying greater than
20% responsivity in malignant melanomas (independently on BRAF
status) with more than 10 years survival in some patients [52] have
shown the potential of the “immune checkpoint” therapies in a deadly
cancer such as malignant melanoma, for which the survival achieved
using single or combined targeted BRAF therapy is still below one
year. The following demonstration of the additive effect between
CTLA- 4 and PD-1 blocking combinations in melanoma patients with
>50% response and >80% tumor regression, has further established
the effectiveness of immune checkpoint strategy in the treatment
of Malignant Melanoma [53-55]. Additional TCR response comodulating factors have been identified such as LAG3, TIM3, TIGIT,
VISTA, ICOS, OX40, GITR, 4-IBB, CD40, CD27 and their cellular ligands
[56]. The clarification of their exact role in cancer immune-evasion
in the micro-environmental cellular context will likely provide valid
novel strategies to fine tune future immune-therapy in malignant
melanoma and other tumor types. In this context, is not surprising
that Immune signature in the two largest cohort studies available to
date [17,34] has demonstrated a predictive value which is consensual
to the one shown by the overall Tumor Mutation Burden (TMB)
towards predicting patient survival, therefore suggesting immune
signature profiling to be a parameter to be taken in consideration
both in translational research and clinical setting for melanoma.
The findings disclosing mechanisms causing checkpoint inhibitors
resistance in melanoma are summarized in Table I, and a graphic
summary of essential key infiltrating T-Cells interactions in the
melanoma tumor microenvironment is also provided in Figure 2.