Ci a ion: Sánchez-Molina, S.;
Figue ola-Bou, E.; Sánchez-Ma gale ,
V.; de la C uz-Me ino, L.; Mo a, J.; de
Ála a Casado, E.; Ga cía-Domínguez,
D.J.; Hon ecillas-P ie o, L. Ewing
Sa coma Mee s Epigene ics,
Immunology and Nanomedicine:
Mo ing Fo wa d in o No el
The apeu ic S a egies. Cance s 2022,
14, 5473. h ps://doi.o g/10.3390/
cance s14215473
Academic Edi o s: S e an Bu dach,
U a Di ksen, Poul H. So ensen and
Ve ecchia F anck
Recei ed: 26 Sep embe 2022
Accep ed: 3 No embe 2022
Published: 7 No embe 2022
Publishe ’s No e: MDPI s ays neu al
wi h ega d o ju isdic ional claims in
published maps and ins i u ional a il-
ia ions.
Copy igh : © 2022 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
This a icle is an open access a icle
dis ibu ed unde he e ms and
condi ions o he C ea i e Commons
A ibu ion (CC BY) license (h ps://
c ea i ecommons.o g/licenses/by/
4.0/).
cance s
Re iew
Ewing Sa coma Mee s Epigene ics, Immunology and
Nanomedicine: Mo ing Fo wa d in o No el
The apeu ic S a egies
Sa a Sánchez-Molina 1,2,† , Elisabe Figue ola-Bou 1,2,† , Víc o Sánchez-Ma gale 3,
Luis de la C uz-Me ino 4, Jaume Mo a 1,2 , En ique de Ála a Casado 5,6,7,* ,
Daniel JoséGa cía-Domínguez 3,4,‡ and Lou des Hon ecillas-P ie o 3,4,*,‡
1De elopmen al Tumo Biology Labo a o y, Ins i u de Rece ca San Joan de Déu, Hospi al San Joan de Déu,
Esplugues de Llob ega , 08950 Ba celona, Spain
2
Pedia ic Cance Cen e Ba celona, Hospi al San Joan de Déu, Esplugues de Llob ega , 08950 Ba celona, Spain
3
Clinical Labo a o y, Depa men o Medical Biochemis y and Molecula Biology, School o Medicine, Vi gen
Maca ena Uni e si y Hospi al, Uni e si y o Se ille, 41009 Se ille, Spain
4Oncology Se ice, Depa men o Medicines, School o Medicine, Vi gen Maca ena Uni e si y Hospi al,
Uni e si y o Se ille, 41009 Se ille, Spain
5Ins i u e o Biomedicine o Se ille (IBiS), Hospi al Uni e si a io Vi gen del Rocío/CSIC/Uni e si y o
Se ille/CIBERONC, 41013 Se ille, Spain
6Pa hology Uni , Hospi al Uni e si a io Vi gen del Rocío/CSIC/Uni e si y o Se ille/CIBERONC,
41013 Se ille, Spain
7Depa men o No mal and Pa hological Cy ology and His ology, School o Medicine, Uni e si y o Se ille,
41009 Se ille, Spain
*Co espondence: [email p o ec ed] (E.d.Á.C.); [email p o ec ed] (L.H.-P.)
† These au ho s con ibu ed equally o his wo k as he i s au ho s.
‡ These au ho s con ibu ed equally o his wo k as he las au ho s.
Simple Summa y:
Ewing Sa coma ea men is adi ionally based on chemo he apy, su ge y, and
adio he apy. Al hough hese s anda d o ca e egimens a e e icien a ea ly disease s ages, many
pa ien s ail o espond app op ia ely, which has p omp ed he sea ch o mo e e icacious and speci ic
ea men s. A deepe unde s anding o he basic molecula mechanisms unde lying he biology o
bo h umo cells and he umo mic oen i onmen , as well as ad ances in d ug deli e y, has led
o he de elopmen o di e en app oaches o imp o e he ea men in Ewing Sa coma pa ien s.
Thus, epigene ic, and immuno he apy-based d ugs, along wi h nano echnology deli e y s a egies,
ep esen no el p eclinical and clinical s udies in he ea men o Ewing Sa coma. In his e iew,
we p o ide a comp ehensi e o e iew o hese eme ging he apeu ic s a egies and summa ize he
po en ial o he la es p eclinical and clinical ials in Ewing Sa coma esea ch. Finally, we unde line
he alue and u u e di ec ions o hese new ea men s.
Abs ac :
Ewing Sa coma (EWS) is an agg essi e bone and so issue umo ha mainly a ec s
child en, adolescen s, and young adul s. The s anda d he apy, including chemo he apy, su ge y,
and adio he apy, has subs an ially imp o ed he su i al o EWS pa ien s wi h localized disease.
Un o una ely, his mul imodal ea men emains elusi e in clinics o hose pa ien s wi h ecu en
o me as a ic disease who ha e an un a o able p ognosis. Consis en ly, he e is an u gen need o
ind new s a egies o pa ien s ha ail o espond o s anda d he apies. In his ega d, in he las
decade, ea men s a ge ing epigene ic dependencies in umo cells and he immune sys em ha e
eme ged in o he clinical scena io. Addi ionally, ecen ad ances in nanomedicine p o ide no el
deli e y d ug sys ems, which may add ess challenges such as side e ec s and oxici y. The e o e,
he apeu ic s a egies s emming om epigene ics, immunology, and nanomedicine yield p omising
al e na i es o ea ing hese pa ien s. In his e iew, we highligh he mos ele an EWS p eclinical
and clinical s udies in epigene ics, immuno he apy, and nano he apy conduc ed in he las i e yea s.
Cance s 2022,14, 5473. h ps://doi.o g/10.3390/cance s14215473 h ps://www.mdpi.com/jou nal/cance s
Cance s 2022,14, 5473 2 o 28
Keywo ds: Ewing Sa coma; epigene ic; immuno he apy; nano he apy
1. In oduc ion
Ewing Sa coma (EWS) is a a e and highly agg essi e bone and so issue umo ha
a ec s child en, adolescen s, and young adul s wi h a peak o incidence in he second
decade o li e. The p ognosis o EWS has imp o ed conside ably, wi h cu en mul imodal
he apy including chemo he apy, su ge y, and adia ion, wi h a 65–70% cu e a e o
localized disease. Howe e , olde pa ien s (>18 yea s), me as a ic pa ien s a diagnosis,
and pa ien s wi h ea ly elapsing umo s s ill ha e a poo p ognosis, wi h a 5-yea su i al
a e o less han 30% [
1
,
2
]. The e o e, he highe he apeu ic challenge emains on how o
con ol he sys emic disease and imp o e he su i al a es, especially in hose pa ien s
wi h wo se p ognosis.
EWS umo cells a e cha ac e ized by a usion gene in ol ing one membe o he FET
amily o genes and one o he ETS amily o ansc ip ion ac o s, EWSR1-FLI1 being he
mos common [
1
,
3
]. Fusion genes ha e been demons a ed o be essen ial o umo igenesis
and, he e o e, a e a ac i e he apeu ic a ge s ha can be add essed h ough di ec and
indi ec molecula a ge ed app oaches [
4
]. Ne e heless, he lack o speci ic enzyma ic
ac i i y o EWSR1-FLI1 challenges a di ec a ge ed pha macological inhibi ion. Mo eo e ,
indi ec inhibi ion o oncogene ac i i y by he pe u ba ion o downs eam a ge s, al hough
i has p esen ed success ul in eg a ion in p eclinical models, emains elusi e in clinics [5].
Ad ances in he molecula mechanisms unde lying he epigene ic emodeling o
ch oma in media ed by he usion oncogene and he immune sys em ha e led o he
de elopmen o no el he apeu ic app oaches. Epigene ic changes d i en by EWSR1-FLI1
ha e been epo ed in he umo igenesis o EWS. Indeed, EWSR1-FLI1 ewi es ch oma in
and ep og ams gene exp ession causing bo h induc ion and ep ession o selec ed gene
pa hways [
6
–
8
]. The e o e, epigene ic-based ea men s p o ide a p ominen op ion o
ea ing his agg essi e umo by e e sing he e ec in he epigenome induced by he
usion gene. Mo eo e , based on he expe ience gained om adul cance , immuno he apy
s udies ha e been ansla ed o pedia ic umo s including EWS.
The e is a p essing equi emen o de elop a ge ed he apies o d ug ca ie s ha can
deli e he apeu ic agen s wi h highe e iciency o lowe he dosage needed and minimize
side e ec s. On his basis, nano echnology plays a p ominen ole in mode n medicine,
by po en ially o e coming he de iciencies o con en ional me hods o adminis e ing
chemo he apy and ul ima ely imp o ing clinical ou comes [9].
In his a icle, we will e ise he ongoing p eclinical and clinical s udies o he las i e
yea s ocusing on epigene ics, immuno he apy and nano he apy in EWS.
2. Epigene ic and Immuno he apy-Based T ea men s in EWS: Mo ing Fo wa d in
Ta ge ed The apies
The ul ima e knowledge o he basic aspec s o he epigene ics and immuno he apy
o cance has made signi ican s ides, leading o he de elopmen o a wide a ie y o
new he apeu ic agen s. He e, we summa ize he newes epigene ic and immune-based
ea men s in EWS.
2.1. Epigene ic The apy
Epigene ics encompasses he e e sible molecula p ocesses a ec ing ch oma in ha
de ine cellula iden i y by main aining on and o s a es o ansc ip ion wi hou al e a ions
in he DNA sequence. Upon sequencing s udies, di e en g oups epo ed EWS as a
umo wi h pauci y in he mu a ional a e, implica ing epigene ics behind EWSR1-FLI1
as a umo igenic ac o [
10
]. As a esul , many publica ions ha e shed ligh on he ole o
he EWS epigenome bo h in he unde s anding o he molecula mechanisms in ol ed in
Cance s 2022,14, 5473 3 o 28
umo de elopmen and in he iden i ica ion o no el a ge s o new and combina ional
he apies [11].
Epigene ics is c i ical o induce he p ope en i onmen o EWSR1-FLI1 es ablishmen ,
as cells wi h highe plas ici y will p o ide mo e signi ican oppo uni ies o ep og am-
ming by he oncogene [
12
,
13
]. Besides, he oncogene ha in e ac s di ec ly wi h DNA
p esen s sca olding p ope ies ha media e p o ein–p o ein in e ac ions wi h impo an
epigene ic egula o s o ch oma in s uc u e, ewi ing he comple e epigenome and, ul i-
ma ely, hei exp ession p og ams [
14
]. EWSR1-FLI1 beha es as a pionee ac o by di ec ly
ec ui ing ch oma in emodele s o GGAA mic osa elli es, whe e i induces he o ma ion
o de no o ac i e supe -enhance s in egions ha we e p e iously ep essed [
11
]. Finally,
he ep essi e ole o he oncogene is desc ibed by i s capabili y o displace endogenous
ansc ip ion ac o s [
7
]. Unde s anding he epigene ic mechanisms ha pe mi cance cells
o quickly adap , and hei e e sibili y, he e o e, cons i u es a g ea oppo uni y o he
de elopmen o new s a egies o ea cance [
15
]. The ollowing sec ions will ocus on
hose epigene ic d ugs ha can be ansla ed in o he clinics, which include a ge ing DNA
me hyla ion, nucleosome emodele s, his one pos - ansla ional modi ica ions and hei
modi ie s (Table 1).
Table 1.
Summa izing he open clinical ials (las 5 yea s) a ge ing epigene ic ac o s. Sou ce:
ClinicalT ials.go (accessed on 1 Sep embe 2022).
Molecula
Mechanism
Molecula
Ta ge D ug Clinical T ial
Iden i ie Pa ien s Phase S a us/Re
DNA
me hyla ion IDH I odesinib NCT04195555
Ad anced Solid
Tumo s, Lymphoma, o
His iocy ic diso de s
wi h IDH1 mu a ions
II Rec ui ing
Nucleosome
emodeling LSD1/NURD
Seclidems a
+ opo ecan and
cyclophos-
phamide
NCT03600649
Ewing Sa coma (EWS);
Myxoid Liposa coma;
Sa comas wi h
FET- amily
ansloca ion
I Rec ui ing
Seclidems a NCT05266196
EWS; Myxoid
Liposa coma;
Desmoplas ic Small
Round Cell Tumo ;
Ex askele al Myxoid
Chond osa coma;
Angioma oid Fib ous
His iocy oma; Clea
Cell Sa coma;
Myoepi helial Tumo ;
Low G ade
Fib omyxoid Sa coma;
Scle osing Epi helioid
Fib osa coma
I/II En olling
INCB059872 NCT03514407 Re ac o y o elapsed
EWS Ib Te mina ed
INCB059872 NCT02712905
Solid Tumo s and
Hema ologic
Malignancy
I/II Te mina ed
Cance s 2022,14, 5473 4 o 28
Table 1. Con .
Molecula
Mechanism
Molecula
Ta ge D ug Clinical T ial
Iden i ie Pa ien s Phase S a us/Re
SWI/
SNF
T abec edin +
adia ion NCT05131386
Os eosa coma;
Chond osa coma;
EWS;
Rhabdomyosa coma;
Desmoplas ic Small
Round Cell Tumo
II Rec ui ing
T abec edin +
i ino ecan NCT04067115 EWS I Rec ui ing
Lu binec edin
wi h o wi hou
i ino ecan
NCT05042934 Me as a ic and
ecu en EWS I/II Wi hd awn
Lu binec edin +
i ino ecan NCT02611024
Ad anced Solid
Tumo s; Glioblas oma;
So Tissue Sa coma
(Excluding GIST)
Endome ial
Ca cinoma;
Epi helial O a ian;
Ca cinoma;
Meso helioma; Gas-
oen e opanc ea ic
Neu oendoc ine
Tumo ;
SCLC; Gas ic
Ca cinoma;
Panc ea ic
Adenoca cinoma;
Colo ec al Ca cinoma;
Neu oendoc ine
Tumo s
I/II Rec ui ing
His one w i e EZH2 Tazeme os a NCT03213665
Relapsed o e ac o y:
B ain umo s; Solid
Tumo s; non-Hodgkin
Lymphoma; his iocy ic
diso de s wi h EZH2,
SMARCB1, o
SMARCA4 gene
mu a ions
II Ac i e, no
ec ui ing
His one e ase HDAC Vo inos a +
chemo he apy NCT04308330
EWS;
Rhabdomyosa coma;
Wilms Tumo ;
Neu oblas oma;
Hepa oblas oma; Ge m
Cell Tumo
I Rec ui ing
His one eade BET
BMS-986158
and
BMS-986378
NCT03936465 Pedia ic Cance I Rec ui ing
2.1.1. DNA Me hyla ion
DNA me hyla ion a cy osine (5-me hylcy osine, 5mC) is an essen ial p ocess in em-
b yonic de elopmen and cell di e en ia ion [
16
]. Dis up ion o he DNA me hyla ion
pa e n is a common ai o di e en cance s, including EWS, whe e hype me hyla ion o
key genes co ela es wi h mo e agg essi e beha io and hypome hyla ion was epo ed in
ac i e enhance s [
17
,
18
]. DNA me hyl ans e ases (DNMT) and en-ele en ansloca ion
Cance s 2022,14, 5473 5 o 28
(TET) me hylcy osine dioxygenases, esponsible o DNA deme hyla ion, ha e been majo
a ge s o epigene ic d ug de elopmen . Despi e hei high e iciency, DNMT inhibi o s
(DNMTi), such as azaci idine and deci abine, p esen ed oxici y in phase I clinical ials
and low doses in combina ion wi h o he agen s we e u he es ed [
19
]. Recen ly, he
no el non-nucleoside DNMTi MC3343 has been desc ibed o induce a speci ic deple ion o
DNMT1 ha induces DNA damage wi hou al e a ions in DNA me hyla ion [20].
Besides, non-epigene ic d ugs we e epo ed o a ec TET enzymes and his one
deme hylases. Mu a ions ha dis up isoci a e dehyd ogenase IDH1/2 enzyma ic unc-
ion p oduce a educ ion in
α
-ke oglu a a e (
α
KG) and an inc ease in he oncome aboli e
2-hyd oxyglu a a e (2HG). In pa icula , 2HG inhibi s TET enzymes esul ing in DNA
hype me hyla ion; hus, d ugs inhibi ing mu an IDH1/2 eac i a e
α
KG and es o e
me hyla ion le els [
6
]. On his basis, i odesinib, an inhibi o o mu a ed IDH1, is ac u-
ally in phase II clinical ial o e ac o y and ecu en pedia ic solid umo s including
EWS (NCT04195555).
2.1.2. Nucleosome Remodeling
Nucleosome emodeling e e s o he ATP-dependen mul ip o ein complexes ha
a ec nucleosome posi ioning and s uc u e, in luencing ansc ip ion egula ion. Among
hese complexes, EWSR1-FLI1 ec ui s he nucleosome emodeling and deace ylase (NuRD)
complex. This complex con ains his one deace ylases (HDAC), lysine speci ic deme hylase
1 (LSD1) and ch omodomain-helicase-DNA-binding p o ein 3/4 (CHD3/4) and di ec ly
binds o EWSR1-FLI1 p omo ing ansc ip ional ep ession in EWS [
21
]. The inhibi ion o
LSD1 wi h he non-compe i i e e e sible LSD1 inhibi o s HCI-2509 and HCI-2528 was
e ec i e in a ge ing EWS cell lines, while hei e iciency was dependen on EWSR1-FLI1
exp ession [
21
]. HCI-2509 delayed umo g ow h in mono he apy [
22
] and i s e iciency
was no al e ed by he p e ious inhibi ion o EWS cell lines wi h he i e e sible inhibi o
GSK-LSD1, sugges ing ha HCI-2509 dis up s he LSD1 in e ac ion wi h EWSR1-FLI1 [
23
].
Ne e heless, he la es s udies ha e epo ed LSD1 colocaliza ion a EWSR1-FLI1 ac i e
supe -enhance s, co ela ing wi h HCI-2509 dis up ion no only o ep ession bu also gene
ac i a ion [
24
]. SP-2577 (seclidems a ), ano he LSD1 inhibi o , inhibi ed he g ow h o
h ee ou o eigh EWS xenog a models [
25
]. A p esen , he e a e ou clinical ials: (i) a
phase I e alua ing he sa e y-dose escala ion and expansion o seclidems a wi h opo ecan
and cyclophosphamide in pa ien s wi h elapsed o e ac o y EWS (NCT03600649); (ii) a
phase I/II as a con inua ion o a p e ious one, which allows he pa ien con inued access
o he d ug (NCT05266196); (iii) a phase I s udy e alua ing he sa e y and p elimina y
an i umo ac i i y o INCB059872, ano he selec i e and o al LDS1 inhibi o , in e ac o y
o elapsed EWS pa ien s (NCT03514407); and (i ) a dose-escala ion and dose-expansion
s udy o INCB059872 in ad anced solid malignancies including EWS (NCT02712905).
Among a panel o pedia ic sa coma cell lines, EWS cells we e he mos sensi i e
o abec edin, an an i umo d ug de i ed om he sea squi ha binds o he mino
g oo e o DNA, e e sing he gene signa u e o EWSR1-FLI1 by in e e ence wi h i s an-
sc ip ion ac o ac i i y [
26
]. EWSR1-FLI1 can also ec ui he mammalian swi ch/suc ose
non- e men ing (SWI/SNF) nucleosome emodele o enhance s con aining GGAA mi-
c osa elli es acili a ing ch oma in opening and ac i a ion o EWSR1-FLI1- a ge s [
27
].
La e s udies demons a ed ha abec edin e ic ed he SWI/SNF complex om ch oma in
and edis ibu ed EWSR1-FLI1 wi hin he nucleus, dis up ing i s unc ion as a pionee
ac o [
28
]. Al hough he phase I clinical ial in child en wi h e ac o y solid umo s
concluded ha abec edin was sa e, a phase II s udy was unsuccess ul [
29
,
30
]. A new
phase II clinical ial combines abec edin wi h adia ion in ad anced and me as a ic EWS
(NCT05131386), and ano he h ee e alua e he combina ion o abec edin o i s de i a-
i e lu binec edin wi h i ino ecan based on hei syne gy (NCT04067115, NCT05042934,
NCT02611024 and [31]).
Cance s 2022,14, 5473 6 o 28
2.1.3. His one Modi ica ions and Modi ie s
His one ails unde go a a ie y o pos - ansla ional co alen modi ica ions ha a ec
hei in e ac ion wi h DNA. The di e en his one modi ica ions cons i u e a code whe e
syne gis ic o an agonis ic in e ac ions de e mine ch oma in accessibili y o ansc ip ion
ac o s and ul ima ely ansc ip ion ac i a ion o ep ession [32]. The enzyma ic ac i i ies
behind his his one code in ol e w i e s ha se le hese modi ica ions (including his one
ace yl ans e ases (HAT) o his one me hyl ans e ases (HMT)), e ase s, which elimina e
hem (including his one deme hylases (HDM) o HDAC), and inally, eade s ha ecognize
and media e an epigene ic signal.
His one W i e s: Polycomb G oup and G9a Me hyl ans e ase
The polycomb g oup (PcG) p o eins seg ega e in wo ansc ip ional ep essi e com-
plexes, PRC1 and PRC2. PRC1 con ains he E3 ubiqui in ligase enzyme RING1A o RING1B,
while PRC2 consis s o HMT ac i i y om EZH1 o EZH2. Despi e he ep essi e ole
o PRC1, RING1B has been desc ibed o be a ansc ip ional ac i a o in a ious cance
en i ies [
33
,
34
]. In EWS, RING1B is highly exp essed and is necessa y o he exp ession
o c i ical EWSR1-FLI1 a ge s by acili a ing oncogene ec ui men o ac i e enhance s.
Inhibi ion o au o a kinase (AURK) B by AZD1152 has been p oposed as an excellen
s a egy o impai RING1B ac i i y a ac i e enhance s [
35
]. Mo eo e , EWS cells we e
highly sensi i e o bo h AURKA and B inhibi o s and hei combina ion wi h ocal adhesion
kinase (FAK) inhibi o s educed he umo g ow h in EWS mouse models [36].
The PRC2 subuni EZH2 is o e exp essed in EWS and i s knockdown inhibi ed u-
mo g ow h and me as asis
in i o
[
37
,
38
]. Consequen ly, di e en EZH2 inhibi o s ha e
been e alua ed in EWS in o de o a ge PCR2 ac i i y, such as he non-speci ic inhibi o
3-deazaneplanocin A (DZNep) and he speci ic inhibi o azeme os a . DZNep ea men
p oduced a cell cycle a es
in i o
and g ow h supp ession in EWS mice [
39
]. The ole a-
bili y o azeme os a is being e alua ed in a phase II clinical ial in pedia ic pa ien s wi h
gain o unc ion mu a ions o EZH2 including EWS (NCT03213665). Ne e heless, azeme-
os a showed no ac i i y in ou xenog a models o EWS [
40
]. Besides, EZH2 inhibi o s
combined wi h immuno he apy migh o e a new he apeu ic oppo uni y. I has been
obse ed ha GSK126, ano he selec i e EZH2 inhibi o , as well as azeme os a , enhance
he su ace exp ession o disialoganglioside (GD2) in EWS cell lines, which sensi izes EWS
cells o cy olysis by GD2-speci ic chime ic an igen ecep o (CAR) T-cell immune he apy
(see nex chap e ) [41].
Finally, G9a, an HMT ha dime hyla es H3K9, has been ound o be o e exp essed
in di e en cance ypes. Speci ically, i s o e exp ession in EWS co ela ed wi h poo
p ognosis and me as asis [
42
]. Indeed, he G9a inhibi ion wi h BIX01294 was p o ed
e ec i e in dis up ing mig a ion, in asion, adhesion, colony o ma ion, and asculogenic
mimic y ia he up egula ion o NEU1. Dec ease in me as asis and umo g ow h wi h
BIX01294 was p o en in wo in i o models o EWS me as asis [42].
His one E ase s: Deace ylases and Deme hylases
HDAC an agonize he enzyma ic ac i i y o HAT by emo ing his one ace yla ion.
EWSR1-FLI1 was shown o globally ep ess HAT ac i i y while s imula ing HDAC [
43
].
Consis en ly, se e al HDAC inhibi o s (HDACi) we e sc eened in EWS, including FK228
( omidepsin) and MS-275 (en inos a ), which p esen ed an i umo ac i i y
in i o
and
in i o
in EWS, as well as o inos a (SAHA) and sodium bu y a e (NaB) [
43
–
45
]. Lessnick
e al., showed ha bo h o inos a and deple ion o HDAC2/3 e e sed exp ession pa e ns
o EWSR1-FLI1- ep essed a ge s, indica ing ha he oncogene elies on HDAC o i s
ep essi e ole in ansc ip ion [
21
]. Ne e heless, he i s ini ial p eclinical es ing o
o inos a e ie ed no objec i e esponses o any o he solid umo s es ed, including
EWS [
46
]. Besides, en inos a , a selec i e HDAC1 and HDAC3 inhibi o , signi ican ly
educed umo bu den and inc eased su i al in p eclinical xenog a models inducing cell
Cance s 2022,14, 5473 7 o 28
cycle a es and apop osis. Howe e , only he knockdown o HDAC3 was c i ical o EWS
su i al [47].
Fu he s udies ha e e ealed he po en ial o HDACi in combina ion wi h o he d ugs.
A sc eening o 43 epigene ic d ugs e ealed ha he mos sensi i e agen s in EWS cell lines
we e ela ed o HDAC inhibi ion, being BML-281, a speci ic inhibi o o HDAC6, he d ug
wi h he lowe IC50. BML-281 inc eased ace yla ion le els o speci ici y p o ein 1 (SP1),
educing i s binding o he EWSR1-FLI1 p omo e and causing ep ession o he oncogene
and i s associa ed a ge s [
48
]. Fu he mo e, he combina ion o he HDAC6 inhibi o
ACY-1215 wi h doxo ubicin educed umo g ow h in EWS xenog a s [
48
]. On he o he
hand, HDAC1 and HDAC2 knockou s demons a ed a educ ion in in asi eness and umo
g ow h in xenog a s [
49
]. Since he e ec in umo g ow h esembled EZH2 inhibi ion [
37
],
he HDACi omidepsin was combined wi h he emb yonic ec ode m de elopmen (EED)
inhibi o (A-395), which inac i a es he PRC2 complex. This combina ion ea men was
supe io o mono he apy blocking he p oli e a ion and umo g ow h o SK-N-MC o EW7
xenog a models [
49
]. In addi ion, he combina ion o SAHA wi h HCI-2509 dec eased cell
p oli e a ion, igge ing cell cycle a es and apop osis, educing EWSR1-FLI1 exp ession
by egula ion o he EWSR1 p omo e and al e ing umo g ow h [
50
]. Along he same
line, he combina ion o omidepsin wi h HCI-2509 has also p o ed o be syne gis ic [
51
].
Cu en ly, a phase I clinical ial combining o inos a wi h chemo he apy in e ac o y o
elapsed solid umo s is open (NCT04308330). In e es ingly, HDACi could be chemically
modi ied o ha e a second pha macopho e, like imepinos a , which is a hyb id inhibi o
o phospha idylinosi ol 3-kinase (PI3K) and HDACs. This d ug no only educed EWSR1-
FLI1 p o ein by a ec ing i s s abili y bu also cell iabili y and umo g ow h in sa coma
xenog a models [52].
Rega ding his one deme hyla ion, he Jumonji-domain HDM KDM3B deme hyla es
H3K9me2, and has been desc ibed as a no el oncogene downs eam o EWSR1-FLI1 [
53
].
KDM3B and i s di ec a ge , he cell adhesion molecule MCAM, we e posi i ely implica ed
in cell mig a ion and in asion, and hei knockou educed me as asis
in i o
[
54
]. Indeed,
EWS cell lines we e sensi i e o he pan-selec i e Jumonji HDM inhibi o JIB-04, which in-
c eased me hyla ion le els o H3K4me3, H3K9me2, and H3K27me3 and a ec ed he whole
EWSR1-FLI1 ansc ip ome. JIB-04 induced DNA damage ia CDKN1A and dec eased
umo g ow h in xenog a models [
55
]. Besides, a d ug sc eening e ealed ha EWS cell
lines we e sensi i e o he H3K27me3 deme hylase inhibi o GSK-J4. This d ug sensi ized
EWS cell lines o chemo he apy and syne gized
in i o
wi h he cyclin-dependen kinase
(CDK) 7/12/13 inhibi o THZ1 [
56
]. Ne e heless, hese new epigene ic d ugs ha e no ye
eached in o he clinics.
His one Reade s: B omodomains
The b omodomain and ex a- e minal (BET) amily consis s o ou conse ed mam-
malian membe s (BRD con aining 2 (BRD2), BRD3, BRD4, and BRDT) ha in e ac h ough
b omodomains wi h ace yla ed lysine esidues [
57
,
58
]. The i s BET inhibi o desc ibed
was JQ1, a molecule ha compe i i ely binds o b omodomains, p e en ing he in e ac-
ion be ween BET p o eins and ace yla ed his ones. In EWS, bo h JQ1 and deple ed BRD
p o eins supp essed he EWSR1-FLI1 gene signa u e. Besides, JQ1 comp omised cell p oli -
e a ion, angiogenesis, and umo g ow h in EWS xenog a models [
59
,
60
]. BMS-986158 and
BMS-986378, ano he wo BET inhibi o s, ha e now en e ed clinical ials as in es iga ional
d ugs o e alua ing hei e icacy o pedia ic b ain and solid umo s (NCT03936465).
2.2. Immuno he apy
Immuno he apy is a ea men ha boos s he immune sys em esponse agains cance
o blocks any mechanism ha p e en s an i umo immuni y. The local umo mic oen i-
onmen (TME) and he hos immune sys em de ine he umo immunopheno ype, which
is gene ally di ided in o ho and cold umo s. Whe eas ho umo s esemble an immune-
in lamed pheno ype cha ac e ized by in il a ion o T lymphocy es, cold umo s p esen
Cance s 2022,14, 5473 8 o 28
an immune-dese o immune-excluded pheno ype wi h he absence o exclusion o T-
cells [
61
]. EWS exempli ies an immune cold umo wi h e y poo in il a ion o immune
cells o in lamma o y in il a es due o immune escape, immune p i ilege, o immune
inhibi ion by he TME. Tumo cells esemble a de icien exp ession o human leukocy e
an igens (HLA) ha p e en s ecogni ion o umo -associa ed an igens by e ec o T-cells
and an igen p esen ing cells. Consis en ly, sel o umo - eac i e T-cells ex ac ed om EWS
pa ien s show an exhaus ed pheno ype ha ailed o ac i a e despi e he p esence o high
doses o an igen [
62
]. In he same lines, immune-inhibi o y ligands, such as HLA-G we e
ound locally exp essed on umo cells and on in il a ing lymphocy es, which p omo e
di ec inhibi ion o he immune esponse by na u al kille (NK) cells as well as he induc ion
and expansion o myeloid-de i ed supp esso cells (MDSCs) [
63
,
64
]. Besides, la ge popula-
ions o MDSCs we e shown o inhibi EWS immune esponses o he apy [
65
]. While a
be e unde s anding o he in e play be ween EWS and TME is being de eloped, no el
immuno he apy s a egies a e ocused on inc easing he numbe o T-cells d i ing hem
in o he umo and e e sing he immunosupp essi e TME [
66
]. These he apies include
immune checkpoin inhibi o s, adop i e cell he apy, an ibody-based immuno he apy, and
cance accines, which a e add essed below (Figu e 1).
Figu e 1.
Immune he apies cu en ly explo ed in EWS. (
A
) Immune checkpoin inhibi o s block
he in e ac ion o immune checkpoin molecules (e.g., PD-1 o CTLA-4) wi h i s inhibi o y ligands
o s imula e he immune esponse. (
B
) Adop i e cell he apy in ol es he in usion o modi ied
au ologous T-cells o allogenic NK cells. T-cells can be gene ically modi ied o exp ess a chime ic
an igen ecep o (CAR) speci ic o a umo -associa ed an igen (e.g., EGFR) ha can be ecognized
by majo his ocompa ibili y complex (MHC)-independen mechanisms. In con as , T-cells isola ed
om umo s can be s imula ed wi h an oncoly ic pep ide (e.g., LTX-315) and ein usioned back
o media e an an i umo al MHC-dependen esponse. T ans e o NK cells om heal hy dono s
is based on he inna e abili y o NK cells o kill umo cells h ough a ious mechanisms such as
g anzyme B elease. (
C
) An ibody-based he apies in ol e he use o speci ic an ibodies a ge ing
umo -associa ed an igens (e.g., GD2). (
D
) Cance accines s imula e he immune sys em esponse
o he hos h ough a ious mechanisms. The VIGIL accine in EWS is based on he umo cells
enginee ed o exp ess GM-CSF and a bi unc ional shRNA ha p e en s immunosupp ession by
TGF
β
1-2 elease. Rein usion o hese umo cells, hus, p omo es an igen-p esen a ion and he
adap i e immune esponse.
Cance s 2022,14, 5473 9 o 28
2.2.1. Immune Checkpoin Inhibi o s
Immune checkpoin molecules a e inhibi o y and s imula o y ligand– ecep o pai s
ha exe an inhibi o y o s imula o y e ec on immune esponses. They a e usually
exp essed in T-cells o main ain sel - ole ance and egula e he magni ude o he immune
esponse. Addi ionally, hese molecules ha e been desc ibed as pa icipa ing in immune
e asion in cance [
67
]. Blocking he in e ac ion o checkpoin molecules by immune
checkpoin inhibi o s (ICI) is cu en ly unde esea ch o inc ease T-cell ac i a ion and
p oli e a ion, causing T-cell cy o oxici y owa ds umo cells. ICI ea men ypically a ge s
PD1 o CTLA4 immune checkpoin molecules, which ha e shown p omising clinical
e icacy in a ious solid umo s, including melanoma [
68
,
69
]. Th ee ials ha e s udied
he e icacy o ICI in pedia ic sa comas showing no bene i o EWS pa ien s. In a phase I
ial, ipilimumab (an i-CTLA4) was e alua ed in child en and adolescen s wi h sa coma,
howe e , i showed no ema kable bene i conside ing he small sample size [
70
]. Nex , a
mul icen ic s udy e alua ed pemb olizumab (an i-PD1) in ad anced sa comas, epo ing
an objec i e esponse in only 18 and 5% o so issue and bone sa coma, espec i ely,
al hough no esponse in he 13 EWS pa ien s was obse ed [
71
]. The las ial s udied
he combina ion o bo h an i-PD1 and an i-CTLA4 and con i med he limi ed e icacy o
an i-PD1 in mono he apy, while epo ing modes bene i s o he combina ion in some
sa coma sub ypes beyond EWS (5% and 16% o e all esponse a e, espec i ely) [
72
].
The umo mu a ion bu den con ibu es o he immune ecogni ion o cance cells and,
oge he wi h he exp ession o bo h PD-1 and PD-L1, seem o p edic he esponse o ICI
ea men [
73
,
74
]. On his basis, he low mu a ion a es o EWS and he ac ha hese
umo s ha e a low exp ession o PD1 o i s ligands (25.7% and 19.2%, espec i ely) migh
explain he poo esponse o hese umo s o ICI. Mo eo e , ano he s udy epo ed PD-L1
exp ession in 33% o EWS, which signi ican ly an ico ela ed wi h su i al [75,76].
New he apeu ic s a egies beyond ICI ocus on combining hese agen s. VEGF p o-
mo es an immunosupp essi e mic oen i onmen and con ibu es o ICI esis ance in can-
ce [
77
]. Consis en ly, clinical ials a e combining pemb olizumab wi h VEGFR inhibi o s
(NCT02636725, NCT05182164). The combina ion o pemb olizumab wi h he VEGFR in-
hibi o axi inib has shown low oxici y and p elimina y ac i i y in a phase II ial, al hough
no ema kable esponse was epo ed o EWS pa ien s [
78
] (NCT02636725). Ano he phase
II s udy is assessing he e icacy o combining pemb olizumab wi h cabozan inib, a ecep o
y osine kinase inhibi o , in pa ien s wi h ad anced sa comas (NCT05182164). Addi ionally,
a phase I/II ial wi h sequen ial adminis a ion o ni olumab (an i-PD1) and escala ing
doses o he mTOR-inhibi o ABI-009 has been conduc ed wi h EWS pa ien s in which he
e icacy and sa e y o he ea men will be e alua ed (NCT03190174). The las esul s o
his s udy showed no dose-limi ing oxici ies [
79
]. NKTR-214 is an enginee ed e sion o
he in e leukin 2 ecep o (IL-2R) wi h a polye hylene glycol chain (bempegaldesleukin
o BEMPEG) ha educes IL-2 binding o CD25 o e CD122. Consequen ly, a sus ained
ac i a ion o an i umo CD8
+
T-cells and NK cells, which is associa ed wi h umo eg es-
sion, is p omo ed [
80
]. No el s udies indica e he bene i o combining his he apy wi h
ICI [
81
]. On his basis, a non- andomized wo pa open-label ial is e alua ing he sa e y,
ole abili y, and dose le el o he combina o y ea men o ni olumab wi h BEMPEG, as
well as he e icacy o he combina ion in child en and young adul s wi h ecu en o
e ac o y umo s including EWS (NCT04730349). Howe e , ials wi h his combina ion
ha e been discon inued ecen ly.
Finally, B7 homolog 3 (B7-H3) is a checkpoin inhibi o y p o ein o he B7-CD28 amily
ha is o e exp essed in mul iple cance ypes including os eosa coma, whose exp ession is
associa ed wi h poo su i al [
82
]. Enobli uzumab (MGA271) is a humanized IgG1 mono-
clonal an ibody a ge ing B7-H3 ha is being ialed in child en wi h elapsed o e ac o y
malignan solid umo s wi h high exp ession o B7-H3, including os eosa coma, EWS, neu-
oblas oma, habdomyosa coma, Wilms Tumo and desmoplas ic small ound cell umo s
(NCT02982941). This phase I ial will de e mine i s sa e y, ole abili y, immunogenici y,
and p elimina y an i umo ac i i y in hese umo en i ies.
Cance s 2022,14, 5473 16 o 28
Table 3.
Summa y o NPS used in clinical ials (las 5 yea s) in EWS. Sou ce: ClinicalT ials.go
(accessed on 1 Sep embe 2022).
Molecula
Mechanism In e en ions Clinical T ial
Iden i ie Pa ien s Phase S a us
Oncogene d i e
inhibi ion Biological: pbi-shRNA™
EWS/FLI1 Type 1 LPX NCT02736565 EWS I Ac i e, no
ec ui ing
DNA damage by
opoisome ase
inhibi ion
Oni yde + Talazopa ib
(A m A) o Temozolomide
(A m B) NCT04901702
Recu en Solid Tumo s: EWS;
Hepa oblas oma;
Neu oblas oma; Os eosa coma;
Rhabdomyosa coma; Wilms
Tumo . Re ac o y Solid
Tumo s: EWS; Hepa oblas oma;
Malignan Ge m Cell Tumo ;
Malignan Solid Neoplasm;
Neu oblas oma; Os eosa coma;
Pe iphe al P imi i e
Neu oec ode mal Tumo ;
Rhabdoid Tumo ;
Rhabdomyosa coma
I/II Ac i e, no
ec ui ing
MM-398 (I ino ecan
Suc oso a e Liposome) +
cyclophosphamide NCT02013336
Recu en o Re ac o y Solid
Tumo s: EWS;
Rhabdomyosa coma;
Neu oblas oma; Os eosa coma
I Rec ui ing
Depolyme iza ion o
mic o ubules
(pacli axel)
Nab-pacli axel NCT03275818
Desmoplas ic Small Round Cell,
Adul ; Desmoplas ic Small
Round Cell, childhood; EWS;
Desmoid
II Comple ed
Nab-pacli axel NCT01962103
Neu oblas oma;
Rhabdomyosa coma; EWS;
Epi heliod
Sa coma, So Tissue
Sa coma, Spindle Cell
Melanoma; Melanoma;
Os eosa coma; His iocy oma;
Fib osa coma;
De ma o ib osa coma
I/II Comple ed
[152]
Nab-pacli axel +
Gemci abine NCT03507491 Cance I Rec ui ing
Nab-Pacli axel +
Gemci abine NCT02945800 Os eosa coma; EWS;
Rhabdomyosa coma; So
Tissue Sa coma II Rec ui ing
DNA damage by
in e cala ion,
dis up ion o
opoisome ase-II and
gene a ion o ee
adicals
(doxo ubicin)
Disul i am + Coppe
Glucona e and Liposomal
Doxo ubicin NCT05210374 Relapsed Sa comas (including
EWS) INo ye
ec ui ing
Liposomal Doxo ubicin +
MR-HIFU Hype he mia NCT02557854
EWS; Rhabdomyosa coma;
Wilms Tumo ; Neu oblas oma;
Hepa oblas oma; Ge m Cell
Tumo
I Wi hd awn
Temsi olimus + liposomal
doxo ubicin NCT00949325 Sa coma (including EWS) I/II Comple ed
[153]
Lyso- he mosensi i e
liposomal doxo ubicin
(LTLD) + MR-HIFU
Hype he mia
NCT04791228
EWS; Malignan Epi helial
Neoplasm;
Rhabdomyosa coma; Wilms
Tumo ; Hepa ic Tumo ; Ge m
Cell Tumo
II No ye
ec ui ing
Lyso- he mosensi i e
liposomal doxo ubicin +
Magne ic esonance high
in ensi y ocused
ul asound
NCT02536183
Rhabdomyosa coma; EWS;
Os eosa coma; Neu oblas oma;
Wilms Tumo ; Hepa ic Tumo ;
Ge m Cell Tumo s
I Rec ui ing
Cance s 2022,14, 5473 17 o 28
D ug Deli e y Sys ems
Se e al s udies ha e used NPS o ca y an icance d ugs in o de o imp o e d ug
kine ics and achie e be e he apeu ic esul s. On his basis, a small molecule uncha ac-
e ized compound ML111 was ound o inhibi
in i o
he p oli e a ion o six es ablished
EWS cell lines wi h nanomola po ency [
154
]. Sabei e al., ha e epo ed ha ML111
encapsula ed in o a hyd ophobic co e o PEG-PCL-based polyme ic NPS (ML111-NPS)
was able o in e nalize in o EWS cell lines and speci ically inhibi hei iabili y wi hou
al e ing nonmalignan human cell lines [
155
]. Mo eo e , a syne gis ic e ec on he iabili y
o EWS cells esul ed om combining ML111-NPS wi h inc is ine
in i o
, compa ed o
nonmalignan cells. Mo eo e , a eg ession o EWS umo s was obse ed when using
ML111-NPS
in i o
, bo h in mono he apy and in combina ion wi h inc is ine. No oxici y
e ec s we e iden i ied in mice o gans wi h ML111-NPS alone, and wi h he combina ion
he e was a educ ion o side e ec s associa ed wi h inc is ine [
155
]. Besides, he use o
a hyd olyzed galac omannan (hGM)-based amphiphilic NPS o selec i e in a umo al
accumula ion in pedia ic sa coma was also in es iga ed [
156
]. Coupling o hese NPS wi h
he y osine kinase inhibi o ima inib could a ge glucose anspo e -1 (GLUT-1), bo h in
habdomyosa coma cells and in EWS PDX wi h di e en GLUT-1 exp ession le els wi h
a 7.5% o e iciency [
157
], which make hem a po en ial ool agains GLUT-1-exp essing
umo s. Fu he mo e, Bell e al., employed biomime ic high-densi y lipop o ein (HDL)
NPS. These HDL NPS we e able o bind bo h HDL ecep o s and sca enge ecep o ype
B-1 (SCARB1), dep i ing umo cells HDL and choles e ol, and blocking p oli e a ion in
hedgehog-d i en EWS cells and medulloblas oma [158].
PARP inhibi o s such as alazopa ib (TLZ) o olapa ib did no show ac i i y in
EWS [
159
,
160
], al hough hey po en ia e he ea men wi h he DNA alkyla ing agen
emozolomide (TMZ). A nano o mula ion o TLZ (NanoTLZ) was epo ed o be mo e
e ec i e and well ole a ed
in i o
, while i s combina ion wi h TMZ elici ed an inc ease
in he maximum ole a ed dose o TMZ o EWS ea men [
159
]. Ne e heless, ano he
s udy showed ha he TC71 TLZ- esis an EWS cell line was no a ec ed by equen ly
adminis e ed o al TLZ no a ec ed by he long-ac ing PEGyla ed TLZ [161].
Oni yde (MM-398 o PEP02) is a nanoliposomal o mula ion o he DNA opoiso-
me ase I inhibi o i ino ecan, which is used o ea se e al solid umo s, al hough i
has a complex pha macokine ics [
162
]. Oni yde showed an imp o emen on he an i u-
mo ac i i y, biodis ibu ion, and a educ ion o oxici y in EWS xenog a s compa ed o
he cu en clinical o mula ion o i ino ecan [
162
]. Cu en ly, a ec ui ing phase I clin-
ical ial s udies he highes dose o MM-398 ha can be gi en sa ely when combined
wi h cyclophosphamide in pa ien s wi h ecu en o e ac o y pedia ic solid umo s
(NCT02013336). Indeed, an ac i e phase I ial is being conduc ed wi h combina ions o
oni yde wi h TLZ o TMZ (NCT04901702) o de e mine he highes ole able doses o he
wo combina ions (NCT04901702).
A ecen wo k e alua ed he albumin-bound (nab)-pacli axel NPS in PDXs o EWS,
habdomyosa coma, and os eosa coma [
163
]. These NPS bind o umo cells ha exp ess
SPARC, a sec e ed acidic p o ein and ich in cys eine ha shows a high a ini y o bind
albumin. Nab-pacli axel was less bound in SPARC-knocked down (SPARC-KD) compa ed
o SPARC-WT cells [
163
]. EWS PDX wi h high exp ession o SPARC was associa ed
o accumula ion o nab-pacli axel showing be e d ug esponses compa ed o umo s
wi h lowe SPARC le els. Consis en ly, pedia ic umo s ha exp ess SPARC we e able
o accumula e nab-pacli axel o mo e ex ended pe iods o ime [
163
]. Nab-pacli axel
is being e alua ed in se e al clinical ials including an ac i e phase II clinical ial in
mono he apy o pa ien s wi h EWS and o he umo s (NCT03275818). A comple ed
phase I/II mul icen e ial (NCT01962103, [
152
]) showed in EWS pa ien s ha he o e all
esponse a e was 0%, he disease con ol a e was 30.8% (4 s able disease), he median PFS
was 13.0; and he 1-yea OS a e was 48%. The sa e y o nab-pacli axel in pedia ic pa ien s
was con i med; howe e , limi ed ac i i y was obse ed [
164
]. Finally, wo clinical ials
a e ec ui ing pa ien s wi h pedia ic elapsed and e ac o y solid umo s (NCT03507491);
Cance s 2022,14, 5473 18 o 28
and pa ien s wi h ecu en / e ac o y sa coma (NCT02945800), in which nab-pacli axel
and gemci abine will be gi en. Howe e , he esul s o he EWS a m o NCT02945800
ha e been published. This clinical ial o nab-pacli axel and gemci abine displayed limi ed
ac i i y in a small coho o EWS pa ien s con i ming only one pa ial esponse. Mo eo e ,
wo pa ial esponses a e wo cycles was obse ed, bu due o he side e ec s o he
p og ession o he disease, hese wo pa ien s we e wi hd awn [165]. The esponse a e o
9% was simila o o he s udy in EWS pa ien s ea ed wi h gemci abine and doce axel [
165
].
Liposomal doxo ubicin was designed o inc ease i s he apeu ic e icacy while de-
c easing oxici y. A phase I clinical ial (no ye ec ui ing) pu pose o e alua e disul i-
am wi h coppe glucona e and liposomal doxo ubicin in ea men - e ac o y sa comas
(NCT05210374). Ano he phase I ial is cu en ly unning o de e mine whe he deli e y
o a liposomal doxo ubicin called doxil p io o MR-HIFU (magne ic esonance-guided
high in ensi y ocused ul asound) hype he mia will be sa e o he ea men o pedia ic
and young adul pa ien s wi h ecu en and e ac o y solid umo s. Un o una ely, his
ial is wi hd awn because o he lack o en ollmen (NCT02557854). Also- he mosensi i e
liposomal doxo ubicin (LTLD) is he i s hea -ac i a ed o mula ion o a liposomal d ug
ca ie o be u ilized in human clinical ials. The e a e wo clinical ials wi h EWS pa ien s.
A phase I, ec ui ing ial ha combined LTLD and MR-HIFU in pedia ic e ac o y solid
umo s (NCT02536183); and a phase II ial, in which LTLD wi h MR-HIFU hype he mia
ollowed by abla ion will be s udied in subjec s wi h e ac o y/ elapsed solid umo s
(NCT04791228).
Finally, comple e phase I and II clinical ials showed ha combina ions o liposomal
doxo ubicin and emsi olimus we e sa e and showed e icacy o pa ien s wi h ecu en
sa coma (NCT00949325 and [153,166]).
3.1.2. Ino ganic NPS
Ino ganic NPS a e me al-based (gold, i on, lead, sil e ) and me al oxide-based (alu-
minum oxide, zinc oxide, e c.) pa icles [
146
]. Me al-based NPS o gold and sil e (Au and
Ag NPS, espec i ely) ha e been epo ed o ha e an i umo e ec s [
156
,
167
,
168
]; howe e ,
Ag NPS can induce gene al oxici y in non- a ge o gans [
169
]. These NPS ha e been also
e alua ed in he con ex o EWS a p eclinical le el. Naumann e al., ha e de eloped
Au-NPS whe e selec i e SN-38 ac i a ion in cance cells is media ed by he EWS speci ic
mRNAs BIRC5 (su i in) and EWSR1-FLI1. In his sys em, he gold pa icle is conjuga ed
o he speci ic mRNA whe e he complemen a y SN38-conjuga ed oligonucleo ide anneals.
SN38 elease will be dependen on he p esence o he EWS speci ic mRNA. The iabili y o
EWS cells ea ed wi h SN38-su i in Au-NPS and SN38-EWS/FLI1 Au-NPS was signi i-
can ly educed in ou EWS cell lines and in mu ine xenog a s [
170
]. The an i umo ac i i y
o sil e chlo ide and sil e /sil e chlo ide NPS (AgCl and Ag/AgCl NPS, espec i ely)
has been also in es iga ed in EWS. T ea men o EWS cell lines and a non- umo cell line
wi h Ag NPS caused a educ ion in cell iabili y speci ic o umo cells. Bo h AgCl and
Ag/Ag-NPS inc eased he pe cen age o apop o ic cells and ROS p oduc ion, accompanied
wi h a loss o mi ochond ial memb ane po en ial, and lysosomal damage. These e ec s
we e speci ic o umo cells wi h minimal e ec s shown on heal hy cells [171].
3.1.3. Ca bon-Based Nanoma e ials
Ca bon-based NPS include ulle enes, ca bon nano ibe s, diamonds, ca bon nano ubes,
and g aphene. These NPS display mul iple p ope ies ha make hem sui able o d ug
deli e y sys ems and cance he apy, as well as imaging, biosensing, o diagnosis [
156
]. Al-
haddad e al., in es iga ed he abili y o a siRNA deli e y sys em using diamond NPS [
172
].
These diamond NPS we e coa ed wi h a ca ionic polyme and encapsula ed siRNA o
inhibi EWSR1-FLI1. Because diamond NPS ha e in insic luo escen p ope ies i s in e -
naliza ion in o EWS cell lines was e icacious and could be obse ed di ec ly. Following he
in e naliza ion, EWSR1-FLI1 inhibi ion was obse ed a mRNA and p o ein le els
in i o
.
Finally, cell oxici y was low a e ea men wi h diamond NPS [172].
Cance s 2022,14, 5473 19 o 28
3.1.4. Hyb id NPS
Hyb id NPS a e o med by polyme and o ganic- o ino ganic-based NPS sys ems ha
combine he p ope ies o single sys ems. Consis en ly, hey ha e lowe ci cula ion ime
and bioa ailabili y, mo e s abili y and he apeu ic e icacy, being a iable al e na i e when
compa ed o single sys ems [173,174].
Hyb id polyme iza ion liposomal NPS (HPLNs) has been de eloped an ibody encap-
sula ed wi h i ino ecan (CD99-HPLN/I ). Low doses o his hyb id sys em ha e shown
educed EWS umo s in xenog a mice and comple e umo abla ion, which was mo e e i-
cacious compa ed o oni yde and doxil NPS sys ems. D ug bioa ailabili y was imp o ed
six- old wi h HPLN and encapsula ed i ino ecan wi hou CD99 and wel e- old wi h
CD99-HPLN/I in espec o oni yde. Consis en ly, i ino ecan oxic side e ec s we e mini-
mized [
173
]. Along he same lines, HPLN has been used o deli e siRNA, ASO, o unc-
ional CRISPR-Cas9 sys ems agains he usion oncogene EWSR1-FLI1.
In i o
expe imen s
esul ed in an e icien EWSR1-FLI1 educ ion being he mos e ec i e HPLN/CRISPR-Cas9
sys em. Mo eo e , CD99-HPLN wi h CRISPR-Cas9 agains encapsula ed EWSR1-FLI1,
educed EWS umo g ow h
in i o
[
175
]. Al hough p omising p eclinical esul s, he
po en ial o hese hyb id NPS sys ems emains o be u he e alua ed in clinical s udies.
4. Conclusions and Fu u e Pe spec i es
The s anda d he apy o EWS pa ien s based on cy o oxic chemo he apy and adio-
he apy has eached a pla eau, especially o ha subg oup o pa ien s wi h he wo s
p ognosis. Mo eo e , pa ien s who su i e ace debili a ing and o en li e- h ea ening
heal h consequences as a esul o he high oxici y o hese he apies. The e o e, and espe-
cially conside ing he young age and po en ial li espan o he pa ien s, he e is u gen need
o inding new he apies o imp o e he ou come o hese pa ien s [
176
]. Epigene ic-based
he apies ha e changed he a ge ing ocus om ex acellula and in acellula signaling
o ch oma in, whe e hese pa hways in eg a e egula ing gene exp ession in a e e sible
manne ha o e s he oppo uni y o pheno ype con e sion. The inhibi ion o epigene ic
complexes ha egula e he exp ession and p o ein s abili y o he oncogene i sel , as well as
hose co ac o s ha pa icipa e in he modula ion o i s ac i i y, shows signi ican achie e-
men s in he con ol o he disease in p eclinical s udies. Ne e heless, he epigene ic
d ugs used in clinics ha e epo ed modes an i umo e icacy in mono he apy leading o
he de elopmen o new epigene ic app oaches based on he usage o second gene a ion
d ugs and combina o ial s a egies ha p omo e syne gis ic e ec s. Fu he mechanis ic
app oaches should explo e di e ences in d ug e iciency be ween a ge ing speci ic enzy-
ma ic domains and he e ec s o deple ing he whole p o ein o inducing i s deg ada ion.
Immuno he apy, unlike o he app oaches, induces a he apeu ic esponse no only limi ed
o dis up a single oncogenic e en . Despi e p omising esul s o immuno he apy in adul
umo s, hei applica ion in EWS and o he sa comas has demons a ed poo he apeu ic
ac i i y due o he immune-cold na u e o hese umo s. Howe e , di e en immune s a e-
gies a e being de eloped sea ching o e icien combina ions wi h s anda d o new a ge ed
he apies, including epigene ic d ugs. Besides, o he esea ch s a egies a e ocused on he
de elopmen o mo e a ge ed app oaches and he e e sion o he cold immune landscape
o EWS in o a ho pheno ype. Indeed, ou unde s anding o he c oss alk be ween he
umo and he issue mic oen i onmen as well as he basic aspec s o he ascula u e
and hypoxia o EWS would help u u e di ec ion in immunomodula ion he apies [
177
].
Bo h o epigene ics and immuno he apy, he in oduc ion o CRISPR sc eenings o de ine
no el a ge able umo dependencies will pos ula e p omising combina o ial s a egies
o explo e in clinical ials. On he o he hand, nanomedicine has e ol ed o ace he lack
o speci ici y, d ug esis ance and high oxici y a es o bo h s anda d egimens and hese
new he apeu ic al e na i es. Consis en ly, NPS as d ug deli e y sys ems ha e educed
bo h he oxici y associa ed wi h cy o oxic d ugs and he ole a ed dose. This ac aises he
possibili y o escue he usage o e ec i e d ugs ha we e disca ded in clinics o hei side
e ec s by coupling hem o no el NPS sys ems.
Cance s 2022,14, 5473 20 o 28
Finally, he highly he e ogenei y in EWS umo s p omo e mo e limi a ions ha also
a ec s he ea men . Fi s ly, he possibili y o de eloping an app op ia e
in i o
model,
which could con ibu e o he disc epancy be ween p eclinical and clinical esul s. Howe e ,
la es esea ch in pa ien de i ed o ganoid, which ecapi ula e gene ic and pheno ypic
cha ac e is ics o hei issue o o igin, suppo he inclusion o his models in p eclinical
alida ion as p edic o s o esponse [
178
]. Secondly, he necessi y on inding speci ic and
uni e sally exp essed memb ane bioma ke s ha migh imp o e ea men speci ici y. The
disco e y o new bioma ke s wi h p ognos ic alue and esponse o ea men wi h a mo e
accu a e classi ica ion o pa ien s, would bene i he c ea ion o a speci ic ea men plan,
also e e ed o as pe sonalized medicine, ha migh bene i su i al o EWS pa ien s.
Taken oge he , hese new he apeu ic al e na i es and he mo e e ec i e deli e y o d ugs
by NPS ep esen a new ho izon in ea ing EWS pa ien s, which is expec ed o bene i
pa ien su i al.
Au ho Con ibu ions:
S.S.-M., E.F.-B., D.J.G.-D. and L.H.-P. we e in ol ed in he concep ion and
d a ing o he manusc ip . All au ho s ha e c i ically e iewed he manusc ip and ha e app o ed
he inal e sion o publica ion. All au ho s ha e ead and ag eed o he published e sion o
he manusc ip .
Funding:
This wo k was suppo ed by FIS-FEDER PI2000003 o E.d.Á.C.; L.H.-P. is suppo ed by he
Conseje ía de Salud y Familias, Jun a de Andalucía (RH-0047-2021).
Con lic s o In e es :
The au ho s decla e no con lic o in e es . The au ho s ha e designed and
concei ed bo h igu es. The igu es equi e no copy igh pe mission om hi d pa ies.
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