Small molecule that reverses dexamethasone resistance in t-cell acute lymphoblastic leukemia (T-ALL)

Article abstract of DOI:10.1021/ml500044g

Glucocorticoids are one of the most utilized and effective therapies in treating T-cell acute lymphoblastic leukemia. However, patients often develop resistance to glucocorticoids, rendering these therapies ineffective. We screened 9517 compounds, selected for their lead-like properties, chosen from among 3372615 compounds, against a dexamethasone-resistant T-ALL cell line to identify small molecules that reverse glucocorticoid resistance. We synthesized analogues of the most effective compound, termed J9, from the screen in order to define the scaffolds structure-activity relationship. Active compounds restored sensitivity to glucocorticoids through upregulation of the glucocorticoid receptor. This compound and mechanism may provide a strategy for overcoming glucocorticoid resistance in patients with T-ALL.

Full text of DOI:10.1021/ml500044g

Letter
pubs.acs.org/acsmedchemlett
Open Access on 04/25/2015
Small Molecule that Reverses Dexamethasone Resistance in T‑cell
Acute Lymphoblastic Leukemia (T-ALL)
Alexandra M. Cantley,†,∇ Matthew Welsch,‡ Alberto Ambesi-Impiombato,⊥ Marta Sanchez-Martin,⊥
Mi-Yeon Kim,⊥ Andras Bauer,† Adolfo Ferrando,⊥,#,○ and Brent R. Stockwell*,†,‡,§,∥
†Department of Biological Sciences, ‡Department of Chemistry, and §Howard Hughes Medical Institute, Columbia University, 1208
Northwest Corner Building, 12th Floor, 550 West 120th Street, MC 4846, New York, New York 10027 United States
⊥
∥Department of Systems Biology, Columbia University Medical Center, and Institute for Cancer Genetics, Columbia University,
New York, New York 10032 United States
#Department of Pathology and ○Department of Pediatrics, Columbia University, New York, New York 10032, United States
S
* Supporting Information
ABSTRACT: Glucocorticoids are one of the most utilized and effective
therapies in treating T-cell acute lymphoblastic leukemia. However, patients
often develop resistance to glucocorticoids, rendering these therapies
ineffective. We screened 9517 compounds, selected for their lead-like
properties, chosen from among 3 372 615 compounds, against a dexa-
methasone-resistant T-ALL cell line to identify small molecules that reverse
glucocorticoid resistance. We synthesized analogues of the most effective
compound, termed J9, from the screen in order to define the scaffold’s
structure−activity relationship. Active compounds restored sensitivity to
glucocorticoids through upregulation of the glucocorticoid receptor. This
compound and mechanism may provide a strategy for overcoming
glucocorticoid resistance in patients with T-ALL.
KEYWORDS: T-cell acute lymphoblastic leukemia, dexamethasone, glucocorticoid resistance, NOTCH1
inhibition of NOTCH signaling can antagonize glucocorticoid
resistance in T-ALL.9 CUTLL1 cells are highly NOTCH
dependent and have previously been shown to be dexametha-
sone resistant and thus represent a good model in which to
screen compounds.
cute lymphoblastic leukemia (ALL) is a hematological
Atumor that is characterized by aggressive invasion of the
bone marrow by immature lymphoid progenitors. This disease
is generally managed by aggressive chemotherapy with
glucocorticoids (GCs).1 GCs such as prednisolone or dexa-
methasone are especially cytotoxic to ALL tumors, and the
initial response to such treatments is associated with favorable
disease prognosis.2,3 Conversely, primary glucocorticoid resist-
ance confers a much higher risk of relapse and is particularly
prevalent in T-ALL compared to B-precursor leukemias.
Moreover, resistance to GC therapy is greatly enhanced upon
relapse.4,5 In this context, reversing glucocorticoid resistance
could potentially render these glucocorticoids effective in GC-
resistant patients. As a biological tool, a small molecule that
reverses such resistance could provide insight into the
mechanism of glucocorticoid resistance and thus further
address the problem of drug resistance in ALL.
The goal of our study was to find a small molecule with
suitable pharmaceutical properties that could reverse gluco-
corticoid resistance in T-ALL. We utilized a human T-ALL cell
line with a t(7;9)(q34;q34) translocation expressing a truncated
and constitutively active NOTCH1 (CUTLL1).6 In the past
decade, the prevalence of such activating NOTCH1 mutations
in T-ALL cases has become apparent,7 and interestingly, the
constitutive activity of NOTCH has been shown to cause GC
resistance in normal developing thymocytes.8 Moreover,
We screened 9517 compounds that had been selected for
lead-like properties in CUTLL1 cells in 384-well format.10,11
Starting from 3 372 615 compounds, we used computational
filtering to choose compound molecular properties desired for
lead compounds (Figure S1, Supporting Information).12 Cells
treated with each compound were screened in parallel with cells
treated with both 1 μM dexamethasone and 5.33 μg/mL of
each test compound. Using a cutoff of 30% difference in
viability between the two screening conditions, we identified J9,
a compound that, in combination with dexamethasone,
inhibited cell growth with an EC50 of 28 μM. J9 treatment
alone was significantly less toxic to these cells, with an EC50 of
294 μM, suggesting it either restores sensitivity to dexametha-
sone or is synergistically lethal with dexamethasone. (Figure 1).
To confirm that J9 and dexamethasone cotreatment kills cells
rather than merely decreases alamar blue activity, we examined
levels of cleaved PARP1, a marker for apoptosis. We found that,
Received: January 30, 2014
Accepted: April 25, 2014
Published: April 25, 2014
© 2014 American Chemical Society
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levels of activated NOTCH, as compared to treatment with a
commercially available gamma-secretase inhibitor (Compound
E), which completely abrogated NOTCH cleavage from the
membrane (Figure 2). This suggests that J9 is not a gamma-
secretase inhibitor, but rather has a distinct mechanism of
action.
We were interested in determining whether treatment with
J9 resensitized cells to dexamethasone or instead acted through
a glucocorticoid-receptor-independent pathway. To investigate
these hypotheses, we cotreated J9 + Dex with RU486
(Mifepristone), an estrogen and glucocorticoid receptor
antagonist. The addition of RU486 significantly rescued cells
from J9 + Dex-induced death, revealing the dependence of
cytotoxicity of J9 + Dex treatment on the activity of the
glucocorticoid receptor (Figure 3). We further investigated the
Figure 1. Percent growth inhibition of CUTLL1 cells 48 h after
treatment with J9 or J9 + dexamethasone (1 μM). Structure of
compound J9 is shown on the right.
as expected, treatment with either J9 or dexamethasone alone
induced negligible levels of cleaved PARP1, as compared to
cotreatment of J9 and Dex (Figure 2). This corresponds with
Figure 3. Growth inhibition of J9 + Dex treatment compared to J9 +
Dex + RU486 in CUTLL1 cells. Growth inhibition was measured after
48 h. Dex and RU486 were tested at 1 μM.
importance of this receptor using CUTLL1 cells expressing
high and low levels of exogenous glucocorticoid receptor
(Figures S3 and S4, Supporting Information). Cells with higher
levels of GR were significantly more susceptible to dexametha-
sone treatment, and we found that we could greatly enhance
dexamethasone cytotoxicity by the addition of J9 to these cells.
Conversely, cells with low GR levels were insensitive to
dexamethasone treatment alone, and while they were sensitive
to high concentrations of the J9 + Dex treatment, they were
significantly less sensitive than either the overexpressed GR
cells or CUTLL1 cells. The basal level of sensitivity that is
exhibited by the low GR cells is most likely due to the
incomplete knockdown of the GR, as measured by Western
blot (Figures S3 and S4, Supporting Information). Neither cell
line was sensitive to equal concentrations of J9 alone (Figure
4). To further understand these results, we performed Western
blots comparing GR expression levels in each of these cell lines,
which confirmed that GR protein level (either expressed
exogenously or through treatment with high concentrations of
J9) correlates with sensitivity to dexamethasone (Figure S4,
Supporting Information). These data reinforce the notion that
glucocorticoid receptor expression is required for the lethality
of J9 + Dex cotreatment.
Figure 2. Levels of cleaved PARP after 24 h of treatment with J9 (25
μM), J9 + Dex, or J9 + Dex + RU486. Cleaved-PARP protein
quantification (shown as fold change) shown on bottom. Protein levels
of intracellular NOTCH (ICN) after 24 h of treatment with J9, J9 +
Dex, or Dex + the gamma-secretase inhibitor Compound E (CE).
Both Dex and RU486 were tested at 1 μM. CE was tested at 100 nM.
our observations that J9 + Dex treatment can cause cell death in
as little as 24 h, whereas an equivalent concentration of J9 alone
causes minimal lethality to CUTLL1 cells. We were also
interested in seeing whether J9 was lethal only in the presence
of dexamethasone or acting through a more general cytotoxic
mechanism. We treated CUTLL1 cells with J9 in combination
with a variety of cytotoxic agents acting through diverse
mechanisms and found that J9 did not increase the potency of
any of these other compounds (Figure S2, Supporting
Information). These data further show that J9 activity is
specific to dexamethasone-treated cells, and it does not merely
sensitize cells to any lethal agent.
Since previously it was reported that gamma-secretase
inhibitors act synergistically with dexamethasone to kill
CUTLL1 cells, we tested whether J9 was acting as a gamma-
secretase inhibitor.9 To address this question, we measured
levels of intracellular NOTCH after treatment of J9 or J9 +
Dex. We found that J9 or J9 + Dex treatment did not affect
It has been suggested that decreased expression of the
glucocorticoid receptor is an important mechanism of
resistance to dexamethasone, so we investigated whether J9
might increase glucocorticoid receptor (GR) levels and thus
increase the effectiveness of dexamethasone treatment.13−15 We
treated cells for 24 h with J9 alone or J9 + Dex and found that
GR protein levels in J9 + Dex-treated cells were higher than
treatment with dexamethasone alone. At concentrations less
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in exploring whether J9 + Dex treatment enriched for the same
genes as GC treatment alone. Using microarray gene expression
analysis, we compared CUTLL1 cells treated with dexametha-
sone alone, to cotreatment with Dex and J9. These data showed
a marked increase in the upregulation and downregulation of
Dex-regulated genes, suggesting an increase in dexamethasone
activity upon cotreatment with J9. J9 alone had no effect on
these genes (Figure 6B). We used gene set enrichment analysis
(GSEA)16 to compare our results to a previously generated data
set depicting changes in gene expression upon treatment of
GC-sensitive ALL patients with GCs.17 This analysis revealed
that genes upregulated and downregulated by treatment with J9
+ Dex were highly enriched for genes upregulated and
downregulated after GC treatment in the clinic, further
confirming the relevance of J9 + Dex treatment in the context
of ALL (Figure 6A).
In order to understand further the structure−activity
relationship (SAR) of J9, as well as to develop compounds
with increased potency, we synthesized and tested a number of
structural analogues with single-site modifications compared to
J9 (Table 1). J9 itself was synthesized through the LDA-
facilitated addition of 4-methylpicoline to ethyl cyclopropyl
carboxylate (Scheme 1a). The construction of the pyrimidine
core was then accomplished via aminoformylation of the
resulting ketone with N,N-dimethylformamide dimethyl acetal
(Scheme 1b) and subsequent cyclization with guanidine
hydrochloride (Scheme 1c). These conditions proved to be
robust and allowed for the formation of compounds 2−4, 8−
13, and 14−23 in Table 1, using the corresponding ester,
picoline, or amidine derivative. Compounds 9, 10, 11, and 13
were synthesized from compounds 8 or 12 though either a
Suzuki or Stille coupling (Scheme 1d). Compounds 5 and 6
required an alternative method for making the cyclopropyl
ketone. This was accomplished through converting cyclo-
propylmagnesium bromide to the corresponding cuprate (via
CuBr) followed by the nucleophilic addition to a phenacetyl
chloride derivative (see Supporting Information). Interestingly
even changes as subtle as substituting the cylcopropyl ring for
an isopropyl group (compound 14, Table 1) or movement of
the nitrogen in the pyridine ring from the 4- to 3- position
(compound 3, Table 1) resulted in complete loss of potency,
suggesting a highly specific interaction. While any alterations
made to the J9 core eliminated activity, we found that by
substituting the 3 position of the pyridine ring with either
bromine (J9A) or a furan ring (J9B), (compounds 12 and 13,
Table 1, respectively) gave a 2-fold increase in potency relative
to the parent compound (Figure 7), whereas the other
substitutions we made to this position showed decreased
potency and/or selectivity. The same alterations made at the 2
position (compounds 8 and 11, Table 1) resulted in a loss of
selectivity for dexamethasone treatment. Further exploration of
different moieties added to the 3-position will likely allow for
further increased cytotoxicity in combination with dexametha-
sone without losing specificity.
Figure 4. Growth inhibition by J9 or J9 + Dex in cells expressing high
and low levels of glucocorticoid receptor (GR). Cells treated with 1
μM dexamethasone.
than the EC50, J9 alone did not significantly increase GR
protein levels, a change that was also reflected in transcript
levels after treatment (Figure 5).
Figure 5. Glucocorticoid receptor (GR) protein levels normalized to
α-tubulin (left) and relative mRNA levels after 24 h (right). Cells in
the top panel were treated with 25 μM J9, while those in the bottom
panel were treated with 50 μM J9.
To confirm these results, we examined GR protein levels
after cotreatment of Dex + J9 + RU486, the estrogen receptor
antagonist that rescues from Dex + J9-induced death. We found
that the addition of RU486 to the J9 + Dex treatment
prevented the increase in GR protein levels. We also conducted
identical experiments to determine whether these changes
could be confirmed at the transcript level. We performed RT-
qPCR and found that mRNA levels after 24 h of J9 + Dex
treatment were also increased, a phenomenon that could be
abrogated by the addition of RU486 (Figure 5). We then
treated cells with high concentrations of J9 alone, which we
knew to be lethal, to see if this would induce a change in the
glucocorticoid receptor. We found that the addition of 50 μM
J9 did indeed increase expression of the GR as early as 6 h after
treatment (Figures 5 and S5, Supporting Information). RT-
qPCR analysis of transcript levels did not reflect this same
change, suggesting a post-translational regulation of GR by J9.
As glucocorticoids have been shown to control the
expression of a number of different genes, we were interested
To verify that the two most potent analogues (J9A and J9B)
were acting through the same mechanism as J9, we treated both
J9A and J9B with Dex and RU486 to see if RU486 could
reverse the lethality of J9A/B + Dex, as it did for J9 + Dex.
Indeed we found that at lethal concentrations of J9A + Dex or
J9B + Dex, treatment with RU486 decreased growth inhibition
by up to 60% (J9A) or 50% (J9B) (Figure 8). These results
suggest that these derivatives indeed act through the same
mechanism as the parent compound and that further chemical
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Figure 6. (a) GSEA enrichment plots of genes regulated by glucocorticoids in ALL patients undergoing glucocorticoid therapy, in CUTLL1 cells
treated with Dex + J9 compared to CUTLL1 cells treated with dexamethasone alone. (b) Heat map representation of top enriched genes (leading
edge) in CUTLL1 cells treated with dexamethasone + J9 compared with CUTLL1 cells treated with dexamethasone alone. Each column represents
the expression data in CUTLL1 cells treated with vehicle only (DMSO), dexamethasone alone (1 μM), J9 alone (30 μM), and dexamethasone (1
μM) + J9 (30 μM), for 24 h (in triplicate). The scale bar shows a color-coded differential expression with red indicating higher levels and blue lower
levels of expression.
Table 1. Structure−Activity Relationship Analysis of Compound J9
modification could both enhance potency and maximize
for lead-like properties to identify those that reverse
glucocorticoid resistance. From this screen, we found a
compound, J9, which is lethal to CUTLL1 cells only in the
presence of dexamethasone. Preliminary mechanistic studies
specificity.
In conclusion, we screened in a dexamethasone-resistant
human T-ALL cell line a library of small molecules optimized
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a
Scheme 1
a
(a) LDA (1.1 mol equiv), picoline, 0 °C, 1 h, then desired ester (1.3 mol equiv), 0-25 °C, 6 h, THF; (b) N,N-dimethylformamide dimethyl acetal
(2.0 mol equiv), 90 °C, 5 h, neat; (c) guanidine hydrochloride (1.1 mol equiv), sodium ethoxide (1.1 mol equiv), then iii, 40 °C, 4 h, EtOH; (d)
Pd(PPh3)4 (5%), sodium carbonate (2 M, 5.0 mol equiv), R2-B(OH)2 (1.5 mol equiv), 80 °C, 12 h, dioxane or tributylvinyl tin (1.5 mol equiv), 80
°C, 12 h, dioxane.
Figure 7. Growth inhibition of analogues J9A and J9B treated for 48 h
with and without dexamethasone (1 μm).
Figure 8. Growth inhibition after 24 h treatment of analogues J9A and
J9B with Dex/J9/RU486. Both Dex and RU486 were tested at 1 μM.
revealed that the lethality of J9 + Dex treatment is dependent
on expression of the glucocorticoid receptor and also results in
increased abundance of the receptor. GSEA analysis further
confirmed that cotreatment with dexamethasone and J9
enhances dexamethasone activity and affects gene expression
in a manner consistent with GC-treatment in the clinic. These
data also reveal genes that are regulated by J9 treatment alone,
which may provide insight into the mechanism of action of this
compound. Finally, we performed SAR studies in an effort to
increase the potency of J9 and understand the components of
the molecule that contribute to its specificity. These studies
revealed that minor changes to this structure result in a lack of
activity, suggesting a possible specific molecular recognition
event. Derivatives of this scaffold could help further our
understanding of the mechanisms behind glucocorticoid
resistance and could potentially lead to drugs that resensitize
GC-resistant T-ALL cells to glucocorticoid therapy.
ASSOCIATED CONTENT
* Supporting Information
Experimental procedures for biological assays and syntheses of
compounds. Details of LOC library construction and additional
figures. This material is available free of charge via the Internet
at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*(B.R.S.) E-mail: bstockwell@columbia.edu.
■
Present Address
∇(A.M.C.) Department of Biological Chemistry and Molecular
Pharmacology, Harvard Medical School, Boston, Massachusetts
02115, United States.
Funding
This work was funded by grants from the US National
Institutes of Health (NIH R01CA097061, R01GM085081, R01
CA161061, and RC2 CA148308), the Swim Across America
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(12) Dixon, S. J.; Lemberg, K. M.; Lamprecht, M. R.; Skouta, R.;
Zaitsev, E. M.; Gleason, C. E.; Patel, D. N.; Bauer, A. J.; Cantley, A.
M.; Yang, W. S.; Morrison, B.; Stockwell, B. R. Ferroptosis: an iron-
dependent form of nonapoptotic cell death. Cell 2012, 149 (5), 1060−
1072.
Foundation, the Leukemia and Lymphoma Society, and the
Chemotherapy Foundation.
Notes
The authors declare no competing financial interest.
(13) Ploner, C.; Schmidt, S.; Presul, E.; Renner, K.; Schrocksnadel,
̈
K.; Rainer, J.; Riml, S.; Kofler, R. Glucocorticoid-induced apoptosis
and glucocorticoid resistance in acute lymphoblastic leukemia. J.
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(14) Schmidt, S.; Irving, J. A. E.; Minto, L.; Matheson, E.; Nicholson,
L.; Ploner, A.; Parson, W.; Kofler, A.; Amort, M.; Erdel, M.; Hall, A.;
Kofler, R. Glucocorticoid resistance in two key models of acute
lymphoblastic leukemia occurs at the level of the glucocorticoid
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(15) Geley, S.; Hartmann, B. L.; Hala, M.; Strasser-Wozak, E. M.;
Kapelari, K.; Kofler, R. Resistance to glucocorticoid-induced apoptosis
in human T-cell acute lymphoblastic leukemia CEM-C1 cells is due to
insufficient glucocorticoid receptor expression. Cancer Res. 1996, 56
(21), 5033−5038.
(16) Subramanian, A.; Tamayo, P.; Mootha, V. K.; Mukherjee, S.;
Ebert, B. L.; Gillette, M. A.; Paulovich, A.; Pomeroy, S. L.; Golub, T.
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ACKNOWLEDGMENTS
■
B.R.S. is an Early Career Scientist of the Howard Hughes
Medical Institute. M-Y. K. is a fellow of the Leukemia and
Lymphoma Society. We acknowledge the assistance of Dr. John
Decatur, and the use of Columbia Chemistry NMR core facility
instruments provided by NSF grant CHE 0840451 and NIH
grant 1S10RR025431-01A1. We thank Dr. Y. Itagaki for
assistance with MS analyses.
ABBREVIATIONS
■
ALL, acute lymphoblastic leukemia; T-ALL, T-cell acute
lymphoblastic leukemia; GC, glucocorticoid; GR, glucocorti-
coid receptor; Dex, dexamethasone; CE, compound E; LDA,
lithium di-isopropyl amide; PARP, poly ADP ribose polymerase
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