letrozole or anastrozole, or the estrogen receptor
antagonist fulvestrant) for treatment of
advanced/metastatic HR+/HER2–breast can-
cers in postmenopausal women. Addition of
CDK4/6 inhibitors significantly extended med-
ian progression-free survival ( 78 , 122 – 130 ) and
prolonged median overall survival ( 131 – 134 ).
Moreover, abemaciclib has shown clinical ac-
tivity when administered as a single agent
( 135 ). Consequently, palbociclib, ribociclib, and
abemaciclib have been approved by the US
Food and Drug Administration (FDA) for treat-
ment of patients with advanced/metastatic
HR+/HER2–breast cancer (Box 1). A recent
phase 3 clinical trial, MonarchE, evaluated
abemaciclib plus standard endocrine therapy
in treatment of patients with early-stage, high-
risk, lymph node–positive HR+/HER2–breast
cancer. Addition of abemaciclib reduced the
risk of breast cancer recurrence ( 136 ). This is
in contrast to the similar PALLAS study re-
ported this year, which found no benefit of
adding palbociclib to endocrine therapy for
women with early-stage breast cancer ( 137 ).
Analysis of patient populations in these two
trials may help to explain the different out-
comes. It is also possible that the favorable
outcome of the MonarchE study reflects a
broader spectrum of kinases inhibited by
abemaciclib. The utility of CDK4/6 inhibitors
in early-stage breast cancer remains unclear
and is being addressed in ongoing clinical trials
(PALLAS, PENELOPE-B, EarLEE-1, MonarchE)
( 138 ).Currently, palbociclib is being used in
164 active or recruiting clinical trials, riboci-
clib in 69 trials, and abemaciclib in 98 trials
for more than 50 tumor types ( 139 ). These
trials evaluate combinations of CDK4/6 in-
hibitors with a wide range of compounds
(Table 4). Trials with trilaciclib test the bene-
fitofthiscompoundinpreservingbonemar-
rowandtheimmunesystem.
Resistance to CDK4/6 inhibitors
Although CDK4/6 inhibitors represent very
effective agents in cancer treatment, nearly
all patients eventually develop resistance and
succumb to the disease. Moreover, a substan-
tial fraction of tumors show intrinsic resistance
to treatment with CDK4/6 inhibitors (Fig. 3).
The best-documented mechanism of preex-
isting and acquired resistance is the loss of
RB1 ( 71 , 81 , 140 ). Acquired RB1 loss has been
detected in PDXs ( 141 ), in circulating tumor DNA
(ctDNA) ( 142 , 143 ), and in tumors from patients
treated with CDK4/6 inhibitors ( 144 , 145 ). How-
ever, RB1 mutations are likely subclonal and
are seen in only 5 to 10% of patients ( 143 , 145 ).
IncreasedexpressionofCDK6wasshownto
underlie acquired resistance to CDK4/6 inhib-
itors. Amplification of theCDK6gene and the
resulting overexpression of CDK6 protein were
found in abemaciclib-resistant ER+breast can-
cer cells ( 146 ) and in ctDNA of patients with
ER+breast cancers that progressed during
treatment with palbociclib plus endocrine the-
rapy ( 147 ). Also,CDK4gene amplification con-
ferred insensitivity to CDK4/6 inhibition in
GBM and sarcomas ( 148 – 150 ), whereas over-
expression of CDK4 protein was associated
with resistance to endocrine therapy in HR+
breast cancers ( 79 ).
Resistant breast cancer cells can also up-
regulate the expression of CDK6 through sup-
pression of the TGF-b/SMAD4 pathway by the
microRNA miR-432-5p. In this mechanism,
exosomal expression of miR-432-5p mediates
the transfer of the resistance phenotype be-
tween neighboring cell populations ( 151 ). Another
mechanism of CDK6 up-regulation in ER+breast
cancers is the loss of FAT1, which represses
CDK6 expression via the Hippo pathway. Loss
of FAT1 triggers up-regulation of CDK6 ex-
pression by the Hippo pathway effectors TAZ
and YAP. Moreover, genomic alterations in
other components of the Hippo pathway, al-
though rare, are also associated with reduced
sensitivity to CDK4/6 inhibitors ( 81 ).
Genetic lesions that activate pathways con-
verging on D-type cyclins can cause resistance
to CDK4/6 inhibitors. These include (i)FGFR1/2
gene amplification or mutational activation,
detected in ctDNA from patients with ER+
breast cancers that progressed upon treatment
with palbociclib plus endocrine therapy ( 147 );
(ii) hyperactivation of the MAPK pathway in
resistant prostate adenocarcinoma cells, pos-
sibly due to increased production of EGF by
cancer cells ( 152 ); and (iii) increased secretion
of FGF in palbociclib-resistant KRAS-mutant
NSCLC cells, which stimulates FGFR1 signal-
ing in an autocrine or paracrine fashion, re-
sulting in activation of ERK1/2 and mTOR as
well as up-regulation of D-cyclin, CDK6, and
cyclin E expression ( 153 ). Analyses of longitu-
dinal tumor biopsies from a melanoma patient
revealed an activating mutation in thePIK3CA
gene that conferred resistance to ribociclib plus
MEK inhibitor treatment ( 154 ). It is possible
that these lesions elevate the cellular levels of
active cyclin D–CDK4/6 complexes, thereby in-
creasing the threshold for CDK4/6 inhibition.
Formation of a noncanonical cyclin D1–
CDK2 complex was shown to represent anoth-
er mechanism of acquired CDK4/6 inhibitor
resistance. Such a complex was observed in
palbociclib-treated ER+breast cancer cells and
was implicated in overcoming palbociclib-
induced cell cycle arrest ( 141 ). Also, deple-
tion of AMBRA1 promoted the interaction
of D-cyclins with CDK2, resulting in resistance
to CDK4/6 inhibitors ( 20 , 22 ); it remains to
be seen whether this represents an intrin-
sic or acquired resistance mechanism in hu-
man tumors.
Genetic analyses revealed that activation of
cyclin E can bypass the requirement for cyclin
D–CDK4/6 in development and tumorigenesis( 155 , 156 ).Hence,itcomesasnosurprisethat
increased activity of cyclin E–CDK2 is respon-
sible for a large proportion of intrinsic and
acquired resistance to CDK4/6 inhibitors. Sev-
eral different mechanisms can activate cyclin
E–CDK2 kinase in resistant tumor cells: (i)
Down-regulation of KIP/CIP inhibitors results
in increased activity of cyclin E–CDK ( 54 , 157 ).
(ii) Loss of PTEN expression, which activates
AKT signaling, leads to nuclear exclusion of
p27KIP1.Thisinturnpreventsaccessofp27KIP1
to CDK2, resulting in increased CDK2 kinase
activity ( 144 ). (iii) Activation of the PI3K/AKT
pathway causes decreased levels of p21CIP1. Co-
treatment of melanoma PDXs with MDM2
inhibitors (which up-regulate p21CIP1via p53)
sensitized intrinsically resistant tumor cells to
CDK4/6 inhibitors ( 158 ). (iv) Up-regulation of
cyclin D1 levels triggers sequestration of KIP/
CIP inhibitors from cyclin E–CDK2 to cyclin
D–CDK4/6, thereby activating the former ( 158 ).
(v) Amplification of theCCNE1gene and in-
creased levels of cyclin E1 protein result in
elevated activity of E-CDK2 kinase ( 141 ). (vi)
mTOR signaling has been shown to up-regulate
cyclin E1 (and D1) in KRAS-mutated pancreatic
cancer cells; CDK2 activity was essential for
CDK4/6 inhibitor resistance in this setting
( 159 ). (vii) Up-regulation of PDK1 results in acti-
vation of the AKT pathway, which increases the
expression of cyclins E and A and activates CDK2
( 160 ). (viii) In CDK4/6 inhibitor–resistant mel-
anoma cells, high levels of RNA-binding protein
FXR1 increase translation of the amino acid
transporter SLC36A1. Up-regulation of SLC36A1
expression activates mTORC1, which in turn
increases CDK2 expression ( 161 ). All these
lesions are expected to allow cell proliferation,
despite CDK4/6 inhibition, as a consequence
of the activation of the downstream cell cycle
kinase CDK2.
The role for cyclin E–CDK2 in CDK4/6 in-
hibitor resistance has been confirmed in clini-
cal trials. In patients with advanced ER+breast
cancer treated with palbociclib and letrozole
or fulvestrant, the presence of proteolytically
cleaved cytoplasmic cyclin E in tumor tissue
conferred strongly shortened progression-free
survival ( 71 ). Moreover, analyses of PALOMA-3
trial for patients with ER+breast cancers re-
vealed lower efficacy of palbociclib plus ful-
vestrant in patients displaying high cyclin E
mRNA levels in metastatic biopsies ( 80 ). Am-
plification of theCCNE1gene was detected
in ctDNA of patients with ER+breast cancers
that progressed on palbociclib plus endocrine
therapy ( 147 ). Also, amplification of theCCNE2
gene (encoding cyclin E2) was seen in a frac-
tion of CDK4/6 inhibitor–resistant HR+mam-
mary carcinomas ( 145 , 162 ).
Collectively, these analyses indicate that re-
sistant cells may become dependent on CDK2
for cell cycle progression. Indeed, depletion of
CDK2 or inhibition of CDK2 kinase activity inFasslet al.,Science 375 , eabc1495 (2022) 14 January 2022 11 of 19
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