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Preclinical Evaluation of Synergistic Drug Combinations in Acute Myeloid Leukemia
by
Lianne Emily Rotin
A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy
1.1.2.1 AML Pathogenesis ........................................................................................ 5 1.1.2.2 Epidemiology of AML .................................................................................... 6 1.1.2.3 AML Classification and Prognostication ........................................................ 6 1.1.2.4 AML Management ......................................................................................... 7
1.2 Tyrosine Kinase Inhibitor Therapy in AML ............................................... 10 1.2.1 Targeted Cancer Therapies ............................................................................. 10
1.2.1.1 Tyrosine Kinase Inhibitors ........................................................................... 10 1.2.2 Oncogenic Tyrosine Kinases in AML ............................................................. 11
1.3.1 Bruton’s tyrosine kinase: background & role in signal transduction from the B-cell receptor .................................................................................................... 14
1.3.1.1 BTK domains ............................................................................................... 14 1.3.1.2 BTK expression ........................................................................................... 15 1.3.1.3 BTK: Role in B-cell Maturation .................................................................... 15 1.3.1.4 BTK Signaling in B-Cells ............................................................................. 15
1.3.2 A Rationale for Targeting BTK in B-cell Malignancies ................................. 16 1.3.2.1 Chronic Lymphocytic Leukemia .................................................................. 17 1.3.2.2 Mantle-Cell Lymphoma ............................................................................... 17 1.3.2.3 Waldenström Macroglobulinemia ................................................................ 17
1.3.3 Development of Ibrutinib as a Selective and Irreversible BTK Inhibitor with In Vivo Activity .......................................................................................................... 18 1.3.4 Preclinical and clinical activities of ibrutinib in B-cell cancers ................... 19
1.3.6 A Role for Targeting BTK Beyond B-Cell Cancers ....................................... 29 1.3.6.1 Rheumatoid Arthritis .................................................................................... 29 1.3.6.2 Multiple Myeloma ........................................................................................ 30 1.3.6.3 Acute Myeloid Leukemia ............................................................................. 31 1.3.6.4 Prostate Cancer .......................................................................................... 32
1.4 EGFR inhibitors in AML: Anti-Leukemic Mechanisms of Action and Preclinical and Clinical Activity ....................................................................... 34
1.4.1 Development of Small Molecule EGFR Tyrosine Kinase Inhibitors ............ 34 1.4.2 Expression of EGFR in AML Cells .................................................................. 35 1.4.3 Preclinical EGFR-TKI activity against AML ................................................... 36
1.4.3.1 Differentiation .............................................................................................. 36 1.4.3.2 Cell Cycle Arrest and Cell Death ................................................................. 37
2.1.1 Aim I: Identify compounds that synergize with ibrutinib in AML ................ 49 2.1.2 Aim II: Evaluate the mechanism of synergy between ibrutinib and daunorubicin in AML ................................................................................................ 49 2.1.3 Aim III: Identify compounds that synergize with erlotinib in AML .............. 50
Chapter 3: Ibrutinib synergizes with poly(ADP-ribose) glycohydrolase inhibitors to induce cell death in AML cells via a BTK-independent mechanism ........................................................................................................ 51 3.1 Abstract ....................................................................................................... 52 3.2 Introduction ................................................................................................. 53 3.3 Methods ....................................................................................................... 54
3.4 Results ......................................................................................................... 60 3.4.1 BTK is overexpressed and constitutively active in AML cells .................... 60 3.4.2 AML cell lines are insensitive to chemical BTK inhibition with ibrutinib ... 62 3.4.3 A combination chemical screen with ibrutinib in AML cell lines identifies the PARG inhibitor, ethacridine lactate, as an ibrutinib sensitizer ...................... 64 3.4.4 The ibrutinib-ethacridine combination is preferentially cytotoxic to a subset of primary AML cells compared to normal hematopoietic cells .............. 70 3.4.5 The combination of ibrutinib and ethacridine delays the growth of AML cells in vivo ................................................................................................................ 74 3.4.6 Ethacridine synergizes with other small molecule BTK inhibitors, but not inhibitors of unrelated kinases ................................................................................ 76 3.4.7 Ibrutinib and ethacridine synergize to induce cell death via a ROS-dependent mechanism ............................................................................................. 80 3.4.8 The chemical PARG inhibitor gallotannin also synergizes with ibrutinib to induce cell death by excessive ROS production ................................................... 82 3.4.9 The synergy of ibrutinib with ethacridine is independent of the inhibitory effect on BTK ............................................................................................................. 85
3.5 Discussion ................................................................................................... 88 Chapter 4: Investigating the synergistic mechanism between ibrutinib and daunorubicin in acute myeloid leukemia cells. .............................................. 91 4.1 Abstract ....................................................................................................... 92 4.2 Introduction ................................................................................................. 93 4.3 Methods ....................................................................................................... 94
5.4 Results ....................................................................................................... 114 5.4.1 TEX and OCI-AML2 cell line sensitivity to erlotinib .................................... 114 5.4.2 A high-throughput combination chemical screen identifies erlotinib sensitizers in TEX and OCI-AML2 cells ................................................................ 116 5.4.3 The erlotinib-ethacridine combination synergizes in primary AML cells and other AML cell lines ................................................................................................ 119 5.4.4 Combining erlotinib and ethacridine generates lethal levels of reactive oxygen species ....................................................................................................... 122 5.4.5 Ethacridine synergizes with EGFR-targeting kinase inhibitors ................ 125 5.4.6 TEX and OCI-AML2 cell lines do not express EGFR .................................. 125 5.4.7 Erlotinib potentiates ethacridine accumulation in TEX and OCI-AML2 cells. .................................................................................................................................. 126 5.4.8 High-dose ethacridine treatment mimics ROS production observed from the erlotinib-ethacridine combination. .................................................................. 126
6.1.1 BTK-independent anti-leukemic activity of ibrutinib .................................. 135 6.1.1.1 Ibrutinib potentiates ethacridine accumulation .......................................... 135 6.1.1.2 Synergy between ibrutinib and daunorubicin is mediated by a mechanism unrelated to that of ibrutinib/erlotinib and ethacridine ........................................... 137
6.1.2 Anti-leukemic activity of ethacridine lactate ............................................... 138 6.1.3 Clinical relevance of BTK-independent effects of ibrutinib ....................... 139
7.1.1 Determining the mechanism of ethacridine accumulation by erlotinib and ibrutinib .................................................................................................................... 144
7.1.1.1 ABC transporters ....................................................................................... 144 7.1.2 Determining the relevant target of ethacridine ........................................... 145
7.1.2.1 PARG inhibition ......................................................................................... 145 7.1.2.2 p53 induction and the ribosomal stress pathway ...................................... 146
VEGFR Vascular endothelial growth factor receptor WHO World Health Organization WM Waldenström macroglobulinemia WT Wild type Xid X-lined immunodeficiency XLA X-linked agammaglobulinemia
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Preface
Acute myeloid leukemia (AML) is a hematologic malignancy characterized by the
accumulation of improperly differentiated – and thus nonfunctional – myeloid
lineage cells. The mainstay of first-line therapy for this disease is aggressive
treatment with chemotherapy, which aims to eradicate leukemic cells and to
restore normal hematopoiesis. Unfortunately, this approach is inadequate for the
majority of patients: treatment-related toxicities and drug resistance have
translated to five-year survival rates of 25%. Thus, there is a great need for novel
approaches to AML treatment. One potential strategy for reducing therapy-
associated toxicity and improving efficacy is to combine anti-leukemic drugs with
synergizing agents in order to enhance AML cell sensitivity to these drugs. We
therefore screened drugs with documented preclinical anti-AML activity against
chemical libraries in AML cell lines to identify synergistic drug combination
candidates.
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Chapter 1: Literature Review
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1.1 Acute Myeloid Leukemia
1.1.1 Normal Hematopoiesis
In the traditional model of normal hematopoiesis in humans, maturation of
hematopoietic cells follows a hierarchy in which stem cells differentiate to give
rise to the lineages that produce mature blood cells: hematopoietic stem cells
(HSCs) self-renew or differentiate to multipotent progenitor cells (MPPs), which in
turn give rise to the oligopotent common myeloid (CMP) and common lymphoid
(CLP) progenitor cells. CLPs differentiate to T- and B-lymphocytes and natural
killer cells, while CMPs differentiate into megakaryocyte erythroid progenitors
(MEP) and granulocyte monocyte progenitors (GMP). MEPs ultimately give rise
to erythrocytes and megakaryocytes (from which platelets form), while GMPs
differentiate to infection and pathogen-fighting granulocytes (neutrophils,
eosinophils, and basophils) and monocytes. This model is illustrated in Figure 1-1 (top panel).
Recent work has contested this original model: (Notta et al., 2015) provided
evidence to support a two-tier model of hematopoiesis in the adult bone marrow
wherein multipotent HSCs give rise to unipotent progenitor cells that mature to
form monocytes, granulocytes, erythrocytes, and lymphocytes. Interestingly,
megakaryocytes were found to originate from the multipotent tier, and thus do not
arise from the same progenitors (CMPs) as the rest of the myeloid lineage, as
had previously been thought (Figure 1-1, bottom-right).
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Figure reproduced from Notta et al. (2015), license #3817030184707 Figure 1-1: Hematopoiesis: original and revised models. Top panel: classical model of hematopoiesis. Bottom panel: revised model of hematopoiesis in the adult bone marrow (right) and fetal liver bone marrow (left). Abbreviations: HSC, hematopoietic stem cell; MPP, multipotent progenitor; CMP, common myeloid progenitor; CLP, common lymphoid progenitor; MEP, megakaryocyte erythroid progenitor; GMP, granulocyte-monocyte progenitor; Ly, lymphoid cell; Er, erythroid cell; Mk, megakaryocyte; Gran, granulocyte; Mono, monocyte; My, granulocyte/monocyte
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1.1.2 Acute Myeloid Leukemia
Acute myeloid leukemia (AML) comprises many different hematologic neoplasms
with one unifying feature: the presence of proliferative, clonal, myeloid-lineage
cells that are improperly differentiated and the ensuing absence of one or more
mature myeloid-lineage cell types (reviewed in Döhner et al. (2015)). These
improperly differentiated cells—termed blasts—accumulate in the bone marrow
and peripheral blood. Generally, when the blast percentage in the blood and
bone marrow reaches or exceeds 20%, a diagnosis of AML is made (Vardiman et
al., 2002).
1.1.2.1 AML Pathogenesis
AML results from a series of mutations that cooperate to confer a proliferative
and survival advantage to the leukemic clone. The cells of origin in AML are
leukemic stem cells (LSCs), which develop from alterations in normal
Figure 1-3: Mechanism of PARG activity PARP uses NAD+ as a substrate for PAR synthesis. PAR is assembled as polymers onto proteins. PARG hydrolyzes PAR polymers, releasing ADP-ribose. Reproduced from Feng and Koh (2013), license #3817021464698.
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Chapter 2: Project Rationale and Aims
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2.1 Thesis Aims
Given the limited effectiveness of currently approved therapy, novel approaches
to the treatment of AML are urgently needed for this disease. Identifying clinically
approved drugs that enhance the anti-AML activity of agents that are currently
approved for use in – or under investigation for – the treatment of AML would
provide a new therapeutic strategy that could be rapidly advanced to the clinic.
The central objective of this thesis was to identify synergistically cytotoxic
combinations of approved drugs that preferentially kill AML cells, and to uncover
the mechanisms by which these drug pairs synergize.
2.1.1 Aim I: Identify compounds that synergize with ibrutinib in AML
Several groups have described a potential role for the Bruton’s tyrosine kinase
inhibitor ibrutinib in the treatment of AML. With the goal of enhancing ibrutinib’s
anti-AML activity, we used a combination high-throughput screening approach to
identify ibrutinib-sensitizing agents. We subsequently investigated the synergistic
mechanism between ibrutinib and the top synergistic screen hit, hypothesizing
that this synergistic activity was dependent upon ibrutinib-mediated BTK
inhibition.
2.1.2 Aim II: Evaluate the mechanism of synergy between ibrutinib and daunorubicin in AML
Ibrutinib was previously reported to synergize with daunorubicin in AML cells. We
hypothesized that this synergy was dependent upon ibrutinib-mediated BTK
inhibition. We evaluated the role of BTK in ibrutinib-daunorubicin synergy by
treating BTK-knockdown AML cell lines with daunorubicin. We further evaluated
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this synergistic mechanism by measuring combination-induced reactive oxygen
species production and ibrutinib-mediated intracellular daunorubicin
accumulation.
2.1.3 Aim III: Identify compounds that synergize with erlotinib in AML
The epidermal growth factor receptor (EGFR) inhibitor erlotinib has been
reported to exert modest EGFR-independent anti-AML activity in clinical trials.
We sought to identify erlotinib combination candidates by carrying out a
combination high-throughput chemical screen against this drug in erlotinib-
insensitive AML cell lines. We subsequently delineated the synergistic
mechanism of action between erlotinib and the top synergistically cytotoxic hit
using mass spectrometry.
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Chapter 3: Ibrutinib synergizes with poly(ADP-ribose) glycohydrolase inhibitors to induce cell death in AML cells via a BTK-independent mechanism
This chapter has been published:
Rotin LE, Gronda M, MacLean N, Hurren R, Wang X, Lin F, Wrana J, Datti A, Barber DL, Minden MD, Slassi M, Schimmer AD (2016). Ibrutinib synergizes with poly(ADP-ribose) glycohydrolase inhibitors to induce cell death in AML cells via a BTK-independent mechanism. Oncotarget, 7(3): 2765-2779. doi: 10.18632/oncotarget.6409.
Open-Access License (Creative Commons Attribution License); no permissions required.
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3.1 Abstract
Targeting Bruton's tyrosine kinase (BTK) with the small molecule BTK inhibitor
ibrutinib has significantly improved patient outcomes in several B-cell
malignancies, with minimal toxicity. Given the reported expression and
constitutive activation of BTK in acute myeloid leukemia (AML) cells, there has
been recent interest in investigating the anti-AML activity of ibrutinib. We noted
that ibrutinib had limited single-agent toxicity in a panel of AML cell lines and
primary AML samples, and therefore sought to identify ibrutinib-sensitizing drugs.
Using a high-throughput combination chemical screen, we identified that the
The HT Universal Colorimetric PARG Assay Kit (Trevigen, Gaithersburg, MD)
was used to measure the PARG inhibitor activity of ethacridine. The assay was
carried out as per manufacturer instructions.
3.3.12 Statistical Analysis
All graphed viability data are expressed as mean ± SD. Statistical significance
was determined by the unpaired Student’s t test with Holm-Sidak correction for
multiple comparisons or a one-way ANOVA with Tukey’s post-hoc test for
multiple comparisons. Statistical tests were performed using GraphPad Prism
6.03 software (La Jolla, CA).
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3.4 Results
3.4.1 BTK is overexpressed and constitutively active in AML cells
To determine the relevance of BTK as a therapeutic target in AML, we examined
the protein and mRNA expression of BTK in a panel of AML cell lines. Analysis of
the Cancer Cell Line Encyclopedia (Barretina et al., 2012) demonstrated that
AML cells expressed levels of BTK mRNA similar to B-cell malignancies (Figure 3-1). Likewise, a subgroup of primary AML patient samples had increased BTK
expression (Figure 3-2A). Next, we evaluated the expression of BTK by
immunoblotting in a series of AML cell lines. OCI-AML2, THP1, U937, NB4,
K562, and the stem cell-like AML cell line TEX all expressed BTK, but this protein
was not detectable in KG1a AML cells or Jurkat D1.1 T-ALL cells (Figure 3-2B). Phosphorylation of BTK at Tyr223, a marker of BTK activation (Park et al., 1996;
Rawlings et al., 1996; Wahl et al., 1997), was detected in all cell lines expressing
Figure 3-1: BTK mRNA levels in AML cell lines are similar to those of B-cell
malignancies. BTK mRNA expression AML relative to other cancer cell lines, as reported by the Cancer Cell Line Encyclopedia (Barretina et al., 2012).
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3.4.2 AML cell lines are insensitive to chemical BTK inhibition with ibrutinib
To investigate the effects of BTK inhibition on AML viability and proliferation, we
treated AML cells with increasing concentrations of ibrutinib. Phospho-BTK was
reduced to undetectable levels at doses as low as 1 µM ibrutinib (Figure 3-2C). Compared to the sensitive B-lymphoblastic leukemia cell line, Daudi, the AML
cell lines TEX, OCI-AML2, HL60, and U937 were relatively insensitive to ibrutinib,
with IC50s ranging from 4- to 30-fold higher than Daudi cells in the Alamar Blue
Assay, which measures cell proliferation and viability (Figure 3-2D) and much
higher than the 1 µM concentration required to reduce levels of phospho-BTK.
Similar insensitivity to ibrutinib was seen when measuring cell viability with
Annexin V and PI staining (Figure 3-2E), which measures apoptosis.
Interestingly, KG1a cells lacking detectable expression of BTK were the most
sensitive to ibrutinib compared to other AML cell lines (KG1a IC50 = 2.87 µM by
Alamar Blue assay).
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Figure 3-2: AML cell lines express constitutively active BTK, but are insensitive to
ibrutinib. (A) BTK mRNA expression in primary AML cells was determined by analysis of microarray gene expression (GEO accession code: GSE1159). BTK mRNA expression was examined in 267 patients with AML (Valk et al., 2004). (B) BTK and pBTK-Y223 expression in AML cell lines was determined by immunoblotting. (C) TEX and OCI-AML2 cells were treated with 1 µM ibrutinib for 1 h or 16 h. pBTK-Y223 and BTK expression in cell lysates was detected by immunoblotting. (D, E) AML cell lines were treated with increasing concentrations of ibrutinib over 72 h and cell growth and viability relative to untreated cells was determined by (D) Alamar Blue or (E) Annexin V and PI staining on flow cytometry. Data depict mean relative viability ± SD from a representative experiment performed in triplicate. Data are representative of three (D) or two (E) independent experiments.
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3.4.3 A combination chemical screen with ibrutinib in AML cell lines identifies the PARG inhibitor, ethacridine lactate, as an ibrutinib sensitizer
Given the limited single-agent cytotoxicity in AML cell lines, we sought to
determine whether we could identify drugs that sensitize AML cells to ibrutinib.
To this end, we carried out a high-throughput combination chemical screen with
ibrutinib in both TEX and OCI-AML2 cells. The cell lines were co-treated with
ibrutinib and increasing concentrations of compounds from an in-house chemical
library containing 240 internationally prescribed drugs for a total of 5046 different
assays among the two cell lines in this screen. Following 72 hours of incubation,
cell growth and viability were measured by the sulforhodamine-B (SRB) assay.
Potential synergistic hits were identified using the excess-over-Bliss additivism
(EOBA) formula and average positive EOBA scores for each combination were
rank ordered (Figure 3-3A). Ethacridine lactate and pentamidine were top
synergistic hits common to both TEX and OCI-AML2 cells. We validated both
combinations in these cell lines, but pursued ethacridine lactate over pentamidine
because of its greater synergy with ibrutinib (EOBA scores of up to 0.58 and 0.47
by Alamar Blue in TEX and OCI-AML2, respectively) (Figure 3-3B & 3-3C, Figure 3-4). The ibrutinib-ethacridine combination induced cell death, as
determined by Annexin V and PI staining, but the mechanism of cell death was
caspase-independent (Figure 3-5). Ibrutinib and ethacridine also induced strong
synergistic cytotoxicity in U937, HL60, and K562 leukemia cells, but not in KG1a
cells (Figure 3-6).
Ethacridine lactate is used clinically as a topical antiseptic (O’Meara et al., 2014)
and intra-amniotic abortifacient (Mei et al., 2014). It is a DNA intercalator and
Figure 3-3: The PARG inhibitor ethacridine lactate sensitizes AML cell lines to
ibrutinib.
(A) Ibrutinib was co-treated with 240 drugs in TEX and OCI-AML2 cells for 72h. Growth and viability was determined with the SRB assay and synergy was calculated using the EOBA formula as described in the methods. Compounds were ranked in order of increasing average positive EOBA score. (B, C) TEX and OCI-AML2 cells were combination-treated with ibrutinib and ethacridine for 72 h and cell growth and viability relative to untreated cells was determined by Alamar Blue. (B) Data represent mean
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growth and viability ± SD from a representative experiment performed in triplicate. (C) EOBA synergy scores (shown) were calculated for each of the combinations tested. EOBA values > 0.1 (lightest grey) denote a synergistic combination, while values > 0.5 (darkest grey) denote a strongly synergistic combination. Data represent mean EOBA scores from a representative experiment performed in triplicate.
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Figure 3-4: Combination chemical screen validation for pentamidine. TEX and OCI-AML2 cells were subjected to 72h treatment with concentrations of ibrutinib and pentamidine similar to those tested during the combination chemical screen. Cell growth and viability was measured with the SRB assay, and calculated relative to untreated cells. Data represent mean percent growth and viability ± SD and mean EOBA scores from a single experiment performed in triplicate.
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Figure 3-5: Cell death caused by ibrutinib-ethacridine combination is caspase
independent. Top: TEX and OCI-AML2 cells were subjected to combination ibrutinib (4 µM)-ethacridine (6 µM) treatment in the presence and absence of 50 µM Z-VAD-FMK (caspase inhibitor) for 48h. Viability was subsequently measured with Annexin V and PI staining on flow cytometry and calculated relative to vehicle-treated cells. Bottom: TEX and OCI-AML2 cells were treated at the indicated concentrations of ibrutinib and/or ethacridine for 48h, and induction of apoptosis was measured by Annexin V staining on flow cytometry.
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Figure 3-6: The ibrutinib-ethacridine combination is strongly synergistic in HL60,
U937, and K562, but not KG1a AML cell lines. AML cell lines were treated with increasing concentrations of ibrutinib and ethacridine for 72h. Relative growth and viability was measured with the Alamar Blue assay. Data depict mean growth and viability ± SD and mean EOBA scores from a representative experiment performed in triplicate.
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3.4.4 The ibrutinib-ethacridine combination is preferentially cytotoxic to a subset of primary AML cells compared to normal hematopoietic cells
Having identified the combination of ethacridine and ibrutinib as synergistic in
AML cell lines, we tested the combination in primary AML cells (n = 9) (see Table 3-1 for patient characteristics) and normal hematopoietic cells obtained from
consenting donors of G-CSF mobilized stem cells for allotransplantation (n = 9).
Primary cells were incubated with increasing concentrations of ethacridine and
ibrutinib for 48 hours in Iscove’s Modified Dulbecco’s Medium supplemented with
10% fetal bovine serum, without additional growth factors, and viability was
subsequently measured with Annexin V/PI staining and flow cytometry (Figure 3-7). Similar to the AML cell lines, ibrutinib had minimal single-agent cytotoxicity,
with IC50s exceeding 8 µM in all primary cells. We noted that primary AML cells,
on average, were more sensitive to single-agent ethacridine and combination
ibrutinib-ethacridine treatment compared to normals: a subset of 6 of 9 AMLs
demonstrated greater than 70% cell death from the combination, while only 1 of 9
normals (Normal 2) exhibited similar sensitivity. However, in some normal
samples, the drug combination induced ≥ 50% cell death, suggesting that the
ibrutinib-ethacridine combination may also have toxicity towards some normal
hematopoietic cells.
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Table 3-1: Patient demographics.
Patient Characteristics Sample ID
Diagnosis
Age at Diagnosis Gender Cytogenetics Molecular
130794 AML 73 Male 46,XY[20] Not done 130819 AML, M5a 63 Female 46,XX[20] NPM1+, FLT3-ITD-, FLT3-TKD- 130826 AML, M4 58 Male 46,XY[20] NPM1+, FLT3-ITD+, FLT3-TKD- 130874 AML 69 Male 49,XY,+12,+16,+21[10] Not done 130877 AML 75 Male 48,XY,+9,+13[4]/46,XY[16] Not done 140994 AML 67 Male 45,XY,-7[10] NPM1-, FLT3-ITD-, FLT3-TKD- 141130 AML, M5b 80 Female Inconclusive CBFB-MYH11-
150177 AML 53 Female 42~46,XX,-2,der(3)add(3)(p21)?del(3)(q21q26),del(5)(q12), der(7)t(7;?11)(p13;q13),del(8)(p21),add(11)(q13),-18, add(20)(p13),+3mar[4]
Not done
150256 AML 24 Male 45,XY,der(6;7)t(6;7)(p21;q22)del(6)(q13q21)[17]/46,XY[3] Not done
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Figure 3-7: The ibrutinib-ethacridine combination is preferentially cytotoxic to
primary AML cells over normal hematopoietic cells. (Preceding page) Primary AML and normal hematopoietic cells (G-CSF mobilized peripheral blood stem cells) were treated with ibrutinib, ethacridine, or both in combination for 48 h. Viability was determined by Annexin V and PI staining. Data represent mean percent viability ± SD from a single experiment performed in triplicate. Ibru = ibrutinib, Ethac = ethacridine.
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3.4.5 The combination of ibrutinib and ethacridine delays the growth of AML cells in vivo
To assess the in vivo efficacy and toxicity of ibrutinib in combination with
ethacridine, we evaluated this combination in a mouse model of leukemia. SCID
mice were injected subcutaneously with OCI-AML2 cells. When tumors were
palpable, mice were treated with ibrutinib, ethacridine, or the combination of both
drugs. The combination of ibrutinib and ethacridine decreased the growth of OCI-
AML2 cells more than either drug alone (*P < 0.001 and **P < 0.0001). Of note,
no toxicity from combination treatment was detected as measured by changes in
body weight, behavior or gross examination of the organs at the end of the
experiment (Figure 3-8).
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Figure 3-8: Ibrutinib-ethacridine combination displays anti-AML activity in mice.
1 × 106 OCI-AML2 cells were subcutaneously injected in SCID mice. Eight days after injection, mice were treated with 300 mg/kg of ibrutinib by oral gavage, 20 mg/kg of ethacridine by i.p. injection, a combination of two drugs, or vehicle control (5% DMSO, 20% Cremophor, 0.9% NaCl) by oral gavage on the indicated days. Tumor volume (A) and body weight (B) were monitored over time. Mean ± SEM for tumor volume and mean ± SD for body weight, n = 7. *P < 0.001 and **P < 0.0001 from a two-way ANOVA with Tukey’s posttests comparing all treatment groups at day 18 and 20.
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3.4.6 Ethacridine synergizes with other small molecule BTK inhibitors, but not inhibitors of unrelated kinases
We sought to investigate whether the observed synergy with ethacridine was
specific to ibrutinib or a property common to other BTK inhibitors. We therefore
tested ethacridine in combination with two other BTK inhibitors currently in
clinical trials: CC-292 and ONO-4059. Cell growth and viability was measured 72
hours after incubation by the Alamar Blue assay and EOBA scores were
calculated. CC-292 and ONO-4059 synergized with ethacridine in TEX and OCI-
AML2 cells with efficacy similar to ibrutinib (Figure 3-9).
To further examine the specificity of the synergistic activity of ethacridine, we
sought to determine whether this compound generally sensitized AML cells to
kinase inhibitors. We therefore selected inhibitors of kinase targets bearing
minimal sequence similarity to BTK. Specifically, we tested PIM1/2 and STO-609,
inhibitors of Calcium/calmodulin-dependent protein kinase family members PIM
1/2 and CaMKK, respectively. TEX cells were treated with these compounds in
combination with ethacridine. Synergy was assessed by EOBA calculation
following viability determination at 72 hours with Annexin V and PI staining on
flow cytometry. Neither PIM1/2, nor STO-609 synergized with ethacridine in TEX
cells, with EOBA scores not exceeding 0.03 for either combination (Figure 3-10A).
We also tested the combination of ethacridine with the ABL kinase inhibitor
imatinib and the ABL and SRC family kinase inhibitor, dasatinib. Of note, ibrutinib
is reported to inhibit SRC family kinases (Honigberg et al., 2010) as they share
sequence homology to the TEC kinases. TEX and OCI-AML2 cells were
combination-treated with ethacridine and these kinase inhibitors. Following a 72-
hour incubation, cell growth and viability was determined by the Alamar Blue
assay. The combinations produced primarily additive effects as calculated by the
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EOBA formula (Figure 3-10B & Figure 3-11). Thus, the observed synergy with
ethacridine appears specific for TEC family kinase inhibitors.
Figure 3-9: Ethacridine synergizes with other small-molecule BTK inhibitors. TEX and OCI-AML2 cells were treated with increasing concentrations of ethacridine and (A) CC-292 or (B) ONO-4059 for 72 h. Growth and viability was measured by Alamar Blue and EOBA synergy scores were calculated. Data depict mean percent viability ± SD and mean EOBA scores from a representative experiment performed in triplicate. Data are representative of three independent experiments.
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Figure 3-10: Ethacridine does not synergize with inhibitors of unrelated kinases. (A) TEX cells were combination-treated with ethacridine and PIM1/2 or STO-609 for 72 h. Viability was measured by Annexin V/PI staining and EOBA scores were generated. Combination ibrutinib-ethacridine treatment of TEX cells was included as a positive synergy control for this method of cell viability determination. (B) TEX cells were combination-treated with ethacridine and dasatinib or imatinib for 72 h. Growth and viability was measured by Alamar Blue and EOBA synergy scores were calculated. Data depict mean percent viability (A) or growth and viability (B) ± SD and mean EOBA scores from a representative experiment performed in triplicate. Data are representative of three independent experiments.
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Figure 3-11: Dasatinib and imatinib do not synergize with ethacridine in OCI-
AML2 cells. OCI-AML2 cells were combination-treated with ethacridine and dasatinib (left) or imatinib (right) for 72 h. Cell growth and viability was measured with the Alamar Blue assay, and calculated relative to untreated cells. Synergy was calculated with the EOBA formula. Data represent mean percent growth and viability ± SD and mean EOBA scores from a single experiment performed in triplicate. Data are representative of three independent experiments.
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3.4.7 Ibrutinib and ethacridine synergize to induce cell death via a ROS-dependent mechanism
To investigate the mechanism of synergy between ethacridine and ibrutinib, we
examined ROS (reactive oxygen species) production in AML cells treated with
the drug combination. Using carboxy-H2DCFDA staining and flow cytometry, we
measured total intracellular ROS production after TEX and OCI-AML2 treatment
with ibrutinib, ethacridine or the drug combination. At concentrations that were
associated with synergistic cell death, neither drug alone markedly increased
intracellular ROS production. However, ROS production in live cells was
increased with the drug combination as early as two hours following treatment in
both TEX and OCI-AML2 cells (Figure 3-12A). Moreover, the increased ROS
production was functionally important for the observed cell death, as the addition
of the anti-oxidant α-tocopherol abrogated cytotoxicity from the combination in
both cell lines (Figure 3-12B). The observed increase in ROS following
combination treatment did not appear to be mitochondrial in origin, as MitoSOX
staining did not increase following combination treatment relative to single-agent
treatment (Figure 3-12C). Thus, these findings suggest that the synergistic
cytotoxicity caused by the ibrutinib-ethacridine combination is due to excessive
intracellular, but not mitochondrial, ROS production.
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Figure 3-12: The ibrutinib-ethacridine combination induces cytotoxic levels of
intracellular ROS. (A) TEX and OCI-AML2 cells were treated with ibrutinib, ethacridine, or both in combination for 2, 6 or 24 h. Intracellular ROS was measured by carboxy-H2DCFDA staining and dead cells were excluded by PI staining on flow cytometry. Fold increase in ROS was calculated relative to the geometric means of carboxy-H2DCFDA (FITC) staining in untreated cells. H2O2 treatment served as a positive control for intracellular ROS generation. (B) TEX and OCI-AML2 cells were pre-treated with α-tocopherol prior to a 48 h incubation with ibrutinib and/or ethacridine. Viability was measured by Annexin V and PI staining, and calculated relative to respective untreated controls (+ or − α-tocopherol). (C)TEX and OCI-AML2 cells were treated with ibrutinib, ethacridine, or both drugs in combination for 2, 6 or 24 h. Mitochondrial ROS was measured by MitoSOX Red staining, with dead cell exclusion by Annexin V staining on flow cytometry. Fold increase in mitochondrial ROS was calculated relative to the geometric means of carboxy-H2DCFDA (FITC) staining in untreated cells. Antimycin A (50 µM) treatment served as a positive control for mitochondrial ROS generation.
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Data represent mean fold increase in ROS production ± SD (A, C) or mean viability ± SD (B) from representative experiments performed in triplicate. Data are representative of two (A, C) or three (B) independent experiments. In all panels, *P < 0.05, **P < 0.01; ***P < 0.001; ****P < 0.0001 as determined by one-way ANOVA with Tukey’s post-hoc test for multiple comparisons (A), or unpaired Student’s t test with Holm-Sidak correction for multiple comparisons (alpha = 5%) (B).
3.4.8 The chemical PARG inhibitor gallotannin also synergizes with ibrutinib to induce cell death by excessive ROS production
Ethacridine is a putative PARG inhibitor (Boulikas, 1990; Tavassoli et al., 1985)
and we demonstrated that ethacridine inhibited PARG (Figure 3-13A). Therefore, we evaluated the combination of ibrutinib and gallotannin, another
reported PARG inhibitor (Aoki et al., 1993; Formentini et al., 2008; Tsai et al.,
1992). We treated TEX and OCI-AML2 cells with increasing concentrations of
ibrutinib and gallotannin over 48 hours and then measured viability with Annexin
V and PI staining. The ibrutinib-gallotannin combination was also profoundly
synergistic, yielding EOBA values of up to 0.60 and 0.72 in TEX and OCI-AML2
cells, respectively (Figure 3-13B). Likewise, pre-treatment with α-tocopherol
abrogated ibrutinib-gallotannin cytotoxicity (Figure 3-13C). Pretreatment with the
poly(ADP-ribose) polymerase (PARP) inhibitor olaparib did not rescue
combination-induced cytotoxicity (Figure 3-14). However, olaparib was directly
toxic to the cells, thus potentially obscuring any protective effects.
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Figure 3-13: The PARG inhibitor gallotannin synergizes with ibrutinib (A) Ethacridine’s inhibitory activity against PARG was determined using a cell-free colorimetric assay that measures levels of biotinylated PAR attached to histones in the presence of PARG enzyme. A loss of absorbance at 450 nm correlates with increased PARG activity. Relative PARG activity was calculated by comparing the loss of absorbance at 450nm in the presence of ethacridine to that of no PARG control (maximal absorbance at 450 nm). Data represent mean PARG activity ± SD from a single experiment performed in triplicate. (B) TEX and OCI-AML2 cells were treated with increasing concentrations of ibrutinib and gallotannin for 48 h. Viability was measured by Annexin V and PI staining and EOBA scores were calculated. Data represent mean EOBA scores from a representative experiment performed in triplicate. Data are representative of two (OCI-AML2) or three (TEX) independent experiments. (C) TEX cells were pre-treated with α-tocopherol and subjected to 48 h treatment with ibrutinib and gallotannin. Viability was measured by Annexin V and PI staining and calculated relative to untreated controls. Data represent mean viability ± SD from a representative experiment performed in triplicate. Data are representative of three independent experiments. In all panels, *P < 0.05, **P < 0.01; ***P < 0.001; ****P < 0.0001 as determined by unpaired Student’s t test with Holm-Sidak correction for multiple comparisons (alpha = 5%) (C).
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Figure 3-14: Treatment of TEX and OCI-AML2 cells with olaparib in combination
with ibrutinib and ethacridine. TEX and OCI-AML2 cells were pre-treated with the PARP inhibitor olaparib 4 hours prior to a 72 h incubation with ibrutinib, ethacridine or both in combination at the indicated concentrations. Growth and viability was measured by the Alamar Blue assay and then calculated relative to untreated controls.
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3.4.9 The synergy of ibrutinib with ethacridine is independent of the inhibitory effect on BTK
To determine whether synergy between ibrutinib and ethacridine was due to BTK
inhibition by ibrutinib, we knocked down BTK with shRNA in TEX and OCI-AML2
cells. The reduction of BTK expression was confirmed by immunoblotting (Figure 3-15A) and qPCR (not shown). Knockdown cells were then treated with
increasing concentrations of ethacridine and cell growth and viability was
assessed by Alamar Blue. Despite substantial levels of BTK knockdown by
shRNA, ethacridine treatment of BTK-knockdown cells was no more cytotoxic
than ethacridine treatment of shRNA control cells (Figure 3-154A). These
observations suggest that synergy of ibrutinib with ethacridine is independent of
its inhibitory effect on BTK.
To further examine whether synergy of ibrutinib with ethacridine is due to targets
beyond BTK, we tested the drug combination in Jurkat D1.1 cells, a T-acute
lymphoblastic leukemia cell line that does not express BTK (Figure 3-2B). The
ibrutinib-ethacridine combination synergized in Jurkat D1.1 cells, reaching EOBA
values of 0.25 (Figure 3-15B), further supporting a synergistic mechanism for
ibrutinib and ethacridine beyond AML cell lines analyzed in this study.
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Figure 3-15: Ibrutinib’s synergy with ethacridine is independent of BTK. (A) TEX and OCI-AML2 cells were transduced with 2 different shRNAs targeting BTK or a non-targeting shRNA control in lentiviral vectors. On day 4 post-transduction, cells were treated with ethacridine at concentrations previously shown to synergize with ibrutinib. Growth and viability at 72 h post-ethacridine treatment was determined by Alamar Blue and calculated relative to untreated control. BTK knockdown was confirmed by immunoblotting. (B) Jurkat cells were treated with increasing concentrations of ibrutinib and ethacridine for 72 h. Cell growth and viability was determined by the Alamar Blue assay and synergy was calculated using the EOBA formula. Data depict mean percent growth and viability ± SD from a representative experiment performed in triplicate. Data in (A) and (B) are representative of two independent experiments. In all panels, ns = not significant, based on the results of an unpaired Student’s t test with Holm-Sidak correction for multiple comparisons (alpha = 5%).
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Figure 3-16: Expression of TEC family kinases in AML cell lines. Expression of kinases BMX, TLK, TEC, and ITK in a panel of AML cell lines, Jurkat D1.1 and Daudi cells was determined by immunoblotting.
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3.5 Discussion
The small molecule BTK inhibitor ibrutinib has demonstrated exceptional efficacy
with minimal toxicity in several B-cell cancers. Ibrutinib is currently approved for
clinical use in CLL, mantle-cell lymphoma (MCL) and Waldenström’s
macroglobulinemia (WM), owing to its impressive patient response rates during
recent clinical trials. With widespread BTK expression across B-cell malignancies
and its role as a node in several oncogenic signaling pathways, this cytoplasmic
kinase has emerged as an attractive therapeutic target for B-lymphoid cancers.
Multiple clinical trials investigating ibrutinib alone and in combination for these
diseases are currently underway.
In addition to its expression in B-cell malignancies, BTK is also expressed in
myeloid cell lines and can be activated through mechanisms independent of the
B-cell receptor (Doyle et al., 2007; Kawakami et al., 1994; Oellerich et al., 2015).
Thus, targeting BTK with ibrutinib may have efficacy in myeloid malignancies
such as AML.
In concordance with previous work by Rushworth et al. (2014) and Oellerich et al.
(2015), we demonstrated the expression of constitutively active BTK in several
AML cell lines. However, in contrast to these other studies, in the cell lines we
tested, the cytotoxicity from ibrutinib was likely independent of its effects on BTK.
Supporting this contention, of the tested AML cell lines, KG1a cells were the
most sensitive to ibrutinib and yet lacked detectable BTK by immunoblot.
Moreover, the IC50 in TEX and OCI-AML2 leukemia cells were over 10-fold
higher than the concentration of ibrutinib required to completely repress BTK
phosphorylation and higher than the pharmacologically achievable
concentrations in humans (Appendix 1). Consistent with these observations,
ibrutinib has been reported to induce AML cell death via a BTK-independent
mechanism (Wu et al., 2015).
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Although ibrutinib was largely inactive as a single agent in the tested AML cells,
we successfully sensitized two of the more resistant AML cell lines (TEX, IC50 =
13.01 µM and OCI-AML2, IC50 = 27.44 µM) to ibrutinib by combining the drug
with the putative PARG inhibitor ethacridine. However, the observed synergistic
cytotoxicity was independent of the inhibitory effect of ibrutinib on BTK. These
findings suggest that ibrutinib may also have anti-AML activity that extends to
targets beyond BTK.
In addition to inhibiting BTK, ibrutinib cross-reacts with TEC and SRC family
kinases with similar efficacy (Honigberg et al., 2010). We noted that the SRC
inhibitor dasatinib did not recapitulate ibrutinib’s synergy with ethacridine,
however the BTK inhibitors ONO-4059 and CC-292, which have reported
inhibitory activity against TEC family kinases (Akinleye et al., 2013; Ariza et al.,
2013; Evans et al., 2013; Hendriks et al., 2014; Yoshizawa et al., 2012), did
synergize with ethacridine. We therefore favor the TEC family kinases as likely
targets of ibrutinib in its synergy with ethacridine in AML. To date, 5 TEC family
members have been identified: BTK, BMX, TEC, ITK, and RLK (Schmidt et al.,
2004a). ITK is expressed selectively in T cells and its inhibition by ibrutinib leads
to decreased STAT6, IkBa, JUNB, and NFAT activity, as well as decreased
intracellular calcium release (Dubovsky et al., 2013). BMX is expressed in
hematopoietic progenitor cells and myeloid leukemias (Kaukonen et al., 1996;
Weil et al., 1997) and has been found to mediate STAT3 activation and
subsequent transformation by Src (Tsai et al., 2000). Interestingly, BMX, TEC,
and RLK are all expressed in AML and Jurkat D1.1 cell lines (Figure 3-16). However, further investigations will be required to determine whether these TEC
family members—or different kinases altogether—are targets of ibrutinib that
explain its synergy with ethacridine. One possible strategy for ibrutinib target
determination is a synthetic-lethal human kinome shRNA array, which would
uncover kinases that when individually knocked down induce AML cell line
sensitivity to ethacridine.
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To our knowledge, this work is the first to report on the activity of PARG inhibitors
such as ethacridine and gallotannin in AML. While further work is required to fully
determine the impact of PARG inhibition in AML, it is interesting to speculate
whether some of the single agent activity of ibrutinib in primary AML might be
observed in patients with the lowest basal PARG expression.
The addition of poly(ADP-ribose) (PAR) moieties to target proteins alters their
structure, function, and localization. PAR-ylation of target proteins is mediated by
the PARP (Poly(ADP-ribose) polymerase) family of enzymes, of which PARP-1 is
the most abundant and best characterized (Durkacz et al., 1980; Luo & Kraus,
2012; Virág et al., 2013). PARP adds PAR groups to target proteins, and these
moieties are removed by PARG (Feng & Koh, 2013). Thus, genetic or chemical
inhibition of PARG leads to the accumulation of excess PAR-ylated proteins.
Through the accumulation of excess PAR-ylated proteins, PARG inhibition
reduces the proliferation of malignant cells (Erdélyi et al., 2009; Pan et al., 2012),
sensitizes cells to genotoxic stress (Cortes et al., 2004; Koh et al., 2004; Shirai et
al., 2013) and inhibits cell signaling pathways including NFκB, p38 and ERK (Pan
et al., 2012). Through these and other mechanisms, increased levels of PAR-
ylated proteins may also promote ROS generation (Krenzlin et al., 2012), which
is relevant to our observed mechanism of action of the drug combination.
Though the ibrutinib-PARG inhibitor combination produced striking synergistic
cytotoxicity in AML cell lines, it is important to note that this combination also
induced cytotoxicity in a subset of normal hematopoietic cells (Figure 3-7). This
observation highlights a potential toxicity that would need to be assessed in the
context of clinical trials of these agents.
Thus in summary, through identification of an ibrutinib combination that
sensitizes resistant AML cell lines to this kinase inhibitor, we uncovered a novel
BTK-independent role for ibrutinib in AML. Moreover, we present a potential role
for PARG inhibition as a novel target for combination therapy in AML.
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Chapter 4: Investigating the synergistic mechanism between ibrutinib and daunorubicin in acute myeloid leukemia cells.
This chapter has been published as a Letter to the Editor:
Rotin LE, Gronda M, Hurren R, Wang X, Minden MD, Slassi M, Schimmer AD (2016). Investigating the synergistic mechanism between ibrutinib and daunorubicin in acute myeloid leukemia cells. Leuk Lymphoma. Feb 17:1-5. Doi: 10.3109/10428194.2016.1138292 [Epub ahead of print]
Radioactivity of lysed cells was quantified with a Beckman LS6000IC liquid
scintillation counter, and counts were normalized to cellular protein content.
All other experiments were carried out using materials and procedures previously
described in Chapter 3.3 (pgs 56-61).
4.4 Results & Discussion
We first validated the previously reported synergistic cytotoxicity resulting from
ibrutinib-daunorubicin and ibrutinib-cytarabine combinations in AML cell lines. We
treated the AML cell lines OCI-AML2 with wild type FLT3 (Quentmeier et al.,
2003) and TEX whose FLT3 mutation status is not known with increasing
concentrations of ibrutinib and daunorubicin or cytarabine for 72 hours and
measured cell viability using Annexin V and PI staining on flow cytometry. We
determined the extent of any resultant synergy for each of the combinations
tested with the excess-over-Bliss additivism (EOBA) formula, as previously
described (Borisy et al., 2003). The ibrutinib-daunorubicin combination yielded
synergistic EOBA scores of up to 0.33 in TEX and 0.25 in OCI-AML2 (Figure 4-1). These findings are in line with a previous report describing synergistic killing
activity between ibrutinib and doxorubicin, another anthracycline, in activated B-
cell-like subtype of diffuse large B-cell lymphoma cells (ABC-DLBCL) (Mathews
Griner et al., 2014). Meanwhile, the ibrutinib-cytarabine combination was only
borderline synergistic, producing EOBA scores no greater than 0.11 in TEX and
OCI-AML2 (Figure 4-2). Given that the ibrutinib-daunorubicin combination was
more profoundly synergistic, we decided to investigate this combination further.
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Figure 4-1: Ibrutinib and daunorubicin synergize in TEX and OCI-AML2 cells. TEX and OCI-AML2 cells were treated with ibrutinib (“Ibru”) and daunorubicin alone and in combination for 72h. Viability was measured by Annexin V and PI staining on flow cytometry, then calculated relative to untreated cells (histograms). Excess-over-Bliss additivism (EOBA) scores for each tested combination are shown (tables); values >0.10 (grey) denote a synergistic combination, with higher EOBA values (darker grey) indicating greater synergy. Data represent mean percent viability ± SD and mean EOBA scores from a representative experiment performed in triplicate. Data are representative of two independent experiments.
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Figure 4-2: Combination ibrutinib-cytarabine treatment of TEX and OCI-AML2
cells. TEX and OCI-AML2 cells were treated with ibrutinib (“Ibru”) and cytarabine alone and in combination for 72h. Viability was measured by Annexin V and PI staining on flow cytometry and then calculated relative to untreated cells (histograms). Excess-over-Bliss additivism (EOBA) scores are shown (tables). Data represent mean percent viability ± SD and mean EOBA scores from a representative experiment performed in triplicate.
Figure 4-3: Ibrutinib inhibits BTK phosphorylation. TEX and OCI-AML2 cells were incubated with 1 µM ibrutinib (+) or vehicle (-) for 1h. Cells were lysed and probed for BTK-pY223, a marker of BTK activation, on immunoblot.
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To determine whether the synergy between ibrutinib and daunorubicin was
dependent upon BTK inhibition by ibrutinib, we treated BTK-knockdown TEX and
OCI-AML2 cells with daunorubicin to reproduce the sensitization observed in
combination with ibrutinib. Both cell lines express constitutively active BTK, and
treatment with ibrutinib inhibited BTK phosphorylation (Figure 4-3). We
lentivirally transduced TEX and OCI-AML2 cells with five different BTK-targeting
shRNAs, alongside one non-targeting shRNA control (GFP). On Day 3 following
puromycin selection, transduced cells were treated with increasing
concentrations of daunorubicin for 72 hours, and viability relative to respective
untreated controls was subsequently measured with Annexin V and PI staining.
Despite knockdown of BTK to undetectable levels (Figure 4-4), little to no
sensitization to daunorubicin was seen among five independent shRNA clones
targeting BTK (Figure 4-5). While sensitization to daunorubicin was observed at
single doses of daunorubicin in two shRNA clones targeting BTK, for the majority
of clones and doses of daunorubicin tested, no increased sensitization was
observed despite undetectable levels of BTK. Thus, these findings do not
convincingly support BTK inhibition as the target of ibrutinib that would explain
synergy with daunorubicin. Given that ibrutinib is known to inhibit other kinases,
including SRC family kinases and other TEC family members (Honigberg et al.,
2010), it is possible that off-target kinase inhibition by ibrutinib may explain its
synergy with daunorubicin. Possible BTK-independent explanations for profound
daunorubicin sensitization in clones shRNA-BTK508 and shRNA-BTK2490
include off-target shRNA effects (Manjunath et al., 2009).
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Figure 4-4: BTK knockdown confirmation. BTK knockdown was confirmed by immunoblotting in TEX and OCI-AML2 cells.
Figure 4-5: Daunorubicin treatment of BTK-knockdown cells. TEX and OCI-AML2 cells were transduced with 5 shRNAs targeting BTK alongside a non-targeting shRNA control (GFP) via the PLKO.1 lentiviral vector, which contains a puromycin resistance gene. On day 3 post-puromycin selection, transduced cells were treated with increasing concentrations of daunorubicin for 72h, and viability relative to respective untreated controls was determined by Annexin V/PI staining. Data depict mean percent viability ± SD from representative experiments performed in triplicate. Data are representative of two independent knockdown experiments.
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To address the possibility that a subpopulation of BTK-knockdown cells was
highly BTK-dependent and thus killed off prior to treatment with daunorubicin, we
treated BTK-knockdown TEX cells with ibrutinib and measured viability with
Annexin V and PI staining. Compared to shRNA control, neither shRNA-BTK974,
nor shRNA-BTK1066 was more resistant to ibrutinib at the concentrations
evaluated (Figure 4-6), suggesting that the overall observed lack of daunorubicin
sensitization in BTK knockdown cells was not the result of having selected for a
subpopulation of BTK-independent cells.
One of the mechanisms by which kinase inhibitors have been shown to synergize
with antineoplastic agents is through inhibition of ATP-binding cassette (ABC)
transporter-mediated drug efflux activity (Lainey et al., 2012). Ibrutinib is a
reported inhibitor of the MRP1 (ABCC1) transporter and sensitized MRP1-
overexpressing cell lines to the MRP1 substrate vinblastine by enhancing its
accumulation (Zhang et al., 2014). We sought to determine whether potentiation
of daunorubicin accumulation by ibrutinib might explain the synergistic killing by
this combination. We treated TEX cells with increasing concentrations of
radiolabelled daunorubicin in the presence and absence of ibrutinib, comparing
daunorubicin counts from lysed cells normalized to total protein content. Ibrutinib
failed to increase daunorubicin accumulation (Figure 4-7), suggesting that
inhibiting daunorubicin efflux is unlikely to be the mechanism by which ibrutinib
and daunorubicin synergize in TEX cells.
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Figure 4-6: Ibrutinib treatment of BTK-knockdown TEX cells. TEX shRNA-BTK974 and shRNA-BTK1066 cells were treated with ibrutinib alongside shRNA control and parental TEX cells for 48 hours (starting on day 3 post-puromycin selection). Viability was measured with Annexin V/PI staining and calculated relative to respective untreated controls. Data depict mean percent viability ± SD from representative experiments performed in triplicate. Data are representative of two independent knockdown experiments.
Figure 4-7: Daunorubicin accumulation in the presence or absence of ibrutinib. TEX cells were incubated with radiolabelled daunorubicin in the presence and absence of 8 µM ibrutinib for 3h. Data depict mean radioactive counts ± SD relative to protein content from an experiment performed in triplicate. Data are representative of two independent experiments.
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We have previously noted that ibrutinib synergizes with reactive oxygen species
(ROS)-inducing agents to cause cell death mediated by excessive ROS
production (Rotin et al., 2014). Because daunorubicin is also a ROS-inducer
(Gewirtz, 1999), we sought to determine whether cell death after treatment with
the ibrutinib-daunorubicin combination was ROS-dependent. TEX and OCI-AML2
cells were treated with daunorubicin and ibrutinib with and without the antioxidant
α-tocopherol. Pre-treatment with α-tocopherol dramatically rescued viability in
TEX and OCI-AML2 cells treated with ibrutinib, daunorubicin, or the combination
(Figure 4-8). We also tested whether the ibrutinib-daunorubicin combination
increased total intracellular ROS production by staining cells with carboxy-
H2DCFDA. Mildly increased carboxy-H2DCFDA staining was observed following
a 6-hour combination treatment, and this change was statistically significant
(Figure 4-9). α-tocopherol pre-treatment abrogated the slight increase in
intracellular ROS production (Figure 4-10). Examination of mitochondrial ROS
production with MitoSOX Red staining in combination-treated TEX and OCI-
AML2 cells revealed a small increase in mitochondrial ROS production following
ibrutinib treatment; however no further increases were noted when ibrutinib was
combined with daunorubicin (Figure 4-11).
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Figure 4-8: α-tocopherol rescue of combination-treated TEX and OCI-AML2 cells. TEX and OCI-AML2 cells were treated with 3mM α-tocopherol for 24h prior to a 72h incubation with ibrutinib and daunorubicin (“DNR”) alone and in combination, with α-tocopherol maintained at a concentration of 2.4mM. Viability relative to respective untreated controls was measured with Annexin V and PI staining. Data depict mean percent viability ± SD from an experiment performed in triplicate. Data are representative of two independent experiments.
ROS TEX and OCI-AML2 cells were treated with ibrutinib and daunorubicin (“DNR”) for 6h, then stained with carboxy-H2DCFDA and PI to detect intracellular ROS in live cells by flow cytometry. Fold increase in ROS was calculated relative to the geometric mean fluorescence intensities (GMFI) of carboxy-H2DCFDA staining in untreated live cells. Hydrogen peroxide treatment was included as a positive carboxy-H2DCFDA staining control. Data depict average GMFIs ± SD and are representative of a single experiment performed in triplicate. Data are representative of two independent experiments. In all panels, ns = not significant, *P<0.05, **P<0.01; ***P<0.001; ****P<0.0001, as determined by one-way ANOVA with Tukey’s post-hoc test for multiple comparisons.
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Figure 4-10: Intracellular ROS production following combination ibrutinib-
daunorubicin treatment in the presence or absence of α-tocopherol. TEX and OCI-AML2 cells were treated with 3mM α-tocopherol prior to a 6h incubation with ibrutinib (“Ibru”), daunorubicin (DNR), or both drugs in combination. Cells were subsequently stained with carboxy-H2DCFDA and PI to measure intracellular ROS production in live cells on flow cytometry. Increases in ROS were calculated relative to the average geometric mean fluorescence intensity (GMFI) of carboxy-H2DCFDA staining in untreated TEX or OCI-AML2 cells. Hydrogen peroxide (H2O2) was included as a positive control for intracellular ROS. Data depict mean fold increase in ROS ± SD from an experiment performed in triplicate.
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Figure 4-11: Mitochondrial ROS production following combination ibrutinib-
daunorubicin treatment in TEX and OCI-AML2 cells. TEX and OCI-AML2 cells were treated with ibrutinib (“Ibru”), daunorubicin (DNR), or both drugs in combination for 6h. Cells were stained with MitoSOX Red and Annexin V to measure mitochondrial ROS on flow cytometry. Fold increase in mitochondrial ROS production was calculated relative to the average GMFI of MitoSOX+, Annexin V- staining untreated cells. Antimycin A was included as a positive control for mitochondrial ROS staining. Results depict the mean fold increase in mitochondrial ROS ± SD from an experiment performed in triplicate. Data are representative of two independent experiments.
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A possible explanation for the discrepancy between the degree of viability rescue
by α-tocopherol and induction of ROS following combination treatment is the
length of incubation with drug (6 hours) prior to ROS measurement: previous
studies in ABC-DLBCL cells demonstrated highest ROS production by ibrutinib at
1-2 hours post-treatment, with levels significantly decreasing by four hours
(Dasmahapatra et al., 2013). Alternatively, it is possible that the modest increase
in ROS production following ibrutinib-daunorubicin treatment does not fully
account for the extent of antioxidant-mediated cell rescue: the observed
cytoprotection by α-tocopherol may have been due to an off-target effect. In
support of this possibility, a recent study reported reversal of kinase inhibitor-
mediated apoptosis and cell cycle arrest by α-tocopherol, which is independent of
its antioxidant activity (Pédeboscq et al., 2012).
In conclusion, ibrutinib potentiates the AML cell-killing activity of daunorubicin via
a mechanism that is potentially BTK-independent, and unrelated to enhancement
of intracellular daunorubicin accumulation. Incorporating ibrutinib into treatment
regimens for AML patients, regardless of BTK expression and constitutive
activation status, may be warranted.
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Chapter 5: Erlotinib synergizes with the poly(ADP-ribose) glycohydrolase inhibitor ethacridine in acute myeloid leukemia cells
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5.1 Abstract
Erlotinib is a small-molecule epidermal growth factor receptor (EGFR) inhibitor
that has demonstrated significant EGFR-independent activity against acute
myeloid leukemia (AML) cell lines and primary AML blasts in preclinical studies,
however these findings have not been reproducible in the clinical trial setting.
Combining erlotinib with other antineoplastic agents has been proposed as a
strategy for improving the clinical activity of erlotinib. With the goal of identifying
erlotinib-sensitizing drugs, we screened erlotinib against several chemical
libraries in the erlotinib-insensitive AML cell lines TEX and OCI-AML2, identifying
the poly(ADP-ribose) glycohydrolase inhibitor ethacridine lactate as the top
synergistic hit common to both cell lines. The erlotinib-ethacridine combination
induced synergistic cell death, which was preceded by a profound and lethal
increase in intracellular reactive oxygen species (ROS) production. Using mass
spectrometry, we determined that erlotinib synergized with ethacridine by
potentiating ethacridine accumulation in TEX and OCI-AML2 cells. This
synergistic mechanism of action was confirmed by demonstrating that high-dose
ethacridine treatment mimics the significant increases in ROS observed following
combination erlotinib-ethacridine treatment. Thus, we have identified that erlotinib
promotes the accumulation of select drugs, thereby leading to synergism. In
addition, the potential anti-AML activity of PARG inhibitors warrants further study.
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5.2 Introduction
Erlotinib is a small-molecule epidermal growth factor receptor (EGFR) inhibitor
that reversibly blocks autophosphorylation of C-terminal EGFR tyrosine residues,
inhibiting cell proliferation and inducing apoptosis (Moyer et al., 1997). It is used
in the clinical treatment of non small-cell lung cancer (NSCLC), where EGFR is
often over-expressed or constitutively active, and thus promotes tumor cell
survival and proliferation via PI3K/AKT/mTOR, STAT, and Ras/Raf/MAPK
signaling pathways (Sharma et al., 2007; Siegelin & Borczuk, 2014).
Erlotinib has well-documented preclinical activity against acute myeloid leukemia
(AML) cells, where it induces differentiation (Boehrer et al., 2008a), cell cycle
arrest (Boehrer et al., 2008a; Boehrer et al., 2011; Lainey et al., 2011), and
apoptosis (Boehrer et al., 2008a; Boehrer et al., 2008b), yet EGFR expression in
these cells is absent (Boehrer et al., 2008a; Chan & Pilichowska, 2007;
Stegmaier et al., 2005). Several erlotinib targets have been proposed or reported
to account for its anti-leukemic effects: erlotinib was shown to inhibit SRC family
kinases (SFKs) (Boehrer et al., 2011; Weber et al., 2012), which are
constitutively active in primary AML cells and AML cell lines and mediate mTOR
complex 1 (mTORC1) signaling in these cells (Dos Santos et al., 2008). In line
with these findings, erlotinib was found to inhibit phosphorylation of mTORC1
targets and to induce autophagy in the AML cell line KG-1 (Boehrer et al., 2011).
Erlotinib has also been found to bind directly to Bruton’s tyrosine kinase (BTK)
and to decrease phosphorylation at Y551 (Weber et al., 2012); Y551
phosphorylation is required for activation of this kinase. BTK has been proposed
as a potential therapeutic target in AML because of its role as a mediator of
FLT3-ITD and TLR9 signaling in FLT3-ITD-positive and FLT3-ITD-negative AML
cell lines, respectively (Oellerich et al., 2015).
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Finally, erlotinib was shown to inhibit JAK2 Tyr1007/1008 phosphorylation and
downstream STAT5 phosphorylation at Tyr694 in KG-1 cells (Boehrer et al.,
2008a). JAK2-STAT3/5 signaling has been implicated in AML CD34+ cell survival
and colony formation (Cook et al., 2014).
Clinically, the anti-AML activity of erlotinib has been more modest. While two
case reports described AML remissions in two patients with concomitant NSCLC
and AML (Chan & Pilichowska, 2007; Pitini et al., 2008), subsequent clinical trials
examining the single-agent activity of erlotinib in AML have not yielded equally
remarkable results, with a small minority of patients exhibiting decreased
peripheral blast counts and zero patients achieving complete remission (Sayar et
al., 2015; Thepot et al., 2014). The authors of both studies suggested that
erlotinib may have better clinical utility when administered in combination with
other anti-leukemic agents (Sayar et al., 2015; Thepot et al., 2014).
In light of the limited clinical single-agent activity of erlotinib in AML, its favorable
safety profile (Gordon et al., 2005; Sayar et al., 2015; Soulieres et al., 2004;
Thepot et al., 2014), and previous reports of synergistic interactions with other
antineoplastic agents (Lainey et al., 2013a; Lainey et al., 2013b; Landriscina et
al., 2010), we sought to identify erlotinib combination candidates in AML using a
high-throughput drug screening approach.
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5.3 Materials and Methods
5.3.1 Reagents
Anti-EGFR (#2232) and anti-GAPDH (#2118) antibodies were obtained from Cell
Signaling Technology (Danvers, MA). HRP-conjugated goat anti-rabbit and goat
anti-mouse secondary antibodies were acquired from GE Healthcare
(Buckinghamshire, UK). Erlotinib was obtained from Cayman Chemical (Ann
Arbor, MI). Ethacridine lactate, sulforhodamine-B, α-tocopherol, and hydrogen
peroxide were purchased from Sigma-Aldrich (St. Louis, MO). Imatinib was
obtained from AK Scientific, Inc. (Union City, CA). Drug libraries were obtained
from MicroSource Discovery Systems, Inc. (Gaylordsville, CT), and Sequoia
Research Products (Pangbourne, UK). The kinase inhibitor library was obtained
from the Ontario Institute for Cancer Research, Toronto, Canada.
5.3.2 Cell culture
TEX cells (Warner et al., 2005) were provided by Dr. John Dick (Ontario Cancer
Institute, Toronto, Canada) and maintained in Iscove’s Modified Dulbecco’s
Medium (IMDM) supplemented with 15% fetal bovine serum (FBS)
San Diego, CA), and 2ng/ml IL-3 (R&D Systems, Minneapolis, MN). OCI-AML2
and U937 cells were provided by Dr. Mark Minden (Ontario Cancer Institute,
Toronto, Canada). K562 cells were provided by Dr. Suzanne Kamel-Reid
(Ontario Cancer Institute, Toronto, Canada). OCI-AML2 and K562 cells were
grown in IMDM supplemented with 10% FBS 100 µg/ml penicillin, and 100 U/ml
streptomycin. U937 cells were grown in RPMI 1640 supplemented with 10%
FBS, 100 µg/ml penicillin, and 100 U/ml streptomycin. MDA-468 cells were grown
in RPMI-1640 supplemented with 10% FBS. All cell lines were maintained at
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37°C and 5% CO2.
5.3.3 Primary cells
Primary bulk AML cells and peripheral blood stem cells (PBSCs) from stem cell
donors treated with G-CSF were collected from consenting patients, with the
approval of the University Health Network (Toronto, Canada) institutional review
board. Primary AML cells were isolated by Ficoll density centrifugation and
PBSCs were isolated by apheresis. Cells were maintained in IMDM
supplemented with 10% FBS, 100µg/ml penicillin, and 100U/ml streptomycin, at
37°C and 5% CO2.
5.3.4 Immunoblotting
PBS-washed cells were lysed in 1xLaemmli buffer and protein from whole cell
lysates was quantified with the DC Protein Assay (Biorad Laboratories,
Mississauga, ON, Canada). Lysates were resolved by SDS-PAGE and
transferred to a PVDF membrane. Following blocking in 5% milk-TBST for 1 hour
at room temperature, membranes were incubated with primary antibody on a
rocker overnight at 4°C. Following a 1-hour incubation with secondary antibody
(GE Healthcare, Buckinghamshire, UK), proteins were detected by
chemiluminescence.
5.3.5 Cell viability assays
Cells were treated with drugs alongside vehicle control (DMSO) in 96- or 384-
well plates over 48 or 72 hours. Cell growth and viability was measured with the
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sulforhodamine-B assay, performed as previously described (LaPointe et al.,
2005), or the Alamar Blue assay, as per manufacturer’s instructions (Life
Technologies, Carlsbad, CA). Cell viability was also measured by Annexin V and
PI staining on a BD CantoII 96w (BD Biosciences, San Jose, CA) flow cytometer,
and analyzed using FlowJo version 7.6.5 (TreeStar, Ashland, OR). For both
methods, cell viability was calculated relative to that of vehicle controls.
5.3.6 High-throughput combination drug screening & excess-over-Bliss additivism synergy calculations
High-throughput combinatorial drug screening was carried out as previously
described (Rotin et al., 2016b). The excess-over-Bliss additivism formula (Borisy
et al., 2003), which calculates the degree of cell killing unaccounted for by the
added cytotoxicities of each individual drug, was applied as previously described
(Rotin et al., 2016b).
5.3.7 Reactive oxygen species measurement
Intracellular ROS production in live drug-treated cells was measured by staining
cells with 10 µM carboxy-H2DCFDA (Molecular Probes/Life Technologies,
Eugene, OR) for 30 minutes at 37°C and 5% CO2 and subsequent staining with
propidium iodide. ROS production in stained live cells was detected with a BD
Fortessa LSR X20 (BD Biosciences, San Jose, CA) flow cytometer, and
calculated relative to the geometric mean fluorescence intensity of untreated live
cells.
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5.3.8 Mass spectrometry
Chromatographic separations were carried out on an Acquity UPLC BEH C18
(2.1 X 50 mm, 1.7 µm) column using ACQUITY UPLC II system. The mobile
phase was 0.1% formic acid in water (solvent A) and 0.1% formic acid in
acetonitrile (solvent B). A gradient starting at 95% solvent A going to 5% in 4.5
min., holding for 0.5 min., going back to 95% in 0.5 min. and equilibrating the
column for 1 min. was employed. A Waters Synapt G2S QTof mass
spectrometer equipped with an atmospheric pressure ionization source was used
for mass spectrometric analysis. MassLynx 4.1 was used for data analysis.
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5.4 Results
5.4.1 TEX and OCI-AML2 cell line sensitivity to erlotinib
To ascertain erlotinib sensitivity in the AML cell lines TEX and OCI-AML2, we
subjected these cells to treatment with increasing concentrations of erlotinib for
72 hours, and subsequently measured relative growth and viability using the
sulforhodamine-B (SRB) assay. The average erlotinib IC50s were 8.99µM and
15.61µM in TEX and OCI-AML2, respectively (Figure 5-1). These erlotinib IC50
values are significantly higher than clinically achievable concentrations
(Appendix 1) and far greater than the nanomolar-range IC50 values reported in
the NSCLC cell lines HCC827, HCC4006, HCC4011, H3255, and H292, using
the same assay (Gao et al., 2014).
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Figure 5-1: AML cell line sensitivity to erlotinib. (A) TEX and OCI-AML2 cells were treated with increasing concentrations of erlotinib for 72h. Growth and viability was measured using the SRB assay and calculated relative to vehicle-treated cells. Results depict average percent growth and viability ± SD from a representative experiment performed in triplicate. Data are representative of three independent experiments.
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5.4.2 A high-throughput combination chemical screen identifies erlotinib sensitizers in TEX and OCI-AML2 cells
With the goal of identifying synergistic combination candidates for erlotinib, we
screened this drug against three chemical libraries—the Microsource Discovery
Systems International Drug and Natural Product (“NatProd”) collections, and a
312-compound library from Sequoia Research Products—in TEX and OCI-AML2
cells. Erlotinib at its IC10 and IC25 was combined with increasing concentrations of
1,352 drugs for a total of 16,230 different assays in two cell lines. Treated cells
were incubated for 72 hours and relative growth and viability was measured with
the SRB assay. These viability data were then used to determine synergy based
on the excess-over-Bliss additivism (EOBA) formula (Borisy et al., 2003), which
calculates the difference between observed and predicted killing by a given drug
combination, with a greater positive difference indicating stronger synergy.
Compounds were plotted in order of increasing positive EOBA scores for each
drug library, in both cell lines (Figure 5-2). The most profoundly synergistic hits
from each library were individually validated using a broader range of
concentrations to more thoroughly evaluate synergy. Ethacridine lactate, a
poly(ADP-ribose) glycohydrolase (PARG) inhibitor (Bernardi et al., 1997;
Boulikas, 1990; Tavassoli et al., 1985) and abortifacient (Hou et al., 2010), was
validated as the top synergistic hit common to both TEX and OCI-AML2 cells,
generating EOBA scores of up to 0.79 in TEX and 0.69 in OCI-AML2 (Figure 5-3, top). Validation of two other screen hits, apigenin and berberine, are included
for comparison (Figure 5-3, bottom).
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Figure 5-2: Erlotinib sensitizers in TEX and OCI-AML2 cells. Erlotinib was screened against 1,352 drugs from three chemical libraries (Sequoia, International, and Natural Products A, B and C). Following a 72h incubation, EOBA synergy scores were calculated from relative growth and viability values determined by the SRB assay. Compounds were ranked in order of increasing positive EOBA score.
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Figure 5-3: Validation of synergistic hits. Synergistic hits (shown: ethacridine, apigenin, and berberine) were validated in TEX and OCI-AML2 cells using broader concentration ranges. Cells were combination-treated for 72h and percent growth and viability was measured with the SRB assay (graphs). Synergy was calculated according to EOBA criteria (tables), with EOBA values >0.1 (lightest grey) denoting a synergistic combination, while values >0.5 (darkest grey) denoting a profoundly synergistic combination. Results depict mean percent growth and viability ± SD (graphs) or mean EOBA scores (tables) from a single experiment performed in triplicate.
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5.4.3 The erlotinib-ethacridine combination synergizes in primary AML cells and other AML cell lines
To determine whether the observed synergy between erlotinib and ethacridine
extended beyond TEX and OCI-AML2 cells, we combination-treated U937 and
K562 leukemia cells over 72 hours and measured relative growth and viability
with the SRB assay. Combination erlotinib-ethacridine treatment yielded EOBA
scores in the profoundly synergistic range (>0.50) at concentrations of erlotinib
that were clinically relevant (Figure 5-4A). We also examined this combination in
5 primary AML samples and 2 samples of normal hematopoietic cells derived
from healthy volunteers donating G-CSF-mobilized peripheral blood stem cells
for allogeneic transplant (denoted as “Normal”) (see Table 5-1 for patient
demographics). For this assay, cells were incubated with increasing
concentrations of erlotinib, ethacridine, or both in combination for 48 hours.
Viability relative to untreated (vehicle) controls was measured with Annexin V
and propidium iodide (PI) staining on flow cytometry (Figure 5-4B). None of the
primary samples or normals, with the exception of AML130183, were sensitive to
single-agent erlotinib treatment. Single-agent ethacridine sensitivity was variable
amongst primary AML cells, however all were more sensitive to ethacridine killing
compared to normals. The erlotinib-ethacridine combination was synergistic in all
5 primary AML samples, most strikingly in AML130208 and AML130237, which
yielded EOBA scores in the >0.30 range (Figure 5-4C). In contrast, the
combination was not synergistic in either of the normals; the highest EOBA score
calculated was 0.11, which was only observed in one pairing (Figure 5-4C).
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Figure 5-4: Erlotinib and ethacridine synergize in additional AML cell lines and
primary AML blasts. U937, K562, primary AML blasts, and PBSCs were combination-treated with erlotinib and ethacridine for 72h (A) or 48h (B). Viability was determined by the SRB assay (A) or Annexin V and PI staining (B). Graphs depict (A) mean percent growth and viability or (B) mean percent viability ± SD from a single experiment performed in triplicate. Tables (A, C) represent mean EOBA values.
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Table 5-1: AML patient demographics.
Patient Demographics Sample ID Diagnosis Age at
Diagnosis Gender Cytogenetics Molecular
130177 AML, M4Eo 62 Male 46,XY,inv(16)(p13.1q22)[8]/46,XY[2] CBFB-MYH11 +,KIT-
130183 AML 69 Male 46,XY[20] NPM1+, FLT3-ITD+, FLT3-TKD-
intracellular ROS production. (A) TEX and OCI-AML2 cells were combination-treated with erlotinib and ethacridine for 24h. Intracellular ROS was measured with carboxy-H2DCFDA staining and dead cells were excluded by PI staining. Fold increase in ROS production was calculated relative to the geometric mean fluorescence intensity (GMFI) of vehicle-treated cells. H2O2 was included as a positive intracellular ROS control. Results depict mean fold increase in GMFI ± SD from a representative experiment performed in triplicate. Data are representative of two independent experiments. (B) TEX and OCI-AML2 cells were treated with 3mM α-tocopherol for 24h, then treated with the erlotinib-ethacridine combination for the following 48h (with α-tocopherol maintained at 2.4mM). Viability was measured with Annexin V and PI staining on flow cytometry, and calculated relative to respective (± α-tocopherol) controls. Data represent mean percent viability ± SD from an experiment performed in triplicate. These data are representative of two independent experiments. (C) TEX cells were combination-treated with erlotinib and gallotannin for 48h. Viability was determined by Annexin V and PI staining. Graph depicts mean percent
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viability ± SD from a single experiment performed in triplicate. Table represents mean EOBA values from the same experiment. Data are representative of at least three independent experiments.
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5.4.5 Ethacridine synergizes with EGFR-targeting kinase inhibitors
Given that erlotinib has been proposed to inhibit other kinases in its activity
against AML cells (Boehrer et al., 2008a; Boehrer et al., 2011; Weber et al.,
2012), we sought to determine whether off-target kinase inhibition by erlotinib
was responsible for its synergy with ethacridine. We therefore screened
ethacridine against an in-house kinase inhibitor library, comprising 480 kinase
inhibitors with a broad range of kinase targets and varying degrees of kinase
specificity. OCI-AML2 cells were incubated with ethacridine along with two
different concentrations of each kinase inhibitor for 72 hours, for a total of 2,883
different assays. Relative growth and viability was subsequently measured with
the SRB assay and synergy scores were again calculated with the EOBA formula
and plotted in order of increasing synergy score (Figure 5-6A). Erlotinib was the
second most synergistic hit, which served as further validation for our initial
combination screen. Interestingly, 4 of the 5 top synergistic hits (GW583340,
erlotinib, GW2974, and WHI-P 154) were reported EGFR inhibitors. Furthermore,
the clinically approved EGFR inhibitors gefitinib and lapatinib were also identified
as synergistic hits, with EOBA values reaching 0.18 and 0.14, respectively.
5.4.6 TEX and OCI-AML2 cell lines do not express EGFR
The observed synergy between ethacridine and multiple EGFR inhibitors
prompted us to investigate whether erlotinib might inhibit EGFR to synergize with
ethacridine in TEX and OCI-AML2 cells. We evaluated total EGFR expression in
these cell lines by immunoblot and were unable to detect expression of this
kinase in either cell line (Figure 5-6B). Likewise, EGFR expression was absent
in K562 and U937 cells, which also demonstrated erlotinib-ethacridine synergy
(Figure 5-4A). Thus, these chemical EGFR inhibitors likely synergize with
ethacridine via a common, EGFR-independent mechanism.
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5.4.7 Erlotinib potentiates ethacridine accumulation in TEX and OCI-AML2 cells.
One property common to several small molecule EGFR inhibitors is their potent
inhibition of ATP-binding cassette (ABC) transporter efflux activity, and thus their
ability to enhance the cellular accumulation of—and sensitivity to—their
substrates. We therefore investigated whether erlotinib potentiates ethacridine
accumulation in AML cells. We treated TEX and OCI-AML2 cells with 5 µM
ethacridine in the presence and absence of erlotinib for one hour. Cells were
lysed and contents were quantified by LC-MS/MS. Ethacridine concentrations
increased nearly 2-fold in the presence of as little as 1 µM erlotinib in both cell
lines (Figure 5-6C). In contrast, imatinib (which does not synergize with
ethacridine (Figure 5-7)) did not potentiate ethacridine accumulation in either cell
line (Figure 5-6C).
5.4.8 High-dose ethacridine treatment mimics ROS production observed from the erlotinib-ethacridine combination.
To determine whether erlotinib-mediated intracellular ethacridine accumulation
could be responsible for excessive ROS production, we treated TEX and OCI-
AML2 cells with high concentrations of ethacridine and measured changes in
ROS production. Cells were incubated for 24 hours with 15 µM and 20 µM
ethacridine to mimic the increase in ethacridine accumulation observed in the
presence of 3 µM erlotinib, alongside combination-treated cells. ROS production
in live cells was measured by carboxy-H2DCFDA and PI staining on flow
cytometry, and calculated relative to vehicle-treated controls. High-dose
ethacridine increased ROS production more than 2-fold in TEX cells, and greater
than 3-fold in OCI-AML2 cells (Figure 5-6D), which was comparable to the
increase in ROS production observed with combination erlotinib-ethacridine
treatment (Figure 5-5A). These findings suggest that erlotinib-mediated
127
ethacridine accumulation is the mechanism that explains synergistic cell death
caused by excessive ROS production.
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Figure 5-6: Erlotinib enhances ethacridine accumulation in TEX and OCI-AML2
cells. (A) Ethacridine was screened against a 480-compound kinase inhibitor library in OCI-AML2 cells. Cells were treated for 72h. Growth and viability was measured with the SRB assay and synergy scores were calculated with the EOBA formula. Compounds were ranked in order of increasing positive synergy score. (B) EGFR expression in a panel of AML cell lines was detected by immunoblotting, with the MDA-468 cell line included as an EGFR-positive control. (C) TEX and OCI-AML2 cells were treated with ethacridine in the presence and absence of erlotinib for 1h. Cells were lysed and analyzed by LC-MS. Imatinib was included as a negative (non-synergizing) control. Data depict mean ethacridine accumulation ± SD from an experiment performed in triplicate. Data are representative of three (TEX) or two (OCI-AML2) independent experiments. (D) TEX and
129
OCI-AML2 cells were treated with high-dose ethacridine or erlotinib and ethacridine in combination for 24h. Intracellular ROS was measured with carboxy-H2DCFDA staining (with PI exclusion of dead cells) and calculated relative to GMFI of vehicle-treated cells. Data depict mean fold increase in GMFI ± SD from a representative experiment performed in triplicate, and are representative of two independent experiments.
130
Figure 5-7: Imatinib does not synergize with ethacridine in TEX and OCI-AML2
cells. TEX and OCI-AML2 cells were treated with increasing concentrations of imatinib and ethacridine alone and in combination. Relative growth and viability following a 72h incubation was measured with the Alamar Blue assay. Synergy was calculated with the EOBA formula. Graphs depict mean percent growth and viability ± SD from an experiment performed in triplicate. Tables depict mean EOBA scores from the same experiment. Data are representative of three independent experiments.
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5.5 Discussion
The impressive EGFR-independent antileukemic activity of erlotinib reported in
preclinical studies has not translated to significant responses in the clinical
setting, with two clinical trials reporting negligible or no single-agent erlotinib
activity against AML. The first, a Phase I/II clinical trial evaluating erlotinib use in
30 high-risk MDS and AML patients who had previously failed azacytidine
treatment, reported no responses to erlotinib treatment in those with AML (n=12)
(Thepot et al., 2014). The second study, which assessed erlotinib treatment in
nine patients with treatment-naïve AML and two patients with relapsed or
refractory AML, reported disease progression in all 11 patients, however two
patients demonstrated a significant decrease in peripheral blasts (Sayar et al.,
2015). Failure of erlotinib as a single-agent therapeutic strategy for this disease
in clinical trials has prompted interest in the investigation of this tyrosine kinase
inhibitor (TKI) as a combination candidate for AML therapy.
With the goal of identifying novel erlotinib combination candidates, we screened
this TKI against several drug libraries in two non-EGFR expressing AML cell
lines, which had erlotinib sensitivities in the micromolar range. The PARG
inhibitor ethacridine lactate was the most synergistic hit common to both TEX
and OCI-AML2 cells. We determined that synergy was due to erlotinib
potentiation of intracellular ethacridine accumulation, as evidenced by the fact
that high-dose ethacridine treatment recapitulated the lethal increase in ROS
production observed following combination erlotinib-ethacridine treatment.
Erlotinib has been previously shown to synergize with other drugs in AML cell
lines and primary AML blasts: erlotinib potentiated ATRA- and vitamin D3-
mediated differentiation of HL60 cells, an effect attributed to erlotinib’s inhibition
of SRC and p38MAPK phosphorylation (Lainey et al., 2013b). Erlotinib and
gefitinib, another EGFR inhibitor, were also found to synergistically block cell
132
proliferation and induce apoptosis when combined with the hypomethylating
agent azacytidine, owing to erlotinib- and gefitinib-mediated potentiation of
intracellular azacytidine accumulation (Lainey et al., 2013a). Finally, erlotinib and
gefitinib were shown to sensitize KG-1 AML cells to antineoplastic agents such
as etoposide and doxorubicin by enhancing their accumulation through
simultaneous inhibition of P-glycoprotein (P-gp), multidrug resistance protein 1
(MRP1), and breast cancer resistance protein (BCRP)-mediated drug efflux
activity (Lainey et al., 2012). EGFR inhibitors have been extensively shown to
enhance the cytotoxicity of antineoplastic drugs through inhibition of ABC
transporter-mediated drug efflux (Dai et al., 2008; Kuang et al., 2010; Shi et al.,
2007).
While our study did not address the mechanism of intracellular ethacridine
uptake or the mechanism by which it is extruded from AML cells, the fact that the
concentrations of erlotinib required for synergy with ethacridine are in line with
those required for erlotinib synergy with other antineoplastic agents (1 to 10 µM
range (Lainey et al., 2012; Lainey et al., 2013a)) would strongly suggest that
erlotinib inhibition of one or more ABC transporters accounts for lethal levels of
ethacridine accumulation in TEX and OCI-AML2 cells.
Given the capacity of erlotinib to promote the accumulation of other drugs, it is
surprising that the combination chemical screen against erlotinib did not yield a
greater number of validated synergistic hits common to both TEX and OCI-AML2
cells. This observation may suggest that in these AML cell lines, erlotinib may
inhibit a more restricted number of transporters. This observation may also
highlight the potential therapeutic relevance of PARG inhibition in AML. PARG
hydrolyzes poly(ADP-ribose) (PAR) polymers, which are synthesized by
poly(ADP-ribose) polymerases (PARPs) in response to DNA damage and other
cellular stresses. PARG therefore quenches PARP-elicited signals, which drive
processes such as DNA repair or cell death.
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With the exception of our own previous report describing the synergistic
cytotoxicity between chemical PARG inhibitors and the Bruton’s tyrosine kinase
inhibitor ibrutinib (Rotin et al., 2016b), the role of PARG inhibition in AML has not
been investigated. PARG inhibition has been found to sensitize other cancer cell
lines to DNA-damaging and oxidative stress-inducing agents by failing to reduce
cellular PAR levels following PARP activation. Excessive PAR accumulation
induces cell death by necrosis (NAD+ is a substrate for PAR synthesis, thus
cellular ATP stores become depleted), or parthanatos (excess PAR triggers
nuclear translocation of apoptosis-inducing factor). We therefore hypothesize that
erlotinib-mediated ethacridine accumulation induces lethal levels of PAR
accumulation due to profound PARG inhibition.
In summary, we have identified the PARG inhibitor ethacridine as a novel
combination candidate for erlotinib in AML. Erlotinib synergizes with ethacridine
by potentiating its intracellular accumulation. The impact of PARG inhibition as a
therapeutic strategy in AML warrants further investigation.
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Chapter 6: General Discussion & Conclusion
135
6.1 Discussion
The goal of this project was to further elucidate the role of BTK as a therapeutic
target in AML, through the identification of drugs that sensitize AML cells to killing
by ibrutinib. We determined that both ethacridine lactate and daunorubicin
synergized with ibrutinib, however neither synergistic interaction was dependent
upon ibrutinib-mediated BTK inhibition. Further work investigating the striking
synergy between ethacridine and the kinase inhibitor erlotinib, a known inhibitor
of ATP-binding cassette (ABC) transporter-mediated drug efflux, shed light on
the possibility that ibrutinib also likely synergizes with ethacridine via this
mechanism. The mechanism by which ibrutinib and daunorubicin synergize is not
yet known.
6.1.1 BTK-independent anti-leukemic activity of ibrutinib
In demonstrating that ethacridine or daunorubicin treatment of BTK-knockdown
AML cell lines does not recapitulate the synergy observed when these drugs are
combined with ibrutinib, and that ibrutinib and ethacridine synergized in cells
lacking BTK protein expression, we provided evidence to suggest that ibrutinib
has anti-AML activity that extends beyond BTK inhibition.
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Appendix 1
Clinically achievable concentrations of tyrosine kinase inhibitors
Drug Dose (oral)
Approximate steady-state
plasma concentration
Reference
Ibrutinib 840mg/d 450nM (peak) Byrd et al. (2013)
Imatinib 400mg/d 5.3µM (peak) 2.4µM (trough) Peng et al. (2005)