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Overcoming Resistance to Tumor-Targeted and Immune-Targeted Therapies

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Overcoming Resistance to Tumor-Targeted

and Immune-Targeted Therapies
Mihaela Aldea1, Fabrice Andre1,2,3, Aurelien Marabelle3,4, Semih Dogan2, Fabrice Barlesi1,5,
and Jean-Charles Soria3,4

aBstRact Resistance to anticancer therapies includes primary resistance, usually related to

lack of target dependency or presence of additional targets, and secondary resist-
ance, mostly driven by adaptation of the cancer cell to the selection pressure of treatment. Resistance
to targeted therapy is frequently acquired, driven by on-target, bypass alterations, or cellular plasticity.
Resistance to immunotherapy is often primary, orchestrated by sophisticated tumor–host–microen-
vironment interactions, but could also occur after initial efficacy, mostly when only partial responses
are obtained. Here, we provide an overview of resistance to tumor and immune-targeted therapies and
discuss challenges of overcoming resistance, and current and future directions of development.

Significance: A better and earlier identification of cancer-resistance mechanisms could avoid the use
of ineffective drugs in patients not responding to therapy and provide the rationale for the admin-
istration of personalized drug associations. A clear description of the molecular interplayers is a
prerequisite to the development of novel and dedicated anticancer drugs. Finally, the implementation
of such cancer molecular and immunologic explorations in prospective clinical trials could de-risk the
demonstration of more effective anticancer strategies in randomized registration trials, and bring us
closer to the promise of cure.

intRoduction
The therapeutic success of tumor- and immune-targeted
therapies has revolutionized the way we understand the biology
of tumors and radically changed the therapeutic landscape of
cancers. Spectacular tumor responses were seen with targeted
therapy in oncogene-addicted cancers, and unprecedented long-
term remissions in some patients treated with immunotherapy.
Despite this immense progress, advanced cancer is ultimately
lethal for most patients due to treatment resistance.
Cancer resistance is classified into two broad categories:
primary resistance, with an early tumor progression, without
prior tumor response, and secondary (acquired) resistance,
which occurs after initial tumor responses (1–3). Although
1
Department of Medical Oncology, Gustave Roussy, Villejuif, France.
2
INSERM U981, PRISM Institute, Gustave Roussy, Villejuif, France. 3Paris
Saclay University, Saint-Aubin, France. 4Drug Development Department,
Gustave Roussy, Villejuif, France. 5Aix Marseille University, CNRS, INSERM,
CRCM, Marseille, France.
Note: Supplementary data for this article are available at Cancer Discovery
Online (http://cancerdiscovery.aacrjournals.org/).
Corresponding Author: Jean-Charles Soria, Gustave Roussy Cancer Cam-
pus, Villejuif, 94805, France. Phone: 331-4211-4016; E-mail: Jean-Charles.
Soria@gustaveroussy.fr
Cancer Discov 2021;11:874–99
doi: 10.1158/2159-8290.CD-20-1638
©2021 American Association for Cancer Research.
the two types of resistance can occur with both tumor and
immune-targeted drugs, it is more common to see acquired
resistance to tumor-targeted therapies and primary resistance
to immune-targeted therapies, where there is an important
proportion of patients without response to therapy in many
cancers (3–6). For targeted agents and notably EGFR tyros-
ine kinase inhibitors (TKI), the Jackman criteria have been
accepted by the medical community for defining acquired
resistance (7). No such definition exists for immunotherapy,
despite ongoing efforts from the Society for Immunotherapy
of Cancer (SITC; ref. 8).
Finding predictive biomarkers of resistance has important
implications for cancer care. It may avoid giving an ineffective
treatment to patients whose tumors do not respond (9–11) or
guide therapeutic choices to overcome resistance. In the case
of targeted therapy, several biomarkers of resistance are vali-
dated and routinely used. For immunotherapy, the focus has
been primarily to find biomarkers associated with a positive
predictive value of response [PD-L1, microsatellite instabil-
ity, tumor mutational burden (TMB), etc.]. Therefore, data
are still sparse, and there is no approved, clinically validated
resistance biomarker to guide treatment selection (although
the absence of PD-L1 expression is de facto rendering some
patients with cancer not eligible for anti–PD-L1 therapies).
Here, we provide an overview of the main concepts of
tumor resistance to cancer cell–, stromal cell–, and immune
cell–targeted therapies, applicable across tumor types. We
provide examples of the clinical and preclinical evidence of

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Overcoming Resistance to Targeted Therapy and Immunotherapy
tailoring treatment beyond progression, the challenges faced
today, and potential directions of research.
Mechanisms of Resistance to Cancer
Cell–Targeted Therapy
A targeted therapy may fail because of drug resistance or
lack of drug target, or following inadequate drug exposure.
Here we will focus on mechanisms of drug resistance when
the target is expressed and validated. An overview of mecha-
nisms of resistance to targeted therapy is reported in Fig. 1.
Primary Resistance to Targeted Therapy
Primary resistance is mostly related to the lack of target
dependency or the presence of additional targets, in the con-
text of tumor heterogeneity. Biomarkers of primary resistance
to a certain drug are (i) insensitive variants of the target [e.g.,
EGFR insertions in exon 20 and the majority of EGFR inhibi-
tors (EGFRi) in EGFR-mutated non–small cell lung cancer
(NSCLC)]; (ii) aberrations of oncogenic pathways connecting
to the target (e.g., RAS mutations and EGFR inhibition in
metastatic colorectal cancer; refs. 9, 10); (iii) activating muta-
tions downstream to the target (RB1 mutations and CDK4/6
inhibitors in estrogen receptor–positive/HER2-negative breast
cancer; ref. 12); or (iv) activation of parallel oncogenic path-
ways (e.g., de novo ALK rearrangement and EGFR mutation in
NSCLC and single-agent EGFR/ALKi; Fig. 1A). The latter is
a very rare situation where it is critical to establish the domi-
nant driver if single-agent inhibition is planned. In NSCLC,
de novo ALK and EGFR aberrations were found to colocalize
in the same tumor cell population and possibly in the same
cellular clone in 1.3% of NSCLC cases (13). The dominant
driver receptor seems to be more frequently the EGFR muta-
tion, as patients tend to benefit more from EGFRi than from
ALKi. The sensitivity to TKI appears to be related to the dif-
ferential phosphorylation of EGFR and ALK, but the clinical
validity and utility of testing phosphorylation levels of these
proteins is currently unknown (13, 14). Another example is
the co-occurrence of EGFR exon 19 deletions with nondisrup-
tive TP53 exon 8 mutations, which have been associated with
primary resistance to EGFRi in NSCLC (15).
Gene Silencing
Despite the identification of a targetable genomic alteration,
drugs may be ineffective in the case of gene silencing, when
the target lacks its protein expression. An analysis focused on
50 putative drivers comparing whole-exome tumor (somatic)/
normal (germline) sequencing with whole-transcriptome
sequencing in 1,417 primary tumors revealed that nearly
13% of the somatic single-nucleotide variants (SNV) were
­unexpectedly not transcribed as RNA. SNVs with high tran-
scription rates included TP53, PIK3CA, and KRAS, whereas
those with lower transcription rates included ALK, CSF1R,
ERBB4, FLT3, GNAS, HNF1A, KDR, PDGFRA, RET, and SMO
alterations. Interestingly, the higher the mutational load, the
higher the number of silenced variants (16). Another example
of gene silencing is posttranscriptional editing with RNA
silencing by miRNA, RNA interference, and small interfering
RNA (17).
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Short-Term Adaptation to Targeted Therapies
The inhibition of feedback loops by targeted therapies can
lead to unintended compensatory overactivation of upstream
pathways. In PIK3CA-mutant estrogen receptor (ER)–positive
breast cancer, increased ER activity occurs early upon PI3K
inhibition and limits the antitumor activity of single-agent
PI3K inhibitors (18). Also, mTOR and dual PI3K/mTOR inhib-
itors suppress mTORC1 and S6K, which normally send inhibi-
tory signals via the insulin/IGFR and other receptor tyrosine
kinases (RTK). Once these feedback loops are released, resist-
ance may occur via upstream activation of PI3K, AKT, and
ERK, which circumvents the antitumoral effects of PI3K/AKT/
mTOR inhibitors. Treatment-induced hyperglycemia induces
systemic hyperinsulinemia, which may activate PI3K signal-
ing despite continued suppression with PI3K inhibitors. The
ketogenic diet or SGLT2 inhibitors that lower blood glycemia
were shown to inhibit the insulin feedback, decrease mTORC1
signaling in the tumor, and enhance the efficacy of PI3K inhib-
itors (19). Another example is anti-BRAF monotherapy that
fails in BRAFV600E-mutated colorectal cancer due to feedback
activation of EGFR, as opposed to melanoma and NSCLC (20).
Similarly, the modest benefit of sotorasib in KRASG12C-mutated
colorectal cancer as compared with NSCLC is explained by
EGFR dependency and signaling rebound kinetics (21). MEK
inhibitors also induce PI3K/AKT activation via EGFR, due
to the cross-talk between the PI3K and RAS pathways (22),
and HER2 TKIs induce HER3 upregulation as a compensa-
tory mechanism of PI3K/AKT inhibition (23). The transla-
tional control of gene expression may also mediate resistance,
exemplified by the formation of the eukaryotic translation
initiation complex eIF4F, which regulates translation of many
oncogenic pathways, including RAS–MAPK and PI3K–mTOR.
The abnormal expression of eIF4F has been linked to resist-
ance to HER2, BRAF, and MEK inhibitors (24, 25). Another
example of short-term adaptation is nicely illustrated by a pre-
clinical study investigating KRASG12C inhibitors. First-in-class
KRASG12C inhibitors bind only to the inactive guanosine-5′-
triphosphate state of the oncoprotein. Upon MAPK suppres-
sion by KRASG12C inhibition, some quiescent cells can produce
new, active/­­drug-insensitive KRAS, in an EGFR–SHP2- and
AURK-dependent manner (26).
Acquired Resistance to Targeted Therapy
The main mechanisms of acquired resistance include: (i)
on-target resistance alterations; (ii) bypass alterations in the
same pathway or connecting pathways; (iii) alterations that
cause a phenotypic transformation of the tumor [epithelial-
to-mesenchymal transition (EMT), small cell transformation
of NSCLC, squamous transformation of adenocarcinomas];
and (iv) loss of target and target dependency (Fig. 1B; refs.
4, 6, 27–29).
On-Target Mechanisms of Resistance
On-target mechanisms of resistance include secondary
mutations, target amplifications, or alternative splicing of the
protein kinase mRNA. Following TKIs, “second-site muta-
tions” are often seen in the intracellular kinase domain of
the protein. They reduce drug affinity for the target by weak-
ening the chemical bonds between the kinase and the drug
CANCER DISCOVERY 10TH ANNIVERSARY ISSUE | 875
REVIEW Aldea et al.

A Primary resistance to targeted therapy

a. b. Anti-EGFR
EGFR antibody
Wild-type EGFR EGFR exon 20
insertion

C-helix RAS PI3K

Insertion
RAF AKT

MEK mTOR
Inactive Active
ERK

c. EGFRmut ALK fusion d. CDK4/6 inhibitor

P P CDK4/6
P
Cyclin D
RAS PI3K JAK
P G2
RB1 S
RAF AKT STAT
RB1
MEK mTOR E2F M
G1
Free
Cell-cycle
ERK E2F
progression

B Acquired resistance to targeted therapy

EGFRmut
i. Activating Activating + second-site ii.
mutation mutation
Drug STOP
Target ALK fusion

Drug
RAS PI3K JAK
ATP
RAF AKT STAT
Amplification Alternative splicing MEK mTOR

ERK

iii. iv.
Epithelial Mesenchymal Loss of target
EMT

Adenocarcinoma Squamous Small cell

Inhibitory
signal

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Overcoming Resistance to Targeted Therapy and Immunotherapy REVIEW

(e.g., EGFRC797S), induce conformational changes of the
kinase (gatekeeper mutations, e.g., BCR–ABLT315I, EGFRT790M,
ALKL1196M, ROS1L2026M, RETV804M/L, and TRKAF589L) or cause
direct steric hindrance to drug binding (solvent-front muta-
tions, e.g., EGFRG796, ALKG1202R, ROS1G2032R, ROS1D2033N, RETG810,
TRKAG595R, TRKBG639R, TRKCG623R; xDFG mutations, for exam-
ple, ROS1G2101A/C, TRKAG667S, or TRKCG696A; refs. 30–41). As a
consequence, some drugs with similar structure are susceptible
to cross-resistance. Second-site mutations have been prepon-
derantly described with TKIs targeting membrane receptors
(RTKi; EGFR, cKIT, FGFR, MET, NTRK1–3, and RET) or cyto-
solic fusion proteins (ALK, ROS1, and BCR–ABL), whereas only
exceptionally in case of intracellular protein kinases derived
from oncogenic mutations. A single case of a secondary BRAF
mutation (BRAFL514V) has yet been reported (42). Interestingly,
the ALK fusion variant seems to affect acquired resistance,
with more ALK resistance mutations in patients with variant
3 than those with variant 1, particularly ALKG1202R (43). Treat-
ment with mAbs may result in mutations of the extracellular
domain of the protein, which impair antibody binding (e.g.,
EGFR extracellular domain variants after cetuximab and pani-
tumumab; refs. 44, 45). Mutations affecting the ligand binding
domain may occur after endocrine therapy, leading to consti-
tutive receptor activation (e.g., ESR1-activating mutations in
endocrine treatment–resistant breast cancer, or androgen recep-
tor splice variants, such as AR-V7, in enzalutamide/abiraterone-
resistant prostate cancer; refs. 46, 47).
Alternative Splicing. Aberrantly spliced proteins can mediate
resistance via enhanced dimerization (e.g., splice variants of
BRAFV600E that occur in 13% to 30% of patients with mela-
noma failing BRAF inhibitors (BRAFi; ref. 48) or by constitu-
tive protein activation in the absence of ligand (e.g., androgen
receptor splice variants in prostate cancer; ref. 47).
Target Amplification. Exemplified by EGFR amplification
emerging after EGFR inhibitors, BRAF amplification after
BRAFi, or androgen receptor amplification in hormone-
resistant prostate cancer, target amplification may limit the
effectiveness of the drug by exceeding its inhibition capacity
(49–51). In addition to the amplified mutant allele, high-level
gene amplification of the wild-type allele can also induce
resistance (51, 52). Another process that drives resistance is
oncogene amplification on extrachromosomal DNA (ecDNA).
These circular structures lacking centromers undergo an un-
equal segregation during mitosis, which causes increased copy-
number amplification with enhanced oncogene expression.
This results in aggressive tumor behavior, poor prognosis,
and drug resistance, but the exact underpinning mechanisms
remain largely unknown (53). In experimental models of
glioblastoma, resistance to EGFR TKI occurred through the
loss of extrachromosomal (ec) EGFRvIII DNA, whereas drug
withdrawal was followed by reemergence of clonal EGFR on
ecDNA. This unexpected twist illustrates how EGFRvIII levels
are modulated by ecDNA according to the tumor’s needs (54).
Loss or Activation of Mirror Protein. An illustrative example
of “mirror proteins” is PI3K and PTEN proteins, with their
divergent role on PIP3 formation. In PI3K signaling, the trans-
formation of PIP2 to PIP3 is a necessary step for signal trans-
duction, which is promoted by PI3K and negatively regulated
by PTEN. In PIK3CA-mutated breast tumors treated with
PI3Kα inhibitors, resistance may emerge via a progressive loss
of PTEN expression, which abrogates the pathway inhibition.
Preclinical studies point out that concomitant use of PI3Kα
and PI3K p110β blockade might reverse resistance (55).
Bypass Resistance Mechanisms
There are three major oncogenic signaling pathways that
drive cell growth and proliferation: the PI3K/AKT/mTOR
(PI3K pathway), RAS/RAF/ERK (MAPK pathway), and STAT/
JAK pathways. Oncogenic drivers frequently signal through
the same pathways: EGFR, IGFR1, FGRF2, and HER2 signal
via the PI3K and MAPK pathways and ALK, ROS1, MET, and
cKIT via all three oncogenic pathways, whereas oncogenic
BRAF signals exclusively through the MAPK pathway. Under
targeted therapy, bypass tracks could eventually lead to the
abnormal activation of the downstream pathway or connect-
ing signaling pathways. This will ensure a sustained abnormal
signaling despite continuous target inhibition. Notably, the
more potent target inhibitors are used, the more frequently
bypass tracks are likely to develop (56). Also, despite their
apparent diversity, bypass tracks follow similar patterns due
to the common signaling of targets. In the case of RTK inhi-
bition, one common event among drivers is the activation of
parallel RTK. For instance, in NSCLC, EGFR inhibition may
result in the emergence of MET, ERBB amplification, FGF2–
FGFR1 loop mutations, IGF1R activation, and fusion events
(28, 57, 58); ALK inhibition could lead to increased EGFR acti-
vation or KIT amplification (52, 56); MET exon 14 inhibition
could generate EGFR, HER2 amplifications (59). Examples of
downstream pathway reactivation include MAPK pathway
activation via BRAF, NRAS, and KRAS alterations following
RTK inhibition in NSCLC (60–63), MAP2K1/2 and MITF alter-
ations upon BRAFi in melanoma (64–66), NF1-inactivating
mutations associated with endocrine therapy resistance in ER+
breast cancer (67) or PI3K activation via PTEN loss in breast
cancer, and PIK3CA or AKT alterations as acquired resistance
to RTK inhibitors in NSCLC, to anti-EGFR mAbs in colorectal
cancer, or to BRAFi in melanoma (51, 64, 68–70).

Figure 1. A, Examples of primary resistance. (a) Insensitive variants of the target: The EGFR exon 20 insertion promotes a rigid structure that “pushes”
the C-helix inward and prevents it from adopting the outward, inactive conformation. Subsequently, the permanently active EGFR protein preserves
a good affinity for ATP and does not enhance affinity for first-generation EGFR inhibitors, which are inactive in this case. (b) Aberrations of pathways
connecting to the target: In colorectal cancer, the activation of the RAS pathway, located downstream of the transmembrane EGFR receptor, limits the
activity of anti-EGFR mAbs. (c) Concomitant activity of parallel RTK: ALK and EGFR may rarely coexist (1.3% in NSCLC) in the same tumor biopsy and
limit benefit from single-agent treatment. The dominant activity seems to be harbored by the protein with the highest phosphorylation level. (d) Activat-
ing mutations downstream to the target: Deleterious mutations of RB1 encoding the RB1 protein located downstream of the intracellular CDK4/6 protein
impair the activity of CDK4/6 inhibitors and lead to cell-cycle progression through the free form of E2F. B, Examples of acquired resistance. (i) On-target
resistance alterations; (ii) bypass alterations by the activation of parallel RTK; (iii) phenotypic transformation of the tumor, including EMT, small-cell
transformation, and squamous transformation of adenocarcinomas; (iv) loss of target.

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Among bypass tracks, acquired fusions were recently dis-
covered at EGFRi failure in EGFR-mutated NSCLC (71). The
most frequently reported are the RET fusion (46%), followed
by ALK (26%), NTRK1 (16%), and FGFR3 (11%; refs. 71–73).
They were identified at higher rates after third-generation
EGFRi than with first- or second-generation EGFRi (16% vs.
3%, respectively), when assessed in tissue biopsies by whole-
exome sequencing (WES)/RNA sequencing (RNA-seq; ref.
72). As opposed to the classic oncogenic drivers, they fre-
quently harbor uncommon 5′ partners, such as STRN for the
ALK fusion and CCDC6 and NCOA4 for the RET fusion (71,
74). Fusions have been reported in other tumors where EGFR
inhibition is routinely used, such as colorectal and head and
neck cancers (75–77). Acquired fusions are not limited to
EGFR inhibition, as they have also been recently described in
metastatic ER+ breast cancer as a putative resistance mecha-
nism to endocrine therapy (78).
Lineage Plasticity
Phenotypic switching, or cell plasticity, is a tumor-escape
mechanism that allows cells with the same genotype to
acquire diverse phenotypes in response to adverse tumor
microenvironment (TME), such as hypoxia, inflammation,
or exposure to targeted therapy. In the latter, this enables
cells to proliferate independently of initial oncogenic driv-
ers and promotes tumor resistance. For instance, resistant
drug-exposed cells have been shown to regain sensitivity to
the same drug after drug holidays, which suggests that non-
genomic mechanisms of resistance are involved (79, 80). Cells
could lose their epithelial phenotype and acquire mesenchy-
mal characteristics, a process called EMT. This is induced by
epigenetic modifications, such as upregulation of the histone
methyltransferase enhancer of zeste homologue 2 (EZH2) or
the RE1-silencing transcription factor (REST). Transformed
cells feature increased migration and invasive potential, being
highly refractory to targeted therapy. Proposed mechanisms
are decreased levels of proapoptotic proteins and increased
drug efflux due to upregulated ABC-binding cassette trans-
porters. EMT has been described at failure of EGFRi in
EGFR-mutated NSCLC (81). Lineage plasticity was also
shown in breast cancer failing fulvestrant, where ARID1A-
inactivating mutations promoted a phenotypic switch from
ER-dependent luminal cells to ER-independent basal-like/
stem-like cells (82). Cell transdifferentiation, a phenotypic
transformation from adenocarcinoma to squamous or neu-
roendocrine carcinoma, has also been reported in 3% to 14%
of patients with EGFR-mutated NSCLC after EGFRi and in
around 17% of prostate cancer failing abiraterone/enzalu-
tamide. Neuroendocrine transformation is preceded by the
concomitant inactivation of TP53 and RB1, but this is not
enough to induce lung cell transformation. Additional fac-
tors are necessary, such as MYC, BCL2 overexpression, and
AKT overactivation (83, 84).
Loss of Target or Target Dependencies
Repairing Tumor Vulnerabilities. In tumors with DNA dam-
age repair alterations, cells rely on PARP-mediated DNA
repair to survive. In tumors with frameshift or nonsense
mutations of BRCA1/2, PALB2, and RAD51C/D treated with
PARP inhibitors (PARPi), reversion mutations could restore
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Aldea et al.
the original open reading frame and thus regain protein func-
tion (85, 86). Reversion mutations emerge as a mechanism of
resistance to platinum agents as well, being more frequently
found in platinum-refractory/resistant than in platinum-
sensitive ovarian tumors. In patients with high-grade ovarian
carcinoma, the detection of reversion BRCA mutations in the
liquid biopsy before the start of rucaparib has been correlated
with a lower progression-free survival (PFS; ref. 87). Another
example of repairing tumor vulnerabilities is the loss of tar-
get. For instance, the loss of PARP1 function by mutations
in the DNA binding domain of PARP or by increased PARyla-
tion of PARP, which prevent PARP trapping, drives resistance
to PARPi (29). Also, primarily ER+ breast tumors may lose
the estrogen receptor at relapse, which is predictive for poor
response to subsequent endocrine therapy (88).
Other Mechanisms of Resistance
Upregulation of genes encoding P-glycoprotein efflux
pumps could result in increased efflux of drugs outside the
tumor cell, if drugs are substrates of the P-glycoprotein. Some
examples are PARPi or ALKi (with the exception of alectinib;
refs. 29, 89).
Transient adaptive mutability has recently been described
in colorectal cancer cell lines. In response to EGFRi and BRAFi,
cancer cells experienced a downregulation of m ­ ismatch-repair
proteins, decreased homologous recombination (HR) profi-
ciency, and increased oxidative stress production. As a result,
cells transiently increased their mutational load as a stress-
response mechanism. This process was reversed once the
tumor regained its abnormal growth capacity (90). Currently,
there is no clinical evidence for this process.
Epigenetic Changes
Aberrant epigenetic modifications to the genome occur
independently of the DNA sequence and could mediate drug
resistance in addition to mutational processes. Epigenetic
changes have been incriminated as key players in the induc-
tion of dormant, quiescent cells, which are mostly nondivid-
ing cells. These cells, called persister cells, constitute a small
fraction of the tumor bulk that acquire stem cell features and
survive despite high drug exposure or unfavorable micro-
environmental conditions. They display a high expression
of the KDM5A gene, which encodes a histone demethylase,
resulting in reduced H3K4 methylation. The knockdown of
KDM5A was shown to reverse the resistance phenotype and
diminished the development of drug-tolerant persisters and
their expansion in cell cultures (79).
Secretion of Soluble Growth Factors
Cancer-associated stromal cells may promote drug resistance
by secretion of soluble factors, such as hepatocyte growth fac-
tor (HGF; ref. 83). HGF has been shown to confer innate resist-
ance to BRAFi in BRAF-mutant melanoma, colorectal cancer, or
glioblastoma cell lines (91), and to promote resistance to alec-
tinib in ALK-positive NSCLC (92). HGF was able to activate
the MET receptor, even in the absence of MET amplifications
or activating mutations. As opposed to the selective ALKi
alectinib, this phenomenon was not observed with crizotinib,
because of its dual inhibition of ALK and MET (92).
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Overcoming Resistance to Targeted Therapy and Immunotherapy
Increased Mutagenesis
The apolipoprotein B mRNA-editing catalytic polypeptide-
like (APOBEC) enzymes, responsible for DNA cytosine deami-
nation, are an important source of mutagenesis that fuel
cancer diversity and subclonal evolution (93–95). APOBEC3B
family member has been found to be upregulated in more than
half of all cancers, and the APOBEC mutational signature has
been shown to be the most prevalent in cancer after aging sig-
natures (96, 97). Moreover, abnormal APOBEC function can
generate late driver mutations as subclonal events (94).
Standard and Investigational
Drugs for Overcoming Resistance
to Targeted Therapy
Overcoming Primary Resistance
Promising results from phase I/II studies are currently avail­
able in the case of EGFR exon 20 insertions, with favor­
able objective response rate (ORR) and duration of response
with mobocertinib (TAK-788), a selective inhibitor of EGFR
and HER2 exon 20 insertion mutations (98), and with ami-
vantamab (JNJ-61186372), a bispecific anti-EGFR–cMET
antibody (99). Bispecific antibodies reduce the expression
of EGFR and MET on the cell surface by promoting their
internalization with subsequent downregulation by lysoso-
mal degradation. They also induce tumor cell apoptosis in
a BIM- and caspase-dependent fashion and show antibody-
dependent cell-mediated cytotoxicity, with natural killer (NK)
cells as effectors (100).
To overcome short-term adaptation mechanisms to tar-
geted therapies, there is a need for concomitant blockade of
feedback loops, such as blocking ER in addition to PIK3CA
in PI3KCA-mutated ER+ breast cancer (101) or EGFR in
addition to BRAF and probably KRAS in BRAFV600E- and
KRASG12C-mutated colorectal cancer, respectively (21, 102).
Overcoming On-Target Resistance
Second-site mutations should be treated with drugs that
have a different structure or a different binding and that are
not subject to the same resistance alteration as the failing
drug. In the case of TKIs, therapeutic strategies include the
switch between drugs of different generations that differ in
size and binding affinity, the switch between type I and II
ATP-competitive inhibitors, an alternance between allosteric
inhibitors and ATP-competitive inhibitors (Supplementary
Table S1; refs. 32–38, 59, 103–112), and, more recently, the
use of bispecific antibodies (113).
For example, in EGFR-mutated NSCLC, the EGFRT790M-
resistant mutation located in the ATP-binding cleft of the
kinase domain is able to restore the ATP affinity of the kinase.
As a result, direct ATP competitors, first- and second-­generation
EGFRi, become ineffective (30). Osimertinib, a third-genera-
tion EGFRi, covalently binds to the C797 residue of the ATP-
binding site and bypasses the change of the T790 residue (114).
Nevertheless, tertiary resistance mutations may emerge under
osimertinib, mainly at the C797 binding site, and further pre-
vent the covalent binding of osimertinib. Other third-genera-
tion EGFRi with similar structures (e.g., rociletinib, olmutinib,
and narzatinib) are likely to show cross-resistance with osimer-
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tinib (81). There are currently no approved treatments for these
tertiary mutations, but very encouraging results were reported
with amivantamab (JNJ-61186372) in the phase I CHRYSA-
LIS trial, where patients with NSCLC failing third-generation
EGFR inhibitors achieved partial responses in 10 of 47 cases,
including 4 with EGFRC797S mutation (113).
In NTRK fusion–positive tumors, the second-generation
TRK inhibitors selitrectinib and repotrectinib are able to over-
come resistance to first-generation TRK inhibitors (entrectinib
and larotrectinib) induced by solvent-front substitutions (39,
108). In preclinical models, TRK xDFG mutations (TRKAG667
and TRKCG696) were shown to mediate resistance to both first-
and second-generation TRK inhibitors (type I inhibitors that
bind to the active conformation state of the kinase), by stabi-
lizing the kinase in an inactive DFG-out conformation. How-
ever, type II inhibitors (cabozantinib, ponatinib, and foretinib)
were able to overcome this type of resistance, as the inactive
state of the kinase facilitates their access to the allosteric “back
pocket” of the DFG motif (115).
Another strategy is inducing degradation of target pro-
teins, exemplified by selective ER degraders (SERD) for ER
downregulation, or proteolysis-targeting chimeras (PROTAC)
that act in conjunction with the ubiquitin-proteasome sys-
tem (116, 117). Although the technology of PROTACs is still
maturing, this is highly promising for undruggable, resistant
targets (117).
Overcoming Bypass Resistance
Bypass tracks warrant a dual blockade of the founder
mutation and the acquired resistance alteration. This strategy
has proved to be safe and effective in the phase I TATTON
trial evaluating savolitinib, a MET inhibitor, in combina-
tion with osimertinib in EGFR-mutated NSCLC (118). In
individual case reports, acquired fusions have also benefited
from a dual blockade (refs. 56, 61, 63, 71, 73, 119–124; Sup-
plementary Table S2). Also, many drug combinations have
proved to reverse resistance in in vitro studies, but clinical
validation is pending (Supplementary Table S2). The ongo-
ing phase II ORCHARD study (NCT03944772) evaluates
combination strategies for bypass tracks in patients with
NSCLC failing first-line osimertinib. Bispecific antibodies
are also promising treatment strategies. In preclinical stud-
ies on wild-type and mutant EGFR NSCLC with c-MET
pathway activation, the bispecific antibody targeting EGFR
and c-MET (JNJ-61186372) demonstrated antitumor activ-
ity through target inhibition, enhanced a­ ntibody-dependent
cell-mediated cytotoxicity, and FcγRIIIa binding (125). A
phase I clinical trial is currently ongoing (NCT02609776).
The prevention of acquired resistance is another valuable
strategy. In BRAFV600E-mutated melanoma or NSCLC, MEKi
are combined with BRAFi to prevent MAPK pathway reac-
tivation, which frequently occurs under single-agent BRAFi
(64, 126). Interestingly, aside from increasing efficacy and
preventing resistance, the combination of MEKi and BRAFi
also decreases cutaneous toxicities observed with paradoxical
MAPK activation under BRAFi monotherapy (127, 128).
Lineage Plasticity
Histologic transformation is treated with chemotherapy or
chemoimmunotherapy. Potential novel therapies suggested
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Hypoxia

Bevacizumab
VEGFA
VEGFR
TKI
T helper HIF1α
17

IL17
Proangiogenic factors CAFs, M2-TAM IL6/STAT3 Acquisition High apelin
VEGF recruitment activation of stemness
APLNR
Angiopoietins
PDGF G-CSF Proangiogenic
FGF1/2 factors IL6 MAPK signaling
HGF blockade
EPC EC
MDSC VEGFA
MAPKi
POLR1D1 amplification High IL8 expression Vasculogenic mimicry Normal vessel co-option

Bevacizumab Sunitinib

VEGFA STOP

IL8
VEGFA

Chr 13 CRC cell

IL8 blockade
proliferation

Figure 2. Potential mechanisms of resistance to antiangiogenic drugs and proposed ways of overcoming resistance. Chr, chromosome; CRC, colorectal
cancer; EC, endothelial cell; EPC, endothelial progenitor cell; FGF, fibroblast growth factor; HGF, hepatocyte growth factor; IL, interleukin; MAPKi, MAPK
inhibitors; M2-TAM, M2 tumor–associated macrophages; MDSC, myeloid-derived suppressor cells; TKI, tyrosine kinase inhibitors.

by preclinical data are targeting factors involved in lineage
plasticity, such as blocking epigenetic factors (e.g., EZH2
inhibitors), antiapoptotic proteins (e.g., navitoclax, a BCL2
inhibitor), or stem cell markers (e.g., SOX2 inhibitors; refs.
83, 84).
Other
Overcoming resistance to PARPi is a novel field where only
preclinical data are available so far. In tumors with restored
HR or restored replication fork protection, the combina-
tion of PARPi with ATR inhibitors (ATRi) might overcome
PARPi resistance by hindering ATR-dependent mechanisms
of proper HR/replication fork functionality. Moreover, the
association between PARPi and ATRi might resensitize cells
with combined disruption of BRCA1 and TP53BP1 or REV7
to PARPi (85).
As multiple oncogenic pathways may use the same key
regulators of mRNA translation, the inhibition of these
regulators may overcome resistance emerging from the inhi-
bition of their upstream pathways. An example is the case
of eIF4E for RAS/RAF and PI3K pathways, where resistance
to BRAF, MEK inhibitors, or combinations, either ERK-
dependent or ERK-independent, results in increased eIF4E
complex formation, with subsequent signal transduction. It
is hypothesized that combinations of BRAF signaling with
drugs targeting eIF4E complex formation might overcome
most of the resistance mechanisms arising from BRAF/MEK
inhibition (25).
Mechanisms of Resistance to
Antiangiogenic Targeted Therapies
Resistance to antiangiogenic drugs is still poorly under-
stood. Important interplayers are both the tumor and the
TME, with treatment-induced hypoxia influencing both
compartments. Tumor cells adapt to VEGF blockade and
hypoxia by redundancy in angiogenic signaling and activa-
tion of compensatory signaling pathways of angiogenesis
(Fig. 2; ref. 129). This explains why antiangiogenic drugs
targeting different tyrosine kinases are effective even in
sequential use (130). Hypoxia promotes the recruitment of
­cancer-associated fibroblasts (CAF) or M2–tumor-associated
macrophages (M2-TAM), which can drive angiogenesis by
activation of endothelial cells and their bone marrow–derived
precursors (131). Stromal cell–mediated resistance to anti-
VEGF therapy includes an increase of T helper (TH) type 17
tumor infiltration with release of IL17. In murine models,
this resulted in higher G-CSF secretion by CAFs and G-CSF–
dependent myeloid-derived suppressor cell (MDSC) activa-
tion and recruitment in the TME. Consequently, there was

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Overcoming Resistance to Targeted Therapy and Immunotherapy
a secretion of G-CSF–inducible angiogenic factors indepen­
dent of VEGF. Pharmacologic inhibition of TH17 rendered
treatment with VEGF antibodies effective, suggesting that
immunomodulation might have a role in overcoming resist-
ance of antiangiogenic drugs (132). Hypoxia also drives
cancer cells to undergo metabolic changes to face nutrient
starvation and poor oxygenation. It may even promote the
acquisition of stem-like properties, with increased VEGF
production, enhanced migratory properties, and aggressive-
ness, thus limiting treatment efficacy (133–135). Secondary
resistance with anti-VEGF treatment was associated with
increased expression of apelin (APLN) in preclinical xenograft
models of ovarian cancer. Patients with ovarian cancer and
high APLN expression had a significantly shorter disease-
free survival than those with a low APLN expression (136).
APLN binds to APLN receptor and activates MAPK signaling,
thus resulting in endothelial progenitor cell proliferation
and new vessel formation (137). Also, in vitro experiments
showed that resistance to VEGF inhibitors may be medi-
ated by IL6/STAT3 activation and overcome by IL6 blockade
(138). Bevacizumab resistance has been associated in two
cases with a focal amplification on chromosome 13q12.2,
reported in 8.7% of patients with metastatic colorectal cancer.
This is explained by the amplification of POLR1D1, which
acts as a potential driver and upregulates VEGFA, an impor-
tant promoter of angiogenesis (139). In the case of TKI,
patients with renal cancer refractory to sunitinib were found
to have increased IL8 expression. This was confirmed in xeno-
graft models, where sunitinib-resistant tumors had high IL8
tumor secretion, whereas IL8 blockade resensitized tumors
to sunitinib (140).
Sustained tumor angiogenesis may occur via VEGF-inde-
pendent pathways, including normal vessel co-option or
vasculogenic mimicry. In glioblastoma, hypoxia induced by
bevacizumab may rapidly trigger adaptive mechanisms and
drug resistance via vasculogenic mimicry, a process where
tumor cells acquire endothelial-like properties and develop
vessel-like structures (141). In response to antiangiogenic
drugs, primary resistance occurs most likely in tumors with
alternative neovascularization, whereas in tumors with high
VEGF dependency, such as in clear cell renal cell carcinoma,
resistance is often acquired (142).
Mechanisms of Resistance to
Immune-Targeted Therapies
Approved immune-targeted therapies for cancer include
antagonistic mAbs targeting immune checkpoints such as
CTLA4, PD-1, and PD-L1 with the aim of modulating the
antitumor T-cell immune response. The mechanisms of
resistance of such T-cell immune modulatory drugs can, to
some extent, speak to the mechanisms of resistance to bispe-
cific T-cell engagers and chimeric antigen receptor T (CAR-T)
cells approved for CD19+ B-cell leukemias and lymphomas.
Resistance to immune-checkpoint targeted immunotherapies
[immune checkpoint blockade (ICB)] has been reported to be
driven by genomic and nongenomic mechanisms, where the
tumor–host–microenvironment relationship has an essential
role. Defects or alterations in the processes orchestrating this
relationship could be divided into three broad categories,
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which relate to the cancer cell–intrinsic biology, the impact of
this cancer cell on the phenotype of the TME, and the biology
of the host (refs. 2, 3, 5, 143; Fig. 3).
Cancer Cell–Intrinsic Mechanisms of Resistance
Neoantigen Loss
The proof that neoantigen presentation drives immuno-
therapy sensitivity lies in cancers with secondary resistance to
immunotherapy, which lose their initial antigen presentation
(Fig. 3A). Following adoptive T-cell transfer therapy, it was
shown that melanoma may undergo dedifferentiation with loss
of the melanocyte differentiation antigen (144). Of note, neo-
antigen loss has been described even in early-stage, untreated
melanoma and NSCLC, caused by promoter hypermethylation
of genes coding neoantigens, or by copy-number loss or chro-
mosomal deletions of truncal alterations (145–147).
Defective Neoantigen Presentation
Disrupted antigen presentation may affect ICB efficacy.
For instance, a comparison between squamous lung car-
cinoma and adenocarcinoma with similar predicted neo-
antigens showed that squamous lung carcinoma is more
prone to immune escape by downregulating HLA class I
genes (148). Other examples are loss of function (LOF) of
β2-microglobulin, another component of MHC class I; dys-
regulation of proteins involved in the proteolytic degradation
of antigens (PSMB5); of transporters that pump the anti-
genic fragments across the endoplasmic reticulum (TAP1,
TAP2, and TAPBP); or disruption of IFN–JAK–STAT signal-
ing, which normally promotes MHC I expression on the cell
surface (refs. 147, 149, 150; Fig. 3B and C). Interestingly,
only B2M LOF mutations were found to be significantly
enriched in nonresponders versus responders in two inde-
pendent cohorts, in contrast to mutations in IFNGR1, JAK1,
JAK2, STAT2, and TAP1/2 found in both nonresponders and
responders (150). Nevertheless, B2M mutations were reported
in 24% of microsatellite instability–high (MSI-H) colorectal
cancers, where they did not preclude response to ICB (151).
B2M LOF or downregulation has also been shown to cause
acquired resistance to ICB in melanoma, NSCLC, and MSI-H
colorectal cancer (152–154).
Neoantigen Intratumoral Heterogeneity
T-cell responses are effectively elicited by clonal neoantigens,
as opposed to subclonal neoantigens and intratumoral hetero-
geneity (ITH; ref. 148). This may explain why TMB, which does
not distinguish between clonal and subclonal neoantigens,
is only imperfectly correlated with ICB responses. Notably,
patients lacking a durable benefit under anti–PD-1 were found
to have higher ITH scores than patients with durable clinical
benefits. The acquisition of subclonal neoantigens is induced
by abnormal APOBEC activity (mutational signature 2) and
favored by chemotherapy and radiation. This might contribute
to immune escape by the outgrowth of T cells that act against
only a limited tumor cell fraction (94, 148, 155).
IFN Signaling Defects
Copy-number alterations and mutations within the IFNγ
pathway have been identified in 75% of melanomas with
primary resistance to anti-CTLA4 inhibitors (156, 157).
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Cancer cell Cancer cell and/or Tumor stroma Host

stroma

Primary Primary Primary Primary

PD-L1 = 0 TGFβ Organ location (visceral) LDH
TMBlo IL6 CD3/CD8 (d)NLR
MSS (CRC, ...) IL8 PD-L1 Eosinophils
EGFRmut (NSCLC) Microbiome
ALK fusion (NSCLC)
STK11mut Secondary
WNT/β-catenin TIM3 upregulated on
upregulation CD4/8
PTEN loss

Secondary
JAK1/2mut (melanoma)
IFNGR1/2 (melanoma)
IRF1mut
B2Mmut
Loss of heterozygosity
Subclonal elimination

A Neoantigen loss B Defects of the antigen processing C Abnormal IFNγ signaling

machinery

Gene silencing
T cell T cell
MHC-I
INFγ
MHC-I with receptor
PD-L1
antigen STOP STOP

MHC-I JAK STOP

B2M JAK
Tumor cell Antigen Proteasome
+
Tumor STAT
cell IFN genes
TAP
A T G C GA C T
Dedifferentiation

D Aberrant genomic signaling E Coinhibitory checkpoints F Immunosuppressive TME

Immunosuppressive TIGIT Treg

cytokines
WNT WNT BTLA
Frizzled
MDSC
LAG3

TIM3

M2-TAM TGFβ
CTLA4
β-catenin
PD-1 IL10 IL35

Figure 3. Most commonly described mechanisms driving resistance to immunotherapy. A, Neoantigen loss. B, Defects of the antigen processing
machinery. C, Abnormal IFNγ signaling. D, Aberrant genomic signaling. E, Coinhibitory checkpoints. F, Immunosuppressive TME. CRC, colorectal cancer;
IL, interleukin; IFNy, interferon gamma; MDSC, myeloid-derived suppressor cells; MHC I, major histocompatibility complex I; M2-TAM, tumor-associated
macrophages type 2; MSS, microsatellite-stable; mut, mutation; Treg, regulatory T cells.

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Overcoming Resistance to Targeted Therapy and Immunotherapy
Less frequently, LOF of JAK1 and JAK2 has also been shown
to abrogate IFNγ signaling and IFN-induced inhibition of
growth, described in both primary and acquired resistance
to ICB (152, 156, 158, 159). Deleterious JAK1 and JAK2
mutations are enriched in tumors with MSI, notably in
endometrial cancer (35% of immune-naïve patients had JAK1
mutations; refs. 158, 160, 161). Interestingly, JAK2 and type
I IFN genes are frequently codeleted with CDKN2A, due to
their relative proximity on chromosome 9p, suggesting that
CDKN2A loss may be a biomarker indirectly associated with
immunotherapy resistance (162, 163). Patel and colleagues
identified 13 IFNγ-induced genes and three TNFα-induced
genes that could foster resistance to ICB (149). Also, the LOF
of the APLNR gene, which normally encodes a rhodopsin-like
receptor with roles in blood pressure regulation, has recently
been shown to reduce immune response by IFNγ modulation
via JAK1 (149). IFN secretion might also be impaired when
STING and/or cGAS expression is silenced through epige-
netic hypermethylation processes (164).
Oncogenic Signaling with T-cell Exclusion
Tumors lacking CD8+ T cells are termed “cold tumors”
and have been associated with a lack of response to ICB. Such
tumors do not display an inflammatory, chemokine signature
and generally lack negative regulators, such as PD-L1, regula-
tory T cells (Treg), or indoleamine-2,3-dioxygenase (IDO; ref.
165). The lack of effector T-cell infiltration could be mediated
by specific oncogenic signals, such as tumor-intrinsic active
β-catenin signaling, loss of PTEN, STK11/LKB1, or KEAP1 aber-
rations, or because of IFN signaling defects (JAK1/2 and B2M
LOF mutations; refs. 166, 167). Active β-catenin signaling was
shown to promote T-cell exclusion by failure of T-cell priming,
with resistance to anti–PD-L1/anti-CTLA4 mAb therapy in a
melanoma mouse model (166), as well as resistance to T-cell
adoptive transfer and vaccination (168). The activators of the
β-catenin pathway could be activating mutations of CTNNB1,
overexpression of specific WNT ligands or Frizzled receptors,
or inactivating mutations of pathway inhibitors, such as the
AXIN1 gene (Fig. 3D; ref. 166). Biallelic loss of PTEN was
reported to mediate resistance to ICB (169, 170), possibly
by inducing an increased expression of immunosuppressive
cytokines and the recruitment of suppressive immune cells via
VEGFR (171–173). STK11/LKB1 and KEAP1-mutant or -defi-
cient NSCLC has been associated with impaired IFN signaling
(167). Patients with NSCLC presenting STK11/KEAP1 aberra-
tions had inferior PFS with combined chemoimmunotherapy
compared with those without, and there has been no observed
benefit from the addition of ICB to chemotherapy (174). The
prognostic effect of STK11–KEAP1 was further confirmed in a
large real-world data cohort of patients with advanced NSCLC,
but not its predictive value (175). Notably, various KEAP1-
driven comutations (STK11, SMARCA4, and PBRM1) have been
associated with a lack of response to ICB despite a high TMB
in lung adenocarcinoma (176). In KRAS-mutant NSCLC cell
lines with STK11/LKB1 loss, STING expression was markedly
silenced by hyperactivation of DNMT1 and EZH2, thus facili-
tating immune escape and explaining why such tumors are
often refractory to ICB (177).
Certain clonal driver alterations associate with an increased
immunosuppressive TME and low CD8+ T cell/Treg ratios in
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the tumor (156). In a retrospective study of 551 patients with
oncogene-addicted NSCLC receiving ICB monotherapy, some
patients experienced tumor regression, but generally the effi-
cacy was lower as compared with the KRAS group. Notably,
patients with ALK-positive NSCLC did not obtain any objec-
tive response (178). Immunotherapy responses seem to differ
between EGFR mutation subtypes when compared with EGFR
wild-type patients, with lower ORR and overall survival for
EGFR exon 19 deletion and similar outcomes for EGFRL858R
substitution. A possible explanation is the higher TMB
observed among EGFRL858R-mutated tumors as compared
with EGFR exon 19–mutated tumors (179). Also, oncogenic
signaling, notably the RAS–MAPK and PI3K–mTOR path-
ways, stimulates the eIF4F eukaryotic translation initiation
complex, which regulates the translation of STAT1 mRNA
and mediates the induction of PD-L1 expression (180).
Tumors with increased levels of chromosomal instability
activate the cGAS–STING pathway through chronic release
of cytosolic DNA, but they may escape the immune system
by suppressing downstream type I IFN signaling and instead
upregulating the alternative NF-κB pathway. This leads to
paradoxical protumorigenic effects and the recruitment of
immunosuppressive cells (164, 181). A similar immune eva-
sion may occur in HR-deficient tumors, where high levels
of DNA damage may activate the ATM–TRAF6 alternative
STING pathway, resulting in IL6 and TGFβ production, with
M2-TAM and Treg recruitment (182, 183).
An interesting biomarker is polybromo-1 (PBRM1) gene
LOF mutations, clinically validated as a predictor of ICB
response in clear cell renal cell carcinoma in an independent
cohort of patients treated with nivolumab within a rand-
omized clinical trial (184). Nevertheless, in a retrospective
analysis of 2,764 patients with NSCLC who received ICB as
a second or later line of therapy, patients with PBRM1 muta-
tions (n = 84 patients, 3%) had worse survival than patients
with wild-type PBRM1, despite associating with a significantly
higher TMB (185).
Resistance to Lysis of Metastatic Tumor Cells
In a prostate cancer model, both early and metastatic
tumors induced cytotoxic T cells after irradiation, but only
primary cancer cells were preponderantly eliminated by
immunotherapy, whereas metastatic cells were resistant to
cytotoxic T cell–induced cell lysis. Despite IFN-induced MHC I
expression on metastatic cells, these cells maintained resist-
ance to lysis (186).
Stromal Mechanisms of Resistance
Location of Metastasis
In patients with melanoma, the presence of visceral metas-
tasis was shown to be associated with primary resistance
to anti–PD-L1, as opposed to lung, lymph node, and soft-
tissue metastases (187). In patients with both melanoma
and NSCLC, patients with liver metastases had worse clinical
outcomes with ICB, compared with patients without liver
metastases (188, 189). Liver metastases exhibit an immu-
nosuppressive TME, and it has been suggested that they
may even promote systemic immunosuppression (188). In
melanoma, patients with liver metastases had higher levels of
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eotaxin 2, interferon gamma-induced protein 10 (IP10) and
IL8, MMP8 and HIF1α, as compared with patients without
liver metastases (188). Brain metastases also display an immu-
nosuppressive TME, with low tumor-infiltrating lympho-
cytes (TIL), increased M2-TAM and neutrophils, inhibition of
dendritic cell maturation, TH1, and leukocyte extravasation
signaling pathways, as well as a significant reduction in the
proinflammatory cell adhesion molecule vascular cell adhe-
sion protein 1 (VCAM1), responsible for leukocyte adhesion
to inflammatory sites (190–192).
Expression of Multiple Inhibitory Regulators
In addition to PD-1 and CTLA4 expression, infiltrating
CD8+ T cells may have increased expression of T-cell immu-
noglobulin mucin-3 (TIM3), lymphocyte-activation gene 3
(LAG3), and B and T lymphocyte attenuator (BTLA; Fig.
3E). These regulators are also expressed by other immune
cells, such as Tregs or cells of innate immunity (193). The
upregulation of coinhibitory molecules has been described
as a mechanism of acquired resistance to single-agent anti–
PD-1/PD-L1, causing an impairment of T cells to respond to
polyclonal activation. It has been shown that multiple inhibi-
tory checkpoints were acquired gradually, with PD-1 as an
early event and LAG3/BTLA expression as a late event. This
mechanism could drive resistance to ICB even in PD-1 high-
expressing tumors (194, 195).
Exhaustion of Cytotoxic T Cells
The chronic exposure of cytotoxic T cells to high antigen
load might severely impair their inflammatory and cytotoxic
function, from a gradual loss of effector function to an
exhausted phenotype (194). This exhausted phenotype can
present abnormal DNA methylation (196), high PD-1, and
LAG3 expression (148).
Immunosuppressive Microenvironment
Tregs have important roles in preventing autoimmune
reactions by secretion of immunosuppressive cytokines, such
as TGFβ, IL10, and IL35. When present in the TME, they
restrict T-cell responses (197). TGFβ may also be released by
cancer cells or stromal fibroblasts (198). It has been found
to be increased in the bone marrow from bone metastasis
compared with the normal bone, which might explain the
low benefit of ICB in metastatic castration-resistant prostate
cancer (mCRPC) with bone-predominant disease. Moreover,
in mice with bone mCRPC, the association of anti-CTLA4
and anti-TGFβ increased TH1 cells and decreased Tregs in
the tumor-infiltrating T-cell population. The combination
of drugs resulted in an improved tumor response and over-
all survival of mice in comparison with either drug alone
(199). Innate immune cells, MDSCs, and M2-TAM have also
emerged as major regulators of immune responses in cancer,
and they have been shown to negatively affect the activity
of ICB, adoptive T-cell therapy, or dendritic cell vaccination
(Fig. 3F; ref. 3).
Metabolic Pathways
Under aerobic conditions, tumor cells rapidly process glu-
cose into lactic acid, a process called the Warburg effect. This
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results in increased release of H+ ions into the TME. The
abnormal tumor vasculature is unable to effectively clear H+
ions, which promotes an acidic tumor milieu. Consequently,
TILs and cytokine production are inhibited, while the accu-
mulation of immune-suppressive cells is favored, thus lead-
ing to immune resistance (193, 200). Another mechanism
is the production of the tryptophan-metabolizing IDO by
tumor cells in response to inflammation. IDO has immuno-
suppressive functions that limit effector T-cell activity and
engage mechanisms of immune tolerance (165).
Immune Heterogeneity
In the TRACERx and LATTICe-A cohorts of primary lung
adenocarcinoma, the presence of immune cold regions within
a tumor was shown to be an independent predictor of cancer
relapse, even in the case of an average increased immune infil-
tration of the tumor. The comparison between cold and hot
regions reflected different cancer subclones, possibly due to
immunoediting (201). An increased heterogeneity was found
in the immune microenvironment of different regions of the
same tumor in nearly one third of early NSCLC tumors (145).
Characteristics of the Host
Age and Sex
Growing evidence points out that younger and female
patients respond less to immunotherapy than older and
male patients. It has been shown that younger and female
patients are prone to stronger immunoediting early in their
disease evolution, with depletion of potentially immunogenic
mutant peptides and MHC presentation of less immuno-
genic, more invisible peptides to the immune system (202).
Microbiota
Specific gut bacteria are associated with ICB response and/
or toxicity. For instance, in patients with melanoma treated
with ICB, baseline gut microbiota enriched with Faecalibacte-
rium and other Firmicutes was associated with enhanced anti-
tumor responses (203, 204). Also, a molecular mimicry was
recently observed between tumor MHC class I antigens and
an enterococcal prophage, which correlated with long-term
responses under anti–PD-1 therapy in renal and lung cancers
(205). The composition of gut microbiota may affect immune
responses of distant lesions, especially via the blood circula-
tion of microbial metabolites produced in the colon through
bacterial fermentation of dietary fibers. High blood levels of
short-chain fatty acids, such as butyrate and propionate, have
been shown to associate with CTLA4 blockade resistance and
with an increased proportion of Tregs. In patients treated
with ipilimumab, high blood levels of butyrate limited the
accumulation of memory and ICOS+ CD4+ T cells, as well as
IL2 impregnation (206).
Host–Tumor Interactions
Blood Immune Cells
Relative eosinophil count (REC) <1.5% was associated with
a worse survival in patients with melanoma treated with pem-
brolizumab, as compared with REC ≥ 1.5% (187). Interestingly,
lymphopenia per se is not associated with primary resist-
ance to anti–PD-L1 but rather the proportion among other
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Overcoming Resistance to Targeted Therapy and Immunotherapy
cells (187, 207), notably neutrophils, within a neutrophil-
to-lymphocyte ratio (NLR; refs. 208–210). The negative
impact of NLR seems to be limited to patients with stable
disease (SD) and not to patients with progression or objective
response as best response (211). Also, the proportion of neu-
trophils among total leukocytes minus neutrophils (dNLR),
combined with lactate dehydrogenase (LDH) in the Lung
Immune Prognostic Index (LIPI) score, has been associated
with primary resistance to anti–PD-L1 (212).
Blood Cytokines
IL6 has prognostic value, with high IL6 levels being cor-
related with a worse survival in patients with metastatic mela-
noma treated with ICB or chemotherapy (213). IL8 has been
related to the level of infiltrating myeloperoxidase-positive
(MPO+) and/or CD15+ monocytes and neutrophils in tumors
(214). IL8-high patients, as defined by serum IL8 values above
23 pg/mL, represent around 25% to 30% of patients with
advanced cancer. Interestingly, IL8 serum levels are independ-
ent from PD-L1 tumor expression by IHC and TMB.
LDH
Serum LDH value above normal threshold is a strong pre-
dictor of primary resistance to ICB (187, 209, 215, 216). LDH
leads to accumulation of lactates in the TME, which leads
to modifications of immune cells toward a tolerogenic phe-
notype (217) and negatively affects the type I IFN pathway
by inhibition of retinoic acid–inducible gene 1 (RIG1; refs.
218, 219).
Of note, patterns of primary resistance differ among
tumor types even for the same immunotherapy (Supple-
mentary Table S3; refs. 145, 157, 158, 167, 174, 178, 185,
220, 221). Tumor types with a low likelihood of response
to ICB, for instance, sarcoma or prostate cancer, are usually
cold tumors with a low TMB. Responses may be exception-
ally seen, for instance in sarcomas with B cell–rich tertiary
lymphoid structures (221), or MSI-H or CDK12 biallelic
alterations in prostate cancer (222, 223). In these cases, pre-
dictors of response should be searched. Conversely, in tumor
types with a high likelihood of response to ICB, such as
melanoma, Hodgkin lymphoma, and MSI-H tumors, predic-
tors of resistance should be interrogated, and these usually
consist of defects of antigen presentation and IFN signaling.
Concerning acquired resistance, defects of antigen presenta-
tion, loss of antigen, IFN signaling defects, and overexpres-
sion of coinhibitory checkpoints seem to be the central
players in progression, without evident differences among
tumor types at the actual state of knowledge (144, 146, 147,
152, 153, 159, 170, 195).
Overcoming Resistance
to Immunotherapy
Overcoming resistance to immunotherapy is guided by sev-
eral main pillars: (i) increase tumor visibility to the immune
system, (ii) enhance T-cell infiltration, (iii) remove barriers
from T-cell intratumoral trafficking, (iv) enhance immune
system function, (v) address genomic and epigenetic abnor-
malities, and (vi) adapt to the host (Fig. 4).
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Increase Tumor Visibility to the Immune System
Making the tumor visible to the immune system is essential
for T-cell activation and migration into the TME. One major
strategy is the induction of “immunogenic cell death” by
tumor cytotoxic therapies, including chemotherapy, irradia-
tion, targeted therapies, epigenetic drugs, or oncolytic viruses
or peptides (193, 224). Following cell death, tumor antigens,
proinflammatory cytokines, damage-associated molecular
patterns (DAMP), calreticulin, and ATP are released in the
TME (Fig. 4A). This may favor antitumor immune responses
by recruiting antigen-presenting cells (e.g., dendritic cells),
and inducing their maturation (e.g., HLA-I, CD80/86 upregu-
lation) and subsequent T-cell activation (e.g., upregulation of
costimulatory checkpoints; refs. 224, 225). DNA lesions may
activate the cGAS–STING pathway, which can increase the
immunogenicity of the tumor by activating the type I IFN
pathway (164). In addition, some chemotherapies can deplete
some suppressive immune cells such as Tregs or MDSCs
(193). Chemoimmunotherapy improved survival outcomes
in comparison with chemotherapy alone in advanced lung
cancer, regardless of PD-L1 expression (226). Interestingly, it
seems to reduce the risk of early progression seen with ICB
alone. It is unknown if a shorter administration of chemo-
therapy would be more effective when combined with ICB in
order to prevent lymphopenia or the generation of subclonal
antigens. Possibly, the next step will be the administration
of antibody–drug conjugates (ADC) in combination with
ICBs, as they would spare lymphocytes. Successful examples
of combining antiangiogenic drugs with immunotherapy
are especially seen in renal cell carcinoma (e.g., axitinib plus
pembrolizumab, axitinib with avelumab), where in 90% of
cases there is a deregulation of the VHL gene, which regulates
HIF1α and HIF2α (227). Also, antiangiogenic drugs have
been shown to modulate the expression of inhibitory check-
points on CD8+ T cells and the levels of Treg-infiltrating
tumors (228, 229). Radiotherapy has been shown to induce
an abscopal effect, first described in a case report in 2012
(230), but this effect has not been reproduced in large studies.
Concerns were raised on the effectiveness of single-site irra-
diation, as it fails to address intertumoral and immune het-
erogeneity (225, 231). New strategies of combining ICB with
multiple site irradiation are being tested (225). It has been
suggested that nodal irradiation should be avoided when
used to enhance ICB effects, as it may compromise T-cell
proliferation and activation (232). Furthermore, radiation-
induced STING activation may cause resistance by recruiting
MDSCs via the CCR2 pathway, for which a possible abrogat-
ing strategy might be the use of anti-CCR2 antibodies (233).
Oncolytic viruses are used to preferentially infect cancer cells,
where they trigger immune responses by viral replication,
cancer cell release of DAMPs, pathogen-associated molecular
patterns, and type I IFNs, with subsequent increase of TILs
and even induction of abscopal effects (234–236). Oncolytic
peptides may act by mitochondrial membrane permeabiliza-
tion, which triggers a caspase-independent necrosis (237).
Enhance T-cell Infiltration
In addition to poor tumor immunogenicity, the lack
of T-cell infiltration could be caused by inefficient T-cell
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A Increase tumor visibility B Enhance T-cell infiltration C Remove TME barriers

Cytotoxic therapies
Chemotherapy Antigens Abnormal
Radiotherapy vasculature,
Targeted therapy Cytokines hypoxia
Oncolytic virus DAMPs

CAR-T cell
Antiangiogenic drugs

Cancer cell Calreticulin

ATP Vasculature
normalization
Immunogenic cell death
TCR
Immune cell

D Enhance T/NK cell function E Unfavorable genomics/epigenetics F Adapt to the host

+ + STOP
A T G C G A C T
Manipulate microbiota

Anti-TIGIT TLR9 agonist Anti-TGFβ

Anti-LAG3 proinflammatory Anti CSF1R Abnormal IFN Abnormal β-catenin
Anti-VISTA cytokines Anti-CCR5 signaling NK agonists?
Anti-TIM3 Anti-CXR2/CXCR Older
INFγ Biallelic PTEN loss male
STING agonists PI3K inhibitors? Younger
Specific oncogenes female
?

Effector T cell NK cell M2-TAM

Epigenetic changes
Epigenetic drugs Drug combinations? Monotherapy?
OX40 agonist

Figure 4. Overcoming resistance to immunotherapy. A, Increase tumor visibility. B, Enhance T-cell infiltration. C, Remove TME barriers. D, Enhance
T-cell/NK-cell function. E, Unfavorable genomics/epigenetics. F, Adapt to the host.

priming or the lack of T-cell attraction. STING agonists,
which activate the STING pathway, promote tumor secre-
tion of type I IFN and proinflammatory cytokines (164).
The role of chemokines on ICB to attract intratumor T cells
is being investigated in early-phase clinical trials, as certain
chemokines (e.g., CXCL13) may mediate the recruitment of T
cells into the TME (238). The induction of tertiary lymphoid
structure formation in tumors is being investigated using
cytokines, chemokines, antibodies, antigen-presenting cells,
or synthetic scaffolds (239). Other strategies include vaccines
and adoptive cellular therapies (Fig. 4B). Engineered T cells
with CAR or transgenic T-cell receptors (TCR) have shown
modest activity in solid tumors so far, possibly explained
by tumor heterogeneity, which limits the effectiveness of
strategies targeting one single antigen, the lack of cancer-
specific targets for CARs, and the difficulty in matching HLA
antigen for TCRs (240–242). Promising results were seen in
patients with neuroblastoma treated with CAR-T cells target-
ing di­sialoganglioside GD2, showing the potential of CAR-T
cells in solid tumors, when the target is adequate (243).
Why Combination with ICB Is Important
Once non–T cell–inflamed tumors have T-cell infiltrates,
IFNγ signaling will be activated, with subsequent expression
of inhibitory mechanisms, such as PD-L1. For instance, in
mCRPC, ipilimumab failed to induce durable responses in
most patients despite being able to increase T-cell infiltra-
tion in the tumors. This was caused by the overexpression of
compensatory inhibitory pathways, PD-L1 and VISTA (244).
A similar escape mechanism was observed in patients treated
with dendritic vaccines, which led to a significant increase
in PD-L1 expression (245). This provides the rationale that
bringing T cells into the tumor must be accompanied by a
blockade of emerging inhibitory pathways.
Remove Barriers from Intratumoral
T-cell Trafficking
Leaky blood vessels coupled with defective lymphatic drain-
age cause an elevated interstitial pressure in the TME, which
may impair drug and immune cell entry. Moreover, TME cells
exposed to hypoxia produce various cytokines with immu-
nosuppressive effects (VEGF, TGFβ, and prostaglandin E),
whereas tumor cells might acquire highly aggressive stemness
features (193, 246). In addition to promoting immunogenic
cell death, antiangiogenic drugs may normalize tumor vascu-
lature and increase T-cell infiltration in the TME (Fig. 4C).
The spatial distance between T cells and tumor cells might
be minimized by bispecific antibodies that bind T cells with
tumor cells. For instance, an early success was seen with the
bispecific antibody blinatumomab, approved in the t­ reatment

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Overcoming Resistance to Targeted Therapy and Immunotherapy
of B-cell leukemia, which acts by binding CD3 on T cells and
CD19 on B cells (247).
Enhance Immune System Function
Enhancing immune functions focuses on promoting the
infiltration and function of effector T cells and antigen-
presenting cells on inhibiting immunosuppressive cells such
as Tregs and MDSCs, or modifying cytokine production or
cellular metabolism (Fig. 4D; ref. 193).
Boosting effector T cells can be achieved by concomitant
blockade of inhibitory regulatory checkpoints or by activat-
ing stimulatory pathways (193–195). The combination of
anti–PD-1/PD-L1 agents with anti-CTLA4 mAbs was the first
proof of increased efficacy compared with monotherapy in
several cancer types, albeit with the cost of increased toxic-
ity (5). Ongoing clinical trials investigate the combination
of anti–PD-1/PD-L1 or anti-CTLA4 with inhibitors of other
immune-checkpoint regulators, such as LAG3 or TIGIT (5,
193). Recently, the phase II CITYSCAPE trial evaluating
the anti-TIGIT mAb tiragolumab plus the PD-L1 inhibitor
atezolizumab in patients with NSCLC with positive PD-L1
showed an improved ORR and PFS for the combination
therapy, as compared with atezolizumab alone. Efficacy was
higher in the high–PD-L1 subgroup (248). Phase III trials are
ongoing (SKYSCRAPER-01 and SKYSCRAPER-02).
T-cell function can also be enhanced by removing immu-
nosuppressive signals. In case of immunosuppressive TME,
inhibitors of M2-TAM or ICB combinations with anti-TGFβ
therapies may be used (156).
Both innate immunity and adaptive immunity may be stim-
ulated by proinflammatory cytokines (249–251), as nicely illus-
trated in a phase I trial investigating an engineered IL2 receptor
agonist (bempegaldesleukin) in combination with nivolumab
in immunotherapy-naïve patients with advanced solid tumors.
Longitudinal biopsies revealed increased T-cell infiltration and
clonality in patients responding to therapy, with no expansion
of Tregs in the tumor (249). In preclinical models, IL15 agonist
or recombinant human IL2 used in combination with ICB was
shown to be more effective than either monotherapy by stimu-
lating both CD8+ T cells and NK cells (250, 251).
Innate immunity may be stimulated by intratumoral
administration of Toll-like receptor 9 (TLR9) agonists (CpG
olidonucleotides, e.g., SD-101; refs. 252, 253), double-stranded
RNA viruses triggering TLR3 and RNA helicases (254), or by
STING/cGAS activation (164). This might be a rational strat-
egy for tumors that lose MHC, as MHC I–deficient cells are
unable to present the antigen to effector T cells, but they are
susceptible to NK cell–dependent cytotoxicity (255).
Address Genomic and Epigenetic Abnormalities
In mice bearing melanomas with PTEN loss, the addi-
tion of a PIK3β inhibitor to anti–PD-1 blockade was shown
to increase intratumoral T-cell infiltration and to improve
tumor control as compared with each treatment alone (171).
Abnormal IFN signaling is addressed by combinations
of ICB with IFNγ or STING agonists (Fig. 4E; ref. 156).
JAK inhibition was also tested in a phase I trial, but proved
unsuccessful when combined with pembrolizumab, being
associated with reduced peripheral T-cell activation and no
significant changes in intratumoral Treg infiltration (256).
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Conversely, PD-1 blockade resistance caused by JAK1/2 and
B2M inactivating mutations has been successfully reversed in
murine models by TLR9 agonists and CD122-preferential IL2
pathway agonists, respectively (255).
The potential synergy between epigenetic drugs and ICB is
also being investigated by addressing the epigenetic modulation
of tumor neoantigens, chemokines, or inhibiting activation-
induced cell death of T cells (193). For example, promising results
were seen in the ENCORE-601 phase Ib/II trial in patients with
melanoma, NSCLC, or mismatch repair–proficient (pMMR)
colorectal cancer treated with pembrolizumab in association
with a histone deacetylase inhibitor who obtained tumor
responses even after prior immunotherapy failure (257).
Adapt to the Host
Younger and female patients may be better candidates for
drug combinations, as opposed to older and male patients
with higher chances of response to ICB (202). Manipulat-
ing microbiota is undergoing investigation for enhancing
immune responses or managing immune-related adverse
events (Fig. 4F; ref. 258). Studies investigating ICB combined
with LDH inhibitors might be of interest for patients with
high LDH levels, as experimental data showed antitumor
effects of LDHA inhibitors by inducing reactive oxygen spe-
cies and apoptosis (259).
Current Challenges with Tumor-
and Immune-Targeted Therapies
and Future Perspectives
Improving the Characterization of Resistance
Tumors may contain cells that follow different evolu-
tionary tracks, including drug-sensitive cells, drug-tolerant
persister cells, fully resistant cells, and partially sensitive or
weakly resistant cells (79, 260). Tumor response is dictated
by the tumor fraction harbored by these cell subtypes, which
makes a deeper tumor characterization essential for antici-
pating resistance and improving treatment choices (Fig. 5A
and B). We are currently limited in our ability to distinguish
between the clonal expansion of preexistent resistant cells
and acquired resistance (e.g., EGFRT790M might also be found
in patients with treatment-naïve NSCLC; ref. 1). Treatment
effectiveness may differ in these cases, with earlier treatment
failure when resistant cells are present at baseline. To improve
the characterization of tumor heterogeneity, single-cell analy-
sis emerged as a technological breakthrough that may reveal
clonal relationships with high precision (261, 262). However,
the use of single-cell analysis in clinical practice is still limited
by its cost and duration of sample processing.
Liquid biopsy, increasingly used in routine practice, is able
to depict tumor heterogeneity and to reveal the spatial hierar-
chy and temporal distribution of cancer alterations. It effec-
tively identifies resistance alterations (71, 75), and it may
perform better than tissue in the identification of complex,
polyclonal alterations (263, 264). Also, liquid biopsy may
be used to monitor resistance during treatment or minimal
residual disease after curative treatments. However, when cir-
culating tumor DNA shedding is low (e.g., low tumor burden,
isolated central nervous system progression, lung-only ­disease,
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A Tumor cell Primary

composition resistance Resistance

Sensitive cells

Partially sensitive/
weakly resistant cells Acquired
resistance
Mutational and
Fully resistant cells epigenetic changes
Drug-tolerant
persister cells
Drug Time
start

Natural evolution (mutagenesis, epigenetic changes)

Tumor composition in time

B
Early stage Advanced stage Drug resistance Complex resistance

Mutagenesis Mutagenesis Mutagenesis

Tumor mutational burden, subclonal diversity, intratumoral heterogeneity, genomic complexity

Block clonal Block multiple drivers; Block targetable Selective delivery of

drivers earlier prioritize clonal >> resistance alterations; unselective drugs
subclonal, rank alterations use drug combinations if
needed

Inhibit mutagenesis
Promote mutagenesis
Adapt treatment: clonal and subclonal
Eliminate persister cells
monitoring with sequential therapy
Monitor and treat
minimal residual disease

PREVENT Resistance TREAT

Drug selectivity

Figure 5. A, Evolutionary tracks of resistance. B, Proposed strategies to defer and overcome tumor resistance.

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Overcoming Resistance to Targeted Therapy and Immunotherapy
or certain histologic subtypes such as urological cancers; refs.
265–267), more sensitive techniques are required (e.g., whole-
genome sequencing of circulating free DNA; ref. 268).
In the case of immunotherapy, nongenomic mechanisms
of resistance need to be addressed in conjunction with the
genomic profile, where the main limitation is the lack of
validated tools to provide a comprehensive view (2). An
interesting approach is the investigation of genomic or tran-
scriptomic signatures. The tumor–host–microenvironment
interaction could leave specific genomic/transcriptomic
scars, which could be identified by DNA-seq and RNA-seq.
Examples include the T cell–inflamed TME signature, the
transcriptional signatures of fibroblasts, endothelial cells,
TGFβ signaling, and MDSCs and regulatory Tregs (156). The
comparison of genomic/transcriptomic signatures between
responsive and nonresponsive tumors might identify pre-
dictors of resistance. For instance, the innate anti–PD-1
resistance signature (IPRES) was found to be correlated
with primary resistance to PD-1 blockade in patients with
melanoma. IPRES displays enhanced expression of genes
involved in mesenchymal transition, extracellular matrix
remodeling, angiogenesis, cell adhesion, and wound healing.
Notably, the same signature could occur in melanomas with
activated MAPK signaling following treatment with TKIs,
which could explain in some cases the cross-resistance with
anti–PD-1 therapy (269). Another challenge is capturing the
heterogeneity of TME immune infiltration, which might be
addressed by single-cell analysis and radiomic signatures
(e.g., the radiomic signature of CD8-infiltrating T cells; refs.
261, 262, 270).
Nongenomic mechanisms of resistance may also have an
important role in tumors failing targeted therapies, especially in
the absence of clear bona fide genomic alterations responsible
for resistance. In addition to genomic analyses, the integration
of a proteomic and epigenetic characterization may expand our
knowledge and further improve treatment tailoring.
Defer Resistance and Treat Earlier
A growing tumor develops novel alterations and exhibits
increasing genomic instability, clonal diversity, and tumor
heterogeneity, which enhance drug resistance even in the
absence of prior treatments (Fig. 5B; refs. 12, 181, 271). It is
possible that an earlier drug administration might be more
effective than a later administration. In favor of this hypoth-
esis is the efficacy of tamoxifen in the adjuvant therapy of
localized breast cancer, and osimertinib in the first-line set-
ting of advanced EGFR-mutated NSCLC or even in the adju-
vant setting, where it showed an impressive reduction in the
risk of relapse (272–274). The lesser subclonal neoantigens
and lower tumoral burden in earlier stages may also favor an
earlier use of immunotherapy.
Novel-generation drug use in the up-front setting may
prevent the emergence of on-target resistance mechanisms
seen with older-generation drugs, as successfully exemplified
in the FLAURA trial with osimertinib versus first-genera-
tion EGFRi in patients with treatment-naïve EGFR-mutated
advanced NSCLC (273). However, in this trial and others,
deferring resistance alterations is not the only factor respon-
sible for improved clinical outcomes, as the increased intrac-
ranial activity of newer-generation drugs has an essential role
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in oncogene-addicted cancers with increased central nervous
system (CNS) tropism (275–277). Thus, it would be interest-
ing to investigate the role played by deferring resistance when
comparing drugs that have similar CNS activity.
Other strategies to prevent resistance are the simultaneous
inhibition of co-occurring drivers, proved to be effective and
safe in the I-PREDICT trial (278), or the concomitant block-
ade of two proteins of the same oncogenic pathway for which
cancer cells show a strong dependency (279). To maximize the
expected outcomes, selected treatment should target the pre-
dominant cell populations within the tumor, prioritize clonal
rather than subclonal target inhibition, and prioritize accord-
ing to the highest level of actionability of the targets. In the
case of sequential therapies, monitoring clonal and subclonal
alterations may guide subsequent treatment based on the
dominant resistant cell population (Fig. 5B).
Treating Multiple, Highly Resistant Alterations
The treatment of complex, polyclonal aberrations is a major
challenge. The sequential use of TKIs may result in a stepwise
accumulation of mutations that highly increase drug resist-
ance (56, 263, 280). Compound on-target alterations may
induce resistance even to third-generation TKIs. Also, multi-
ple bypass tracks or on-target and bypass alterations might
co-occur in the same patient (59). In these cases, it is currently
unknown if targets should be ranked for treatment prior-
itization, or if drug combinations should be used. With the
increasing number of acquired alterations, multidrug resist-
ance would probably need different strategies. One example
is the administration of nonselective cytotoxic therapies with
preferential delivery in the tumor, such as ADCs. The selective
intratumoral accumulation of ADCs is based on their ability
to target proteins overexpressed in the tumor (281). The novel
HER2-targeted ADC trastuzumab deruxtecan recently showed
increased efficacy in heavily pretreated HER2-expressing or
HER2-mutant solid tumors (282). As tumor heterogeneity is
likely to be increased in this stage, the use of ADCs that harbor
a bystander effect is likely to have more efficacy.
Manipulating the Mutational Process Might
Be More Important than Treating the Genomic
Alteration That Mediates Resistance
If preventing resistance is more effective than treating
resistance, targeting the mutational process that drives can-
cer progression might have a paramount importance. Block-
ing mutagenesis by “therapy by hypomutation” (e.g., A3B
inhibition of the APOBEC complex) would be expected to
prevent subclone diversification and thus limit the aggres-
siveness of tumors and drug resistance (94, 283). The thera-
peutic targeting of ecDNA would share a similar purpose and
is worth further investigation (53). In advanced cancers, such
strategies should be evaluated in conjunction with the block-
ade of tumor drivers, where it may increase the treatment
duration on targeted treatments, while in early cancer stages
potentially as maintenance therapy after local treatments.
A reverse strategy is the use of “therapy by hypermutation,”
by enhancing mutagenesis until cell destabilization and cell
death. This could be useful in advanced, pretreated APOBEC-
high tumors that already achieved hard-to-treat genomic
alterations. Strategies may use agonists of A3, PARP, and ATR
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inhibitors (94, 283). A similar strategy may be used in tumors
with high chromosomal instability, by using agents that
further increase genomic instability (e.g., WEE1 inhibitors in
conjunction with DNA-damaging agents).
Demonstrate the Missing Links between
Oncogenic Stresses and Immune Suppression
Active research is needed to deepen the current under-
standing of immune exclusion and suppression associated
with certain oncogenic pathways, as to be able to reverse this
phenomenon and to integrate immunotherapy in the thera-
peutic armamentarium of oncogene-addicted tumors. In this
domain, research is still in its infancy. Examples of putative
players are the expression of CD73 in EGFR-mutated cancers
that might predict ICB responses (284), the PTEN-loss PI3K
pathway activation that induces PD-L1 expression and pro-
motes resistance to T-cell lysis (285), or the INFα/β-induced
p53 gene activation with subsequent cell apoptosis (286).
Associations between ICB and epigenetic drugs should be
used in a personalized fashion, guided by epigenetic modi-
fications. Epigenetic alterations would need clinical studies
to interrogate and validate a potential predictive role for ICB
response, as certain mutations have been correlated with ICB
response, especially the loss of SWI/SNF components (287,
288). Also, genomic ITH might determine different immune
infiltration patterns. For instance, tumor subregions with
copy-number gain in chromosome 7 were associated with
unfavorable immune composition, with a lack of leukocyte
infiltration and presence of activated neutrophils (289). The
identification of the molecular-associated immune response
in the tumor might be crucial for anticipating resistance pat-
terns and personalizing treatment.
Personalizing Treatment Selection
with Immunotherapy
There is an urgent need to personalize the administration
of immunotherapy, as the blinded use of ICB and investiga-
tional immunotherapies is most likely to fail in a significant
proportion of cases where the target is absent. The most illus-
trative example is the administration of PD-1/PD-L1 inhibi-
tors in tumors with no T-cell infiltration. Another example
is the combination of anti–PD-1 with anti-IDO that showed
promising antitumor activity in phase I–II trials on bio-
marker-unselected patients, but the phase III trial showed no
benefit of the combination in comparison with single-agent
PD-1 inhibitor (290). Would the results be the same in selected
patients with high IDO–expressing tumors? Also, strategies
for enhancing the function of effector T cells are likely to
work in “hot” tumors with T-cell infiltration, but are they
effective in tumors featuring immune exclusion or minimal
immune cell infiltrates? MHC I–deficient cells are unable to
present antigen to effector T cells, but they are susceptible to
NK cell–dependent cytotoxicity, which provides a rationale
for NK-cell stimulation in such cases (255). Such tumors are
also good candidates for CAR-T cell therapy, as CAR-T cells
are able to recognize antigens without MHC, whereas TCR
therapies could better address tumors with lower antigen
densities within the TME (193). Similarly, investigational
agents aiming to overcome acquired resistance to immuno-
therapy are unlikely to succeed without targeting the specific
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Aldea et al.
resistance mechanism. Also, as opposed to targeted thera-
pies, advancements in immunotherapeutic drug discovery
and research are slowed down by the intrinsic limitations of
current preclinical models for testing, which need an intact
human TME in order to be useful (291).
Finding better tumor-agnostic biomarkers to predict
immunotherapy response is essential to find patients
responding to therapy in tumor types not expected to ben-
efit. Tertiary lymphoid structures, recently shown to highly
correlate with immunotherapy responses even in tumors with
low mutational burden, warrant further investigation in a
pan-tumor setting (221). Resistance biomarkers need further
validation and ranking, based on their magnitude of predic-
tion. For example, resistance mechanisms found in nonre-
sponding tumors that were highly expected to respond, such
as MSI-high tumors, should have a high prediction value of
resistance in other tumor types as well.
Patient “immunograms,” first proposed by Blank and col-
leagues (292), should be further developed to integrate the
current state of knowledge (Fig. 6A) and guide treatment
choice (Fig. 6B). Trials investigating a personalized approach
with combination therapies based on biomarkers are ongo-
ing (e.g., ADVISE, NCT03335540; PIONeeR, NCT03833440).
The preliminary results of the PIONeeR trial suggest a predic-
tive value for PD-L1 density on all cell types, PD-L1 tumor
expression, infiltrating cytotoxic T cells, and immunosup-
pressive cell density. Of note, nonresponding patients with
high PD-L1 tumors actually had low PD-L1 density on all cell
types (tumor and stromal; ref. 293).
Better Define Acquired Resistance
to Immunotherapy
An accepted set of criteria to define acquired resistance to
immunotherapy is essential for future drug development.
Many new immunotherapy compounds have failed because
they enroll in clinical trials both patients with primary resist-
ance and patients with acquired resistance to ICB. The lack
of consensus for defining resistance means that patients
with acquired resistance are defined by some as only patients
with partial response/complete response versus others who
also account for stable disease (SD) for a certain period. It
is totally unclear if SD for 3 or 6 months truly represents a
sensitive patient who then acquires resistance. In order to
increase the likelihood of response to new immunotherapy
molecules, we should probably focus more on patients who
truly responded to ICB initially and then developed acquired
resistance. Such patients may still have inflamed tumors that
can be amenable to a new immunomodulation.
Aim to Cure: The Role of Surgery or Local Ablative
Therapies in the Treatment of Metastatic Disease
In oligoprogressive tumors, local therapies are used, as
they allow the pursuit of an effective treatment for the rest of
the lesions and improve survival outcomes (294–296). They
are also discussed in tumor boards in the case of dissociated
response under ICB, but prospective studies are still pending
to confirm benefit (297). The choice between surgery or local
ablative therapies still needs refinement, especially in selected
oligoprogressive patients with a long disease control under
treatment. Surgery remains the most effective treatment for
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Overcoming Resistance to Targeted Therapy and Immunotherapy REVIEW

A B Predicted response to
anti–PD-1/PD-L1 therapy
1
Tumor foreignness 14 2
Tumor mutational burden
13 3

Clonal neoantigen 12 4
burden
Low neoantigen
Favorable genomics intratumoral heterogeneity 5
1 11
14 2
Favorable microbiota 6
General immune status
Favorable TME 13 3
Lymphocyte count
No hypoxia, no spacial 10 7
distance between tumor
12 4 Low immune 8
cells and TILs 9
heterogeneity

Tumor sensibility to 5 TIL Secondary resistance to anti–PD-1/PD-L1

immune effectors 11 therapy
MHC 6
IFN signaling Tertiary lymphoid
structures 1
14 2 1
14 2
Absence of inhibitory 10 7
13 3
tumor metabolism Absence of coinhibitory 13 3
LDH, glucose checkpoints
8 LAG3, VISTA, TIGIT, etc. 12 4 12
utilization, IDO 9 4

Absence of Absence of 5 Loss of 5

soluble inhibitors PD-L1 11 MHC 11
6 6
IL6, CRP
Loss of
immune
10 7
10 7 infiltration
Expression
8
9 of LAG3

Anti– Anti-LAG3 antibody

NK-cell
PD-1/PD-L1
stimulators
antibody

Figure 6. Cancer immunogram adapted with permission from Blank et al. (292). A, Ideal features of an “immunogenic” tumor. B, Immunograms cor-
responding to a theoretical patient illustrating features predictive of response to anti–PD-1/PD-L1 antibodies, followed by two examples of secondary
resistance with potential tailored treatments. Red text is used to illustrate the changes made to the initial cancer immunogram of Blank et al.

ITH, hypoxic tumors, and persister cells, and these might be
the exact triggers behind progression. However, the selection
of the ideal candidates is challenged by the difficulty of predict-
ing the behavior of the rest of the lesions. Innovative methods
predicting relapse, based on genomics and radiomics, would
be desired. Also, radiotherapy could be modulated based on
the tumor genomic and microenvironmental features, as well
as its immune infiltration. Another direction of research is to
investigate the role of a “consolidation” therapy for patients
responding to treatment in a heterogeneous manner. The less
responsive lesions might exhibit genomic/immune heteroge-
neity and be the culprit of subsequent local or distant progres-
sion. In the case of ICB, the addition of a local treatment of
liver metastases might also be beneficial, especially when con-
sidering recent hypotheses of systemic immunosuppression
induced by liver metastases, which might impair ICB efficacy.
non-financial support, and other from Pfizer, Merck Serono, and
Novartis outside the submitted work. F. Barlesi reports personal
fees from AstraZeneca, Bayer, Bristol-Myers Squibb, Boehringer-
Ingelheim, Eli Lilly Oncology, F. Hoffmann-La Roche Ltd, Novartis,
Merck, Mirati, MSD, Pierre Fabre, Pfizer, Seattle Genetics, and
Takeda outside the submitted work. J.-C. Soria reports personal fees
from Relay Therapeutics, and other from AstraZeneca, Daiichi San-
kyo, Gritstone, and Hookipa Pharmaceuticals during the conduct of
the study. No other disclosures were reported.
Acknowledgments
The figures are previously unpublished original works, created
by Mihaela Aldea and Semih Dogan with BioRender.com, for the
express purpose of publication in this Cancer Discovery review.
Received November 9, 2020; revised January 13, 2021; accepted
February 1, 2021; published first April 2, 2021.

Conclusion
Having all this in mind, it is clear that despite major pro-
gress in cancer care, drug resistance evolves dynamically and
remains the most important barrier to achieving cure. A deeper
understanding of the processes underlying resistance and more
potent drugs and innovative strategies are urgently needed.
Authors’ Disclosures
F. Andre reports grants from Novartis, Roche, Daiichi, Eli Lilly,
Astra Zeneca, and Pfizer outside the submitted work. A. Marabelle
reports grants, personal fees, non-financial support, and other from
AstraZeneca, BMS, MSD, and Roche/Genentech and personal fees,
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Mihaela Aldea, Fabrice Andre, Aurelien Marabelle, et al.

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