Simultaneous Drug Targeting of the Promoter MYC G-Quadruplex and

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Simultaneous Drug Targeting of the Promoter MYC G‑Quadruplex and BCL2 i‑Motif in Diffuse Large B‑Cell Lymphoma Delays Tumor Growth Samantha Kendrick,*,† Andrea Muranyi,‡ Vijay Gokhale,∥ Laurence H. Hurley,§ and Lisa M. Rimsza†,⊥ †

Department of Pathology, University of Arizona, 1501 North Campbell Avenue, Tucson, Arizona 85724, United States Ventana Medical Systems, Inc., 1910 Innovation Park Drive, Tucson, Arizona 85755, United States § College of Pharmacy, University of Arizona, 1703 East Mabel Street, Tucson, Arizona 85721, United States ⊥ Mayo Clinic, 13400 East Shea Boulevard, Scottsdale, Arizona 85259, United States ∥ BIO5 Institute, University of Arizona, 1657 EAST HELEN STREET, Tucson, Arizona 85721, United States ‡

S Supporting Information *

ABSTRACT: Secondary DNA structures are uniquely poised as therapeutic targets due to their molecular switch function in turning gene expression on or off and scaffold-like properties for protein and small molecule interaction. Strategies to alter gene transcription through these structures thus far involve targeting single DNA conformations. Here we investigate the feasibility of simultaneously targeting different secondary DNA structures to modulate two key oncogenes, cellular-myelocytomatosis (MYC) and B-cell lymphoma gene-2 (BCL2), in diffuse large B-cell lymphoma (DLBCL). Cotreatment with previously identified ellipticine and pregnanol derivatives that recognize the MYC G-quadruplex and BCL2 i-motif promoter DNA structures lowered mRNA levels and subsequently enhanced sensitivity to a standard chemotherapy drug, cyclophosphamide, in DLBCL cell lines. In vivo repression of MYC and BCL2 in combination with cyclophosphamide also significantly slowed tumor growth in DLBCL xenograft mice. Our findings demonstrate concurrent targeting of different DNA secondary structures offers an effective, precise, medicine-based approach to directly impede transcription and overcome aberrant pathways in aggressive malignancies.

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cyclopenta[a]phenanthren-3-ol (NSC-59276, IMC-76,14,15 76), respectively, through interaction with their respective promoter region DNA secondary structure (Figure 1E,F). The ellipticine derivative 05 stabilizes the MYC G-quadruplex and maintains an “off-switch” DNA conformation.13 In contrast, 76, a pregnanol derivative, stabilizes the BCL2 flexible hairpin, which is in equilibrium with the i-motif structure located on the DNA strand complementary to the BCL2 G-quadruplex and thereby selects for an alternative DNA conformation from the “on-switch”.14,15 In both cases, we used ChIP analysis to show the expected effects on the occupancy of the Gquadruplex- or i-motif-binding proteins at the promoter elements and, in the case of 05, demonstrated using an exonspecific assay that the MYC G-quadruplex is required for drug action.13,15 These DNA secondary structure-targeting agents led to the repression of MYC or BCL2 transcription and an increased etoposide sensitivity of chemotherapy-resistant Burkitt’s and mantle lymphoma cell lines and a mantle cell

INTRODUCTION

Duplex DNA is capable of adopting noncanonical secondary structures that arise from guanine-rich and cytosine-rich sequences: the G-quadruplex and the i-motif, respectively.1,2 These secondary DNA structures act as molecular switches, turning gene expression on or off. Consistent with this function, genome-wide analyses have detected DNA secondary structureforming sequences within close proximity to transcription start sites in 43% of promoter regions, which are mostly oncogene promoters, including cellular myelocytomatosis (MYC) and Bcell lymphoma gene-2 (BCL2).3−7 Our group, along with others, characterized the MYC G-quadruplex and BCL2 i-motif structures that form within each promoter region (Figure 1A,B) and the interplay of these structures with nuclear proteins (Figure 1C,D).8−15 In addition, we recently discovered small molecules from a high-throughput screen of the NCI Diversity Set I (1990 compounds) that specifically modulate either MYC or BCL2 expression, i.e., ellipticine [9-(dimethylaminoethoxy)dihydrochloride (NSC-338258, GQC-05, 1 3 05)] or (3S,5S,8R,9S,10S,13S,14S)-17-(1-(6-methoxy-2H-benzo[e][1,3]oxazin-3(4H)-yl)ethyl)-10,13-dimethylhexadecahydro-1H© 2017 American Chemical Society

Received: February 22, 2017 Published: June 12, 2017 6587

DOI: 10.1021/acs.jmedchem.7b00298 J. Med. Chem. 2017, 60, 6587−6597

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Figure 1. Proposed model for targeting the DNA secondary structure formation within the MYC and BCL2 gene promoter regions with 05 and 76. (A) The MYC promoter region consists of a G-rich element that forms a G-quadruplex structure from three stacked G-tetrads composed of four GHoogsteen base pairs.19−21 (B) The C-rich sequence upstream of the BCL2 promoter region is capable of adopting an i-motif structure with three and one-half sets of two intercalated hemiprotonated C+−C base pairs.14 (C) During transcription NM23-H2 can unfold the MYC G-quadruplex, resulting in single-stranded DNA to which CNBP can bind and activate MYC transcription. (D) The nuclear protein hnRNP LL can initiate conformational changes in the BCL2 i-motif, leading to transcription activation. (E) Binding of 05 stabilizes the MYC G-quadruplex, resulting in transcription repression.21−23 (F) 76 sequesters the flexible hairpin form of this dynamic element and inhibits transcription.24,25

lymphoma xenograft mouse model.14,15 It is likely that targeting DNA secondary structures offers a strategy to minimize resistance because mutations within coding regions that alter protein conformation or post-translational modifications and confer resistance do not affect these structures within oncogene promoter regions. Thus, these structures potentially provide an advantage over protein-directed therapeutic approaches. In diffuse large B-cell lymphoma (DLBCL), the most common non-Hodgkin’s lymphoma, there is a particularly aggressive form of the disease characterized by concurrent overexpression of MYC and BCL2 oncogenes that occurs in at least 30% of patients. Patients with dual MYC and BCL2 protein-positive DLBCL experience a rapid disease progression that is often refractory to the current immunochemotherapy regimen consisting of rituximab monoclonal antibody, cyclophosphamide (CPA), doxorubicin, vincristine, and prednisone. The majority of patients survive less than five years due to the lethal combination of resistance to cell death and high cell proliferation.16−20 Currently there are no effective strategies to treat these patients. Even preclinically there is no approach to directly and specifically modulate MYC expression, although the broad bromodomain and extra-terminal domain inhibitor tert-butyl 2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2f ][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate (JQ1, 1) has been shown to indirectly lower MYC expression.21 Similarly, most anti-BCL2 therapies have not been clinically useful except for the BH-3 mimetic 4-[4-[[2-(4-chlorophenyl)-4,4-dimethylcyclohexen-1-yl]methyl]piperazin-1-yl]-N-[3-nitro-4-(oxan-4ylmethylamino)phenyl]sulfonyl-2-(1H-pyrrolo[2,3-b]pyridin-5yloxy)benzamide (ABT-199, venetoclax, 199) found to inhibit BCL2 protein activity.22 However, resistance to compounds 1

and 199 and other BH-3 mimetics has been observed in patients and anticipated in preclinical models.22−27 Thus far, investigations have focused on individually targeting these potent oncogenes, but this may not be clinically relevant for DLBCL patients with double-positive tumors and may be less effective in circumventing drug resistance and relapse. Despite the presently available pharmacologic inhibitors of MYC and BCL2 in preclinical and clinical studies, issues with resistance and specificity demand continued, active research focused on promising new approaches to target these critical oncogenes in this aggressive disease. We sought to directly and simultaneously target MYC and BCL2 transcription through DNA secondary structures to induce chemosensitization using DLBCL as a model. Herein we apply our novel therapeutic strategy of inhibiting oncogenes at the transcriptional level to address the coincident expression of MYC and BCL2 known to confer an aggressive tumor cell phenotype and chemotherapy resistance in DLBCL. For the first time, we demonstrate that concurrent targeting of the MYC and BCL2 DNA secondary structures inhibits gene expression and subsequently protein expression and sensitizes dualexpressing DLBCL cell lines to CPA. CPA was selected for this proof-of-concept because it is a major component of the DLBCL standard chemotherapy regimen that induces DNA damage via nonspecific interaction with DNA as an alkylating agent, leads to mitochondrial BCL2-dependent apoptotic cell death, and is highly toxic. This chemosensitization resulted in a reduced concentration of CPA required to lower viability and to induce apoptosis in lymphoma cells, as well as to inhibit tumor growth in xenograft mice. With the increasing appreciation for the genetic complexity of lymphoma, targeted 6588

DOI: 10.1021/acs.jmedchem.7b00298 J. Med. Chem. 2017, 60, 6587−6597

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Figure 2. MYC targeting with 05 in DLBCL in vitro and in vivo models. (A) MYC mRNA (left panel) and protein (right panel) expression in DLBCL cell lines as detected by qPCR and Western blot. (B) Effect of 24 h treatment with 05 on MYC mRNA and protein expression. (C) Tumor burden of mice treated with CPA alone or cotreated with 05 as measured by tumor volume. Arrows indicate the 5 days of drug administration. MYC mRNA expression is relative to TBP and compared to levels in either the HT or untreated cells. Western blots are representative, with actin as a loading control and DMSO used as vehicle/diluent control. *P < 0.05, **P < 0.01, and ***P < 0.0001 for triplicate experiments.

independently, defined the mechanism for these drug-like molecules to silence MYC and BCL2 gene expression, and demonstrated their potential to increase chemotherapy sensitivity in non-Hodgkin’s lymphoma cells.13−15 In order to assess the efficacy of our novel approach to simultaneously target the MYC G-quadruplex and BCL2 i-motif for dual transcription repression, we utilized DLBCL as a model due to

therapy in conjunction with existing chemotoxic drugs may be important in management of these aggressive lymphomas that depend on specific oncogenic pathways for survival.

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RESULTS

Previous work from our group identified compounds that target the structures within the MYC and BCL2 promoter regions 6589

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Figure 3. BCL2 targeting with 76 in DLBCL cell lines. (A) BCL2 mRNA (left panel) and protein (right panel) expression in DLBCL cell lines as detected by qPCR and Western blot. (B) Effect of 24 h treatment with 76 on BCL2 mRNA and protein expression. BCL2 mRNA expression is relative to TBP and compared to levels in either the HT or untreated cells. Western blots are representative, with actin as a loading control and DMSO used as vehicle control. *P < 0.05, **P < 0.01, and ***P < 0.0001 for triplicate experiments.

strated that 05 effectively down-regulates MYC in a Burkitt’s lymphoma cell line, RAJI, after 24 h of treatment using concentrations below the cell viability IC50 so as not to inhibit ≥50% of cell growth.13 Here we also performed cell viability assays with 05 and treated our DLBCL cell lines with increasing concentrations based on the IC50 values for 24 h (Supporting Information, Figure S2 and Table S1). Treatment with 05 resulted in a substantial knock-down of up to 90% of MYC mRNA that was dose-dependent in the SUDHL4 and U2932 cells and occurred at the highest concentration of 3 μM in the VAL and HT cells, with corresponding decreases at the protein level as shown by Western blot (Figure 2B). Since the repression of MYC by 05 can occur as early as 6 h in the RAJI cell line,13 we tested whether this immediate effect would be present in the DLBCL cell lines and found that a similar, potent lowering of MYC occurred in all cell lines (Supporting Information, Figure S3). Compound 05 exhibits a considerable specificity for the MYC G-quadruplex and transcription inhibition relative to other oncogenes with promoters

the frequent concurrent overexpression and clinical significance of both oncogenes. DLBCL cell lines were characterized for MYC and BCL2 translocation and amplification status, and four cell lines were selected to represent the varying molecular mechanisms of MYC and BCL2 deregulation observed within patient tumors (Supporting Information, Figure S1). The SUDHL4 (BCL2 translocation-positive), VAL (BCL2 and MYC translocation-positive), and U2932 (MYC translocation-positive; BCL2 amplification-positive) cells display different levels of MYC and BCL2 expression at the mRNA and protein levels (Figures 2A and 3A). The HT cell line with no MYC or BCL2 genomic abnormalities, no detectable levels of BCL2, and little MYC (Figures 2A and 3A) was used for comparison to the dual-expressing cell lines. Our initial studies confirmed the inhibition of MYC and BCL2 in vitro using the DNA secondary structure-interactive small molecules 05 and 76 within these DLBCL cells. Targeting the MYC G-Quadruplex Inhibits Gene Expression in Vitro and in Vivo. We previously demon6590

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Figure 4. Dual-targeting of BCL2 and MYC increases the efficacy of CPA in DLBCL cell lines. (A) Percent cell viability determined by an MTS cytotoxicity assay following 96 h treatment with CPA alone, with 76 or 05, and with both 76 and 05. CPA concentration for the combination treatments were 5 mM for SUDHL4 and VAL and 10 mM for HT and U2932 cells. Significance considered at P ≤ 0.006 for CPA only comparisons to the nine possible combination treatments and P ≤ 0.03 for the CPA+76 or CPA+05 compared to the triple combination. (B) Percent caspase-3 activity following 24 h treatment with 1.25 mM CPA alone or in combination with 76 and/or 05. Significance considered at P ≤ 0.02 for CPA only comparisons to the three possible combination treatments. Data represent three replicates, with the exception of HT and SUDHL4 in panel B, which ́ k−Bonferroni multiple test correction applied. *P < represent four replicates. Comparisons were conducted using the Student’s t test with the Šidá 0.05 and **P < 0.01.

consisting of potential DNA secondary structures.13 While not a comprehensive transcriptome analysis, these data included both competition binding and expression assays with duplex DNA and VEGF, Hif1α, hTERT, PDGFRβ, PDGF-A, BCL2, and MYC G-quadruplex-forming DNA. In the present study we also observed significant MYC repression with little to no effect on BCL2 mRNA levels within the same cell lysates (Supporting Information, Figure S4A). Albeit, at the highest concentration 05 decreased BCL2 mRNA in SUDHL4 cells, presumably due to the slight interaction with the BCL2 G-quadruplex shown in a competition assay.13 The ultimate goal of the current work is

to simultaneously down-regulate both BCL2 and MYC; thus, this only strengthens the likelihood for a combined effect and the advantage of using these compounds. Compound 05 was never tested in a xenograft mouse model, unlike the BCL2 i-motif-interactive small molecule 76. Therefore, our first in vivo study involved 05 alone in combination with CPA, a component of the multichemotherapy DLBCL regimen, to determine the maximum tolerated dose (MTD) and the independent effects on tumor growth and MYC levels prior to combining with 76. As indicated in Figure 2C, both 2.5 and 5 mg/kg 05 with 50 mg/kg CPA significantly 6591

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reduced tumor burden in comparison to diluent-treated severe combined immunodeficient (SCID) mice with U2932 DLBCL xenografts. In contrast, there was a trend for slowed tumor growth with CPA alone (Figure 2C). However, at these doses, the addition of 05 potentiated CPA for only up to 10 days after the first day of treatment. Importantly, tumors from mice treated with 05 and CPA were compared to diluent-treated mice or mice treated with CPA alone for MYC mRNA level and found to have significantly less MYC expression (Figure 2C). As expected, there was no effect on BCL2 mRNA levels (Supporting Information, Figure S5A). There was no effect on mean mouse weight, and LD50 was not reached at either concentration of 05 in combination with CPA (Supporting Information, Figure S5B,C). Targeting the BCL2 i-Motif Down-Regulates BCL2 in DLBCL Cell Lines. Compound 76 specifically targets the BCL2 i-motif when examined in parallel with mutant, MYC, and VEGF i-motif, BCL2 G-quadruplex, and duplex-forming sequences and does not affect MYC or VEGF mRNA levels in Burkitt’s lymphoma, mantle cell lymphoma, and breast carcinoma cell lines.14 Consistent with these findings, 76 did not alter MYC expression in the DLBCL cell lines (Supporting Information, Figure S4B) and significantly decreased BCL2 mRNA levels by 11−37% in SUDHL4, VAL, and U2932 cells following 24 h treatment with 1 and 3 μM (Figure 3B). There was no effect in the non-BCL2-expressing HT cell line. The targeting of BCL2 gene expression also resulted in a concomitant decrease in protein expression (Figure 3B). Although the down-regulation of BCL2 expression by 76 is less than that observed for MYC in response to 05, the average 20% loss of BCL2 is most likely sufficient to facilitate chemosensitization and to a similar level as achieved by the protein inhibitor 4-[4-[[2-(4-chlorophenyl)phenyl]methyl]piperazin-1-yl]-N-[4-[[(2R)-4-(dimethylamino)-1-phenylsulfanylbutan-2-yl]amino]-3-nitrophenyl]sulfonylbenzamide (ABT737), as previously noted in other non-Hodgkin’s lymphoma cell lines.14 The dependence of cancer cells on BCL2 is known to “prime” the cells for death such that even small levels of inhibition skew the pro- and anti-apoptotic ratio. With the balance tipped toward pro-apoptotic proteins, cells then require less stimuli to induce cell death and allow for chemosensitivity when coupled with a chemotherapeutic agent.28 Concurrent Treatment with MYC and BCL2 Transcription Inhibitors Enhances the Cytotoxicity of CPA in Vitro. We then sought to determine whether simultaneous knock-down of MYC and BCL2 with both 05 and 76 would lead to an increased potency of CPA by cotreating DLBCL cells. We determined the effects of 05 and 76 combined treatment on cell viability and CPA IC50 using the MTS cytotoxicity assay as previously described.29−31 Initially, we conducted the cell viability assay with increasing concentrations of CPA to determine the IC50 of CPA alone relative to untreated cells [Figure 4A (black bars) and Table 1]. A CPA concentration above the IC50 (5 mM for SUDHL4 and VAL, 10 mM for HT and U2932) was then selected to combine with known concentrations of both inhibitors that repress MYC and BCL2 expression (1−3 μM). We observed in all four DLBCL cell lines that the addition of 05 and 76 at 3 μM to CPA at subIC50 significantly (P < 0.01) decreased cell viability by more than 50% (Figure 4A, red bars). Specifically, the SUDHL4, VAL, and U2932 MYC/BCL2-expressing cell lines exhibited a 30−46% decrease in cell viability compared to CPA alone, with a concomitant 1.6−2-fold lowering of CPA IC50 (Figure 4A and

Table 1. Cyclophosphamide (CPA) IC50 in the Absence and Presence of the BCL2 Inhibitor (76) and/or the MYC Inhibitor (05) CPA IC50 (mM) cell line HT SUDHL4 VAL U2932

CPA 10.5 5.9 5.9 12.2

± ± ± ±

1.6 0.2 0.3 0.3

CPA+76a 5.9 4.9 3.5 8.7

± ± ± ±

1.6 0.4 0.4b 0.7b

CPA+05a 6.2 5.1 5.2 9.6

± ± ± ±

1.2 0.5 0.8 1.5

CPA+76+05a 7.3 3.6 2.9 6.7

± ± ± ±

0.8 0.2c 0.7b 0.8c

Concentrations of 76 and 05 are 3 μM. bP < 0.01 for comparison with CPA alone IC50. cP < 0.001 for comparison with CPA alone IC50. a

Table 1). The HT cells, which express MYC only, also displayed lower cell viability (27%); however, there was no measurable reduction in IC50 of CPA (Figure 4A and Table 1). In further support, when the dual inhibition of MYC and BCL2 was compared to the independent targeting with 05 or 76 as single combinations to enhance CPA cytotoxicity, there was a notable and moderately synergistic reduction in cell viability in the SUDHL4 and U2932 cells (Figure 4A). Synergy was determined by calculating the relative risk ratio, where any value less than 1.0 indicates synergy.32 The relative risk ratio values for SUDHL4 and U2932 cells were 0.86 and 0.82, respectively. Synergy for dual inhibition was not observed for the VAL double-hit (BCL2 and MYC translocation-positive) lymphoma cell line, as these cells displayed more of an effect after BCL2 inhibition alone, which may indicate a greater dependence on the BCL2 oncogenic and/or apoptotic pathways. The BCL2 inhibitor 76 at 2 and 3 μM was able to potentiate the toxicity of 5 mM CPA up to 45% with a corresponding 1.7-fold decrease in CPA IC50 [Figure 4A (blue bars) and Table 1]. However, we still observed that a lower concentration of CPA could reduce cell viability and increase apoptosis with 76 and 05 cotreatment [Figure 4A (red bars) and Figure 4B]. Although the HT cells responded to the combined treatment of 76 at the highest concentration and 10 mM CPA by a 22% decrease in cell viability, there was no effect on CPA IC50 [Figure 4A (blue bars) and Table 1]. Inhibition of MYC alone did not sensitize any of the DLBCL cells to CPA [Figure 4A (green bars)]. These findings suggest that MYC and BCL2 repression together at sub-IC50 concentrations leads to maximum chemosensitization. The observed lowering of cell viability in the presence of both inhibitors in combination with CPA is most likely due to an increase in apoptosis, as evidenced by the increase in caspase-3 activity (Figure 4B). With the exception of 05 with CPA in the VAL cell line, treatment of each inhibitor alone with CPA did not result in a significant increase in caspase-3 activity in comparison to cells treated with CPA only. However, in all dual-expressing cell lines, cotreatment of both 05 and 76 with CPA induced caspase-3 activity by 30% to almost 100% from the activity with CPA alone (Figure 4B). Dual Repression of BCL2 and MYC Leads to Chemosensitization of in Vivo Tumors. For utility in sensitizing aggressive lymphoid tumors that dual-express MYC and BCL2, we examined the efficacy of 05 and 76 combination therapy to inhibit tumor growth in an in vivo model of SCID U2932 DLBCL xenograft mice. We carried over the 5 mg/kg 05 from the initial in vivo study (Figure 2C) to a subsequent experiment where we also tested a 10 mg/kg 05 dose and included 76 at 10 mg/kg, a previously established efficacious dose.14 While the mice were able to tolerate the 5 mg/kg 05 dose when 76 was 6592

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added, with or without CPA, to the treatment regimen, the LD50 was reached and surpassed in mice treated with the 10 mg/kg 05 combinations (Supporting Information, Figure S5D,E). The lowered LD50 at the higher dose of 05 suggests alternative mechanisms may be elicited at this high concentration that negatively impact mouse survival in addition to the on-target effect of MYC G-quadruplex stabilization and MYC inhibition. However, the 10 mg/kg concentration of 05 when cotreated with 76 and CPA resulted in a notable decrease of tumor MYC expression relative to untreated mice and those treated with CPA only as well as a detectable inhibition of BCL2 (Supporting Information, Figure S5F). We then conducted a follow-up study to evaluate effects on tumor burden using concentrations of 05 below the LD50, 5 and 7.5 mg/kg, and also reduced the dose of CPA from 50 to 30 mg/kg. The cotreatment of mice with 05 (7.5 mg/kg) and 76 (10 mg/kg) in combination with CPA delayed tumor growth and decreased tumor size by 50%, 46%, and 37% at 9, 12, and 15 days after the first day of drug administration compared to the diluent-treated mice and 50%, 67%, and 44% smaller tumors relative to those treated with CPA only (Figure 5A). Overall, there was a prominent decrease in tumor burden in these mice as determined by area under the curve (Figure 5B), and the tumors displayed a lower mRNA expression of MYC and BCL2 (Figure 5C). The corresponding protein levels from representative mouse tumors are provided in Figure S6 of the Supporting Information. Interestingly, knock-down of MYC and BCL2 with 05 and 76 resulted in no effect on tumor burden unless CPA was present (Figure 5) indicating the two transcription inhibitors act as chemosensitizing agents at these concentrations. The tumor burden curves for all treatment groups are shown in Figure 7A of the Supporting Information. None of the various drug regimens resulted in a significant loss of mouse weight, and while survival for mice treated with 7.5 mg/kg 05 was reduced, LD50 was not reached (Supporting Information, Figure 7B,C).

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DISCUSSION AND CONCLUSIONS These are the first studies to demonstrate that two different promoter DNA secondary structures can be targeted at the same time for direct transcription inhibition that leads to a change in a cellular phenotype. Analogous to the varied folding patterns exhibited by proteins and similar conformational features within families of proteins, there are certain structural characteristics conserved among G-quadruplexes and i-motifs and features that confer diversity. The loop regions and the number of stacked G-tetrads or cytosine+−cytosine base pairs provide a complexity of structures based on the specific nucleotide sequence and allow for selective recognition by nuclear proteins and small molecules. For example, the unique feature of a broken tetrad at the 3′-end of the PDGFRβ promoter G-quadruplex33 and the cooperative folding mechanism that leads to distinct kinetic properties of the hTERT secondary structure34 allow for additional selectivity. Furthermore, the development of specific antibodies that demonstrate selectivity for the c-KIT2 or telomeric G-quadruplex structures over other G-quadruplexes and duplex DNA, along with several lines of evidence that show protein recognition for specific Gquadruplex folding patterns such as nucleolin, supports that these structures have the potential to serve as selective therapeutic targets.6 Here, small compounds shown to have high affinity for the MYC G-quadruplex and the BCL2 i-motif lower MYC and BCL2 gene and consequent protein expression.

Figure 5. Simultaneous BCL2 and MYC repression sensitizes DLBCL tumor cells to CPA and inhibits tumor growth in mice. (A) Tumor burden of U2932 xenograft mice treated with CPA alone, 76 and 05, or CPA with 76 and 05 as measured by tumor volume (n = 5−8). Arrows indicate the 5 days of drug administration. (B) Comparison of the area under the curve for the tumor burden of each treatment combination or control (n = 5−8; solid black bars represent alternative treatment combinations for which curves are shown in Figure S6A of the Supporting Information). (C) qPCR analysis for BCL2 (top) or MYC (bottom) mRNA expression relative to TBP compared to levels in diluent-treated tumors (n = 2−3). *P < 0.05 and **P < 0.01.

Following the concurrent knock-down, we found similar levels of chemosensitization with a notable enhancement of CPA efficacy on cell viability and apoptosis in different DLBCL cell lines, regardless of the mechanism underlying the high levels of MYC and BCL2, including gene translocation or amplification. 6593

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the University of Arizona Genetics Core (Tucson, AZ) using the PowerPlex 16 System (Promega), which consists of forensic-style 15 autosomal STR loci, including 13 CODIS DNA markers (nine of the standard loci collected by ATCC and DSMZ), amelogenin, and a mouse-specific locus, every 6−10 months (Supporting Information, Table S2). Western Blot. Expression of MYC and BCL2 proteins was evaluated in cell line or tumor lysates at basal levels or following treatment with 05 and/or 76. Protein lysates were obtained and assayed for the presence of MYC or BCL2 as previously described.12 Exceptions include the use of 20 μg of total protein and the antibodies used for BCL2 (mouse, 610538, BD Laboratories) and MYC (rabbit, ab32072, Abcam) detection at 1:1000. Actin (mouse, MAB1501 from Millipore, and rabbit, PA1-183 from Thermo Fisher Scientific) was used as a loading control. Untreated and DMSO-treated cells or tumors were used as experimental controls for comparison. Real-Time Quantitative PCR. Total RNA was isolated from the same cell line or tumor preparations as for protein lysates with the Roche High Pure RNA isolation kit. Reverse transcription and realtime quantitative PCR (qPCR) were performed using the Bio-Rad iScript kit and the Bio-Rad Probe Supermix, respectively, on the BioRad CFX1000 Touch thermal cycler. All kits were used according to the manufacturer’s protocol. The Ct values obtained were normalized to TBP (TATA-binding protein) and compared to the untreated controls. TaqMan probes were used for BCL2 (Hs00608023_m1), MYC (Hs00153408_m1), and TBP (Hs00427620_m1). Dual ISH Assays. The BCL2 and MYC dual ISH probes were previously designed by Ventana Medical Systems, Inc. (VMSI) (Tucson, AZ). The dual ISH assays were performed on a VENTANA BenchMark ULTRA automated staining system in collaboration with VMSI as previously described.20,42 Cell lines were pelleted and placed into a paraffin block (Tissue Acquisition and Cellular/Molecular Analysis Shared Resource, University of Arizona), and 4 μm cuts were subjected to either the BCL2 break-apart, BCL2 amplification, or MYC amplification assay. Stained sections were viewed using an Olympus BX-61 microscope system (Olympus America Inc., Center Valley, PA) at 60× magnification (Plan Fluor, Nikon Instruments Inc., Melville, NY). Images were captured with a Nikon DS-Fil digital CCD camera and DS-L2 imaging controller (Nikon). On the basis of previously published guidelines, cell lines were scored translocation positive (+) if at least 15% of cells within a 20-informative-cell count displayed distinctly separate black (DNP, 5′-end of BCL2) and red (DIG, 3′-end of BCL2) signals.5 The FISH assays to detect MYC translocation were performed at the Mayo Cytogenetics Core, as a MYC translocation CISH probe set is unavailable at this time (Rochester, MN). Immunohistochemistry Staining and Evaluation. Paraffin slides were prepared from blocks cut at 4 μm and stained using a VENTANA BenchMark XT instrument. The primary antibodies VMSI Clone SP66 (rabbit, 790-4604) and Epitomics Clone Y69 (rabbit, 1472-1) were used to detect BCL2 and MYC following previous protocols.20,42 Briefly, the BCL2 antibody was used as supplied and incubated for 32 min at 37 °C while the MYC antibody was used at a 1:50 dilution and incubated for 36 min. Protein was detected with an OptiView DAB Detection Kit (760-700, VMSI). All slides were counterstained with hematoxylin for 4 min. Cut-offs for positivity were at least 30% of cells (within a 100 cell count) with moderate to strong DAB brown staining for BCL2 and 40% of cells with any DAB brown staining for MYC, as previously described.20,42 Cytotoxicity Assay. The percent cell viability of the cell lines was determined by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) colorimetric assay as per the manufacturer’s protocol (Promega). In brief, 100 000 cells were set and incubated with the compounds alone or in combination for 96 h. The IC50 values were calculated using the Boltzmannsigmoidal equation with variable slope in the GraphPad Prism software version 6 (GraphPad Software). Caspase-3 Activity Assay. Caspase-3 activity was measured by the ApoAlert Caspase-3 plate-based or reagent-based assay (Clontech) as per the manufacturer’s specifications.

The ability of the DNA secondary structure-targeting molecules to also exhibit a concerted effect in delaying tumor growth in an animal model demonstrates the potential for this novel approach to repress oncogene expression and sensitize this type of exceedingly difficult-to-treat lymphoma. In using these compounds as chemosensitizing agents at concentrations below their cytotoxic levels, lowering MYC and BCL2 expression could serve as an effective adjunct therapy to enhance the existing treatment regimen for dose-reduction, limit toxicity, and overcome first-line resistance. Cooperation of MYC and BCL2 deregulation is necessary for nearly full penetrance of lymphoma development and mortality in transgenic mice when compared to the overexpression of either oncogene alone.35 Not surprisingly, dual expression of MYC and BCL2 in DLBCL patients confers an aggressive clinical course with poor prognosis relative to patients with MYC- or BCL2-only tumors. In cells where the intrinsic apoptotic pathway is unperturbed, CPA, an alkylating agent, triggers cell death via DNA damage. However, in cells with elevated MYC and BCL2, this cell death signal is overcome because cell cycle checkpoints are bypassed and pro-apoptotic factors are blocked. This creates a phenomenon referred to as oncogene addiction, in which MYC inactivation causes preferential tumor cell death.36,37 Thus, inhibiting BCL2 alongside MYC has the potential to synergistically reduce the threshold for initiating the apoptotic pathway and to sensitize malignant cells to lower concentrations of DNA damaging agents such as CPA. However, it is important to also consider that while not fully understood, hyperelevated MYC expression can paradoxically induce apoptosis through regulation of p53, in which BCL2 inhibition and apoptosis is a downstream consequence. Therefore, dual inhibition may minimize this MYC pro-apoptotic function and dampen synergistic effects.38−41 The synergy observed in the SUDHL4 and U2932 cell lines, which harbor p53 mutations, but not in cells such as VAL with wild-type p53 supports this hypothesis and suggests that dual inhibition can chemosensitize tumors when MYC cannot exert is pro-apoptotic activity and BCL2 inhibition is then sufficient to induce sensitization when the MYC p53-induced apoptotic pathway is intact. Our findings provide insight into future studies aimed at developing DNA secondary structure therapeutic agents. On the basis of these favorable proof-of-principle studies, optimization of the preclinical activity and chemical development of the 05 and 76 lead compounds for testing with other drug combinations to determine an optimal multitargeted therapeutic strategy for selection of a clinical candidate is ongoing.

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EXPERIMENTAL SECTION

Compounds. Compounds 05 and 76 were obtained from the National Cancer Institute (NCI) Developmental Therapeutics Program (DTP). The purity of these compounds was confirmed by HPLC to be ≥95% (Supporting Information, Figures S8 and S9). CPA was used in the monohydrate form (Sigma, C7068), which has proven in vitro cytotoxic activity despite the prodrug form.29−31 Cell Line Maintenance and Authentication. The SUDHL4 and VAL cell lines were previously obtained from Dr. Staudt (NCI) and Dr. Rossi (University of Rochester), respectively. The U2932 and HT cells were purchased from the DSMZ (ACC633) and the American Type Culture Collection (CRL2260), respectively. All cells were cultured in 10% FBS, 5% penicillin/streptomycin-supplemented RPMI, except the SUDHL4 cells, which were cultured in 15% FBS according to ATCC recommendations. Cell lines were tested for mycoplasma every 6 months (data not shown) and authenticated by 6594

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Xenograft SCID Mouse Study. The in vivo studies were performed by The University of Arizona Cancer Center Experimental Mouse Shared Resource according to the American Association for Laboratory Animal Care guidelines under protocols approved by the University of Arizona Institutional Animal Care and Use Committee and those published by the National Institutes of Health (NIH publication no. 85-23, revised 1985). Mice were housed in microisolater cages (Allentown Caging Equipment Co.) and maintained under specific pathogen-free conditions. The mice ate NIH31 irradiated pellets (Tekland Premier) and drank autoclaved water. Mice were screened each month by ELISA serology for mycoplasma, mouse hepatitis virus, pinworms, and Sendai virus. Inhouse female or male mice 6−8 weeks of age were bled (50 μL) by cheek pouch puncture to determine mice immunoglobulin levels using ELISA, and mice with levels ≤20 μg/mL were used. SCID mice (n = 8 per treatment group) were injected with U2932 diffuse large B-cell lymphoma cells (10 × 106 cells/100 μL sterile saline, mycoplasma free, viability >91%) subcutaneously in the left flank. 76 and/or 05 in the absence or presence of CPA was administered by intraperitoneal injection every day for a total of 5 days. The control group received all three compound diluents, and the groups receiving two compounds concurrently also were administered the diluent of the absent compound. As tumors developed, subcutaneous tumors were measured for tumor volume estimation (cm3 or mm3) in accordance with the formula a2 × b/2, where a equals the smallest diameter and b is the largest diameter. The mice were pair-matched into the different control and drug groups by sorting the mice evenly based on tumor volume measurement. The Grubbs’ (maximum normed residual test) was used to detect outliers in each treatment group. Two outliers from the initial study with 05 and CPA only were found and excluded (n = 1 per group; CPA+05 2.5 mg/kg and CPA+05 5.0 mg/kg). Statistical Analysis. Statistical analyses were performed with GraphPad Prism software version 6 (GraphPad Software). Significance (P < 0.05) was evaluated using a two-tailed Student’s t test unless ́ k−Bonferroni correction multiple testing occurred, and then the Šidá for multiple comparisons was applied. Significance was considered at P ≤ 0.006 for CPA only comparisons to the nine possible combination treatments or P ≤ 0.03 for the CPA+76 or CPA+05 to the triple combination in Figure 4A, and at P ≤ 0.02 for CPA only comparisons to the three possible combination treatments in Figures 4B and 5. Data are represented as mean ± standard error from at least three independent experiments.

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Author Contributions

S.K. designed and performed all the experiments, except for the dual-chromogenic ISH and FISH assays, analyzed and interpreted data, and drafted the manuscript. A.M. performed the dual-chromogenic ISH assays and edited the manuscript. V.G. and L.H.H. synthesized compound 05, contributed to data interpretation, and edited the manuscript. L.M.R. supervised the research, contributed to data interpretation, and edited the manuscript. All authors have given approval to the final version of the manuscript. Notes

The authors declare the following competing financial interest(s): A.M. is an employee of Ventana Medical Systems, V.G. and L.H.H. have equity ownership in Reglagene. All other authors declare no potential conflicts of interest.

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ACKNOWLEDGMENTS S.K. was supported by a Lymphoma Research Foundation PostDoctoral Fellowship (2558183). The University of Arizona Experimental Mouse Shared Resource is supported by the National Institutes of Health (NIH) Cancer Center Support Grant (P30 CA023074). We thank the Mayo Cytogenetics Core, Director Patricia T. Greipp, D.O., and the individual technologists Darlene Knutson, Sara Kloft-Nelson, and Ryan Knudson for performing the FISH analysis. We thank David Bishop, Project Coordinator for L.H.H., for proofreading and editing the final version of the manuscript and figures.

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ABBREVIATIONS USED BCL2, B-cell lymphoma gene-2; ChIP, chromatin immunoprecipitation; CPA, cyclophosphamide; DLBCL, diffuse large Bcell lymphoma; FISH, fluorescent in situ hybridization; ISH, in situ hybridization; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; MYC, cellular myelocytomatosis; SCID, severe combined immunodeficient; TBP, TATA-binding protein; qPCR, quantitative PCR

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b00298. Figures showing in situ hybridization characterization of the DLBCL cells for MYC and BCL2 translocations and gene amplification; cell viability MTS curves; qPCR data for MYC expression following 6 h treatment with 05; qPCR data for MYC and BCL2; mean mouse weight, survival curves, qPCR, Western blot data from in vivo studies; and purity determination of the compunds and tables summarizing the IC50 of 05 and 76 and STR data for authentication of DLBCL cell lines (PDF) Molecular formula strings (CSV)

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REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*Telephone: (520) 626-9884. E-mail: [email protected]. edu. ORCID

Samantha Kendrick: 0000-0002-7782-7415 Laurence H. Hurley: 0000-0002-8522-450X 6595

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