2012 EDUCATIONAL BOOK - American Society of Clinical Oncology

Mechanism of Resistance Drug a
MAPK Bypass
In addition to persistent MAPK pathway activation, alternative
signaling pathways may confer resistance, as evidenced
by the discovery of several receptor tyrosine kinases
(RTKs) associated with this phenomenon. In particular,
the RTKs HER2, AXL, platelet-derived growth factor-beta
(PDGFRß), and insulin-like growth factor-1 receptor
(IGF1R) have been linked to vemurafenib resistance in
preclinical studies. 12,14,20 In addition, PDGFRß and IGF1R
were activated and phosphorylated in some tumors that
developed resistance to vemurafenib. 14,20 These RTKs seem
to operate independently of MAPK signaling (Fig. 1), since
ectopic expression of these RTKs tended not to show MAPK
pathway reactivation. The extent to which these RTK alterations
are sufficient to confer clinical resistance remains
unclear, because overexpression of PDGFRß and IGF1R did
not confer resistance in some preclinical studies, nor did the
administration of imatinib resensitize melanoma cells that
exhibited PDGFRß upregulation. 12,21 Thus, their resistance
effects may manifest in certain molecular contexts or in
cooperation with as-yet unmeasured microenvironmental
effects. The RTK activation phenomena may also relate to
the relief of feedback inhibition that may result from therapeutic
inhibition of activated oncoproteins. 22
Although the majority of robust resistance mechanisms
characterized thus far tend to occur through downstream
pathway reactivation, there is some evidence that ERKindependent
mechanisms may also play a role. The ERKindependent
effects of RTKs such as PDGFR and IGF1R
constitute a line of evidence in this regard. In addition,
reports of disease progression with persistent suppression
of MEK/ERK activity have begun to emerge. 23 The mechanisms
of ERK bypass remain poorly characterized and may
gain increased importance if MEK inhibitors gain a role in
the treatment of BRAF-mutant melanoma.
Anticipating Resistance to Combined
RAF/MEK Inhibition
The preponderance of MEK/ERK-dependent resistance
mechanisms provides a strong rationale for the use of RAF
and MEK inhibitors in combination. Indeed, clinical trials of
combined RAF/MEK inhibition are ongoing. Several resistance
mechanisms described above should in principle exhibit
sensitivity to MEK inhibition (e.g., NRAS mutation,
Table 1. Summary of Studies Probing Resistance to BRAF Inhibitors
Type of Study Confirmed In Vivo? Reference
In Vitro Studies
NRAS mutation (Q61K, G12D) PLX4032 Drug-resistant clones Yes 14
KRAS mutation (K117N) PLX4032 Stepwise selection No 15
p61BRAF(V600E) PLX4032 Drug-resistant clones Yes 16
CRAF overexpression PLX4720 Systematic ORF screen No 12
CRAF overexpression AZ628 Drug-resistant clones No 13
COT/MAP3K8 amplification PLX4720 Systematic ORF screen Yes 12
IGF1R activation SB-590885 Drug-resistant clones Yes 20
PDGFR activation PLX4032 Drug-resistant clones Yes 14
MEK mutation (many) AZD6244, PLX4032 Random mutagenesis No 25
In Vivo Studies
MEK mutation (C121S) PLX4032 Targeted massively parallel sequencing N/A 24
Abbreviations: IGF1R, insulin-like growth factor-1 receptor; MEK, mitogen-activated protein kinase kinase; ORF, open reading frame; PDGFR, platelet-derived growth
factor.
a PLX4032 is vemurafenib. PLX4720 is the preclinical equivalent of PLX4032. AZ628 inhibits BRAF(V600E) and wild-type CRAF. SB-590885 is a BRAF(V600E) kinase
inhibitor. AZD6244 is a MEK inhibitor. The generation of drug-resistant clones (continuous exposure at one dose) or stepwise selection (steadily increasing drug
concentration) is described in the text.
682
GOETZ AND GARRAWAY
COT/MAP3K8 overexpression/copy gain, BRAF alternative
splicing). On the other hand, activating mutations in MEKs
might be predicted to confer resistance to combined inhibitor
exposure (Fig. 1). Indeed, clinical evidence has already
begun to emerge in support of this hypothesis. A C121S
mutation in MEK1 was discovered in a clinical specimen
resistant to vemurafenib, and expression of C121S MEK1
in a vemurafenib-sensitive cell line conferred resistance to
both RAF and MEK inhibition. 24 Thus, mutations in MEK
that occur clinically can confer resistance to both RAF and
MEK inhibition—and additional preclinical studies have
indentified several other mutations in MEK that were crossresistant
to RAF and MEK inhibitors. 24,25 In principle,
mutations in MEK would still be vulnerable to ERK inhibition.
Toward this end, selective ERK inhibitors have
recently entered clinical trials. The role (and safe administration)
of such agents in either up-front or salvage therapy
for BRAF-mutant melanoma will undoubtedly emerge as an
area of active investigation.
Methods of Therapeutic Resistance Discovery
The most common method for examining resistance involves
generation of drug-resistant subclones using cancer
cell lines in vitro. This is accomplished by either slowly
increasing drug concentrations over time (stepwise selection)
or through continuous incubation in high doses of a
drug (Table 1). After several months, the clones are analyzed
for mechanisms of resistance. Toward this end, changes in
gene expression, protein modification, or point mutations
can all lead to development of resistance. These changes
may sometimes be difficult to disambiguate; alternatively,
multiple mechanisms may contribute. Moreover, stepwise
selection may not accurately model the acquisition of resistance,
since tumors are not exposed to an increasing gradient
of a drug over a period of weeks or months. Therefore, an
alternative method for in vitro selection of drug-resistant
subclones involves continuous exposure at high drug doses.
This approach has proved useful in studies of resistance to
vemurafenib in vitro.
Several other techniques, such as random mutagenesis
and systematic screens, are useful to probe resistance in
vitro. These methods can speed up development of resistance
(weeks instead of months) and may inform distinct
spectra of resistance mechanisms. Random mutagenesis is