Kirjassa, joka kertoo aktuellista syöpäterapiasta englanniksi, on artikkeli
Shaokun Shu et Kornelia Polyak, BET Bromodomain Proteins as Cancer Therapeutic Targets.
Kirjoitin siitä muutamia muistiinpanoja ja kuvia. Mutta kun nyt haen netistä löydän myös asiasta seuraavaa:
PMCID: PMC4854653
NIHMSID: NIHMS742790
Response and resistance to BET bromodomain inhibitors in triple negative breast cancer (TNBC).
(TNBC tarkoittaa ER-, PR- HER2- (Puuttua estrogeenireseptori (ER)ilmentymä, puuttuu progesteronireseptori (PR) ilmentymä ja puuttuu HER2reseptori-ilmentymä (HER2 = Human Epidermal Growth Factor Receptor 2)
Tavallisin BC tyyppi on luminaalinen A tai B ja ne ovat ER+, PR+ (70%)
Sitten on vielä HER2+, jossa on ERBB2 onkogeeniamplifikaatio).
Otan nettiartikkelista abstraktin tähän blogiin:
Triple negative breast cancer (TNBC) is a heterogeneous and clinically aggressive disease for which there is no targeted therapy1-3.
BET bromodomain inhibitors, which have shown efficacy in several models of cancer4-6, have not been evaluated in TNBC. These inhibitors displace BET bromodomain proteins such as BRD4 from chromatin by competing with their acetyllysine (AcK) recognition modules, leading to inhibition of oncogenic transcriptional programs7-9.
BET bromodomain inhibitors, which have shown efficacy in several models of cancer4-6, have not been evaluated in TNBC. These inhibitors displace BET bromodomain proteins such as BRD4 from chromatin by competing with their acetyllysine (AcK) recognition modules, leading to inhibition of oncogenic transcriptional programs7-9.
Here we report the preferential sensitivity of TNBCs to BET bromodomain inhibition in vitro and in vivo,
establishing a rationale for clinical investigation and further
motivation to understand mechanisms of resistance. In paired cell lines
selected for acquired resistance to BET inhibition from previously
sensitive TNBCs, we failed to identify gatekeeper mutations, new driver
events or drug pump activation. BET-resistant TNBC cells remain
dependent on wild-type BRD4, which supports transcription and cell
proliferation in a bromodomain-independent manner. Proteomic studies of
resistant TNBC identify strong association with MED1 and
hyper-phosphorylation of BRD4 attributable to decreased activity of
PP2A, identified here as a principal BRD4 serine phosphatase. Together,
these studies provide a rationale for BET inhibition in TNBC and present
mechanism-based combination strategies to anticipate clinical drug
resistance.
To explore non-oncogene addiction to BRD4 in breast cancer, we studied a series of BET bromodomain inhibitors (BBI) across breast cell lines reflecting transcriptionally-defined breast cancer subtypes: luminal, HER2+, and TNBC2,10 as well as MCF10A and MCF12A basal/mesenchymal immortalized mammary epithelial cells (Supplementary Table 1). Potent inhibitory effects were observed preferentially in TNBC lines, compared to more resistant luminal lines (Fig. 1a). Analysis of potency of drug response and subtype or known driver mutations identified the basal subtype as the only significant association (p=0.0475) (Supplementary Table 1 and D.N.S.). BRD4 dependency was confirmed by RNAi and phenocopied BBI (Extended data Fig. 1a-c). JQ1 or BRD4 knock-down induced growth inhibition resulted in G1 arrest and apoptosis (Extended Data Fig. 1d-g). Expression of factors described to mediate JQ1 effect (MYC) or required for TNBC growth (JAK2/STAT3) showed no clear association with JQ1 sensitivity (Extended Data Fig. 1h and Extended Data Fig. 2a,b). JQ1 treatment of TNBC cells induced significant morphologic changes consistent with induction of senescence confirmed by β-galactosidase staining and luminal differentiation evidenced by changes in the expression of basal and luminal markers (Extended Data Fig. 2c-d and Fig. 1b).
( JQ1 = thienotriazolodiazepine)
Extending the translational significance of these findings,
we evaluated the ability of JQ1 to inhibit tumor growth in murine TNBC
xenografts. Two week treatment efficiently inhibited established tumor
growth from SUM159 and MDA-MB-231 lines, and patient-derived primary
human TNBC xenografts (Fig. 1c and Extended Data Fig. 2e,f).
Down-regulation of BRD4 using two independent TET-inducible shRNAs
produced even more pronounced effects leading to complete tumor
regression and failure to regrow even after discontinuing doxycycline
treatment (Fig. 1c and Extended Data Fig. 2g). Evidence of BBI-induced basal-to-luminal differentiation was confirmed in vivo (Extended Data Fig. 2f,h).
Using integrated epigenomic analysis (Supplementary Table 2),
we identified the direct transcriptional targets of BBI in TNBC. BBI
binding was identified at active promoter and enhancer regions using
ChemSeq11
for biotinylated JQ1 (Bio-JQ1) enrichment and ChIP-seq for
acetyl-histone (H3K27ac) and BRD4 enrichment, with the three marks
showing near perfect co-localization (Fig. 1d and Extended Data Fig. 3a). BBI efficiently displaced chromatin-bound BRD4 in treated SUM159 (Fig. 1e and Extended Data Fig. 3b) and in SUM149 cells (Extended Data Fig. 3c).
To identify biologically relevant, direct targets of BBI in SUM159 and
SUM149 cells, we quantified binding of Bio-JQ1 and BRD4 genome-wide and
found strong enrichment at 219 and 159 super-enhancers, respectively
(SEs; Fig. 1f and Extended Data Fig. 3d and Supplementary Table 3)8,9,12,13.
TFs with known roles in breast cancer, such as POU5F1B/MYC14 and HIF1α15,
were evident among top SE-associated genes in both lines. Kinetic
effects of JQ1 treatment on gene expression demonstrated preferential
SE-associated gene down-regulation (Fig. 1g and Extended Data Fig. 3e,f).
Expression changes were observed within 3 hours after JQ1 treatment
and, as expected, more genes were significantly down- than up-regulated (Extended Data Fig. 3g-j, and Supplementary Table 4). Unsupervised Metacore16
analysis of JQ1 affected target pathways revealed down-regulation of
regulatory and effector genes in anti-apoptotic and JAK/STAT signaling
pathways (Extended Data Fig. 3k).
These data support selective disruption of SE-associated genes by JQ1,
leading to deregulation of coordinated transcriptional pathways involved
in cell proliferation, invasion, and survival.
Dissecting
resistance to targeted therapy is critical to elucidate mechanisms of
drug and target action, and to suggest approaches to treat or anticipate
drug resistance in patients. Therefore, we established BBI-resistant
TNBC cell lines by long-term culture of both SUM159 and SUM149 cells in
escalating JQ1 doses. Low (0.5 μM) and high (2.0 μM) doses of JQ1
severely impaired proliferation of parental SUM159 and SUM149 lines,
reducing viable cells after 6 days (Fig. 2a and Extended Data Fig. 3l). In contrast, JQ1-resistant cells (SUM159R and SUM149R) proliferated linearly, even in high JQ1 doses (20 μM) (Fig. 2a and Extended Data Fig. 3l). BBI-resistance is not attributable to drug export, as MDR1 and other transporters are not transcriptionally up-regulated (Extended Fig. 4a), co-incubation with MDR1 inhibitors (verapamil) had no effect (Extended Data Fig. 4b), and structurally divergent BBIs are equally inactive as JQ1 (Fig. 2b).
Further support is provided by the equivalent chromatin engagement of
BRD4 in sensitive and resistant cells, demonstrated by ChemSeq with
Bio-JQ1 (Extended Data Fig. 4c).
Notably, BBI-resistant TNBC cells retain sensitivity to compounds from
orthogonal active drug classes, such as CXCR2 and JAK2 inhibitors17; establishing specific resistance to BBIs (Extended Data Fig. 4d).
Adaptive drug resistance was not attributable to outgrowth of a minor
subpopulation of pre-existing resistant cells, as 10 independent single
cell-derived clones showed similar resistance profiles to pooled SUM159R
cells (Extended Data Fig. 4e). Similar results were obtained in vivo, as SUM159R derived xenografts derived were JQ1 unresponsive (Extended Data Fig. 4f).
In all resistant TNBC populations studied, exome sequencing failed to
identify alterations in BET bromodomain-encoding genes (e.g.,
gatekeepers) or known driver genes (parallel pathway activation; Supplementary Table 5).
Absent new genetic alterations, we explored the plausibility
of an epigenomic mechanism of resistance. Differential enhancer analysis
revealed a significant gain of SEs in resistant SUM159R cells (ChemSeq;
Fig. 2c and Supplementary Table 6). The gain of Bio-JQ1 SEs was associated with enrichment for BRD4 binding to these genomic loci (Fig. 2d) and increased transcription of associated genes (Fig. 2e). An upstream/intragenic region of H3k27ac at the BCL-xL locus featured prominently among top gained SEs in SUM159R (Fig. 2f), consistent with increased BCL-xL mRNA and protein expression in resistant cells (Supplementary Table 7, Extended Data Fig. 4g).
Functionally, cells with acquired resistance to BBI featured a
concordant switch in JQ1 anti-apoptotic response based on dynamic BH3
profiling18,19 (Extended Data Fig. 4h).
Observing
emergent enhancers in resistant cells, we assessed whether
BBI-resistant TNBC cells retained non-oncogene addiction to BRD4.
Notably, we observed loss of SUM159R cell viability upon BRD4 knockdown (Extended Data Fig. 5,b).
Together these studies establish persistence of BRD4 addiction despite
resistance to bromodomain inhibition, establishing the plausibility of
bromodomain-independent recruitment of BRD4 to enhancers in
BBI-resistant TNBCs. To test this hypothesis, we performed BRD4 ChIP-seq
on sensitive and resistant cells with and without JQ1. JQ1 neither
displaced BRD4 from chromatin in SUM159R (Fig. 2g), nor meaningfully influence epigenome structure by H3K27ac ChIP-seq (Extended Data Fig. 5c-g). Notably, several luminal markers (FOXA1, CD24, and luminal cytokeratins) were elevated in SUM159R cells in cell culture and in vivo (Extended Data Fig. 5h,i),
supporting a model whereby resistance arises via essential BRD4
recruitment to chromatin in a bromodomain-independent manner. Similar
observations were made in SUM149R cells and in TNBC cells inherently
resistant to JQ1 (Extended Data Fig. 3h-j; Extended Data Fig. 6a-d), suggesting a general mechanism of epigenomic resistance to BBI.
To
disclose potential differences in BRD4-associated complexes between
sensitive and resistant SUM159 cells, we performed quantitative
proteomics using RIME (rapid immunoprecipitation mass spectrometry of
endogenous proteins)20
with and without JQ1. Analysis of BRD4-associated proteins identified
relative enrichment of MED1 and BRD3 in JQ1-treated resistant cells (Fig. 3a, Extended Data Fig. 7, and Supplementary Table 8).
BRD4 immunoprecipitation followed by immunoblot for MED1 and BRD3
revealed that JQ1 efficiently displaced BRD4 from MED1 in sensitive
cells, but not in resistant cells (Fig. 3b), a result confirmed in SUM149 and BBI-resistant SUM149R, as well as inherently resistant TNBC and luminal lines (Extended Data Fig. 8a).
Though elevated BRD3 abundance was observed in SUM159R, increased
association of BRD4 and BRD3 was not confirmed by immunoblot, (Fig. 3b).
To assess functionally whether increased recruitment of BRD4 to
chromatin by MED1 underlies resistance to JQ1, we expressed an exogenous
bromodomain-inactivated mutant (BDmut) with concomitant knock-down of
endogenous BRD4 (Extended Data Fig. 8b,c).
Downregulation of endogenous BRD4 decreased cell growth both in
parental and resistant cells, which was rescued by enforced expression
of wild type BRD4 (Fig. 3c).
BDmut BRD4 expression failed to rescue parental SUM159 cells, but
supported growth of JQ1-resistant SUM159R consistent with an evident
bromodomain-independent mechanism of BRD4 recruitment (Fig. 3c).
Next, we assessed the sensitivity of cells expressing BDmut BRD4 to
JQ1, observing increased sensitivity to JQ1 In parental SUM159 cells
exogenously expressing BDmut (Fig. 3d). In contrast, expression of BDmut BRD4 in SUM159R cells rescued the anti-proliferative effect of JQ1 (Fig. 3e),
although this could partially be due to the slower growth of BDmut
expressing cells. Together, these studies suggest BBI-resistance is
associated with increased binding of BRD4 to MED1, in a
bromodomain-independent manner unaffected by JQ1.
Mechanism of BBI-resistance in TNBCs
A
recent study reported that the stability and nuclear localization of
BRD4 is increased with phosphorylation by casein kinase II (CK2)21.
To explore the contribution of BRD4 phosphorylation to BBI-resistance,
we performed immunoblot analysis in parental and resistant cells and
found a marked increase of phospho-BRD4 (pBRD4) in resistant cells (Fig. 4a and Extended Data Fig. 8d). Small-molecule inhibition of CK2 decreased BRD4 phosphorylation in SUM159 and SUM159R cells (Extended Data Fig. 8e).
These results imply BRD4 hyperphosphorylation in resistant cells either
due to increased phosphorylation by CK2 or, alternatively, to decreased
dephosphorylation by an as yet unidentified BRD4 phosphatase. We
therefore first analyzed CK2 activity in parental and resistant cells by
performing pan-CK2 substrate immunoblots and detected no significant
differences in CK2 activity (Extended Data Fig. 8f).
Regulation and relevance of BRD4 phosphorylation
Inactivation
of the PP2A phosphatase tumor suppressor gene occurs commonly in breast
cancer and is associated with therapy resistance22; PP2A also often opposes CK2 function23,24.
Thus, we investigated whether PP2A may dephosphorylate BRD4 and whether
decreased PP2A activity could lead to BBI-resistance. Downregulation of
PP2A catalytic subunit (PP2CA) in SUM149 and SUM159 cells led
to increased BRD4 phosphorylation, establishing PP2A as a previously
unrecognized BRD4 phosphatase (Fig. 4b), further supported by pharmacologic inhibitors of PP2A that showed similar effects (Extended Data Fig. 8g).
To strengthen the link between PP2A activity and BBI-resistance, we
tested the JQ1 sensitivity of SUM149 cells following the knock-down of
PP2A C subunit and determined that downregulation of PP2A decreased JQ1
sensitivity (Fig. 4c). We have collaboratively reported phenothiazine compounds as activators of PP2A enzymatic activity25.
Thus, we analyzed pBRD4 levels in SUM159R, SUM149R, and other cell
lines after short-term treatment with phenothiazine (PTZ) and detected
rapid dephosphorylation of BRD4 (Fig. 4d and Extended Data Fig. 8h). Combined treatment with PTZ and JQ1 overcame BBI-resistance in SUM159R cells (Fig. 4e).
To investigate the functional role of BRD4 hyperphosphorylation in
BBI-resistance, we analyzed whether BRD4 phosphorylation influences MED1
binding. Indeed, SUM159R cells treated with CK2 inhibitor or PTZ both
lead to decreased MED1 abundance in BRD4 immunoprecipitations,
suggesting that pBRD4 binds MED1 more efficiently than BRD4 (Fig. 4f,g).
To
functionally assess the role for BRD4 phosphorylation in BBI-resistance
and MED1 binding, we generated BRD4 constructs encoding mutants that
cannot be phosphorylated by CK2 (7 serine to alanine substitutions; “7A
mutant”) or mimic constitutive phosphorylation (7 serine to aspartate
substitutions; “7D mutant”). We first assessed the ability of these
constructs to rescue effects of endogenous BRD4 knock-down in stable
cell lines (Extended Data Fig. 8b,c).
We observed expression of both 7D and 7A mutants supporting the growth
of both parental SUM159 and JQ1-resistant SUM159R cells (Fig. 3d).
Next, we analyzed MED1 binding and subcellular localization of 7A and
7D mutants +/− JQ1. We found that the 7A mutant displays weaker MED1
binding compared to WT BRD4 and completely dissociates after JQ1 whereas
the 7D mutant seems to have higher affinity for MED1 that unaffected by
JQ1 treatment (Fig. 4h and Extended Fig. 8i).
Lastly, we assessed the sensitivity of cells expressing 7A or 7D mutant
BRD4 to JQ1. In parental SUM159 cells exogenously expressed 7D mutant
BRD4 decreased sensitivity to JQ1 whereas the 7A mutant slightly
increased sensitivity (Fig. 4i).
In contrast, expression of 7A mutant BRD4 in SUM159R cells restored JQ1
sensitivity whereas the 7D mutant showed a modest decrease. These
results strongly support the hypothesis that hyperphosphorylation of
BRD4 arises from decreased PP2A activity in BBI resistant cells leading
to increased binding of BRD4 to MED1, recruitment to chromatin and
decreased responsiveness to bromodomain inhibition.
To
explore the clinical relevance of phospho-BRD4 (pBRD4) in BET
inhibitor-naive TNBC, we performed immunofluorescence analysis of a
tissue microarray (TMA) featuring of 89 patient-derived TNBC specimens.
First, we validated the pBRD4 immunofluorescence assay by comparing
xenografts derived from SUM159 and SUM159R cell lines and detected
significantly higher pBRD4 in SUM159R cells (Extended Data Fig. 9a). We detected strong pBRD4 staining among a subset of TNBCs (Extended Data Fig. 9b,c),
and variable staining overall that was not correlated with expression
of the androgen receptor (AR) and basal cytokeratins (bCK; Extended Fig. 9d,e) and it was not significantly associated with disease outcomes (Supplementary Table 9 and Extended Data Fig. 9f).
To
extend the translational relevance of our findings, we conducted
synergy studies of JQ1 with molecules targeting BCL-xL (ABT737), a
gained super-enhancer in SUM159R cells, and modulators of BRD4
phosphorylation, the CK2 inhibitor CX-4945 and the PP2A activator
perphenazine (PPZ). We observed significant synergy between JQ1 and all
three compounds studied (Extended Data Fig. 10), establishing a rationale for combination studies of BBI in TNBC to improve response and to anticipate BBI resistance.
BRD4
inhibition has demonstrated efficacy in disparate models of cancer in a
rapidly expanding literature. Despite apparent resistance in the vast
majority of tumor types, as we observed here in TNBC, mechanisms of
BBI-resistance have not been mechanistically explained. As this research
was in review, two studies reported moderate emergent resistance to BBI
in murine AML associated phenotypically with a stem-like state and WNT
pathway activation26,27.
Interestingly, in our study TNBCs with more basal/stem cell-like
features and WNT pathway activation are more sensitive to BET
inhibition, whereas resistant disease emerges as epigenomic adaptation
to a more differentiated luminal phenotype. Our findings of persistent
BET bromodomain dependency despite BBI-resistance, as well as pBRD4
staining in resistant disease should be studied in these murine AML
models and further in human leukemia.
Integrating
approaches in epigenomics, proteomics, and chemical biology, we provide
an example of epigenomic drug resistance by an epigenetic mechanism,
where in BBI resistant cells, decreased PP2A activity leads to
hyperphosphorylated BRD4, which binds more strongly to MED1,
facilitating a bromodomain-independent chromatin recruitment mechanism.
This research proposes putative combination strategies to anticipate and
overcome BBI resistance, including pairing with BCL-xL inhibitors
(e.g., ABT-737) or CK2 inhibitors, and guides the development of
second-generation BBIs that disrupt BET function via orthogonal
biophysical or biochemical actions. More immediately, the robust
efficacy observed in pre-clinical models supports the development of BET
inhibition in TNBC alone, and in combination with mechanism-based
targeted therapies.
METHODS
Cell lines and breast tumor tissues
Breast
cell lines were obtained from the ATCC and Dr. Steve Ethier, University
of Michigan, Ann Arbor, MI (SUM series). Cells were cultured in media
recommended by the provider, their identity was confirmed by STR
analysis, and they were regularly tested for mycoplasma. Breast tumor
samples were collected using protocols approved by the DF/HCC
Institutional Review Board, informed consent was obtained from all
patients. Tumors were minced with razor blades and digested with
stirring for 3-4 hours at 37°C in DMEM/F12 with 2 mg/mL BSA, 2 mg/mL
collagenase type IV, and 2 mg/mL hyaluronidase. After digestion, cells
were filtered through 500-micron mesh, washed in DMEM/F12 with 5% FBS,
frozen in DMEM/F12 with 5% FBS and 10% DMSO, and stored in liquid
nitrogen for subsequent xenograft studies. PDX IDC50 was derived from a
primary tumor of highly invasive metaplastic TNBC resistant chemo and
radiation therapy leading to the rapid death of the patient. Exome
sequencing of the tumor and xenograft identified numerous mutations
including heterozygous frameshift mutation in PTEN (chr10_89701964-89701964_A) and CDH1
chr16_67400242-67400242_C). PDX EL-12-58 was derived from a liver
metastasis of a heavily pretreated basal-like TNBC, Oncopanel mutation
testing identified homozygous mutations in BRCA2 (p.S1970*), TP53 (p.I232fs), TSC2, FLT3, and ROS1, and lower frequency mutations in RAD21, JAK3, ARID1B, ARID1A, KDM6A.
High-Throughput Screening of BET Bromodomain Inhibitors in breast cell line panel
We
tested a panel of compounds (synthesized in the Bradner lab) in 40
human breast cell lines in a 384-well format at 2,000 cells per well
using a semi-automated screen essentially as described5. Cell viability at 72 hr was evaluated using ATPlite (Perkin Elmer).
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