Graphical abstract
graphic file with name fx1.jpg
[47]Open in a new tab
Highlights
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AhR is a targetable tumor promoter in MYCN-amplified neuroblastoma
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AhR positively regulates MYCN in MYCN-amplified neuroblastoma cell
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AhR antagonists and retinoids induce durable differentiation of
neuroblastoma cells
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Cell biology; Stem cells research; Cancer
Introduction
Neuroblastoma is a malignancy arising from cells of the developing
sympathetic nervous system and is the most common extracranial tumor in
children.[48]^1^,[49]^2 Despite intensive, multi-modality treatments,
approximately 50% of patients with high-risk neuroblastoma die of
progressive or recurrent disease.[50]^1^,[51]^2^,[52]^3^,[53]^4
Moreover, long-term survivors develop a long list of side effects from
the treatment, exacerbating the morbidity associated with high-risk
neuroblastoma and underscoring the need for better therapeutic
approaches.[54]^4 While retinoic acid therapies have provided clinical
benefit by inducing differentiation of neuroblastoma cells in patients
with minimal residual disease, many patients eventually experience
relapses due to insurgence of therapy
resistance[55]^3^,[56]^5^,[57]^6^,[58]^7 and escaping the
differentiation imparted by retinoids. The mechanisms that contribute
to escape from retinoid-mediated differentiation are not fully
understood.
MycN is a member of the Myc family of basic-helix-loop-helix zipper
transcription factors, and its expression is mainly restricted to
embryonic development.[59]^8 MycN is a major transcriptional driver of
neuroblastoma progression, and its amplification is present in 40–50%
of high-risk patients. Amplification of MYCN is an unfavorable factor
for survival and correlates with poor patient prognosis and poor
response to retinoic acid treatment.[60]^9^,[61]^10^,[62]^11
The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription
factor that is kept inactive in the cytoplasm through interaction with
chaperone proteins and upon ligand binding translocates to the nucleus
to modulate gene expression.[63]^12 While AhR was originally discovered
for its role in mediating xenobiotic metabolism,[64]^13^,[65]^14 recent
studies have demonstrated that AhR plays an important role in cancer
biology. However, its exact functions in tumors are quite
controversial, as previous studies have demonstrated that AhR can act
as an oncogene in some
settings[66]^15^,[67]^16^,[68]^17^,[69]^18^,[70]^19^,[71]^20 but as a
tumor suppressor in
others,[72]^21^,[73]^22^,[74]^23^,[75]^24^,[76]^25^,[77]^26 sometimes
with conflicting or contradictory
reports.[78]^18^,[79]^19^,[80]^23^,[81]^27^,[82]^28^,[83]^29
Additionally, AhR has been shown to promote, suppress, or have no
effect on the expression of Myc family proteins depending on the cancer
cell type and cellular context.[84]^15^,[85]^21^,[86]^30 The role of
AhR as a regulator of MYCN-amplified neuroblastoma progression remains
incompletely characterized.
While no AhR antagonists are currently approved for cancer treatment, a
few of them are either FDA-approved for different indications (i.e.,
clofazimine) or currently in clinical trials (i.e., BAY-2416964).
Importantly, clofazimine has been safely used in children with leprosy,
multi-drug resistant tuberculosis, and other infections with no adverse
effects.[87]^31^,[88]^32
Here, we report that AhR acts as a tumor promoter in the context of
MYCN-amplified neuroblastoma, partly through MycN regulation but also
via MycN-independent suppression of differentiation. Furthermore, we
show that AhR pharmacological antagonism with clofazimine potentiates
retinoic acid treatment efficacy in vitro and in vivo. Thus, our study
identifies AhR as a potential therapeutic target in MYCN-amplified
neuroblastoma.
Results
AhR acts as a tumor promoter in MYCN-amplified neuroblastoma
Immunoblot analysis of a panel of human MYCN-amplified and
non-amplified cell lines revealed a positive correlation in protein
expression between AhR and MycN and an inverse correlation with cMyc
([89]Figure 1A). MycN and cMyc are known to have opposite patterns of
expression in neuroblastoma[90]^33^,[91]^34^,[92]^35 and our data open
the possibility that AhR may play different roles in MYCN-amplified
versus non-amplified neuroblastoma. AhR expression levels may not
necessarily represent levels of AhR activity.[93]^36 To assess whether
AhR transcriptional activity (which assesses AhR’s functionality) has
any correlation with neuroblastoma survival, we chose two human
MYCN-amplified neuroblastoma cell lines (BE2C and Kelly) that showed
detectable expression of AhR protein ([94]Figure 1A) and depleted AHR
with two lentiviral shRNA constructs that we previously
validated[95]^15 or their corresponding non-silencing control
([96]Figure 1B). We then performed RNA-seq and extracted a signature of
123 genes that were commonly upregulated (shAHR_UP) and 135 genes that
were commonly downregulated (shAHR_DOWN) by the two shAHR constructs in
both cell lines, with an absolute fold change ≥1.5 and adj. p < 0.05
([97]Figure S1; [98]Table S1). We overlapped the “shAHR_UP” and
“shAHR_DOWN” gene lists with recently reported “favorable” and
“unfavorable” survival gene signatures from 11 neuroblastoma
patients’ datasets[99]^37 that are hosted on the R2 database. These
datasets contain both MYCN-amplified and non-amplified patients, with
the non-amplified representing most of the cases. The shAHR_UP genes
significantly overlapped with genes found in the favorable signatures,
while the shAHR_DOWN genes significantly overlapped with genes in the
unfavorable signatures ([100]Figure 1C). At the same time,
interrogation of two widely used datasets, SEQC and “Therapeutically
Applicable Research to Generate Effective Targets project” (TARGET),
revealed inconsistent association between AHR mRNA levels (split on
median) and the overall survival of patients ([101]Figures S2A–S2F). In
the SEQC dataset a positive correlation between AHR levels and survival
was detected when analyzing the entirety of the set, similarly to what
was previously reported,[102]^26 as well as in the non-MYCN-amplified
subset ([103]Figures S2C and S2E), but no difference in survival was
observed with MYCN-amplified patients ([104]Figure S2A). Interestingly,
analysis of the TARGET dataset did not reveal any association
independently of the stratification ([105]Figures S2B, S2D, and S2F).
Together, these data suggest that AhR transcriptional activity is
detrimental to the survival of neuroblastoma patients and could
potentially have better prognostic value than AHR mRNA levels.
Figure 1.
[106]Figure 1
[107]Open in a new tab
AhR associates with MycN and with poorer prognosis in neuroblastoma
(A) Immunoblot for AhR, MycN, and c-Myc in a panel of neuroblastoma
cell lines with or without MYCN amplification. Tubulin is used as
loading control.
(B) Immunoblot of BE2C and Kelly cells transduced with two independent
shAHR constructs and their corresponding non-silencing control vector.
Actin or GAPDH are used as loading control.
(C) Overlaps between genes from our shAHR_UP and shAHR_DOWN gene
signatures and published favorable and unfavorable gene signatures
derived from 11 neuroblastoma datasets. Statistics performed with
hypergeometric test. Jaccardi index was used to compare the similarity.
See also [108]Figures S1 and [109]S2, [110]Tables S1 and [111]S2.
We assessed the consequences of AHR depletion in MYCN-amplified
neuroblastoma cells and found that the clonogenic potential of BE2C and
Kelly cells depleted of AHR, either genetically or pharmacologically
with the AhR antagonist clofazimine (CLF)[112]^15 ([113]Figures 2A and
2B), as well as their invasive capability ([114]Figures 2C and 2D) were
reduced compared to control cells. Pharmacological inhibition of AhR
with three additional antagonists, including the classical AhR
antagonist CH-223191,[115]^38 BAY-2416964 (clinical trials
[116]NCT04069026 and [117]NCT04999202), and KYN-101[118]^39 yielded
similar results ([119]Figure S3A).
Figure 2.
[120]Figure 2
[121]Open in a new tab
AhR is a tumor promoter in MYCN-amplified neuroblastoma
(A) Representative clonogenic assay (out of 3) of BE2C and Kelly cells
transduced with two independent shAHR constructs and their
corresponding non-silencing control vector or treated with the AhR
antagonist clofazimine (CLF, 2 μM Kelly, 4 μM BE2C).
(B) Quantification of (A) performed with ImageJ. Statistics by
two-tailed Student’s t test. Data are average −/+ SD (n = 3).
(C) Invasion assay of MYCN-amplified human neuroblastoma BE2C and Kelly
cells transduced with two independent shAHR constructs and their
corresponding non-silencing control vector. Data are average −/+ SD
(n = 4). Statistics by two-tailed Student’s t test.
(D) Invasion assay of BE2C and Kelly cells treated for a total of 48hrs
with CLF (4 μM BE2C, 2 μM Kelly). Data are average −/+ SD (n = 4).
Statistics by two-tailed Student’s t test.
(E) BE2C or (F) Kelly cells transduced as in (D) were injected SQ in
the right flank of NSG mice (n = 10 for BE2C and n = 6 for Kelly).
Equal numbers of females and males were used. Tumors were measured
twice/week. Animals were humanely euthanized when a tumor in any group
reached the limits set by IACUC protocol. Data are average −/+ SEM.
Statistics by two-way ANOVA test. See also [122]Figure S3.
Conversely, AHR ectopic expression in the same cells led to a ∼2-fold
increase in invasion ([123]Figure S3B), supporting a pro-tumorigenic
function of AhR in MYCN-amplified neuroblastoma cells. Interestingly,
previous reports have suggested that AhR acts as a tumor suppressor in
neuroblastoma cells, especially those of non-MYCN-amplified
origin.[124]^25 AHR ectopic expression in human non-MYCN-amplified
SK-N-SH and SH-SY5Y cells resulted in a reduction in clonogenic growth
([125]Figure S3C) and a slight reduction in invasion capability
([126]Figure S3D), corroborating previous findings,[127]^25^,[128]^26
as well as in a reduction in cMYC protein levels ([129]Figure S3E)
while MycN was virtually undetectable. Thus, AhR appears to have a
previously unrecognized dual role in neuroblastoma, as a tumor promoter
or tumor suppressor depending on the MYCN-amplification status.
Finally, BE2C and Kelly cells depleted of AHR failed to grow tumors
in vivo when implanted subcutaneously (SQ) into the flank of
immunocompromised NOD SCID-gamma (NSG) mice ([130]Figures 2E and 2F).
Altogether, these data strongly suggest that AhR acts as a tumor
promoter in MYCN-amplified neuroblastoma.
AhR suppresses differentiation of MYCN-amplified neuroblastoma cells
Gene Ontology (GO) analysis of the aforementioned RNA-seq showed a
significant upregulation of neuronal differentiation signatures in both
BE2C and Kelly cells upon AHR depletion (including genes such as
STMN4[131]^40 and EFNA2,[132]^41 [133]Table S1), with concomitant
suppression of processes associated with cell cycle progression
([134]Figures 3A and [135]S4A; [136]Table S2). These findings were
further confirmed at a morphological level, with AHR depletion causing
cellular morphological changes, consistent with induction of neuronal
differentiation, such as the appearance of cell neurite projections
([137]Figures 3B and 3C). Immunostaining for bona fide neurite markers
tubulin beta 3 (Tubb3)[138]^42^,[139]^43 and neurofilament
(Nefl)[140]^44 confirmed the nature of the neurites ([141]Figures S4B
and S4C). Similar morphological changes were observed upon AhR
pharmacological inhibition with CLF, CH-223191, BAY-2416964, and
KYN-101 in BE2C cells ([142]Figure S4D). Finally, AHR-depleted cells
were found to accumulate in G[0]/G[1] phase ([143]Figure 3D) which is
conducive to differentiation.[144]^45
Figure 3.
[145]Figure 3
[146]Open in a new tab
AhR suppresses differentiation
(A) GSVA pathway enrichment analysis for several neuronal-related
pathways from RNA-seq of BE2C and Kelly cells transduced with two
independent shAHR constructs and their corresponding non-silencing
control vector.
(B) Representative phase contrast images of cells as in (A). Scale bar
is 100 μm. Arrows point to neurites.
(C) Quantification of % cells with neurites, average neurites/cell, and
average neurite length from 3 independent experiments as in (A). Data
are average −/+ SD. Statistics by two-tailed Student’s t test
∗p < 0.05; ∗∗p < 0.001; ∗∗∗p < 0.0001.
(D) Cell cycle analysis of cells as in (A). Data are average −/+ SD of
3 independent experiments. Statistics by two-tailed Student’s t test.
∗p < 0.05. See also [147]Figures S4 and [148]S5.
Inhibition of MycN in MYCN-amplified cells is also known to induce
differentiation.[149]^46 AHR depletion by shRNA in both cell lines
resulted in reduced MYCN RNA ([150]Figure S5A) and protein
([151]Figure S5B) levels, while AhR activation with its prototypical
ligand TCDD did not cause any significant changes in MycN levels
([152]FigureS5C). Consistently, AHR depletion resulted in
down-regulation of Myc-related signaling, as evidenced by GSVA and gene
set enrichment analysis (GSEA) pathway enrichment analysis of
differentially expressed genes ([153]Figures S5D and S5E), as well as
down-regulation of established MYCN target genes such as MDM2[154]^47
and PTMA[155]^48 ([156]Figure S5F). Similarly, AhR pharmacological
inhibition with the four antagonists described previously caused a
reduction in MycN levels ([157]Figure S5G). To investigate whether
AhR’s effects on differentiation are linked to its ability to regulate
MycN, we depleted BE2C and Kelly cells of MYCN via siRNA, with or
without concomitant ectopic expression of AHR ([158]Figure S5H). While
MYCN depletion induced morphological changes and neurites outgrowth, as
previously reported,[159]^46 AHR over-expression was able to inhibit
this process ([160]Figures S5I and S5J).
These data suggest a unique role for AhR in the regulation of
neuroblastoma differentiation.
AhR is involved in chromatin remodeling
It is well-established that cellular differentiation is characterized
by changes in chromatin accessibility.[161]^49 To further examine how
AhR regulates the differentiation of MYCN-amplified neuroblastoma
cells, we performed Assay for Transposase-Accessible Chromatin
(ATAC)-sequencing in BE2C and Kelly cells with or without AHR
depletion. Although most ATAC-seq peaks were found to be promoter
proximal, as expected with this method ([162]Figure 4A, top six rows),
when we assessed the relative distribution of the “gained” and “lost”
open regions in AHR-depleted cells, we found that most of the
differentially accessible regions (DARs) were residing distal (10–100
Kb) to transcription start sites (TSSs) ([163]Figure 4A, bottom four
rows).
Figure 4.
[164]Figure 4
[165]Open in a new tab
AhR is involved in chromatin remodeling at distal regions
(A) Peaks of accessible chromatin across 3 replicates/sample (top 6
rows) and differentially accessible regions (DARs, bottom 4 rows)
identified by ATAC-seq in Kelly and BE2C cells transduced with control
vector or depleted of AHR.
(B) ChromHMM analysis based on ChIP-seq data in Kelly cells identifies
active enhancers as highly enriched in DARs upon AhR knockdown.
(C) Significant overlap between lost DARs and super-enhancers (SEs)
defined in Kelly cells.
(D) Significant motifs identified in 21 lost DARs that overlap with
SEs. Red bars indicate TFs associated with less differentiated
phenotype. GIGGLE analysis to compare DARs with known binding sites for
over 9,000 transcription factors in Kelly (E) or BE2C (F) cells.
In order to gain insight into what types of genomic regions exhibited
altered open chromatin in response to knockdown of AHR, we performed
ChromHMM[166]^50 using publicly available ChIP-seq data from Kelly
cells ([167]GSE138314
)[168]^51 using the histone marks H3K4me1 (enhancer region), H3K4me3
(promoter region), H3K27me3 (repressive state), and H3K27ac (active
state) to characterize the DARs from our own Kelly ATAC-seq. The
analysis revealed that both gained and lost DARs were highly enriched
for active enhancers ([169]Figure 4B), with active enhancers making up
only ∼10% of total genomic space in Kelly cells but making up ∼45% and
75% of genomic space among the gained and lost DARs, respectively.
These data suggest a strong enhancer reprogramming effect upon AHR
knockdown. Given that super-enhancers (SEs) are known to drive
transcription factors governing cell identity,[170]^52^,[171]^53 we
first went back to ChIP-seq data from Kelly cells
([172]GSE138314)[173]^51 and defined active super enhancers (SE) using
the active histone mark H3K27ac with the ROSE (Rank Ordering of Super
Enhancers) algorithm.[174]^54 We next compared regions that lost
accessibility upon AHR knock-down (lost DARs) with active SEs
identified in Kelly cells and found a significant overlap
([175]Figure 4C). Motif analysis revealed that 18/21 of these
overlapping regions contained binding sites for AP-2 (a master
regulator of the mesenchymal phenotype in neuroblastoma) and that 7 of
the 11 transcription factor motifs found drive mesenchymal
transcriptional programs in neuroblastoma[176]^55 ([177]Figure 4D, red
bars).
To gain a broader view of the transcription factors whose access to
chromatin may be altered upon AHR knockdown, we used the GIGGLE genomic
inquiry tool to overlap all of the DARs identified by ATAC-seq with
CistromeDB and found that lost DARs were enriched in known binding
regions for Myc and MycN (and their binding partner MAX) as well as
with those for AP-2, in agreement with the aforementioned analysis
([178]Figures 4E and 4F). On the other hand, gained DARs aligned
strongly with binding regions for PHOX2B, GATA2/3, and RARA
([179]Figures 4E and 4F) which are known drivers of neuronal
differentiation.[180]^56^,[181]^57
Together, these findings indicate that knockdown of AHR alters
chromatin accessibility at regions distal to transcriptional start
sites and enriches for predicted enhancers and SEs. Moreover, regions
that lose accessibility strongly enrich for binding of transcription
factors of the MYC and AP-2 family known to promote a mesenchymal
phenotype, while regions that gain accessibility enrich for RARA and
other drivers of neuronal differentiation. These observations at the
genomic level are consistent with the phenotypic and morphological
changes observed in [182]Figure 2 upon knockdown or antagonism of AhR,
as well as the global gene expression changes observed.
These findings suggest that antagonism or loss of AhR leads to enhancer
reprogramming, thus affecting the differentiation potential of
MYCN-amplified neuroblastoma cells.
AhR inhibition synergizes with retinoic acid therapy in MYCN-amplified
neuroblastoma
The aforementioned ATAC-seq findings raised the possibility that AHR
depletion could affect the response to retinoic acid-based therapy.
This was further supported by detection of induction of established
all-trans retinoic acid (ATRA) target genes upon AHR depletion in our
RNAseq, such as SCG2,[183]^58 NAV2,[184]^59 CREB5,[185]^60
NBL1,[186]^60 and FZD7[187]^60 ([188]Figure 5A). To investigate this,
BE2C and Kelly cells depleted or not of AHR were treated with ATRA and
we found that AHR depletion augmented retinoic acid-induced
differentiation, as measured by percent of cells with neurites, average
number of neurites per cell, and average neurite length relative to
treatment with retinoic acid alone ([189]Figures S6A–S6D). Moreover,
AHR depletion potentiated the ATRA-induced suppression of MycN
([190]Figures S6E and S6F).
Figure 5.
[191]Figure 5
[192]Open in a new tab
Clofazimine and ATRA are synergistic in vitro
(A) Normalized expression of selected ATRA-induced genes from the
RNAseq analysis.
(B) Representative phase contrast images of BE2C and Kelly cells
treated with 4 μM CLF, 10 μM ATRA, or a combination of the two for
7 days. Scale bar is 100 μm.
(C) Representative clonogenic assay (out of 3) of BE2C and Kelly cells
treated with 1 μM CLF, 2 μM (Kelly) or 5 μM (BE2C) ATRA, or a
combination of the two.
(D) Quantification of (C) performed with ImageJ. Statistics by
two-tailed Student’s t test. Data are average −/+ SD (n = 3).
(E) Representative immunoblot for MycN in BE2C and Kelly cells treated
as in (A). Actin or tubulin are used as loading control.
Quantifications performed with ImageJ: MycN signal was normalized to
that of the corresponding loading control and then normalized to the
value of DMSO.
(F) CompuSyn-based analysis of synergy between CLF and ATRA in BE2C and
Kelly cells. Cells were treated with several combinations of the drugs,
centered around their IC[50] (CLF 0-6 μM; ATRA 0-80 μM) for 48 hrs.
Surviving cells were stained with methylene blue. Most of the
combinations have a CI below 1 indicating synergy. See also
[193]Figure S6.
Consistently, CLF-mediated AhR antagonism and ATRA treatment induced
stronger morphological changes in cells when combined than singularly
([194]Figure 5B), as well as almost completely abolished colony
formation at doses at which the single drugs were not as effective
([195]Figures 5C and 5D). Similar to the genetic depletion, the
combination of CLF and ATRA resulted in a stronger suppression of MycN
levels than control or treatment with either drug alone
([196]Figure 5E).
To test for potential synergy between CLF and ATRA, BE2C and Kelly
cells were treated with several combinations of the drugs, centered
around their IC[50] and the proportions of surviving cells were
analyzed with CompuSyn. The analysis revealed a strong synergistic
effect of CLF and ATRA in suppressing the growth of BE2C and Kelly
cells, as indicated by a combination index (CI) of less than 1
([197]Figure 5F).
To assess the degree of persistence of differentiation upon drug
treatment, cells were treated with CLF, ATRA, their combination, or
DMSO as vehicle control for 10 days to induce differentiation. At that
point (day 0 in [198]Figure S7), cells were released from the drugs and
cultured in complete media alone for up to 25 days. Control cells
continued to grow regularly and needed to be split every 2–3 days;
clofazimine-treated cells resumed growth and de-differentiated
morphology within a couple of days and, similar to control, needed to
be split every 2–3 days. ATRA-treated cells took about a week to
restart growing with normal doubling time and morphology. The CLF+ATRA
cells maintained the differentiated morphology throughout the 25 days
and did not resume growth ([199]Figure S7). These data suggest that the
combination of CLF and ATRA is more efficient in inducing a stable
differentiation of cells.
To test whether the combination would also be more effective in an
in vivo setting, we pretreated cells with either drug alone, their
combination, or vehicle control, to mimic minimal residual disease
settings as previously reported.[200]^61 Equal numbers of live cells
were implanted SQ in NSG mice, with no further treatment. While vehicle
control cells and single drug-treated cells formed rapidly growing
tumors, the combination strongly suppressed tumor growth
([201]Figures 6A and 6B). Statistical analysis comparing the
combination group to control or either single drug showed a
statistically significant (p = 0.001) difference, indicating a
synergistic effect ([202]Figure 6A). Tumors sections were stained for
Tubb3 and MycN. CLF and ATRA as single agents showed an increase in
staining intensity for the differentiation marker Tubb3
([203]Figure 6C) and a reduction in MycN ([204]Figure 6D). Their
combination was significantly more potent at inducing this
differentiation phenotype, in agreement with the almost negligible
growth of the xenografts in mice ([205]Figures 6A and 6B).
Figure 6.
[206]Figure 6
[207]Open in a new tab
Clofazimine and ATRA are synergistic in vivo
(A) BE2C cells treated with 6 μM CLF, 10 μM ATRA, or a combination of
the two for 7 days were injected SQ in the right flank of NSG mice (n =
10, equal numbers of females and males were used). Tumors were measured
with a caliper twice/week. Animals were humanely euthanized when a
tumor in any group reached the limit set by IACUC protocol. Data are
average −/+ SEM. Statistics by two-way ANOVA test. Representative tumor
pictures are shown.
(B) Representative images of tumors excised at the endpoint of (A).
(C and D) Representative IHC images and quantification of tumor
sections from (A) stained for Tubb3 (C) and MycN (D). Data are average
−/+ SD (n = 6). Statistics by two-tailed Student’s t test. See also
[208]Figure S7.
Overall, our study unveils a previously undisclosed pro-tumorigenic
function of AhR in MYCN-amplified neuroblastoma, centered on its
ability to suppress differentiation. These functions pass partly
through regulation of chromatin accessibility, which negates retinoic
acid-induced signaling and supports MycN functions. Most importantly,
our data highlight AhR as a potential target in the treatment of
MYCN-amplified neuroblastoma, whereby AhR antagonism may prime the
cells to be responsive to retinoic acid treatment and synergize with
retinoid-based therapy. As a few AhR antagonists are already in
clinical trials and/or FDA-approved for other interventions, our data
provide the basis for their repurposing into future pre-clinical and
clinical trials for MYCN-amplified neuroblastoma.
Discussion
The tumorigenic role of AhR in the nervous system and particularly in
neuroblastoma is only beginning to emerge and remains largely
understudied and controversial, with different groups reporting
different effects of AhR activation or inhibition on neuroblastoma cell
fate.[209]^25^,[210]^26^,[211]^62^,[212]^63^,[213]^64^,[214]^65^,[215]^
66 Our findings revealed that in MYCN-amplified neuroblastoma, AhR acts
as a tumor promoter, supporting tumor growth, positively regulating
MycN levels, and maintaining cells in an undifferentiated state.
Similarly, AhR activation by low doses of its prototypical ligand TCDD
has been shown to enhance neuroblastoma cell migration[216]^66 and
parental exposure to various AhR ligands has been loosely linked to
higher odds of neuroblastoma development in their offspring.[217]^67
On the other hand, AHR ectopic expression has been suggested to induce
differentiation in mouse Neuro2a cells, which do not otherwise express
endogenous AhR[218]^63 and miR-124-mediated suppression of AhR has been
linked to suppression of differentiation in SK-N-SH cells.[219]^64
However, miR-124 is actively up-regulated during induction of
differentiation in SH-SY5Y cells[220]^68 (which are a derivative of
SK-N-SH) thus adding to the controversy. Wu et al.[221]^25 reported AhR
to be a tumor suppressor in neuroblastoma through MycN downregulation:
these studies mostly interrogated AhR function and regulation of MycN
expression in non-MYCN-amplified neuroblastoma, where MycN is not a
driver of the disease and in cells that, according to our results and
published ones[222]^26^,[223]^69 do not have detectable levels of
endogenous AhR protein.
In our hands, AHR overexpression in these cell systems leads to
downregulation of cMyc, which is highly expressed in non-MYCN-amplified
cells and contributes to their tumorigenicity. Consistently, upon AHR
depletion we observed a reduction in clonogenic growth and invasion of
non-MYCN-amplified cells, while we could not detect MycN protein
levels. In order to observe neurite outgrowth, Wu et al.[224]^25 kept
AHR-overexpressing cells in constant selective antibiotic for a month,
which may have led to unintended consequences in terms of cell stress
or mutations and may explain some of the discrepancies with our data.
In our current work, only 48-72 hrs of antibiotic selection after
lentiviral delivery of shAHR were applied and neurite outgrowth, whose
nature was confirmed by immunofluorescence for neurite markers, was
observed within a week.
Altogether, these results highlight a previously unrecognized opposing
role of AhR in different neuroblastoma subtypes, similar to what has
been reported in different breast cancer
subtypes.[225]^18^,[226]^19^,[227]^70^,[228]^71 The data obtained in
non-MYCN-amplified cells suggest that AhR’s anti-tumor functions in
these settings are independent of MycN and may depend instead on other
nuclear factors and receptors being expressed differently between the
subtypes, such as cMYC. This hypothesis is also consistent with the
notion that AhR can act very differently during organismal development
and during tumor progression. For instance, a recent paper reported
novel endogenous ligands of AhR that mediate neural development in
zebrafish[229]^72 and previous work suggested that crossing AHR
knock-out mice with the TRAMP model results in promotion of prostate
carcinogenesis[230]^73 as well as colon carcinogenesis in the Apc/Kras
mice background.[231]^74 However, AhR manipulation in cancer cells from
both prostate and colon cancer suggest a tumor promoting role of
AhR.[232]^65^,[233]^75 Thus, the role of the nuclear milieu in terms of
factors and co-factors potentially influencing AhR activity needs to be
explored in depth and may help better understand the shift in functions
of AhR and shed light on some of the controversies surrounding AhR
biology.
AhR is normally sequestered in an inactive form in the cytoplasm and
becomes active once it is released and allowed to translocate to the
nucleus where it activates gene transcription. Thus, AhR levels per se
(at RNA or protein level) are not necessarily indicative of its
activity. Wu et al. reported that AHR mRNA levels (stratified by
average level of expression) associated with differences in patients’
survival when interrogating the SEQC dataset[234]^26; however, this
relationship does not exist when restricting the analysis to
MYCN-amplified patients in this same dataset nor in the
“Therapeutically Applicable Research to Generate Effective Targets
project” (TARGET) independently of patients’ stratification. Since the
anti-tumor effects of AhR were proposed to be through its ability to
suppress MYCN[235]^25^,[236]^26 but MycN is not a driver in
non-MYCN-amplified neuroblastoma, the significance of this association
needs to be reevaluated as what these data altogether suggest is that
AhR tumor suppressive roles in non-MYCN-amplified neuroblastoma might
be through other factors (such as possibly cMYC). Similarly, the
survival analyses between AhR protein expression and different types of
neuroblastoma (undifferentiated, differentiating, and
ganglioneuroblastoma)[237]^26 will need to be re-evaluated separating
cohorts on median or average expression of AhR rather than scanning for
the best separation groups, as well as analyzing the correlation with
nuclear localized (active) AhR rather than total. Several AhR
transcriptional activity gene signatures have been
described[238]^17^,[239]^36; however, they were mainly derived from
studies in epithelial cells. In order to obtain neuroblastoma-relevant
genes, we created a signature from genes that were consistently
regulated upon AHR depletion with two independent shRNA constructs in
two MYCN-amplified human neuroblastoma cell lines, thus enhancing the
stringency of the analysis. Interestingly, this gene signature strongly
aligns with a favorable outcome in several distinct patients’
datasets,[240]^37 which yet contain >70% non-MYCN-amplified cases.
Thus, it will be important in the future to carefully evaluate the
effects of AhR antagonism on patient-derived organoids or xenografts to
assess any confounding effect due to tumor heterogeneity.
ATAC-seq experiments revealed that AhR can affect chromatin
accessibility at distal regions, in agreement with previous reports of
AhR’s ability to promote DNA hypermethylation[241]^76^,[242]^77 and to
participate in epigenetic regulatory complexes.[243]^78^,[244]^79
Neuroblastoma has been shown to be composed of interchangeable
populations of mesenchymal and adrenergic subtypes.[245]^53^,[246]^55
The mesenchymal subtype is considered to be more undifferentiated and
therapy-resistant and is enriched post-treatment and in relapsing
tumors.[247]^53^,[248]^55 The adrenergic subtype is more committed and
therapy-responsive.[249]^53^,[250]^55 The transcription factors PHOX2B,
GATA2/3, and HAND1/2 are associated with the SE circuitry that defines
the adrenergic lineage and control neuronal specification and
differentiation during development.[251]^55^,[252]^80^,[253]^81 The
levels of these transcription factors need to be tightly regulated in
cells as high levels are deleterious and correlate with poor
survival.[254]^82^,[255]^83^,[256]^84 Consistently Zimmerman
et al.[257]^85 reported GATA3 and PHOX2B to be downregulated during
ATRA-induced differentiation. Thus, dosage of these transcription
factors appears to be critical where low levels may promote
differentiation while too high levels drive neuroblastoma progression.
In our RNAseq data, we see a trend toward small but significant
downregulation of PHOX2B and GATA3 upon AHR-downregulation, although
this is not fully consistent among constructs. At the same time, we see
opening of chromatin loci bound by these factors when cells are
depleted of AHR. Thus, it is possible that reduced levels of PHOX2B and
GATA3 binding at newly opened regions may help drive differentiation,
consistent with their biological role during differentiation.
Notably, we found that AHR depletion induced opening at regions
commonly bound by RARα and restricted access to regions commonly bound
by MycN. These data imply that AHR depletion would suppress MycN
related signaling and potentially prime the cells for retinoic
acid-mediated signaling and induction of differentiation. Indeed,
RNA-seq analysis yielded results consistent with this hypothesis, which
was further validated morphologically and by immunostaining.
Interestingly, AHR ectopic expression was able to counteract the
differentiation caused by MYCN depletion, suggesting that AhR may work
independently of (or perhaps in parallel with) MycN in regard to
differentiation. These findings add to the growing complexity and
controversy on the topic of the AhR-Myc family interaction and
cross-regulation.[258]^21^,[259]^25^,[260]^26^,[261]^30^,[262]^86^,[263
]^87^,[264]^88^,[265]^89
Treatment of patients with high-risk MYCN-amplified neuroblastoma
remains a clinical challenge, as relapses resulting from the residual
tumor cells’ ability to overcome differentiating therapies such as
retinoic acid occur in more than 50% of patients.[266]^90 We find that
the retinoic acid effects are potentiated when AhR is inhibited either
genetically or pharmacologically, suggesting a potential synergy
between AhR inhibition and retinoid treatment to achieve a more durable
response. Consistently, injection of cells pre-treated with the
combination of CLF and ATRA, in order to mimic minimal residual disease
settings occurring in patients, prevented the growth of tumor xenograft
in mice suggesting that the differentiation imparted by both drugs is
more durable and less easy to overcome than the one imparted by the
individual drugs. The AhR antagonist CLF is already FDA-approved for
the treatment of leprosy, drug-resistant tuberculosis, and other
infections and has been safely used in children with no adverse
effects[267]^31^,[268]^32^,[269]^91^,[270]^92 and our studies showed
that CLF synergistically enhances the effectiveness of retinoic acid in
MYCN-amplified neuroblastoma. Of the two novel AhR antagonists used in
this study, BAY-2416964 is currently in Phase I clinical trials for
advanced solid tumors ([271]NCT04069026 and [272]NCT04999202) and
KYN-101 demonstrated anti-tumor efficacy in pre-clinical murine models
of melanoma.[273]^93 Importantly, the differentiation imparted by the
combination of CLF and ATRA on cultured cells was sustained for up to
25 days post-drug withdrawal, while the single drug treated cells
resumed growth within a week, consistent with the in vivo data. Thus,
it will be important to thoroughly evaluate the synergy between
treatment with AhR antagonists and retinoids in multiple pre-clinical
models of the disease as a means to ensure a more durable response in
patients.
Limitations of the study
Our study showed that pre-treatment of cells with clofazimine and
retinoic acid to mimic induction of minimal residual diseases reduce
their growth in vivo and maintain the cells in a differentiated-like
status for longer. However, it will be important to use a better model
of minimal residual disease mimicry, such as implanting naive cells,
letting tumors grow, treat them with conventional therapy until
regressed, and then administer the drug combination. Similarly, it will
be important to test the drug combination in a relevant model of the
disease, such as the TH-MYCN mice, where the effects from and upon the
immune system are also kept in consideration.
STAR★Methods
Key resources table
REAGENT or RESOURCE SOURCE IDENTIFIER
Antibodies
__________________________________________________________________
Rabbit monoclonal to MycN (D1V2A) Cell Signaling Cat# 84406,
RRID:[274]AB_2800038
Rabbit monoclonal to AhR (D5S6H) Cell Signaling Cat# 83200,
RRID:[275]AB_2800011
Rabbit polyclonal to c-Myc Cell Signaling Cat# 9402,
RRID:[276]AB_2151827
Rabbit monoclonal to beta-tubulin-III (D71G9) Cell Signaling Cat# 5568,
RRID:[277]AB_10694505
Rabbit polyclonal to neurofilament-L (C28E10) Cell Signaling Cat# 2837,
RRID:[278]AB_823575
Mouse monoclonal to AhR (A-3) Santa Cruz Biotechnology Cat# sc-133088,
RRID:[279]AB_2273721
Mouse monoclonal to alpha-tubulin (B-5-1-2) Sigma Aldrich Cat# T6074,
RRID:[280]AB_477582
HRP-beta actin Proteintech HRP-60008, RRID:[281]AB_2819183
HRP-GAPDH Proteintech Cat# HRP-60004, RRID:[282]AB_2737588
Alexa Fluor 568 (Red) -conjugated goat-anti-rabbit antibody Thermo
Fisher Scientific Cat# A21069, RRID:[283]AB_1056360
__________________________________________________________________
Chemicals, peptides, and recombinant proteins
__________________________________________________________________
Alexa Fluor 488 (Green)-conjugated phalloidin Thermo Fisher Scientific
Cat# A12379
Hoechst-33342 Invitrogen Cat#H3570
Clofazimine Sigma Aldrich Cat#C8895
CH-223191 Sigma Aldrich Cat#C8124
all-trans-retinoic acid (ATRA) Sigma Aldrich Cat#R2625
BAY-2416964 Selleckchem Cat#S8995
KYN-101 Aobious Cat# [284]AOB11039
LipoD293 SignaGen Cat# SL100668
Hexadimethrine bromide Sigma Aldrich Cat#H9268
PureLink DNase Set Thermo Fisher Scientific Cat#12185010
__________________________________________________________________
Critical commercial assays
__________________________________________________________________
PureLink RNA Mini kit Thermo Fisher Scientific Cat#12183018A
High-Capacity cDNA Reverse Transcription kit Thermo Fisher Scientific
Cat# 4368814
Hema3 Staining Kit Fisher Scientific Cat#22-123869
8.0 μm Biocoat Matrigel-Coated Invasion Chambers Corning Cat#354480
__________________________________________________________________
Deposited data
__________________________________________________________________
RNAseq This Paper GEO; [285]GSE224037
ATACseq This Paper GEO:[286]GSE224390
[287]GSE224037 +
[288]GSE224390 This Paper GEO Super-Series: [289]GSE224391
__________________________________________________________________
Experimental models: Cell lines
__________________________________________________________________
Human MYCN-amplified neuroblastoma Kelly Dr. Katerina Gurova N/A
Human MYCN-amplified neuroblastoma BE2C Dr. Katerina Gurova N/A
Human MYCN-amplified neuroblastoma IMR-32 Dr. Michael Higgins N/A
Human non-MYCN-amplified neuroblastoma SK-N-SH Dr. Michael Higgins N/A
Human non-MYCN-amplified neuroblastoma SH-SY5Y Dr. Michael Higgins N/A
Human non-MYCN-amplified neuroblastoma SHEP Dr. Michelle Haber N/A
Human non-MYCN-amplified neuroblastoma NBL-S Dr. Michelle Haber N/A
HEK293-T Dr. Irwin Gelman N/A
__________________________________________________________________
Experimental models: Organisms/strains
__________________________________________________________________
Mouse: NOD-SCID Gamma (NSG) Roswell Park Comprehensive Cancer Center
Colony N/A
__________________________________________________________________
Oligonucleotides
__________________________________________________________________
siRNA pools against MYCN Santa Cruz Biotechnology Cat#sc-36003
Primer for MYCN amplification qRT-PCR_FWD 5′ CACAAGGCCCTCAGTACCTC 3′
This Paper N/A
Primer for MYCN amplification qRT-PCR_REV 5′ ACCACGTCGATTTCTTCCTC 3′
This Paper N/A
Primer for RPS20 amplification qRT-PCR_FWD 5′ AAGGATACCGGAAAAACACCC 3′
This Paper N/A
Primer for RPS20 amplification qRT-PCR_REV 5′ TTTACGTTGCGGCTTGTTAGG 3′
This Paper N/A
__________________________________________________________________
Recombinant DNA
__________________________________________________________________
pLVp-SV4-puro lentiviral vector Dr. Peter Chumakov N/A
pLKO-GFP lentiviral vector Sigma Aldrich SHC005
pLKO-shAHR #1 lentiviral vector Sigma Aldrich TRCN0000245285
pLKO-shAHR#2 lentiviral vector Sigma Aldrich TRCN000021258
pCMV-VSV-G Stewart et al. RNA (2003) Apr; 9(4):493-501 Addgene Plasmid
# 8454
pCMV-psPAX2 Dr. Irwin Gelman N/A
__________________________________________________________________
Software and algorithms
__________________________________________________________________
ImageJ [290]https://imagej.nih.gov/ij/index.html
R v3.6–4.0 [291]https://www.r-project.org/
[292]https://www.r-project.org/
DESeq2 Love et al.[293]^65
[294]https://bioconductor.org/packages/release/bioc/html/DESeq2.html
Bowtie2 Zhang et al.[295]^70
DiffBind Stark et al.[296]^71
GIGGLE Layer et al.[297]^72
CistromeDB Mei et al.[298]^73
ChromHMM Ernst et al.[299]^38
GenomicRanges Lawrence et al.[300]^75
ROSE Whyte et al.[301]^42
[302]http://younglab.wi.mit.edu/super_enhancer_code.html
HOMER v4.11 [303]http://homer.ucsd.edu/homer/
Graphpad Prism V9.3.1 Graphpad Software [304]https://www.graphpad.com/
ModFit ModFit LT 5.0 software
QuantStudio Real-Time PCR Software v1.3 Applied Biosystems
[305]https://www.thermofisher.com/us/en/home/global/forms/life-science/
quantstudio-6-7-flex-software.html
GeneSys Syngene
[306]https://www.syngene.com/software/genesys-rapid-gel-image-capture/
CompuSyn [307]https://www.combosyn.com/
R2: Genomics Analysis and Visualization Platform Genomics Analysis and
Visualization Platform ([308]http://r2.amc.nl) [309]http://r2.amc.nl
[310]Open in a new tab
Resource availability
Lead contact
* •
Further information and requests for resources and reagents should
be directed to and will be fulfilled by the lead contacts, Anna
Bianchi-Smiraglia (Anna.Bianchi-Smiraglia@RoswellPark.org).
Materials availability
* •
This study did not generate new unique reagents.
Data and code availability
* •
All sequencing data reported here (RNAseq and ATACseq) are
available in the NCBI Gene Expression Omnibus (GEO) database and
are publicly available as of the date of publication. Accession
numbers are listed in the [311]key resources table.
* •
This paper does not report original code.
* •
Any additional information required to reanalyze the data reported
in this paper is available from the [312]lead contacts upon
request.
Experimental model and study participant details
Mouse models
All experiments involving animals were approved by the Institutional
Animal Care and Use Committee. (IACUC) and listed under protocol 1450M.
Kelly or BE2C cells (1 x 10^6) with indicated manipulations were
resuspended in 100
[MATH: μ :MATH]
L of Matrigel (Corning, Corning, NY, USA) and inoculated subcutaneously
into the right flank of equal numbers of males and females 6-8-week-old
NOD-SCID Gamma mice, bred and housed at the Division of Laboratory
Animal Resources (Roswell Park Comprehensive Cancer Center, Buffalo,
NY, USA). As neuroblastoma affects almost equally males and females
(1.2:1 ratio) equal numbers of male and female mice were used. No sex
difference emerged during the studies. Tumor volumes were recorded
twice/week and mice were humanely euthanized when a tumor volume
reached 2 cm^3 or when a tumor became ulcerated. No animals were
excluded from the study since all animals developed palpable tumors
within 2 weeks of subcutaneous inoculation of cells and no animals
developed significant morbidity before the end of the study.
Cell lines
MYCN-amplified Kelly (female) and BE2C (male) cells were a kind gift
from Dr. Katerina Gurova (Roswell Park Comprehensive Cancer Center,
Buffalo, NY, USA). MYCN-amplified IMR32 (male) cells and
non-MYCN-amplified SK-N-SH (female) and SH-SY5Y (female) cells were a
kind gift from Dr. Michael Higgins (Roswell Park Comprehensive Cancer
Center, Buffalo, NY, USA). Non-MYCN-amplified SHEP (female) and NBL-S
(male) cells were a kind gift from Drs. Michelle Haber and Murray
Norris (Children’s Cancer Institute, Sydney, Australia). HEK-293T cells
were a kind gift from Dr. Irwin Gelman (Roswell Park Comprehensive
Cancer Center, Buffalo, NY, USA). Kelly, BE2C, and IMR32 cells were
cultured in RPMI media (Invitrogen, Carlsbad, CA, USA); SK-N-SH,
SH-SY5Y, SHEP, NBL-S, and HEK293T were cultured in DMEM media
(Invitrogen). Both media were supplemented with 10% fetal bovine serum
(Invitrogen) and antibiotic-antimycotic (Invitrogen). All cell lines
were authenticated via short tandem repeat sequencing at the Roswell
Park Genomics Shared Resource between November 2019 and March 2023 and
routinely tested for mycoplasma contamination.
Method details
Lentiviral infections
Transfection of plasmids was performed using LipoD293 (SignaGen,
Frederick, MD, USA) into HEK293T cells along with the pCMV-VSV-G vector
(Addgene, Cambridge, MA, USA) and the pCMV-psPAX2 vector (a kind gift
from Dr. Irwin Gelman, Roswell Park Comprehensive Cancer Center,
Buffalo, NY, USA). The pLVp-SV4-puro lentiviral vector was obtained
from Dr. Peter Chumakov (Cleveland Clinic, Cleveland, OH, USA).
pLKO-GFP and shRNA toward AhR were purchased from Sigma: shAhR #1
TRCN0000245285; shAhR #2 TRCN000021258.
All lentiviral infections were performed as previously
described.[313]^15 Briefly, HEK293T cells were transfected with
LipoD293 and target plasmid in the presence of packaging plasmids
according to the manufacturer’s protocol. The media was refreshed after
8hrs and lentiviral supernatant was harvested at 48hrs, filtered with a
0.45 μm filter and syringe, and transduced to cells in the presence of
8
[MATH: μ :MATH]
g/mL hexadimethrine bromide (Sigma, St. Louis, MO, USA).
Cell cycle analysis
Approximately 2 × 10^6 cells were washed with PBS, harvested, and fixed
in ice-cold 70% ethanol. Cells were washed twice with PBS, resuspended
in staining buffer (100 mM Tris pH 7.4, 150 mM NaCl, 1 mM CaCl[2],
0.5 mM MgCl[2], 0.1% Nonidet P-40) with RNase A (10
[MATH: μ :MATH]
g/mL), and incubated at 37°C for 30 min. Cells were resuspended in
fresh staining buffer with propidium iodide solution (1
[MATH: μ :MATH]
g/mL). Samples were acquired on an LSR Fortessa Becton Dickinson flow
cytometry analyzer at the Roswell Park Comprehensive Cancer Center
Flow & Image Cytometry Shared Resource Facility using a 535/617 nm
filter. Cell cycle analysis was conducted using ModFit LT 5.0 software
(Verity Software House, Topsham, ME, USA).
Immunofluorescence
Immunofluorescence staining was performed as previously
described.[314]^15 Briefly, cells were grown on glass coverslips, fixed
in 4% paraformaldehyde (PFA) in PBS, permeabilized in 0.01% Triton
X-100 in PBS and blocked in 3% milk in PBS. Primary antibody incubation
was carried out in 1% milk in PBS at room temperature, and secondary
antibody and phalloidin staining were carried out in 0.5% milk in PBS
at RT. Nuclei were stained with Hoechst 33258. Images were acquired
with a Zeiss Axio Observer z.1 inverted microscope equipped a Zeiss Mrm
camera and AxioVision 4.8 software.
RNA-seq
Total cellular RNA was isolated using the PureLink RNA Mini kit with
on-column DNase treatment (Thermo Fisher Scientific). Sequencing
libraries were prepared with the TruSeq Stranded mRNA kit (Illumina)
from 500 ng total RNA following manufacturer’s instructions.
PCR-amplified libraries were pooled in an equimolar fashion, loaded
into a 75-cycle NextSeq Reagent Cartridge, and single-end sequencing
performed on a NextSeq 500 (Illumina) following the manufacturer’s
recommended protocol. Genome alignments and feature counting were
performed at the Roswell Park Comprehensive Cancer Center’s Genomics
Shared Resource. Raw reads were mapped to the human reference genome
(GRCh38.p13) using STAR.[315]^94 Raw feature count normalization and
differential expression analysis were carried out using DESeq2.[316]^95
Differential expression rank order was used for subsequent
GSEA,[317]^96 performed using the cluster profile package in R. Gene
sets queried included the Hallmark, Canonical pathways, and GO
Biological Processes Ontology collections available through the
Molecular Signatures Database (MSigDB).[318]^97 For select pathways,
per sample enrichment was calculated via ssGSEA, performed using the
GSVA package.[319]^98 Overlaps of DEG lists across companions were
calculated by hypergeometric testing. All analyses were performed using
R statistical software, version 4.1.1.
ATAC-seq
Samples for ATAC-seq were prepared using a protocol by Buenrostro
et al.,[320]^99 with minor modifications. Briefly, cells (5 × 10^3) in
triplicates were collected in cold PBS and spun down. Pellets were
gently resuspended in 50μL cold lysis buffer and nuclei were spun at
2,100 rpm, 10 min at 4°C. Supernatant was discarded and pellets were
placed on ice for the transposition reaction. ATAC-seq libraries were
prepared by incubating nuclei pellets with TD 2x buffer and TDE1
transposase by Illumina for 30 min at 37°C, followed by sample
purification with Qiagen MinElute PCR purification kit. Transposed DNA
fragments were amplified with 10 PCR cycles using a PCR primer
(Ad1_noMX,IDT) and a barcoded PCR primer (Ad_index primer, IDT) with a
NEB Next High Fidelity 2x PCR Master Mix (NEB). Libraries were
sequenced with an Illumina NextSeq 500 Platform with 75 bps paired-end
reads. Reads were aligned to the human (hg19) reference genome with
Bowtie2 tool and called peaks using MACS3.[321]^100 DARs were
determined using DiffBind.[322]^101 To find potential transcription
factor binding enrichment within DARs, we utilized GIGGLE[323]^102 to
query the complete human transcription factor ChIP-seq dataset
collection in Cistrome DB.[324]^103 Putative co-enriched factors were
identified by assessment of the number of time a given factor was
observed in the top 200 most enriched datasets relative to the total
number of datasets for that factor in the complete Cistrome DB (>1.2 FC
enrichment over background).
ChromHMM
Previously published ChIP-seq data from Kelly cells ([325]GSE138314
)[326]^51 was reprocessed and reanalyzed as previously
detailed.[327]^104 We applied ChromHMM[328]^50 to learn the regulatory
chromatin states in Kelly cells using the histone marks H3K4me1
(enhancer region), H3K4me3 (promoter region), H3K27me3 (repressive
state), and H3K27ac (active state). We produced a new 13-chromatin
state model which was further collapsed into 6 broad regulatory
chromatin states based on histone mark signal intensity, human genomic
region annotations, and TSS genomic neighborhoods: Quiescent/No Signal,
Repressed/Polycomb (defined by H3K27me3), Bivalent/Poised Enhancer
(defined by distal H3K4me1 and H3K27me3), Bivalent/Poised Promoter
(defined by proximal H3K4me3 and H3K27me3), Active Enhancer (defined by
distal H3K4me1 and H3K27ac), and Active Promoter (defined by proximal
H3K4me3 and H3K27ac). Enrichment of ATAC-seq peaks within Kelly
ChromHMM-defined regulatory states was performed using
GenomicRanges[329]^105 with a max overlap of 200 bp. Enrichment was
defined as the percentage of overlapping regions proportional to the
total genomic space defined in Kelly cells. All analyses were
undertaken using the R platform for statistical computing (version 4.1
or later) using library packages implemented in Bioconductor or using
the indicated software packages implemented in Java>1.6 and Python3.
Super-enhancers identification
Using the same reprocessed and reanalyzed ChIP-seq data from Kelly
cells ([330]GSE138314
),[331]^51 we defined active SEs (SE) using the active histone mark
H3K27ac with the ROSE (Rank Ordering of SEs) algorithm.[332]^54 We
distinguished typical enhancers from active SEs using the
ChromHMM-defined enhancer states and our previously published ROSE
parameters.[333]^104 All analyses were undertaken using the R platform
for statistical computing (version 4.1 or later) using library packages
implemented in Bioconductor or using the indicated software packages
implemented in Java>1.6 and Python3.
Motif analysis
Known and de novo transcription factor motif enrichment was performed
using HOMER v4.11 software utilizing the findMotifsGenome.pl command
using default parameters. All analyses were undertaken using the R
platform for statistical computing (version 4.1 or later) using library
packages implemented in Bioconductor or using the indicated software
packages implemented in Java>1.6 and Python3.
Quantitative real-time PCR
Total cellular RNA was isolated using the PureLink RNA Mini kit with
on-column DNase treatment (ThermoFisher Scientific, Waltham, MA, USA).
cDNA was prepared using the High-Capacity cDNA Reverse Transcription
kit (Thermo Fisher Scientific). Quantitative real time PCR was
performed on a QS6 Fast Real-Time PCR machine (Thermo Fisher
Scientific) using Power Up SYBR Green Master Mix (Thermo Fisher
Scientific) using the following human site-specific primers listed
below. Data were analyzed using the QuantStudio Real-Time PCR Software
(Thermo Fisher Scientific).
MYCN FWD: 5′ CACAAGGCCCTCAGTACCTC 3’
MYCN REV: 5′ ACCACGTCGATTTCTTCCTC 3’
RPS20 FWD: 5′ AAGGATACCGGAAAAACACCC 3’
RPS20 REV: 5′ TTTACGTTGCGGCTTGTTAGG 3’
Colony formation assays
Cells (500/well) were seeded in 6-well plates in triplicate in 2 mL of
media. Media was replenished with or without indicated drug treatments
every 2–3 days. After 3–4 weeks, cells were fixed and stained with 0.5%
methylene blue in a 1:1 methanol: water solution and imaged once dry.
Invasion assays
Invasion assays were performed as previously described.[334]^106
Briefly cells were harvest by trypsinization and resuspended in
serum-free media. 1x10^5 cells were seeded into the top compartment of
8.0
[MATH: μ :MATH]
m Biocoat Matrigel-coated invasion chambers (Corning, Corning, NY, USA)
in duplicates. Complete media with 10% FBS was used as a
chemoattractant in the bottom compartment. Cells were incubated at 37°C
for 24hrs and bottom membranes were fixed and stained with the Hema3
kit (Fisher Scientific) according to the manufacturer’s protocol. Cells
were counted from 5 different view-fields per transwell.
Immunoblotting
Whole cell extracts were prepared in RIPA buffer (50 mM Tris-HCl,
150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium
dodecyl sulfate (SDS), 10% glycerol, 2 mM EDTA). Samples were resolved
on 10% polyacrylamide gels and transferred to nitrocellulose membranes
(Biorad, Hercules, CA, USA). Membranes were incubated overnight at 4°C
with primary antibody diluted in blocking buffer. Appropriate
HRP-conjugated secondary antibodies (Bio-Rad, Hercules, CA, USA) were
used at 1:5,000 dilution in blocking buffer for 1 h at RT. Signals were
visualized with BioRad chemiluminescence reagents and a GeneGnome XRQ
NPC system (Syngene, Frederick, MD, USA).
Neurite analysis
Cells were treated with the indicated drugs or transduced with the
indicated vectors and images were acquired using a Nikon Eclipse Ts2
inverted microscope and NIS Elements software (Melville, NY, USA).
Quantification of percentage of cells with neurites, average neurites
per cell, and average neurite length was carried out using the NeuronJ
plugin[335]^107 of ImageJ (National Institutes of Health, Bethesda, MD,
USA).
Immunohistochemistry staining
Tissues were fixed in 10% buffered formalin for 24 h prior to
processing. Tissues were embedded in paraffin and sectioned at 5
microns. Slides were de-paraffinized in several baths of xylene and
then rehydrated in graded alcohols followed by ddH[2]O. Slides were
incubated in 1x pH 6 citrate buffer (Invitrogen Cat #00–5000) for
20 min. Slides were incubated in 3% H[2]O[2] for 15 min. To block
non-specific binding, tissues were incubated with 10% normal goat serum
for 10 min, followed by avidin/biotin block (Vector Labs, Newark, CA,
USA; Cat#SP-2001). Primary antibodies b3-Tubulin (1:400) from Cell
Signaling Cat#5568, and N-Myc (1:800) from Cell Signaling Cat#51705
were diluted in 1% BSA solution and incubated for 30 min at room
temperature, followed by the biotinylated Goat anti Rabbit secondary
antibody (Vector Labs #BA-1000) for 15 min. For signal enhancement, ABC
reagent (Vector Labs Cat #PK 6100) was applied for 30 min. To reveal
endogenous peroxidase activity, slides were incubated with DAB
substrate (Dako Cat #K3467) for 5 min and then counterstained with DAKO
Hematoxylin for 20 s. Slides were dehydrated through several baths of
graded alcohols and xylenes and then coverslipped. Images were acquired
on a Leica Biosystem microscope with a 20X lens and a Flexacam C1
camera. Signal quantification was performed with ImageJ.
Quantification and statistical analysis
Experiments were repeated at least three independent times (exact n is
indicated in figure legends). Statistical analysis was performed using
Student’s t test within Prism version 9 software (GraphPad, San Diego,
CA), unless otherwise noted. A two-tailed p value < 0.05 was considered
statistically significant for analyses. For mouse studies, the log
tumor sizes were modeled as a function of treatment group, time, their
two-way interaction, and random mouse effects using a linear mixed
model. Tumor growth rates were compared between treatment groups using
tests about the appropriate contrasts of model estimates. All model
assumptions were verified graphically and all analyses were performed
in SAS v9.4 (Cary, NC) at a significance level of 0.05.
Acknowledgments